TW201803888A - Specific modification of antibody with IgG-binding peptide capable of specifically and easily modifying the antibody - Google Patents

Abstract

The present invention relates to an IgG-binding peptide, an IgG-binding peptide modified with a cross-linking agent, a complex of IgG and the IgG-binding peptide modified with a cross-linking agent, and a method for producing the complex. Furthermore, a peptide is characterized in comprising the following formula I: (X1-3)-C-(X2)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-(X1-3), wherein each X is independently an arbitrary amino acids residue except the cysteine, C is a cysteine residue, H is a histidine residue, Xaa1 is a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, 2-amine suberic acid or diaminopropionic acid, G is a glycine acid residue, Xaa2 is a glutamic acid residue or an aspartic acid residue, L is a leucine residue, V is a valine residue, and W is a tryptophan residue. The expressed amino acids sequence that includes 13-17 amino acids residues can be combined with humanity IgG and/or rabbit IgG.

Description

Specific decoration of antibodies by IgG-binding peptides

The invention relates to an IgG-binding peptide, an IgG-binding peptide modified with a cross-linking agent, a complex of the IgG-binding peptide modified with a cross-linking agent and IgG, and a method for producing the complex.

In the past, antibodies have been used to detect target molecules in a large variety of research and development, and it has become extremely important in the industry as a detection reagent or diagnostic drug. In addition, antibodies have attracted attention as medicines for treating diseases due to their specificity for target molecules.

As a chemical modification to add functions to antibodies, enzymes such as alkaline phosphatase (AP) or peroxidase (HRP) (non-patent documents 1 to 2) have been used, and iodinated or chelated compounds for radioisotopes have been used. (Non-Patent Document 3) and modification using low-molecular compounds such as biotin (Non-Patent Document 4). These modifications are mainly carried out through the amino group of lysine or the thiol group of cysteine, and the activated carboxyl group, etc. Although these are specific to functional groups, they are not site-specific. The modification of the antibody to the antigen-binding site caused problems such as a decrease in the activity of the antibody, or difficulties in controlling the number of compounds to be bound. Regarding the antibody drug complex (ADC) of antibody medicine (non-patent documents 5 to 6) that has appeared in recent years, the anticancer agent site also binds to the antibody non-specifically, so the activity of the antibody itself is weakened. The difficulty in controlling the number of combinations leads to problems such as that the subsequent formulation steps become complicated.

In order to overcome such a problem, an antibody having a specific functional group specifically introduced at a use site is performed, and the antibody is modified. For example, by using genetic engineering An unnatural amino acid (Non-Patent Documents 7 to 9) or free cysteine (Non-Patent Documents 10 to 11) is introduced into a specific site, and a specific site is modified. In addition, it has been reported that natural or artificially introduced specific glutamine in antibodies is modified by using transglutaminase (TG) as a target (Non-Patent Documents 12 to 13). The structure of the introduced compound or the space environment of the target glutamine residue is greatly affected. Furthermore, modification techniques that use the sugar chain on the Fc of the antibody as a target are also used (Non-Patent Documents 14 to 15). Although these methods are site-specific, oxidative modification of the sugar chain is required, so there are changes in the reaction steps. Get complicated and so on. In this way, although the industry has gradually developed site-specific antibody modification technologies, in most cases it is necessary to modify the antibody engineering of the antibody itself. Considering the reduced function of the antibody accompanying the modification or the high cost of development, Not necessarily an advantageous approach.

[Prior technical literature] [Non-patent literature]

[Non-Patent Document 1] Imagawa, M. et al., Journal of Applied Biochemistry, 1982, 4, pp. 41-57

[Non-Patent Document 2] Hashida, S et al., Journal of Applied Biochemistry, 1984, 6, pp. 56-63

[Non-Patent Document 3] Rodwell, J. D. et al., Proceedings of the National Academy of Sciences of the United States of America, 1986, 83, pp. 2632-2636

[Non-Patent Document 4] Hermanson, G. T., Bioconjugate Techniques, The third edition, Elsevier, USA, 2013

[Non-Patent Document 5] Lewis Phillips, G. D. et al., Cancer Research, 2008, 68, pp. 9280-9290

[Non-Patent Document 6] Boyraz, B. et al., Current Medical Research and Opinion, 2013, 29, pp. 405-414

[Non-Patent Document 7] Axup, J. Y. et al., Proceedings of the National Academy of Sciences of the United States of America, 2012, 109, pp. 16101-16106

[Non-Patent Document 8] Tian, F. et al., Proceedings of the National Academy of Sciences of the United States of America, 2014, 111, pp. 1766-1771

[Non-Patent Document 9] Zimmerman, E. S. et al., Bioconjugate chemistry, 2014, 25, pp. 351-361

[Non-Patent Document 10] Shen, B. Q. et al., Nature Biotechnology, 2012, 30, pp. 184-189

[Non-Patent Document 11] Bernardes, G. J. et al., Nature Protocols, 2013, 8, pp. 2079-2089

[Non-Patent Document 12] Dennler, P. et al., Bioconjugate Chemistry, 2014, 25, pp. 569-578

[Non-Patent Document 13] Jeger, S. et al., Angewandte Chemie 2010, 49, pp. 9995-9997

[Non-Patent Document 14] Bejot, R et al., J. Labelled. Compd. Rad., 2012, 55, pp. 346-353

[Non-Patent Document 15] Zhou, Q. et al., Bioconjugate Chemistry, 2014, 25, pp. 510-520

Therefore, there is a need for a method that can specifically and easily modify antibodies.

The present inventors have reported on peptides that specifically or selectively bind to IgG (hereinafter referred to as "IgG-binding peptides") (see WO2013 / 027796 and WO2008 / 054030). In order to solve the above problems, the inventors based on the IgG binding peptide and The X-ray crystal structure of the IgG Fc complex was analyzed, and the relationship between the position of each amino acid of the IgG-binding peptide and the position of each amino acid of IgG Fc in the bound state was studied in detail. It was further found that, by introducing an amino acid capable of binding to a cross-linking agent into a peptide, and modifying the amino acid with a cross-linking agent, an IgG-binding peptide specifically modified by a cross-linking agent site was prepared, and this was used The IgG-binding peptide can modify IgG, thus completing the present invention.

That is, the present invention includes the following aspects.

(1) A peptide characterized by comprising the following formula I: (X _1-3 ) -C- (X ₂ ) -H- (Xaa1) -G- (Xaa2) -LVWC- (X _1-3 ) ( I)

(Wherein each of X is independently any amino acid residue other than cysteine, C is a cysteine residue, H is a histidine residue, Xaa1 is an lysine residue, a half Cysteine residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, G is a glycine residue, and Xaa2 is a glutamic acid residue or Asparagine residue, L is leucine residue, V is valine residue, and W is tryptophan residue)

The amino acid sequence shown contains 13 to 17 amino acid residues, and can bind to human IgG and / or rabbit IgG.

(2) The peptide according to the above (1), characterized by comprising the following formula II: (X _1-3 ) -C- (Xaa3)-(Xaa4) -H- (Xaa1) -G- (Xaa2) -LVWC- (X _1-3 ) (II)

(Wherein each of X is independently any amino acid residue other than cysteine, C is a cysteine residue, H is a histidine residue, Xaa1 is an lysine residue, a half Cysteine residue, aspartic acid residue, glutamic acid residue Group, 2-aminosuberic acid, or diaminopropionic acid, G is a glycine residue, Xaa2 is a glutamate residue or an asparagine residue, L is a leucine residue, and V is Valine residue, W is tryptophan residue, Xaa3 is alanine residue, serine residue or threonine residue, and Xaa4 is tyrosine residue or tryptophan residue)

The amino acid sequence shown contains 13 to 17 amino acid residues, and can bind to human IgG and / or rabbit IgG.

(3) The peptide according to (1) or (2) above, characterized in that it comprises the following formula III: (X _1-3 ) -CAYH- (Xaa1) -GELVWC- (X _1-3 ) (III)

(Wherein each of X is independently any amino acid residue other than cysteine, C is a cysteine residue, A is an alanine residue, Y is a tyrosine residue, and H is Histamine residue, Xaa1 is lysine residue, cysteine residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, G Is a glycine residue, E is a glutamic acid residue, L is a leucine residue, V is a valine residue, and W is a tryptophan residue)

The amino acid sequence shown contains 13 to 17 amino acid residues, and can bind to human IgG and / or rabbit IgG.

(4) The peptide according to any one of (1) to (3) above, wherein when the number is 17 amino acid residues, each of the numbers 1 to 3 and 15 to 17 from the N-terminus The amino acid residue is No. 1 amino acid residue = S, G, F, or None, and the No. 2 amino acid residue = D, G, A, S, P, homocysteine, or None, No. 3 amino acid residues = S, D, T, N, E, or R, No. 15 amino acid residues = S, T, or D, No. 16 amino acid residues = H, G, Y , T, N, D, F, homocysteine or none, amino acid residue No. 17 = Y, F, H, M or none.

(5) The peptide according to the above (4), which comprises any amino acid sequence of 1) to 15) below, wherein Xaa1 is an lysine residue, a cysteine residue, or aspartic acid Residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, Xaa2 is homocysteine.

1) DCAYH (Xaa1) GELVWCT (serial number 1)

2) GPDCAYH (Xaa1) GELVWCTFH (serial number 2)

3) RCAYH (Xaa1) GELVWCS (serial number 3)

4) GPRCAYH (Xaa1) GELVWCSFH (serial number 4)

5) SPDCAYH (Xaa1) GELVWCTFH (serial number 5)

6) GDDCAYH (Xaa1) GELVWCTFH (serial number 6)

7) GPSCAYH (Xaa1) GELVWCTFH (serial number 7)

8) GPDCAYH (Xaa1) GELVWCSFH (serial number 8)

9) GPDCAYH (Xaa1) GELVWCTHH (serial number 9)

10) GPDCAYH (Xaa1) GELVWCTFY (serial number 10)

11) SPDCAYH (Xaa1) GELVWCTFY (serial number 11)

12) SDDCAYH (Xaa1) GELVWCTFY (serial number 12)

13) RGNCAYH (Xaa1) GQLVWCTYH (serial number 13)

14) G (Xas2) DCAYH (Xaa1) GELVWCT (Xaa2) H (serial number 36)

15) RRGPDCAYH (Xaa1) GELVWCTFH (serial number 37)

(6) The peptide according to (1) or (2) above, which is characterized by comprising the following formula IV: DC- (Xaa3)-(Xaa4) -H- (Xaa1) -G- (Xaa2) -LVWCT ( IV)

(In the formula, D is an aspartic acid residue, C is a cysteine residue, H is a histidine residue, and Xaa1 is an lysine residue, a cysteine residue, and an aspartic acid residue. Group, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, G is a glycine residue, Xaa2 is a glutamic acid residue or asparagine residue, and L is leucine Acid residues, V is a valine residue, W is a tryptophan residue, T is a threonine residue, Xaa3 is an alanine residue or a threonine residue, and Xaa4 is a tyrosine residue or The amino acid sequence represented by tryptophan residue) includes 13 amino acid residues, and can bind to human IgG and / or rabbit IgG.

(7) The peptide according to the above (6), which comprises an amino acid sequence of any of 1) to 4) below, wherein Xaa1 is an lysine residue, a cysteine residue, or aspartic acid Residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid.

1) DCTYH (Xaa1) GNLVWCT (serial number 14)

2) DCAYH (Xaa1) GNLVWCT (serial number 15)

3) DCTYH (Xaa1) GELVWCT (serial number 16)

4) DCAWH (Xaa1) GELVWCT (serial number 17)

(8) A peptide characterized by comprising the following formula V: D-C- (Xaa2)-(Xaa3)-(Xaa4)-(Xaa1) -G- (Xaa5) -L- (Xaa6) -W-C-T (V)

(Where D is an aspartic acid residue, C is a cysteine residue, G is a glycine residue, L is a leucine residue, W is a tryptophan residue, and T is a threonine Acid residue, Xaa1 is lysine residue, cysteine residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, Xaa2 is propylamine Acid residue, serine residue or threonine residue, Xaa3 is tryptophan residue or tyrosine residue, Xaa4 is histamine residue, arginine residue, serine residue or Threonine residue, Xaa5 is a glutamic acid residue, an asparagine residue, an arginine residue, or an aspartic acid residue, and Xaa6 is an isoleucine residue or a valine residue The amino acid sequence represented by) contains 13 amino acid residues and is capable of binding to human IgG and / or rabbit IgG.

(9) The peptide according to any one of (1) to (8) above, wherein the peptide forms a disulfide bond between two cysteine (C) residues on the outer side or two on the outer side of the peptide The sulfur group in the cysteine residue uses the following formula:

The linkers indicated are linked.

(10) The peptide according to any one of (1) to (9) above, which is obtained by using a labeling substance. Line tag.

(11) The peptide according to any one of (1) to (10) above, which is bound with a pharmaceutical agent.

(12) The peptide according to any one of (1) to (11) above, wherein Xaa1 is an lysine residue.

(13) The peptide according to any one of (1) to (12) above, wherein Xaa1 is modified with a crosslinking agent.

(14) The peptide according to the above (13), wherein the cross-linking agent is selected from the group consisting of DSG (disuccinimidyliminoglutarate), DSS (disuccinimidyliminosuberate), and DMA (Dimethyl adipimidate dihydrochloride), DMP (dimethylheptimimidate dihydrochloride), DMS (dimethyloctimidate dihydrochloride), DTBP (3 , 3'-dithiobispropionimide dimethyl dihydrochloride) and DSP (dithiobissuccinimide iminopropionate).

(15) The peptide according to the above (14), wherein the cross-linking agent is DSG (disuccinimidyliminoglutarate) or DSS (disuccinimidyliminosuberate).

(16) A complex which is a complex of a peptide and IgG as described in any one of (13) to (15) above, and which is obtained by a cross-linking reaction between a peptide modified with a cross-linking agent and IgG form.

(17) A method for producing a complex of a peptide and an IgG, comprising mixing the peptide described in any one of (13) to (15) above with the IgG to cross-link the peptide modified with a cross-linking agent with the IgG Steps of the joint reaction.

(18) A pharmaceutical composition comprising the peptide according to any one of the above (1) to (15) or the complex according to the above (16).

(19) A method for producing a peptide in which two or more cysteine residues are linked using a linker, which comprises a peptide comprising two or more cysteine residues and the following formula:

The compounds represented (in the formula, R ₁ and R ₂ are each independently an arbitrary halogen atom) are mixed to obtain sulfur groups in two or more cysteine residues using the following formula:

Steps of peptides represented by the indicated linkers.

(20) The method according to the above (19), wherein in the compound, R ₁ and R _{2 are the} same and are Cl, Br, or I.

(21) The method according to the above (19) or (20), wherein the peptide is a peptide specified in any one of the above (1) to (8) and (10) to (15).

The cross-linker-modified IgG-binding peptide of the present invention can be added to IgG in a short period of time with almost no side reactions. Therefore, by binding various compounds to the IgG-binding peptide, various compounds can be used specifically and Easy modification of IgG. In addition, the IgG-binding peptide modified by the cross-linking agent of the present invention can directly bind to wild-type IgG and the like without modifying the sequence of the antibody molecule, and therefore does not cause a reduction in the function accompanying the genetic modification of the antibody molecule. Thus, various compounds can be bound to antibodies at a lower cost. Furthermore, the introduced compound can be bound to an IgG binding peptide in advance, and the binding reaction between the IgG binding peptide and the antibody can be performed under appropriate reaction conditions. Therefore, the step of directly reacting the introduced compound with IgG does not need to be previously performed. The necessary complex reaction can prevent the reduction of the function of the antibody caused by the reaction.

FIG. 1 (A) shows the structure of a complex of an IgG-binding peptide (C35A-3 / 15: DCAYHRGELVWCT (SEQ ID NO: 33)) and a human IgG Fc. The IgG-binding peptides are represented by a space-filled model, the IgG Fc is represented by a band model, and the sugar chain of Fc is represented by a line model. Figure 1 (B) shows the IgG structure modified by DSG (disuccinimidylglutarate). Model of the cross-linked structure of the peptide (C35A-3 / 15 (R8K): DCAYHKGELVWCT (SEQ ID NO: 34)) and IgG Fc. The backbone of the peptide is represented by a band model. Peptide-Lys8 represents the 6th lysine residue of C35A-3 / 15 (R8K), and peptide-Tyr6-Gly9 represents the 4th tyrosine residue of C35A-3 / 15 (R8K) to 7th glycine Amino acid residues. In addition, Fc-Lys248 represents Lys248 of Fc according to EU number, and Fc-Pro247-Asp249 represents Pro247 to Asp249 of Fc according to EU number.

Figure 2 shows SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis) of a mixture of labeled IgG-binding peptides and various proteins (A) and Western blot ( B) Results. In the figure, DSG indicates that an IgG-binding peptide that reacts with DSG (disuccinimidyl glutarate) is used for the test, and DSS (disuccinimidylsuberate) indicates that it will react with DSS (di Succinimide imino suberate) IgG-binding peptide was used for the test. In the figure, hIgG indicates human IgG, hIgA indicates human IgA, and HAS indicates human serum albumin.

FIG. 3 shows the results obtained by ELISA (Enzyme-Linked Immunosorbent Assay) based on the reaction mol ratio (A) and the reaction time (B) in the reaction between the labeled IgG-binding peptide and IgG. DSS R8K 0min is a trivalent HCl (pH 7.0) added to the labeled IgG-binding peptide of 10 times the molar ratio of IgG to block the NHS group and added to the wells. No DSS R8K is a biotinylated IgG-binding (R8K) peptide that does not bind to DSS. No pep indicates a control without peptide addition.

FIG. 4 shows the results obtained by measuring the reactivity of labeled IgG-binding peptides to each protein (hIgA, hIgG, BSA (bovine serum albumin)) using size exclusion chromatography. (A) shows the results obtained by measuring the reactivity of the DSS-modified IgG-binding peptide, and (B) shows the results obtained by measuring the reactivity of the DSG-modified IgG-binding peptide.

Fig. 5 (A) shows the Fc solution of human IgG added with a molar ratio of 0.5, 1.0, 2.0, or 5.0 to human IgG and DSG-modified IgG-binding peptide dissolved in DMF and stirred Then, the result of liquid chromatography after reacting at room temperature. FIG. 5 (B) shows the unreacted state (peak 2), the addition of one peptide (peak 3), and two when a human IgG is reacted with a DSG-modified IgG-binding peptide at each mole ratio. Changes in the amount of peptide appendage (peak 4).

Figure 6 shows the DSG dissolved in DMF added to the Fc solution of human IgG prepared at pH 4.0 (A), pH 5.5 (B), or pH 7.0 (C) in DMF. The modified IgG-binding peptide was stirred at room temperature, and reacted at room temperature. The reaction was unreacted after 1, 5, 10, or 30 minutes (peak 2), an addition to the peptide (peak 3), and 2 Changes in the amount of peptide addition (peak 4).

Figure 7A shows DSG-modified biotinylated IgG-binding peptide (biotinylated IgG-binding peptide) or biotinylated anti-human IgG mouse antibody (anti-hIgG mAb-biotin-labeled), and PE-labeled streptavidin (SA-PE labeling) The result obtained by detecting the binding of 4D5-Fc antibody to the HER2 antigen on breast cancer cell line SK-BR3. Figure 7B shows the results of the same experiment (biotinylated IgG binding peptide + SA-PE labeling, or anti-hIgG mAb-biotin labeling + SA-PE labeling) without the addition of 4D5-Fc antibody, and the use of 4D5-Fc antibody Results were compared with PE-labeled anti-human IgG mouse antibodies (anti-hIgG mAb-PE labeling) as a positive control.

FIG. 8A shows an azide peptide antibody and a dibenzocyclooctyne-maleimide (Dibenzocyclooctyne-maleimide) VHH (variable domain of heavy chain of heavy-chain antibody). The three main peaks (a, b, and c) obtained as a result of ion exchange chromatography after linking by clicking the reaction. FIG. 8B shows the results obtained by analyzing each of the obtained peaks by SDS-PAGE in a reduced state. Band 1 indicates the result obtained by electrophoresis of anti-HER2 human IgG antibody, band 2 indicates the result obtained by electrophoresis of anti-HER2 human IgG antibody-azide peptide, and band 3 indicates the peak a (unreacted anti-HER2). The result obtained by electrophoresis of human IgG antibody), band 4 represents the result obtained by electrophoresis of peak b (anti-HER2 human IgG antibody-1 valence VHH), and the band 5 represents the result of peak c (anti-HER2 human IgG antibody- 2 price VHH) The results obtained by performing electrophoresis, band 6 indicates the results obtained by electrophoresis of VHH, and band 7 indicates the results obtained by electrophoresis of molecular weight markers.

Figures 9A ~ C show the use of 7-AAD staining to remove cell components from dead cells, and the use of anti-HER2 human IgG antibody (Figure 9A), anti-IgA receptor VHH (C-terminal HIS tag attached) (Figure 9B), Anti-HER2 human antibody-1 valent VHH (C-terminally attached HIS tag) (Figure 9C) was used as the primary antibody, using a biotinylated anti-HIS antibody with a final concentration of 50 nM + PE-labeled SA (Serum Albumin, serum albumin) The mixture was used as a secondary antibody, and SK-BR3 cells expressing HER2 were analyzed by FACS (Fluorescence Activated Cell Sorting). Figures 9D to F show the use of anti-HER2 human antibody (Figure 9D), anti-IgA receptor VHH (C-terminal HIS tag attached) (Figure 9E), and anti-HER2 human antibody-1 valent VHH (Figure 9F) as primary antibodies, and PE-labeled anti-human IgG polyclonal antibody was used as a secondary antibody, and the results obtained by detecting the binding to HL60 cells that induced differentiation by 1.3% of DMSO (Dimethyl sulfoxide) and highly expressed IgA receptors were obtained. .

Figure 10-1 shows the culture of SK-BR3 cells in the presence of a 0-10 nM agent (Herceptin or the antibody-drug complex prepared in Example 11). The cell assay kit was used and the absorbance (Abs.) Was used. The number of cells obtained after 72 hours was evaluated. In the figure, BG (Back ground, shading) indicates a control without added cells. FIG. 10A shows the effect of anti-HER2 antibody-DM1 * 1 on SK-BR3 cells, and FIG. 10B shows the effect of anti-HER2 antibody-DM1 * 2 on SK-BR3 cells.

Figure 10-2 is the figure following figure 10-1. FIG. 10C shows the effect of anti-HER2 antibody-DM1 * 1 on C6 cells, and FIG. 10D shows the effect of anti-HER2 antibody-DM1 * 2 on C6 cells.

FIG. 11 shows a synthetic scheme of an IgG-binding peptide having an SS cross-linked structure using dichloroacetone prepared in Example 12. FIG.

FIG. 12 shows the results obtained by culturing SK-BR3 cells in the presence of a 0-500 nM agent and evaluating the number of cells after 72 hours using a cell measurement kit. Figure 12A The results are shown in the case where Herceptin or VcMMAE is added, and FIG. 12B shows the results in the case where Herceptin or the antibody-drug complex prepared in Example 12 is added.

FIG. 13A shows the results of electrophoresis based on SDS-PAGE for various IgG antibodies of human, mouse, rabbit, and rat (band 1: label, band 2: trastuzumab (IgG1), region Band 3: human IgG1, band 4: human IgG2, band 5: human IgG3, band 6: human IgG4, band 7: mouse IgG1, band 8: mouse IgG2b, band 9: mouse IgG3 (Zone 10: rabbit IgG (multiple antibody), zone 11: rat IgG1, zone 12: rat IgG2b, zone 13: rat IgG2c). FIG. 13B shows the results obtained by transferring the gel after electrophoresis to a PVDF (polyvinylidene fluoride, polyvinylidene fluoride) film, and using Western blotting using biotin-labeled IgG-binding peptide and HRP-labeled streptavidin. .

<IgG-binding peptide>

As used herein, "IgG" refers to mammals, primates such as humans and chimpanzees, experimental animals such as rats, mice, and rabbits, livestock animals such as pigs, cattle, horses, sheep, and goats, and The IgG of pets such as dogs and cats is preferably human IgG (IgG1, IgG2, IgG3, or IgG4). The IgG in this specification is further preferably human IgG1, IgG2, or IgG4, or rabbit IgG, and particularly preferably human IgG1, IgG2, or IgG4.

In one aspect, the present invention relates to a peptide, which is characterized by comprising the following formula I: (X _1-3 ) -C- (X ₂ ) -H- (Xaa1) -G- (Xaa2) -LVWC- (X _1-3 ) (I)

(Wherein each of X is independently any amino acid residue other than cysteine, C is a cysteine residue, H is a histidine residue, Xaa1 is an lysine residue, a half Cysteine residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, G is a glycine residue, Xaa2 is a glutamic acid residue or an asparagine residue, L is a leucine residue, V is a valine residue, and W is a tryptophan residue)

The amino acid sequence shown contains 13 to 17 amino acid residues, and can bind to human IgG and / or rabbit IgG.

In the above formula, the expression of X _1-3 at the N-terminus or C-terminus means that an independent and arbitrary amino acid residue X other than cysteine (C or Cys) exists continuously for 1 to 3, and the amine constituting it The amino acid residues are the same or different residues, and it is preferred that the residues include all three sequences that are not the same. Similarly, X ₂ also means that there are two independent and arbitrary amino acid residues X other than cysteine (C or Cys), and the amino acid residues constituting it are the same or different residues, It is preferred to include a sequence in which the two consecutive amino acid residues are not the same.

The two cysteine residues of formula I can be combined by disulfide bonds to form a cyclic peptide. Generally, in a peptide of Formula I, the two outer cysteine residues are bound by a disulfide bond. Alternatively, in the peptide of formula I, the sulfur group in the two outer cysteine residues can also use the following formula:

The linkers indicated are linked. The dotted line portion in the above formula means a bonding portion with a sulfur group. This linker is more stable to reduction reactions and the like than ordinary disulfide bonds. This peptide can be produced, for example, by the method described in the following "Method for producing a peptide in which a cysteine residue is linked by a linker".

The peptides represented by the formulae I 'and I "in the amino acid sequence of the peptide of the formula I and further specifically the amino acid residue X are shown below.

That is, the peptide represented by Formula I 'is characterized by comprising (X _1-3 ) -C- (X ₁ ) -YH- (Xaa1) -GNLVWC- (X _1-3 ) (I')

(Wherein each of X is independently any amino acid residue other than cysteine, C is a cysteine residue, Y is a tyrosine residue, H is a histidine residue, Xaa1 Is an amino acid residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid, or a diaminopropionic acid, and G is a glycine residue, N is asparagine residue, L is leucine residue, V is valine residue, and W is tryptophan residue)

The amino acid sequence shown contains 13 to 17 amino acid residues, and can bind to human IgG and / or rabbit IgG.

The peptide represented by Formula I "is characterized by comprising (X _1-3 ) -CA- (X ₁ ) -H- (Xaa1) -GELVWC- (X _1-3 ) (I")

(Wherein each of X is independently any amino acid residue other than cysteine, C is a cysteine residue, A is alanine residue, H is a histamine residue, and Xaa1 is Leucine residue, cysteine residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, G is a glycine residue, E Is a glutamic acid residue, L is a leucine residue, V is a valine residue, and W is tryptophan residue)

The amino acid sequence shown contains 13 to 17 amino acid residues, and can bind to human IgG and / or rabbit IgG.

In addition, a peptide represented by the formula II in which the amino acid residue X is specified in the amino acid sequence of the peptide of the formula I is shown below.

That is, the peptide represented by Formula II is characterized by including (X _1-3 ) -C- (Xaa3)-(Xaa4) -H- (Xaa1) -G- (Xaa2) -LVWC- (X _1-3 ) ( II)

(Wherein each of X is independently any amino acid residue other than cysteine, C is a cysteine residue, H is a histidine residue, Xaa1 is an lysine residue, a half Cysteine residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, G is a glycine residue, and Xaa2 is a glutamic acid residue or Asparagine residue, L is leucine residue, V is valine residue, W is tryptophan residue, Xaa3 is alanine residue, serine residue or threonine residue, And Xaa4 is a tyrosine residue or a tryptophan residue)

The amino acid sequence shown contains 13 to 17 amino acid residues, and can bind to human IgG and / or rabbit IgG.

In the case where 17 amino acid residues are set in the amino acid sequence of the peptide of the formula I ', formula I "and formula II described above, Nos. 1 and 2 and 16 and 17 from the N-terminus The amino acid residue X can also be deleted. This peptide contains 13 amino acids in length.

The so-called "in the case of 17 amino acid residues" as used in the present specification is used to refer to peptide residues of formula I when the amino acid residue of a peptide is referred to by the amino acid number. The 17-residue N-terminus, which is the longest amino acid in length, is numbered from No. 1 to No. 17 in order to simplify the expression.

In addition, a peptide represented by the formula III in which the amino acid residue X is specified in the amino acid sequence of the peptide of the formula I is shown below.

That is, the peptide represented by Formula III is characterized by including (X _1-3 ) -CAYH- (Xaa1) -GELVWC- (X _1-3 ) (III)

(Wherein each of X is independently any amino acid residue other than cysteine, C is a cysteine residue, A is an alanine residue, Y is a tyrosine residue, and H is Histamine residue, Xaa1 is lysine residue, cysteine residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, G Is a glycine residue, E is a glutamic acid residue, L is a leucine residue, V is a valine residue, and W is a tryptophan residue)

The amino acid sequence shown contains 13 to 17 amino acid residues, and can bind to human IgG and / or rabbit IgG.

In the case where 17 amino acid residues are set in the amino acid sequence of the peptide of the above formula III, the first and second amino acids from the N-terminus, and the amino acid residues 16 and 17 are X It can also be deleted, and this peptide contains 13 amino acids in length.

Furthermore, when the amino acid residues other than the cysteine (C) of the amino acid sequence of each of the above-mentioned peptides are set to 17 amino acid residues, from 1 to 3, Each amino acid residue of No. 5, 6, 15-17 is preferably selected from the following. Here, the capital letters Horse alphabet is a single character expression of amino acid: No. 1 amino acid residue = S, G, F or none, No. 2 amino acid residue = D, G, A, S, P, homocysteine Amino acid or none, amino acid residue No. 3 = S, D, T, N, E or R, amino acid residue No. 15 = S, T or D, amino acid residue No. 16 = H, G, Y, T, N, D, F, homocysteine or none, amino acid residue No. 17 = Y, F, H, M or none.

Amino acid residue No. 5 = A or T, and amino acid residue No. 6 = Y or W.

In addition, peptides represented by the formula IV in which the amino acid residue X is specified in the amino acid sequence of the peptide of the formula I are shown below.

That is, the peptide represented by Formula IV is characterized by including D-C- (Xaa3)-(Xaa4) -H- (Xaa1) -G- (Xaa2) -L-V-W-C-T (IV)

(In the formula, D is an aspartic acid residue, C is a cysteine residue, H is a histidine residue, and Xaa1 is an lysine residue, a cysteine residue, and an aspartic acid residue. Group, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, G is a glycine residue, Xaa2 is a glutamic acid residue or asparagine residue, and L is leucine Acid residues, V is a valine residue, W is a tryptophan residue, T is a threonine residue, Xaa3 is an alanine residue or a threonine residue, and Xaa4 is an amino acid sequence represented by a tyrosine residue or a tryptophan residue) containing 13 amino acid residues, and can bind to human IgG and / or rabbit IgG.

Some specific examples of peptides of formula I are listed in the following 1) to 19), but are not limited by them: 1) DCAYH (Xaa1) GELVWCT (sequence number 1), 2) GPDCAYH (Xaa1) GELVWCTFH (sequence number 2), 3) RCAYH (Xaa1) GELVWCS (serial number 3), 4) GPRCAYH (Xaa1) GELVWCSFH (serial number 4), 5) SPDCAYH (Xaa1) GELVWCTFH (serial number 5), 6) GDDCAYH (Xaa1) GELVWCTFH ( Sequence numbers 6), 7) GPSCAYH (Xaa1) GELVWCTFH (Sequence number 7), 8) GPDCAYH (Xaa1) GELVWCSFH (Sequence number 8), 9) GPDCAYH (Xaa1) GELVWCTHH (Sequence number 9), 10) GPDCAYH (Xaa1) GELVWCTFY (serial number 10), 11) SPDCAYH (Xaa1) GELVWCTFY (serial number 11), 12) SDDCAYH (Xaa1) GELVWCTFY (serial number 12), 13) RGNCAYH (Xaa1) GQLVWCTYH (serial number 13), 14) G ( Xaa2) DCAYH (Xaa1) GELVWCT (Xaa2) H (serial number 36), 15) RRGPDCAYH (Xaa1) GELVWCTFH (serial number 37), 16) DCTYH (Xaa1) GNLVWCT (serial number 14), 17) DCAYH (Xaa1) GNLVWCT (Sequence number 15), 18) DCTYH (Xaa1) GELVWCT (sequence number 16), and 19) DCAWH (Xaa1) GELVWCT (sequence number 17), (where Xaa1 is an lysine residue, a cysteine residue Group, aspartic acid residue, glutamic acid residue, 2-aminooctyl Diacid, or diaminopropionic acid, Xaa2 is homocysteine, preferably homocysteine forms disulfide bonds with each other).

As preferred specific examples of the peptide of formula I, 1) DCAYH (Xaa1) GELVWCT (sequence number 1), 2) GPDCAYH (Xaa1) GELVWCTFH (sequence number 2), 13) RGNCAYH (Xaa1) GQLVWCTYH (sequence number) 13), 14) G (Xaa2) DCAYH (Xaa1) GELVWCT (Xaa2) H (Serial Number 36), and 15) RRGPDCAYH (Xaa1) GELVWCTFH (Serial Number 37)

(In the formula, Xaa1 is an lysine residue, and Xaa2 is a homocysteine, and it is preferred that the cysteines and / or the homocysteines form disulfide bonds with each other).

The peptide of the present invention is characterized by comprising the following formula V: D-C- (Xaa2)-(Xaa3)-(Xaa4)-(Xaa1) -G- (Xaa5) -L- (Xaa6) -W-C-T (V)

(Where D is an aspartic acid residue, C is a cysteine residue, G is a glycine residue, L is a leucine residue, W is a tryptophan residue, and T is a threonine Acid residue, Xaa1 is lysine residue, cysteine residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, Xaa2 is propylamine Acid residue, serine residue or threonine residue, Xaa3 is tryptophan residue or tyrosine residue, Xaa4 is histamine residue, arginine residue, serine residue or Threonine residue, Xaa5 is a glutamic acid residue, an asparagine residue, an arginine residue, or an aspartic acid residue, and Xaa6 is an isoleucine residue or a valine residue The amino acid sequence represented by) contains 13 amino acid residues as a generalized primary structure, and can interact with human IgG and / or rabbit IgG Combined.

The two cysteine residues of formula V can be combined by disulfide bonds to form a cyclic peptide. Generally, the two cysteine residues on the outside of the peptide of formula V are bound by disulfide bonds. Alternatively, in the peptide of formula V, the sulfur group in the two outer cysteine residues can also use the following formula:

The linkers indicated are linked. The dotted line portion in the above formula means a bonding portion with a sulfur group. This linker is more stable to reduction reactions and the like than ordinary disulfide bonds. This peptide can be produced, for example, by the method described in the following "Method for producing a peptide in which a cysteine residue is linked by a linker".

Some specific examples of peptides of formula V are listed in the following 20) ~ 31), but are not limited by them: 20) DCTYT (Xaa1) GNLVWCT (sequence number 18), 21) DCAYT (Xaa1) GNLVWCT (sequence number 19), 22) DCSYT (Xaa1) GNLVWCT (serial number 20), 23) DCTWT (Xaa1) GNLVWCT (serial number 21), 24) DCTYH (Xaa1) GNLVWCT (serial number 22), 25) DCTYR (Xaa1) GNLVWCT ( (Serial numbers 23), 26) DCTYS (Xaa1) GNLVWCT (serial number 24), 27) DCTYT (Xaa1) GNLVWCT (serial number 25), 28) DCTYT (Xaa1) GELVWCT (serial number 26), 29) DCTYT (Xaa1) GRLVWCT (serial number 27), 30) DCTYT (Xaa1) GDLVWCT (serial number 28), and 31) DCTYT (Xaa1) GNLIWCT (SEQ ID NO: 29), (where Xaa1 is an lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, 2-aminooctyl Diacid, or diaminopropionic acid).

As described above, the peptide of the above formula related to the present invention is characterized by having at least two cysteine (C) residues separated in each amino acid sequence, and The cysteine residues are arranged in a way that disulfide bonds are formed between the residues. The preferred peptide is also that two cysteine residues are combined by a disulfide bond to form a cyclic peptide, and each cysteine The residue has 1 or 2 amino acid residues other than cysteine on the N-terminal side and C-terminal side. When there are 1 or 2 amino acid residues on the N-terminal side and the C-terminal side of each cysteine residue, the case where it is set to 17 amino acid residues is 1 to 1 from the N-terminus. 2. Each amino acid residue of Nos. 16 to 17 is exemplified above.

As described above, in the peptide of the present invention, Xaa1 is an amino acid and a diamine group of proteins such as lysine residue, cysteine residue, aspartic acid residue, and glutamic acid residue. Non-proteins such as propionic acid and 2-aminosuberic acid constitute an amino acid, and preferably an amino acid residue. Xaa1 is preferably capable of being modified with the following crosslinking agent. In the present specification, the "non-protein constituting amino acid" refers to an amino acid that cannot be used to constitute a protein in a living body. In order to improve the site specificity when the peptide of the present invention is modified with a cross-linking agent, the peptide of the present invention preferably has absolutely no or almost no (e.g., only one or two) in the sequence of Residues. For example, in the case where Xaa1 is an lysine residue, the peptide of the present invention preferably has no or almost no lysine residue in the sequence other than Xaa1.

The binding affinity of the peptide of the present invention to human IgG is about 10 times or more, preferably about 50 times or more, and more preferably about 200 times compared to the binding affinity of other human immunoglobulins (IgA, IgE, IgM). Times more. The dissociation constant (Kd) related to the binding of the peptide of the present invention to human IgG can be determined by surface plasmon resonance spectrum analysis (for example, using the BIACORE system), for example, it is less than 1 × 10 ^-1 M ~ 1 × 10 ^-3 M, preferably less than 1 × 10 ^-4 M, and more preferably less than 1 × 10 ^-5 M.

The IgG-binding peptide of the present invention binds to the Fc region of IgG. The IgG-binding peptide of the present invention is as shown in the following examples. In the above Xaa1, a specific region with the IgG Fc, that is, the Lys248 residue according to the Eu number in the human IgG Fc (hereinafter, also briefly described in this specification) "Lys248", which corresponds to the 18th residue of human IgG CH2 (SEQ ID NO: 30) or Lys246 residue (hereinafter, abbreviated as "Lys246" in this specification, which corresponds to human IgG CH2 (SEQ ID NO: 30) Residue 16), preferably Lys248 is close.

The peptides of the present invention can be prepared by conventional peptide synthesis methods such as liquid phase synthesis method and solid phase synthesis method, peptide synthesis using an automatic peptide synthesizer, etc. (Kelley et al., Genetics Engineering Principles and Methods, Setlow, JKeds ., Plen μm Press NY. (1990) Vol. 12, p. 1-19; Stewart et al., Solid-Phase Peptide Synthesis (1989) WHFreeman CO .; 1985) 82: p. 5132, "Creative Chemistry Experiment Course 1 Protein IV" (1992), edited by the Japan Biochemical Society, Tokyo Chemical Co., Ltd.). Alternatively, the peptide may be produced by a genetic recombination method, a phage display method, or the like using a nucleic acid encoding the peptide of the present invention. For example, DNA (deoxyribonucleic acid) encoding the amino acid sequence of the peptide of the present invention can be incorporated into a expression vector, the expression vector can be introduced into a host cell, and the cell can be cultured to produce a target peptide. . The produced peptides can be prepared by common methods such as gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse phase column chromatography, and high performance liquid chromatography (HPLC). Analysis), such as chromatography, ammonium sulfate separation, ultrafiltration, and immunoadsorption methods.

In peptide synthesis, for example, amino acids that are intended to protect the α-amino group to be bonded to each amino acid (whether natural or unnatural) and functional groups other than α-carboxyl groups, and -A peptide bond formation reaction is performed between the amine group and the α-carboxyl group. Generally, the carboxyl group of the amino acid residue at the C-terminus of the peptide is bound to the solid phase in advance via an appropriate spacer or linker. Selectively remove the protective group at the amino terminal of the dipeptide thus obtained, and remove it from the next amino acid A peptide bond is formed between the α-carboxyl groups. This operation is carried out continuously to produce side-group protected peptides, and finally all protecting groups are removed and separated from the solid phase. The details of the type of the protecting group, the protecting method, and the peptide binding method are described in detail in the aforementioned literature.

The production by the genetic recombination method can be performed, for example, by inserting the DNA encoding the peptide of the present invention into an appropriate expression vector, introducing the vector into an appropriate host cell, and culturing the cell, either inside or outside the cell. The method is performed by recovering the target peptide. The vector is not limited, and examples include vectors such as plastids, phages, adherent plastids, phagemids, and viruses.

The plastid vector is not limited, and examples include plastids derived from E. coli (for example, pET22b (+), pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), plastids derived from Bacillus subtilis (e.g. pUB110, pTP5, etc.), and yeast-derived plastids (eg, YEp13, YCp50, etc.).

The phage vector is not limited, and examples thereof include T7 phage display vectors (T7Select10-3b, T7Select1-1b, T7Select1-2a, T7Select1-2b, T7Select1-2c, etc. (Novagen)), and lambda phage vectors (Charon4A, Charon21A, EMBL3). , EMBL4, λgt10, λgt11, λZAP, λZAPII, etc.). The viral vector is not limited, and examples thereof include retroviruses, adenoviruses, adeno-associated viruses, vaccinia virus, and animal viruses such as Sendai virus, and insect viruses such as baculovirus. The adhesive plastid carrier is not limited, and examples thereof include Lorist 6, Charomid 9-20, and Charomid 9-42.

The phagemid vector is not limited, and examples thereof include pSKAN, pBluescript, pBK, and pComb3H. The vector may be capable of expressing the target DNA in a manner including a regulatory sequence, a selection marker for selecting a vector containing the target DNA, a multiple selection site for inserting the target DNA, and the like. Such regulatory sequences include a promoter, a promoter, a terminator, an S-D sequence or a ribosome binding site, an origin of replication, a poly A site, and the like. As the selection marker, for example, an ampicillin-resistant gene or a neomycin-resistant gene can be used. Factor, concomycin resistance gene, and dihydrofolate reductase gene. The host cells used to introduce the vector are bacteria such as E. coli or Bacillus subtilis, yeast cells, insect cells, animal cells (such as mammalian cells), and plant cells, and the transformation or transfection of such cells includes calcium phosphate Method, electroporation method, lipofection method, particle gun method, and PEG (Polyethylene glycol, polyethylene glycol) method. The culture of the transformed cells is performed according to a usual method used for the culture of the host organism. For example, the culture medium of microorganisms such as E. coli or yeast cells contains a carbon source, a nitrogen source, an inorganic salt, and the like that can be metabolized by the host microorganism.

In order to facilitate the recovery of the peptide of the present invention, it is preferred that the peptide produced by expression is secreted outside the cell. This can be performed by binding DNA encoding a peptide sequence capable of secreting a peptide from the cell to the 5 'end side of DNA encoding a peptide of interest. The fusion peptide transferred to the cell membrane is cleaved by the signal peptidase, secretes the target peptide and is released into the culture medium. Alternatively, the target peptide accumulated in the cell may be recovered. In this case, the cells are physically or chemically destroyed, and the target peptide is recovered using a protein purification technique.

Therefore, the present invention further relates to a nucleic acid encoding the peptide of the present invention. Here, the nucleic acid includes DNA or RNA (RibonucleicAcid, ribonucleic acid) (for example, mRNA (Messenger RNA, messenger ribonucleic acid)).

When the IgG-binding peptide of the present invention is fused with other proteins, after preparing the IgG-binding peptide and other proteins separately, a linker can be used to fuse the IgG-binding peptide with the protein if necessary, or by genetic recombination if necessary. Add appropriate linkers and make them as fusion proteins. In this case, it is preferable to prepare a fusion protein in such a manner that the binding property between the IgG-binding peptide and IgG of the present invention is not impaired.

<Peptide Modified with Crosslinking Agent>

In one aspect, the aforementioned IgG-binding peptide in the present invention is preferably modified with a cross-linking agent.

As described above, the IgG-binding peptide of the present invention is as shown in the following examples. In Xaa1, the specific region with the IgG Fc, that is, the human IgG Fc is encoded according to Eu. Lys248 or Lys246, preferably Lys248 is close. Therefore, by using a cross-linking agent to modify the Xaa1 of the IgG-binding peptide of the present invention and cross-linking it with IgG, it can be between Xaa1 of the IgG-binding peptide and Lys248 or Lys246, preferably Lys248 of the IgG Fc. Site-specific topographic cross-linked structure. As described above, by modifying Xaa1 of the IgG-binding peptide of the present invention with a cross-linking agent and various compounds, and performing a cross-linking reaction with IgG, various compounds can be specifically and simply introduced into IgG. In addition, according to the present invention, since a compound can be introduced through an IgG-binding peptide, a compound having various structures can be introduced into IgG. Furthermore, the method of the present invention has the advantage of lowering the possibility of reducing the function of the antibody because the product obtained has a higher yield and is not accompanied by modification of the antibody itself.

The IgG-binding peptide of the present invention can also be used for animal IgG, preferably mammalian IgG. In this case, as long as it is a practitioner who has read the present specification, the position in the IgG to which the IgG-binding peptide of the present invention binds can be obtained by pairing the sequence of, for example, human IgG with the sequence of IgG of another animal. Easily specified.

In the present invention, the so-called "crosslinking agent" is a chemical substance used to link the IgG-binding peptide of the present invention with IgG Fc by covalent bonding. The cross-linking agent of the present invention can be appropriately selected as long as it is a practitioner, and it can be set to have at least two amino acids (e.g., lysine residue, cysteine residue, asparagine) Compounds at the sites where amino acid residues, glutamic acid residues, 2-aminosuberic acid, or diaminopropionic acid, and arginine are bonded. The examples are not limited, and examples thereof include DSG (disuccinimidyl glutarate, disuccinimidyl glutarate), DSS (disuccinimidyl suberate, disuccinimidyl suberate), etc. More than succinimide imino crosslinkers, DMA (dimethyl adipimidate‧2 HCl, dimethyl adipimidate dihydrochloride), DMP (dimethyl pimelimidate‧2 HCl, dimethylpimelimidate ‧ HCl Ester dihydrochloride), DMS (dimethyl suberimidate‧2 HCl, dimethyl suberimidate dihydrochloride), and other crosslinking agents containing preferably two or more imine moieties, and DTBP ( dimethyl 3,3'- dithiobispropionimidate‧2HCl, 3,3'-dithiobispropionimide dimethyl dihydrochloride) and DSP (dithiobis (succinimidyl propionate), dithiobissuccinimide propionate) have SS bonds Of crosslinking agent.

The IgG-binding peptide of the present invention can also be modified by other functional substances, such as antibodies such as IgA or VHH, labeling substances, and / or other agents. IgG-binding peptides can be linked to other functional substances by methods known to practitioners, such as the reaction of azide and dibenzocyclooctyne, or the reaction of cis-butenylimine and thiol. And so on. In the case of labeling with a labeling substance, the IgG-binding peptide of the present invention forms a complex with IgG, whereby IgG can be detected or quantified via the labeling substance. The labeling substance is not limited, and includes, for example, a fluorescent pigment, a chemiluminescent pigment, a radioisotope (such as a radioactive iodine or a chelate of a radioisotope metal ion, such as DOTA (1,4,7,10-tetraazacyclododecane-1,4,7 , 10-tetraacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or deferoxamine chelate), and biotin and GFP (green Fluorescent proteins such as fluorescent proteins, luminescent proteins, and enzymes such as peroxidase. Examples of preferred labeling substances are luciferins and fluorescein derivatives such as FITC (Fluorescein Isothiocyanate), Rhodamine derivatives such as rhodamine and tetramethyl rhodamine, and fluorescent pigments such as Texas Red. In the case where the peptide of the present invention is modified with other drugs, the drug is not limited. Examples include auristatin such as auristatin E, Maytansine, Emtansine, and doxorubicin. ), Bleomycin (Bleomycin), or derivatives thereof; and drugs that can bind to receptors on the blood-brain barrier and move to the central nervous system, or can bind to cancer cells to make antibodies Targeting agents such as drugs moving into cells. When a pharmaceutical agent is linked, the IgG-binding peptide of the present invention can improve the therapeutic effect of a disease by forming a complex with IgG used as a pharmaceutical antibody, for example.

The cross-linking agent-modified IgG-binding peptide of the present invention can be performed, for example, by using an IgG-binding peptide and a cross-linking agent obtained according to the method described in the above item of "IgG-binding peptide". Made by reaction. In this case, it is necessary to specifically modify the side chain of the amino acid residue of the aforementioned Xaa1 in the IgG-binding peptide, and this case can be performed, for example, by selecting a type of Xaa1 and a combination of a cross-linking agent. For example, DSS or DSG-containing crosslinkers containing succinimide will react with the side chain of the lysine residue and the primary amine present at the N-terminus of the polypeptide. Therefore, by blocking the N-terminus of the IgG-binding peptide And react with DSS or DSG, DSS or DSG can be used to specifically modify only the side chain of the lysine residue. As long as it is a practitioner, a combination of such an amino acid residue and a crosslinking agent can be appropriately selected.

The cross-linking agent-modified IgG-binding peptide of the present invention can also be produced, for example, by performing peptide synthesis using an amino acid residue modified with a cross-linking agent. Similarly, when a IgG-binding peptide is modified with a labeling substance and / or other drugs, the labeled substance and / or can be prepared by peptide synthesis using an amino acid residue to which these modifications are added. IgG-binding peptides modified by other agents.

<Crosslinking reaction>

In one aspect, the present invention relates to a method for producing a complex of IgG-binding peptide and IgG including the step of mixing the IgG-binding peptide modified with a cross-linking agent of the present invention with IgG. According to this step, a cross-linking reaction can be generated between the IgG-binding peptide modified with the cross-linking agent and the IgG. The cross-linking reaction can be generated specifically at the site between the amino acid residue of Xaa1 of the IgG-binding peptide and Lys248 or Lys246, preferably Lys248 of IgG Fc.

The conditions for this mixing step are not particularly limited as long as they are performed under conditions in which a crosslinking reaction occurs between the IgG-binding peptide and IgG of the present invention. For example, the reaction can be performed by mixing the IgG-binding peptide of the present invention and IgG in an appropriate buffer at room temperature (for example, about 15 ° C to 30 ° C). In this mixing step, an appropriate amount of a catalyst to promote the cross-linking reaction may be added as needed.

The mixing ratio of the IgG-binding peptide and IgG of the present invention in this mixing step is not particularly limited. The molar ratio of the IgG-binding peptide to IgG of the present invention can be set to, for example, 1: 1 to 20: 1, preferably 2: 1 to 20: 1, or 5: 1 to 10: 1.

The mixing time (reaction time) in this mixing step is not limited as long as a cross-linking reaction occurs between the IgG-binding peptide of the present invention and IgG, and it can be set to, for example, 1 minute to 5 hours, preferably 10 minutes. ~ 2 hours or 15 minutes ~ 1 hour.

The method for producing a complex of the IgG-binding peptide and IgG of the present invention may optionally further include the step of separating impurities such as unreacted IgG-binding peptide, IgG, and reagents from the mixture having undergone the above steps, The complex is refined. This step can be performed by a method known in the art, such as gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse phase column chromatography, and chromatography such as HPLC.

<Complex>

In one aspect, the invention relates to a complex of an IgG-binding peptide and IgG of the invention. The complex can be formed by the above-mentioned crosslinking reaction. Therefore, the present invention preferably relates to an IgG-binding peptide in which the amino acid residue of Xaa1 of the IgG-binding peptide is specifically bound to the IgG Fc Lys248 or Lys246, preferably Lys248, via a crosslinker site. Complex with IgG.

The complex system of the present invention is formed by a site-specific cross-linking reaction, so the cross-linking reaction is less likely to have a negative effect on the activity of IgG. In addition, by linking the modified IgG-binding peptide to IgG, new functionality can be added to IgG. For example, if an IgG-binding peptide modified with a labeling substance is bound to IgG, IgG can be detected or quantified via the labeling substance. Examples of the labeling substance are as described above, and therefore description thereof is omitted here. For example, if the IgG-binding peptide modified with a drug is bound to IgG as a pharmaceutical antibody, the therapeutic effect of IgG disease can be improved. Examples of medicines are as described above, and therefore descriptions are omitted here.

<Pharmaceutical composition or diagnostic agent>

In one aspect, the present invention relates to a pharmaceutical composition or diagnosis comprising the above-mentioned IgG-binding peptide, the above-mentioned cross-linker-modified IgG-binding peptide, or the above-mentioned cross-linker-modified IgG-binding peptide and IgG complex Agent. When it is contained in a pharmaceutical composition, the IgG-binding peptide is preferably modified with the above-mentioned agent, for example, and when it is contained in a diagnostic agent, The IgG-binding peptide is preferably modified with, for example, the above-mentioned labeling substance.

The diseases to be targeted for the pharmaceutical composition and diagnostic agent of the present invention are not limited, and examples include diseases or disorders that can be targeted by antibodies, and preferably cancer, inflammatory diseases, infectious diseases, and neurodegenerative diseases. disease.

The pharmaceutical composition of the present invention can be administered by oral or parenteral administration (for example, intravenous injection, intramuscular injection, subcutaneous administration, intraperitoneal administration, rectal administration, or transmucosal administration, etc.). In addition, the pharmaceutical composition of the present invention can be prepared into an appropriate dosage form depending on the route of administration. Specifically, it can be prepared into various preparations such as granules, lozenges, pills, capsules, syrups, emulsions, suspensions, intravenous injections, arterial injections, or injections, drips, external preparations, or suppositories for intramuscular injection. form. As for the administration method and dosage form, as long as it is a practitioner, it can be appropriately selected according to the sex, age, weight, symptoms, etc. of the patient.

The pharmaceutical composition of the present invention can be formulated according to a commonly used method (for example, see Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA), and can also include a pharmaceutically acceptable carrier or additive.

Examples of carriers and pharmaceutical additives that can be included in the pharmaceutical composition of the present invention include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, Sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, sansyl gum, acacia, casein, Agar, polyethylene glycol, diglycerin, glycerol, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, and permitted as pharmaceutical additives Surfactants and so on.

The actual additive is selected from the above alone or appropriately combined according to the dosage form of the pharmaceutical composition of the present invention, but is not limited to these. For example, for preparations for injection In this case, the IgG-binding protein or the complex of IgG-binding protein and IgG of the present invention can be used in a solution such as physiological saline, buffer solution, glucose solution, etc., and a container adsorption preventing agent such as Tween can be added to the solution. 80, Tween 20, gelatin, human serum albumin, etc. Alternatively, in order to prepare a dosage form that is dissolved and reconstituted before use, it may be freeze-dried. As a stabilizer for freeze-drying, for example, sugar alcohols such as mannitol and glucose and / or sugars may be used.

The effective administration amount and administration interval of the pharmaceutical composition of the present invention can be appropriately selected according to the sex, age, weight, and symptoms of the patient.

The period of administration of the pharmaceutical composition of the present invention can be administered either preventively or therapeutically before or after the clinical symptoms of the above-mentioned diseases occur.

<Method for producing a peptide in which cysteine residues are linked by a linker>

In one aspect, the present invention relates to a method for producing a peptide in which a cysteine residue is linked using a linker. The method includes the following steps, that is, a peptide comprising two or more, preferably two cysteine residues and the following formula:

The compounds represented (in the formula, R ₁ and R ₂ are each independently an arbitrary halogen atom) are mixed to obtain two or more, preferably two sulfur groups in the cysteine residue using the following formula:

A peptide formed by the indicated linkers. The dotted line portion in the above formula means a bonding portion with a sulfur group. The peptides linked by cysteine residues using this linker are effective It should wait more stable than peptides linked by usual disulfide bonds.

Among the above compounds, R ₁ and R _{2 are} preferably selected from the group consisting of F, Cl, Br, and I, and further preferably selected from the group consisting of Cl, Br, and I. R ₁ and R _{2 are} preferably the same, and more preferably R ₁ and R ₂ are both Cl.

The conditions of the mixing step in this method are not particularly limited as long as they are conditions that cause a linking reaction between the cysteine residues of the peptide. For example, the reaction can be performed by mixing the peptide and the compound in an appropriate buffer, such as a buffer solution containing guanidine chloride, at room temperature (for example, about 15 ° C to 30 ° C). This mixing step may be carried out by adding an appropriate amount of a catalyst that promotes the linking reaction as needed.

The mixing ratio of the peptide to the compound in the mixing step of the method is not particularly limited. The molar ratio of the peptide to the compound can be set to, for example, 1: 0.2 to 1:10, preferably 1: 0.5 to 1: 5, or 1: 1 to 1: 2.

The mixing time (reaction time) in this mixing step is not limited as long as a linking reaction occurs between the cysteine residues of the peptide, and it can be, for example, 1 minute to 5 hours, preferably 10 minutes to 2 Hour or 15 minutes ~ 1 hour.

The method of the present invention may optionally further include a step of separating impurities such as unreacted peptides and compounds from the mixture having undergone the above steps, and purifying the peptide to which the cysteine residue is linked. This step can be performed by a method known in the art, such as gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse phase column chromatography, and chromatography such as HPLC.

The type of the peptide used in this method is not particularly limited as long as it is a cysteine residue that can be linked using the above-mentioned compound, and examples thereof include the IgG-binding peptides described in this specification or those described in the specification of WO2013 / 027796. Documented peptide. Examples of peptides described in the specification of WO2013 / 027796 include peptides in which the Xaa1 residue of the IgG-binding peptide described in this specification is replaced with an arginine residue (R).

[Example]

[Example 1: Analysis of X-ray crystal structure of IgG-binding peptide and IgG complex]

<Method>

(1) Preparation of IgG-binding peptide solution

A sequence with G (HC) DCAYHRGELVWCT (HC) H-NH ₂ (SEQ ID NO: 31, where HC is homocysteine, No. 4) was prepared by a peptide solid-phase synthesis method based on the F-moc method and according to a common method. The two Cys of No. 14 and No. 14 and the two homocysteine of No. 2 and No. 16 form disulfide bonds in the molecule respectively). A powder of 0.8 mg of the prepared IgG-binding peptide was dissolved in 24 μL of 100% dimethylsulfene (Wako Pure Chemical Industries, Ltd.) to prepare an IgG-binding peptide solution.

(2) Preparation of complex of Fc and IgG binding peptide

Humans were treated with Papain (manufactured by Roche) in a 20 mmol / L phosphate buffer (pH 7.0) containing 10 mM EDTA (Ethylene Diamine Tetraacetic Acid) and 1 mM L-cysteine at 37 ° C. The hinge part of IgG (Zhongwai Pharmaceutical) was severed. Next, human IgG Fc was purified using a cation exchange column (TSKgel SP5-PW (Tosoh)) at a flow rate of 1 mL / min in a 20 mM sodium acetate buffer (pH 5.0) using a gradient of 0-0.3M NaCl. 63 μL of a solution containing 16 mg / mL of human IgG Fc (0.1M sodium chloride (Wako Pure Chemical Industries, Ltd.), 0.04M 2-morpholine ethanesulfonic acid (Wako Pure Chemical Industries, Ltd.) (pH 6.0)) and the above (1) 2 μL of the IgG-binding peptide solution prepared in the above was mixed to prepare a complex solution of Fc and IgG-binding peptide.

(3) Fabrication of crystals of Fc and IgG binding peptide complex

Crystals of the complex of Fc and IgG-binding peptides can be obtained by the dripping vapor diffusion method. That is, using HydoraII + (manufactured by Matrix Corporation) as an automatic instrument for crystallization, 0.3 μL of a complex solution of the Fc and IgG-binding peptide prepared in the above (2) and a crystallization agent (20% polyethylene glycol 3350 ( Sigma-Aldrich), 0.2 M potassium iodide (Wako Pure Chemical Industries, Ltd.) (pH 6.9)) 0.3 μL were mixed on the S1 well of a smart crystallizing plate (manufactured by VERITAS) to prepare crystallized drops. 70 μL of the above-mentioned crystallization agent was dispensed as a stock solution. Leverage PowerSeal CRISTAL VIEW (manufactured by Greiner Bio-One) sealed the plate and left it in a constant temperature bath at 20 ° C for about 2 weeks to obtain crystals.

(4) Collection of X-ray diffraction intensity data of crystals of the complex of Fc and IgG binding peptide

Move the crystals obtained in (3) to the stabilized mother liquor (22% polyethylene glycol 3350, 0.2M potassium iodide, 0.1M sodium chloride, 25% glycerol (w / v), 0.04M 2-morpholine ethyl In sulfonic acid (pH 6.0)), it was rapidly frozen under a nitrogen gas flow at -170 ° C, and the X-ray diffraction data was measured by a vibration method. The X-ray was performed at a wavelength of 1 angstrom and a vibration angle of 1 ° / frame. Next, the diffraction intensity data processing program HKL2000 (manufactured by HKL Research) was used to process the diffraction intensity data with a resolution of 3.0 angstroms. As a result, the space group of the crystal was P21, and the lattice constants were a = 66.1 angstrom, b = 60.5 angstrom, c = 69.5 angstrom, α = γ = 90 °, and β = 101.3 °. The completeness of the obtained information was 99.9% and Rmerge was 13.8%.

(5) Determination of the crystal structure of the complex of Fc and IgG binding peptide

For DCAYHRGELVWCT (sequence number 33), the Phaser based on the molecular replacement method was used to determine the phase of the diffraction intensity data obtained in (4) above using Phaser, a program included in CCP4 (Collaborative Computational Project Number 4). The search model of the molecular replacement method is a model of the Fc part registered in the protein database (PDB, URL: http: //www.rcsb.0rg/pdb/) with the PDB registration code: 1DN2. As a result, a model of one molecule was found in the asymmetric unit. Then, the structure refinement using the structure refinement program Refmac5 included in CCP4 and the modification of the model using X-tal view as a model building program were repeatedly implemented to obtain Fc and IgG binding peptides (DCAYHRGELVWCT (SEQ ID NO: 33)) The crystalline structure of the complex. An electron density equivalent to the IgG-binding peptide was observed at the peptide-binding site of the Fc. The R-factor is 0.216, which is an indicator of the correctness of the crystal structure. Furthermore, the Rfree factor calculated based on the structural factor equivalent to 5% of total reflection that was not added to the calculation in the stage of refinement was 0.317.

(6) Production of cross-linked structure model

Based on the X-ray crystallized structure, a cross-linked structure model was made on the computational science software MOE (Molecular Operating Environment). After replacing the 6th amino acid of DCAYHRGELVWCT (SEQ ID NO: 33) with Lys, the form of linking the epsilon amino group of Lys with the epsilon amino group of Lys No. 248 of antibody Fc will use DSG or DSS The obtained crosslinked structure is modeled.

<Result>

As shown in FIG. 1A, it is believed that the IgG-binding peptide binds to the junction region of the CH2 and CH3 domains that overlaps with the binding site of protein A, and uses the IgG-binding peptide Fc-III (DeLano, WL et al., Science) , 2000, 287, pp. 1279-1283) in a similar manner to IgG. The characteristic interaction between IgG-binding peptide and Fc is the carboxylate of the guanidine group on the side chain of residue 8 of IgG-binding peptide Arg with 2.91 Angstroms and the side chain of Glu380 (based on EU numbering, the same below) of Fc Combined. The side chain of the Glu380 is bound to the intramolecular salt-binding network in the human IgG Fc by binding with the Lys248 salt, and the Arg8 of the IgG-binding peptide and the Lys248 of the Fc are approached through the interaction with the Glu380 of the Fc. Therefore, it was conceived to replace residue No. 8 of IgG-binding peptide Arg with Lys, and use a cross-linking agent to form the peptide's Lys8 with the amino group of the side chain of Lys248 of the antibody in a form similar to the network structure of the salt binding Perform cross-linking. In fact, based on the complex structure of IgG-binding peptide and human IgG Fc, DSG (disuccinimidylglutarate) or DSS (disuccinimidyl suberate) is produced. As a result of the model of the cross-linked structure, it was thought that a cross-linking agent could be introduced in the space without accompanying deformation of the main chain structure of the antibody (FIG. 1B).

[Example 2: Preparation and characteristics of labeling peptide]

<Method>

Biotin or 5 / 6TAMURA succinimide (AnaSpec, Inc.) (fluorescent pigment) amine-modified amine-PEG4 synthesis peptide GPDCAYHXGELVWCTFH (SEQ ID NO. 2) (Chemical) is synthesized by the Fmoc solid-phase synthesis method according to a common method. After removing the protective group, at pH In the aqueous solution of 8.5, an intramolecular SS bond was formed under oxidizing conditions. Reverse phase HPLC was used, and a gradient of 10% to 60% acetonitrile in 0.1% TFA (Trifluoroacetic acid) was used with a flow rate of 1.0 ml / min. Dissolution is performed to purify a peptide having an intramolecular SS bond.

100 μL of a DMF (Dimethylformamide, dimethylformamide) solution containing 1 mM purified IgG-binding peptide was mixed with 100 μL of a 100 mM solution of DSS or DSG (Thermo Fisher Scientific) in acetonitrile, and then reacted at room temperature. one night. After diluting the reactant to 2.5 times with 0.1% TFA, spray it to μ Bondasphere 5C18 100 angstroms (diameter 3.9mm × 150mm) manufactured by Waters, and dissolve it in a gradient containing 4% to 60% acetonitrile of 0.1% TFA. . Regarding the addition of a crosslinking agent to the obtained product, an LC-mass spectrometer (Acquity SQD UPLC system, Waters Corp.) connected to a BEH300 C18 (1.7 μm, diameter 2.1 mm × 50 mm) column was used to contain 0.1% formic acid. A gradient of 4% to 60% of acetonitrile was dissolved and confirmed by measurement of the molecular weight of the peak.

The affinity analysis of the obtained labeled reagent peptide was performed by adding 1 / 10th of 1M Tris-HCl (pH = 7.0) for 15 minutes to hydrolyze the NHS group, and then carried out by the following method . In 0.4M EDC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and 0.1M sulfo- NHS (sulfo-N-hydroxysuccinimide, sulfo-N-hydroxysuccinimide) was mixed in equal amounts, and the sensor wafer was sprayed onto a CM5 sensor wafer set at BIAcoreT200 (GE healthcare) at a flow rate of 10 μl / ml. After 7 minutes, the sensor wafer was activated, and the IgG was immobilized under the condition of pH 4.0 (10 mM Na acetate) so that the amount of immobilization became 4000 to 5000 in terms of RU value. While using HBS-EP buffer (0.01M HEPES, 0.15M NaCl, 0.005% Tween 20, 3mM EDTA, pH 7.0), spray a peptide at a concentration of 10nM to 2μM at a flow rate of 50μl / ml for 180 seconds to monitor binding The reaction was followed by washing with a buffer solution for 600 sec. The dissociation reaction was measured. The analysis of the combined parameters was performed using BIAevalution T100 software.

<Result>

In order to study whether the introduction of the cross-linked structure will affect the specificity and affinity of the IgG-binding peptide, SPR (Surface Plasmon Resonance, Surface Plasmon Resonance) analysis was used to determine the binding of the IgG-bound peptide with the cross-linked structure to the IgG Force (Table 1). Substituted the arginine residue of residue 8 with lysine-based IgG-binding peptide (hereinafter also referred to as type I (R8K)) for human IgG with an affinity of 131 nM (Kd), and the IgG-binding peptide before replacement (hereinafter (Also known as type I), the affinity is reduced by 10 times. Among those obtained by binding each cross-linking agent to a type I (R8K) peptide, about the affinity for human IgG, the type I (R8K) -DSG-OH is about 330 nM (Kd), and the type I (R8K) -DSS -OH was about 390 nM (Kd), and no significant decrease in affinity due to binding of the crosslinking agent was found. Regardless of the peptide, since it has an affinity of μM or less in terms of Kd value, it is considered that sufficient specificity can be achieved.

[Example 3: Specific modification of human IgG-Fc using IgG-binding peptide]

<Method>

In the same manner as in Example 2, a labeling reagent peptide modified with DSS or DSG for the IgG-binding peptide (TypeI (R8K)) with biotin-PEG4 attached to the N-terminus was prepared and reacted with human IgG Fc. To study the labeling reaction of human IgG Fc. That is, the excess IgG-binding peptide (R8K) (200 pmol / 5 μL in 0.1% TFA) after reacting with DSS or DSG was purified in a reverse-phase column by the same method as in Example 2, and then under reduced pressure. The acetonitrile was removed, and then about 1/8 of 0.5M Na ₂ HPO _{4 was added} for neutralization, and immediately added to protein samples (hIgG (Chinese and foreign pharmaceuticals), hIgA (Athens Research & Technology), HSA ( Sigma-Aldrich), or serum (obtained from a healthy person) (40 pmol / 5 μL each, the serum is 10 times diluted with PBS (Phosphate-buffered Saline, phosphate buffer)), and the final After measuring the amount to 20 μL in PBS, the solution was left at room temperature for 5 minutes. After that, 1 μl of 1M Tris-HCl (pH = 7.0) was added to stop the reaction, and then 4 × SDS sample solution 6.7 μl and 2-mercaptoethanol 1.4 μl (final 5%) were added, and treated at 95 ° C. for 10 minutes, and thereafter SDS-PAGE was performed using a pre-made gel SuperSep ^™ Ace, 5-20% (Wako Pure Chemical Industries, Ltd. ⁾ . The gel after electrophoresis was transferred to a PVDF membrane using a Hoefer Semiphor TE70 Trans-Blot System at 35 mA for 60 minutes, and then blocked with 0.5% BSA (Bovine Serum Albumin, bovine serum albumin). Biotinylated peptide-labeled proteins were detected using SA-coupled HRP (1000-fold dilution, Vector Laboratories) with a chemiluminescence reagent (ImmunoStar (registered trademark) Basic, Wako Pure Chemical Industries, Ltd.).

<Result>

As shown in FIG. 2B, in the Western blot method, a band that is regarded as a complex is observed only when reacting with IgG. Therefore, it is learned that the IgG-binding peptides that reacted with DSG or DSS did not react with IgA or HAS, Proteins other than IgG in the serum bind to and selectively bind to IgG.

[Example 4: Study on reaction conditions of IgG-binding peptide to IgG]

<Method>

(1) Response to Morse ratio

In the wells of a 96-well microplate (Nunc (registered trademark) MaxiSorp), each protein (IgG (Chinese and foreign pharmaceutical), IgA (Athens Research & Technology), or bovine gelatin (Wako Pure Chemical Industries, Ltd.)) (50ng (0.33pmol) ) / μl / well) 0.1M NaHCO ₃ solution was added to the dish and left at room temperature for one night, so that each protein was adsorbed on the surface of the dish, and blocked with 0.5% BSA, and then into each well. DSG-modified biotinylated IgG-binding peptide (0,1,2,5,10 in molar ratio) prepared in the same manner as in Example 2 was added, and 3 μL of 1M Tris-HCl was added after 1 hour. (pH 7.0) and the reaction was stopped. Add 50 μL of SA-HRP (Vector Laboratories) diluted 2000-fold with 0.5% BSA and react at room temperature for one hour, then wash with 0.1% PBST (Phosphate-buffered Saline with Tween 20, phosphate buffer + Tween 20) Clean 5 times, and then develop HRP (Horseradish Peroxidase, horseradish peroxidase) for color development using TMB (Tetramethyl benzidine, tetramethyl benzidine) solution (Wako Chemicals) for 5 minutes, then read the plate by ELISA The instrument (model 680 micro-disk reader (Bio-Rad)) measures the absorbance at 450 nm.

(2) Research on reaction time

To hIgG (50ng) obtained by immobilizing a 50ng / 50μL solution at 4 ° C overnight, a DSG-modified biotinylated IgG-binding peptide was added at a molar ratio of 2 to the reaction time (0 ~ 60). The reaction was stopped by adding 3 μL of 1M Tris-HCl (pH 7.0) within 3 minutes. The detection of binding was performed in the same manner as in (A).

<Result>

DSS was used to label the IgG-binding peptide and ELISA was used to study the reaction efficiency depending on the difference in the number of moles and reaction time with the antibody (Figure 3). That is, the molar ratio of the IgG-binding peptide immobilized on a plastic plate was changed from 1 to 10 and reacted with hIgG. As a result, saturation was found at approximately molar ratio 5. Therefore, it is considered that if a molar reagent with a molar ratio of about 5 is added, , The labeling of the antibody is sufficient (Figure 3A). Although very weak binding was found in the non-DSS-modified biotinylated IgG-binding (R8K) peptide (NO DSS R8K), it is believed that the binding activity was formed by utilizing non-covalently bonded peptides. Furthermore, even if the labeled IgG Peptide binding reagents, no binding to other proteins (hIgA, bovine gelatin or BSA used as a blocking agent) was detected at all.

Next, the reaction time when IgG was reacted with the IgG-binding peptide at a molar ratio of 1: 2 was investigated. As a result, saturation was found at about 15 minutes, and the reaction was considered to be almost complete at 15 minutes (Fig. 3B).

Based on the above results, it was shown that the IgG-binding peptide of the present invention modified with a cross-linking agent specifically binds to IgG in a short time.

[Example 5: Fc labeling using fluorescent IgG-binding peptide]

<Method>

IgG (Chinese and foreign pharmaceutical), IgA (Athens Research & Technology), or BSA (Sigma-Aldrich) (15 μg: 100 pmol in terms of IgG conversion) and the DSG cross-linked peptide or DSS cross-linked peptide prepared according to Example 2 ( 500 pmol) was reacted in 200 μL at room temperature for 60 min, and 10 μL of 1M Tris-HCl (pH = 7.0) was added to stop the reaction. Thereafter, size exclusion chromatography was performed under conditions of SuperdexTM 200 10 / 30GL diameter 1.0 cm × 30 cm (GE Healthcare); flow rate: 0.3 ml / min; electrophoresis buffer: PBS pH 7.4, and a fluorescence detector RF was used -10A (Shimadzu Corporation) (excitation light: 541 nm fluorescence: 565 nm) was measured.

<Result>

The labeled IgG-binding peptide reacted with DSS or DSG was reacted with the protein at a molar ratio of 1: 5 to each protein for 60 min at room temperature, and analyzed by size exclusion chromatography. No matter what kind of labeled IgG-binding peptide (DSS or DSG) is used, the specificity of the reactivity to IgG is the same degree, and no fluorescent labeling of other proteins such as hIgA or BSA is detected (Figure 4) ). Based on the above, it is known that no matter what kind of IgG-binding peptide is produced, it has high specificity, and human IgG can be fluorescently labeled.

[Example 6: Analysis of Modification of Fc Using IgG Binding Peptide (pH 4.5)]

<Method>

Add 200 μL of 0.5, 1.0, 2.0, 5.0 μL (0.5, 1.0, 2.0, 5.0 in molar ratio) of human IgG (Chinese and foreign pharmaceuticals) Fc solution (20 μM, 0.1M acetic acid buffer pH 4.5) and dissolve in DSG-modified IgG-binding peptide (RGNCAYHXGQLVWCTYH (SEQ ID NO: 35), X is lysine) (4mM) in DMF, which was modified by the same method as in Example 2. After rapid stirring, it was reacted at room temperature for 15 minutes 10 μL of 1M Tris-HCl (pH 7.0) was added to stop the reaction. 50 μL of the reaction was sprayed onto an NGC chromatography system (Bio-Rad) connected to a Shodex IEC SP-825 column, and 25 mM acetate buffer (pH 4.5) was applied to 25 mM acetate buffer containing 1 M NaCl. Dissolution was performed with a gradient of pH (4.5), and protein dissolution was monitored by absorbance at 215 nm. Each of the obtained peaks was fractionated and used for molecular weight measurement by LC / MS (Liquid Chromatography-Mass Spectrometry).

20 μL of the obtained peak fraction was injected into Shimadzu LCMS-8030 connected to a Waters ACQUITY UPLC BEH C8 (1.7 μm 2.1 mm × 100 mm) column, and then 4% acetonitrile containing 0.1% formic acid to 60% containing 0.1% formic acid A gradient of% acetonitrile was used for dissolution. Mass spectral analysis of the eluted peaks was performed and the mass was calculated by deconvolution of the polyvalent ion peaks using analytical software.

<Result>

Human IgG11-Fc and DSG-modified IgG binding peptide (4mM, biotin-PEG4-RGNCAYHXGQLVWCTYH-NH ₂ ; molecular weight 2760, X is lysine with DSG, 2 Cys form intramolecular SS bond) The ear ratio is 0.5, 1.0, 2.0, or 5.0. The results are shown in Figure 5A. The peaks (peak 2) and two peaks 3 and 4 of the original dissolution position of human IgG11-Fc appear. DSGized IgG-binding peptide). In order to identify these molecular species, LCMS analysis was performed. The IgG1-Fc before the reaction was eluted at the position of peak 1 in the ion exchange chromatogram, and a value of 55084 was obtained in the LCMS analysis. The LCMS analysis of the peaks 2, 3, and 4 after the reaction was performed, and the values of 55087, 57735 (55087 + 2648), and 60384 (55087 + 5297) were obtained, respectively. According to this situation, it is known that peak 2 after the reaction is unreacted Fc, and peaks 3 and 4 are those in which one and two peptides are bound to the Fc, respectively.

FIG. 5B is a graph showing changes in the amount of unreacted (peak 2), one peptide addition (peak 3), and two peptide additions (peak 4) when reacting at each mole ratio. Into a person. For example, it can be seen that when the reaction is performed at a molar ratio of 1: 1, the unreacted material also becomes 20% or less. If the molar ratio is 1: 2, the unreacted material is 10% or less and the yield is extremely high. Moreover, in the case of an excessive molar ratio of 1: 5, the production ratio of the appendages of the two peptides also relatively increased, but no Fc with more than two peptides was detected on the ion exchange chromatogram. Therefore, it is known that the labeling reaction is very specific.

[Example 7: Effect of pH and reaction time on Fc response using IgG-binding peptide]

<Method>

1.0 μL (1.0 in molar ratio) of 200 μL of an Fc solution of human IgG prepared at pH 4.0 (25 mM acetate buffer), pH 5.5 (25 mM acetate buffer), or pH 7.0 (PBS) The DSG-modified IgG-binding peptide (4 mM) dissolved in DMF prepared in Example 5 was quickly stirred and then reacted at room temperature. Add 10 μL of 1M Tris-HCl (pH 7.0) at 1, 5, 10, or 30 minutes after the reaction to stop the reaction. Spray 50 μL of the reactant to the NGC chromatography system connected to the Shodex IEC SP-825 column. (Bio-Rad), a gradient of 25 mM acetate buffer (pH 4.5) to 25 mM acetate buffer (pH 4.5) containing 1 M NaCl was used for dissolution, and protein dissolution was monitored using an absorbance at 215 nm. The ratio of each peak is calculated based on the obtained chromatogram.

<Result>

As shown in FIG. 6, it was found that in any of the tested pH values of 4.0, 5.5, and 7.0, the labeling reaction was earlier, and more than 90% of the reaction was completed within 1 minute. In addition, at a pH of 4.0, the residual amount of unreacted substances exceeds 40% and the reaction yield is low, especially the yield of the two peptides (peak 4) is about 15%, which is in line with other pH values. This situation is relatively low (35-40%). In the case of pH 5.5 and 7.0, the yield of unreacted substances is also low from 10% to less than 20%, and it is known that the reaction proceeds efficiently. As the difference between pH 5.5 and 7.0, it can be seen that the yield of Wave 4 has a tendency to decrease slightly in the case of pH 7.0.

[Example 8: FACS analysis of single-chain Fv-Fc antibody using IgG-binding peptide using fluorescent labeling]

<Method>

Lipofectamine 2000 was used to transfect pcDNA3.1 / Zeo (+) containing scFv-Fc from Her2 scFv (4D5) and Fc gene into HEK293 cells. After 5 days of culture, protein A column was used. The scFv-Fc secreted into the culture medium is purified to prepare a 4D5-Fc antibody (a single-chain Fv pure Fusion protein of 4D5 and Fc having specificity to HER2). Next, 1.0 μg of the prepared 4D5-Fc antibody was diluted in 10 μL of PBS containing 3% BSA, and the N-terminal biotinylated IgG-binding peptide DS-modified by the same method as in Example 2 was used. (Biotin-PEG4-RGNCAYHXGQLVWCTYH (SEQ ID NO: 35), X is a DSG lysine, and 2 Cys form an intramolecular SS bond) 0.16 μg (20 pmol) were mixed and reacted for 10 minutes. This reaction was added to a breast cancer cell line SK-BR3 (purchased from ATCC) (5.0 × 10 ⁵ cells) dispersed in 100 μL of PBS containing 10% FBS, and left at 4 ° C. for 30 minutes. After washing once with PBS containing 3% BSA and suspending in 100 μL of PBS containing 3% BSA, 0.01 μg (0.2 pmol) of PE-labeled streptavidin (Vector Laboratories) was added and left at 4 ° C for 30 minutes. minute. After washing once with PBS containing 3% BSA again, it was dispersed in 100 μL of PBS containing 3% BSA, and after adding 10 μL of 7-AAD reactive dye (Beckman Coulter), it was left for 15 minutes. 400 μL of PBS was added to disperse, and after passing through a 35 μm sieve (Corning), analysis was performed on an S3e ^™ cell sorter (Bio-Rad).

<Result>

The results obtained by detecting the binding of the 4D5-Fc antibody to the HER2 antigen on the breast cancer cell line SK-BR3 by DSG-modified biotinylated IgG-binding peptide and PE-labeled streptavidin are shown in FIG. 7A. As a control, it is also shown that a biotinylated anti-human IgG mouse antibody (anti-hIgG mAb-biotin labeling) (0.01 μg) is used instead of a DSG-modified biotinylated IgG binding peptide (biotinylated IgG binding peptide). Results of flow cytometry analysis in the case (in either case, only cell components other than dead cells stained by 7-AAD staining were used for analysis). In both systems, there was almost no difference in fluorescence intensity. Therefore, it was learned that SG-modified biotinylated IgG-binding peptides can be specifically labeled with human Fc and used for FACS staining of single-chain Fv-Fc, etc. . On the other hand, the system without the addition of 4D5-Fc was also studied as a negative control (Figure 7B), and the system without the addition of SG-modified biotinylated IgG-binding peptide (anti-hIgG mAb-biotin labeling + SA-PE labeling) , And anti-hIgG mAb-PE labeling) Similarly, no shift in fluorescence intensity was observed at all, so it was learned that in the case of a DSG-modified biotinylated IgG-binding peptide alone, non-cytokines were not produced. Specific modification.

[Example 9: Formation of conjugate of anti-IgA receptor VHH using IgG-binding peptide and human IgG antibody]

<Method>

DSG-modified N-terminal azide IgG-binding peptide (azide-PEG4-GPDCAYHXGELVWCTFH (sequence number 2), X is a lysine acid modified by DSG, 2 Cys form an intramolecular SS bond, and the C-terminal is aminated ) Was prepared by the same method as in Example 2. The peptide was dissolved in DMSO at a concentration of 10 mM, and 20 μL of the solution was added to 8 mL of a 16.6 μM anti-HER2 human IgG antibody (Chinese and foreign pharmaceutical) solution dissolved in 25 mM acetic acid buffer (pH 5.0) (peptide and antibody Molar ratio = 1: 1.5), and allowed to react for 5 hours at room temperature. After the reaction, the azide anti-HER2 human IgG antibody (1) was eluted on a column (1 mL) of CIMmultus ^TM SO3-1 (SHOWA DENKO) with a NaCl gradient of 0 to 1M in 25 mM acetate buffer (pH 5.0). Valence azide antibody, mixture of bivalent azide antibody).

The alpaca-derived anti-IgA receptor VHH antibody pure line 2b1-L9 (with the HIS tag attached to the C-terminus) was affinity purified using the HIS tag attached to the C-terminus to the expression expressed by E. coli HB2151. Specifically, after the phagemid vector pKSTV03 having the VHH gene was introduced into E. coli HB2151, selection was performed in a 2TYAG culture plate, and culture was performed overnight in a 2TYA liquid culture medium at 37 ° C. After 10 mL of this culture solution was added to 500 mL of 2TYA and cultured at 37 ° C. for 1 hour, 500 μL of 1M IPTG was added and the culture was shaken for 16 hours. The centrifuged cells were suspended in 10 mL of TES buffer (0.2M Tris-base, 0.5mM EDTA, 0.5M sucrose), and left to stand on ice for 2 hours. 20 mL of TES buffer diluted to 4 times was added to resuspend it, and it was left to stand on ice for 1 h and then centrifuged to recover the supernatant component (homogeneous component). Supernatant was purified from VHH in affinity column (His trap excel, GE Healthcare) using Profinia (BioRad) purification system (about 2mL / min for flow rate, binding and dissolution, 2mL / min for washing, about Buffer (Buffer), 0.5M NaCl, 20mM sodium phosphate for the equilibration buffer system, 0.5M NaCl, 20mM sodium phosphate for the washing buffer system, and 500mM imidazole, 0.5M NaCl, 20mM sodium phosphate for the dissolution buffer system) . Then, in PBS (pH 7.4) in the presence of 0.1 mM DTT (Dithiothreitol, dithiothreitol) at room temperature for 1 hour after reduction treatment, in an IEC SP-825 (Shodex) column ( 8.0 mm × 75 mm) was purified by using a gradient of 0 to 1 M NaCl in 10 mM acetic acid buffer (pH 4.5). 200 μL of a reduction-treated VHH solution (41.2 μM, pH 4.5) and 870 μM dibenzocyclooctyne (DBCO) -cis-butenedifluorene imine (10 μM) dissolved in 10 mM acetic acid buffer (pH 4.5) Click Chemistry Tools) was mixed with 42 μL (1: 4.4 in molar ratio), and allowed to react at room temperature for 1 hour. 290 μL of dibenzocyclooctyne-cis-butene difluorinated VHH (22 μM) and 116 μL of azide antibody solution (17 μM, pH 4.5) prepared by the above method Mixing (3.3: 1 in mole ratio) was carried out, and it was made to react at 4 degreeC for 14 hours. Reactant IEC SP-825 (Shodex) column (8.0 mm x 75 mm) was purified by dissolving it in a 10 mM acetic acid buffer (pH 4.5) using a gradient of 0 to 1 M NaCl. After reduction, the purified components were separated by SDS-PAGE on a 5-20% gradient gel Super Sep Ace (WAKO), and then protein stained by CBB.

<Result>

The result of ion-exchange chromatography after the click reaction of the azide peptide antibody and the dibenzocyclooctyne-cis-butene difluorinated VHH was 3 c) The main peak (Figure 8A). The results obtained by analyzing each of them in the reduced state by SDS-PAGE are shown in FIG. 8B. In a (band 3), a 50 kDa derived from the H chain and a 25 kDa derived from the L chain were observed in the same manner as the original IgG (band 1). In b (band 4), although there is no change in the band of the L chain, in addition to the original heavy chain (about 50 kDa) of the IgG antibody (band 1), a new position of about 80 kDa at about the same concentration was also observed. The belt. In c (zone 5), although the band of the L chain remained unchanged, the band of the original heavy chain (about 50 kDa) disappeared, and only a band of about 80 kDa was observed. According to these circumstances, it is known that a is an IgG antibody with no VHH attached, b is an IgG antibody (anti-HER2 human antibody-1 valent VHH) linked to one VHH, and c is an IgG antibody (anti-HER2) linked to two VHH Human antibody-2 is VHH).

According to the above situation, it is known that by using a IgG-binding peptide reagent, a click reaction between an azide group introduced into an IgG antibody and a dibenzocyclooctynyl group introduced into a VHH low-molecular antibody, a low-molecular antibody (VHH Etc.) linked to an IgG antibody.

[Example 10: Analysis of antigen binding by FACS of a conjugate of an anti-IgA receptor VHH and an anti-HER2 human IgG antibody via an IgG-binding peptide]

<Method>

HL60 cells (obtained from JCRB) were added to RPMI1640 medium (Life Technologies) containing 10% FBS and 100 units / mL penicillin G and 100 μg / mL streptomycin sulfate (Life Technologies), and induced differentiation for 6 days. About SK-BR3 cells (purchased from ATCC), in McCoy's 5A (Life Technologies) medium containing 10% FBS, 100 units / mL penicillin G, and 100 μg / mL streptomycin sulfate at 37 ° C in 5% CO ₂ After culturing in an incubator, the cells were detached by trypsin-EDTA (Life Technologies) and recovered. Each 2 × 10 ⁵ cells were dispersed in 200 μL of PBS containing 3% BSA, and the primary antibody (anti-HER2 human IgG antibody, anti-IgA receptor VHH prepared in Example 9) was added so that the final concentration became 200 nM. (C-terminal HIS tag attached), or the anti-HER2 human antibody-1 valence VHH (C-terminal HIS tag attached) prepared in Example 9, and left at 4 ° C for 30 minutes. After washing once, add 200 μL of cell fluid dispersed in PBS containing 3% BSA 1) Biotinylated anti-HIS-tagged antibody (MBL Life Science) + PE-labeled SA (final concentration 50nM) (Vector Laboratories), or 2) PE-labeled anti-human IgG polyclonal antibody (Affimetrix eBioscience) (final concentration 13nM) as secondary antibodies, and left at 4 ° C for 30 minutes. After washing once, 10 μL of 7-AAD reactive dye (Beckman Coulter) was added to 200 μL of the cell solution dispersed in PBS containing 3% BSA and left for 15 minutes, then 800 μL of PBS was added and passed through a 35 μm sieve The analysis was performed on a S3e ^™ cell sorter (Bio-Rad) after Corning.

<Result>

Use 7-AAD staining to remove cell components from dead cells, and use anti-HER2 human IgG antibody (Figure 9A), anti-IgA receptor VHH (C-terminally attached HIS tag) (Figure 9B), anti-HER2 human antibody- Monovalent VHH (H-tag attached to the C-terminus) (Figure 9C) was used as a primary antibody, and a mixture of biotinylated anti-HIS antibody + PE-labeled SA was used as a secondary antibody against SK-BR3 with high expression of HER2 The cells were analyzed by FACS, and the obtained results are shown in Figs. 9A to 9C. In the case of FIG. 9C, a large fluorescence shift was observed, and thus it was known that the anti-HER2 antibody in the prepared anti-HER2 human antibody-1 valence VHH has binding activity to SKBR-3 cells.

On the other hand, anti-HER2 human antibody (Figure 9D), anti-IgA receptor VHH (C-terminal HIS tag attached) (Figure 9E), and anti-HER2 human antibody-1 valence VHH (Figure 9F) were used as primary antibodies and used PE-labeled anti-human IgG polyclonal antibodies are used as secondary antibody pairs. The binding of DMSO to HL60 cells expressing IgA receptors at a high level of 1.3% was detected, and the results obtained are shown in Figures 9D to F. That is to say, binding to HL60 is only observed in the anti-HER2 human antibody-1 valence VHH of FIG. 9F. Therefore, it is known that the VHH in the anti-HER2 human antibody-1 valence VHH retains the antigen to the IgA receptor. Binding activity. Furthermore, some fluorescence shifts in FIG. 9D indicate that a small amount of HER2 is expressed on differentiated HL60 cells, but the fluorescence intensity in FIG. 9F is much larger than that based on the fluorescence intensity based on the binding, so it is considered that it can be Ignore the effect on HER2 binding.

[Example 11: Inhibition of cell proliferation of an antibody drug complex using an IgG-binding peptide]

<Method>

N-terminal amine group modified with cis-butenylimide ethoxylated succinimide ester cis-butenimide-PEG4 synthesis peptide RGPPDCAYHXGELVWCTFH (SEQ ID NO: 37: peptide in SEQ ID NO: 2 The N-terminus is obtained by adding two Args, X is an lysine, and the C-terminus is imidized). It is synthesized by the Fmoc solid-phase synthesis method and according to a common method. After removing the protecting group, an intramolecular SS bond was formed in an aqueous solution of pH 8.5 under oxidizing conditions. Reverse-phase HPLC was used with a flow rate of 1.0 ml / min and 10% to 60% acetonitrile containing 0.1% TFA. Gradient dissolution refines peptides with intramolecular SS bonds. To 40 μL of the peptide (18.5 mM) dissolved in DMSO was added 24 μL of DM-1 (emtansine (XDCExplorer), 50 mM) similarly dissolved in DMSO (molar ratio of peptide to DM-1 = 1: 1.6), Further, 3.4 μL of pyridine (final concentration 5%) was added, and the mixture was allowed to react at 50 ° C. for 3 hours. Then, 80 μL of DSG (500 mM) dissolved in acetonitrile was added and reacted at 50 ° C. for 3 hours, and a cross-link was formed between the cis-butenylimide group of the IgG-binding peptide and the thiol group of DM-1. structure. The entire amount was diluted in 10 ml of 10% acetonitrile containing 0.1% TFA, and the centrifuged supernatant was injected into an Insertsustain C18 column (7.6 mm 1 × 250 mm, GL Science) to 10% to 0.1% TFA. A gradient of 70% acetonitrile was used for dissolution. Mass analysis of the eluted substance was performed, and the target substance was recovered, and then the solvent was removed, followed by freeze-drying.

0.56 μL of DSG-modified maleimide-PEG4ized IgG-binding peptide reagent linked to 12.0 mM DM-1 dissolved in DMSO and 6.8 μM dissolved in 10 mM acetate buffer (pH 5.5) 1 mL of anti-HER2 human antibody (Chinese and foreign pharmaceutical) was mixed and allowed to react for 30 minutes at room temperature (molar ratio of peptide to antibody = 1: 1). The DM-1 modified human antibody (antibody-drug complex, ADC) prepared by the above method was used in a cationic ion exchange column Shodex SP825 (8.0mm × 75mm, Shodex) using a 10 mM acetate buffer solution (pH 5.5) ) From 0M to 1.0M NaCl gradient elution and purification. After the two peaks (peaks A and B) other than the unreacted antibody were collected, desalting and concentration were performed on a Vivaspin (10000 Da critical, Sartorius) by a 3000 g centrifugation operation. The obtained sample was measured by MALDI-TOF-MAS autoflex speed TOF / TOF-KG (Bruker Daltonics). The peak A was increased by 3553 (theoretical value 3535) compared with the original anti-HER2 human antibody, and the peak B was compared with the original anti-HER2 antibody. HER2 human antibody increased by 7092 (theoretical value 7070). Therefore, it was confirmed that one cis-butenediimine-PEG4 IgG-binding peptide to which DM-1 was linked was introduced (anti-HER2 antibody-DM1 * 1), and 2 (anti-HER2 antibody-DM1 * 2).

SK-BR3 cells (purchased from ATCC) or C6 cells (obtained from JCRB) were seeded in each well of a 96-well cell culture plate to contain 10,000 cells / 100 μL to contain 10% FBS, 100 units / mL penicillin G, and 100 μg. / mL of streptomycin sulfate in McCoy's 5A (Life Technologies) medium. After incubating in a 5% CO ₂ incubator at 37 ° C. for 24 hours, 100 μL of a medium containing the antibody-drug complex (ADC) prepared in the above manner was added at each concentration, and then in a CO ₂ incubator at 37 ° C. Incubate for 72 hours. To each well, 10 μL of the cell-counting Kit-8 (Tongren Chemical) was added, and the cells were incubated in a CO ₂ incubator at 37 ° C. for 2 hours, and then the absorbance at 450 nm was measured using a plate reader.

<Result>

In order to evaluate the inhibitory effect of the prepared ADC on the proliferation of breast cancer cell line SK-BR3, SK-BR3 cells were cultured in the presence of ADC of 0-10nM, and The cell assay set evaluated the number of cells after 72 hours (Figure 10). The anti-HER2 antibody-DM1 * 1 and anti-HER2 antibody-DM1 * 2 prepared this time showed a significant cell proliferation inhibitory activity at a concentration of 0.4nM or more for SK-BR3, which expresses HER2. On the other hand, in C6 cells that did not express HER2, no cell proliferation inhibition was observed in the concentration range of the antibody-drug complex used. According to the above circumstances, it is known that an antibody-drug complex covalently bonded via an IgG-binding peptide can exhibit effective cell proliferation inhibitory activity on cancer cell lines.

[Example 12: Cell proliferation inhibition of an antibody drug complex by an IgG-binding peptide having an SS cross-linked structure obtained using dichloroacetone]

<Method>

N-terminal acetylated RRC (Acm-protected) -PEG4 synthesis peptide GPDCAYHXGELVWCTFH (sequence number 2, X is lysine, C-terminated amidation) is on peptide synthesis beads (Rink-amide-Chemmatrix resin, Biotage) It was synthesized by the Fmoc solid-phase synthesis method and according to a common method. After the peptide was cut out from the resin and deprotected, the peptide was obtained (Fig. 11, a). 65 mg (15.6 μmol) of the obtained peptide was dissolved in 5 mL of a phosphate buffer solution (pH = 7.3) containing 6 M Gn‧HCl, and 1,3-dichloro-2-acetone dissolved in 120 μL of acetonitrile was added thereto. (2.9 mg, 23.4 μmol, 1.5 equal moles), and stirred at room temperature. After 1 hour, the completion of the reaction was confirmed by HPLC analysis, and the reaction solution was directly purified by HPLC to obtain a cyclized peptide (FIG. 11, b, 33 mg, 7.8 μmol, 50% yield). To this cyclized peptide was added silver acetate (30.8 mg, 184.5 μmol) suspended in a 90% aqueous acetic acid solution (8.8 mL), and stirred at room temperature under light-shielding for 5 hours. Dithiothreitol (DTT: 352 mg, 2.3 mmol) was added, the resulting precipitate was removed by centrifugation, and the obtained supernatant was purified by HPLC to obtain a cyclized peptide (Figure 11, c , 20.5 mg, 5.2 μmol, yield 67%).

The cyclized peptide prepared in the above manner and 27 mM VcMMAE (cis-butenyldiiminohexanyl-valinic acid-citrulline-p-aminobenzyl) similarly dissolved in DMSO Ethoxycarbonyl-monomethyl auristatin E (MedChem Express) 18 μL (Molar ratio of peptide to VcMMAE = 1: 1.4) was added to 6 μL of a solution (60 mM) dissolved in DMSO, and pyridine was further added 1.2 μL (final concentration 5%), and allowed to react at 50 ° C. for 3 hours. Then, 25 μL of DSG (500 mM) dissolved in acetonitrile was added and reacted at 50 ° C. for 3 hours. The entire amount was diluted in 10 ml of 10% acetonitrile containing 0.1% TFA, and the centrifuged supernatant was injected into an Insertsustain C18 column (7.6 mm × 250 mm, GL Science) to contain 10% to 80% of 0.1% TFA. A gradient of% acetonitrile was used for dissolution. Mass analysis of the eluted substance was performed to recover the target substance, and then the solvent was removed, followed by freeze-drying.

5.4 μL of 5.0 mM DSG modified N-terminal acetylated RRC-PEG4 IgG-binding peptide reagent (R8K) dissolved in DMSO and 6.8 μM anti-HER2 human IgG dissolved in 10 mM acetate buffer (pH 5.5) Antibodies (Chinese and foreign pharmaceuticals) 1 mL were mixed and allowed to react for 15 hours at room temperature (molar ratio of peptide to antibody = 1: 4). The VcMMAE modified human IgG antibody (antibody drug complex, ADC) prepared by the above method was used in a cationic ion exchange column Shodex SP825 (8.0mm × 75mm, Shodex) using a 10 mM acetate buffer solution (pH 4.5) A gradient of 0M to 1.0M of NaCl was eluted and purified. The main peak other than the unreacted antibody was fractionated, and then desalted and concentrated on a Vivaspin (10,000 Da critical, Sartorius) by a 3000 g centrifugation operation. The obtained sample was measured by MALDI-TOF-MAS autoflex speed TOF / TOF-KG (Bruker Daltonics). The mass was increased by 3941 (theoretical value 4178) compared to the original anti-HER2 human antibody, so it was confirmed that 1 N-terminal acetofluorene-RRC-PEG4 IgG-binding peptide (R8K) of VcMMAE.

SK-BR3 cells (purchased from ATCC) or C6 cells (obtained from JCRB) were seeded in each well of a 96-well cell culture plate to contain 10,000 cells / 100 μL to contain 10% FBS, 100 units / mL penicillin G, and 100 μg. / mL of streptomycin sulfate in McCoy's 5A (Life Technologies) medium. After culturing in a 5% CO ₂ incubator at 37 ° C. for 24 hours, 100 μL of a medium containing antibody drug complex (ADC) at each concentration was added, and then cultured in a CO ₂ incubator at 37 ° C. for 72 hours. To each well, 10 μL of a cell-counting Kit-8 (following human chemistry) was added, and the cells were incubated in a CO ₂ incubator at 37 ° C. for 2 hours, and then the absorbance at 450 nm was measured using a plate reader.

<Result>

In order to evaluate the inhibitory effect of the prepared ADC on the proliferation of breast cancer cell line SK-BR3, SK-BR3 cells were cultured in the presence of 0-500 nM ADC, and the cell number was measured after 72 hours using a cell measurement kit. Evaluation (Figure 12). Regarding the used anticancer agent VcMMAE itself, proliferation inhibition was observed only at a level of 250 nM or more (Figure 12A). In the ADC prepared this time, for SK-BR3, which expresses HER2, at a concentration of 0.4 nM or more A significant cell proliferation inhibitory activity was shown (Fig. 12B), and the cell proliferation inhibitory activity was increased by about 500 times by the conjugate with the antibody. On the other hand, such inhibition of cell proliferation was not observed only in the original anti-HER2 human antibody. According to the above circumstances, it is known that an antibody-drug complex covalently bonded via an IgG-binding peptide can exhibit an effective cell proliferation inhibitory activity on a cancer cell line.

[Example 13: Labeling evaluation using IgG-binding peptides for various IgGs]

<Method>

3.15 μL of PBS was added to 1.25 μL (equivalent to 2.5 μg of antibody) of various antibody solutions (14 μM) in humans, mice, rabbits, and rats, and mixed with DSG-modified N by the same method as in Example 2. Terminal biotinylated IgG-binding peptide biotin-PEG4-RGNCAYHXGQLVWCTYH (sequence number 35, X is DSG-ized lysine, 2 Cys form intramolecular SS bond) in DMSO solution (118 μM) 0.65 μL, at room temperature The reaction was allowed to wait for 30 minutes (Molar ratio of antibody to peptide = 1: 4). To this reaction solution (5.0 μL) was added 5.0 μL of SDS-PAGE sample buffer (4 ×), 0.6 μL of 2-mercaptoethanol, 9.35 μL of ultrapure water, and the mixture was heated at 95 ° C. for 10 minutes. SDS-PAGE was performed on a gradient gel (Super Sep ^™ Ace 5-20%, Wako Chemicals). The gel system uses CBB (Coomassie brilliant blue, Coomassie brilliant blue) for protein staining, and the protein system transferred from the gel to the PVDF membrane is used for Western Blot. That is, the PVDF (Polyvinylidene fluoride) film was blocked with 0.5% BSA, and the HRP-labeled streptavidin (Vector Laboratories) was reacted at room temperature for 1 hour, and chemiluminescence was used. Detection reagent Chemi-Lumi One (NACALAI TESQUE) and detection using a chemiluminescence imager ChemDock (BioRad). The antibodies used as labels are described below. Human IgG1 (Clone ID: CB1), human IgG2 (Clone ID: CB2), human IgG3 (Clone ID: CB3), human IgG4 (Clone ID: CB4), mouse IgG1 (Clone ID: CB5), mouse IgG2b ( Clone ID: CB8) and mouse IgG3 (Clone ID: CB9) were purchased from Crown Bioscience Inc. Rat IgG1 (Clone #: 43414) and IgG2b (Clone #: 141945) were purchased from R & D, IgG2c (Clone Name: SB68b) was purchased from LifeSpan BioScience, and rabbit IgG was purchased from Thermo Scientific.

<Result>

As shown in FIG. 13, dark bands were confirmed for human monoclonal IgG1, human IgG2, and human IgG4 to the same extent as trastuzumab (anti-HER2 humanized IgG1 antibody). Among the animal antibodies used, rabbit IgG antibodies were significantly stained. Based on the above, it is known that the labeling of human IgG1, 2, and 4 and rabbit IgG antibodies can be efficiently performed using the labeling of the IgG-binding peptide.

[Industrial availability]

Using the IgG-binding peptide of the present invention, various compounds linked to the IgG-binding peptide can be added to the Fc of IgG in a short time and with almost no side reactions. This makes it possible to specifically and easily modify IgGs used as detection reagents, diagnostic drugs, medicines, and the like with various compounds.

All publications, patents, and patent applications cited in this specification are incorporated directly into this specification by reference.

<110> Kagoshima University

<120> Specific modification of antibodies by IgG-binding peptides

<130> PH-6549-TW

<160> 37

<170> PatentIn version 3.5

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<223> IgG-binding peptide

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<222> (8) .. (8)

<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

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<212> PRT

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<221> Unclassified characteristics

<222> (6) .. (6)

<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

<400> 14

<210> 15

<211> 13

<212> PRT

<213> Artificial

<220>

<223> IgG-binding peptide

<220>

<221> Unclassified characteristics

<222> (6) .. (6)

<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

<400> 15

<210> 16

<211> 13

<212> PRT

<213> Artificial

<220>

<223> IgG-binding peptide

<220>

<221> Unclassified characteristics

<222> (6) .. (6)

<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

<400> 16

<210> 17

<211> 13

<212> PRT

<213> Artificial

<220>

<223> IgG-binding peptide

<220>

<221> Unclassified characteristics

<222> (6) .. (6)

<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

<400> 17

<210> 18

<211> 13

<212> PRT

<213> Artificial

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<223> IgG-binding peptide

<220>

<221> Unclassified characteristics

<222> (6) .. (6)

<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

<400> 18

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<212> PRT

<213> Artificial

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<223> IgG-binding peptide

<220>

<221> Unclassified characteristics

<222> (6) .. (6)

<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

<400> 19

<210> 20

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<212> PRT

<213> Artificial

<220>

<223> IgG-binding peptide

<220>

<221> Unclassified characteristics

<222> (6) .. (6)

<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

<400> 20

<210> 21

<211> 13

<212> PRT

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<223> IgG-binding peptide

<220>

<221> Unclassified characteristics

<222> (6) .. (6)

<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

<400> 21

<210> 22

<211> 13

<212> PRT

<213> Artificial

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<223> IgG-binding peptide

<220>

<221> Unclassified characteristics

<222> (6) .. (6)

<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

<400> 22

<210> 23

<211> 13

<212> PRT

<213> Artificial

<220>

<223> IgG-binding peptide

<220>

<221> Unclassified characteristics

<222> (6) .. (6)

<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

<400> 23

<210> 24

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<212> PRT

<213> Artificial

<220>

<223> IgG-binding peptide

<220>

<221> Unclassified characteristics

<222> (6) .. (6)

<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

<400> 24

<210> 25

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<212> PRT

<213> Artificial

<220>

<223> IgG-binding peptide

<220>

<221> Unclassified characteristics

<222> (6) .. (6)

<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

<400> 25

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<212> PRT

<213> Artificial

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<223> IgG-binding peptide

<220>

<221> Unclassified characteristics

<222> (6) .. (6)

<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

<400> 26

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<212> PRT

<213> Artificial

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<400> 27

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<212> PRT

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<223> IgG-binding peptide

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<221> Unclassified characteristics

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<400> 28

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<212> PRT

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<223> IgG-binding peptide

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<221> Unclassified characteristics

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<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

<400> 29

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<220>

<221> Unclassified characteristics

<222> (2) .. (2)

<223> Xaa is homoserine

<220>

<221> Unclassified characteristics

<222> (16) .. (16)

<223> Xaa is homoserine

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<212> PRT

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<223> IgG-binding peptide

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<222> (8) .. (8)

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<400> 32

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<223> IgG-binding peptide

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<223> IgG-binding peptide

<220>

<221> Unclassified characteristics

<222> (8) .. (8)

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<400> 35

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<220>

<221> Unclassified characteristics

<222> (2) .. (2)

<223> Xaa is homoserine

<220>

<221> Unclassified characteristics

<222> (8) .. (8)

<223> Xaa is lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid, or diaminopropionic acid

<220>

<221> Unclassified characteristics

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<400> 37

Claims (21)

A peptide characterized by comprising the following formula I: (X _1-3 ) -C- (X ₂ ) -H- (Xaa1) -G- (Xaa2) -LVWC- (X _1-3 ) (I) ( In the formula, each of X is independently any amino acid residue other than cysteine, C is a cysteine residue, H is a histidine residue, Xaa1 is an lysine residue, a cysteine Glycine residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, G is a glycine residue, and Xaa2 is a glutamate residue or a Asparagine residues, L is a leucine residue, V is a valine residue, and W is a tryptophan residue) represented by an amino acid sequence containing 13 to 17 amino acid residues, It can bind to human IgG and / or rabbit IgG. The peptide of claim 1, comprising the following formula II: (X _1-3 ) -C- (Xaa3)-(Xaa4) -H- (Xaa1) -G- (Xaa2) -LVWC- (X _1-3 ) (II) (wherein each of X is independently an amino acid residue other than cysteine, C is a cysteine residue, H is a histamine residue, and Xaa1 is an lysine Residues, cysteine residues, aspartic acid residues, glutamic acid residues, 2-aminosuberic acid, or diaminopropionic acid, G is a glycine residue, and Xaa2 is glutamine Acid residue or asparagine residue, L is leucine residue, V is valine residue, W is tryptophan residue, Xaa3 is alanine residue, serine residue or threonine Acid residue, and Xaa4 is a tyrosine residue or a tryptophan residue) represented by an amino acid sequence containing 13 to 17 amino acid residues, and can bind to human IgG and / or rabbit IgG. If the peptide of claim 1 or 2 comprises the following formula III: (X _1-3 ) -CAYH- (Xaa1) -GELVWC- (X _1-3 ) (III) (wherein each of X is independently Any amino acid residue other than cysteine, C is a cysteine residue, A is an alanine residue, Y is a tyrosine residue, H is a histamine residue, and Xaa1 is an amine residue Acid residue, cysteine residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, G is a glycine residue, and E is a bran Amino acid residues, L is a leucine residue, V is a valine residue, and W is a tryptophan residue) represented by an amino acid sequence comprising 13 to 17 amino acid residues, and Capable of binding to human IgG and / or rabbit IgG. The peptide according to any one of claims 1 to 3, wherein in the case of 17 amino acid residues, each of the amino acid residues Nos. 1 to 3 and 15 to 17 from the N-terminus is the first No. amino acid residue = S, G, F or none, No. 2 amino acid residue = D, G, A, S, P, homocysteine or none, No. 3 amino acid residues = S, D, T, N, E, or R, No. 15 amino acid residues = S, T, or D, No. 16 amino acid residues = H, G, Y , T, N, D, F, homocysteine or none, amino acid residue No. 17 = Y, F, H, M or none. The peptide of claim 4, which comprises any of the amino acid sequences of 1) to 15) below, wherein Xaa1 is an lysine residue, a cysteine residue, an aspartic acid residue, or a glutamic acid Residue, 2-aminosuberic acid, or diaminopropionic acid, Xaa2 is homocysteine, 1) DCAYH (Xaa1) GELVWCT (sequence number 1) 2) GPDCAYH (Xaa1) GELVWCTFH (sequence number 2) 3) RCAYH (Xaa1) GELVWCS (serial number 3) 4) GPRCAYH (Xaa1) GELVWCSFH (serial number 4) 5) SPDCAYH (Xaa1) GELVWCTFH (serial number 5) 6) GDDCAYH (Xaa1) GELVWCTFH (serial number 6) 7) GPSCAYH (Xaa1) GELVWCTFH (Serial Number 7) 8) GPDCAYH (Xaa1) GELVWCSFH (Serial Number 8) 9) GPDCAYH (Xaa1) GELVWCTHH (Serial Number 9) 10) GPDCAYH (Xaa1) GELVWCTFY (Serial Number 10) 11) SPDCAYH ( Xaa1) GELVWCTFY (serial number 11) 12) SDDCAYH (Xaa1) GELVWCTFY (serial number 12) 13) RGNCAYH (Xaa1) GQLVWCTYH (serial number 13) 14) G (Xaa2) DCAYH (Xaa1) GELVWCT (Xaa2) H (serial number 36) 15) RRGPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 37). The peptide of claim 1 or 2, comprising the following formula IV: D-C- (Xaa3)-(Xaa4) -H- (Xaa1) -G- (Xaa2) -L-V-W-C-T (IV) (wherein, D is an aspartic acid residue, C is a cysteine residue, H is a histidine residue, Xaa1 is an lysine residue, a cysteine residue, an aspartic acid residue, and glutamine Acid residue, 2-aminosuberic acid, or diaminopropionic acid, G is a glycine residue, Xaa2 is a glutamate residue or an asparagine residue, and L is a leucine residue, V is a valine residue, W is a tryptophan residue, T is a threonine residue, Xaa3 is an alanine residue or a threonine residue, and Xaa4 is a tyrosine residue or a tryptophan residue The amino acid sequence represented by the amino group contains 13 amino acid residues, and is capable of binding to human IgG and / or rabbit IgG. The peptide according to claim 6, which comprises any amino acid sequence of 1) to 4) below, wherein Xaa1 is an lysine residue, a cysteine residue, an aspartic acid residue, or a glutamic acid Residue, 2-aminosuberic acid, or diaminopropionic acid, 1) DCTYH (Xaa1) GNLVWCT (SEQ ID NO: 14) 2) DCAYH (Xaa1) GNLVWCT (SEQ ID NO: 15) 3) DCTYH (Xaa1) GELVWCT ( SEQ ID NO: 16) 4) DCAWH (Xaa1) GELVWCT (SEQ ID NO: 17). A peptide characterized by comprising the following formula V: DC- (Xaa2)-(Xaa3)-(Xaa4)-(Xaa1) -G- (Xaa5) -L- (Xaa6) -WCT (V) (where, D is an aspartic acid residue, C is a cysteine residue, G is a glycine residue, L is a leucine residue, W is a tryptophan residue, T is a threonine residue, Xaa1 is an lysine residue, a half Cysteine residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, Xaa2 is alanine residue, serine residue or threonine Residue, Xaa3 is tryptophan residue or tyrosine residue, Xaa4 is histamine residue, arginine residue, serine residue or threonine residue, Xaa5 is glutamate residue , Asparagine residue, arginine residue, or aspartic acid residue, and Xaa6 is an isoleucine residue or a valine residue) represented by 13 amino acid residues Amino acid sequences and are capable of binding to human IgG and / or rabbit IgG. The peptide of any one of claims 1 to 8, wherein the peptide forms a disulfide bond between the two outer cysteine (C) residues, or one of the two cysteine residues on the outer side of the peptide Sulfur group uses the following formula:

The linkers indicated are linked. The peptide according to any one of claims 1 to 9, which is labeled with a labeling substance. The peptide according to any one of claims 1 to 10, which is combined with a medicament. The peptide of any one of claims 1 to 11, wherein Xaa1 is an lysine residue. The peptide of any one of claims 1 to 12, wherein Xaa1 is modified with a cross-linking agent. The peptide according to claim 13, wherein the cross-linking agent is selected from the group consisting of DSG (disuccinimidyliminoglutarate), DSS (disuccinimidyliminosuccinate), DMA (adipic acid) Dimethyl Dihydrochloride), DMP (Diheptanedioimidic Acid Dihydrochloride), DMS (Dioctimedioic Acid Dimethyl Dihydrochloride), DTBP (3,3'-disulfide Group consisting of dimethyl dipropionimidate dihydrochloride) and DSP (dithiobissuccinimide iminopropionic acid). The peptide according to claim 14, wherein the cross-linking agent is DSG (disuccinimidyliminoglutarate) or DSS (disuccinimidyliminosuccinate). A complex, which is a complex of the peptide and IgG according to any one of claims 13 to 15, and is formed by a cross-linking reaction of a peptide modified with a cross-linking agent and IgG. A method for producing a complex of a peptide and an IgG, comprising the step of mixing the peptide according to any one of claims 13 to 15 with the IgG and subjecting the cross-linking agent-modified peptide to the IgG. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 15 or the complex according to claim 16. A method for producing a peptide in which two or more cysteine residues are linked using a linker, which comprises a peptide comprising two or more cysteine residues and the following formula:

The indicated compounds (wherein R ₁ and R ₂ are each independently an arbitrary halogen atom) are mixed to obtain sulfur groups in two or more cysteine residues using the following formula: [化 3]

Steps of peptides represented by the indicated linkers. The method according to claim 19, wherein in the above compounds, R ₁ and R _{2 are the} same and are Cl, Br, or I. The method according to claim 19 or 20, wherein the peptide is a peptide as defined in any one of claims 1 to 8 and 10 to 15.