Photo-crosslinking affinity peptide, conjugate and application thereof

Abstract

The invention provides a photo-crosslinking affinity peptide, an antibody conjugate and application of the photo-crosslinking affinity peptide and the antibody conjugate, and relates to the technical field of biology. The photo-crosslinking affinity peptide contains an affinity peptide Z34C or a fragment obtained after the sixth site of a variant of the affinity peptide Z34C is mutated into a photo-crosslinking amino acid residue. The photo-crosslinking affinity peptide has the advantages of high connection efficiency and good stability, and is helpful for realizing directional formation of conjugates without engineering modification of molecules to be formed into conjugates, and is simple and easy to operate.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a photocrosslinked affinity peptide, antibody conjugate, and its applications. Background Technology

[0002] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] Antibody site-specific conjugation methods have been extensively studied in antibody therapy and diagnostics. However, most of these methods require engineered modification or alteration of the antibody, making them technically complex and subject to numerous uncontrollable factors, hindering their widespread adoption and application. For example: (1) THIOMAB TM The technology requires the design of additional cysteine ​​at specific sites on the antibody through genetic engineering. In the process of antibody expression and assembly, the additional cysteine ​​is prone to disulfide bond mismatch with the natural cysteine ​​of the antibody, resulting in an incorrect antibody structure. Moreover, the free sulfhydryl group of the additional cysteine ​​is prone to oxidation during the expression process; (2) Enzyme-mediated methods, such as sorting enzymes, bacterial transglutaminase and SPY Catcher, require the introduction of tags at specific sites on the antibody through genetic engineering, which is technically complex; (3) Non-natural amino acid site-directed incorporation technology based on genetic code expansion requires the establishment of an expression platform that can recognize and express TAG amber stop codons, which is technically difficult; (4) Chemically selective modification, such as antibody disulfide bond reconstruction technology, requires the reduction treatment of antibody disulfide bonds, which involves many uncontrollable factors and uneven coupling sites.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a photocrosslinking affinity peptide and a conjugate containing the same, so as to improve the defects of conjugates in the prior art.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] In a first aspect, a photocrosslinked affinity peptide (PAP) is provided, comprising a fragment containing a 6th position of affinity peptide Z34C or a variant thereof mutated to a photocrosslinked amino acid residue.

[0008] In a second aspect, a modified photocrosslinking affinity peptide is provided, said modified photocrosslinking affinity peptide being the photocrosslinking affinity peptide of the first aspect modified with at least one extension for connecting with other molecules.

[0009] Thirdly, the application of the photocrosslinking affinity peptide described in the first aspect, or the modified photocrosslinking affinity peptide described in the second aspect, in the preparation of linkers or conjugates is provided.

[0010] Fourthly, a conjugate is provided, the conjugate comprising (i) and (ii):

[0011] (i) the photocrosslinking affinity peptide described in the first aspect, and / or the modified photocrosslinking affinity peptide described in the second aspect;

[0012] (ii) First functional molecule;

[0013] The photocrosslinked affinity peptide is covalently linked to the first functional molecule.

[0014] Fifthly, a method for preparing the conjugate described in the fourth aspect is provided, comprising reacting the first functional molecule with the photocrosslinking affinity peptide described in the first aspect, or with the modified photocrosslinking affinity peptide described in the second aspect, under photocrosslinking conditions.

[0015] The sixth aspect also provides the application of the conjugates described in the fourth aspect, or the preparation method described in the fifth aspect, in the preparation of pharmaceuticals or diagnostic reagents.

[0016] In a seventh aspect, a pharmaceutical composition is also provided, comprising the photocrosslinking affinity peptide described in the first aspect, or the modified photocrosslinking affinity peptide described in the second aspect, or the conjugate described in the fourth aspect.

[0017] In an eighth aspect, a detection reagent or kit is also provided, comprising the photocrosslinking affinity peptide described in the first aspect, or the modified photocrosslinking affinity peptide described in the second aspect, or the conjugate described in the fourth aspect. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The reaction mechanism is photoinduced by 4-benzoyl-L-phenylalanine;

[0020] Figure 2 The mass spectrometry results are those of PAP at different mutation sites after photocrosslinking with human IgG antibody in Example 2.

[0021] Figure 3The results are mass spectrometry findings of PAP1 and different subtypes of mouse IgG antibody after photocrosslinking in Example 3.

[0022] Figure 4 The results of non-reducing mass spectrometry detection after photocrosslinking of PAP1 with rabbit IgG antibody in Example 3 are shown.

[0023] Figure 5 The results of reduction mass spectrometry detection after photocrosslinking of PAP1 with rabbit IgG antibody in Example 3;

[0024] Figure 6 This refers to the non-reducing detection mass spectrometry of PAP1 after photocrosslinking with human IgG antibody in Example 3;

[0025] Figure 7 The results of reduction mass spectrometry detection after photocrosslinking of PAP1 with human IgG antibody in Example 3;

[0026] Figure 8 The results of mass spectrometry analysis of the thermostability of the antibody-affinity peptide conjugate 35C-17-PAP1 in Example 4 are shown. Detailed Implementation

[0027] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] The articles “a / an,” “one,” and “the” in this text include plural references unless the context clearly indicates otherwise.

[0029] In this document, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0030] The term "photocrosslinking" refers to the process by which a molecule containing photocrosslinking-active components undergoes covalent crosslinking with groups of neighboring molecules under suitable light conditions, forming a complex.

[0031] In this paper, the term "photocrosslinked affinity peptide" refers to an affinity peptide that can react with neighboring molecules to form chemical bonds under light conditions. An affinity peptide is a peptide segment that has an affinity for a target molecule.

[0032] In this article, the term "affinity peptide Z34C" refers to a peptide derived from Staphylococcus aureus protein A. The Staphylococcus aureus protein A z-domain has three α-helices, of which helix1 and helix2 bind directly to the antibody, while helix3 does not. Removal of helix3 reduces affinity by 10%. -5 The concentration was changed from 10 nM to 1 mM. To reduce the number of amino acids in the Z-domain without affecting affinity, in 1997, James A. Wells et al., based on structural analysis and mutation site design, used phage display technology for affinity screening, obtaining the affinity peptide Z34C, with the amino acid sequence: FNMQCQRRFYEALHDPNLNEEQRNAKIKSIRDDC (SEQ ID NO. 11). The term "affinity peptide Z34C" in this article includes wild-type or variants that retain its activity; the amino acid sequence of the wild-type is shown in SEQ ID NO. 11.

[0033] In this document, the term "conjugate" refers to a compound formed by the linkage of at least two molecules. Each molecule is independently formed, including but not limited to at least one of sugars, proteins, polypeptides, lipids, and nucleic acids. The terms "first functional molecule" and "second functional molecule" independently refer to the molecules mentioned above used to form the conjugate.

[0034] Typically, a natural, complete antibody consists of two heavy (H) chains and two light (L) chains. Based on the presence or absence of α, δ, ε, γ, and μ heavy chains, antibodies can be classified into five main categories or isotypes: IgA, IgD, IgE, IgG, and IgM. Several major antibody categories can also be subclassed, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain), etc. Each heavy chain consists of a variable region (VH) and first, second, third, and fourth (optionally) constant regions (CH1, CH2, CH3, CH4, respectively). Mammalian light chains can be divided into λ or κ chains, and each light chain consists of a variable region (VL) and a constant region (CL). The variable regions of the light and heavy chains determine antigen binding. Each chain typically contains three highly variable regions called "complementary determinant regions (CDRs)". The CDRs for light chains include LCDR1, LCDR2, and LCDR3, while the CDRs for heavy chains include HCDR1, HCDR2, and HCDR3.

[0035] The heavy chain was cleaved near the N-terminus of the interchain disulfide bond using papain, yielding three fragments: two identical antigen-binding fragments (Fab) and one crystallizable fragment (Fc). In this paper, the "Fc region" refers to the C-terminal region of the antibody heavy chain, which contains at least a portion of the constant region. The amino acid sequence of the Fc region in this paper is derived from the natural sequence and / or variants of the natural sequence.

[0036] In this article, when the molecule forming a conjugate with the photocrosslinking affinity peptide contains the Fc region of an antibody, the Fc region of the antibody can be a complete antibody Fc region or a partial fragment derived from the antibody Fc region.

[0037] The antibodies used in this article for photocrosslinking affinity peptides are used in the broadest sense, covering a variety of antibody structures, including but not limited to monoclonal or polyclonal antibodies, monospecific or multispecific antibodies, naturally derived antibodies or artificially modified or constructed antibodies; artificially modified or constructed antibodies include but are not limited to chimeric antibodies, humanized antibodies, single-chain antibodies, or antibodies derived from a combination of partial domains of the aforementioned artificially modified or constructed antibodies and partial domains of natural antibodies.

[0038] In this article, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include amino acids encoded by the genetic code and their modified forms, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common natural amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid analogs typically have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0039] In this paper, the term "photocrosslinked amino acid residue" refers to an amino acid that can covalently crosslink with amino acid residues of a neighboring protein under suitable light conditions. "Photocrosslinked amino acid residue" can include both natural and non-natural amino acids.

[0040] In this article, the term "multispecific antibody" refers to an antibody molecule that can bind to multiple (two or more) different antigenic epitopes of the same antigen or multiple (two or more) different antigens.

[0041] In this paper, the term "linker" refers to the part used to connect any two molecules, which enables at least two molecules to combine to form a conjugate.

[0042] In this document, the term "signaling substance" refers to a substance that can provide a detectable signal that can be directly observed by the naked eye or detected by conventional instruments acceptable in the art. The signaling substance can provide a signal directly, such as color (e.g., colloidal gold, colored microspheres), fluorescence (fluorescent molecules), magnetism, radiation, or luminescence; or it can provide a signal indirectly through a reaction in which the signaling substance participates, such as catalyzing a specific substrate reaction to produce any of the above signals.

[0043] In this document, the term "linker" refers to a chemical moity that may contain about 2 to about 50 atoms or 4 to about 30 atoms (excluding hydrogen), and may contain a chain of 2 to about 30 atoms or 3 to about 20 atoms, each of which is independently selected from the group generally consisting of carbon, oxygen, sulfur, nitrogen, and phosphorus. In some instances, some or all of the linker may be part of a linked molecule, such as, but not limited to, amino acid residues on a poly(amino acid). The number of heteroatoms in the linker may be 0 to about 20, or 1 to about 15, or about 2 to about 10. The linker may be aliphatic or aromatic. When heteroatoms are present, oxygen is generally present as an oxo or oxygen group bonded to carbon, sulfur, nitrogen, or phosphorus; nitrogen is generally present as a nitro, nitroso, or amino group generally bonded to carbon, oxygen, sulfur, or phosphorus; sulfur is similar to oxygen; and phosphorus is generally bonded to carbon, sulfur, oxygen, or nitrogen as a phosphonate mono- or diester and a phosphate mono- or diester. Common functional groups that form covalent bonds between the linker and the molecule to be coupled include alkylamines, amidines, thioamides, ethers, ureas, thioureas, guanidines, azo compounds, thioethers and carboxylic acid esters, sulfonates and phosphate esters, amides and thioesters.

[0044] In most cases, the linking group further has a linking functional group (functional group for reacting with the group), including nonoxocarbonyl groups of nitrogen and sulfur analogs, phosphate ester groups, amino groups, alkylating agents such as halo- or toluenesulfonyl alkyl groups, oxy (hydroxyl or sulfur analogs, mercapto) oxocarbonyl groups (e.g., aldehydes or ketones), or reactive olefins such as vinyl sulfones or α-, β-unsaturated esters. These functional groups can be linked to amine groups, carboxyl groups, reactive olefins, alkylating agents, such as bromoacetyl groups. When an amine is linked to a carboxylic acid or its nitrogen derivative or phosphoric acid, amides, amidines, and phosphoramides can be formed. When a thiol is linked to an activated olefin, a thioether is formed. When a thiol is linked to an alkylating agent, a thioether is formed. When an aldehyde and an amine are linked under reducing conditions, an alkylamine is formed. When a ketone or aldehyde and a hydroxylamine (including its derivatives, wherein a substituent replaces the hydrogen of the hydroxyl group) are linked, an oxime functional group (=NO-) is formed. When a carboxylic acid or phosphoric acid and an alcohol are linked, an ester is formed. When the connecting functional group is, for example, a click chemical group, including methyltetrazine, trans-cyclooctene, azide, dibenzocyclooctene, tetrazine, alkyne, cyclopropanecyclooctene, and cyclopropene, it can be connected to a pair of orthogonal click chemical groups.

[0045] In this document, the term "pharmaceutical composition" refers to a form in which the biological activity of the active ingredient is permitted and which does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the composition is administered.

[0046] In this article, "pharmaceutically acceptable carriers" can include any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents.

[0047] In this article, “tumor-associated antigen” refers to any effective target molecule acceptable in the art for cancer diagnosis and treatment that is expressed at a higher level on the surface of cancer cells than in non-cancer cells.

[0048] In this paper, the "AVI tag" is a short peptide tag consisting of 15 amino acid residues, which can be biotinylated by biotin ligase by attaching a biotin to a lysine residue, both in vivo and in vitro, thereby achieving protein biotinylation. The "SPY tag" contains 13 amino acid residues and can spontaneously react with the Lys residue on the SPYCatcher in the SPYTag / SPYCatcher system to form an isopeptide covalent bond. The "LPXTG tag" is a sorting enzyme recognition motif.

[0049] In a first aspect, a photocrosslinked affinity peptide (PAP) is provided, comprising a fragment containing a 6th position of affinity peptide Z34C or a variant thereof mutated to a photocrosslinked amino acid residue.

[0050] The affinity peptide Z34C can bind to the antibody Fc to form a complex. In this invention, the glutamine Q at position 6 of Z34C is mutated to a photocrosslinked amino acid residue, which is the photocrosslinked affinity peptide PAP of this invention. Under light excitation, the photocrosslinked amino acid residue on the photocrosslinked affinity peptide PAP can be covalently linked to the methionine at position 252 of the antibody to form a site-specific complex.

[0051] In an optional embodiment, the affinity peptide Z34C comprises the amino acid sequence shown in SEQ ID NO.11; or comprises a mutated amino acid sequence of the amino acid sequence shown in SEQ ID NO.11.

[0052] SEQ ID NO. 11: FNMQCXRRFYEALHDPNLNEEQRNAKIKSIRDDC.

[0053] In an optional embodiment, variants of the affinity peptide Z34C include at least one amino acid residue in the amino acid sequence shown in SEQ ID NO.11 that has undergone a substitution mutation, deletion mutation, or insertion mutation.

[0054] In an optional embodiment, the amino acid sequence shown in SEQ ID NO.11 is used as the initial sequence, and the mutation sites include at least one mutation at positions 5, 9, 17, 19 and 34 of the affinity peptide Z34C.

[0055] In an optional implementation, a substitution mutation occurs at position 5 and / or position 19; optionally, position 5 is substituted with Q, and optionally, position 19 is substituted with K.

[0056] In an optional implementation, an insertion mutation occurs at the 9th position; alternatively, an A is inserted at the 9th position.

[0057] In an optional implementation, a deletion mutation occurs at position 17 and / or position 34.

[0058] In an optional embodiment, variants of the affinity peptide Z34C include amino acid sequences as shown in any of SEQ ID NO. 12 to 15.

[0059] SEQ ID NO.12: FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD;

[0060] SEQ ID NO.13: FNMQCQRRFYEALHDPNLKEEQRNAKIKSIRDDC;

[0061] SEQ ID NO.14: FNMQCQRRFYEALHDPLNEEQRNAKIKSIRDDC;

[0062] SEQ ID NO. 15: FNMQCQRRAFYEALHDPNLNEEQRNAKIKSIRDDC.

[0063] In an optional embodiment, the amino acid sequence of the photocrosslinked affinity peptides A1 to A6 (positions 1 to 6 of the amino acid sequence) is FNMQX1X2 (SEQ ID NO.16); X1 is Q or C, and X2 is a photocrosslinked amino acid residue.

[0064] In an optional embodiment, the photocrosslinking affinity peptide comprises the amino acid sequence shown in SEQ ID NO.1, or comprises a mutated amino acid sequence of the amino acid sequence shown in SEQ ID NO.1, wherein the mutation does not occur at position 6 of SEQ ID NO.1.

[0065] SEQ ID NO.1: FNMQCX3RRFYEALHDPNLNEEQRNAKIKSIRDDC, where X3 is a photocrosslinked amino acid residue.

[0066] In an optional embodiment, the mutated amino acid sequence of the amino acid sequence shown in SEQ ID NO.1 includes at least one amino acid residue that has undergone substitution, deletion, or insertion mutations.

[0067] In an optional embodiment, the amino acid sequence shown in SEQ ID NO.1 is used as the base sequence, and the mutation sites include at least one mutation at positions 5, 9, 17, 19 and 34 of the amino acid sequence shown in SEQ ID NO.1.

[0068] In an optional implementation, a substitution mutation occurs at position 5 and / or position 19; optionally, position 5 is substituted with Q, and optionally, position 19 is substituted with K.

[0069] In an optional implementation, an insertion mutation occurs at the 9th position; alternatively, an A is inserted at the 9th position.

[0070] In an optional implementation, a deletion mutation occurs at position 17 and / or position 34.

[0071] In an optional embodiment, the photocrosslinking affinity peptide comprises an amino acid sequence as shown in any of SEQ ID NO. 2 to 5.

[0072] SEQ ID NO.2: FNMQQX4RRFYEALHDPNLNEEQRNAKIKSIRDD;

[0073] SEQ ID NO.3: FNMQCX5RRFYEALHDPNLKEEQRNAKIKSIRDDC;

[0074] SEQ ID NO.4: FNMQCX6RRFYEALHDPLNEEQRNAKIKSIRDDC;

[0075] SEQ ID NO5: FNMQCX7RRAFYEALHDPNLNEEQRNAKIKSIRDDC;

[0076] X4, X5, X6, and X7 are each independently photocrosslinked amino acid residues.

[0077] In an optional embodiment, the photocrosslinked amino acid residue is a non-natural amino acid residue with a photocrosslinked group in its side chain. The non-natural amino acid residue includes benzophenone-alanine, 4-benzoyl-L-phenylalanine, L-luciferine, L-luciferase, 3,4-difluorophenylalanine, 4,4,4-trifluoro-L-valine, 5-fluoro-L-tryptophan, 5,5,5-trifluoro-L-leucine, or 3-amino-5-azidobenzoylcarbonyl-L-lysine.

[0078] In an optional embodiment, the non-natural amino acid residue is 4-benzoyl-L-phenylalanine. 4-benzoyl-L-phenylalanine B is a photocrosslinked amino acid that, under ultraviolet 365 nm excitation, can react with amino acids containing CH groups in their side chains to form carbon-carbon double bonds, such as... Figure 1 As shown.

[0079] In an optional embodiment, the amino acid sequence of the photocrosslinked affinity peptide is as shown in any one of SEQ ID NO. 1 to 5, and the photocrosslinked amino acid residue is 4-benzoyl-L-phenylalanine. That is, the amino acid sequence of the photocrosslinked affinity peptide is as shown in any one of SEQ ID NO. 6 to 10.

[0080] The photocrosslinked affinity peptide provided in the first aspect of the embodiments can be linked to other molecules by light irradiation. Molecules modified with this photocrosslinked affinity peptide facilitate the simple and easy-to-operate linking of other molecules to the modified molecule without requiring engineering modifications, thus achieving directional coupling. In some optional embodiments, when the photocrosslinked affinity peptide is linked to an antibody, the linkage efficiency can reach 100%, and the DAR value can reach 1.5. Furthermore, the photocrosslinked affinity peptide exhibits good stability after being linked to an antibody, and can be stably stored at 2–8°C for at least 22 months.

[0081] In a second aspect, a modified photocrosslinking affinity peptide is provided, said modified photocrosslinking affinity peptide being the photocrosslinking affinity peptide of the first aspect modified with at least one extension for linking with other molecules. When said photocrosslinking affinity peptide is modified with two or more extensions, the extensions may be the same or different, the difference including all extensions being different or at least one extension being different from the others.

[0082] In an optional embodiment, the extension includes a click chemical group, which includes one or more of azide, alkyne, trans-cyclooctene, tetrazine, BCN (bicyclo[6.1.0]nonyne), DBCO (dibenzocyclooctene), and DIBO (4-dibenzocycloocteneol).

[0083] In an optional embodiment, the extension includes one or more of maleimide, biotin, thiol, AVI tag, SPY tag, and LPTGS tag.

[0084] In an optional embodiment, each of the extended portions is independently modified at the 2nd, 4th, 8th, 11th, 16th, 17th, 19th, 21st, 22nd, 25th, 29th, 33rd or 34th of the photocrosslinking affinity peptide.

[0085] In an optional embodiment, each of the extended portions is independently modified with N2, Q4, R8, E11, P16, N17, N19, E21, Q22, A25, S29, D33 or C34 of the photocrosslinking affinity peptide.

[0086] In an optional embodiment, each of the extended portions is independently modified with E11, N17, N19 or C34 of the photocrosslinking affinity peptide.

[0087] Thirdly, the application of the photocrosslinking affinity peptide described in the first aspect, or the modified photocrosslinking affinity peptide described in the second aspect, in the preparation of linkers or conjugates is provided.

[0088] In an optional embodiment, the conjugate contains at least one Fc region of an antibody.

[0089] In an optional embodiment, the conjugate contains an antibody.

[0090] In an optional embodiment, the conjugate contains an intact antibody or a heavy chain antibody;

[0091] In an optional embodiment, the conjugate includes a drug or a detection reagent;

[0092] In an optional implementation, the drug includes an antibody-drug conjugate.

[0093] Fourthly, a conjugate is provided, the conjugate comprising (i) and (ii):

[0094] (i) the photocrosslinking affinity peptide described in the first aspect, and / or the modified photocrosslinking affinity peptide described in the second aspect;

[0095] (ii) First functional molecule;

[0096] The photocrosslinked affinity peptide is covalently linked to the first functional molecule.

[0097] In an optional embodiment, the first functional molecule contains amino acid residues for covalently linking with the photocrosslinked amino acid residues.

[0098] In an optional embodiment, the photocrosslinked affinity peptide is covalently linked to the first functional molecule via photocrosslinked amino acid residues.

[0099] In an optional embodiment, the photocrosslinked affinity peptide is covalently linked to the first functional molecule via a carbon-carbon double bond.

[0100] In an optional embodiment, the first functional molecule includes a protein or a polypeptide.

[0101] In an optional embodiment, the first functional molecule contains at least one Fc region of an antibody.

[0102] In an optional embodiment, the first functional molecule contains 1, 2, 3 or more antibody Fc regions, and the conjugate may have some or all of the antibody Fc regions covalently linked to the photocrosslinked affinity peptide.

[0103] In an optional embodiment, the antibody Fc region includes methionine at position 252 of the antibody Fc.

[0104] In an optional embodiment, the amino acid sequence of each antibody Fc region is independently derived from a portion or all of the constant region sequence of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. In an optional embodiment, each antibody Fc region is the Fc region of an IgG antibody.

[0105] In an optional embodiment, the amino acid sequence of each antibody Fc region is independently derived from the Fc region sequence of one or more antibodies selected from humans, rabbits, mice, rats, guinea pigs, hamsters, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, and monkeys.

[0106] In an optional embodiment, the amino acid sequence of the Fc region of the antibody is derived from the Fc region sequence of a human or rabbit.

[0107] In an optional embodiment, the photocrosslinked affinity peptide is covalently linked to the Fc region of the antibody via photocrosslinked amino acid residues.

[0108] In an optional implementation, the first functional molecule includes an antibody.

[0109] In an optional implementation, the first functional molecule includes an intact antibody or a heavy chain antibody.

[0110] In an optional embodiment, the antibody includes a human antibody, a humanized antibody, or a chimeric antibody with a constant region derived from human origin.

[0111] In an optional embodiment, the humanized antibody includes a humanized mouse antibody or a rabbit antibody.

[0112] In an optional embodiment, the chimeric antibody may include a mouse or rabbit antibody whose Fc region is replaced with a human Fc region.

[0113] In an optional embodiment, the amino acid sequence of the Fc region of the antibody is derived from the sequence of the Fc region of a human IgG antibody or from the sequence of the Fc region of a rabbit IgG antibody.

[0114] In an optional embodiment, the antibody is a human IgG antibody or a rabbit IgG antibody; more optionally, the antibody is a complete antibody molecule.

[0115] In an optional embodiment, the first functional molecule is covalently linked to 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) of the photocrosslinked affinity peptides.

[0116] In an optional embodiment, when the conjugate contains more than one photocrosslinking affinity peptide and / or a modified photocrosslinking affinity peptide, the photocrosslinking affinity peptides in the conjugate may be the same or different. The difference includes that the photocrosslinking affinity peptides are not identical to each other, or that at least one photocrosslinking affinity peptide is different from all other photocrosslinking affinity peptides. The difference includes differences in the amino acid sequence and / or photocrosslinking amino acid residues of the photocrosslinking affinity peptides. When the conjugate contains at least two modified photocrosslinking affinity peptides, the derivatives of each modified photocrosslinking affinity peptide may be the same or different. Differences in the extensions include differences in the type, number, and modification site of the extension.

[0117] In an optional embodiment, the conjugate further contains at least one second functional molecule, which is linked to the first functional molecule via the photocrosslinking affinity peptide.

[0118] In an optional embodiment, at least one second functional molecule is connected to the extension via a linker;

[0119] In an optional embodiment, the second functional molecule includes a drug and / or a signaling substance. The second functional molecule may have the functions of both a drug and a signaling substance, or have one of the functions of a drug or a signaling substance.

[0120] In an optional embodiment, the drug includes one or more of topoisomerase II inhibitors, DNA cross-linking cytotoxic agents, alkylating agents, taxanes, and anthracyclines.

[0121] In optional embodiments, exemplary signaling agents include, but are not limited to, one or more of fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

[0122] In optional embodiments, exemplary fluorescent dyes include, but are not limited to, at least one of fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, and protein dyes and their derivatives.

[0123] In optional embodiments, exemplary enzymes include, but are not limited to, any one of horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0124] In optional embodiments, exemplary radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 At least one of F.

[0125] In optional embodiments, exemplary chemiluminescent reagents include, but are not limited to, at least one of luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0126] In optional embodiments, exemplary nanoparticle markers include, but are not limited to, any one of colloids, organic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0127] In optional embodiments, exemplary colloids include, but are not limited to, at least one of colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0128] In optional embodiments, exemplary colloidal metals include, but are not limited to, at least one of colloidal gold, colloidal silver, and colloidal selenium.

[0129] In an optional embodiment, the conjugate includes the following structure:

[0130] Ab(-PAP-XLW) D ;

[0131] Ab is an antibody, PAP is the photocrosslinked affinity peptide, X is the extension, L is the linker, and W is the second functional molecule. The second functional molecule W is connected to the extension X on the photocrosslinked affinity peptide PAP through the linker L. The RAP in each structural unit (-PAP-XLW) is directly covalently linked to Ab.

[0132] D is the number of structural units (-PAP-XLW), and D is a positive integer greater than 0;

[0133] In an optional implementation, 1≤D≤8, for example, but not limited to 1, 2, 3, 4, 5, 6, 7 or 8.

[0134] In an optional implementation, D is 1 or 2.

[0135] Fifthly, a method for preparing the conjugate described in the fourth aspect is provided, comprising reacting the first functional molecule with the photocrosslinking affinity peptide described in the first aspect, or with the modified photocrosslinking affinity peptide described in the second aspect, under photocrosslinking conditions.

[0136] In an optional embodiment, the photocrosslinking conditions include ultraviolet light irradiation.

[0137] In an optional implementation, the ultraviolet light wavelength is 365nm.

[0138] The sixth aspect also provides the application of the conjugates described in the fourth aspect, or the preparation method described in the fifth aspect, in the preparation of pharmaceuticals or diagnostic reagents.

[0139] In an optional implementation, the drug includes an antibody-drug conjugate.

[0140] In a seventh aspect, a pharmaceutical composition is also provided, comprising the photocrosslinking affinity peptide described in the first aspect, or the modified photocrosslinking affinity peptide described in the second aspect, or the conjugate described in the fourth aspect.

[0141] In an optional embodiment, the pharmaceutical composition comprises the conjugate described in the fourth aspect, wherein the first and / or second functional molecules in the conjugate target tumor-associated antigens.

[0142] In an optional embodiment, the pharmaceutical composition comprises the conjugate described in the fourth aspect, the conjugate containing a first functional molecule and a second functional molecule, the first functional molecule comprising an antibody and the second functional molecule comprising a drug. In a further optional embodiment, the antibody targets a tumor-associated antigen.

[0143] In optional embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient. The acceptable carrier and pharmaceutically acceptable excipient may be any conventional carrier and / or excipient known in the art. Examples of carriers include, but are not limited to, any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents; examples of excipients include, but are not limited to, fillers, disintegrants, preservatives, solubilizers, and emulsifiers.

[0144] In optional embodiments, the pharmaceutical composition may further include one or more active pharmaceutical ingredients with other therapeutic effects, including but not limited to one or more of the following: chemotherapeutic agents, anticancer drugs, radiotherapy agents, immunotherapy agents, antiangiogenic agents, targeted therapy agents, cell therapy agents, gene therapy agents, hormone therapy agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, and cytokines.

[0145] In an eighth aspect, a detection reagent or kit is also provided, comprising the photocrosslinking affinity peptide described in the first aspect, or the modified photocrosslinking affinity peptide described in the second aspect, or the conjugate described in the fourth aspect.

[0146] In an optional embodiment, the detection reagent or kit contains the conjugate described in the fourth aspect, wherein the first functional molecule and / or the second functional molecule in the conjugate is a signaling molecule.

[0147] In an optional embodiment, the detection reagent or kit contains the conjugate described in the fourth aspect, wherein the first functional molecule and / or the second functional molecule in the conjugate targets the target molecule to be detected.

[0148] In an optional embodiment, the detection reagent or kit contains the conjugate described in the fourth aspect, the conjugate containing a first functional molecule and a second functional molecule, the first functional molecule including an antibody that targets the target molecule to be detected, and the second functional molecule including a signaling substance, thereby achieving the detection purpose through the detection signal provided by the signaling substance.

[0149] In optional embodiments, the detection reagent or kit further contains detection reagents, including but not limited to one or more of the following: buffer reagents, salts, secondary antibodies, chromogenic substrates, blocking solutions, washing solutions, solvents, elution solutions, coupling agents, negative controls, positive controls, standards, quality control materials, and markers. Those skilled in the art can formulate other reagents in the reagent or kit according to the corresponding detection methods, and this invention does not limit this.

[0150] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0151] Example 1

[0152] I. Synthesis of photocrosslinking affinity peptide PAP:

[0153] The sequences of the photocrosslinked affinity peptides are shown in Table 1. PAPs were synthesized using the standard Fmoc solid-phase peptide synthesis method, purified to >95% by reversed-phase HPLC, and then lyophilized.

[0154] Table 1. Sequences of photocrosslinking affinity peptides

[0155]

[0156] PAP1 has a 4-benzoyl-L-phenylalanine at position 6 (B), with a disulfide bond formed between C5 and C34, using PAP1 as the baseline sequence. PAP2 does not contain a disulfide bond; its C at position 5 is mutated to Q, and its C at position 34 is deleted. PAP3 contains a disulfide bond, with a substitution mutation at position 19. PAP4 contains a disulfide bond, with a deletion mutation at position 17. PAP5 contains a disulfide bond, with an insertion mutation at position 9. PAP6 has a 4-benzoyl-L-phenylalanine at position 7 (B), with a disulfide bond formed between C5 and C34. PAP7 has a 4-benzoyl-L-phenylalanine at position 4 (B), with a disulfide bond formed between C5 and C34.

[0157] II. Affinity Test:

[0158] Fresh 50 mM N-hydroxysuccinimide (NHS) and 200 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were mixed in a 1:1 ratio and injected into Flow cell 1 and Flow cell 2 for 420 s (10 μL / min) to activate the surface active coupling sites of the sensor chip CM5 (BIACORE, Inc.). Then, 20 μg / ml mouse IgG1 & 3 and mouse IgG were added. 2a and mouse IgG 2b; and 10 μg / ml human IgG and rabbit IgG (diluted with NaAc pH=4.0) were injected into Flow cell 2 for 420s (10 μL / min) to achieve a coupling response of more than 5000RU. Finally, the remaining active coupling sites on the chip surface were blocked by injecting 1 mol / L ethanolamine hydrochloride into Flow cell 1 and Flow cell 2 for 420s (10 μL / min).

[0159] kd(RU) was calculated on a CM5 chip using Biacore 8K plus (BIAcore, Inc., Piscataway, NJ) with 8 response units. For kinetic measurements, PAP2 (0.3 μM to 20 μM) was injected sequentially at a flow rate of approximately 30 μl / min at 25 °C with two-fold dilutions. Since the antigen and antibody followed a fast binding-fast dissociation model, affinity was used to fit association and dissociation sensor maps. All data were processed using Biacore 8K plus Evaluation software to calculate the association rate (kon) and dissociation rate (koff), and the equilibrium dissociation constant (Kd) was calculated as the koff / kon ratio.

[0160] Table 2. Affinity of photocrosslinked affinity peptides

[0161]

[0162] Table 2 shows that the affinity peptide PAP2 does not contain disulfide bonds, and its affinity is reduced by 3-fold compared to PAP1. PAP3, PAP4, and PAP5 all contain disulfide bonds, and compared to PAP1, they have undergone substitution mutations, deletion mutations, and insertion mutations, respectively. These mutations have little effect on affinity. The mutation positions of 4-benzoyl-L-phenylalanine in PAP6 and PAP7 are different from those in PAP1. In PAP6, the R at position 7 is mutated to B, and in PAP7, the Q at position 4 is mutated to B, while in PAP1, the Q at position 6 is mutated to B.

[0163] Example 2

[0164] Effect of different mutation sites on coupling efficiency of affinity peptides

[0165] I. Experimental Materials:

[0166] 1. Antibody: 35C-17, targeting cTnI (cardiac troponin I), Fc humanized mouse monoclonal IgG antibody, mass concentration 7.83 mg / mL, molar concentration 52.2 μM.

[0167] 2. Photocrosslinking affinity peptides: The molar concentrations of PAP1, PAP2, PAP3, PAP4, PAP5, PAP6 and PAP7 are all 2.4 mM.

[0168] II. Experimental Procedure:

[0169] Take a PCR tube and add 20mM pH7.4 PB, antibody, and photocrosslinking affinity peptide in the order listed in the table below. Shake to mix.

[0170] The reaction system is as follows:

[0171] Table 3 Response parameters of PAP at different mutation sites

[0172] Photocrosslinking affinity peptide name PAP Affinity peptide: antibody molar ratio 5 Total reaction volume (μL) 50 Antibody working concentration (mg / mL) 4 Antibody addition volume (μL) 25.5 Working concentration (μM) of photocrosslinking affinity peptide 134 Volume of photocrosslinked affinity peptide added (μL) 2.8 20 mM pH 7.4 PB (μL) 21.7

[0173] A 36nm LED UV lamp box (18W power) was placed in a 4℃ refrigerator. Two PCR tubes containing the prepared reaction system were placed in the UV lamp box, and the illumination time was set to [number] hours. After the reaction, non-reducing mass spectrometry was used for detection.

[0174] III. Experimental Results:

[0175] Mass spectrometry results as follows Figure 2 As shown, PAP1, PAP2, PAP3, PAP4, and PAP5 have a mutant B at Q6; after photocrosslinking, the antibody molecular weight increases. PAP6 and PAP7 have mutants B at R7 and Q4 respectively; after photocrosslinking, the antibody molecular weight does not increase. Figure 2 As shown, PAP6 and PAP7 cannot be conjugated with the antibody. The Fc-humanized mouse monoclonal antibody 35C-17 contains two affinity peptide binding sites at its Fc terminus. After conjugation of 35C-17 with the affinity peptide PAP, the molecular weight increases, by a factor of 1 and 2, respectively, relative to the molecular weight of the affinity peptide. Based on the mass spectrometry results, the conversion efficiency of the conjugated products and the percentage of each conjugated product were further calculated, and the results are shown in Table 6.

[0176] Table 4. Mass spectrometry data analysis of different mutation sites

[0177]

[0178] The conjugation efficiency of affinity peptides PAP1–5 was 100%, with slight differences in DAR values. The DAR value is calculated as: Affinity peptide 1 × Content percentage + Affinity peptide 2 × Content percentage, reflecting the distribution of the two conjugated products. A larger DAR value indicates a higher proportion of conjugated products from affinity peptide 2, and vice versa. The DAR values ​​of PAP2 and PAP5 were slightly lower than the other three PAPs, but the difference was not significant. All PAPs from different mutants could efficiently form adducts with the antibody.

[0179] Example 3

[0180] Evaluation of conjugation efficiency of different types of antibodies:

[0181] I. Experimental Materials:

[0182] 1. The antibody information used in this experiment is shown in the table below:

[0183] Table 5 Information on different types of antibodies

[0184] name Antibody Fc type concentration molar concentration cTnI 30C-12-21 <![CDATA[Mouse IgG1]]> 9.12 60 cTnI 30C-9-1 <![CDATA[Mouse IgG 2a > 9.6 64 cTnI 30C-119 <![CDATA[Mouse IgG 2b > 5.45 36 P24 30P24-25 Rabbit IgG 6.45 42 cTnI 35C-17 Human IgG 7.83 52

[0185] 2. Photocrosslinking affinity peptide: PAP1, molar concentration 2.4mM.

[0186] II. Experimental Procedure:

[0187] Take 5 PCR tubes and add 20mM pH 7.4 PB, antibody, and photocrosslinking affinity peptide sequentially according to the table below. Vortex to mix. The reaction system is as follows:

[0188] Table 6 Parameters of Different Types of Antibodies

[0189] antibody types <![CDATA[Mouse IgG1]]> <![CDATA[Mouse IgG 2a > <![CDATA[Mouse IgG 2b > rabbit people Affinity peptide: antibody molar ratio 5 5 5 5 5 Total reaction volume (μL) 50 50 50 50 50 Antibody working concentration (mg / mL) 4 4 4 4 4 Antibody addition volume (μL) 22 20 37 31 26 Volume of photocrosslinked affinity peptide added (μL) 3 3 3 3 3 20 mM pH 7.4 PB (μL) 25 27 10 16 21

[0190] A 36nm LED UV lamp box (18W power) was placed in a 4℃ refrigerator. Two PCR tubes containing the prepared reaction system were placed in the UV lamp box, and the illumination time was set to [number] hours. After the reaction, the reaction products of different mouse IgG subtypes with PAP were detected using non-reduction mass spectrometry. The reaction products of rabbit IgG and human IgG with PAP were detected using both non-reduction and reduction mass spectrometry.

[0191] III. Experimental Results:

[0192] Mass spectrometry detection results as follows Figures 3-7 As shown in Table 7, the mass spectrometry data of different types of antibodies are statistically analyzed. The results show that the conjugation efficiency of rabbit IgG antibody and human IgG antibody with PAP1 can reach more than 1.6.

[0193] Table 7. Statistical analysis of mass spectrometry data for different types of antibodies

[0194]

[0195] Example 4

[0196] Antibody-affinity peptide stop-conjugate stability:

[0197] The antibody-affinity peptide conjugate 35C-17-PAP1, at a concentration of 4 mg / mL, with a total volume of 200 μL, was divided into five equal portions of 40 μL each. One portion was stored at 4°C, and the other four portions were incubated at 37°C. These portions were removed on days 1, 3, 7, and 14 and then stored at 4°C. Mass spectrometry analysis was performed on all five samples. The results are shown below. Figure 8As shown, the antibody affinity peptide 35C-17-PAP1, stored at 4°C, has two molecular weights: one for antibodies binding one PAP1 and the other for antibodies binding two PAP1s. After being stored at 37°C for different days, the molecular weight and relative strength of the antibody affinity peptide remained unchanged. According to the Arrhenius equation, heating at 37°C for one day is equivalent to storing the product at 2–8°C for 1.6 months. Therefore, the antibody affinity peptide can be stably stored at 2–8°C for at least 22 months.

[0198] The mass spectrometry detection methods in Examples 2-4 are as follows:

[0199] 1. For non-reduced intact molecular weight mass spectrometry detection, the sample is diluted to 1 mg / mL with ddH2O and injected directly.

[0200] Reduced molecular weight mass spectrometry (MMS) detection, denaturation conditions: 50 mM DTT, 57 °C, 45 min. Add 95 μl of 100 μg monoclonal antibody to a 0.6 ml EP tube, then add 5 μl of 1 M DTT to achieve a final concentration of 50 mM. Incubate in a metal bath at 57 °C with shaking for 45 min. After incubation, aspirate the solution into a sample vial for injection.

[0201] 2. Detection using liquid chromatography-mass spectrometry (LC-MS):

[0202] Liquid phase:

[0203] Table 8 Liquid Phase Parameters

[0204]

[0205] Mass spectrometry section:

[0206] Table 9 Mass Spectrometry Parameters

[0207]

[0208] 3. Data analysis: Deconvolution is performed using relevant software to obtain the corresponding graph.

[0209] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A photocrosslinking affinity peptide, characterized in that, A fragment containing the affinity peptide Z34C or a variant thereof with a mutation at position 6 followed by a photocrosslinked amino acid residue.

2. The photocrosslinking affinity peptide according to claim 1, characterized in that, The amino acid sequence of the photocrosslinked affinity peptides A1 to A6 is FNMQX1X2; X1 is Q or C, and X2 is a photocrosslinked amino acid residue.

3. The photocrosslinking affinity peptide according to claim 1, characterized in that, The photocrosslinking affinity peptide comprises the amino acid sequence shown in SEQ ID NO.1, or comprises a mutated amino acid sequence of the amino acid sequence shown in SEQ ID NO.1, wherein the mutation does not occur at position 6 of SEQ ID NO.1; Optionally, the mutation includes a substitution mutation, deletion mutation, or insertion mutation of at least one amino acid residue; Optionally, the amino acid sequence shown in SEQ ID NO.1 is used as the base sequence, and the mutation sites include at least one mutation at positions 5, 9, 17, 19 and 34 of the amino acid sequence shown in SEQ ID NO.1; Optionally, a substitution mutation occurs at position 5 and / or position 19; Optionally, the 5th bit is replaced with Q; Optionally, the 19th position is replaced with K; Optionally, an insertion mutation occurs at position 9; Optionally, insert an A in the 9th position; Optionally, a deletion mutation occurs at position 17 and / or position 34; Optionally, the photocrosslinking affinity peptide comprises any of the amino acid sequences shown in SEQ ID NO. 2 to 5; Optionally, the amino acid sequence of the photocrosslinked affinity peptide is as shown in any one of SEQ ID NO. 1 to 5.

4. The photocrosslinking affinity peptide according to claim 1, characterized in that, The photocrosslinked amino acid residues are non-natural amino acid residues with photocrosslinking groups in their side chains; The non-natural amino acid residues include benzophenone-alanine, 4-benzoyl-L-phenylalanine, L-luciferine, L-luciferase, 3,4-difluorophenylalanine, 4,4,4-trifluoro-L-valine, 5-fluoro-L-tryptophan, 5,5,5-trifluoro-L-leucine, or 3-amino-5-azidobenzoylcarbonyl-L-lysine; Optionally, the amino acid sequence of the photocrosslinked affinity peptide is as shown in any one of SEQ ID NO. 6 to 10.

5. A modified photocrosslinking affinity peptide, characterized in that, The photocrosslinking affinity peptide according to any one of claims 1 to 4 is modified to have at least one extension for connecting with other molecules.

6. The modified photocrosslinking affinity peptide according to claim 5, characterized in that, The extension includes click chemical groups; Optionally, the click chemical group includes one or more of azide, alkyne, trans-cyclooctene, tetrazine, bicyclo[6.1.0]nonyne, dibenzocyclooctene, and 4-dibenzocyclooctynol; Optionally, the extension may include one or more of maleimide, biotin, thiol, AVI tag, SPY tag, and LPTG tag.

7. The modified photocrosslinking affinity peptide according to claim 5 or 6, characterized in that, Each of the aforementioned extension portions is independently modified at the 2nd, 4th, 8th, 11th, 16th, 17th, 19th, 21st, 22nd, 25th, 29th, 33rd, or 34th of the photocrosslinking affinity peptide; Optionally, each of the extended portions is independently modified with N2, Q4, R8, E11, P16, N17, N19, E21, Q22, A25, S29, D33 or C34 of the photocrosslinking affinity peptide; Optionally, each of the extended portions is independently modified with E11, N17, N19 or C34 of the photocrosslinking affinity peptide.

8. The use of the photocrosslinking affinity peptide according to any one of claims 1 to 4, or the modified photocrosslinking affinity peptide according to any one of claims 5 to 7, in the preparation of linkers or conjugates; Optionally, the conjugate contains at least one Fc region of an antibody; Optionally, the conjugate contains an antibody; Optionally, the conjugate contains a complete antibody or a heavy chain antibody; Optionally, the conjugate includes a drug or a detection reagent; Optionally, the drug includes an antibody-drug conjugate.

9. A conjugate, characterized in that, Contains (i) and (ii): (i) the photocrosslinking affinity peptide according to any one of claims 1 to 4, and / or the modified photocrosslinking affinity peptide according to any one of claims 5 to 7; (ii) First functional molecule; The photocrosslinked affinity peptide is covalently linked to the first functional molecule.

10. The conjugate according to claim 9, characterized in that, The photocrosslinked affinity peptide is covalently linked to the first functional molecule via photocrosslinked amino acid residues; Optionally, the first functional molecule contains amino acid residues for covalently linking with the photocrosslinked amino acid residues; Optionally, the first functional molecule includes a protein or a polypeptide; Optionally, the first functional molecule contains at least one Fc region of an antibody; Optionally, the photocrosslinked affinity peptide is covalently linked to the Fc region of the antibody via photocrosslinked amino acid residues; Optionally, the first functional molecule includes an antibody; Optionally, the first functional molecule includes an intact antibody or a heavy chain antibody; Optionally, the first functional molecule is covalently linked to 1 to 8 of the photocrosslinking affinity peptides.

11. The conjugate according to claim 9 or 10, characterized in that, It also contains at least one second functional molecule, which is linked to the first functional molecule via the photocrosslinking affinity peptide; Optionally, at least one second functional molecule is connected to the extension via a linker; Optionally, the second functional molecule includes a drug and / or a signaling molecule; Optionally, the drug includes one or more of topoisomerase II inhibitors, DNA cross-linking cytotoxic agents, alkylating agents, taxanes, and anthracyclines; Optionally, the signaling material includes one or more of the following: fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

12. The conjugate according to claim 11, characterized in that, Includes the following structure: Ab(-PAP-X-L-W) D ; Ab is an antibody, PAP is the photocrosslinked affinity peptide, X is the extension, L is the linker, and W is the second functional molecule. The second functional molecule W is connected to the extension X on the photocrosslinked affinity peptide PAP through the linker L. The RAP in each structural unit (-PAP-XLW) is directly covalently linked to Ab. D is the number of structural units (-PAP-XLW), and D is a positive integer greater than 0; Alternatively, 1 ≤ D ≤ 8; Optionally, D can be 1 or 2.

13. The method for preparing the conjugate according to any one of claims 9 to 12, characterized in that, This includes reacting the first functional molecule with the photocrosslinking affinity peptide according to any one of claims 1 to 4, or with the modified photocrosslinking affinity peptide according to any one of claims 5 to 7, under photocrosslinking conditions.

14. The preparation method according to claim 13, characterized in that, It also includes linking the second functional molecule to the photocrosslinking affinity peptide.

15. The conjugate according to any one of claims 9 to 12, or the preparation method according to claim 13 or 14, in the preparation of a drug or a diagnostic reagent; Optionally, the drug includes an antibody-drug conjugate.

16. A pharmaceutical composition, characterized in that, It comprises the photocrosslinking affinity peptide according to any one of claims 1 to 4, or the modified photocrosslinking affinity peptide according to any one of claims 5 to 7, or the conjugate according to any one of claims 9 to 12.

17. A detection reagent or kit, characterized in that, It comprises the photocrosslinking affinity peptide according to any one of claims 1 to 4, or the modified photocrosslinking affinity peptide according to any one of claims 5 to 7, or the conjugate according to any one of claims 9 to 12.

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