Conjugate and use thereof

Abstract

The present invention relates to a conjugate. The conjugate comprises a chimeric immunoglobulin and a conjugation partner, wherein the chimeric immunoglobulin comprises an antigen-binding fragment and a leucine zipper, and the leucine zipper is linked to the terminus of the antigen-binding fragment.

Description

Conjugates and their applications

[0001] Priority information

[0002] This application claims priority to Chinese patent applications filed on November 22, 2024, with application number 202411693093.0 entitled "Conjugates and Applications Thereof" and filed on May 21, 2025, with application number 202510665641.7 entitled "Conjugates and Applications Thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biological detection. Specifically, it relates to conjugates and their applications. More specifically, it relates to conjugates, chimeric immunoglobulins, methods for preparing conjugates, methods for detecting antigens in samples, and the use of conjugates in immunoassays. Background Technology

[0004] The leucine zipper is a characteristic structure discovered as a motif for protein secondary structure (Science. 1988 Jun 24; 240(4860): 1759-64.). The leucine zipper has a basic backbone in which leucine residues appear every seven amino acids in an amino acid sequence that tends to form an α-helix. These leucine residues are located on the same side of the α-helix, allowing two protein molecules to form a dimer through hydrophobic interactions between these leucine residues, resembling a zipper.

[0005] Chimeric immunoglobulins are recombinant proteins created by linking genes of proteins from different sources to an immunoglobulin gene at the genetic level and expressing them in eukaryotic or prokaryotic expression systems. By chimerizing single-chain immunoglobulins with complementary interaction domains (CIAGs), polymeric immunoglobulins can be prepared. The stability of chimeric immunoglobulins depends on the binding strength of the CIAGs, such as leucine zippers. Leucine zippers, through their unique hydrophobic interactions, can facilitate the formation of stable dimers from two single-chain antibodies (scFvs), enhancing antibody binding ability. Recombinant antibodies linked by leucine zippers exhibit greater stability in serum and higher protective potency compared to their corresponding single-chain antibodies. In *E. coli*, the use of leucine zippers accelerates the binding of heavy and light chains, forming functional Fab fragments. This technique, known as "Zipbody," significantly improves the efficiency of active Fab fragment formation in *E. coli*. Leucine zippers can also be used to prepare bispecific antibodies, which can simultaneously recognize two different antigens, significant for immunotherapy and diagnostics.

[0006] However, the application of chimeric immunoglobulins containing leucine zippers in the field of immunoassay still needs further development and improvement. Summary of the Invention

[0007] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a conjugate. According to embodiments of the present invention, the conjugate comprises a chimeric immunoglobulin and a conjugate chaperone. The chimeric immunoglobulin comprises an antigen-binding fragment and a leucine zipper, the end of which is connected to the leucine zipper. The leucine zipper has a motif represented as (Leu-X1-X2-X3-X4-X5-X6)n, where X1, X2, X3, X4, X5, and X6 are each independently selected from any amino acid, and when n is an integer not less than 2, the sequence X1-X2-X3-X4-X5-X6 may be the same or different. The conjugate according to embodiments of the present invention has improved activity for the detection of target antigens.

[0008] The "leucine zipper" described in this application refers to an α-helical domain capable of mediating protein dimerization through periodically repeating leucine residues. Its core feature is that in an amino acid sequence that tends to form an α-helix, the first position of every seven amino acids (i.e., a heptapeptide repeating unit) is typically a leucine residue. These leucine residues at the beginning of the heptapeptide appear once every two turns (7 ÷ 3.6 ≈ 2) in the α-helix and are arranged on one side of the helix as a hydrophobic "ridge." Two such α-helices interlock like the teeth of a zipper through hydrophobic interactions between their leucine ridges, forming stable parallel or antiparallel coiled-coil dimers.

[0009] Based on the above core features, the leucine zipper sequence involved in this application is described as follows:

[0010] (1) SEQ ID NO:1(GCN4),2(c-Jun),3(v-Fos),6(GCN4),7(GCN4): These are well-known and characteristic leucine zipper sequences in the art. Taking SEQ ID NO:1(RMKQLEDKVEELLSKNYHLENEVARLKKLVGER) as an example, its leucine zipper core region can be divided into continuous heptapeptide repeating units: (Leu-Glu-Asp-Lys-Val-Glu-Glu)-(Leu-Leu-Ser-Lys-Asn-Tyr-His)-(Leu-Glu-Asn-Glu-Val-Ala-Arg)-(Leu-Lys-Lys-Leu-Val-Gly-Glu). The first amino acid of each unit is leucine (Leu), which perfectly matches the sequence characteristics of a leucine zipper.

[0011] (2) SEQ ID NO:4 (MYC) and SEQ ID NO:5 (MAX): MYC and MAX proteins are known transcription factor protein pairs that form heterodimers via leucine zipper structures. SEQ ID NO:4 and 5 contain the complete domains responsible for dimerization in these proteins. Those skilled in the art can identify the leucine zipper regions by performing secondary structure prediction and alignment analysis on these sequences. For example, SEQ ID NO:4 contains a typical leucine zipper motif, and a portion of its sequence (such as "...VLERGRRNELKRSFFAL...") can be divided into segments conforming to heptapeptide repeating patterns. Therefore, peptides containing these sequences can perform the function of a leucine zipper in the context of this application, i.e., mediating the dimerization of chimeric immunoglobulins.

[0012] The above statement "the sequences X1-X2-X3-X4-X5-X6 are the same or different" refers to whether the sequence "X1-X2-X3-X4-X5-X6" is the same as a whole.

[0013] According to embodiments of the present invention, the conjugate may further include at least one of the following additional technical features:

[0014] According to an embodiment of the present invention, n≥2.

[0015] According to an embodiment of the present invention, n≥4.

[0016] According to embodiments of the present invention, n is 4 to 8. The inventors have found that when n ≥ 4, especially 4 to 8, the conjugate exhibits higher detection activity against the target antigen.

[0017] According to an embodiment of the present invention, the motif represented by (Leu-X1-X2-X3-X4-X5-X6)n has an active group.

[0018] According to embodiments of the present invention, the number of active groups refers to the total number of amino acid residues in the leucine zipper sequence whose side chains contain amino, carboxyl, hydroxyl, or thiol groups.

[0019] In this application, the active groups on the leucine zipper can be derived from its natural amino acid sequence, or they can be introduced or modified using mature protein engineering methods in the art. For example, by utilizing the active groups in the natural sequence, a suitable natural leucine zipper sequence can be selected, which itself contains a sufficient number and appropriate positions of active amino acid residues. For example, the GCN4 leucine zipper (SEQ ID NO:1) contains multiple lysine (Lys, K) and glutamic acid (Glu, E) residues, and its side chains provide amino and carboxyl groups as active groups, respectively. Active groups can be introduced through site-directed mutagenesis: when a specific type, number, or position of active groups is required, site-directed mutagenesis can be used to modify the selected leucine zipper sequence. This method is a conventional technique in the art, and the specific steps are as follows:

[0020] a. Target site selection: Without hindering the formation of the α-helix and dimerization function of the leucine zipper, positions X1 to X6 in the heptapeptide repeat unit are selected as mutation sites. Preferably, polar or charged amino acids are mutated to amino acids with the desired active groups.

[0021] b. Mutation design: To introduce an amino group, the target site can be mutated to lysine (Lys,K) or arginine (Arg,R); to introduce a carboxyl group, it can be mutated to glutamic acid (Glu,E) or aspartic acid (Asp,D); to introduce a hydroxyl group, it can be mutated to serine (Ser,S), threonine (Thr,T) or tyrosine (Tyr,Y); to introduce a thiol group, it can be mutated to cysteine ​​(Cys,C).

[0022] c. Mutation procedures: using overlap extension PCR or based on commercially available kits (e.g.) The method (purchased from Agilent Technologies) involves mutating the nucleic acid sequence encoding the target leucine zipper. The mutated nucleic acid molecule is then cloned into an expression vector and expressed and purified according to the method described in the preparation examples of this application to obtain a chimeric immunoglobulin with customized active groups.

[0023] Using the methods described above, those skilled in the art can flexibly design and prepare leucine zippers with the desired active group characteristics, and are not limited to natural sequences.

[0024] According to an embodiment of the present invention, the X1-X2-X3-X4-X5-X6 sequence has no more than 3 active groups.

[0025] According to embodiments of the present invention, the ratio of the number of active groups in the motif (Leu-X1-X2-X3-X4-X5-X6)n to n is (1-3):1. The inventors have found that when the number of active groups is within this range, the conjugate exhibits higher detection activity against the target antigen.

[0026] According to an embodiment of the present invention, the leucine zipper has active groups, and the ratio of the number of active groups in the leucine zipper to n is (1-5):1.

[0027] According to embodiments of the present invention, the reactive group or active group includes at least one selected from amino, carboxyl, hydroxyl, or thiol groups. The statement that the motif or leucine zipper has an active group refers to the presence of an active group on the side chain of the amino acid in the motif or leucine zipper. According to specific embodiments of the present application, the active group can be selected from at least one of amino, carboxyl, hydroxyl, or thiol groups; the amino acid containing an amino group in the side chain can be selected from lysine (K) or arginine (R); the amino acid containing a carboxyl group in the side chain can be selected from glutamic acid (E) or aspartic acid (D); the amino acid containing a hydroxyl group in the side chain can be selected from threonine (T), serine (S), or tyrosine (Y); and the amino acid containing a thiol group in the side chain can be selected from cysteine ​​(C) or methionine (M).

[0028] According to a specific embodiment of this application, the active group is an amino group, and the amino acid containing an amino group in the side chain is lysine.

[0029] According to an embodiment of the present invention, the number of active amino groups in the leucine zipper is 5 to 10.

[0030] For example, the number of active amino groups in the leucine zipper is 5, and n is 5.

[0031] For example, the number of active amino groups in the leucine zipper is 5, and n is 4.

[0032] For example, the number of active amino groups in the leucine zipper is 6, and n is 4.

[0033] For example, the number of active amino groups in the leucine zipper is 10, and n is 8.

[0034] According to an embodiment of the present invention, the motif represented by (Leu-X1-X2-X3-X4-X5-X6)n includes at least one active group.

[0035] According to an embodiment of the present invention, the motif represented by (Leu-X1-X2-X3-X4-X5-X6)n includes 1, 2, 3, 4, 5, 6, 7 or 8 active groups.

[0036] According to specific embodiments of this application, the number of active groups in the motif (Leu-X1-X2-X3-X4-X5-X6)n is 4 to 8.

[0037] According to a specific embodiment of this application, the number of active groups in the motif (Leu-X1-X2-X3-X4-X5-X6)n is 4, and n is 4.

[0038] According to a specific embodiment of this application, the number of active groups in the motif (Leu-X1-X2-X3-X4-X5-X6)n is 5, and n is 5.

[0039] According to a specific embodiment of this application, the number of active groups in the motif (Leu-X1-X2-X3-X4-X5-X6)n is 8, and n is 8.

[0040] In this paper, the term "leucine zipper" refers to a motif that mediates protein dimerization, in which a leucine residue appears every 7 amino acids in a motif that tends to form an α-helix structure. These leucine residues are located on the same side of the α-helix structure, so that two protein molecules can form a dimer with a zipper-like shape through hydrophobic interactions between these leucine residues.

[0041] It should be understood that the motif represented by (Leu-X1-X2-X3-X4-X5-X6)n tends to form an α-helix structure, with a leucine residue every 7 amino acids on the α-helix. The leucine residues are arranged on one side of the α-helix, forming a hydrophobic "zipper".

[0042] According to an embodiment of the present invention, the leucine zipper further comprises a head sequence and / or a tail sequence, wherein the head sequence is connected to the N-terminus of the sequence (Leu-X1-X2-X3-X4-X5-X6)n, and the tail sequence is connected to the C-terminus of the sequence (Leu-X1-X2-X3-X4-X5-X6)n. It should be understood that the head sequence and / or tail sequence can be any sequence, as long as it does not affect the formation of the "zipper" structure of the motif shown above in (Leu-X1-X2-X3-X4-X5-X6)n.

[0043] It should be understood that Leu in (Leu-X1-X2-X3-X4-X5-X6)n includes not only Leu itself, but also Leu's equivalent amino acids. For example, existing literature has pointed out that replacing Leu with its conserved amino acid Ile does not affect its formation of the leucine zipper.

[0044] According to an embodiment of the present invention, the Leu-X1-X2-X3-X4-X5-X6 sequence is taken from a sequence on GCN4, c-Jun, v-Fos, MYC, MAX, or C / EBP protein.

[0045] According to an embodiment of the present invention, the Leu-X1-X2-X3-X4-X5-X6 sequence is taken from a sequence on GCN4, c-Jun, or v-Fos protein.

[0046] According to an embodiment of the present invention, the Leu-X1-X2-X3-X4-X5-X6 sequence is selected from LEDKVEE, LLSKNYH, LENEVAR, LKKLVGE, LEEKVKT, LKAQNSE, LASTANM, LREQVAQ, LKQKVMN, LQAETDQ, LEDKKSA, LQTEIAN, LLKEKEK, and LEFILAA.

[0047] According to an embodiment of the present invention, the motif represented by (Leu-X1-X2-X3-X4-X5-X6)n is taken from a motif on GCN4, c-Jun, v-Fos, MYC, MAX, or C / EBP protein.

[0048] According to an embodiment of the present invention, the motif represented by (Leu-X1-X2-X3-X4-X5-X6)n is taken from a motif on GCN4, c-Jun, or v-Fos protein.

[0049] According to an embodiment of the present invention, the basal sequence represented by (Leu-X1-X2-X3-X4-X5-X6)n is selected from LEDKVEELLSKNYHLENEVARLKKLVGE, LEEKVKTLKAQNSELASTANMLREQVAQLKQKVMN and LQAETDQLEDKKSALQTEIANLLKEKEKLEFILAA.

[0050] According to an embodiment of the present invention, the leucine zipper comprises at least one selected from GCN4, c-Jun, v-Fos, MYC, MAX, C / EBP, or fragments thereof.

[0051] According to an embodiment of the present invention, the leucine zipper comprises at least one selected from GCN4, c-Jun, v-Fos, or fragments thereof.

[0052] It should be understood that the fragment includes at least the motif (Leu-X1-X2-X3-X4-X5-X6)n that forms the leucine zipper structure in the aforementioned protein.

[0053] The inventors discovered that when the leucine zipper includes at least one of GCN4, c-Jun, v-Fos, or fragments thereof, the conjugate exhibits higher detection activity against the target antigen. Furthermore, the introduction of the leucine zipper does not increase the leucine zipper-related background.

[0054] It should be understood that when homomeric immunoglobulins are formed, the leucine zipper is selected from GCN4 or a fragment thereof; when heteromeric immunoglobulins are formed, the leucine zipper is selected from c-Jun or a fragment thereof, and v-Fos or a fragment thereof.

[0055] According to embodiments of the present invention, the leucine zipper has an amino acid sequence shown in any one of SEQ ID NO: 1 to 7 or has an amino acid sequence that is at least 80% identical to the amino acid sequence shown in any one of SEQ ID NO: 1 to 7. The variant (at least 80% identical) still has the leucine zipper structure and active groups shown in any one of SEQ ID NO: 1 to 7.

[0056] According to an embodiment of the present invention, the leucine zipper has a substitution of a conserved amino acid based on the amino acid sequence shown in any one of SEQ ID NO: 1 to 7.

[0057] In this document, "replacement of conserved amino acids" does not significantly affect or alter the binding properties of conjugates containing that amino acid sequence. Substitution can be introduced into the conjugates of this invention using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved amino acid substitution refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the conjugates of the present invention can be replaced by other amino acid residues from the same side chain family, and the retained function of the modified conjugates can be tested using the functional assay methods described herein. Preferably, the number of conservative modifications does not exceed one or two.

[0058] According to an embodiment of the present invention, the leucine zipper is based on the amino acid sequence shown in any one of SEQ ID NO: 1 to 7, with arginine residues replaced by lysine residues.

[0059] According to an embodiment of the present invention, the C-terminus of the antigen-binding fragment is connected to the N-terminus of the leucine zipper.

[0060] According to an embodiment of the present invention, the chimeric immunoglobulin further includes a linker peptide, the N-terminus of which is linked to the C-terminus of the antigen-binding fragment, and the C-terminus of which is linked to the N-terminus of the leucine zipper. The linker peptide can further improve the flexibility of the chimeric immunoglobulin, which is more conducive to the independence of the "zipper" structure, or the exposure of active side chain groups such as amino groups on the leucine zipper in the chimeric immunoglobulin, further improving the activity for detecting the target antigen.

[0061] According to embodiments of the present invention, the linker peptide is a rigid linker peptide or a flexible linker peptide.

[0062] According to an embodiment of the present invention, the amino acid sequence of the linker peptide is selected from (GGGGS)n, (GGGS)n, (GGS)n, (GS)n or (G)n, wherein n is selected from 1, 2, 3, 4, 5 or 6.

[0063] According to embodiments of the present invention, the antigen-binding fragment includes a selection from monoclonal antibodies, bispecific or multispecific antibodies.

[0064] According to embodiments of the present invention, the antigen-binding fragment includes at least one selected from F(ab')2, F(ab)2, Fab', Fab antibody, full-length natural antibody, and Fv antibody.

[0065] According to embodiments of the present invention, the antigen-binding fragment is selected from IgG, IgA, IgD, IgM and IgE.

[0066] In this article, the term "Fab antibody" has its usual meaning, referring to an antibody or fragment containing only Fab molecules, which consists of the VH and CH1 of the heavy chain and a light chain, with the light and heavy chains linked by disulfide bonds.

[0067] In this article, the term "Fab' antibody" has its usual meaning, referring to an antibody or fragment containing only Fab' molecules, which consists of the VH, CH1, and hinge of the heavy chain, and the light chain, which are linked by disulfide bonds.

[0068] In this article, the term "F(ab')2 antibody" has its usual meaning, referring to the F(ab') portion of two antigens linked together by disulfide bonds.

[0069] In this article, the term "Fv antibody" has its usual meaning, referring to an antibody or fragment consisting only of light chain variable regions (VL) and heavy chain variable regions (VH) linked by non-covalent bonds.

[0070] According to an embodiment of the present invention, the antigen-binding fragment includes a heavy chain, the heavy chain including a first heavy chain and a second heavy chain, the ends of the first heavy chain and the second heavy chain being respectively connected to the leucine zipper.

[0071] According to an embodiment of the present invention, the antigen-binding fragment includes a heavy chain, which is composed of a first heavy chain and a second heavy chain, the ends of which are respectively connected to the leucine zipper.

[0072] According to an embodiment of the present invention, the leucine zippers connected to the ends of the first heavy chain and the second heavy chain may be the same or different.

[0073] According to an embodiment of the present invention, the antigen-binding fragment comprises a light chain.

[0074] According to an embodiment of the present invention, the antigen-binding fragment includes a heavy chain and a light chain; the ends of the heavy chain and the light chain are respectively connected to the leucine zipper, and the leucine zipper connected to the end of the light chain may be the same as or different from the leucine zipper connected to the end of the heavy chain.

[0075] According to an embodiment of the present invention, the light chain includes a light chain constant region (CL).

[0076] According to an embodiment of the present invention, the light chain further includes a light chain variable region (VL).

[0077] According to an embodiment of the present invention, the first heavy chain or the second heavy chain can be a natural full-length heavy chain or a truncated heavy chain of a natural full-length heavy chain.

[0078] According to an embodiment of the present invention, the antigen-binding fragment comprises a heavy chain and a light chain; the heavy chain comprises a first heavy chain and a second heavy chain, both of which include a VH-CH1-hinge region, and the light chain comprises a VL-CL. The leucine zippers attached to the ends of the first and second heavy chains are identical. For example, the leucine zipper may be GCN4.

[0079] According to an embodiment of the present invention, the antigen-binding fragment is F(ab')2.

[0080] Unless otherwise specified, the immunoglobulin element connection methods described in this application, such as "VH-CH1-hinge region," are all in the direction from the N-terminus to the C-terminus from left to right. The same applies to others.

[0081] According to an embodiment of the present invention, both the first heavy chain and the second heavy chain include a VH-CH1-hinge region-CH2-CH3-tail peptide, and the light chain includes VL-CL. The leucine zippers attached to the ends of the first heavy chain and the second heavy chain are identical. For example, the leucine zipper may be GCN4.

[0082] According to an embodiment of the present invention, the antigen-binding fragment is a full-length natural antibody, such as a full-length natural antibody of IgG, IgA, or IgD.

[0083] According to an embodiment of the present invention, both the first heavy chain and the second heavy chain comprise a VH-CH1-CH2-CH3-CH4-tail peptide, and the light chain comprises VL-CL. The leucine zippers attached to the ends of the first heavy chain and the second heavy chain are identical. For example, the leucine zipper may be GCN4.

[0084] According to an embodiment of the present invention, the antigen-binding fragment is a full-length natural antibody, such as a full-length natural antibody of IgM or IgE.

[0085] According to an embodiment of the present invention, both the first heavy chain and the second heavy chain are full-length natural heavy chains, and the light chain is a full-length natural light chain. The leucine zipper attached to the ends of the first heavy chain and the second heavy chain is the same. For example, the leucine zipper may be GCN4.

[0086] According to an embodiment of the present invention, both the first heavy chain and the second heavy chain comprise VH-CH1, and the light chain comprises VL-CL. The leucine zippers attached to the ends of the first heavy chain and the second heavy chain are identical. For example, the leucine zipper may be GCN4.

[0087] According to an embodiment of the present invention, the antigen-binding fragment is F(ab)2.

[0088] According to an embodiment of the present invention, both the first heavy chain and the second heavy chain include VH-CH1-hinge region-CH2, and the light chain includes VL-CL. The leucine zipper connected to the ends of the first heavy chain and the second heavy chain is the same; for example, the leucine zipper is GCN4.

[0089] According to an embodiment of the present invention, both the first heavy chain and the second heavy chain are VH-CH1-hinge region-CH2, and the light chain is VL-CL. The leucine zipper connected to the ends of the first heavy chain and the second heavy chain is the same; for example, the leucine zipper is GCN4.

[0090] According to embodiments of the present invention, the antigen-binding fragment comprises a heavy chain and a light chain; the heavy chain comprises VH-CH1, and the light chain comprises VL-CL, wherein the leucine zippers attached to the ends of the heavy chain and the light chain are different. For example, the leucine zippers may be c-Jun and v-Fos.

[0091] According to an embodiment of the present invention, the antigen-binding fragment is Fab.

[0092] According to an embodiment of the present invention, the antigen-binding fragment comprises a heavy chain and a light chain; the heavy chain comprises VH, and the light chain comprises VL, wherein the leucine zippers attached to the ends of the heavy chain and the light chain are different. For example, the leucine zippers are c-Jun and v-Fos.

[0093] According to an embodiment of the present invention, the antigen-binding fragment is Fv.

[0094] It should be understood that different VH and VL can be selected depending on the target antigen.

[0095] It should be understood that the hinge region serves at least to facilitate the formation of disulfide bonds between the heavy and light chains, for example, by containing at least one carbon atom. Alternatively, the hinge region serves at least to facilitate maintaining flexibility, for example, by containing at least one phosphorus atom.

[0096] According to an embodiment of the present invention, the chimeric immunoglobulin is further attached to a protein tag at its end.

[0097] According to embodiments of the present invention, the protein tag is selected from Spytag tag, His tag, Flag tag, GST tag, MBP tag, SUMO tag and C-Myc tag.

[0098] It should be understood that those skilled in the art may add protein tags to the ends of proteins, usually the C-terminus, for purposes such as facilitating subsequent purification of recombinantly expressed proteins. Attaching these small fragment tags to the ends will not have a substantial impact on the performance of the protein.

[0099] According to an embodiment of the present invention, the protein tag is selected from Spytag tags.

[0100] According to an embodiment of the present invention, the amino acid sequence of the Spytag tag is shown in SEQ ID NO: 14.

[0101] According to an embodiment of the present invention, the chimeric immunoglobulin further includes a linker peptide, the N-terminus of which is connected to the C-terminus of the leucine zipper, and the C-terminus of which is connected to the N-terminus of the protein tag.

[0102] According to embodiments of the present invention, the linker peptide is a rigid linker peptide or a flexible linker peptide.

[0103] According to an embodiment of the present invention, the amino acid sequence of the linker peptide is selected from (GGGGS)n, (GGGS)n, (GGS)n, (GS)n or (G)n, wherein n is selected from 1, 2, 3, 4, 5 or 6.

[0104] According to an embodiment of the present invention, the species source of the heavy chain is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, or humans.

[0105] According to an embodiment of the present invention, the conjugation partner is chemically coupled to the active group on the chimeric immunoglobulin.

[0106] According to an embodiment of the present invention, the active group is an amino group.

[0107] According to an embodiment of the present invention, the mate is a marker.

[0108] According to embodiments of the present invention, the aforementioned markers refer to a class of substances that have properties such as luminescence, color development, and radioactivity that can be directly observed by the naked eye or detected or probed by instruments. These properties enable qualitative or quantitative detection of the corresponding target objects.

[0109] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0110] For example, the marker is selected from enzymes, luminescent substances, fluorescent substances, colored substances, radioactive substances, colloids, or combinations thereof.

[0111] According to embodiments of the present invention, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0112] According to embodiments of the present invention, the luminescent material includes, but is not limited to, luminol and its derivatives, luciferin, luteolin and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxate and its derivatives.

[0113] According to embodiments of the present invention, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0114] According to embodiments of the present invention, the radioactive isotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0115] According to embodiments of the present invention, the colloid includes, but is not limited to, dispersed dyes, dye-labeled microspheres, and latexes.

[0116] According to an embodiment of the present invention, the fused partner is a combined spouse.

[0117] For example, conjugated couples include biotin, streptavidin, or avidin.

[0118] According to an embodiment of the present invention, the mate is a solid-phase carrier.

[0119] For example, the solid support is selected from microspheres, plates, or membranes.

[0120] In optional embodiments, the solid-phase carrier includes, but is not limited to, magnetic microspheres, plastic microspheres, and plastic microparticles.

[0121] According to embodiments of the present invention, it is preferable for the conjugate partner to be a marker (such as AE) rather than a solid carrier (such as microspheres).

[0122] According to embodiments of the present invention, when the conjugate partner is a marker (such as an AE), it exhibits superior performance, such as a better signal-to-noise ratio, compared to when it is a solid carrier (such as microspheres).

[0123] According to embodiments of the present invention, the conjugation partner is directly or indirectly covalently coupled to the active group on the chimeric immunoglobulin.

[0124] According to embodiments of the present invention, the conjugation partner is directly or indirectly coupled to the chimeric immunoglobulin via covalent bonds, which is superior to other forms of chemical bonding, such as non-covalent interaction coupling.

[0125] According to an embodiment of the present invention, "indirectly coupled via covalent bonds" refers to a covalent bond connection between the coupling partner and the coupling intermediate, or between the coupling intermediate and the coupling intermediate, or between the coupling intermediate and the chimeric immunoglobulin.

[0126] According to an embodiment of the present invention, the coupling is non-directional coupling. According to an embodiment of the present invention, non-directional coupling between the conjugation chaperone and the chimeric immunoglobulin is superior to directional coupling.

[0127] Directed coupling technology refers to coupling at specific sites on an antibody, while non-directed coupling technology usually involves random coupling at multiple sites on the antibody, including active groups such as lysine residues or cysteine ​​residues.

[0128] In a second aspect, the present invention provides a chimeric immunoglobulin defined in the foregoing conjugate. Further details will not be repeated here.

[0129] In a third aspect, the present invention provides a method for using a nucleic acid molecule, a vector, a host cell, or the aforementioned chimeric immunoglobulin, wherein the nucleic acid molecule encodes the aforementioned chimeric immunoglobulin; the vector contains a nucleic acid molecule encoding the aforementioned chimeric immunoglobulin; the host cell contains the aforementioned nucleic acid molecule or the vector; and the method comprises culturing the aforementioned host cell.

[0130] In one aspect of the invention, a nucleic acid molecule is proposed.

[0131] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. The nucleic acid sequences in this application include DNA or RNA forms.

[0132] In another aspect, the present invention provides a vector. According to embodiments of the present invention, the vector carries the aforementioned nucleic acid molecule. When the aforementioned nucleic acid molecule is ligated to the vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself or can be exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecule and the control elements can be operably linked. In this document, "operably linked" means ligating a foreign gene to the vector so that the control elements within the vector, such as transcription control sequences and translation control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors can be, for example, plasmids, bacteriophages, etc. After the vector of some specific embodiments of the present invention is introduced into suitable recipient cells, the aforementioned conjugates and immunoglobulins can be effectively expressed under the mediation of a regulatory system, thereby achieving the in vitro large-scale acquisition of the aforementioned conjugates and immunoglobulins.

[0133] According to embodiments of the present invention, the vector includes a selection from eukaryotic expression vectors or prokaryotic expression vectors.

[0134] According to an embodiment of the present invention, the vector is a plasmid expression vector.

[0135] In another aspect, the present invention provides a host cell. According to embodiments of the invention, the host cell comprises: carrying the aforementioned nucleic acid molecule or the aforementioned vector; or expressing the aforementioned chimeric immunoglobulin. Using this host cell, under suitable conditions, the aforementioned chimeric immunoglobulin can be efficiently expressed intracellularly.

[0136] According to embodiments of the present invention, a method for preparing the above-mentioned chimeric immunoglobulin is provided. According to embodiments of the present invention, the method includes the following steps: fusing a nucleotide fragment encoding an antigen-binding fragment with a nucleotide fragment encoding a leucine zipper, subcloning the fusion fragment into a vector, and then expressing the resulting expression plasmid in a host cell to obtain the chimeric immunoglobulin.

[0137] According to an embodiment of the present invention, the host cell is a prokaryotic cell or a eukaryotic cell.

[0138] According to an embodiment of the present invention, the host cell is a eukaryotic cell.

[0139] In a fourth aspect, the present invention provides a method for preparing the aforementioned conjugate. According to an embodiment of the invention, the method comprises: chemically coupling the above-described chimeric immunoglobulin or the chimeric immunoglobulin prepared by the above method with a conjugate chaperone to obtain the conjugate.

[0140] According to an embodiment of the invention, the conjugation partner is the conjugation partner defined in the conjugation of the first aspect.

[0141] According to an embodiment of the present invention, the conjugation chaperone is covalently coupled to the active group on the chimeric immunoglobulin.

[0142] According to an embodiment of the present invention, the coupling is a non-directional coupling.

[0143] In a fifth aspect, the present invention provides a method for detecting a target antigen in a sample to be tested. According to an embodiment of the present invention, the method includes: contacting the sample to be tested with a conjugate prepared by the above-described method or a conjugate described above, wherein an antigen-binding fragment in the conjugate binds to the target antigen in the sample to be tested.

[0144] In a sixth aspect, the present invention provides the use of the aforementioned conjugate, the conjugate prepared by the aforementioned method, the aforementioned chimeric immunoglobulin, or the chimeric immunoglobulin prepared by the aforementioned method in the preparation of immunoassay products.

[0145] In a seventh aspect, the present invention provides a kit or test strip comprising the conjugate described above, the conjugate prepared by the method described above, the chimeric immunoglobulin described above, or the chimeric immunoglobulin prepared by the method described above.

[0146] According to an embodiment of the present invention, the test strip further includes at least one of a sample pad, a conjugate pad, a reaction membrane, and an absorbent pad, wherein the reaction membrane is provided with a detection area and a quality control area; the conjugate or chimeric immunoglobulin is coated on the detection area and / or dropped onto the conjugate pad.

[0147] According to embodiments of the present invention, the kit further comprises at least one of the following: sample pretreatment reagents (such as sample purification and enrichment reagents, lysis buffers, etc.), washing solutions (such as water, etc.), buffers (such as PBS or Tris, etc.), and colorimetric reagents for signal substances.

[0148] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0149] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0150] Figure 1 shows the correlation results between the chimeric immunoglobulin of Example 1 of the present invention and the Roche-assay specimen. Detailed Implementation

[0151] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0152] It should be noted that 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 technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0153] In this document, the terms “comprising,” “including,” or “having” are open-ended expressions, meaning they include the contents specified in this application but do not exclude other contents.

[0154] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0155] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York). and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm by Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm by Smith et al. (1981) Adv. Appl. Math. 2: 482; and the similarity search method by Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444. The Smith-Waterman algorithm (Meth. Mol. Biol.) .70:173-187 (1997). And the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996)). Or GAP, BESTFIT, BLAST Altschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA. And the CLUSTAL PC / Gene program provided by Intelligenetics, Mountain View, California.

[0156] In this document, the term "having at least 80% identity" can mean having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence similarity. The sequence identity described in this application can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. All amino acid sequences mentioned in this application are shown from the N-terminus to the C-terminus.

[0157] In this document, the term "having at least 90% identity" can mean having sequence similarity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%.

[0158] In this document, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule that contains one or more nucleotide or amino acid differences (mutations) that differ from a reference sequence. This difference can be a substitution, deletion, or insertion of one or more amino acids.

[0159] This invention relates to the generation of chimeric antibodies containing leucine zipper sequences and the preparation of conjugates. The conjugates embodied in this invention are particularly suitable for detecting antigens with diagnostic potential.

[0160] Common immunoglobulin (Ig) monomers consist of two identical light chain ("L") polypeptides and two identical heavy chain ("H") polypeptides. The four chains are linked by disulfide bonds in a "Y" configuration. At the base of the Y, the two H chains are linked by covalent disulfide bonds. The light chain, from N-terminus to C-terminus, includes a variable region and a CL region. The heavy chain, from N-terminus to C-terminus, includes a variable region and a constant region.

[0161] In this paper, the CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide are all components of the heavy chain constant region, which is located at the C-terminus of the heavy chain of the antibody molecule. Each heavy chain constant region, from the N-terminus to the C-terminus, includes the CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide. Different types of antibodies (such as IgG, IgA, IgM, etc.) have different amino acid sequences and structures in their heavy chain constant regions, but they all have relatively conserved structural features. These conserved structures enable the heavy chain constant regions to perform their biological functions. The heavy chain constant regions, CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide of different species and subclasses are well known in the art, and their amino acid sequences can be determined based on bioinformatics databases, such as the IMGT database (https: / / www.imgt.org / IMGTrepertoire / Proteins / ). It should be understood that different bioinformatics databases or software may not have completely consistent results in the division and sequence identification of constant regions. However, those skilled in the art have a general and unified understanding of the concept, division and sequence identification of constant regions and their segments. Therefore, the constant regions that those skilled in the art can identify and divide using common knowledge and ordinary methods are all within the scope of protection of this invention.

[0162] For example, the amino acid sequence of the corresponding segment (such as the IgM CH2 region) divided by the IMGT database can be used as the reference sequence. The start or end position of the reference sequence can be moved forward by several amino acid residues (i.e., moved to the IgM CH1 region) or backward by several amino acid residues (i.e. moved to the IgM CH3 region) to obtain a sequence of the corresponding segment that is longer or shorter than the reference sequence.

[0163] In this document, the term "CH1 region" refers to a relatively conserved region located at the N-terminus of the constant region of the heavy chain of an antibody, immediately following the variable region of the heavy chain. The specific amino acid sequence is not limited and is within the scope of protection of this application. CH1 regions of different species and alternative subclasses are well known.

[0164] In this document, the term "hinge region" refers to a polypeptide that links the CH1 and CH2 domains within the constant region of the heavy chain of an antibody. This region is rich in proline and therefore readily stretchable and flexible. Hinge regions are typically dimers, consisting of two polypeptides with the same amino acid sequence. Specific amino acid sequences are not limited and are all within the scope of protection of this application. Hinge regions of different species and subclasses are well known.

[0165] In this document, the term "CH2 region" refers to the second constant region of the antibody's heavy chain constant region, a relatively conserved region located after the CH1 region. The specific amino acid sequence is not limited and is within the scope of protection of this application. CH2 regions of different species and alternative subclasses are well known.

[0166] In this document, the term "CH3 region" refers to the third constant region of the antibody heavy chain constant region, a relatively conserved region located after the CH2 region. The CH3 region of IgM contains cysteine ​​residues involved in polymer formation; the specific amino acid sequence is not limited and is within the scope of protection of this application. The CH3 regions of different species and alternative subclasses are well known.

[0167] In this document, the term "CH4 region" refers to the fourth constant region of the heavy chain, a relatively conserved region located after the CH3 region. The specific amino acid sequence is not limited and is within the scope of protection of this application. CH4 regions of different species are well-known.

[0168] In this paper, the term "tailplate" refers to a short peptide sequence at the end of the CH3 or CH4 region of an antibody. Tailplates of different species are well known.

[0169] In this paper, the "CL region" refers to the constant region of the antibody's light chain, located at the C-terminus of the light chain, immediately following the variable region (VL). The CL region provides stability to the overall antibody structure by binding to the CH1 region of the heavy chain and plays a crucial role in antibody folding. The specific amino acid sequence is not limited and is within the scope of protection of this application. CL regions of different species and subtypes are well-known.

[0170] This invention utilizes a pair of peptides containing the aforementioned motif (referred to as leucine zipper A and leucine zipper B). Leucine zipper A and leucine zipper B have high affinity and form a leucine zipper. Leucine zipper A contains a heptapeptide with leucine residues, thus it can form a leucine zipper. Leucine zipper B also contains one leucine residue at every seven residues. The sequences of leucine zipper A and leucine zipper B can be the same or different, and their lengths are not particularly limited. In principle, leucine zipper A and leucine zipper B are of the same length, but their lengths can differ as long as a leucine zipper structure can be formed.

[0171] This invention unexpectedly revealed that the aforementioned chimeric immunoglobulin is suitable for various immunoassay scenarios and exhibits unexpectedly high activity. Furthermore, it demonstrates an improved signal-to-noise ratio or reduced background and / or does not increase the leucine zipper-related background due to the introduction of the leucine zipper.

[0172] The amino acid sequence involved in this application is as follows:

[0173] The embodiments of the present invention will now be described in further detail, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0174] Unless otherwise specified, the practice of this disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.C. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994) and "Current Protocols in Immunology" (JEColigan et al., ed., 2011), each of which is explicitly incorporated herein by reference.

[0175] The present application's solution will be explained below with reference to preparation examples and embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0176] Preparation Example

[0177] This experiment uses the preparation of anti-CA724 antibody as an example to demonstrate the preparation scheme and performance testing scheme of antibody and conjugate.

[0178] In this embodiment, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor RNA extraction kit was purchased from TOYOBO. BD SMART TM The RACE cDNA Amplification Kit and pMD-18T vector were purchased from Takara. The plasmid extraction kit was purchased from Tiangen Biotech. Primer synthesis and gene sequencing were performed by a gene sequencing company. The cell line secreting the Anti-CA724 monoclonal antibody was an existing cell line from our company, which was revived for later use.

[0179] 1.1 Preparation of Anti-CA724 Antibody Gene

[0180] mRNA was extracted from the cell line secreting Anti-CA724 monoclonal antibody, and the DNA product was obtained by RT-PCR. The DNA product was then inserted into the pMD-18T vector and transformed into DH5α competent cells. After the cells grew, four positive clones of the heavy chain and four positive clones of the light chain genes were collected and sent to a gene sequencing company for sequencing.

[0181] 1.2 Sequence analysis of the variable region gene of the Anti-CA724 antibody

[0182] The gene sequences obtained from the sequencing were analyzed in the IMGT antibody database, and the VNTI11.5 software was used to confirm that the genes amplified by both heavy and light chain primer pairs were correct.

[0183] 1.3 Construction of recombinant antibody expression plasmid

[0184] Based on the above antibody variable region gene sequencing results, VL and VH gene-specific primers for the Anti-CA724 antibody were designed, with restriction endonuclease cleavage sites and protective bases at both ends, respectively. The light chain gene fragment and heavy chain gene fragment with leucine zipper were amplified by PCR.

[0185] The gene fragments were double-digested with restriction endonucleases, and the 3.4A vector was double-digested with restriction endonucleases. After purification and recovery of the fragments and vectors, the above genes were ligated into the 3.4A expression vectors to obtain recombinant expression plasmids with heavy and light chains containing leucine zippers, respectively.

[0186] 1.4 Expression of recombinant antibody samples

[0187] HEK293 cells were revived early and passaged to a 200 mL volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cells / mL, cell viability >95%; centrifuge to wash cells, rehydrate with culture medium, and adjust cell density to 2.9 × 10⁶ cells / mL. 6 Cells / mL were used as cell dilution buffer. Recombinant expression plasmids and transfection reagent dilution buffers for the heavy and light chains obtained in step 1.3 were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. The cells were then incubated in a 35°C incubator at 120 rpm with 8% CO2 for 13 days. After centrifugation, the supernatant was collected, purified, and the corresponding chimeric antibody was obtained.

[0188] 1.5 Activity identification

[0189] 1.5.1 Labeling and Detection Procedures for Acridine Ester (AE)

[0190] Tagging process:

[0191] 1) Buffer Replacement: Dialyze or replace the buffer system of the chimeric immunoglobulins obtained in 1.4 using a dialysis bag or ultrafiltration centrifuge tube with the coupling buffer (e.g., 0.1M borate buffer at pH 8.5, or 0.1M phosphate-buffered saline (PBS) at pH 7.2-7.4). This step aims to remove impurities (such as ammonium ions, Tris, etc.) that may interfere with the coupling reaction and to provide a suitable pH environment for subsequent reactions. Dialyze for at least 2 hours, changing the coupling buffer every 45 minutes during this period.

[0192] 2) Reaction:

[0193] a. Take a certain amount of the buffer-replaced antibody solution into a centrifuge tube.

[0194] b. The reaction is carried out with a mother liquor of acridine ester (NHS-AE) activated by N-hydroxysuccinimide (NHS). The NHS-activated AE mother liquor can be obtained commercially (e.g., from NHS-AE derivatives purchased from companies such as Shanghai Yisheng Biotechnology Co., Ltd. and Sigma-Aldrich), or prepared by conventional methods in the art: reacting an acridine ester carboxylic acid derivative with N-hydroxysuccinimide (NHS) in anhydrous dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) in the presence of carbodiimide (e.g., EDC). The mother liquor is typically prepared as a 1-10 mM stock solution using anhydrous DMSO.

[0195] c. The amount of NHS-AE added is typically 5-20 times the molar amount of the antibody (e.g., 10 equivalents). The reaction is carried out at room temperature (25°C) and in the dark, with gentle vortexing or shaking for 15-30 minutes.

[0196] 3) Termination and purification: Add glycine (Gly) solution (prepared with the same buffer system as the coupling buffer) to the reaction system to a final concentration of 10-50 mM to quench unreacted NHS esters, and continue the reaction at room temperature for 15-30 minutes.

[0197] 4) Purification: After the reaction, the protein-AE conjugate is purified by dialysis, gel filtration chromatography (e.g., using a PD-10 desalting column), or ultrafiltration centrifugation, followed by purification with storage buffer (e.g., PBS pH 7.4 containing 0.1% BSA or 1% trehalose as a stabilizer) to completely remove unreacted free AE, glycine, and other small molecule impurities. For dialysis, frequent medium changes are necessary, for example, once every 30-60 minutes on the first day (at least 4 times), and once every 60 minutes on the second day after overnight incubation (at least 4 times).

[0198] 5) Recovery and storage: Take out the purified Ab-AE conjugate solution and determine the protein concentration. It can be mixed with glycerol to a final concentration of 50% (v / v) and stored at -20℃ for a long time, or directly aliquoted and stored at -80℃.

[0199] Detection procedure: 1) Sample loading reaction: Add 30 μL of the sample to be tested, 50 μL of Ab-bio working solution, and 50 μL of SA-magnetic bead working solution, and react at 37℃ for 10 min; 2) Sample loading reaction: Add 50 μL of the Ab-AE working solution prepared above, and react at 37℃ for 5 min; 3) Wash three times with 1*TBST; 4) Add pre-excitation solution A + excitation solution B, and perform detection on the Yingkai Shine i2910 fully automated chemiluminescence immunoassay analyzer to detect the relative luminescence intensity (RLU).

[0200] 1.5.2 Biotin Indirect Coating and Detection Procedures:

[0201] (Biotin) Indirect Coating Process: The obtained antibody is coated with Zeba... TM A desalting column (10K MWCO) was substituted with PBS (containing 100 mM PB, 50 mM sodium chloride, pH 8.0, and 5 mM EDTA). NHS-LC-BIO was dissolved in DMSO to prepare an 8 mM solution. 20 eq of Bio was added to the antibody solution, and the reaction was carried out at 25°C for 2 hours. Excess reagents were removed by desalting, and the biotinylated antibody was stored at 4°C for later use.

[0202] Detection procedure: 1) Sample loading reaction: Add 30 μL of the sample to be tested, 50 μL of the Ab-bio working solution prepared above, and 50 μL of SA-magnetic bead working solution, and react at 37℃ for 10 min; 2) Sample loading reaction again: Add 50 μL of Ab-AE working solution and react at 37℃ for 5 min; 3) Wash three times with 1*TBST; 4) Add pre-excitation solution A + excitation solution B, and perform detection on the Yingkai Shine i2910 fully automated chemiluminescence immunoassay analyzer to detect the relative luminescence intensity (RLU).

[0203] 1.5.3 AP Labeling and Detection Procedures

[0204] The labeling process is the same as the AE labeling steps described above. Detection procedure: Add the sample to be tested, then add the first antibody coated with magnetic microparticles and the Ab-AP prepared above. Detect using Thermo Scientific. TM Luminoskan TM The Ascent chemiluminescence analyzer was used to measure the RLU values ​​at different concentrations.

[0205] 1.5.4 Coating process and detection process for magnetic microspheres:

[0206] Coating Procedure: 1) Take 10 mg of carboxyl magnetic beads, magnetically separate them, add 900 μL of MES (15 mM, pH 6.0) buffer, mix the magnetic beads, add 100 μL of EDC (10 mg / mL, dissolved in MES (15 mM, pH 6.0)), vortex to mix, and react on a roller at 25°C for 0.5 hours. 2) After the reaction is complete, magnetically separate them, add MES (15 mM, pH 5.5) buffer, add the coating antibody (Ab), vortex to mix, and react on a roller for 2 hours. 3) After the reaction is complete, magnetically separate them, add 1 mL of TBS (Tris 20 mM, 50 mM sodium chloride, BSA, pH 7.4) buffer, vortex to mix, and react at 25°C for 16 hours. 4) After the reaction is complete, perform magnetic separation, add 1 mL of TBS (Tris 20 mM, 50 mM sodium chloride, BSA, pH 7.4) buffer, and vortex to mix. Obtain the coated Ab-magnetic beads and store them.

[0207] Detection procedure: 1) Sample loading reaction: Add 30 μL of the sample to be tested and 50 μL of Ab-magnetic bead working solution, react at 37℃ for 10 min; 2) Sample loading reaction: Add 50 μL of the Ab-AE working solution prepared above, react at 37℃ for 5 min; 3) Wash three times with 1*TBST; 4) Add pre-excitation solution A + excitation solution B, and perform detection on the Yingkai Shine i2910 fully automated chemiluminescence immunoassay analyzer to detect the relative luminescence intensity (RLU).

[0208] Example 1: Anti-CA724 chimeric antibody

[0209] A full-length anti-CA724 antibody (full-length heavy chain and full-length light chain as shown in SEQ ID NO: 8 and 9, respectively) and a chimeric antibody were prepared according to the preparation example. A conjugate labeled with AE was prepared according to preparation example 1.5.1 and its activity was determined. The structure of the anti-CA724 chimeric antibody is a leucine zipper (GCN4) attached to the C-terminus of the heavy chain of F(ab)'2 immunoglobulin (the heavy chain includes the VH-CH1-hinge region and the light chain includes the VL-CL). The amino acid sequence of the F(ab)'2 immunoglobulin heavy chain is shown in SEQ ID NO: 10, the amino acid sequence of the light chain is shown in SEQ ID NO: 9, the leucine zipper attached to the C-terminus of the heavy chain is SEQ ID NO: 1, and the amino acid sequence of the heavy chain attached to the leucine zipper is shown in SEQ ID NO: 11.

[0210] The activity test results of the full-length anti-CA724 antibody and the chimeric anti-CA724 antibody are shown in Table 1.

[0211] Table 1: Comparison of anti-CA724 antibody activities

[0212] The data above shows that the F(ab)'2-leucine zipper has an average activity that is about 39% higher than that of the full-length antibody.

[0213] The inventors also simultaneously used a Roche reagent kit to detect CA724 in the samples, and the correlation with samples of the same Roche value is shown in Figure 1. The results showed that the detection correlation of the full-length control was R0.05. 2 =0.87, the detection correlation of F(ab)'2-leucine zipper can reach R. 2 =0.95.

[0214] Example 2: Anti-TNFα chimeric antibody

[0215] Anti-TNFα full-length antibody (full-length heavy chain and full-length light chain as shown in SEQ ID NO: 16 and 17, respectively), F(ab)'2 type antibody and chimeric antibody were prepared according to the preparation examples. Conjugates labeled with AE were prepared according to preparation example 1.5.1 and their activity was measured. The structure of the anti-TNFα chimeric antibody is F(ab)'2 type immunoglobulin linked to a leucine zipper. The amino acid sequence of the F(ab)'2 type immunoglobulin heavy chain is shown in SEQ ID NO: 18, the amino acid sequence of the light chain is shown in SEQ ID NO: 17, the leucine zipper linked to the C-terminus of the heavy chain is GCN4, and the amino acid sequence of the heavy chain linked to the leucine zipper is shown in SEQ ID NO: 19.

[0216] Among them, the activity test results of the full-length anti-TNFα antibody, the F(ab)'2 type immunoglobulin against TNFα, and the chimeric anti-TNFα antibody showed that the F(ab)'2-leucine zipper had an average activity that was about 20% higher than that of the full-length antibody.

[0217] Table 2: Comparison of anti-TNFα antibody activities

[0218] Example 3: Anti-cTnI chimeric antibody

[0219] A full-length anti-cTnI antibody (full-length heavy chain and full-length light chain as shown in SEQ ID NO: 12 and 13, respectively) and a chimeric antibody (F(ab)'2-leucine zipper-protein tag) were prepared according to the preparation example. The protein tag sequence is shown in SEQ ID NO: 14, the heavy chain sequence of the chimeric antibody is shown in SEQ ID NO: 15, and the light chain sequence is shown in SEQ ID NO: 13. A conjugate directly coated with magnetic beads was prepared according to preparation example 1.5.4, and its activity was measured. The results showed that the activity of the chimeric antibody was higher than that of the full-length antibody.

[0220] Table 3: Comparison of activities of anti-cTnI chimeric antibodies

[0221] Example 4: Anti-T4 chimeric antibody

[0222] Anti-T4 full-length antibody (full-length heavy chain and full-length light chain as shown in SEQ ID NO: 20 and 21, respectively), F(ab)'2 type antibody and chimeric antibody were prepared according to the preparation example. The amino acid sequence of the heavy chain linked to the leucine zipper is shown in SEQ ID NO: 22.

[0223] Bio-coated conjugates were prepared and their activity was determined according to Preparation Example 1.5.2; AP-labeled conjugates were prepared and their activity was determined according to Preparation Example 1.5.3; and conjugates directly coated with magnetic beads were prepared and their activity was determined according to Preparation Example 1.5.4.

[0224] The results showed that F(ab)'2-leucine zipper-coated magnetic microspheres, Bio-coated, or AP-labeled antibodies all exhibited higher activity than the full-length antibody. Furthermore, the activity and signal-to-noise ratio of the labeled antibody (such as AP) were superior to those of the Bio-coated antibody, while the activity and signal-to-noise ratio of the Bio-coated antibody were superior to those of the magnetic microsphere-coated antibody.

[0225] In addition, the inventors constructed chimeric antibodies with different structures and tested their performance. The structures and properties of the chimeric antibodies are as follows:

[0226] Chimeric antibody 1: The heavy chain is a full-length heavy chain, and the light chain is a full-length light chain. The leucine zipper attached to the C-terminus of the full-length heavy chain is GCN4 (the leucine zipper with the sequence shown in SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 7). Its (full-length antibody-leucine zipper) activity is higher than that of the full-length antibody.

[0227] Chimeric antibody 2: The heavy chain is VH-CH1, and the light chain is VL-CL. The leucine zipper attached to the C-terminus of the heavy chain is the same, both being GCN4 (sequence as shown in SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO: 7). Its (F(ab)2-leucine zipper) activity is higher than that of the full-length antibody.

[0228] Chimeric antibody 3: The heavy chain is VH-CH1, and the light chain is VL-CL. The leucine zippers attached to the ends of the heavy and light chains are different. The C-terminus of the heavy chain is connected to c-Jun (sequence as shown in SEQ ID NO: 2), and the C-terminus of the light chain is connected to v-Fos (sequence as shown in SEQ ID NO: 3). Its (Fab-leucine zipper) activity is higher than that of Fab and more than half that of the full-length antibody.

[0229] Chimeric antibody 4: The heavy chain is VH and the light chain is VL. The leucine zippers attached to the ends of the heavy and light chains are different. The C-terminus of the heavy chain is connected to c-Jun and the C-terminus of the light chain is connected to v-Fos. Its activity (Fv-leucine zipper) is higher than that of Fab and more than half that of the full-length antibody.

[0230] It can be seen that conjugates prepared from chimeric immunoglobulins with different antibody structures and different leucine zippers all exhibit enhanced activity.

[0231] Example 5: Anti-BNP chimeric antibody

[0232] Full-length anti-BNP antibodies (full-length heavy chain and full-length light chain as shown in SEQ ID NO: 23 and 24, respectively), Fab, F(ab)'2 type antibodies, and corresponding chimeric antibodies were prepared according to the preparation examples. Conjugates labeled with AE were prepared according to Preparation Example 1.5.1, and their activities were compared at equimolar antibody concentrations. The heavy chain of the Fab-leucine zipper chimeric antibody is shown in SEQ ID NO: 25, and the light chain in SEQ ID NO: 26; the heavy chain of the F(ab)'2-leucine zipper chimeric antibody is shown in SEQ ID NO: 27, and the light chain in SEQ ID NO: 24. The results showed that the activity of the Fab-leucine zipper chimeric antibody was not only much higher than that of Fab, but also about 24% higher than that of the full-length antibody; the activity of the F(ab)'2-leucine zipper chimeric antibody was not only much higher than that of F(ab)'2, but also about 85% higher than that of the full-length antibody.

[0233] Table 4-1: Comparison of anti-BNP antibody activities

[0234] Table 4-2: Proportion of Increased Anti-BNP Antibody Activity

[0235] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0236] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A conjugate, characterized in that, The invention includes a chimeric immunoglobulin and a conjugation partner. The chimeric immunoglobulin comprises an antigen-binding fragment and a leucine zipper, wherein the end of the antigen-binding fragment is connected to the leucine zipper; wherein the leucine zipper has a motif shown as (Leu-X1-X2-X3-X4-X5-X6)n, where X1, X2, X3, X4, X5, and X6 are each independently selected from any amino acid, n≥2, and the sequences X1-X2-X3-X4-X5-X6 may be the same or different.

2. The conjugate according to claim 1, characterized in that, The n≥4.

3. The conjugate according to claim 1, characterized in that, The value of n is 4 to 8.

4. The conjugate according to claim 1, characterized in that, The motif represented by (Leu-X1-X2-X3-X4-X5-X6)n has an active group.

5. The conjugate according to claim 4, characterized in that, The active group includes at least one selected from amino, carboxyl, hydroxyl, or thiol groups.

6. The conjugate according to claim 1, characterized in that, The number of active groups in the motif (Leu-X1-X2-X3-X4-X5-X6)n is 4 to 8.

7. The conjugate according to claim 1, characterized in that, The number of active groups in the motif (Leu-X1-X2-X3-X4-X5-X6)n is 4, and n is 4.

8. The conjugate according to claim 1, characterized in that, The number of active groups in the motif (Leu-X1-X2-X3-X4-X5-X6)n is 5, and n is 5.

9. The conjugate according to claim 1, characterized in that, The number of active groups in the motif (Leu-X1-X2-X3-X4-X5-X6)n is 8, and n is 8.

10. The conjugate according to any one of claims 1 to 9, characterized in that, The Leu-X1-X2-X3-X4-X5-X6 sequence is derived from sequences on GCN4, c-Jun, or v-Fos proteins.

11. The conjugate according to any one of claims 1 to 9, characterized in that, The motif represented by (Leu-X1-X2-X3-X4-X5-X6)n is derived from a motif on the GCN4, c-Jun, or v-Fos protein.

12. The conjugate according to claim 1, characterized in that, The leucine zipper includes at least one selected from GCN4, c-Jun, v-Fos, or fragments thereof.

13. The conjugate according to claim 1, characterized in that, The leucine zipper has an amino acid sequence selected from any one of SEQ ID NO:1 to 7 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NO:1 to 7.

14. The conjugate according to any one of claims 1 to 13, characterized in that, The C-terminus of the antigen-binding fragment is connected to the N-terminus of the leucine zipper.

15. The conjugate according to claim 1, characterized in that, The chimeric immunoglobulin further includes a linker peptide, the N-terminus of which is linked to the C-terminus of the antigen-binding fragment, and the C-terminus of which is linked to the N-terminus of the leucine zipper.

16. The conjugate according to any one of claims 1 to 15, characterized in that, The antigen-binding fragment includes at least one selected from F(ab')2, F(ab)2, Fab', Fab antibody, full-length natural antibody, and Fv antibody.

17. The conjugate according to any one of claims 1 to 16, characterized in that, The antigen-binding fragment includes a heavy chain; the heavy chain includes a first heavy chain and a second heavy chain, the ends of which are respectively connected to the leucine zipper.

18. The conjugate according to claim 17, characterized in that, The leucine zippers attached to the ends of the first and second heavy chains may be the same or different.

19. The conjugate according to claim 17, characterized in that, The antigen-binding fragment also includes a light chain.

20. The conjugate according to any one of claims 1 to 19, characterized in that, The antigen-binding fragment includes a heavy chain and a light chain; the ends of the heavy chain and the light chain are respectively connected to the leucine zipper, and the leucine zipper connected to the end of the light chain may be the same as or different from the leucine zipper connected to the end of the heavy chain.

21. The conjugate according to any one of claims 1 to 19, characterized in that, The antigen-binding fragment includes a heavy chain and a light chain; the heavy chain includes a first heavy chain and a second heavy chain, both of which include a VH-CH1-hinge region, and the light chain includes a VL-CL. The leucine zippers attached to the ends of the first and second heavy chains are identical.

22. The conjugate according to claim 21, characterized in that, The leucine zipper is GCN4.

23. The conjugate according to claim 21, characterized in that, Both the first heavy chain and the second heavy chain include a VH-CH1-hinge region-CH2-CH3-tail peptide, and the light chain includes VL-CL. The leucine zippers attached to the ends of the first heavy chain and the second heavy chain are the same.

24. The conjugate according to claim 23, characterized in that, The leucine zipper is GCN4.

25. The conjugate according to claim 21, characterized in that, Both the first heavy chain and the second heavy chain include VH-CH1, and the light chain includes VL-CL. The leucine zippers attached to the ends of the first heavy chain and the second heavy chain are the same.

26. The conjugate according to claim 25, characterized in that, The leucine zipper is GCN4.

27. The conjugate according to claim 21, characterized in that, Both the first heavy chain and the second heavy chain include VH-CH1-hinge region-CH2, and the light chain includes VL-CL. The leucine zippers connected to the ends of the first heavy chain and the second heavy chain are the same.

28. The conjugate according to claim 27, characterized in that, The leucine zipper is GCN4.

29. The conjugate according to any one of claims 1 to 19, characterized in that, The antigen-binding fragment includes a heavy chain and a light chain; the heavy chain includes VH-CH1, and the light chain includes VL-CL, wherein the leucine zippers attached to the ends of the heavy chain and the light chain are different.

30. The conjugate according to claim 29, characterized in that, The leucine zipper is c-Jun and v-Fos.

31. The conjugate according to any one of claims 1 to 19, characterized in that, The antigen-binding fragment comprises a heavy chain and a light chain; the heavy chain comprises VH and the light chain comprises VL, wherein the leucine zippers attached to the ends of the heavy chain and the light chain are different.

32. The conjugate according to claim 31, characterized in that, The leucine zipper is c-Jun and v-Fos.

33. The conjugate according to claim 1, characterized in that, The chimeric immunoglobulin also has a protein tag attached to its end.

34. The conjugate according to claim 33, characterized in that, The protein tags are selected from Spytag tags, His tags, Flag tags, GST tags, MBP tags, SUMO tags, and C-Myc tags.

35. The conjugate according to any one of claims 1 to 34, characterized in that, The conjugation chaperone is chemically coupled to the active group on the chimeric immunoglobulin; the conjugation chaperone is a marker.

36. The conjugate according to claim 35, characterized in that, The marker is selected from enzymes, luminescent substances, fluorescent substances, colored substances, radioactive substances, colloids, or combinations thereof.

37. The conjugate according to claim 35, characterized in that, The fused partner is a combined spouse.

38. The conjugate according to claim 37, characterized in that, Coupled agonists include biotin, streptavidin, or avidin.

39. The conjugate according to claim 1, characterized in that, The mate is a solid-phase carrier.

40. The conjugate according to claim 39, characterized in that, The solid support is selected from microspheres, plates, or membranes.

41. The conjugate according to claim 4, characterized in that, The conjugation partner is directly or indirectly covalently coupled to the active group on the chimeric immunoglobulin.

42. A chimeric immunoglobulin defined in any one of claims 1 to 41.

43. A nucleic acid molecule, a vector, a host cell, or a method for preparing the chimeric immunoglobulin of claim 42, wherein the nucleic acid molecule encodes the chimeric immunoglobulin of claim 42; the vector contains a nucleic acid molecule encoding the chimeric immunoglobulin of claim 42; the host cell contains the aforementioned nucleic acid molecule or vector; and the method comprises culturing the aforementioned host cell.

44. A method for preparing the conjugate according to any one of claims 1 to 41, comprising: The chimeric immunoglobulin of claim 42 or the chimeric immunoglobulin prepared by the method of claim 43 is coupled with a conjugate chaperone to obtain the conjugate.

45. The method according to claim 44, characterized in that, The conjugation chaperone is covalently coupled to the active group on the chimeric immunoglobulin.

46. ​​The method according to claim 45, characterized in that, The coupling is a non-directional coupling.

47. A method for detecting antigens in a sample to be tested, characterized in that, include: The sample to be tested is contacted using the conjugate according to any one of claims 1 to 41 or the conjugate prepared by the method of claim 44, wherein the antigen-binding fragment in the conjugate binds to the antigen in the sample to be tested.

48. Use of the conjugate according to any one of claims 1 to 41, the conjugate prepared by the method of claim 44, the chimeric immunoglobulin according to claim 42, or the chimeric immunoglobulin prepared by the method of claim 43 in the preparation of an immunoassay product.

49. A reagent kit or test strip, characterized in that, The kit or test strip comprises the conjugate according to any one of claims 1 to 41, the conjugate prepared by the method of claim 44, the chimeric immunoglobulin according to claim 42, or the chimeric immunoglobulin prepared by the method of claim 43.

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