Immunoglobulin and use thereof

Abstract

The present application belongs to the field of biotechnology. Disclosed is an immunoglobulin, comprising a heavy chain and a light chain, wherein the heavy chain sequentially comprises, from the N-terminus to the C-terminus, a heavy chain variable region, an IgG CH1 region, an IgG hinge region, an IgM CH2 region, and an IgM CH3-CH4 region; the light chain sequentially comprises, from the N-terminus to the C-terminus, a light chain variable region and a CL region; and an N mutation is present on the immunoglobulin, and is located in the framework region of the variable region or the IgG CH1 region.

Description

Immunoglobulins and their uses

[0001] Priority information

[0002] This application claims priority to Chinese Patent Application No. 202410940103X, filed on July 12, 2024, entitled "Immunoglobulin and its uses", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of biotechnology, specifically relating to an immunoglobulin and its uses, and more specifically to an immunoglobulin, isolated nucleic acid, vector, host cell, method for preparing immunoglobulin, immunoglobulin conjugate, reagent or kit and its uses, method for detecting antigens in samples to be tested, and method for increasing the expression level of immunoglobulin. Background Technology

[0004] Immunoglobulins (Ig), also known as immunoglobulin molecules or antibodies, are important molecules in the human immune system, secreted by immune cells (mainly B cells). They have the ability to recognize and bind foreign substances (antigens) and are a key component of the body's immune response. The basic structure of immunoglobulins consists of a tetrapeptide chain, including two identical light chains (L chains) and two identical heavy chains (H chains), which are linked by disulfide bonds to form a peptide chain molecule, constituting the basic structure of the immunoglobulin molecule. Immunoglobulins bind to antigens through their variable regions (VH and VL) to exert their biological functions, such as activating complement and binding to Fc receptors on the cell surface.

[0005] Immunoglobulins can be classified into five classes: IgG, IgA, IgM, IgD, and IgE, each with its specific function and characteristics. IgG has a Y-shaped structure and is a monomeric structure. IgM is a multivalent antibody secreted by B cells; in its secreted form, it is a polymer composed of five or six Y-shaped monomers, with 10 or 12 antigen-binding sites and extremely high affinity. However, current research on immunoglobulins is limited. Summary of the Invention

[0006] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides an immunoglobulin and its uses.

[0007] In one aspect of this application, an immunoglobulin is provided. The immunoglobulin comprises a heavy chain and a light chain. The heavy chain, from N-terminus to C-terminus, sequentially includes a heavy chain variable region, an IgG CH1 region, an IgG hinge region, an IgM CH2 region, and an IgM CH3-CH4 region. The light chain, from N-terminus to C-terminus, sequentially includes a light chain variable region and a CL region. The immunoglobulin exhibits an N mutation, which is located at one of the following positions or a combination thereof: HFR3 in the heavy chain variable region, HFR4 in the heavy chain variable region, LFR3 in the light chain variable region, or the IgG CH1 region.

[0008] In another aspect of this application, an isolated nucleic acid is proposed. According to an embodiment of this application, the isolated nucleic acid encodes the aforementioned immunoglobulin.

[0009] In another aspect, this application provides a vector. According to an embodiment of this application, the vector contains the aforementioned isolated nucleic acid.

[0010] In another aspect, this application provides a host cell. According to embodiments of this application, the host cell contains the aforementioned isolated nucleic acid or the aforementioned vector.

[0011] In another aspect, this application provides a method for preparing an immunoglobulin. According to embodiments of this application, the method includes preparation using the aforementioned host cell expression method.

[0012] In another aspect, this application proposes an immunoglobulin conjugate. According to embodiments of this application, the immunoglobulin conjugate comprises the aforementioned immunoglobulins.

[0013] In another aspect of this application, a reagent, test strip, or kit is provided. According to embodiments of this application, the reagent, test strip, or kit includes the aforementioned immunoglobulin or the aforementioned immunoglobulin conjugate.

[0014] In another aspect of this application, the use of the aforementioned immunoglobulin or the aforementioned immunoglobulin conjugate in the preparation of immunoassay products is proposed.

[0015] In another aspect of this application, a method for detecting antigens in a sample to be tested is provided. According to an embodiment of this application, the method includes: a) contacting the aforementioned immunoglobulin, the aforementioned immunoglobulin conjugate, or the aforementioned reagent, test strip, or kit with an antigen in the sample to be tested under conditions sufficient to induce an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, and determining whether the sample to be tested contains an antigen based on the presence of the immune complex.

[0016] In another aspect of this application, a method for improving the expression level or assembly efficiency of immunoglobulins is proposed, wherein the immunoglobulins comprise heavy chains and light chains, the heavy chains comprising, from the N-terminus to the C-terminus, a heavy chain variable region, an IgG CH1 region, an IgG hinge region, an IgM CH2 region, and an IgM CH3-CH4 region; and the light chains comprising, from the N-terminus to the C-terminus, a light chain variable region and a CL region, comprising: mutating one or more amino acids N in the immunoglobulins, wherein the N mutation is located at the same position as the N mutation in the aforementioned immunoglobulins.

[0017] Additional aspects and advantages of this application 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 this application. Attached Figure Description

[0018] 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:

[0019] Figure 1 is the original SEC image of the anti-cTnI-mouse IgG2b heterozygous recombinant antibody in Example 1 of this application, wherein Figure 1A is wild type, Figure 1B is modified group 1, and Figure 1C is modified group 2;

[0020] Figure 2 is the original SEC image of the anti-Helicobacter pylori-mouse IgG1 hybrid recombinant antibody in Example 1 of this application, wherein Figure 2A is wild type, Figure 2B is modified group 1, Figure 2C is modified group 2, and Figure 2D is modified group 3;

[0021] Figure 3 is the original SEC image of the anti-HIV P24-rabbit IgG heterozygous recombinant antibody in Example 1 of this application, where Figure 3A is wild type and Figure 3B is modified group 1;

[0022] Figure 4 shows the activity detection results of the anti-Helicobacter pylori-mouse IgG1 heterozygous recombinant antibody in Example 1 of this application in the colloidal test. Detailed Implementation

[0023] 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.

[0024] 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.

[0025] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0026] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0027] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0028] 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.

[0029] 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: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and 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, including but 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 CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0030] In this document, the term "having at least 80% sequence similarity" 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 similarity 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.

[0031] In this document, the term "having at least 90% sequence similarity" can mean having 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% sequence similarity.

[0032] 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.

[0033] In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into and / or between cells or hosts. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell or host. Typically, by culturing a suitable cell or host containing the vector, the vector can produce the desired expression product.

[0034] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of this application and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0035] This application provides a detailed description of immunoglobulins and their uses.

[0036] Immunoglobulins

[0037] In one aspect of this application, an immunoglobulin is provided. The immunoglobulin comprises a heavy chain and a light chain. The heavy chain, from N-terminus to C-terminus, sequentially includes a heavy chain variable region, an IgG CH1 region, an IgG hinge region, an IgM CH2 region, and an IgM CH3-CH4 region. The light chain, from N-terminus to C-terminus, sequentially includes a light chain variable region and a CL region. The immunoglobulin exhibits an N mutation, which is located at the following positions or combinations thereof: HFR3 in the heavy chain variable region, HFR4 in the heavy chain variable region, LFR3 in the light chain variable region, or the IgG CH1 region. The immunoglobulin of this application has the advantage of high expression levels.

[0038] In this paper, the term "N mutation" refers to the mutation of a non-asparagine residue into an asparagine residue (N).

[0039] According to embodiments of this application, the immunoglobulin may further include at least one of the following technical features:

[0040] According to an embodiment of this application, the numbering of the CH1 region is obtained by sequentially numbering the amino acid sequence shown in the CH1 region from the N-terminus to the C-terminus.

[0041] The division of the CH1 region in the constant region of immunoglobulins of different species and types in this application is shown in the table below:

[0042] Based on the CH1 region defined in the table above, the first amino acid counted from the N-terminus to the C-terminus of CH1 is the "first amino acid of the CH1 region". It should be understood that the "first amino acid of the CH1 region" also includes positions equivalent to 1, and the determination of equivalent positions can be made through sequence alignment.

[0043] According to embodiments of this application, the N mutation is located at the following positions or combinations thereof: 1) the first amino acid in the CH1 region; 2) the tenth amino acid in the HFR3 region; 3) the sixth amino acid in the HFR4 region; 4) the eleventh amino acid in the LFR3 region.

[0044] According to an embodiment of this application, the N mutation is located at one of the following positions: 1) the first amino acid in the CH1 region; 2) the tenth amino acid in the HFR3 region; 3) the sixth amino acid in the HFR4 region; 4) the eleventh amino acid in the LFR3 region.

[0045] In an optional embodiment of this application, the N mutation is located at the first amino acid in the CH1 region.

[0046] In an optional embodiment of this application, the N mutation is located at the 10th amino acid in the HFR3 region.

[0047] In an optional embodiment of this application, the N mutation is located at the 6th amino acid in the HFR4 region.

[0048] In an optional embodiment of this application, the N mutation is located at the 11th amino acid in the LFR3 region.

[0049] According to an embodiment of this application, the HFR3 region is obtained by dividing it with reference to the Kabat definition.

[0050] According to an embodiment of this application, the HFR3 region is numbered sequentially from the N-terminus to the C-terminus of the amino acid sequence shown in the HFR3 region.

[0051] According to an embodiment of this application, the HFR4 region is obtained by dividing it with reference to the Kabat definition.

[0052] According to an embodiment of this application, the HFR4 region is numbered sequentially from the N-terminus to the C-terminus of the amino acid sequence shown in the HFR4 region.

[0053] According to an embodiment of this application, the LFR3 region is obtained by dividing it with reference to the Kabat definition.

[0054] According to an embodiment of this application, the LFR3 region is numbered sequentially from the N-terminus to the C-terminus of the amino acid sequence shown in the LFR3 region.

[0055] In this document, the term "frame region" or "FRs" includes heavy chain frame regions and light chain frame regions, referring to the regions of the antibody heavy chain variable region and light chain variable region other than the CDRs. The heavy chain frame region can be further subdivided into adjacent regions separated by CDRs, including HFR1, HFR2, HFR3, and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by CDRs, including LFR1, LFR2, LFR3, and LFR4 frame regions. Furthermore, the heavy chain variable region is obtained by connecting the following CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0056] Unless otherwise specified, the partitioning of variable regions in this document is based on the KABAT definition. The KABAT definition method is well-known in the art (see the definition system described in Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983)). Based on the KABAT-defined partitioning, this application exemplarily demonstrates the division of HFRs in the variable regions of different species and types of immunoglobulins, as shown in the table below:

[0057] Based on the HFR3 classification in the table above, the 10th amino acid counted from the N-terminus to the C-terminus of HFR3 is the "10th amino acid of the HFR3 region". It should be understood that the "10th amino acid of the HFR3 region" also includes positions that are equivalent to 10, and the determination of equivalent positions can be made through sequence alignment.

[0058] Based on the partitioning defined by KABAT, this application exemplarily illustrates the partitioning of LFRs in the variable regions of different types of immunoglobulins, as detailed in the table below:

[0059] Based on the LFR3 classification in the table above, the 11th amino acid counted from the N-terminus to the C-terminus of LFR3 is the "11th amino acid of the LFR3 region". It should be understood that the "11th amino acid of the LFR3 region" also includes positions that are equivalent to 11, and the determination of equivalent positions can be made through sequence alignment.

[0060] According to embodiments of this application, the immunoglobulin is derived from cattle, horses, pigs, sheep, mice, rats, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

[0061] According to embodiments of this application, the heavy chain variable region, IgG CH1 region, IgG hinge region, IgM CH2-CH3-CH4 region, light chain variable region, and CL region may be of the same or different species origin.

[0062] According to embodiments of this application, the IgG is selected from IgG1, IgG2a, IgG2b, IgG3, or IgG4.

[0063] According to embodiments of this application, the CL is selected from the κ type or the λ type.

[0064] In one optional embodiment of this application, the CL is selected from mouse κ type, rabbit κ type, or rabbit λ type.

[0065] According to embodiments of this application, the amino acid sequence of the CH1 region is as shown in any one of SEQ ID NO:1 to 3, or has at least 80% identity with it.

[0066] Sequence comparison showed that the amino acid sequence shown in SEQ ID NO:1 and the amino acid sequence shown in SEQ ID NO:2 had 86% identity.

[0067] Mouse IgG1:

[0068] Mouse IgG2b:

[0069] Rabbit IgG:

[0070] According to embodiments of this application, the amino acid sequence of the CH1 region is shown in any one of SEQ ID NO: 1 to 3.

[0071] It should be noted that in this application, "the amino acid sequence as shown in SEQ ID NO:A" includes the amino acid sequence of SEQ ID NO:A or the amino acid sequence of SEQ ID NO:A with conservative modifications, all of which are within the scope of protection of this application. For example, "the amino acid sequence of the CH1 region as shown in any one of SEQ ID NO:1 to 3" means that the CH1 region is the amino acid sequence of SEQ ID NO:1 to 3 or the amino acid sequence of SEQ ID NO:1 to 3 with conservative modifications, all of which are within the scope of protection of this application.

[0072] In this document, "conservatively modified amino acid sequences" refers to amino acid modifications that do not significantly affect or alter the binding properties of antibodies containing that amino acid sequence. These modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this application using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions involve replacing 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 (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine). Therefore, one or more amino acid residues in the CDR region of the antibody of this application can be replaced by other amino acid residues from the same side chain family, and the retained function of the modified antibody can be tested using the functional assay methods described herein. Exemplarily, conservative modifications are made in numbers not exceeding 80% of the total, preferably not exceeding 90%. In this article, "conserved modified amino acid sequences" also include naturally occurring amino acid modifications. "Naturally occurring mutations" refer to mutations caused by changes in alleles due to individual differences and other reasons during the natural mutation process of antibodies.

[0073] According to embodiments of this application, the amino acid sequence of the HFR3 region is as shown in any one of SEQ ID NO:4 to 6, or has at least 80% identity with it.

[0074] Sequence comparison showed that the amino acid sequence shown in SEQ ID NO:4 and the amino acid sequence shown in SEQ ID NO:5 had 91% identity.

[0075] HFR3 of mouse IgG1: KATLTADKSNSTAYMQLSSLTSEDSAVYYCAR (SEQ ID NO:4).

[0076] HFR3 of mouse IgG2b: KATLTVDKSNSTAFMELSSLTSEDSAVYYCAR (SEQ ID NO:5).

[0077] HFR3 of rabbit IgG: RSTITRNTNNNTVTLKMTSLTAADTATYFCAR (SEQ ID NO:6).

[0078] According to embodiments of this application, the amino acid sequence of the HFR3 region is shown in any one of SEQ ID NO:4 to 6.

[0079] It should be noted that "the amino acid sequence of the HFR3 region as shown in any one of SEQ ID NO:4 to 6" means that the HFR3 region is an amino acid sequence of SEQ ID NO:4 to 6 or an amino acid sequence of SEQ ID NO:4 to 6 with conservative modifications, both of which are within the scope of protection of this application.

[0080] According to embodiments of this application, the amino acid sequence of the LFR3 region is as shown in any one of SEQ ID NO:7 to 9, or has at least 80% identity with it.

[0081] Mouse κ type LFR3: GVPARFSGSGNGTSYSLTISRVEAEDAATYYC (SEQ ID NO:7).

[0082] LFR3 of mouse λ type: GVPARFSGSLNGDKAALTITGAQTEDEAIYFC (SEQ ID NO:8).

[0083] Rabbit κ type LFR3: GVPSRFEGSGNGTEFTLTISDLECADAATYYC (SEQ ID NO:9).

[0084] According to embodiments of this application, the amino acid sequence of the LFR3 region is shown in any one of SEQ ID NO:7 to 9.

[0085] It should be noted that "the amino acid sequence of the LFR3 region as shown in any one of SEQ ID NO:7 to 9" means that the LFR3 region is an amino acid sequence of SEQ ID NO:7 to 9 or an amino acid sequence of SEQ ID NO:7 to 9 with conservative modifications, both of which are within the scope of protection of this application.

[0086] According to embodiments of this application, the amino acid sequence of the HFR4 region is as shown in any one of SEQ ID NO:77-79, or has at least 80% identity with it.

[0087] Sequence comparison showed that the amino acid sequences shown in SEQ ID NO:77 and SEQ ID NO:78 had 82% identity.

[0088] Sequence comparison showed that the amino acid sequences shown in SEQ ID NO:78 and SEQ ID NO:79 had 82% identity.

[0089] HFR4 of mouse IgG1: WGQGTNLTVSS (SEQ ID NO:77).

[0090] HFR4 of mouse IgG2b: WGQGTNVTVSA (SEQ ID NO:78).

[0091] HFR4 of rabbit IgG: WGPGTNVTVSS (SEQ ID NO:79).

[0092] According to embodiments of this application, the amino acid sequence of the HFR4 region is shown in any one of SEQ ID NO:77-79.

[0093] It should be noted that "the amino acid sequence of the HFR4 region as shown in any one of SEQ ID NO:77 to 79" means that the HFR4 region is an amino acid sequence of SEQ ID NO:77 to 79 or an amino acid sequence of SEQ ID NO:77 to 79 with conservative modifications, both of which are within the scope of protection of this application.

[0094] According to embodiments of this application, the amino acid sequences of HFR1 / HFR2 / HFR4 in the heavy chain variable region are as shown in SEQ ID NO:11-13, SEQ ID NO:14-16 or SEQ ID NO:17-19, respectively, or have at least 80% identity with them.

[0095] It should be noted that the amino acid sequences SEQ ID NO:11-13, SEQ ID NO:14-16 or SEQ ID NO:17-19 in the HFRs of the above-mentioned heavy chain variable region refer to the amino acid sequences of SEQ ID NO:11-13, SEQ ID NO:14-16 or SEQ ID NO:17-19 or the amino acid sequences of SEQ ID NO:11-13, SEQ ID NO:14-16 or SEQ ID NO:17-19 in a conservative modified form, and are all within the protection scope of this application.

[0096] According to embodiments of this application, the amino acid sequences of HFR1 / HFR2 / HFR4 in the heavy chain variable region are shown as SEQ ID NO:11-13, SEQ ID NO:14-16, or SEQ ID NO:17-19, respectively.

[0097] According to embodiments of this application, the amino acid sequences of HFR1 / HFR2 / HFR4 in the heavy chain variable region are shown in SEQ ID NO:11-13, respectively.

[0098] According to embodiments of this application, the amino acid sequences of HFR1 / HFR2 / HFR4 in the heavy chain variable region are shown in SEQ ID NO:14-16, respectively.

[0099] According to embodiments of this application, the amino acid sequences of HFR1 / HFR2 / HFR4 in the heavy chain variable region are shown in SEQ ID NO:17-19, respectively.

[0100] According to embodiments of this application, the amino acid sequences of HFR1 / HFR2 / HFR3 / HFR4 in the heavy chain variable region are as shown in SEQ ID NO:11, 12, 4 and 13, respectively, or have at least 80% identity with them; or

[0101] The amino acid sequences of HFR1 / HFR2 / HFR3 / HFR4 in the heavy chain variable region are as shown in SEQ ID NO:14, 15, 5 and 16, respectively, or have at least 80% identity with them; or

[0102] The amino acid sequences of HFR1 / HFR2 / HFR3 / HFR4 in the heavy chain variable region are as shown in SEQ ID NO:17, 18, 6 and 19, respectively, or have at least 80% identity with them.

[0103] According to embodiments of this application, the amino acid sequences of HFR1 / HFR2 / HFR3 / HFR4 in the heavy chain variable region are shown in SEQ ID NO: 11, 12, 4 and 13, respectively; or

[0104] The amino acid sequences of HFR1 / HFR2 / HFR3 / HFR4 in the heavy chain variable region are shown in SEQ ID NO: 14, 15, 5 and 16, respectively; or

[0105] The amino acid sequences of HFR1 / HFR2 / HFR3 / HFR4 in the heavy chain variable region are shown in SEQ ID NO:17, 18, 6 and 19, respectively.

[0106] According to embodiments of this application, the amino acid sequences of HFR1 / HFR2 / HFR3 / HFR4 in the heavy chain variable region are shown in SEQ ID NO:11, 12, 4 and 13, respectively.

[0107] According to embodiments of this application, the amino acid sequences of HFR1 / HFR2 / HFR3 / HFR4 in the heavy chain variable region are shown in SEQ ID NO:14, 15, 5 and 16, respectively.

[0108] According to embodiments of this application, the amino acid sequences of HFR1 / HFR2 / HFR3 / HFR4 in the heavy chain variable region are shown in SEQ ID NO:17, 18, 6 and 19, respectively.

[0109] According to embodiments of this application, the amino acid sequences of LFR1 / LFR2 / LFR4 in the light chain variable region are as shown in SEQ ID NO:20-22, SEQ ID NO:23-25, or SEQ ID NO:26-28, respectively, or have at least 80% identity with them.

[0110] It should be noted that the amino acid sequences SEQ ID NO:20~22, SEQ ID NO:23~25, SEQ ID NO:26~28 or SEQ ID NO:29~31 in the above-mentioned light chain variable regions refer to the amino acid sequences of SEQ ID NO:20~22, SEQ ID NO:23~25, SEQ ID NO:26~28 or SEQ ID NO:29~31, or the amino acid sequences of SEQ ID NO:20~22, SEQ ID NO:23~25, SEQ ID NO:26~28 or SEQ ID NO:29~31 in a conservatively modified form, and are all within the scope of protection of this application.

[0111] According to embodiments of this application, the amino acid sequences of LFR1 / LFR2 / LFR4 in the light chain variable region are shown as SEQ ID NO:20-22, SEQ ID NO:23-25, or SEQ ID NO:26-28, respectively.

[0112] According to embodiments of this application, the amino acid sequences of LFR1 / LFR2 / LFR4 in the light chain variable region are shown in SEQ ID NO:20-22, respectively.

[0113] According to embodiments of this application, the amino acid sequences of LFR1 / LFR2 / LFR4 in the light chain variable region are shown in SEQ ID NO:23-25, respectively.

[0114] According to embodiments of this application, the amino acid sequences of LFR1 / LFR2 / LFR4 in the light chain variable region are shown in SEQ ID NO:26-28, respectively.

[0115] According to embodiments of this application, the amino acid sequences of LFR1 / LFR2 / LFR3 / LFR4 in the light chain variable region are as shown in SEQ ID NO:20, 21, 7 and 22, respectively, or have at least 80% identity with them; or

[0116] The amino acid sequences of LFR1 / LFR2 / LFR3 / LFR4 in the light chain variable region are as shown in SEQ ID NO:23, 24, 8 and 25, respectively, or have at least 80% identity with them; or

[0117] The amino acid sequences of LFR1 / LFR2 / LFR3 / LFR4 in the variable region of the light chain are as shown in SEQ ID NO:26, 27, 9 and 28, respectively, or have at least 80% identity with them.

[0118] According to embodiments of this application, the amino acid sequences of LFR1 / LFR2 / LFR3 / LFR4 in the light chain variable region are shown in SEQ ID NO:20, 21, 7 and 22, respectively; or

[0119] The amino acid sequences of LFR1 / LFR2 / LFR3 / LFR4 in the variable region of the light chain are shown in SEQ ID NO: 23, 24, 8 and 25, respectively; or

[0120] The amino acid sequences of LFR1 / LFR2 / LFR3 / LFR4 in the variable region of the light chain are shown in SEQ ID NO:26, 27, 9 and 28, respectively.

[0121] According to embodiments of this application, the amino acid sequences of LFR1 / LFR2 / LFR3 / LFR4 in the light chain variable region are shown as SEQ ID NO:20, 21, 7 and 22, respectively.

[0122] According to embodiments of this application, the amino acid sequences of LFR1 / LFR2 / LFR3 / LFR4 in the light chain variable region are shown in SEQ ID NO:23, 24, 8 and 25, respectively.

[0123] According to embodiments of this application, the amino acid sequences of LFR1 / LFR2 / LFR3 / LFR4 in the light chain variable region are shown in SEQ ID NO:26, 27, 9 and 28, respectively.

[0124] According to the embodiments of this application, the IgG hinge region can be a partial hinge region or a complete hinge region. Due to different methods of dividing the hinge region, the length of the resulting hinge region varies. The function of the hinge region is at least to facilitate the formation of disulfide bonds between heavy and light chains.

[0125] According to embodiments of this application, the amino acid sequence of the IgG hinge region is as shown in any one of SEQ ID NO:49 to 51, or has at least 80% identity with it.

[0126] EPSGPISTINPC (SEQ ID NO:49);

[0127] VPRDC (SEQ ID NO:50);

[0128] APSTCSKPMCPPPELPGG (SEQ ID NO:51).

[0129] According to embodiments of this application, the amino acid sequence of the IgG hinge region is shown in any one of SEQ ID NO:49-51.

[0130] According to an embodiment of this application, the amino acid sequence of the IgG hinge region is shown in SEQ ID NO:49.

[0131] According to an embodiment of this application, the amino acid sequence of the IgG hinge region is shown in SEQ ID NO:50.

[0132] According to an embodiment of this application, the amino acid sequence of the IgG hinge region is shown in SEQ ID NO:51.

[0133] According to embodiments of this application, the amino acid sequence of the IgM CH2 region is as shown in any one of SEQ ID NO:52-53, or has at least 80% identity with it.

[0134] Mouse IgM CH2 region:

[0135] Rabbit IgM CH2 region:

[0136] According to embodiments of this application, the amino acid sequence of the IgM CH2 region is shown in any one of SEQ ID NO:52-53.

[0137] According to an embodiment of this application, the amino acid sequence of the IgM CH2 region is shown in SEQ ID NO:52.

[0138] According to an embodiment of this application, the amino acid sequence of the IgM CH2 region is shown in SEQ ID NO:53.

[0139] According to embodiments of this application, the amino acid sequence of the IgM CH3-CH4 region is as shown in any one of SEQ ID NO:54-55, or has at least 80% identity with it.

[0140] Mouse IgM CH3-CH4 region:

[0141] Rabbit IgM CH3-CH4 region:

[0142] According to embodiments of this application, the amino acid sequence of the IgM CH3-CH4 region is shown in any one of SEQ ID NO:54-55.

[0143] According to an embodiment of this application, the amino acid sequence of the IgM CH3-CH4 region is shown in SEQ ID NO:54.

[0144] According to an embodiment of this application, the amino acid sequence of the IgM CH3-CH4 region is shown in SEQ ID NO:55.

[0145] According to embodiments of this application, the amino acid sequence of the CL region is as shown in any one of SEQ ID NO:56 to 58, or has at least 80% identity with it.

[0146] GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO:58). According to embodiments of this application, the amino acid sequence of the CL region is shown in any one of SEQ ID NO:56 to 58.

[0147] According to an embodiment of this application, the amino acid sequence of the CL region is shown in SEQ ID NO:56.

[0148] According to an embodiment of this application, the amino acid sequence of the CL region is shown in SEQ ID NO:57.

[0149] According to an embodiment of this application, the amino acid sequence of the CL region is shown in SEQ ID NO:58.

[0150] In embodiments of this application, the heavy chain variable region further includes HCDRs, and / or the light chain variable region further includes LCDRs.

[0151] In this document, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this application, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0152] The HCDRs and LCDRs of this application can be adjusted according to the antigen they bind to. This application does not impose any special limitations on them, and all are within the scope of protection of this application. The inventors have discovered that, for the immunoglobulin of this application, the HCDRs and LCDRs of the heavy chain variable region and the light chain variable region do not affect the production of the immunoglobulin. As long as an N mutation exists on the immunoglobulin, and the location of the N mutation meets the requirements defined above in this application, the prepared immunoglobulin has the advantage of high expression levels.

[0153] In one example embodiment of this application, the heavy chain variable regions HCDR1, HCDR2 and HCDR3 are shown as IYVLH (SEQ ID NO:59), YINPYIDGTKYNEKFKG (SEQ ID NO:60) and SGYGNYGLAWLAY (SEQ ID NO:61), respectively, and the light chain variable regions LCDR1, LCDR2 and LCDR3 are shown as RSSTGAVTTSNYAN (SEQ ID NO:62), GSNNRAP (SEQ ID NO:63) and ALVYSNNWV (SEQ ID NO:64), respectively.

[0154] In one example embodiment of this application, the heavy chain variable regions HCDR1, HCDR2 and HCDR3 are shown as SYWMH (SEQ ID NO:65), YINPSTGNTEYDQRFKG (SEQ ID NO:66) and DFDTYYDSFDS (SEQ ID NO:67), respectively, and the light chain variable regions LCDR1, LCDR2 and LCDR3 are shown as RASSSVLYIH (SEQ ID NO:68), ATSNLAS (SEQ ID NO:69) and QQWSSNPFT (SEQ ID NO:70), respectively.

[0155] In one example embodiment of this application, the heavy chain variable regions HCDR1, HCDR2 and HCDR3 are shown as RNTMS (SEQ ID NO:71), AISIYADTHYASWAKS (SEQ ID NO:72) and DDVYSAWGRLNI (SEQ ID NO:73), respectively, and the light chain variable regions LCDR1, LCDR2 and LCDR3 are shown as QASQYISDYLS (SEQ ID NO:74), RASTLES (SEQ ID NO:75) and QSADYRSSLA (SEQ ID NO:76), respectively.

[0156] It should be noted that the HCDRs and LCDRs mentioned above are all classified with reference to the Kabat definition method. In an optional example of this application, HCDRs and LCDRs may also be defined by Chothia, AbM, Contact, or IMGT. Those skilled in the art can clearly map this Kabat definition method to any variable region sequence without relying on any experimental data outside the sequence itself. As stated herein, "Kabat definition method" refers to the numbering system described by Kabat et al., USD ept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). HCDRs and LCDRs defined by different definition methods are also within the scope of protection of this application.

[0157] According to embodiments of this application, the immunoglobulin is a monomeric immunoglobulin, a polymeric immunoglobulin, or a mixture thereof.

[0158] Unless otherwise specified herein, immunoglobulins may be monomeric immunoglobulins, polymeric immunoglobulins, or mixtures thereof, and their specific types are not limited, all of which are within the scope of protection of this application. The inventors of this application have discovered that any immunoglobulin containing the N mutation described in this application can significantly increase immunoglobulin production.

[0159] In this paper, the term "monomer immunoglobulin" refers to a Y-shaped structure consisting of two heavy chains and two light chains. Each heavy chain, from the N-terminus to the C-terminus, includes a heavy chain variable region, an IgG CH1 region, an IgG hinge region, an IgM CH2 region, and an IgM CH3-CH4 region. Each light chain, from the N-terminus to the C-terminus, includes a light chain variable region and a CL region.

[0160] In this article, the term "polyimmunoglobulin" refers to an immunoglobulin complex composed of multiple monomeric immunoglobulins linked by disulfide bonds or specific linker chains (such as the J chain).

[0161] According to embodiments of this application, the immunoglobulin is a polymeric immunoglobulin, which comprises 5 or 6 monomeric immunoglobulins. The inventors of this application have discovered that when the immunoglobulin is a polymeric immunoglobulin, not only can the expression level of the immunoglobulin be increased, but the assembly efficiency of the immunoglobulin can also be improved.

[0162] In this paper, the term "assembly efficiency" refers to the proportion of polymeric immunoglobulins in total immunoglobulins.

[0163] According to embodiments of this application, the polymeric immunoglobulin is obtained by polymerizing 5 or 6 monomeric immunoglobulins.

[0164] This application discloses an immunoglobulin. The immunoglobulin comprises a heavy chain and a light chain. The heavy chain, from N-terminus to C-terminus, sequentially includes a heavy chain variable region, an IgG CH1 region, an IgG hinge region, an IgM CH2 region, and an IgM CH3-CH4 region. The light chain, from N-terminus to C-terminus, sequentially includes a light chain variable region and a CL region. The immunoglobulin contains an N mutation, located at the first amino acid of the CH1 region, the tenth amino acid of the HFR3 region, or the eleventh amino acid of the LFR3 region. The amino acid sequence of the CH1 region is shown in any one of SEQ ID NO: 1–3; the amino acid sequence of the HFR3 region is shown in any one of SEQ ID NO: 4–6; and the amino acid sequence of the LFR3 region is shown in any one of SEQ ID NO: 7–9.

[0165] Furthermore, those skilled in the art will understand that the features and advantages described above for immunoglobulins also apply to this immunoglobulin, and will not be repeated here.

[0166] This application discloses an immunoglobulin. The immunoglobulin comprises a heavy chain and a light chain. The heavy chain, from N-terminus to C-terminus, sequentially includes a heavy chain variable region, an IgG CH1 region, an IgG hinge region, an IgM CH2 region, and an IgM CH3-CH4 region. The light chain, from N-terminus to C-terminus, sequentially includes a light chain variable region and a CL region. The immunoglobulin contains an N mutation, which is located at the first amino acid of the CH1 region, the tenth amino acid of the HFR3 region, or the eleventh amino acid of the LFR3 region.

[0167] The amino acid sequence of the CH1 region is shown in any one of SEQ ID NO: 1 to 3;

[0168] The amino acid sequence of the HFR3 region is shown in any one of SEQ ID NO:4 to 6;

[0169] The amino acid sequence of the LFR3 region is shown in any one of SEQ ID NO: 7 to 9;

[0170] The amino acid sequences of HFR1 / HFR2 / HFR3 / HFR4 in the heavy chain variable region are shown in SEQ ID NO: 11, 12, 4 and 13, respectively; or the amino acid sequences of HFR1 / HFR2 / HFR3 / HFR4 in the heavy chain variable region are shown in SEQ ID NO: 14, 15, 5 and 16, respectively; or the amino acid sequences of HFR1 / HFR2 / HFR3 / HFR4 in the heavy chain variable region are shown in SEQ ID NO: 17, 18, 6 and 19, respectively.

[0171] The amino acid sequences of LFR1 / LFR2 / LFR3 / LFR4 in the light chain variable region are shown in SEQ ID NO:20, 21, 7 and 22, respectively; or the amino acid sequences of LFR1 / LFR2 / LFR3 / LFR4 in the light chain variable region are shown in SEQ ID NO:23, 24, 8 and 25, respectively; or the amino acid sequences of LFR1 / LFR2 / LFR3 / LFR4 in the light chain variable region are shown in SEQ ID NO:26, 27, 9 and 28, respectively.

[0172] This application discloses an immunoglobulin. The immunoglobulin comprises a heavy chain and a light chain, and the immunoglobulin contains an N mutation located at the following position or an equivalent position:

[0173] 1) Located at position 76 (the 10th amino acid in the HFR3 region), position 117 (the 6th amino acid in the HFR4 region), or position 123 (the 1st amino acid in the CH1 region) of the heavy chain shown in SEQ ID NO:38 (mouse IgG2b);

[0174] 2) Located at position 76 (the 10th amino acid in the HFR3 region), position 115 (the 6th amino acid in the HFR4 region), or position 121 (the 1st amino acid in the CH1 region) of the heavy chain shown in SEQ ID NO:43 (mouse IgG1);

[0175] 3) Located at position 74 (the 10th amino acid in the HFR3 region), position 114 (the 6th amino acid in the HFR4 region), or position 120 (the 1st amino acid in the CH1 region) of the heavy chain shown in SEQ ID NO:48 (rabbit IgG);

[0176] 4) Located at position 69 (the 11th amino acid of the LFR3 region mutation) of the light chain (mouse λ type) shown in SEQ ID NO:37;

[0177] 5) Located at position 66 (the 11th amino acid of the LFR3 region mutation) of the light chain (mouse κ type) shown in SEQ ID NO:41;

[0178] 6) Located at position 67 (the 11th amino acid of the LFR3 region mutation) of the light chain (rabbit κ type) shown in SEQ ID NO:46.

[0179] In one example embodiment of this application, the HCDR1, HCDR2 and / or HCDR3 sequences in the heavy chain are replaced with other arbitrary sequences of HCDR1, HCDR2 and / or HCDR3.

[0180] In one example embodiment of this application, the LCDR1, LCDR2 and / or LCDR3 sequences in the light chain are replaced with other arbitrary sequences of LCDR1, LCDR2 and / or LCDR3.

[0181] In one example embodiment of this application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and / or LCDR3 sequence is defined by any one or a combination of multiple definition methods such as Kabat, Chothia, IMGT, AbM, or Contact.

[0182] The CDR definition method is well known. CDRs defined under different methods can be obtained using software (such as Abysis).

[0183] In one example embodiment of this application, the heavy chain has at least 80% identity with SEQ ID NO:38, SEQ ID NO:43, or SEQ ID NO:48.

[0184] In one example embodiment of this application, the light chain has at least 80% identity with SEQ ID NO:37, SEQ ID NO:41, or SEQ ID NO:46.

[0185] Furthermore, those skilled in the art will understand that the features and advantages described above for immunoglobulins also apply to this immunoglobulin, and will not be repeated here.

[0186] Isolated nucleic acids, vectors, and host cells

[0187] In another aspect of this application, an isolated nucleic acid is proposed. According to an embodiment of this application, the isolated nucleic acid encodes the aforementioned immunoglobulin.

[0188] According to an embodiment of this application, the isolated nucleic acid is DNA.

[0189] It should be noted that those skilled in the art will understand that the isolated nucleic acids mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases, the complementary strand is also disclosed. Furthermore, the nucleic acid sequences isolated in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0190] In another aspect, this application proposes a vector. According to an embodiment of this application, the vector contains the aforementioned isolated nucleic acid. When the isolated nucleic acid is ligated to the vector, the isolated nucleic acid can be directly or indirectly connected to control elements on the vector, as long as these control elements can control the translation and expression of the isolated nucleic acid. Of course, these control elements can be directly derived from the vector itself, or they can be exogenous, i.e., not derived from the vector itself. Naturally, the isolated nucleic acid and the control elements can be operatively ligated.

[0191] In this article, "operably ligated" refers to ligating a foreign gene to a vector, enabling the control elements within the vector, such as transcriptional and translational control sequences, to perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include plasmids and bacteriophages. According to some specific embodiments of this application, after the vector is introduced into suitable recipient cells, the aforementioned immunoglobulin expression can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of immunoglobulins.

[0192] In some specific embodiments of this application, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0193] In some specific embodiments of this application, the vector is a lentiviral vector.

[0194] In one optional embodiment of this application, the expression vector is a plasmid expression vector.

[0195] In another aspect, this application provides a host cell. According to embodiments of this application, the host cell contains the aforementioned isolated nucleic acid or the aforementioned vector. Using this host cell under suitable conditions, the aforementioned immunoglobulins can be effectively expressed within the host cell.

[0196] According to an embodiment of this application, the host cell is obtained by introducing it into the host cell via the vector described in the fourth aspect.

[0197] It should be noted that the host cell in this application is not particularly limited and can be a prokaryotic cell, a eukaryotic cell, or a bacteriophage. The prokaryotic cell can be *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*, etc. The aforementioned eukaryotic cells include fungi such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, and *Trichoderma*, insect cells such as armyworms, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0198] According to an embodiment of this application, the host cell is a eukaryotic cell.

[0199] According to embodiments of this application, the host cell is a mammalian cell, including but not limited to BHK cells, CHO cells, NSO cells or COS cells, and does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0200] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the immunoglobulins described in this application. Those skilled in the art will readily understand that suitable conditions for the expression of the immunoglobulins include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the immunoglobulins based on the specific environment of their laboratory.

[0201] Methods for preparing immunoglobulins

[0202] In another aspect, this application provides a method for preparing an immunoglobulin. According to embodiments of this application, the method includes preparation using the aforementioned host cell expression method.

[0203] Immunoglobulin conjugates, reagents or kits

[0204] In another aspect, this application proposes an immunoglobulin conjugate. According to embodiments of this application, the immunoglobulin conjugate comprises the aforementioned immunoglobulins.

[0205] According to embodiments of this application, the above-mentioned immunoglobulin conjugate may further include at least one of the following technical features:

[0206] According to embodiments of this application, the immunoglobulin conjugate further includes a conjugation portion, which is conjugated to the immunoglobulin.

[0207] In one optional embodiment of this application, the coupling portion can be linearly coupled to the immunoglobulin or indirectly coupled. The specific connection method is not limited and is within the protection scope of this application.

[0208] In one optional embodiment of this application, the coupling portion of the immunoglobulin conjugate can be directly labeled with immunoglobulins. Compared to indirect labeling, the preparation method of immunoglobulin conjugates obtained by directly labeling immunoglobulins is simpler and more convenient.

[0209] In an optional embodiment of this application, the indirect connection in the immunoglobulin conjugate may be that the marker is directly conjugated to a hydrophilic carrier (exemplary bovine serum albumin BSA, KLH, OVA, etc.) and indirectly connected to the immunoglobulin through a heterobifunctional adapter.

[0210] According to embodiments of this application, the coupling portion is selected from biotin, biotin derivatives, markers, or solid-phase carriers.

[0211] In one optional embodiment of this application, the aforementioned marker refers to a type of substance that has properties such as luminescence, color development, and radioactivity that can be directly observed by the naked eye or detected or probed by instruments. Through these properties, qualitative or quantitative detection of the corresponding target can be achieved.

[0212] In one optional embodiment of this application, the markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0213] 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 application.

[0214] In one optional embodiment of this application, the fluorescent dye includes, but is 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. Cy5, Cy5.5, Cy3, etc. or similar), 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, etc. or similar) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0215] In one optional embodiment of this application, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0216] In one optional embodiment of this application, the radioactive isotope includes, but is not limited to, [other radioactive isotopes]. 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 F.

[0217] In one optional embodiment of this application, the chemiluminescent reagent includes, but is not limited to, 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.

[0218] In one optional embodiment of this application, the nanoparticle-type markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0219] In one alternative embodiment of this application, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latexes.

[0220] In one optional embodiment of this application, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0221] In one optional embodiment of this application, the colloidal metal is colloidal gold.

[0222] In one optional embodiment of this application, the antibody conjugate further includes a solid-phase carrier conjugated to the antibody.

[0223] In one alternative embodiment of this application, the solid support is selected from microspheres, plates, and membranes.

[0224] In one optional embodiment of this application, the solid carrier includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0225] In another aspect of this application, a reagent, test strip, or kit is provided. According to embodiments of this application, the reagent or kit includes the aforementioned immunoglobulin or the aforementioned immunoglobulin conjugate.

[0226] According to embodiments of this application, the above-mentioned reagents, test strips, or kits may further include at least one of the following technical features:

[0227] In one optional embodiment of this application, the kit further includes sample pretreatment reagents (such as sample purification and enrichment reagents, lysis buffers, etc.), washing solutions (such as water), buffers (such as PBS or Tris, etc.), and chromogenic reagents for signaling substances (such as ECL or DAB if the signaling substance is horseradish peroxidase).

[0228] In one optional embodiment of this application, the kit further includes a sample pad, a conjugation pad, a reaction membrane, and an absorption pad, wherein the reaction membrane is provided with a detection area and a quality control area.

[0229] In one alternative embodiment of this application, the kit may further include another antibody, which may be a polymer or a monomer.

[0230] In one optional embodiment of this application, the immunoglobulin can be immobilized on a solid phase, including a nitrocellulose membrane, latex, magnetic beads, or ELISA plate.

[0231] In one optional embodiment of this application, the immunoglobulin can be labeled with signaling substances such as nanoparticles, chemiluminescent substances, fluorescent substances, radioactive substances, colloids, and enzymes.

[0232] In one optional embodiment of this application, the test strip includes 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 immunoglobulin is coated on the detection area and / or dropped onto the conjugate pad.

[0233] It is easy to understand that when both the detection area and the conjugation pad contain the immunoglobulin, the antigenic epitopes bound to their IgG variable regions are different, and the heavy chain constant regions (IgG CH1 region, IgG hinge region, IgM CH2 region and IgM CH3-CH4 region) in the immunoglobulin can be the same or different.

[0234] According to embodiments of this application, the above-mentioned immunoglobulin conjugates and kits may further include at least one of the following technical features:

[0235] In some embodiments, the solid-phase carrier in the kit or immunoglobulin conjugate is a plastic, microparticle, or membrane carrier; the plastic may be polystyrene; the microparticles may be magnetic microparticles; and the membrane carrier may be a nitrocellulose membrane, a glass cellulose membrane, or a nylon membrane.

[0236] In some embodiments, the solid support is selected from test tubes, EP tubes, porous plates, chromatography columns, and micro-reaction plate recesses.

[0237] In this application, the term "microparticle" can refer to a sphere, near-sphere, cube, polyhedron, or irregular shape. The diameter of the microsphere is preferably 10 nm to 1 mm, for example, 100 nm, 500 nm, 1 μm, 10 μm, 100 μm, or 500 μm; more preferably 400 nm to 10 μm.

[0238] The microparticles are preferably magnetic microparticles, which contain magnetic materials. The magnetic materials can be metals (metallic elements or alloys), nonmetals, or composites of metals and nonmetals. Examples of metals include iron, aluminum, nickel, and cobalt; examples of nonmetals include ferrite nonmetals (preferably Fe2O3 or Fe3O4 magnetic nanoparticles); and examples of composites of metals and nonmetals include neodymium iron boron rubber magnetic composite materials.

[0239] Multiwell plates are preferably ELISA plates, which can contain 8, 16, 32, 48, 64, 96 or more wells.

[0240] use

[0241] In another aspect of this application, the use of the aforementioned immunoglobulin or the aforementioned immunoglobulin conjugate in the preparation of immunoassay products is proposed.

[0242] method

[0243] In another aspect of this application, a method for detecting antigens in a sample to be tested is proposed. According to an embodiment of this application, the method includes: a) contacting the aforementioned immunoglobulin, the aforementioned immunoglobulin conjugate, or the aforementioned reagent or kit with an antigen in the sample to be tested under conditions sufficient to induce an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, and determining whether the sample to be tested contains an antigen based on the presence of the immune complex.

[0244] According to embodiments of this application, the antigen detection method described above may further include at least one of the following technical features:

[0245] According to embodiments of this application, the presence of the immune complex indicates the presence of the antigen in the sample to be tested.

[0246] According to an embodiment of this application, the immune complex further includes a second antibody, which binds to the antibody.

[0247] According to embodiments of this application, the immune complex further includes a second antibody that binds to the antigen.

[0248] The method for detecting the antigen can be any method known to those skilled in the art, for example, detecting the antigen with an additional antibody against the antigen (which is typically labeled with a signaling substance, or detected with a secondary antibody labeled with a signaling substance) after the antigen has been contacted with the solid-phase carrier (double antibody sandwich).

[0249] Alternatively, conventional antibodies can be used as antibodies coated on a solid phase and co-incubated with the antigen to be detected, and the immunoglobulin can be used as a free detection antibody (which is usually labeled with a signal substance, or detected by a secondary antibody labeled with a signal substance).

[0250] Alternatively, the solid-phase carrier can be used in conjunction with the immunoglobulin to detect the antigen, in which case the two are paired antibodies.

[0251] The signaling substance can be any one of the following: fluorescent substance, quantum dot, digoxigenin-labeled probe, biotin, radioactive isotope, radioactive contrast agent, paramagnetic ion fluorescent microsphere, electron-dense material, chemiluminescent label, ultrasound contrast agent, photosensitizer, colloidal gold, or enzyme. In some embodiments, the signaling substance is colloidal gold, fluorescein, fluorescent microsphere, acrid ester, horseradish peroxidase, alkaline phosphatase, or β-galactosidase.

[0252] In another aspect of this application, a method for improving the expression level or assembly efficiency of immunoglobulins is proposed, wherein the immunoglobulins comprise heavy chains and light chains, the heavy chains comprising, from N-terminus to C-terminus, a heavy chain variable region, an IgG CH1 region, an IgG hinge region, an IgM CH2 region, and an IgM CH3-CH4 region; the light chains comprising, from N-terminus to C-terminus, a light chain variable region and a CL region, comprising: mutating one or more amino acids N in the immunoglobulins, wherein the N mutation is located at the same position as the N mutation in the aforementioned immunoglobulins;

[0253] Optionally, the immunoglobulin is a monomeric immunoglobulin, a polymeric immunoglobulin, or a mixture thereof.

[0254] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this 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.

[0255] In the following examples, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The pMD 18T vector was purchased from Takara; the plasmid extraction kit was purchased from Tiangen Pharmaceuticals; primer synthesis and gene sequencing were performed by Invitrogen.

[0256] Example 1: Preparation of IgG / IgM heterozygous recombinant antibody

[0257] 1. The construction of expression plasmids for IgG / IgM hybrid recombinant antibodies is described in Example 2 of CN 114621350 A, with the only difference being the amino acid sequence (at least one of the heavy chain variable region, light chain variable region, and CH1 region). The amino acid sequences of the light and heavy chains of each IgG / IgM hybrid recombinant antibody are shown in the table below:

[0258] The specific amino acid sequences in the table above are shown below:

[0259] 2. For the expression, purification and preparation of IgG / IgM hybrid recombinant antibodies, please refer to Example 3 of CN 114621350 A.

[0260] 3. The purity of each IgG / IgM hybrid recombinant antibody purified in step 2 was identified using HPLC-SEC (High Performance Liquid Chromatography-Size Exclusion Chromatography). Specifically, 20 μg of purified antibody was taken, and the antibody purification was determined according to the method in the table below:

[0261] The yields of each IgG / IgM heterozygous recombinant antibody were calculated, and the results are shown in Figures 1 to 3. The antibody yields and corresponding purities after expression of each IgG / IgM heterozygous multimer recombinant antibody are shown in Tables 1 to 3.

[0262] Table 1: Antibody yield and corresponding purity after expression of anti-cTnI-mouse IgG2b heterozygous recombinant antibody

[0263] As shown in the table above, in terms of yield, compared with the wild type, the yield improvement of modified group 1 and modified group 2 is significant, with modified group 1 showing the highest yield, reaching 8.5 times. In terms of assembly efficiency, modified groups 1 to 2 all improved assembly efficiency. Overall, both modified groups 1 and 2 showed significant improvements in expression levels and assembly efficiency.

[0264] Table 2: Antibody yield and corresponding purity after expression of anti-Helicobacter pylori-mouse IgG1 heterozygous recombinant antibody

[0265] As shown in the table above, in terms of yield, both modified groups 1 and 3 significantly improved yield, with modified group 3 showing the highest improvement of 18-fold. Regarding assembly efficiency, both modified groups 1 and 3 improved assembly efficiency, with modified group 3 showing the best improvement. Overall, modified group 3 significantly improved both expression levels and assembly efficiency. Wild-type and modified group 3 will be selected for activity testing in the next step.

[0266] Table 3: Antibody yield and corresponding purity after expression of anti-HIV P24-rabbit IgG heterozygous recombinant antibody

[0267] As shown in the table above, the expression level and assembly efficiency of the modified group were significantly improved.

[0268] 4. Fluorescence activity

[0269] 4.1. Antibody labeling: Take 100 μL of 1% fluorescent microspheres (Merck, Eu-030), add 900 μL of activation buffer, mix well, centrifuge to remove the supernatant, add 1 mL of activation buffer, sonicate to mix well, then add activator (EDC NHS), vortex and mix in the dark for 20 min, centrifuge to remove the supernatant, add an equal volume of conjugation buffer to the microspheres, sonicate to mix well, add 0.2-0.4 mg of labeled antibody, vortex and mix in the dark for 3 h, finally add blocking buffer for blocking, vortex and mix in the dark for 45 min to stop labeling, centrifuge to remove the supernatant, reconstitute the microspheres with microsphere preservation solution, sonicate to mix well, and store at 4℃ for use.

[0270] 4.2. Preparation of microsphere working solution: Dilute the antibody marker to a final concentration of 10-20% with microsphere diluent, and then spray the marker onto the glass fiber using a spray pad.

[0271] 4.3. Preparation of dried microsphere pads: Place the sprayed fluorescent pads in a 50℃ oven and dry for more than 2 hours.

[0272] 4.4. Sample pad treatment: Dilute the blocking agent to 0.4 mg / ml with sample pad diluent, spread it on glass fiber, and dry it in a 50°C oven overnight.

[0273] 4.5. NC membrane coating: Dilute the coating antibody to 1.0 mg / ml with coating diluent and then coat the antibody; dry in an oven at 50°C overnight.

[0274] 4.6. Preparation of fluorescent chromatography strips: The fluorescent chromatography strips are cut into strips using a strip cutter, assembled, and then sample is added for detection.

[0275] 4.7. Detection: (1) Quality control: recombinant antigen; original IgG, see CN 114621350 A, i.e., antibodies obtained naturally through hybridoma or phage technology before polymer modification. (2) Detection method: After adding the sample for 15 minutes, the instrument is judged. Among them, the fluorescence activity results of cTnI-mouse IgG2b hybrid recombinant antibody are shown in Table 4.

[0276] Table 4: Activity results of cTnI-mouse IgG2b heterozygous recombinant antibody

[0277] As shown in the table above, the modified antibody activity was increased by more than 50% compared to the original IgG.

[0278] 5. Preparation of colloidal gold test strips

[0279] 5.1. Preparation of nitrocellulose membranes

[0280] Preparation of nitrocellulose membrane: Dilute the coating antibody to 1-5 mg / ml with coating buffer and streak it as the T line, which is the detection line, with the T line close to the colloidal gold end; dilute the secondary antibody to 1-5 mg / ml with coating buffer and streak it as the C line, which is the control line, with the C line close to the absorbent pad. Dry at 37°C and encapsulate for later use.

[0281] 5.2. Preparation of colloidal gold and gold-labeled monoclonal antibodies.

[0282] 1) Preparation of colloidal gold

[0283] Dilute 1% chloroauric acid to 0.01% with double-distilled deionized water, bring to a boil on an electric stove, and add 2 ml of 1% trisodium citrate per 100 ml of 0.01% chloroauric acid. Continue boiling until the liquid turns bright red, then stop heating. After cooling to room temperature, replenish the lost water. The prepared colloidal gold should be pure, clear, and free of precipitates and floating matter, and has a shelf life of one week.

[0284] 2) Preparation of colloidal gold-labeled antibodies

[0285] Adjust the pH of the colloidal gold to 8.2 using 0.1M potassium carbonate. Add labeled antibody at a ratio of 8–10 μg antibody / ml colloidal gold. Mix with a magnetic stirrer for 30 min. Add BSA to a final concentration of 1% while stirring, and let stand for 1 hour. Centrifuge at 13000 rpm and 4°C for 30 min. Discard the supernatant. Wash the precipitate twice with labeled wash and preservation buffer. Resuspend the precipitate in one-tenth of the initial colloidal gold volume of labeled wash and preservation buffer. Store at 4°C for one week.

[0286] 3) Preparation of gold-labeled pads

[0287] The gold-labeled pads were immersed in blocking solution for 30 minutes and then dried at 37°C. The prepared gold-labeled antibodies were then evenly spread on the gold-labeled pads, with 20 square centimeters per milliliter of solution. The pads were then freeze-dried, encapsulated, and stored at 4°C for later use.

[0288] 4) Preparation of the sample pad for the test strip

[0289] Immerse the sample pad in the blocking solution (containing BSA) for 30 minutes, then dry it at 37°C, seal it, and store it at 4°C for later use.

[0290] 5) Assembly of test strips

[0291] The absorbent pad (purchased from Millipore), nitrocellulose membrane, gold label pad, and sample pad were placed on a non-absorbent support sheet and cut into 3mm wide strips. Ten strips were packaged together, a desiccant was added, and the packages were vacuum sealed to obtain the test strip.

[0292] 5.3. Application of Colloidal Gold

[0293] The intensity of the red precipitate line indicates the strength of the reaction; the deeper the red, the stronger the reaction, and vice versa. The strength of the reaction is represented by a combination of the letter C and a number; the smaller the number after C, the stronger the reaction, and vice versa. No red precipitate line is represented by B.

[0294] The colloidal activity results of the anti-Helicobacter pylori antibody-mouse IgG1 hybrid recombinant antibody are shown in Figure 4 and Table 5.

[0295] Table 5: Colloidal gold detection results of anti-Helicobacter pylori antibody-mouse IgG1 heterozygous recombinant antibody modified group 3

[0296] Note: The lower the colorimetric value (C), the higher the activity; + indicates a high C value of 0.5; ++ indicates a high C value of 0.75.

[0297] As shown in Figure 4 and Table 5, the modified group had more than 1C higher activity than the original IgG.

[0298] In summary, the first amino acid in the CH1 region, the tenth amino acid in the HFR3 region, and the eleventh amino acid in the LFR3 region can significantly improve the yield and assembly efficiency of polyimmunoglobulins, and further enhance detection activity.

[0299] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. 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.

[0300] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. An immunoglobulin, comprising a heavy chain and a light chain, wherein the heavy chain, from the N-terminus to the C-terminus, sequentially comprises a heavy chain variable region, an IgG CH1 region, an IgG hinge region, an IgM CH2 region, and an IgM CH3-CH4 region; and the light chain, from the N-terminus to the C-terminus, sequentially comprises a light chain variable region and a CL region, characterized in that, The immunoglobulin has an N mutation, which is located at the following positions or combinations thereof: HFR3 in the heavy chain variable region, HFR4 in the heavy chain variable region, LFR3 in the light chain variable region, or the IgG CH1 region.

2. The immunoglobulin according to claim 1, characterized in that, The N mutation is located at the following positions or combinations thereof: 1) The first amino acid in the CH1 region; 2) The 10th amino acid in the HFR3 region; 3) The 6th amino acid in the HFR4 region; 4) The 11th amino acid in the LFR3 region.

3. The immunoglobulin according to any one of claims 1 to 2, characterized in that, The immunoglobulin meets at least one of the following conditions: (1) The immunoglobulin is derived from cattle, horses, pigs, sheep, mice, rats, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese or humans; (2) The heavy chain variable region, IgG CH1 region, IgG hinge region, IgM CH2-CH3-CH4 region, light chain variable region and CL region are of the same or different species origin; (3) The IgG is selected from IgG1, IgG2a, IgG2b, IgG3 or IgG4; (4) The CL is selected from the κ type or the λ type.

4. An isolated nucleic acid, characterized in that, The isolated nucleic acid encodes the immunoglobulin according to any one of claims 1 to 3.

5. A carrier, characterized in that, The carrier contains the isolated nucleic acid as described in claim 4.

6. A host cell, characterized in that, The host cell contains the isolated nucleic acid as described in claim 4 or the vector as described in claim 5.

7. A method for preparing immunoglobulin, characterized in that, This includes preparations made using host cell expression as described in claim 6.

8. An immunoglobulin conjugate, characterized in that, The immunoglobulin conjugate comprises the immunoglobulin according to any one of claims 1 to 3.

9. The immunoglobulin conjugate according to claim 8, characterized in that, The immunoglobulin conjugate further includes a conjugation portion, which is conjugated to the immunoglobulin.

10. The immunoglobulin conjugate according to claim 9, characterized in that, The coupling portion is selected from biotin, biotin derivatives, markers, or solid-phase carriers.

11. The immunoglobulin conjugate according to claim 10, characterized in that, The markers are selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

12. A reagent, test strip, or kit, characterized in that, The reagents, test strips, or kits comprise the immunoglobulins according to any one of claims 1 to 3 or the immunoglobulin conjugates according to any one of claims 8 to 11.

13. Use of the immunoglobulin according to any one of claims 1 to 3, or the immunoglobulin conjugate according to any one of claims 8 to 11, in the preparation of immunoassay products.

14. A method for detecting antigens in a sample to be tested, characterized in that, include: a) Under conditions sufficient to induce an antibody / antigen binding reaction, the immunoglobulin of any one of claims 1 to 3, the immunoglobulin conjugate of any one of claims 8 to 11, or the reagent, test strip, or kit of claim 12 is brought into contact with the antigen in the sample to be tested to form an immune complex. and b) Detect the presence of the immune complex, and determine whether the sample to be tested contains an antigen based on the presence of the immune complex.

15. The antigen detection method according to claim 14, characterized in that, The antigen detection method meets at least one of the following conditions: (1) The presence of the immune complex indicates the presence of the antigen in the sample to be tested; (2) The immune complex further includes a second antibody, which binds to the antibody; (3) The immune complex further includes a second antibody, which binds to the antigen.

16. A method for improving the expression level or assembly efficiency of an immunoglobulin, wherein the immunoglobulin comprises a heavy chain and a light chain, and the heavy chain comprises, from the N-terminus to the C-terminus, a heavy chain variable region, an IgG CH1 region, an IgG hinge region, an IgM CH2 region, and an IgM CH3-CH4 region. The light chain, from the N-end to the C-end, sequentially includes a light chain variable region and a CL region, characterized in that, include: One or more amino acid N mutations are made in the immunoglobulin, wherein the position of the N mutation is the same as the position of the N mutation in the immunoglobulin according to any one of claims 1 to 3.

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