Anti-cd68 antibodies, their production and use

Abstract

The application relates to the field of biological medicine, in particular to an anti-CD68 antibody and preparation and application thereof, wherein the anti-CD68 antibody has heavy chain complementarity determining regions 1-3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively, and light chain complementarity determining regions 1 and 3 as shown in SEQ ID NO:4 and SEQ ID NO:5 respectively, and a light chain complementarity determining region 2 with LVS. Compared with the CD68 antibody proposed in the related art, the anti-CD68 antibody proposed in the application has stronger binding specificity to CD68 and lower reaction background, thereby realizing high-accuracy detection of CD68.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to an anti-CD68 antibody and its preparation and application. Background Technology

[0002] CD68 (Macrosialin), also known as LD110, LAMP4, SRD1, etc., is a highly glycosylated transmembrane glycoprotein that is highly expressed in human monocytes and tissue macrophages, primarily located in lysosomes and endosomes. CD68 is one of the major antigens of monocytes and macrophages and a marker of myeloid monocytes. CD68 is a member of the mucin-like molecule family of the blood system, the LAMP family of lysosome / endosome-associated membrane glycoproteins, and the scavenger receptor family.

[0003] Because CD68 is primarily located in the lysosomes and endosomal membrane systems of macrophages, its high expression in these cells makes it a marker for macrophages and monocytes. However, it is not entirely a surface marker—although CD68 is mainly an lysosomal protein, some CD68 is also expressed on the cell surface, especially in certain activated states or specific cell types. Furthermore, CD68 can also serve as a diagnostic marker for diseases such as tumors / cancers. For example, it can be used as a marker for two types of tumor-associated macrophages (TAMs): classically activated type 1 (M1) and alternatingly activated type 2 (M2), thereby detecting tumor growth and cancer progression and contributing to clinical diagnosis and prognosis in cancer.

[0004] Currently, most CD68 antibodies used both domestically and internationally are polyclonal antibodies, while monoclonal antibodies are costly to prepare and have poor stability. Furthermore, research on commonly used monoclonal antibodies in existing technologies (such as the CD68 antibody frequently cited on the Citeab website, https: / / www.citeab.com / ) has revealed that these antibodies generally exhibit non-specific binding when detecting CD68 (e.g., in immunohistochemistry), resulting in high binding background and thus affecting the accuracy of CD68 detection.

[0005] Therefore, there is an urgent need to develop CD68 antibodies with high binding specificity and low reaction background. Summary of the Invention

[0006] This application addresses at least one of the problems of the related technology in the following aspects.

[0007] The first aspect of this application provides an anti-CD68 antibody or antigen-binding fragment, the anti-CD68 antibody or antigen-binding fragment comprising: a heavy chain complementarity-determining region 1 as shown in SEQ ID NO: 1; a heavy chain complementarity-determining region 2 as shown in SEQ ID NO: 2; a heavy chain complementarity-determining region 3 as shown in SEQ ID NO: 3; a light chain complementarity-determining region 1 as shown in SEQ ID NO: 4; a light chain complementarity-determining region 2 having an LVS; and a light chain complementarity-determining region 3 as shown in SEQ ID NO: 5.

[0008] In some embodiments, the anti-CD68 antibody or antigen-binding fragment includes: heavy chain framework regions 1-4 as shown in SEQ ID NO: 6-9 and light chain framework regions 1-4 as shown in SEQ ID NO: 10-13, respectively.

[0009] In some embodiments, the anti-CD68 antibody or antigen-binding fragment includes at least one of the following: a) the anti-CD68 antibody or antigen-binding fragment has a heavy chain variable region as shown in SEQ ID NO: 14 and / or a light chain variable region as shown in SEQ ID NO: 15; and b) has an amino acid sequence with at least 85% identity compared to a), the CD68 antibody or antigen-binding fragment being capable of specifically binding to CD68.

[0010] In some embodiments, the antibody or antigen-binding fragment comprises Fab. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, VHH, and diabody antibodies.

[0011] The second aspect of this application provides a polynucleotide that encodes an anti-CD68 antibody or antigen-binding fragment as described in any embodiment of the first aspect of this application.

[0012] A third aspect of this application provides an expression vector comprising the polynucleotides described in any embodiment of the second aspect of this application.

[0013] The fourth aspect of this application provides a recombinant cell comprising a polynucleotide as described in any embodiment of the second aspect of this application or an expression vector as described in any embodiment of the third aspect of this application.

[0014] The fifth aspect of this application provides an anti-CD68 antibody conjugate, which is prepared from an anti-CD68 antibody or antigen-binding fragment as described in any embodiment of the first aspect of this application, and an antigen and / or a labeling molecule. Optionally, the labeling molecule is a fluorescent labeling molecule, an enzyme, a metal ion, or an isotope.

[0015] A sixth aspect of this application provides a kit comprising an anti-CD68 antibody or antigen-binding fragment as described in any embodiment of the first aspect of this application, a polynucleotide as described in any embodiment of the second aspect of this application, an expression vector as described in any embodiment of the third aspect of this application, recombinant cells as described in any embodiment of the fourth aspect of this application, and / or an anti-CD68 antibody conjugate as described in any embodiment of the fifth aspect of this application.

[0016] In some embodiments, the kit is an IHC kit, an ELISA kit, an IF kit, or a Western Blot kit.

[0017] The seventh aspect of this application proposes the use of anti-CD68 antibodies or antigen-binding fragments as described in any embodiment of the first aspect of this application or kits as described in any embodiment of the sixth aspect of this application in any of the following: a. detecting CD68 protein; b. preparing reagents or reagent kits for detecting CD68 protein; optionally, the detection is an immunoassay, which may be one or more of IHC, Western Blot, IF, and ELISA.

[0018] In some embodiments, the reagent or reagent kit is used for the diagnostic detection of the CD68 antigen.

[0019] The embodiments of this application achieve the following beneficial effects:

[0020] This application proposes a novel CD68 monoclonal antibody derived from mouse hybridoma. Compared with existing CD68 antibodies, the proposed CD68 antibody exhibits higher binding specificity and extremely low reaction background, thereby enabling more accurate detection in various related CD68 detection applications, such as through immunohistochemistry (IHC), Western blotting, immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA). This allows for more accurate and sensitive marker detection, clinical diagnosis, and prognosis assessment of biological samples and individuals. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The gel imaging results of the anti-CD68 antibody according to an embodiment of this application are shown;

[0023] Figure 2 The results of affinity assays for anti-CD68 antibodies according to embodiments of this application are shown.

[0024] Figure 3A The results of IHC detection of various normal tissues using the commercially available CD68 monoclonal antibody KP1 according to embodiments of this application;

[0025] Figure 3B The results of IHC detection of various normal tissues using the anti-CD68 antibody proposed in this embodiment, according to the embodiments of this application;

[0026] Figure 4A The above describes the IHC detection results of tonsil tissue using the commercially available CD68 monoclonal antibody KP1 according to embodiments of this application. Figure 3A Enlarged view of part of the image;

[0027] Figure 4B The above describes the IHC detection results of tonsil tissue using the anti-CD68 antibody proposed in this embodiment, according to an example of this application. Figure 3B A magnified view of a portion of the image. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0029] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0030] Antibody

[0031] In this article, the term "antibody" refers to an immunoglobulin molecule capable of binding to a specific antigen. It consists of two lighter light chains (L chains) and two heavier heavy chains (H chains), linked by disulfide bonds to form a tetrapeptide chain. The amino acid sequence at the N-terminus of the peptide chain varies considerably and is called the variable region (V region), while the carboxyl terminus (C-terminus) is relatively stable and changes very little, and is called the constant region (C region). The V regions of the L and H chains are referred to as VL and VH, respectively.

[0032] Within the variable region, certain areas exhibit a higher degree of variation in amino acid composition and sequence, known as hypervariable regions (HVRs). These hypervariable regions are the sites where antigens and antibodies bind, and are therefore also called complementary determinant regions (CDRs). Both the heavy chain and light chain variable regions contain three CDRs.

[0033] The anti-CD68 antibody proposed in this application can specifically bind to CD68 and can be used to directly detect CD68, or to prepare CD68 detection reagents or kits, so as to achieve more accurate and sensitive CD68 detection, clinical diagnosis and treatment related to CD68 and prognosis.

[0034] In some embodiments, the anti-CD68 antibody or antigen-binding fragment has heavy chains CDR1-3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and light chains CDR1 as shown in SEQ ID NO: 4 and CDR3 as shown in SEQ ID NO: 5, wherein the light chain CDR2 sequence is LVS. In this document, "antigen-binding fragment" refers to an amino acid fragment with the ability to specifically bind to the CD68 antigen. In this document, anti-CD68 antibody may also be referred to as CD68 antibody, both referring to immunoglobulin molecules capable of binding to the CD68 antigen.

[0035] Heavy chain CDR1: GFTFSSFG (SEQ ID NO: 1)

[0036] Heavy chain CDR2: ISGGSSNI (SEQ ID NO: 2)

[0037] Heavy chain CDR3: ARSWPPDY (SEQ ID NO: 3)

[0038] Light chain CDR1: QSLLDSEGKTY (SEQ ID NO: 4)

[0039] Light Chain CDR2: LVS

[0040] Light chain CDR3: WQGTHFPQT (SEQ ID NO: 5)

[0041] In some embodiments, the anti-CD68 antibody or antigen-binding fragment has heavy chain framework regions (FRs) 1-4 as shown in SEQ ID NO: 6-9, respectively.

[0042] Heavy chain FR1: EVKLVESGGGLVQPGGSRKLSCAAS (SEQ ID NO: 6);

[0043] Heavy chain FR2: MHWVRQAPEKGLEWVAY (SEQ ID NO: 7);

[0044] Heavy chain FR3: YYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYC (SEQ ID NO: 8)

[0045] Heavy chain FR4: WGQGTTLTVSS (SEQ ID NO: 9).

[0046] In some embodiments, the anti-CD68 antibody or antigen-binding fragment has light chains FR1-4 as shown in SEQ ID NO: 10-13 respectively:

[0047] Light chain FR1: DIVMTQSPLTLTVTIGQPASISCKSS (SEQ ID NO: 10);

[0048] Light chain FR2: LSWLLQRPGQSPKRLIY (SEQ ID NO: 11);

[0049] Light chain FR3: KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC (SEQ ID NO: 12);

[0050] Light chain FR4: FGGGTKLEIK (SEQ ID NO: 13).

[0051] In some embodiments, the anti-CD68 antibody or antigen-binding fragment has a heavy chain variable region as shown in SEQ ID NO: 14 and a light chain variable region as shown in SEQ ID NO: 15. In other embodiments, the heavy chain variable region sequence of the antibody or antigen-binding fragment has one or more conserved amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 14, for example, one, two, three, four, five, six, seven, eight, nine, ten, or more conserved amino acid substitutions. In some embodiments, the light chain variable region sequence of the antibody or antigen-binding fragment has one or more conserved amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 15, for example, one, two, three, four, five, six, seven, eight, nine, ten, or more conserved amino acid substitutions. These conserved amino acid substitutions do not alter the biological function of the antibody or antigen-binding fragment. In some specific embodiments, these conserved amino acid substitutions can occur on amino acids in the heavy chain and light chain variable regions other than the CDR region.

[0052] Heavy chain variable region amino acid sequence (SEQ ID NO: 14)

[0053] EVKLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISGGSSN IYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARSWPPDYWGQGTTLTVSS (SEQ ID NO: 14).

[0054] Light chain variable region amino acid sequence (SEQ ID NO: 15)

[0055] DIVMTQSPLTLTVTIGQPASISCKSSQSLLDSEGKTYLSWLLQRPGQSPKRLIYLVSKLD SGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK (SEQ ID NO: 15).

[0056] In this document, "conservative amino acid substitution" refers to the substitution of an amino acid by another amino acid with biologically, chemically, or structurally similar residues. Biological similarity means that the substitution does not impair the biological activity of the CD68 antibody or its specific binding to the CD68 antigen. Structural similarity means that the amino acids have side chains of similar length, such as alanine, glycine, or serine, or side chains of similar size. Chemical similarity means that the amino acids have the same charge or are both hydrophilic or hydrophobic. For example, hydrophobic residues such as isoleucine, valine, leucine, or methionine can be substituted for each other. Alternatively, polar amino acids can be used, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, serine replacing threonine, etc. This application is intended to cover antibody or antigen-binding fragments containing these conserved amino acid substitutions.

[0057] In some embodiments, the anti-CD68 antibody or antigen-binding fragment has an amino acid sequence that is at least 70% identical to SEQ ID NO: 14 and / or SEQ ID NO: 15, preferably at least 80%, at least 90%, or at least 95% identical. For example, sequences having sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% may be included, but the percentage is not limited thereto. In the embodiments of this application, the inconsistent amino acids contained in the CD68 antibody or antigen-binding fragment having the same identity as SEQ ID NO: 14 and / or SEQ ID NO: 15 may occur on amino acid sequences other than the CDR region in the heavy chain variable region and the light chain variable region. The difference between it and SEQ ID NO: 14 and / or SEQ ID NO: 15 will not change the biological function of the antibody or antigen-binding fragment, and it still has the activity of specifically binding to CD68.

[0058] In this embodiment, the anti-CD68 antibody or antigen-binding fragment further comprises a constant region, which includes a heavy chain constant region and / or a light chain constant region. The constant region can be derived from various species, such as mice, rats, rabbits, and guinea pigs, but is not limited to these species. In some embodiments, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the light chain constant region includes a light chain constant region selected from κ-type or λ-type. It is understood that, based on specific antibody preparation requirements, those skilled in the art will be able to select suitable constant region sequences for the variable regions described in any of the above embodiments, and these constant region sequences and methods for preparing antibodies containing such constant regions are well known in the art.

[0059] In this application, the anti-CD68 antibody or antigen-binding fragment can be derived from various species, such as mice, rats, rabbits, and guinea pigs, but is not limited to these species. The antibody described herein can be a monoclonal antibody, i.e., an antibody molecule having a single molecular component. Systems and immunological manipulations and techniques for preparing and isolating hybridomas secreting monoclonal antibodies are known in the art. In some embodiments, the anti-CD68 antibody or antigen-binding fragment is derived from mice, such as mouse hybridoma cells.

[0060] In some embodiments, the anti-CD68 antibody or antigen-binding fragment may be of the IgG type, which can specifically recognize cells containing human CD68 protein, thereby being suitable for immunological detection.

[0061] In this embodiment, the anti-CD68 antibody may further include a chimeric antibody having an antibody constant region (preferably) from one species and an antigen-binding site from another species. Furthermore, the antibody may include an antibody having an antigen-binding site from a non-human species and having an original constant region and a framework region.

[0062] In the embodiments of this application, "antigen-binding fragment" refers to an antibody fragment that retains the ability to bind specific antigens, and refers to one or more fragments of an antibody, including, for example: (i) a Fab fragment, a monovalent fragment composed of VL, VH, CL, and CH domains; (ii) an F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by disulfide bonds in a hinge region; (iii) an Fd fragment composed of VH and CH domains; (iv) an Fv fragment composed of the VL and VH domains of an antibody single arm; (v) a single-domain antibody fragment composed of a VH domain (also called a nanobody or VHH antibody); (vi) a separate complementarity-determining region (CDR); and (vii) a binding fragment comprising a combination of two or more separate CDRs, said two or more separate CDRs optionally linked by a synthetic linker. There are also single-chain antibodies (or single-chain Fv(scFv)) formed using recombinant methods to link the two domains (VL and VH) of an FV segment via a synthetic linker.

[0063] In one exemplary embodiment of this application, the antigen-binding fragment may be any one of the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 and diabody, but is not limited thereto.

[0064] Polynucleotides, expression vectors, recombinant cells

[0065] In the process of preparing or obtaining these antibodies or antigen-binding fragments, the corresponding antibodies can be obtained by using polynucleotides that express these antibody or antigen-binding fragments and linking them to different vectors.

[0066] Therefore, embodiments of this application also propose a polynucleotide that encodes an anti-CD68 antibody or antigen-binding fragment as described in any embodiment of this application.

[0067] In some embodiments, the polynucleotide sequence encoding the heavy chain variable region may be as shown in SEQ ID NO: 16:

[0068] gaggtgaagctggtggagtctgggggaggcttagtgcagcctggagggtcccggaaactctcctgtgcagcctctggattcactttcagtagctttggaatgcactgggttcgtcaggctccagagaaggggctggagtgggtcgcatacattagtggtggcagtagtaacatctactatgcagacacagtgaagggccgattcaccatctccagagacaatcccaagaacaccctgttcctgcaaatgaccagtctaaggtctgaggacacggccatgtattactgtgcaagatcatggccccctgactactggggccaaggcaccactctcacagtctcctcag(SEQ ID NO:16)

[0069] In some embodiments, the polynucleotide sequence encoding the light chain variable region may be as shown in SEQ ID NO: 17:

[0070] gacattgtgatgacccagtctccactcactttgacggtcaccattggacaaccagcctccatctcttgcaagtcaagtcagagtctcttagatagtgaaggaaagacatatttgagttggttgttacagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggtacacattttcctcagacgttcggtggaggcaccaagctggaaatcaaac(SEQ ID NO:17)

[0071] In some embodiments, the polynucleotide has at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 16, preferably at least 95%, and more preferably at least 98% or 99%. In at least some embodiments, the polynucleotide has at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 17, preferably at least 95%, and more preferably at least 98% or 99%. These sequences, which are identical to the nucleotide sequences shown in SEQ ID NO: 16 or SEQ ID NO: 17, can express amino acid sequences similar to those in SEQ ID NO: 16 and SEQ ID NO: 17, thereby enabling them to specifically bind to the CD68 antigen and achieve the targeting function of the antibody.

[0072] This invention also provides an expression vector comprising any of the aforementioned polynucleotides. When ligating the polynucleotides to the vector, the polynucleotides 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 polynucleotides. These control elements can be directly derived from the vector itself or be exogenous, i.e., not derived from the vector itself. Of course, the polynucleotides and control elements need to be operatively linked. In this context, "operatively linked" means ligating a foreign gene to the vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Of course, the polynucleotides used to encode the antibody heavy and light chains can be inserted independently into different vectors, but commonly they are inserted into the same vector. Commonly used vectors include plasmids, bacteriophages, lentiviruses, etc.

[0073] This invention also provides a recombinant cell containing the expression vector. The expression vector can be introduced into mammalian cells to construct recombinant cells, which can then be used to express the antibody or antigen-binding fragment provided by this invention. The corresponding antibody can be obtained by culturing these recombinant cells. These usable mammalian cells can be, for example, 293F cells, CHO cells, etc.

[0074] Antibody conjugates

[0075] The anti-CD68 antibody or antigen-binding fragment proposed in this application embodiment can also be used to achieve antigen detection and tracing in the form of antibody conjugates.

[0076] In some embodiments, the anti-CD68 antibody conjugate is prepared from an anti-CD68 antibody or antigen-binding fragment as described in any embodiment of this application, along with an antigen and / or a labeling molecule. In some embodiments, the labeling molecule is a fluorescent labeling molecule (such as fluorescein), an enzyme, a metal ion, or an isotope.

[0077] Reagent test kit

[0078] This application also proposes a kit comprising an anti-CD68 antibody or antigen-binding fragment as described in any embodiment of this application, a polynucleotide encoding the anti-CD68 antibody or antigen-binding fragment, an expression vector containing the polynucleotide, recombinant cells containing the expression vector, and / or an anti-CD68 antibody conjugate.

[0079] In some embodiments, the kit may be an IHC kit, an ELISA kit, an IF kit, or a Western Blot kit.

[0080] It is understood that the kit may also contain other components required for detection, which are well known in the art, and this application is not intended to limit this.

[0081] application

[0082] The embodiments of this application also propose the use of anti-CD68 antibodies or antigen-binding fragments or kits as described in any embodiment of this application in the detection of CD68 protein and / or the preparation of reagents or reagent kits for the detection of CD68 protein.

[0083] In some embodiments, the detection is an immunoassay. In some embodiments, the immunoassay can be one or more of immunohistochemistry (IHC), Western blotting, immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA).

[0084] In some embodiments, the reagents or reagent kits are used for the diagnostic detection of CD68 antigen, so as to perform efficient and accurate detection of CD68 biomarkers and to determine the clinical diagnosis and prognosis related to CD68. For example, CD68 is used as a tumor diagnostic marker to detect the relevant status of tumor-related cells (e.g., TAM) to determine tumor growth and cancer progression.

[0085] In some embodiments, when CD68 is used as a tumor diagnostic marker, the anti-CD68 antibody or antigen-binding fragment can be used to detect one or more of the following tumors: myeloma, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, etc.

[0086] preparation

[0087] This application also provides methods for preparing anti-CD68 antibodies or antigen-binding fragments as described in any embodiment of this application.

[0088] In some embodiments, the method can be achieved by introducing the coding sequence of an anti-CD68 antibody or antigen-binding fragment into an expression vector, transfecting the expression vector into host cells (i.e., recombinant cells), and obtaining the anti-CD68 antibody or antigen-binding fragment from the culture supernatant of the host cells. In some embodiments, the method may further include a purification step.

[0089] The present invention will be explained below 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 the invention. 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 field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0090] Example 1: Screening of positive hybridoma cells after fusion

[0091] The full-length extracellular sequence of CD68 was selected as the antigen (Sinochem, catalog number: 11192-H08B1) and expressed in eukaryotic cells using HEK293 cells. The resulting recombinant protein was used as an immunogen to immunize BALB / c female mice. After four rounds of immunization, with one immunization occurring every 7 days, the dose was 100 μg / mouse. The antiserum titer was detected using an antigen-coated enzyme-linked immunosorbent assay (ELISA). The serum titer of the immunized mice reached 1 million.

[0092] Mice were intraperitoneally injected with an immunogen for intraperitoneal stimulation. Three days after injection, the spleens of the mice were harvested and fused with SP2 / 0 myeloma cells selected from 8-AG using PEG as a fusion agent. The fused cells were then transferred to selective HAT medium (1×HAT) for pressure selection the day after fusion. Seven days after pressure selection, the medium was replaced with 1640 basal medium containing 10% serum. Three days after the medium change, the antibody secretion in the supernatant of the selected live cells was detected by ELISA, yielding CD68-positive hybridoma cells.

[0093] Example 2: Obtaining and purifying CD68 monoclonal antibodies

[0094] 2.1 Preparation of CD68 monoclonal antibody

[0095] The positive hybridoma cells from Example 1 were subcloned using a limiting dilution method. The resulting subclones were detected using ELISA until a single cell colony was observed in each well and the supernatant tested positive. This cell line was then subjected to at least two more rounds of subcloning, ensuring a single cell colony in each well and a positive supernatant test in each round. The obtained subcloned monoclonal lines were selected as stable lines for expansion culture. The supernatant of the stable lines was detected using ELISA. Cell lines with strong antibody secretion capacity (high titer) in the supernatant were selected for passage and expansion culture. The antibody obtained in the supernatant is the CD68 monoclonal antibody.

[0096] 2.2 Purification of CD68 monoclonal antibody

[0097] The stable strains obtained from the above-mentioned expanded culture (with a viability of ≥90%) were domesticated using a one-step method and transferred to a medium containing CDHybridoma Medium (Gibco). TM , 11279023) were cultured in shake flasks for several days until the cells could grow stably in a serum-free culture system.

[0098] The serum-free acclimated cell slurry was collected, centrifuged, and the supernatant was added to a purification column packed with protein A packing material. The column was incubated at room temperature for 10-15 minutes and eluted with 0.1M citric acid to obtain anti-CD68 antibody with high purity.

[0099] 2.3 Detection of CD68 monoclonal antibody purification results

[0100] The purified anti-CD68 antibody was loaded using a 10% protein pregel with both reducing and non-reducing loading methods, and the results are as follows: Figure 1 As shown. By Figure 1 As can be seen, clear bands are visible at 25kDa and 50kDa in the reducing gel, with no other bands observed at other locations, indicating that the antibody purity meets the standard, approximately 90%. Meanwhile, in the non-reducing gel, the bands show that the total molecular weight of the anti-CD68 antibody is approximately 150kDa, consistent with the theoretical size of antibody molecules, proving the successful preparation and purification of the anti-CD68 antibody.

[0101] Example 3: Affinity determination of positive clonal antibodies

[0102] 3.1 The antibody obtained in Example 2 was named anti-CD68-1(9G8) and diluted to 10 μg / mL with 1*PBST;

[0103] 3.2 Dilute the antigen CD68-6his (commercial recombinant protein, source: Sinocare, catalog number: 11192-H08B1) to 500 nM (initial concentration), and then perform 3-fold serial dilutions for a total of 7 dilutions;

[0104] 3.3 Using the K module of BLI (source: Gator-Little Crocodile Biotechnology) and an mFC probe, the KD value was determined. The results are as follows: Figure 2 As shown, koff(1 / s) = 1.02E-003; kon(1 / Ms) = 8.61E+004; KD(M) = koff / kon = 1.18E-008.

[0105] like Figure 2 As shown, the affinity of the anti-CD68-1 (9G8) proposed in this embodiment can reach 1.18E-008M, indicating that the anti-CD68 antibody of this embodiment has extremely high binding specificity to CD68 molecules and can be used in many aspects such as immunohistochemistry.

[0106] Example 4: Sequence characterization of CD68 monoclonal antibody

[0107] This embodiment sequenced the CD68 monoclonal antibody anti-CD68-1 (9G8) proposed in Examples 2 and 3 to characterize its sequence. The specific steps are as follows:

[0108] 4.1 RNA extraction: Total RNA was extracted from serum-free monoclonal cells expressing anti-CD68-1 (9G8) using a kit (Promega: Z3101).

[0109] 4.2 Reverse transcription: The extracted total RNA was reverse transcribed. The reverse transcription system is shown in Table 1. The reverse transcription program was 50℃ for 1 h, 75℃ for 15 min, and 4℃ for ∞.

[0110] Table 1

[0111] reagents Volume (μl) 5×first strand buffer 2 10mM dNTP mix 1 100mM DTT 0.5 5M Betaine 2 1M MgCl2 0.06 100μM TSO 0.1 10μM oligo-dT-30vn (primer) 1 40 U / μl RNase inhibitor 0.25 total RNA 10-100ng 1 mg / mL MMLV reverse transcriptase 0.5 Nuclease-free Water Add to 10

[0112] The oligo-dT-30vn (primer) sequence is: 5'-AAGCAGTGGTATCAACGCAGAGTACT30VN-3' (SEQ ID NO: 18)

[0113] 4.3 Amplification of the variable region: Using the cDNA obtained in 4.2 as a template, the variable region was amplified using the primers in Table 2. The reaction system is shown in Table 3. The reaction program is: 98℃ for 2 min; (98℃ for 10 s, 57℃ for 15 s, 72℃ for 30 s), 28 cycles; 72℃ for 2 min.

[0114] Table 2

[0115]

[0116]

[0117] Table 3

[0118] Components Volume (μl) Template cDNA (30 ng) The injection volume is determined based on the specific cDNA concentration. F-primer (10μM) 0.8 R-primer (10μM) 0.8 Q5 HotStart 2x Master Mix 20 Nuclease-free Water Add to 40

[0119] 4.4 PCR Product Recovery: The PCR products from step 4.3 were electrophoresed on a 1.5% agarose gel. The bands located at 380-400 bp were selected and the products were excised and recovered from the gel (gel recovery kit: EZNA). Gel Extraction Kit (D2500-01), the concentration of the gel recovery product should be ≥20ng / μl, and the OD260 / 280 should be 1.8-2.0.

[0120] 4.5 TA Cloning: Using the TA cloning kit (pMD) TM The recovered PCR products were ligated to the T vector using the 19-T Vector Cloning Kit (Code No. 6013, TAKARA) according to the kit instructions.

[0121] 4.6 Sanger Sequencing: Single clones confirmed as positive after blue-white screening were sent for sequencing. The sequencing primers used were:

[0122] F-primer: GAGCGGATAACAATTTCACACAGG (SEQ ID NO: 44);

[0123] R-primer: CGCCAGGGTTTTCCCAGTCACGAC (SEQ ID NO: 45).

[0124] Sequencing results showed that the heavy chain variable region and light chain variable region sequences of anti-CD68-1(9G8) are as follows:

[0125] EVKLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISGGSSN IYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARSWPPDYWGQGTTLTVSS (heavy chain variable region, SEQ ID NO: 14)

[0126] DIVMTQSPLTLTVTIGQPASISCKSSQSLLDSEGKTYLSWLLQRPGQSPKRLIYLVSKLD SGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK (light chain variable region, SEQ ID NO: 15)

[0127] Example 5: IHC using CD68 monoclonal antibody

[0128] This embodiment uses the anti-CD68 antibody anti-CD68-1 (9G8) characterized in Example 4 to perform IHC on human normal tissue sample sections, and uses the commercially available CD68 monoclonal antibody KP1 (Cat: 916104, Biolegend) as a control to investigate the binding of the anti-CD68 antibody proposed in this embodiment to CD68 in immunohistochemistry. The tissue samples used include colon, tonsils, lung, myocardium, brain, thyroid gland, spleen, pancreas, skeletal muscle, liver, testis, kidney, placenta, mammary gland, esophagus, endometrium, stomach, and skin. The specific experimental procedure is as follows:

[0129] 5.1 Baking slides: Take glass slides containing paraffin sections of various normal human tissue samples and place them in a 68°C constant temperature oven for about 90 minutes.

[0130] 5.2 Dewaxing and dehydration: After the glass slides are baked, they are placed in a dewaxing tank and soaked in xylene three times for 10 minutes each time. Then they are soaked in 100% alcohol for 5 minutes, 95% alcohol for 5 minutes, 85% alcohol for 5 minutes, and 75% alcohol for 5 minutes. Finally, they are rinsed with water three times for 3 minutes each time.

[0131] 5.3 Antigen retrieval: Slowly place the slide into 0.01M citrate buffer (pH 6.0) and heat for 15 min. Allow the reaction system to cool naturally at room temperature. Once cooled to room temperature, remove the slide and rinse it three times with PBS buffer for 5 min each time.

[0132] 5.4 Blocking and inactivating endogenous peroxidase: Add 3% H2O2 to the slide specimen. After completion, slowly rinse the slide three times with PBS buffer, 5 min each time.

[0133] 5.5 Serum blocking: Gently shake off the water on the slide, place it on an incubation box, add normal sheep serum working solution, incubate at room temperature for 10 minutes, and then shake off the water.

[0134] 5.6 Primary antibody incubation: Add 5 μg / ml of the anti-CD68 antibody from Example 1 to a glass slide and incubate overnight at 4°C.

[0135] 5.7 Secondary antibody incubation: The next day, remove the slides, bring them to room temperature, and rinse them three times with PBS buffer for 5 minutes each time. Then, add HRP-labeled goat anti-rabbit secondary antibody working solution to the slides, incubate at room temperature for 10 minutes, remove the slides, and rinse them three times with PBS buffer for 5 minutes each time.

[0136] 5.8 DAB / H2O2 reaction staining: Add freshly prepared DAB staining solution to the slide.

[0137] 5.9 Counterstain with hematoxylin for 2 min, rinse slowly in water for 5 min, allow tissue differentiation for 1 s, rinse with water for 2 min, return to blue with PBS, rinse with water for 2 min, then dehydrate twice in 95% ethanol and anhydrous ethanol, each time for 5 min, and then remove.

[0138] 5.10 Immerse the slide in xylene solution twice, 10 minutes each time, and then seal it with neutral resin.

[0139] 5.11 Observe the staining of the specimen sections under a microscope. The results are as follows: Figures 3A-3B As shown.

[0140] Figure 3A The results of IHC detection using the commercially available CD68 monoclonal antibody KP1 are shown. Figure 3B The results of IHC detection using the anti-CD68 antibody presented in this embodiment are shown. Figure 3A and Figure 3B As can be seen, the staining profile of the antibody proposed in this embodiment is basically the same as that of the positive control, indicating that the antibody proposed in this embodiment can specifically recognize the CD68 target protein, and therefore can be used in various applications targeting CD68, such as tumor marker screening through immune detection. Furthermore, through comparison... Figure 3A and Figure 3B It can be observed that the anti-CD68 antibody proposed in this embodiment exhibits lower non-specific binding, thereby providing extremely low background interference and thus improving the detection accuracy and sensitivity of CD68. This is evident from... Figure 4A and Figure 4B The comparison also shows that: Figure 4A and Figure 4B They are respectively Figure 3A and Figure 3B A magnified image of the staining results of a section of middle tonsil tissue, in which... Figure 4A The results of treatment with the commercially available CD68 monoclonal antibody KP1 are shown. Figure 4B The processing results of anti-CD68-1(9G8) in this embodiment are shown. (By...) Figure 4A and Figure 4B As can be seen, compared with KP1, the anti-CD68-1 (9G8) proposed in this application not only has obvious specific staining, but also shows extremely high binding specificity and extremely low reaction background, thereby effectively improving the detection accuracy of CD68 antigen.

[0141] 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.

[0142] 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. An anti-CD68 antibody or antigen-binding fragment, characterized in that, The anti-CD68 antibody or antigen-binding fragment includes: It has a heavy chain complementarity determination region 1 as shown in SEQ ID NO: 1; It has a heavy chain complementarity determination region 2 as shown in SEQ ID NO: 2; It has a heavy chain complementarity determination region 3 as shown in SEQ ID NO: 3; It has a light chain complementarity determination region 1 as shown in SEQ ID NO: 4; Light chain complementarity determination region 2 with LVS; and It has the light chain complementarity determination region 3 shown in SEQ ID NO:

5.

2. The anti-CD68 antibody or antigen-binding fragment according to claim 1, characterized in that, Includes at least one of the following: a) Having a heavy chain variable region as shown in SEQ ID NO: 14 and / or a light chain variable region as shown in SEQ ID NO: 15; and b) Having an amino acid sequence with at least 85% identity compared to a), the CD68 antibody or antigen-binding fragment is capable of specifically binding to CD68.

3. The anti-CD68 antibody or antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or antigen-binding fragment contains Fab. Optionally, the antigen-binding fragment is selected from any one of the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, VHH and diabody.

4. A polynucleotide encoding an anti-CD68 antibody or antigen-binding fragment as described in any one of claims 1 to 3.

5. An expression vector comprising the polynucleotide as described in claim 4.

6. A recombinant cell comprising the polynucleotide of claim 4 or the expression vector of claim 5.

7. An anti-CD68 antibody conjugate, characterized in that, The conjugate is prepared from an anti-CD68 antibody or antigen-binding fragment as described in any one of claims 1 to 3, and an antigen and / or a labeled molecule. Optionally, the labeling molecule is a fluorescent labeling molecule, an enzyme, a metal ion, or an isotope.

8. A kit comprising an anti-CD68 antibody or antigen-binding fragment as described in any one of claims 1 to 3, a polynucleotide as described in claim 4, an expression vector as described in claim 5, recombinant cells as described in claim 6, and / or an anti-CD68 antibody conjugate as described in claim 7.

9. The kit according to claim 8, wherein the kit is an IHC kit, an ELISA kit, an IF kit, or a Western Blot kit.

10. The use of the anti-CD68 antibody or antigen-binding fragment according to any one of claims 1 to 3, or the kit according to claim 9 or 10, in any one or more of the following: a. Detection of CD68 protein; b. Prepare reagents or reagent kits for detecting CD68 protein; Optionally, the detection is an immunoassay, which may be one or more of IHC, Western Blot, IF, and ELISA. Optionally, the reagent or reagent kit is used for the diagnostic detection of CD68 antigen.

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