Antibodies targeting alb and uses thereof

Abstract

This invention discloses an antibody targeting ALB and its applications. The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively. The anti-ALB monoclonal antibody of this invention can be used to detect the expression level of ALB protein in cells in vitro, exhibiting good specificity, high antibody concentration and purity. It can also be used to analyze the expression level of ALB protein in proteins or tissues in vitro, demonstrating good stability.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an antibody targeting ALB and its applications. Background Technology

[0002] Albumin, also known as albumin, is synthesized by the human liver. A normal daily intake is approximately 12-20g, accounting for about 50% of the total protein in human blood plasma. It is the most important protein in human blood plasma and a fundamental physiological substance essential for maintaining the body's nutrition and osmotic pressure.

[0003] The albumin gene encodes the most abundant protein in human blood. Albumin plays a role in regulating plasma colloid osmotic pressure and serves as a carrier protein for a variety of endogenous molecules, including hormones, fatty acids, metabolites, and exogenous drugs. Furthermore, albumin exhibits esterase-like activity with broad substrate specificity. The encoded preprotein is proteolytically processed to produce the mature protein. The peptide EPI-X4, derived from this protein, is an endogenous inhibitor of the CXCR4 chemokine receptor. Currently, there are relatively few commercially available ALB antibodies, which cannot yet meet market demand. Summary of the Invention

[0004] The technical problem this invention aims to solve is to overcome the lack of stable ALB antibodies with high sensitivity and specificity in existing technologies, and to provide an antibody targeting ALB and its applications. The anti-ALB monoclonal antibody of this invention can be used to detect the expression level of ALB protein in cells in vitro, exhibiting good specificity, high antibody concentration and purity. It can also be used to analyze the expression level of ALB protein in proteins or tissues in vitro, and possesses good stability.

[0005] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0006] A first aspect of the present invention provides an antibody or antigen-binding fragment thereof targeting ALB, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising amino acid sequences HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively; the light chain variable region comprising amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 respectively.

[0007] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:4.

[0008] In some embodiments of the present invention, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:9.

[0009] In some embodiments of the present invention, the antibody is a full-length antibody, Fab, Fab', F(ab')2, scFv, Fv, or a multispecific antibody.

[0010] In this invention, when the antibody is a full-length antibody, the heavy chain constant region of the full-length antibody originates from the heavy chain of a human antibody or a variant thereof, and the light chain constant region of the full-length antibody originates from the κ chain or λ chain of a human antibody or a variant thereof.

[0011] In some specific embodiments of the present invention, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:25 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:25.

[0012] In some specific embodiments of the present invention, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:27 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:27.

[0013] A second aspect of the present invention provides an isolated nucleic acid that encodes an antibody or an antigen-binding fragment thereof as described in the first aspect.

[0014] In some specific embodiments of the present invention, the nucleotide sequence encoding HCDR1 is shown in SEQ ID NO:11, the nucleotide sequence encoding HCDR2 is shown in SEQ ID NO:12, and the nucleotide sequence encoding HCDR3 is shown in SEQ ID NO:13.

[0015] In some specific embodiments of the present invention, the nucleotide sequence encoding LCDR1 is shown in SEQ ID NO:16, the nucleotide sequence encoding LCDR2 is shown in SEQ ID NO:17, and the nucleotide sequence encoding LCDR3 is shown in SEQ ID NO:18.

[0016] In some embodiments of the present invention, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:14.

[0017] In some embodiments of the present invention, the nucleotide sequence encoding the variable region of the light chain is shown in SEQ ID NO:19.

[0018] A third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the second aspect.

[0019] In some embodiments of the present invention, the recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector.

[0020] In some specific embodiments of the present invention, the viral vector is a retroviral vector, a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector.

[0021] A fourth aspect of the present invention provides a cell comprising a recombinant expression vector as described in the third aspect or a nucleic acid as described in the second aspect; or expressing an antibody or an antigen-binding fragment thereof as described in the first aspect.

[0022] In some embodiments of the present invention, the cells are prokaryotic cells or eukaryotic cells.

[0023] In some preferred embodiments of the present invention, the cells are selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or their antigen-binding fragments; the mammalian cells are, for example, HEK293 cells.

[0024] A fifth aspect of the present invention provides a method for preparing an antibody or antigen-binding fragment thereof targeting ALB, the method comprising culturing cells as described in the fourth aspect and obtaining an antibody or antigen-binding fragment thereof targeting ALB from the culture.

[0025] A sixth aspect of the present invention provides a kit comprising an antibody or an antigen-binding fragment thereof as described in the first aspect.

[0026] In some embodiments of the present invention, the kit further includes reagents for detecting the binding of the antibody or its antigen-binding fragment to the antigen.

[0027] A seventh aspect of the present invention provides a method for detecting ALB, the method comprising the step of contacting an antibody or an antigen-binding fragment thereof as described in the first aspect with a sample to be tested.

[0028] In some embodiments of the present invention, the method is for non-diagnostic purposes, such as the development of antibody detection or immunological detection methods in the laboratory.

[0029] The eighth aspect of the present invention provides the use of an antibody or antigen-binding fragment thereof as described in the first aspect, a nucleic acid as described in the second aspect, a recombinant expression vector as described in the third aspect, or a cell as described in the fourth aspect in the preparation of an ALB detection reagent.

[0030] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0031] The reagents and raw materials used in this invention are all commercially available.

[0032] The positive and progressive effects of this invention are as follows:

[0033] The anti-ALB monoclonal antibody of the present invention has high sensitivity, good specificity, high antibody concentration and purity, and can be used for in vitro analysis of ALB protein expression levels in proteins or tissues. It has good stability and has broad application prospects. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the immunofluorescence staining effect of commercially available ALB antibody (1:200) (positive sample).

[0035] Figure 2 This is a schematic diagram of the ALB antibody immunofluorescence staining effect (1:200) (positive sample) in the example.

[0036] Figure 3 This is a schematic diagram of the ALB antibody immunofluorescence staining effect (1:200) (negative sample) in the example.

[0037] Figure 4 This is a flow cytometry image of ALB antibody (1:200) (positive sample) as an example.

[0038] Figure 5 This is a flow cytometry image of ALB antibody (1:200) (negative sample) as an example. Detailed Implementation

[0039] definition

[0040] As used herein, the term "complementarity-determining region" or "CDR region" or "CDR" refers to a region within the antibody variable domain that is sequence-hypervariant and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes and are sequentially numbered from the N-terminus as CDR1, CDR2, and CDR3. Within a given heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of many known antibody CDR assignment systems or combinations thereof. It is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., Journal of Molecular Biology, 273, 927-948 (1997) based on the antibody's three-dimensional structure and the topology of the CDR rings; Kabat et al. (1987) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); the international ImMunoGeneTics database (IMGT) (imgt.cines.fr / ); and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms “CDR” and “complementary determination region” for a given antibody or its region (e.g., variable region) should be understood to encompass the complementary determination region defined by any of the above-described known schemes as described in this invention.

[0041] Antibodies with different specificities (i.e., different binding sites against different antigens) have different core binding receptors (CDRs). However, although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR are directly involved in antigen binding. Minimal overlapping regions can be determined using at least two of the Kabat, Chothia, IMGT, AbM, and Contact methods, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat, Chothia, or AbM may be substituted with conserved amino acid residues.

[0042] As used herein, “percentage (%) sequence identity” or “sequence identity” has a generally accepted definition in the art, referring to the percentage of identical amino acid sequences between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a known algorithm). This can be determined using methods known to those skilled in the art, such as publicly available computer software like BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0043] In this invention, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0044] As used in this article, the term "isolated" refers to substances obtained artificially from their natural state. If an "isolated" substance or component appears in nature, it may be due to an alteration of its natural environment, the isolation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide isolated from this natural state is called "isolated." The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the substance's activity.

[0045] As used in this invention, "vector" refers to a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing said genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0046] As used in this article, the term "host cell" refers to cells that can be used to introduce the vector, including but not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, insect cells such as S2 Drosophila cells or Sf9 cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0047] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0048] Example 1

[0049] The amino acid sequences of the ALB antibodies screened in this embodiment are shown in Table 1. The CDR was defined using Kabat.

[0050] Table 1 Antibody Sequences

[0051]

[0052]

[0053] In the table, VH represents the variable region of the heavy chain, VL represents the variable region of the light chain, CH represents the constant region of the heavy chain, CL represents the constant region of the light chain; HC represents the heavy chain, and LC represents the light chain.

[0054] The signal peptide sequence of the heavy chain in the antibody obtained in this embodiment is as follows:

[0055] MEWPCIFLFLLSVTEGVHS(SEQ ID NO:21)

[0056] The nucleotide sequence encoding it is shown in SEQ ID NO:22.

[0057] The signal peptide sequence of the light chain is as follows:

[0058] MRAPAQIFGFLLLLFPGTRC(SEQ ID NO:23)

[0059] The nucleotide sequence encoding it is shown in SEQ ID NO:24.

[0060] Example 2: Immunofluorescence Application

[0061] This embodiment verifies the antibody's effect using immunofluorescence staining. The immunofluorescence procedure is as follows:

[0062] 1. Remove the fixed hiHep cells from the 24-well plate.[2] After discarding the supernatant, add 300 μl / well of 0.25% Triton X-100 and permeate for 15 min.

[0063] 2. After discarding the supernatant, add 300 μl / well of 3% BSA and seal for 45 min;

[0064] 3. After discarding the supernatant, add 300 μl / well of primary antibody (ALB antibody prepared in Example 1) (1:200) and incubate at room temperature for 2 h;

[0065] 4. After discarding the supernatant, add 500 μl / well of 1×PBS and wash 3 times;

[0066] 5. Add 300 μl / well of secondary antibody (Alexa) 488AffiniPureGoat Anti-Mouse IgG, 115-545-205, Jackson ImmunoResearch (1:400) Incubate at room temperature in the dark for 1 hour;

[0067] 6. After discarding the supernatant, add 500 μl / well of 1XPBS and wash 3 times;

[0068] 7. Add 300 μl / well of DAPI (1:3000) and incubate at room temperature in the dark for 3 min;

[0069] 8. After discarding the supernatant, add 500 μl / well of 1×PBS and wash 3 times. Then add 1 ml of 1×PBS and take a fluorescence photograph.

[0070] like Figure 1 and Figure 2 As shown, the ALB antibody in this embodiment ( Figure 2 When used at a ratio of 1:200, the same staining level as commercially available antibodies (Bethyl, catalog number A80-229A) can be achieved. Figure 1 This indicates that the antibody has higher purity and better sensitivity.

[0071] And as Figure 3 As shown, the ALB antibody in this embodiment showed no signal in the negative control cells, indicating that the antibody has good specificity.

[0072] Example 3: Flow Cytometry Application

[0073] This embodiment verifies the antibody's effect using immunofluorescence staining. The immunofluorescence procedure is as follows:

[0074] 1. Take 8E5 fixed hepG2 cells [1] or UCFT cells [2] Place in a 1.5 mL sterile centrifuge tube;

[0075] 2. Centrifuge and discard the supernatant. Resuspend the cells in 100 μL (PBS + 10% FBS), then add 900 μL of ice-cold methanol, mix well, and permeabilize on ice for 10 minutes.

[0076] 3. After permeabilization, divide the cells in the 1.5ml sterile centrifuge tube into 4 equal portions, centrifuge and discard the supernatant;

[0077] 4. Resuspend the cells in 100 μL (PBS + 10% FBS) for each sample tube, and add the corresponding volume of primary antibody for each sample tube according to the table below;

[0078]

[0079] 5. After mixing the sample with the antibody, incubate at room temperature for 30 minutes;

[0080] 6. After incubation, add 1 mL of (PBS + 2% FBS) to each tube for washing, centrifuge at 6000 rpm for 5 min, remove the supernatant, and repeat the washing process twice.

[0081] 7. After washing, add 100 μL of (PBS + 10% FBS) to each tube to resuspend the cells;

[0082] 8. According to the table above, add the corresponding volume of secondary antibody to each sample tube; after mixing, incubate at room temperature in the dark for 30 minutes;

[0083] 9. After incubation, add 1 mL of (PBS + 2% FBS) to each tube for washing, centrifuge at 6000 rpm for 5 min, remove the supernatant, and repeat the washing process twice.

[0084] 10. After washing, resuspend the cells in 100 μL of PBS for flow cytometry detection and data analysis.

[0085] like Figure 4 As shown, the ALB antibody in this embodiment achieves a good staining effect when used in flow cytometry at a ratio of 1:200, indicating that the antibody has higher purity and better sensitivity.

[0086] And as Figure 5 As shown, the ALB antibody in this embodiment was tested in negative control cells (UCFT cells). [1] The absence of signal indicates good antibody specificity.

[0087] Example 4: Antibody stability test

[0088] The sample used in this embodiment is HepG2 cells. [2] Using ALB antibodies preserved under different conditions, the ALB positivity rate of HepG2 cells was detected by flow cytometry.

[0089] The positive rate of ALB in the samples was detected after storing the antibody in 0.02% ProClin 300 for different times at -20℃, 2~8℃ and 37℃ respectively. The results are shown in Table 2.

[0090] The ALB antibody of this invention maintains good detection sensitivity and stability under storage conditions of -20℃, 2–8℃, and 37℃. Current detection results have been updated to include antibody storage after 60 days. Experimental results show that after 30 days of storage, the ALB positivity rate is not significantly different from the baseline value; after 60 days of storage, the detection results under the three storage conditions are not significantly different, but all show a certain degree of decrease compared to the baseline value. These data indicate that the ALB antibody can be stably stored at 37.0℃ for at least 30 days, which is a significant advantage compared to commercially available antibodies (commercially available antibodies are generally stored at 2–8℃ for 14 days).

[0091] Table 2 Antibody Storage Stability Experiment

[0092]

[0093] Three independent detection experiments were performed on each of the three independent samples (hepG2 and UCFT = 1:1.5 mixed cells). The results are shown in Table 3. The CV (CV% = SD / Average * 100%) of the three detection results were 0.03%, 0.02% and 0.01%, respectively. The small difference coefficients among the independent detections indicate that the antibody has high detection stability.

[0094] Table 3 Antibody stability test

[0095] Sample 1 Sample 2 Sample 3 First test 32.5％ 35.4％ 36.6％ Second test 31.5％ 35.8％ 36.6％ Third test 30.7％ 34.6％ 36.0％ CV 0.03％ 0.02％ 0.01％

[0096] References

[0097] 1. Huang P, Zhang L, Gao Y, He Z, Yao D, Wu Z, et al. Direct reprogramming of human fibroblasts to functional and expandable hepatocytes. Cell stem cell2014;14:370-84.

[0098] 2. Pullinger CR, North JD, Teng BB, et al. The apolipoprotein B gene is constitutively expressed in HepG2 cells: regulation of secretion by oleicacid, albumin, and insulin, and measurement of the mRNA half-life[J]. Journal of Lipid Research, 1989, 30(7): 1065-1077.

[0099] 3. Huang Bo, Zou Guolin, Yang Tianming. Study on the binding effect of doxorubicin with bovine serum albumin [J]. Acta Chimica Sinica, 2002, 60(010):1867-1871.

[0100] 4.Rothschild MA, Oratz M, Schreiber SS. Serum albumin. Am J Dig Dis. 1969Oct; 14(10):711-44.

Claims

1. An antibody or antigen-binding fragment thereof targeting ALB, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. The light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 4; the amino acid sequence of the light chain variable region is an amino acid sequence with at least 80% sequence identity to SEQ ID NO:

9.

3. The antibody or its antigen-binding fragment as described in claim 2, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

9.

4. The antibody or its antigen-binding fragment as described in claim 2, characterized in that, The antibody is a full-length antibody, Fab, Fab', F(ab')2, scFv, or Fv.

5. The antibody or its antigen-binding fragment as described in claim 4, characterized in that, When the antibody is a full-length antibody, the heavy chain constant region of the full-length antibody originates from the heavy chain of the human antibody, and the light chain constant region of the full-length antibody originates from the κ chain or λ chain of the human antibody.

6. The antibody or its antigen-binding fragment as described in claim 5, characterized in that, The amino acid sequence of the heavy chain constant region is an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 25; the amino acid sequence of the light chain constant region is an amino acid sequence with at least 80% sequence identity to SEQ ID NO:

27.

7. The antibody or its antigen-binding fragment as described in claim 6, characterized in that, The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 25, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

27.

8. An isolated nucleic acid, characterized in that, The nucleic acid encodes the antibody or its antigen-binding fragment as described in any one of claims 1-7.

9. The nucleic acid as described in claim 8, characterized in that, The nucleotide sequence encoding HCDR1 is shown in SEQ ID NO: 11, the nucleotide sequence encoding HCDR2 is shown in SEQ ID NO: 12, and the nucleotide sequence encoding HCDR3 is shown in SEQ ID NO: 13; the nucleotide sequence encoding LCDR1 is shown in SEQ ID NO: 16, the nucleotide sequence encoding LCDR2 is shown in SEQ ID NO: 17, and the nucleotide sequence encoding LCDR3 is shown in SEQ ID NO:

18.

10. The nucleic acid as described in claim 9, characterized in that, The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO: 14; the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:

19.

11. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in any one of claims 8-10.

12. A cell, characterized in that, The cells contain the recombinant expression vector as described in claim 11 or the nucleic acid as described in any one of claims 8-10; or express the antibody or its antigen-binding fragment as described in any one of claims 1-7.

13. A method for preparing an antibody or antigen-binding fragment targeting ALB, characterized in that, The method includes culturing the cells as described in claim 12 and obtaining an antibody or antigen-binding fragment thereof targeting ALB from the culture.

14. A reagent kit, characterized in that, The kit comprises an antibody or an antigen-binding fragment thereof as described in any one of claims 1-7.

15. The kit according to claim 14, characterized in that, The kit also includes reagents for detecting the binding of the antibody or its antigen-binding fragment to the antigen.

16. The use of an antibody or antigen-binding fragment thereof as described in any one of claims 1-7, a nucleic acid as described in any one of claims 8-10, a recombinant expression vector as described in claim 11, or a cell as described in claim 12 in the preparation of an ALB detection reagent.

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