Anti-human ALK recombinant antibody and application thereof

Abstract

The invention provides an anti-human ALK recombinant antibody and application thereof, and belongs to the technical field of monoclonal antibodies, the anti-human ALK recombinant antibody comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NO: 1-3 in sequence, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NO: 4-6 in sequence. The anti-human ALK recombinant antibody provided by the invention has high sensitivity and specificity, can provide a reliable detection means in the field of ALK immunodiagnosis, and provides a basis for precise medication treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibodies, and specifically relates to a recombinant antibody against human ALK and its application. Background Art

[0002] About 80% of lung cancers are non-small cell lung cancer (NSCLC). Although the incidence of ALK (anaplastic lymphoma kinase) gene fusion in NSCLC is relatively low, about 5-6%, ALK inhibitors targeting ALK gene mutations have been developed. Currently, three generations of ALK targeted drugs (crizotinib, ceritinib, alectinib) have been used in clinical treatment and have shown very good clinical efficacy. Patients with ALK rearrangement-positive drug resistance and side effects are less likely to have better overall treatment effects. The ALK target is also known as the "diamond target" in the field of NSCLC, so detection of ALK-positive mutations is very necessary.

[0003] However, the choice of diagnostic method for ALK rearrangement detection in clinical practice is still a controversial issue. Our country has approved ALK gene detection companion diagnostic kits of four technical platforms, namely IHC, FISH, RT-PCR, and NGS detection products. Therefore, in unclear cases, at least 2 of the 4 methods should be used to confirm ALK rearrangement. The development of highly sensitive ALK diagnostic antibodies provides an opportunity for standardized immunohistochemistry (IHC) methods to detect ALK-driven tumors. Moreover, compared with FISH and RT-PCR, one of the main advantages of IHC is the direct detection of ALK protein, which is the direct target of ALK inhibitors. IHC has low cost, short turnaround time, and is easy to operate. Therefore, in the 2019 "Expert Consensus on Clinical Practice of ALK Detection in Non-Small Cell Lung Cancer in China", it is strongly recommended to give priority to IHC testing.

[0004] Currently, the ALK antibody products used for immunohistochemical pathological diagnosis sold on the market are mainly monopolized by expensive imported products. The accuracy of Cell Signaling Technology's D5F3 antibody and Abcam's 5A4 antibody are 100% and 95%-99% respectively. Roche VENTANA launched the ALK (D5F3) CDx kit using the D5F3 antibody and optimizing the immunohistochemical detection system. It was approved by CFDA and FDA earlier as a companion diagnostic reagent for targeted drugs crizotinib and ceritinib. my country's research in the field of ALK immunodiagnosis is seriously lagging behind, and domestic antibodies cannot meet the needs of NSCLC patients. Therefore, the development of a strain of ALK antibody with higher sensitivity and specificity can provide a more reliable detection method for many patients and provide a basis for precise drug treatment, which has far-reaching and positive significance. Summary of the invention

[0005] The first object of the present invention is to provide a recombinant antibody against human ALK, which can achieve accurate detection of ALK positive mutations.

[0006] The second object of the present invention is to provide the application of recombinant antibodies against human ALK to achieve accurate detection of ALK-driven tumors based on immunohistochemistry methods.

[0007] To achieve the above first purpose, the technical solution adopted by the present invention is:

[0008] A recombinant antibody against human ALK, comprising VHCDR1, VHCDR2 and VHCDR3 whose amino acid sequences are sequentially shown in SEQ ID NOs: 1 to 3, and VLCDR1, VLCDR2 and VLCDR3 whose amino acid sequences are sequentially shown in SEQ ID NOs: 4 to 6.

[0009] The beneficial effect of the above technical solution is that the present invention provides a recombinant antibody against human ALK, which has good specificity and affinity and can be used as a substitute for Abcam's 5A4 antibody. At the same time, the immunogen design strategy developed by the present invention for the development of monoclonal antibodies against transmembrane proteins can provide a reference for the design of similar transmembrane region proteins.

[0010] Preferably, the antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:7 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:8.

[0011] To achieve the above second purpose, the technical solution adopted by the present invention is:

[0012] Use of recombinant anti-human ALK antibodies in the preparation of ALK in vitro immunoassay reagents or kits.

[0013] The beneficial effect of the above technical solution is that the recombinant antibody of the present invention realizes efficient and accurate detection of ALK protein based on the immunohistochemistry (IHC) method, and through the optimization of specific IHC conditions, the antibody achieves better specificity and sensitivity and can be applied to clinical immunohistochemistry detection.

[0014] More preferably, the working solution of the ALK monoclonal antibody is included, and the concentration of the working solution of the ALK monoclonal antibody is 0.5 μg / mL.

[0015] Further preferably, the secondary antibody reagent in the kit is an ultrasensitive immunochromogenic reagent SD5600, which can enhance the specificity of staining.

[0016] Further preferably, the ALK monoclonal antibody diluent in the kit is a Dako ready-to-use antibody diluent, which can significantly enhance the signal-to-noise ratio of positive staining. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a fusion gene formed by ALK and other genes in Example 1 of the present invention;

[0018] Figure 2 The results of the verification of the five ALK proteins in Example 1 of the present invention using the 5A4 (Abcam) antibody;

[0019] Figure 3 This is the non-small cell lung cancer tissue-2D3 antibody staining profile in Example 3 of the present invention;

[0020] Figure 4 This is the non-small cell lung cancer tissue-5A4 antibody staining pattern in Example 3 of the present invention;

[0021] Figure 5 The staining patterns of the three different secondary antibody systems in Example 4 of the present invention - 2D3 antibody;

[0022] Figure 6 The staining pattern after the two different antibody dilutions are optimized in Example 5 of the present invention - 2D3 antibody;

[0023] Figure 7 This is a staining pattern of one case with inconsistent results in Example 6 of the present invention - 2D3 antibody / 5A4 antibody. DETAILED DESCRIPTION

[0024] The recombinant anti-human ALK antibody of the present invention has high sensitivity and specificity, and can achieve accurate detection of ALK positive mutations based on immunohistochemistry.

[0025] The technical solution of the present invention is further described below in conjunction with specific embodiments. The instruments and equipment involved in the following embodiments are all conventional instruments and equipment unless otherwise specified; the reagents involved are all commercially available conventional reagents unless otherwise specified; the test methods involved are all conventional methods unless otherwise specified.

[0026] Example 1 Acquisition of ALK Antigen

[0027] According to the protein sequence number Q9UM73 published by Uniprot, ALK protein is a single-pass transmembrane protein, with a receptor region outside the cell and a kinase region inside the cell. It is composed of 1620 amino acids and has a molecular weight of 220KDa. According to literature reports, ALK protein usually has three forms of mutation: 1) fusion mutation, the most common mutation of ALK, the protein expressed by gene fusion is abnormally active; 2) point mutation, which occurs relatively less frequently and mainly occurs in the intracellular kinase region, constantly conveying cell growth signals to downstream pathways; 3) amplification mutation, which increases the chance of coupling by increasing the binding with the ligand, thereby increasing the signal that promotes cell growth. Studies have found that among the most typical ALK fusion mutations, there are mainly three types: ① anaplastic large cell lymphoma (ALCL), the 3' part of ALK from chromosome 2 is fused to the 5' part of nucleophosmin NPM from chromosome 5, and the product of NPM-ALK fusion gene has carcinogenic effects; ② non-small cell lung cancer (NSCLC), caused by EML4-ALK fusion mutation; ③ inflammatory myofibroblastic tumor (IMT), caused by TPM3-ALK fusion mutation, the specific mutation mode is as follows Figure 1 As shown, the ALK intracellular domain is present in each fusion protein.

[0028] Therefore, according to the complex mutation forms of ALK protein, the present invention designs 5 different ALK protein sequences as immunogens, in order to obtain an effective immunogen design strategy for the development of transmembrane protein monoclonal antibodies, and after codon optimization of the 5 different nucleotide sequences, the optimized nucleic acid sequences are artificially synthesized, constructed into pET28a or pET32a vectors, and transferred into Escherichia coli for protein expression. After purification by nickel column affinity chromatography, the target protein, i.e., ALK antigen, with a purity of up to 90% is obtained.

[0029] Western Blot verification was performed using Abcam's 5A4 antibody. Figure 2 As shown in the figure, the ALK protein sequence number corresponding to No. 1 is SEQ ID NO: 9; the ALK protein sequence number corresponding to No. 2 is SEQ ID NO: 10; the ALK protein sequence number corresponding to No. 3 is SEQ ID NO: 11; the ALK protein sequence number corresponding to No. 4 is SEQ ID NO: 12; the ALK protein sequence number corresponding to No. 5 is SEQ ID NO: 13. The results of Western Blot test showed that the commercial 5A4 antibody (Abcam) combined with the constructed ALK recombinant proteins 1#, 3# and 4#. It was preliminarily inferred that the above three antigen proteins prepared contained linearized epitopes recognized by the 5A4 antibody, among which the ALK recombinant protein 3# had the best effect, and this antibody development method was more targeted.

[0030] Example 2 Recombinant Antibody Against Human ALK

[0031] This example is the preparation of recombinant antibodies against human ALK, and the specific preparation steps are as follows:

[0032] 1. Animal immunization

[0033] In order to further improve the animal immune effect, CSF2 cytokine was added as an immune partner for immunization. CSF2 is a protein independently constructed and expressed. Immunogen 3# and the immune partner (CSF2 protein) were added to a water-soluble adjuvant to obtain an immune reagent. The concentration of the immune partner in the immune reagent was controlled to be 50μg / mL, and the immune dose of the immunogen was 100μg / rabbit. Then the New Zealand white rabbits were immunized by intramuscular injection in the leg, for a total of three immunizations, each with an interval of 21 days, and the immune dose was the same. On the 14th day after the third immunization, blood was collected from the ear vein to evaluate the immune effect. After achieving the expected effect, the rabbits were subjected to ALK immunogen shock immunization, and 10mL of blood was collected from the ear vein on the fourth day.

[0034] 2. Rabbit peripheral blood collection, PBMCs separation, B lymphocyte sorting and culture

[0035] PBMCs (i.e., peripheral blood mononuclear cells, which can be achieved using a commercial rabbit peripheral blood lymphocyte isolation kit) are isolated from the peripheral blood of rabbits after impact immunization. Subsequently, one or more antibodies against T lymphocyte surface markers CD4, CD8, CD14, CD28, and CD80 are used for rescreening to remove T, monocytes, and other cells.

[0036] Then, activated B lymphocytes were selected using goat anti-rabbit IgG labeled with the fluorescent dye FITC.

[0037] Then, the immunogen ALK was labeled using a commercial biotin labeling kit according to the instructions, and then incubated with a commercial avidin-conjugated fluorescein (one or more of PE, APC, PerCP, PE-Cy7, APC-Cy7) reagent at 37°C for 1 hour (shaking and stirring several times during the period) to obtain the antigen-biotin-avidin screening reagent. When used, it was added according to the cell amount (1×106 cells / 5μL), and the obtained B lymphocytes were positively screened again to obtain antigen-specific B lymphocytes.

[0038] The sorted B lymphocytes were plated and cultured at 1-2 cells / well in a 96-well plate pre-added with trophoblast cells, and one or more cytokines of IL-2, IL-4, IL-6, IL-10, TNF-α, TGF-β, and SAC were added for co-culture.

[0039] 3. ELISA detection of B lymphocyte culture supernatant

[0040] After culturing for 7-12 days, the cell supernatant was taken for ELISA detection. The immunogen ALK protein was coated, and the serum collected from rabbits before immunization was selected as the negative control well, with an OD greater than 2.1 times the negative control well. 450 The read value is defined as positive, and the positive wells screened are selected.

[0041] 4.cDNA acquisition, antibody heavy and light chain gene amplification and sequencing

[0042] The cells in the positive cell wells obtained by screening were lysed, total RNA was extracted and cDNA was obtained by RT-PCR. The heavy chain and light chain gene bands of the antibody in the cell wells were amplified by ordinary PCR using a pair of primers for each heavy chain and light chain, and the primer sequences are shown in SEQ ID NO: 14-SEQ ID NO: 17. The obtained bands were recovered by gel to obtain the antibody heavy and light chain gene products, and a portion of the obtained products was sequenced, and the primer sequences used for sequencing are shown in SEQ ID NO: 18-SEQ ID NO: 19.

[0043] 5. Construction of heavy chain antibody recombinant plasmid and light chain antibody recombinant plasmid

[0044] After analyzing the correctly sequenced antibody heavy and light chain gene sequences, the heavy and light chain variable region sequences were extracted, and the heavy chain variable region and rabbit IgG constant region sequence, and the light chain variable region sequence and rabbit κ chain constant region sequence were respectively inserted into the pcDNA3.4 expression vector for recombination by homologous recombination.

[0045] The DH5α strain containing the pcDNA3.4 plasmid was expanded and cultured for plasmid extraction and double enzyme digestion (EcoRI and HindIII). Plasmid extraction was performed according to the instructions of the Axygen plasmid extraction kit. The double enzyme digestion reaction system was: pcDNA3.4 plasmid 2μg, EcoRI 2μL, HindIII 2μL, Buffer 5μL, ddH2O 40μL; the enzyme digestion conditions were 37℃ water bath, and the time was 2h. The digested system was subjected to agarose gel electrophoresis and the gel was cut to recover the plasmid fragments after double enzyme digestion. This operation was performed according to the instructions of the Axygen gel recovery kit.

[0046] The antibody heavy and light chain variable region gene fragments were obtained by PCR, and the upstream and downstream primer sequences were designed as shown in SEQ ID NO: 20-SEQ ID NO: 23. The PCR amplification cycle was set as follows: I: 98°C 3min; II: 98°C 10s; III: 56°C 10s; IV: 72°C 10s; II-IV: 30 cycles; V: 72°C 5min. The target bands were obtained by 1.5% agarose gel electrophoresis and the target gene fragments were recovered by gel cutting. The operation was also performed according to the instructions of the Axygen gel recovery kit.

[0047] The target gene fragment and the double-enzyme-cut plasmid fragment were homologously recombined using a seamless cloning kit. This step was performed according to the instructions of the Nearshore Seamless Cloning Kit (Cat. No.: NR005-01A).

[0048] After seamless recombination, DH5α competent cells were transformed and screened using ampicillin-resistant LB plates. The cells were cultured in a 37°C incubator for 16-18 hours, and 5 single clones were selected for gene sequencing. The colonies with correct sequencing were expanded and cultured to extract plasmids.

[0049] 6. Plasmid Transfection

[0050] The high-concentration, high-purity antibody heavy and light chain recombinant plasmids obtained through expanded culture were respectively transferred into CHO / HEK293 cells according to the instructions of the PEI transfection reagent. The antibody heavy chain and light chain were pre-mixed at a molar mass of 1:2, and the plasmid was diluted with CHO / HEK293 cell basal medium. At the same time, an equal volume of the medium was used to dilute PEI, plasmid: PEI = 1:3, W / W. Transfected into CHO / HEK293 cells cultured to the logarithmic growth phase. 48 hours after transfection, the antibody titer secreted in the supernatant was detected by indirect ELISA method to determine the cell line with relatively high expression.

[0051] 7. Antibody engineering expression and purification

[0052] The determined cell line was expanded to a volume of 100 mL, the cell growth state was adjusted to the logarithmic growth phase, the plasmid was transfected, the feed was supplemented every other day, and the cell supernatant was harvested on the 6th day after transfection. The cell supernatant was filtered through a 0.22 μm filter and purified by affinity chromatography through a Protein G column. The antibodies obtained by binding and adsorption were eluted with a pH 6.0 citric acid buffer, and the eluate was collected. The pH was rapidly neutralized to between 7.2 and 7.4 using a pH 8.8 Tris-HCl solution. The purified 2D3 antibody was then concentrated by ultrafiltration and centrifugation to a concentration of more than 1 mg / mL to obtain an ALK monoclonal antibody stock solution.

[0053] Example 3 Application of recombinant antibodies against human ALK

[0054] Specifically, it is the application of recombinant antibodies against human ALK in non-small cell lung cancer tissue. The immunohistochemistry detection reagent in this embodiment is an ALK immunohistochemistry detection reagent, that is, the ALK monoclonal antibody stock solution in Example 2 is diluted with antibody diluent (Zhongshan Jinqiao, ZLI-9028) and the working solution concentration is 0.5 μg / mL. Other supporting reagents include antigen EDTA repair solution, hydrogen peroxide blocking solution, horseradish peroxidase-labeled mouse and rabbit universal secondary antibody (SD3208), DAB and its buffer, and hematoxylin.

[0055] The above reagents were used to stain non-small cell lung cancer tissues. The staining process was as follows:

[0056] 1. Paraffin tissue sample sections of non-small cell lung cancer, 3 μm / sheet, baked at 65°C for 2 hours;

[0057] 2. Dewaxing and hydration: The paraffin sections were treated as follows: xylene for 15 min - xylene for 15 min - anhydrous ethanol for 5 min - anhydrous ethanol for 5 min - 90% ethanol for 5 min - 80% ethanol for 5 min - 70% ethanol for 5 min, and then immersed in purified water for 5 min.

[0058] 3. Antigen repair: Heat the EDTA antigen repair solution with pH 9.0 to a boil and put the tissue slices in. Adjust to the micro-boiling mode and time for 20 minutes. After natural cooling for 5 minutes, rinse with running water to cool to room temperature, take out the slices, soak in purified water for 5 minutes, and rinse and soak in TBST for 5 minutes.

[0059] 4. Add peroxidase blocking agent: 100 μL / slide, incubate at room temperature for 5 min, rinse and soak in TBST for 5 min / time, for a total of 2 times.

[0060] 5. Primary antibody incubation: Add the above antibody working solution, 100 μL / plate, incubate at 37°C for 30 min, rinse and soak in TBST for 5 min / time, for a total of 2 times.

[0061] 6. Secondary antibody incubation: add horseradish peroxidase-labeled mouse and rabbit universal secondary antibody, 100 μL / plate, incubate at room temperature for 30 min, rinse and soak in TBST for 5 min / time, for a total of 2 times.

[0062] 7. Color development: add 100 μL / sheet of DAB color developing solution, incubate at room temperature for 4 min, and soak in purified water twice, 5 min each time.

[0063] 8. Restaining: Add 100 μL / sheet of hematoxylin solution, incubate at room temperature for 4 minutes, and rinse with purified water.

[0064] 9. Dehydration and transparency: conventional gradient ethanol dehydration, xylene transparency.

[0065] 10. Seal the slide with neutral gum and observe.

[0066] The staining results of the 2D3 antibody prepared in this example and the 5A4 antibody of Abcam are shown in Figure 2. Figure 3 and Figure 4 As shown in the figure, it can be seen that the staining effect of 2D3 antibody is consistent with that of Abcam's 5A4 antibody.

[0067] Example 4 Application of recombinant antibodies against human ALK

[0068] Specifically, the recombinant antibody against human ALK was studied in different secondary antibody systems. The immunohistochemistry detection reagent in this embodiment is the ALK immunohistochemistry detection reagent, which is the working solution of the ALK monoclonal antibody in Example 2, and its antibody working concentration is 0.5 μg / mL. Other supporting reagents include antigen EDTA repair solution, hydrogen peroxide blocking solution, DAB and its buffer, and hematoxylin.

[0069] Three different secondary antibody reagents were used in the immunohistochemistry experiment (all three reagents are sold by Henan Synotech Biotechnology Co., Ltd.), namely horseradish peroxidase labeled pika universal secondary antibody reagent (SD3208), two-step enhanced immunochromogenic reagent (SD5300), and ultrasensitive immunochromogenic reagent (SD5600), and the staining process was the same as that of Example 3. By testing the compatibility of different secondary antibody reagents with immunohistochemical conditions, a secondary antibody reagent that can achieve the best staining effect was found.

[0070] The staining results of the 2D3 antibody prepared in this example in three different secondary antibody systems are shown in Figure 2. Figure 5 As shown in the figure, it can be seen that the ultrasensitive immunochromogenic reagent (SD5600) has the best staining effect, high staining intensity and clean background, and qualified tissue specificity.

[0071] Example 5 Study of recombinant antibodies against human ALK in different antibody dilutions

[0072] The immunohistochemistry detection reagent in this embodiment is an ALK immunohistochemistry detection reagent, which is the working solution of the ALK monoclonal antibody in Example 2, and its antibody working concentration is 0.5 μg / mL. Other supporting reagents include antigen EDTA repair solution, hydrogen peroxide blocking solution, horseradish peroxidase-labeled mouse and rabbit universal secondary antibody, DAB and its buffer, and hematoxylin.

[0073] Two different antibody diluents were used in the immunohistochemistry experiment, namely antibody diluent 1 (Zhongshan Jinqiao, ZLI-9028) and antibody diluent 2 (Dako ready-to-use antibody diluent), and the staining process was the same as that of Example 3. By testing the compatibility of different antibody diluents with immunohistochemical conditions, an antibody diluent that can achieve the best staining effect was found.

[0074] The staining results of the 2D3 antibody prepared in this example in two different antibody dilutions are shown in Figures 1 and 2. Figure 6 As shown in the figure, it can be seen that the 2D3 antibody staining intensity can be increased to more than 35-40% by using the ultrasensitive immunochromogenic reagent (SD5600) in combination with antibody diluent 2 (Dako ready-to-use antibody diluent), that is, the optimized immunohistochemical staining system can significantly improve the staining intensity, with a clean background and qualified tissue specificity.

[0075] Example 6 Application of recombinant antibodies against human ALK in multiple tissues

[0076] The control group 5A4 antibody and the 2D3 antibody of the present invention were evaluated by immunohistochemistry in multiple tissues in the same manner as above, and the test results are shown in Table 1. The evaluated tissues included non-small cell lung cancer, tonsils, etc., and a total of 225 cases were evaluated.

[0077] Table 1 Comparison of tissue evaluation statistics of 5A4 antibody and 2D3 antibody

[0078]

[0079] As shown in Table 1, in terms of the evaluation of immunohistochemical detection results, the 2D3 antibody in the embodiment detected 41 lung cancer tissues, and the 5A4 antibody in the control group detected 40 cases. The staining pattern of the 1 case with inconsistent results is as follows: Figure 7 As shown. In this embodiment, all lung cancer tissues were stained with HE, and multiple pathologists were invited to jointly read the films to confirm that the 41 tissues detected by the 2D3 antibody of the present invention all showed high-grade lesions in morphology, and all were lung cancer positive tissues. Therefore, the sensitivity of the 2D3 antibody of the present invention is higher, and the positive detection rate of the 2D3 antibody is as high as 100%, which proves that the tissue specificity of the 2D3 antibody is higher, indicating that the recombinant antibody against human ALK of the present invention can be used as a substitute product of Abcam's ALK (5A4).

Claims

1. A recombinant antibody against human ALK, characterized in that: The recombinant anti-human ALK antibody comprises VHCDR1, VHCDR2 and VHCDR3 whose amino acid sequences are sequentially shown in SEQ ID NOs: 1 to 3, and VLCDR1, VLCDR2 and VLCDR3 whose amino acid sequences are sequentially shown in SEQ ID NOs: 4 to 6.

2. The recombinant anti-human ALK antibody according to claim 1, characterized in that The antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:7 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

8.

3. Use of the recombinant anti-human ALK antibody as described in claim 1 or 2 in the preparation of an ALK in vitro immunoassay reagent or kit.

4. The use according to claim 3, characterized in that: The invention also provides a working solution of ALK monoclonal antibody, wherein the concentration of the working solution of ALK monoclonal antibody is 0.5 μg / mL.

5. The use according to claim 3, characterized in that: The secondary antibody reagent in the kit is the ultrasensitive immunochromogenic reagent SD5600.

6. The use according to claim 3, characterized in that: The ALK monoclonal antibody diluent in the kit is a Dako ready-to-use antibody diluent.

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