A recombinant antibody against human ALK and its application

Abstract

This invention provides a recombinant antibody against human ALK and its applications, belonging to the field of monoclonal antibody technology. The recombinant antibody against human ALK of this invention comprises VHCDR1, VHCDR2, and VHCDR3 with amino acid sequences as shown in SEQ ID NO: 1-3, and VLCDR1, VLCDR2, and VLCDR3 with amino acid sequences as shown in SEQ ID NO: 4-6. The recombinant antibody against human ALK of this invention possesses high sensitivity and specificity, providing a reliable detection method in the field of ALK immunodiagnosis and providing a basis for precision medicine treatment.

Description

Technical Field

[0001] This invention belongs to the field of monoclonal antibody technology, specifically relating to a recombinant antibody against human ALK and its applications. Background Technology

[0002] Approximately 80% of lung cancers are non-small cell lung cancer (NSCLC). While the incidence of ALK (anaplastic lymphoma kinase) gene fusions in NSCLC is relatively low, around 5-6%, ALK inhibitors targeting ALK gene mutations have been developed. Currently, three generations of ALK-targeted drugs (crizotinib, ceritinib, and alectinib) are used in clinical treatment and have shown very good clinical efficacy. Patients with ALK rearrangements exhibit less drug resistance and side effects, and have better overall treatment outcomes. The ALK target is considered the "diamond target" in the field of NSCLC, making the detection of ALK-positive mutations essential.

[0003] However, the choice of diagnostic method for ALK rearrangement detection remains controversial in clinical practice. China has approved companion diagnostic kits for ALK gene detection on four technology platforms: IHC, FISH, RT-PCR, and NGS. Therefore, in cases of uncertainty, at least two of these four methods should be used to confirm ALK rearrangement. The development of highly sensitive ALK diagnostic antibodies has provided an opportunity for standardized immunohistochemical (IHC) methods to detect ALK-driven tumors. Moreover, compared to FISH and RT-PCR, one of the main advantages of IHC is the direct detection of the ALK protein, which is a direct target for ALK inhibitors. IHC is also low-cost, has a short turnaround time, and is easy to perform. Therefore, the 2019 Expert Consensus on Clinical Practice of ALK Detection in Non-Small Cell Lung Cancer in China strongly recommends prioritizing IHC detection.

[0004] Currently, the ALK antibody products sold in the market for immunohistochemical pathological diagnosis are mainly monopolized by expensive imported products. Cell Signaling Technology's D5F3 antibody and Abcam's 5A4 antibody have accuracy rates of 100% and 95%-99%, respectively. Roche Ventana, utilizing the D5F3 antibody and optimizing its immunohistochemical detection system, launched the ALK(D5F3)CDx kit, which was among the first to receive approval from the CFDA and FDA as a companion diagnostic reagent for the targeted drugs crizotinib and ceritinib. Research in the field of ALK immunodiagnostics in my country is severely lagging, and domestically produced antibodies cannot meet the needs of NSCLC patients. Therefore, developing an ALK antibody with higher sensitivity and specificity can provide a more reliable detection method for many patients and provide a basis for precision medicine, which has profound and positive significance. Summary of the Invention

[0005] The first objective of this invention is to provide a recombinant antibody against human ALK that enables accurate detection of ALK-positive mutations.

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

[0007] To achieve the first objective mentioned above, the technical solution adopted by this invention is as follows:

[0008] The recombinant antibody against human ALK contains VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NO: 1-3, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NO: 4-6.

[0009] The beneficial effects of the above technical solution are as follows: This invention provides a recombinant antibody against human ALK, which has good specificity and affinity, and can serve as a substitute for Abcam's 5A4 antibody. Furthermore, the immunogen design strategy developed for transmembrane protein monoclonal antibodies in this invention can provide a reference for the design of similar transmembrane proteins.

[0010] Preferably, 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.

[0011] To achieve the second objective mentioned above, the technical solution adopted by the present invention is as follows:

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

[0013] The beneficial effects of the above technical solution are as follows: the recombinant antibody of the present invention achieves efficient and accurate detection of ALK protein based on the immunohistochemical (IHC) method, and through specific IHC condition optimization, the antibody achieves better specificity and sensitivity, and can be applied to clinical immunohistochemical detection.

[0014] More preferably, it includes a working solution of ALK monoclonal antibody, wherein the concentration of the working solution of ALK monoclonal antibody is 0.5 μg / mL.

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

[0016] More preferably, the ALK monoclonal antibody diluent in the kit is Dako ready-to-use antibody diluent, which can significantly enhance the signal-to-noise ratio of positive staining. Attached Figure Description

[0017] Figure 1 This refers to the 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 this invention using the 5A4 (Abcam) antibody;

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

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

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

[0022] Figure 6 The staining pattern optimized from two different antibody dilution solutions in Example 5 of this invention is the 2D3 antibody.

[0023] Figure 7 This is a case of inconsistent staining patterns in Example 6 of the present invention - 2D3 antibody / 5A4 antibody. Detailed Implementation

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

[0025] The technical solution of the present invention will be further described below with reference to specific embodiments. Unless otherwise specified, 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 experimental methods involved are all conventional methods.

[0026] Example 1: Acquisition of ALK antigen

[0027] According to Uniprot's published protein sequence number Q9UM73, ALK protein is a single-transmembrane protein with an extracellular receptor domain and an intracellular kinase domain. It consists of 1620 amino acids and has a molecular weight of 220 kDa. Literature reports indicate that ALK protein typically exhibits three mutational forms: 1) Fusion mutations, the most common type of ALK mutation, resulting in abnormally active protein expression due to gene fusion; 2) Point mutations, which occur less frequently and primarily occur in the intracellular kinase domain, continuously transmitting cell growth signals to downstream pathways; and 3) Amplification mutations, which increase ligand binding and thus coupling opportunities, enhancing cell growth-promoting signals. Studies have found that the most typical ALK fusion mutations are mainly caused by three types: ① Anaplastic large cell lymphoma (ALCL), where the 3' portion of ALK from chromosome 2 is fused to the 5' portion of nucleophosphorus NPM from chromosome 5; the product of the NPM-ALK fusion gene has oncogenic effects; ② Non-small cell lung cancer (NSCLC), caused by EML4-ALK fusion mutations; ③ Inflammatory myofibroblastic tumor (IMT), caused by TPM3-ALK fusion mutations. The specific mutation pathways are as follows... Figure 1 As shown, the ALK intracellular domain appears in each fusion protein.

[0028] Therefore, based on the complex mutational forms of the ALK protein, this invention designed five different ALK protein sequences as immunogens to obtain an effective immunogen design strategy for the development of monoclonal antibodies against transmembrane proteins. After codon optimization of the five different nucleotide sequences, the optimized nucleic acid sequences were artificially synthesized and constructed into pET28a or pET32a vectors, which were then transformed into *E. coli* for protein expression. After purification by nickel column affinity chromatography, the target protein, i.e., the ALK antigen, with a purity of up to 90% was obtained.

[0029] Western blot validation was performed using Abcam's 5A4 antibody, such as... Figure 2 As shown in the figure, the ALK protein sequence number corresponding to 1 is SEQ ID NO: 9; the ALK protein sequence number corresponding to 2 is SEQ ID NO: 10; the ALK protein sequence number corresponding to 3 is SEQ ID NO: 11; the ALK protein sequence number corresponding to 4 is SEQ ID NO: 12; and the ALK protein sequence number corresponding to 5 is SEQ ID NO: 13. Western blot results show that the commercially available 5A4 antibody (Abcam) binds to the constructed recombinant ALK proteins 1#, 3#, and 4#, preliminarily inferring that the prepared three antigen proteins contain linearized epitopes recognized by the 5A4 antibody. Among them, recombinant ALK protein 3# showed the best effect, indicating that this antibody development method is more targeted.

[0030] Example 2: Recombinant Antibody Against Human ALK

[0031] This example demonstrates the preparation of a recombinant antibody against human ALK. The specific preparation steps are as follows:

[0032] 1. Animal immunization

[0033] To further improve the immunization effect in animals, CSF2 cytokine was added as an immune chaperone. CSF2 is a self-constructed and expressed protein. Immunogen 3# and the immune chaperone (CSF2 protein) were added to a water-soluble adjuvant to obtain an immunization reagent. The concentration of the immune chaperone in the immunization reagent was controlled at 50 μg / mL, and the immunization dose of the immunogen was 100 μg / rabbit. New Zealand white rabbits were then immunized via intramuscular injection in the leg, for a total of three immunizations, each 21 days apart, with the same immunization dose. On day 14 after the third immunization, blood was collected from the marginal ear vein to assess the immunization effect. After achieving the expected results, the rabbits were given a pulse immunization with ALK immunogen, and 10 mL of blood was collected from the marginal ear vein on day four.

[0034] 2. Rabbit peripheral blood collection and PBMC isolation, B lymphocyte sorting and culture

[0035] PBMCs (peripheral blood mononuclear cells, which can be obtained using a commercially available rabbit peripheral blood lymphocyte isolation kit) were isolated from the peripheral blood of rabbits after shock immunization. Subsequently, the cells were screened again using one or more antibodies against T lymphocyte surface markers CD4, CD8, CD14, CD28, and CD80 to remove T cells, monocytes, and other cells.

[0036] Activated B lymphocytes were then selected using FITC-labeled goat anti-rabbit IgG.

[0037] Next, using a commercially available biotin labeling kit, the immunogen ALK was labeled according to the instructions. Then, it was conjugated with a commercially available avidin-fluorescein (PE, APC, PerCP, PE-Cy7, APC-Cy7, or one or more of these) reagents and incubated at 37°C for 1 hour (with agitation several times during incubation) to obtain the antigen-biotin-avidin screening reagent. When using this reagent, it was added at a cell density of 1×10⁶ cells / 5 μL to perform a second positive screening on the resulting B lymphocytes, yielding antigen-specific B lymphocytes.

[0038] The sorted B lymphocytes were seeded into 96-well plates with 1-2 cells per well, which were pre-filled with feeder cells. One or more cytokines from 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, cell supernatant was collected for ELISA detection. The cells were coated with the immunogen ALK protein, and serum collected from rabbits before immunization was used as a negative control well, with an OD value 2.1 times larger than the negative control well. 450 The value is defined as positive, and the positive wells selected are chosen.

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

[0042] Cells from the selected positive cell wells were lysed, and total RNA was extracted to obtain cDNA via RT-PCR. Antibody heavy and light chain gene bands from the cells in the wells were amplified using conventional PCR with one primer pair each for the heavy and light chains. The primer sequences are shown in SEQ ID NO: 14-SEQ ID NO: 17. The obtained bands were recovered from the gel to obtain the antibody heavy and light chain gene products. A portion of the products was sequenced, and the primer sequences used for sequencing are shown in SEQ ID NO: 18-SEQ ID NO: 19.

[0043] 5. Construct recombinant plasmids for heavy chain antibodies and light chain antibodies.

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

[0045] DH5α strain containing pcDNA3.4 plasmid was expanded for culture, followed by plasmid extraction and double digestion (EcoRI and HindIII). Plasmid extraction was performed according to the Axygen plasmid extraction kit instructions. The double digestion reaction mixture consisted of: 2 μg pcDNA3.4 plasmid, 2 μL EcoRI, 2 μL HindIII, 5 μL Buffer, and 40 μL ddH2O; digestion was performed at 37°C for 2 hours. The digested plasmid fragments were then subjected to agarose gel electrophoresis and recovered from the gel, following the instructions of the Axygen gel recovery kit.

[0046] The variable region gene fragment of the antibody heavy and light chains was obtained by PCR. The upstream and downstream primer sequences were designed as shown in SEQ ID NO: 20-SEQ ID NO: 23. The PCR amplification cycle settings were: I: 98℃ for 3 min; II: 98℃ for 10 s; III: 56℃ for 10 s; IV: 72℃ for 10 s; II-IV: 30 cycles; V: 72℃ for 5 min. The target band was obtained by 1.5% agarose gel electrophoresis, and the target gene fragment was recovered by gel extraction, following the instructions of the Axygen gel extraction kit.

[0047] Homologous recombination of the target gene fragment and the double-digested plasmid fragment was performed using a seamless cloning kit. This step was performed in accordance with the instructions of the nearshore seamless cloning kit (catalog number: NR005-01A).

[0048] After seamless recombination, the cells were transformed into DH5α competent cells and screened using ampicillin-resistant LB plates. The cells were cultured at 37°C for 16-18 hours, and five single clones from each plate were selected for gene sequencing. Colonies with correct sequencing were expanded and plasmids were extracted.

[0049] 6. Plasmid transfection

[0050] High-concentration, high-purity antibody heavy and light chain recombinant plasmids obtained after expanded culture were transfected into CHO / HEK293 cells according to the PEI transfection reagent instructions. The antibody heavy and light chains were pre-mixed at a molar ratio of 1:2, and the plasmid was diluted with CHO / HEK293 cell basal medium. Simultaneously, an equal volume of the medium was used to dilute PEI (plasmid:PEI = 1:3, w / w). The mixture was then transfected into CHO / HEK293 cells cultured in the logarithmic growth phase. Forty-eight hours after transfection, the antibody titer secreted in the supernatant was detected using an indirect ELISA method to identify cell lines with relatively high expression levels.

[0051] 7. Antibody engineered expression and purification

[0052] The selected cell line was expanded to a volume of 100 mL, and the cell growth state was adjusted to the logarithmic growth phase. Plasmids were transfected, and feeding was performed every other day. Cell supernatant was harvested on day 6 post-transfection. The cell supernatant was filtered through a 0.22 μm filter and purified by affinity chromatography using a Protein G column. The bound antibody was eluted with citrate buffer at pH 6.0, and the eluent was collected. The eluent was rapidly neutralized to pH 7.2–7.4 using Tris-HCl solution at pH 8.8. The purified 2D3 antibody was then concentrated by ultrafiltration and centrifugation to a concentration above 1 mg / mL, yielding the ALK monoclonal antibody stock solution.

[0053] Example 3: Application of recombinant anti-human ALK antibody

[0054] Specifically, this example describes the application of recombinant anti-human ALK antibodies in non-small cell lung cancer tissue. The immunohistochemical detection reagent used in this example is the ALK immunohistochemical detection reagent, which is the working solution of the monoclonal antibody after dilution with antibody diluent (Zhongshan Jinqiao, ZLI-9028) in Example 2. The working solution concentration is 0.5 μg / mL. Other supporting reagents include antigen EDTA retrieval solution, hydrogen peroxide blocking solution, horseradish peroxidase-labeled universal secondary antibody for mice and rabbits (SD3208), DAB and its buffer, and hematoxylin.

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

[0056] 1. Paraffin-embedded tissue sections of non-small cell lung cancer, 3μm / section, baked at 65℃ for 2 hours;

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

[0058] 3. Antigen retrieval: After heating and boiling the EDTA antigen retrieval solution at pH 9.0, place the tissue slide in the solution, adjust to a gentle boiling mode, and time for 20 minutes. After cooling naturally for 5 minutes, rinse with running water to cool to room temperature, remove the slide, soak in purified water for 5 minutes, and then rinse and soak in TBST for 5 minutes.

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

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

[0061] 6. Secondary antibody incubation: Add 100 μL of horseradish peroxidase-labeled universal secondary antibody for mice and rabbits, incubate at room temperature for 30 min, rinse and soak with TBST for 5 min each time, for a total of 2 times.

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

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

[0064] 9. Dehydrated and transparent: Dehydrated with conventional gradient ethanol, and transparent with xylene.

[0065] 10. Observe the mounting with neutral resin.

[0066] The staining results of the 2D3 antibody prepared in this embodiment and the Abcam 5A4 antibody are as follows: Figure 3 and Figure 4 As shown in the figure, the staining effects of the 2D3 antibody and Abcam's 5A4 antibody are quite consistent.

[0067] Example 4: Application of recombinant anti-human ALK antibody

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

[0069] Three different secondary antibody reagents (all three are available from Henan Sainote Biotechnology Co., Ltd.) were used in the immunohistochemical experiments: horseradish peroxidase-labeled universal secondary antibody reagent for mice and rabbits (SD3208), two-step enhanced immunochromatographic reagent (SD5300), and ultrasensitive immunochromatographic reagent (SD5600). The staining process was the same as in Example 3. By testing the compatibility of different secondary antibody reagents with the immunohistochemical conditions, a secondary antibody reagent that achieves the best staining effect was found.

[0070] The staining results of the 2D3 antibody prepared in this embodiment in three different secondary antibody systems are as follows: Figure 5 As shown in the figure, the ultrasensitive immunochromogenic reagent (SD5600) has the best staining effect, with high staining intensity, a clean background, and satisfactory tissue specificity.

[0071] Example 5: Study of recombinant anti-human ALK antibody in different antibody dilution solutions

[0072] The immunohistochemical detection reagent used in this embodiment is the ALK immunohistochemical detection reagent, which is the working solution of the ALK monoclonal antibody in Example 2. The working concentration of the antibody is 0.5 μg / mL. Other supporting reagents include antigen EDTA retrieval 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 immunohistochemical experiments: antibody diluent 1 (Zhongshan Jinqiao, ZLI-9028) and antibody diluent 2 (Dako ready-to-use antibody diluent). The staining process was the same as in Example 3. By testing the compatibility of different antibody diluents with the immunohistochemical conditions, an antibody diluent that achieved the best staining effect was found.

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

[0075] Example 6: Application of recombinant anti-human ALK antibody in multiple tissues

[0076] The control group 5A4 antibody and the 2D3 antibody of this invention were evaluated by immunohistochemical detection in multiple tissues, using the same method as described above. The detection results are shown in Table 1 below. The evaluated tissues included non-small cell lung cancer and tonsils, with a total of 225 cases evaluated.

[0077] Table 1. Statistical analysis of tissue evaluations of 5A4 antibody and 2D3 antibody.

[0078]

[0079] As shown in Table 1, regarding the evaluation of immunohistochemical detection results, the 2D3 antibody in the example detected 41 lung cancer tissues, while the 5A4 antibody in the control group detected 40. One case showed inconsistent results, with the staining pattern as shown below. Figure 7 As shown in the figure. In this embodiment, all lung cancer tissues were stained with hematoxylin and eosin (HE), and after joint review by multiple pathologists, it was confirmed that all 41 tissues detected by the 2D3 antibody of this invention showed high-grade lesions morphologically, and were all lung cancer positive tissues. Therefore, the 2D3 antibody of this invention has higher sensitivity, and the positive detection rate of the 2D3 antibody is as high as 100%, proving that the 2D3 antibody has higher tissue specificity, indicating that the recombinant anti-human ALK antibody of this invention can be used as an alternative product to Abcam's ALK (5A4).

Claims

1. A recombinant antibody against human ALK, characterized in that, The recombinant antibody against human ALK comprises VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NO: 1-3, and VLCDR1, VLCDR2 and VLCDR3 as shown in SEQ ID NO: 4-6; the amino acid in SEQ ID NO: 5 is RAS.

2. The recombinant antibody against human ALK 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. The use of the recombinant anti-human ALK antibody as described in claim 1 or 2 in the preparation of ALK in vitro immunoassay reagents or kits.

4. The application according to claim 3, characterized in that, The working solution includes an ALK monoclonal antibody, wherein the concentration of the ALK monoclonal antibody working solution is 0.5 µg / mL.

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

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