CSPG4-fluorescent protein fusion protein and application thereof in CSPG4 CAR-T cell positive rate detection

Abstract

The invention provides a CSPG4-fluorescent protein fusion protein, which comprises a CSPG4 antigen fragment and a fluorescent protein, and the corresponding sequence is as shown in SEQ ID NO: 1. Compared with the existing detection method, the CSPG4-fluorescent protein fusion protein provided by the invention has the remarkable advantages of low background, high specificity, high accuracy and wide applicability in the aspect of detecting the positive rate of CSPG4 CAR-T cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology, more particularly, it relates to a CSPG4-fluorescent protein fusion protein and its application in CSPG4 CAR-T cell positive rate detection. BACKGROUND

[0002] Cancer is one of the major causes of death worldwide. According to the latest statistics, nearly one sixth of deaths are caused by cancer. Current conventional treatments for tumors include surgery, radiotherapy and chemotherapy, but due to the limitations of the methods, it is difficult to cure malignant tumors. In recent years, immunotherapy for tumors has attracted much attention due to its specificity, high efficiency, low toxicity and other advantages, and is considered to be one of the most promising directions in the field of tumor treatment.

[0003] Chimeric antigen receptor-T (CAR-T) cell therapy is a kind of cell therapy that can target tumor cells by genetically modifying specific antigen recognition domains and T cell activation signals into cells, so that T cells can target tumor cell surface antigens and kill tumor cells. Compared with traditional treatment methods, CAR-T cells have the advantages of high targeting, strong killing ability, and small side effects. One of the key factors for using CAR-T cells for treatment is the selection of effective and safe target points.

[0004] Chondroitin sulfate proteoglycan 4 (CSPG4), also known as melanoma-associated chondroitin sulfate proteoglycan (MCSP) or high molecular weight melanoma-associated antigen (HMW MAA). It is highly expressed in various malignant tumors, such as melanoma, brain glioma, breast cancer, head and neck squamous cell carcinoma, and mesothelial tumor, etc. In contrast, CSPG4 is expressed very low in normal tissues and key organs, and is considered to be an important target for CAR-T cell therapy.

[0005] CAR-T cell positive rate detection is an important part of CAR-T cell preparation. For CSPG4 CAR-T cell positive rate detection, the current methods mainly use Protein L, goat anti-human IgG F(ab')2, and a unique antibody MK2-23 prepared based on scFv 763.74 (Zhili Zheng et al., J Transl Med. 2012, doi: 10.1186 / 1479-5876-10-29; Manuel Wiesinger et al., Cancers (Basel). 2019, doi: 10.3390 / cancers11081198; Koichi Hirabayashi et al., Nat Cancer. 2021, doi: 10.1038 / s43018-021-00244-2). The first two are not specific for CAR-T cell positive rate detection, so the stability and repeatability of the detection are poor, and the third one is only for CAR-T cell positive rate detection based on scFv 763.74, and cannot be used for CAR-T cell positive rate detection prepared by other scFv targeting CSPG4, with narrow application range.

[0006] In summary, there is no broad-spectrum, stable, and accurate detection method for CSPG4 CAR-T cell positive rate at present, therefore, it is a technical problem to be solved by those skilled in the art to provide a CSPG4-fluorescent protein fusion protein with an optimized structure to detect the CSPG4 CAD-T cell positive rate more stably, efficiently, and accurately. SUMMARY

[0007] The technical problem to be solved by the present application is to construct a fluorescent protein-fused CSPG4 antigen protein and apply it to CSPG4 CAR-T cell positive rate detection to solve the technical defects of unstable and inaccurate detection in the prior art.

[0008] The present application provides a CSPG4-fluorescent protein fusion protein comprising a CSPG4 antigen fragment, a linker, a fluorescent protein, and a purification tag, and the corresponding sequence is shown in SEQ ID NO: 1.

[0009] The present application also provides a coding nucleic acid sequence corresponding to the aforementioned CSPG4-fluorescent protein fusion protein and its complementary sequence, which is shown in SEQ ID NO: 2.

[0010] Preferably, in the aforementioned CSPG4-fluorescent protein fusion protein, the fluorescent protein is selected from far-red fluorescent protein mPlum, red fluorescent protein mCherry, tdTomato, mStrawberry, J-Red, DsRed-monomer, mRuby, mApple, mRaspberry, mKate2, orange fluorescent protein mOrange, mKO2, Kusabira Orange2, TagRFP, DsRed, mTangerine, yellow fluorescent protein mCitrine, Venus, mVenus, YPet, EYFP, Topaz, ZsYellowl, mBanana, green fluorescent protein Emerald, EGFP, Superfolder GFP, ZsGreen, cyan fluorescent protein ECFP, CyPet, mCFPm, Cerulean, mTurquoise, mTFPl, ultraviolet fluorescent protein T-Sapphire, blue fluorescent protein EBFP, mTagBFP, mTagBFP2.

[0011] More preferably, in the aforementioned CSPG4-fluorescent protein fusion protein, the fluorescent protein fragment is EGFP protein.

[0012] Preferably, in the aforementioned CSPG4-fluorescent protein fusion protein, the CSPG4 antigen is selected from full-length protein or complete extracellular domain of CSPG4 or partial extracellular domain of CSPG4.

[0013] More preferably, in the aforementioned CSPG4-fluorescent protein fusion protein, the CSPG4 antigen is partial extracellular domain of CSPG4.

[0014] Preferably, the linker between the aforementioned antigen and fluorescent protein is a flexible linker.

[0015] More preferably, the aforementioned linker is selected from (GGGGS)n, (GS)n or (G)n.

[0016] Most preferably, the aforementioned linker is (GGGGS)3.

[0017] Preferably, in the aforementioned CSPG4-fluorescent protein fusion protein, the CSPG4 is located at the N-terminal of the linker and the fluorescent protein is located at the C-terminal of the linker; or the CSPG4 is located at the C-terminal of the linker and the fluorescent protein is located at the N-terminal of the linker.

[0018] More preferably, in the aforementioned CSPG4-fluorescent protein fusion protein, the CSPG4 is located at the C-terminal of the linker and the fluorescent protein is located at the N-terminal of the linker.

[0019] Preferably, in the aforementioned CSPG4-fluorescent protein fusion protein, the C-terminal comprises a purification tag.

[0020] More preferably, the aforementioned CSPG4-fluorescent protein fusion protein, the purification tag is selected from the group consisting of Poly-His tag, Poly-Arg tag, GST tag, MBP tag, FLAG tag, Strep-tag_II tag, Twin-Strep-tag tag, S-tag tag, Avi tag, Halo tag, FC tag or SNAP tag.

[0021] Most preferably, the aforementioned CSPG4-fluorescent protein fusion protein, the purification tag is FLAG tag.

[0022] Preferably, the N-terminal of the aforementioned CSPG4-fluorescent protein fusion protein comprises a secretory signal peptide.

[0023] More preferably, the secretory signal peptide is IL2 signal peptide, and the corresponding sequence is shown in SEQ ID NO: 3.

[0024] More preferably, the nucleic acid sequence encoding the aforementioned secretory signal peptide and its complement is shown in SEQ ID NO: 4.

[0025] The present application also provides a nucleic acid construct comprising the aforementioned secretory signal peptide encoding nucleic acid and CSPG4-fluorescent protein fusion protein encoding nucleic acid.

[0026] Preferably, the viral expression vector is pLVX-IRES-Puro.

[0027] The present application also provides a virus comprising the aforementioned nucleic acid construct.

[0028] Preferably, the virus is lentivirus.

[0029] The present application also provides a cell strain capable of stably expressing the aforementioned CSPG4-fluorescent protein fusion protein.

[0030] Preferably, the mammalian cell strain is HEK293 cell strain.

[0031] The present application also provides a preparation method of the aforementioned CSPG4-fluorescent protein fusion protein, comprising the following steps: chemically synthesizing the amino acid sequence of the aforementioned CSPG4-fluorescent protein fusion protein (i.e. the sequence shown in SEQ ID NO: 1), or using a cell strain stably expressing the aforementioned CSPG4-fluorescent protein fusion amino acid sequence (i.e. the sequence shown in SEQ ID NO: 1) to express the aforementioned CSPG4-fluorescent protein fusion protein.

[0032] The present application also provides a kit for detecting the positive rate of CSPG4 CAR-T cells, comprising the aforementioned CSPG4-fluorescent protein fusion protein.

[0033] The CSPG4-fluorescent protein fusion protein provided by the present application has the following beneficial effects compared with the prior art: The CSPG4-fluorescent protein fusion protein provided by the present application has the following beneficial effects compared with the prior art: BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 It is a pLVX-hCSPG4-fluorescent protein vector map.

[0035] Figure 2 It is a CSPG4-fluorescent protein fusion protein flow cytometry detection of CSPG4 CAR-T cell positive rate results.

[0036] Figure 3 It is a CSPG4-fluorescent protein fusion protein flow cytometry detection of various CSPG4 CAR-T cell positive rate results.

[0037] Figure 4 It is a comparison of the CSPG4-fluorescent protein fusion protein fluorescent protein method, Protein L-PE Streptavidin method and PE anti-human IgG-(Fab')2 method by flow cytometry detection of CSPG4 CAR-T cell positive rate results. DETAILED DESCRIPTION

[0038] The present application will be described in detail below in conjunction with the drawings and examples.

[0039] Example 1, preparation of CSPG4-fluorescent protein fusion protein The CSPG4-fluorescent protein fusion protein provided by the present application has the following beneficial effects compared with the prior art: (1) DNA polymerase (Novagen Phanta EVO Super-Fidelity DNA Polymerase, item number P503) forward primer hCSPG4-4-F (SEQ ID NO: 5) and reverse primer hCSPG4-4-R (SEQ ID NO: 6) were used to amplify the PCR product hCSPG4 (SEQ ID NO: 8) containing homologous arms and hCSPG4-4 (1425-1650 AA) (SEQ ID NO: 7) coding sequence, using LN229 cell cDNA as a template; forward primer EGFP-F (SEQ ID NO: 9) and reverse primer EGFP-R (SEQ ID NO: 10) were used to amplify the PCR product EGFP (SEQ ID NO: 12) containing homologous arms, linker coding sequence and EGFP (SEQ ID NO: 11) coding sequence, using pEGFP-N1 (Shengong pEGFP-N1, item number B540189-0001) as a template; the forward primer IL2-SP-F (SEQ ID NO: 13) and the reverse complementary sequence primer IL2-SP-R (SEQ ID NO: 14) containing homologous arms, Kozak sequence and IL-2 signal peptide (SEQ ID NO: 2) coding sequence were synthesized, and the forward and reverse primers were used to form double-stranded DNA product IL2-SP (SEQ ID NO: 15) by denaturation and annealing; the three fragments were recombined into the pLVX-IRES-Puro (Takara, item number 632183) vector digested by EcoR I and BamH I by using the recombination enzyme (Abclonal MultiF Seamless Assembly Mix recombination enzyme, item number RK21020) premix, and the lentiviral expression vector pLVX-hCSPG4-EGFP (SEQ ID NO. 2) containing the fusion protein of the CSPG4 partial extracellular domain with IL-2 signal peptide at the N-terminus and EGFP connected by (GGGGS) 3 linker was constructed. Figure 1 (2) The pLVX-hCSPG4-EGFP vector obtained in step 1 was co-transfected into HEK293 cells with pMD2.G and psPAX2 at a ratio of 4:3:1. After 48 and 72 hours of transfection, the supernatant of HEK293 cells was collected, filtered by 0.45 μm filter to obtain lentivirus, and the lentivirus was stored at -80℃ for use in infecting HEK293 cells or other cells; (3) The lentivirus obtained in step 2 was used to infect HEK293 cells, and after 48 hours, the puromycin selection obtained a cell strain stably expressing hCSPG4-EGFP; ​(4) 2 x 150 mm hCSPG4-HEK293 stable cell line culture supernatant and cell precipitate, after removing cell debris by centrifugation and incubating with 2 ml Protein A (Repligen CaptivA PriMAB, item number CA-PRI-0100) at 4°C for 3 hours, each 10 ml of lysate was washed 3 times, and the purified protein was eluted with 4 ml of 0.1 M Glycine-HCl (pH 2.7), and immediately neutralized with 600 μl of 0.5 M Tris-HCl (pH 8.0). The eluted protein was replaced and concentrated in 500 μl of 1 x PBS using an ultrafiltration tube to obtain the hCSPG4-EGFP fusion protein; (5) The concentration of the obtained hCSPG4-EGFP fusion protein was determined by BCA method, diluted with PBS to a final concentration of 0.1 mg / ml, and added with a final concentration of 0.01% sodium azide and 10% glycerol, and stored at -20°C for detection of CSPG4 CAR expression.

[0040] Example 2, hCSPG4-EGFP fusion protein detects the positive rate of CSPG4 CAR-T cells Test method and results: (1) From the blood donated by healthy people, mononuclear cells were obtained by Percoll density gradient centrifugation, activated by CD3 and CD28 antibodies, and infected with hCSPG4-LH.28 CAR retrovirus (reference patent CN114249832A) and hCSPG4, mCSPG4-5 (reference patent CN111575241A) to obtain hCSPG4-LH.28 CAR-T, hCSPG4 CAR-T, and mCSPG4-5 CAR-T cells; (2) After 48 hours of infection, 1 x 106 of hCSPG4-LH.28 CAR-T cells and control T cells were centrifuged at 400 g for 3 minutes, the supernatant was discarded, and the cells were resuspended in 100 μl of PBS. The hCSPG4-IgG1-fluorescent protein fusion protein obtained in Example 1 was added at a ratio of 1:100, 1:200, 1:500, 1:1000, and 1:5000, respectively, and incubated at 4°C for 30 minutes; (3) After washing with PBS, the positive rate was detected by Beckman flow cytometry, and the results are shown in Figure 2 and Table 1.

[0041] The results show that: 1. The positive rate of the uninfected control NT cell group is low, indicating that the false positive rate of the hCSPG4-EGFP fusion protein detection is low; 2、And hCSPG4-LH.28 CAR-T cells, the positive rate of hCSPG4-EGFP fusion protein dose gradient, and at 1:200 with the amount of saturation, indicating that hCSPG4-EGFP fusion protein can effectively detect the positive rate of CSPG4 CAR-T cells.

[0042] Table 1. CSPG4 CAR-T cell positive rate The same method, using 1:200 hCSPG4-IgG1-EGFP fusion protein to detect the positive rate of hCSPG4 CAR-T, mCSPG4-5 CAR-T cells, the results are shown in Figure 3 and Table 2.

[0043] Table 2. CSPG4 CAR-T cell positive rate The results show that: hCSPG4-EGFP fusion protein can effectively detect the positive rate of a variety of CAR-T cells targeting CSPG4, wide range of applications.

[0044] Example 3, hCSPG4-EGFP fusion protein method, Protein L-PE Streptavidin method and PE anti-human IgG-(Fab')2 method for detecting hCSPG4-LH.28 CAR cell positive rate comparison Test method and results: (1) The hCSPG4-LH.28 CAR cells prepared by the method of Example 2 were used for comparison of the three detection methods; (2) hCSPG4-IgG1-EGFP fusion protein method according to the steps of Example 2; (3) Protein L-PE Streptavidin method, the collected hCSPG4-LH.28 CAR cells were resuspended in 100 μl PBS containing 1 μg Biotin-Protein L (Kingsway, Catalog: M00097) and incubated at 4°C for 30 minutes. After washing with PBS, the cells were resuspended in 100 μl PBS containing PE Streptavidin (Biolegend, Catalog: 405203) and incubated at 4°C for 30 minutes. After washing with PBS, the positive rate was detected by Beckman flow cytometry; (4) PE anti-human IgG - (Fab')2 method, the collected hCSPG4-LH.28 CAR cells, add 100 μl PBS containing goat F(ab')2 anti-human IgG - (Fab')2 (PE) (Abcam, ab98606) to resuspend the cells, incubate at 4°C for 30 minutes, wash with PBS, and then use Beckman flow cytometry to detect the positive rate. The results are shown in Figure 4 and Table 3.

[0045] Table 3. CSPG4 CAR-T cell positive rate The results show that: 1. The CSPG4-EGFP fusion protein method proposed by the present disclosure has accurate positive rate, low false positive rate and good repeatability, and the results of three repetitions are stable, and the detection positive rate is about 50%, which has obvious advantages compared with the latter two; 2. The Protein L-PE Streptavidin method has high false positive rate and poor repeatability, and the uninfected NT control group has a high positive rate, and the positive rate fluctuates greatly; 3. The PE anti-human IgG - (Fab')2 method can only recognize human IgG - (Fab')2, and cannot recognize hCSPG4-LH.28 CAR after hinge modification, so the application range of CSPG4 CAR positive detection is small, and there is a serious false positive.

[0046] The above is only the preferred embodiment of the present application, and the protection scope of the present application is not limited to the above examples. Any technical solutions falling within the scope of the present application should be considered within the protection scope of the present application. It should be noted that for ordinary skilled persons in the art, some improvements and refinements without departing from the principles of the present application should also be considered within the protection scope of the present application.

Claims

1. A CSPG4-fluorescent protein fusion protein, characterized by, The CSPG4 antigen, a linker, a fluorescent protein, and a purification tag; the fluorescent protein includes far-red fluorescent protein mPlum; further includes red fluorescent protein mCherry, tdTomato, mStrawberry, J-Red, DsRed-monomer, mRuby, mApple, mRaspberry, mKate2; further includes orange fluorescent protein mOrange, mKO2, Kusabira Orange2, TagRFP, DsRed, mTangerine; further includes yellow fluorescent protein mCitrine, Venus, mVenus, YPet, EYFP, Topaz, ZsYellow1, mBanana; further includes green fluorescent protein Emerald, EGFP, Superfolder GFP, ZsGreen; further includes cyan fluorescent protein ECFP, CyPet, mCFPm, Cerulean, mTurquoise, mTFP1; further includes ultraviolet fluorescent protein T-Sapphire, or blue fluorescent protein EBFP, mTagBFP, mTagBFP2.

2. The CSPG4-fluorescent protein fusion protein of claim 1, wherein, The CSPG4 antigen is selected from full-length protein or CSPG4 complete extracellular domain or CSPG4 partial extracellular domain.

3. The fusion protein of claim 2, wherein, A linker is included between the CSPG4 antigen and the fluorescent protein, and the linker is a flexible linker, which includes (GGGGS)n, (GS)n, (G)n; The antigen is located at the N-terminal of the linker, and the fluorescent protein is located at the C-terminal of the linker; or the antigen is located at the C-terminal of the linker, and the fluorescent protein is located at the N-terminal of the linker; one or more purification tags are included at the N-terminal or C-terminal of the fusion protein, and the purification tag includes Poly-His tag, Poly-Arg tag, GST tag, MBP tag, FLAG tag, Strep-tag_II tag, Twin-Strep-tag tag, S-tag tag, Avi tag, Halo tag, FC tag, or SNAP tag; the amino acid sequence of the CSPG4-fluorescent protein fusion protein is shown as SEQ ID NO: 1, and the nucleic acid sequence of the CSPG4-fluorescent protein fusion protein and its complementary sequence is shown as SEQ ID NO:

2.

4. The CSPG4-fluorescent protein fusion protein of claim 3, wherein, The N-terminal of the fusion protein includes a secretory signal peptide, and the secretory signal peptide is an IL2 signal peptide, the amino acid sequence of which is shown as SEQ ID NO: 3, and the nucleotide sequence of the secretory signal peptide and its complementary sequence is shown as SEQ ID NO:

4.

5. A nucleic acid construct, characterized in that, The nucleotide sequence encoding the secretory signal peptide according to claim 6, and the nucleotide sequence encoding the CSPG4-fluorescent protein fusion protein according to claim 5 are included.

6. A virus, characterized in that, The nucleic acid construct according to claim 7 is included, and the virus is a lentivirus.

7. A stable cell line, characterized in that, A stable cell strain which stably expresses the CSPG4-fluorescent protein fusion protein according to claim 1 is a HEK293 cell strain.

8. A method of producing the CSPG4-fluorescent protein fusion protein according to any one of claims 1 to 10, characterized by, The following steps are included: The following steps are included: Chemically synthesizing an amino acid sequence of the CSPG4-fluorescent protein fusion protein according to claim 1, or expressing the CSPG4-fluorescent protein fusion protein according to claim 1 using the stable cell line according to claim 7.

9. Use of the CSPG4-fluorescent protein fusion protein according to claim 1 in the detection of the positive rate of CSPG4 CAR-T cells.

Images