KK-LC-1-targeting binding protein and derivative thereof, kit, and use

Abstract

Provided are a KK-LC-1-targeting binding protein and a derivative thereof, a kit, and a use. The protein sequence of the KK-LC-1-targeting binding protein is as set forth in one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. The molecular weight of the KK-LC-1-targeting binding protein is only about 17 kD, which is about one tenth of that of an antibody, thereby achieving good tissue penetrability. In addition, the production process is simple, and the yield is high. The targeting binding protein has good targeting performance both in vitro and in vivo against tumors showing positive KK-LC-1 expression, can be used for tumor-targeted administration, and is a potential drug for tumor immunotherapy. In addition, a KK-LC-1 serum detection kit is researched and developed by using the targeting binding protein, so that the content of KK-LC-1 in a human body can be quantitatively detected.

Description

A KK-LC-1 targeted binding protein and its derivatives, kit and application Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a KK-LC-1 targeted binding protein, derivatives, a kit and applications thereof. Background Art

[0002] KK-LC-1 (Kita-Kyushu lung cancer antigen-1, also known as CT83 or cxorf61), located on chromosome Xq22, consists of 556 base pairs and is a member of the cancer-testis antigen family. Its molecular weight is 12.784 kDa and it is sublocalized to the cell membrane and cytoplasm. KK-LC-1 has been reported to regulate ALDH1 expression, thereby indirectly mediating the progression of triple-negative breast cancer. Small molecule compounds targeting KK-LC-1 can downregulate ALDH1 expression and thus induce tumor regression, suggesting that KK-LC-1 may play an important regulatory role in tumor development and progression.

[0003] Under physiological conditions, KK-LC-1 has a very narrow expression spectrum, being expressed only in testicular tissue and absent in other healthy tissues. However, in tumor tissue, it has been detected at high levels in a variety of cancer types, including lung, gastric, breast, and liver cancers. This makes it a promising target for targeted therapy. For example, in gastric cancer, KK-LC-1 is expressed in nearly 80% of gastric cancer tissues, but absent in normal gastric tissue. Furthermore, patients with gastric cancer that overexpress KK-LC-1 have a poor prognosis, suggesting that KK-LC-1 is a potential target for gastric cancer treatment. Studies have demonstrated that when a KK-LC-1-targeting peptide-drug conjugate is infused into tumor-bearing mice via the tail vein, tumor growth is significantly suppressed and survival is significantly prolonged in mice subcutaneously inoculated with gastric cancer cells that overexpress KK-LC-1.

[0004] Given that targeted therapy targeting KK-LC-1 has significant effects and low toxic side effects, treatment options targeting KK-LC-1 can be developed in multiple directions, including photosensitivity therapy, antibody-drug conjugates, vaccines, CAR-T, TCR-T, monoclonal antibody drugs, etc. Among them, TCR-T developed by the National Cancer Institute of the United States is undergoing Phase I clinical trials.

[0005] In summary, KK-LC-1 is a potential target in the development of anti-tumor drugs. However, most of the current antibodies targeting KK-LC-1 have the problems of large molecular weight, poor penetration into solid tumors, strong immunogenicity requiring humanization, complex production process, poor stability, high requirements for transportation and storage, and high R&D and use costs. Therefore, the development of low-molecular-weight targeted binding proteins with high affinity and high permeability is particularly critical.

[0006] Meanwhile, existing literature confirms that serum KK-LC-1 expression levels in lung adenocarcinoma patients are positively correlated with tumor burden, suggesting its potential as a useful serum tumor marker. However, there are currently no in vitro diagnostic assays for KK-LC-1 available clinically. Therefore, developing a rapid, accurate, and highly sensitive assay for KK-LC-1 levels in the human body to predict, diagnose, and monitor the prognosis of KK-LC-1-positive tumors would be of great clinical value. Summary of the Invention

[0007] In order to solve the problems in the prior art, the present invention provides a KK-LC-1 targeted binding protein, which can specifically bind to the KK-LC-1 protein and effectively target KK-LC-1-positive tumors in both in vivo and in vitro experiments. On this basis, the present invention provides a derivative of the KK-LC-1 targeted binding protein, a CBA kit for quantitatively detecting KK-LC-1, and the application of the KK-LC-1 targeted binding protein.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] A KK-LC-1 targeting binding protein, the KK-LC-1 targeting binding protein is a protein that can specifically bind to the KK-LC-1 protein, and the protein sequence of the KK-LC-1 targeting binding protein is shown in one of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5.

[0010] Preferably, the amino acid sequence of the KK-LC-1 targeting binding protein is as shown in SEQ ID No. 2: wherein X at positions 30, 32, 33, 35, 41, 42, 63, 65, 66, 68, 74, 75, 96, 98, 99, 101, 107 and 108 is any amino acid.

[0011] A derivative of a KK-LC-1 targeting binding protein comprises a phage, a fluorescent dye coupling product, a chelate, and a radionuclide complex coupling product.

[0012] A kit for KK-LC-1 targeted binding protein is a CBA kit.

[0013] Furthermore, the CBA kit includes a fluorescent magnetic bead-labeled KK-LC-1 targeting binding protein and a biotin-labeled KK-LC-1 targeting binding protein.

[0014] Furthermore, the preparation method of the fluorescent magnetic bead-labeled targeted KK-LC-1 targeting binding protein is as follows: EDC and NHS are added to the chemiluminescent magnetic beads, mixed, and after activation, the magnetic beads are separated using a magnetic stand, the KK-LC-1 targeting binding protein is added to the magnetic beads, mixed, and after the labeling reaction is completed, the magnetic beads are separated using a magnetic stand, a blocking agent is added, and after blocking, the magnetic stand is separated to obtain the final product;

[0015] Furthermore, the preparation method of the biotin-labeled KK-LC-1 targeted binding protein is as follows: adding the KK-LC-1 targeted binding protein to a biotin solution, mixing, and after the labeling reaction is completed, purifying and separating using a PD-10 column to obtain the final product.

[0016] An application of a KK-LC-1 targeting binding protein, wherein the KK-LC-1 targeting binding protein is used to prepare a KK-LC-1 targeting binding protein kit, and the KK-LC-1 targeting binding protein kit is used for quantitative detection of KK-LC-1.

[0017] Furthermore, the method for quantitatively detecting KK-LC-1 by the CBA kit includes the following steps: adding the serum or plasma sample to be tested and the biotin-labeled KK-LC-1 targeting binding protein to the KK-LC-1 targeting binding protein labeled with fluorescent magnetic beads, and shaking and mixing; magnetic separation after the reaction is completed; adding SA-PE antibody, shaking and mixing, magnetic separation after the reaction is completed, detecting the fluorescence intensity, drawing a standard curve, and calculating the concentration of KK-LC-1 in the serum or plasma sample to be tested.

[0018] An application of a KK-LC-1 targeting binding protein, wherein the KK-LC-1 targeting binding protein is used for preparing a KK-LC-1 positive tumor targeting drug.

[0019] Furthermore, the KK-LC-1 positive tumor targeting drug includes:

[0020] Protein-coupled cytotoxic drugs, such as MMAE, PE, maytansine, camptothecin compounds, calicheamicin compounds, etc.;

[0021] Fusion immune cytokines such as IL-2, IL-7, IL-15, IL-21, TNF-α, etc.;

[0022] Immune cell binding proteins such as fused CD3 binding peptides, proteins or antibodies, CD28 binding peptides, proteins or antibodies, CD16A binding peptides, proteins or antibodies, NKp46 binding peptides, proteins or antibodies;

[0023] As well as genetically modified immune cells containing KK-LC-1 targeting binding protein genes and KK-LC-1 targeting binding protein sequences, such as CAR-T, CAR-NK, etc.

[0024] Furthermore, the derivatives of the KK-LC-1 targeting binding protein and various derivative products containing the KK-LC-1 targeting binding sequence are used in the preparation of KK-LC-1 positive tumor targeting drugs.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides a novel, high-affinity, KK-LC-1-specific targeted binding protein with a molecular weight of only approximately 17 kDa, approximately one-tenth that of an antibody. It exhibits excellent tissue penetration, a simple production process, and high yield. The targeted binding protein of the present invention has excellent targeting properties for KK-LC-1-positive tumors both in vivo and in vitro, can be used for tumor-targeted drug delivery, and is a potential drug for tumor immunotherapy. Furthermore, a KK-LC-1 serum detection kit has been developed using the targeted binding protein, which can quantitatively detect KK-LC-1 levels in the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is an absorbance graph of targeted binding proteins 1, 2, 3, 4, and 5 in Example 1;

[0028] FIG2 is a fluorescence image of a cell plate observed under a confocal microscope in Example 2;

[0029] FIG3 is a diagram showing the targeted enrichment of the targeted binding protein in tumor tissue in Example 3;

[0030] FIG4 is a standard curve diagram in Example 4;

[0031] FIG5 is a graph showing the KK-LC-1 levels in the serum of 10 patients in Example 4. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with the embodiments.

[0033] The KK-LC-1 specific targeting binding protein involved in the present invention is synthesized through an Escherichia coli expression system, and has the advantages of simple synthesis, high yield and low cost.

[0034] Example 1: ELISA detection of the binding of the targeted binding protein to the KK-LC-1 molecule:

[0035] 1. Use the E. coli expression system to synthesize the following target binding proteins 2, 3, 4, and 5, and express the negative control target binding protein 1. Purify using a nickel column and remove endotoxins using the GenScript Endotoxin Removal Kit to <0.1EU / ug:

[0036] Targeted binding protein 1 (SEQ ID No. 1 with a histidine tag at the N-terminus)

[0037] Targeted binding protein 2 (SEQ ID No. 2 with a histidine tag at the N-terminus)

[0038] Targeted binding protein 3 (SEQ ID No. 3 with a histidine tag at the N-terminus)

[0039] Targeted binding protein 4 (SEQ ID No. 4 with a histidine tag at the N-terminus)

[0040] Targeted binding protein 5 (SEQ ID No. 5 with a histidine tag at the N-terminus)

[0041] 2. Coat a flat-bottom 96-well plate with KK-LC-1 protein. After blocking, incubate with equal concentrations of target binding proteins 1, 2, 3, 4, and 5 for 2 hours. Wash off each set of target binding proteins with PBS containing 0.5% Tween 20 (0.5% PBST). Add diluted anti-His secondary antibody and continue incubation for 1 hour. Wash off the anti-His secondary antibody with 0.5% PBST, then add TMB solution. Incubate at room temperature in the dark for 2-5 minutes until the solution turns from colorless to blue. Terminate the reaction with 1M dilute hydrochloric acid. The solution will turn from blue to yellow.

[0042] 3. Use a microplate reader to measure the absorbance at OD450;

[0043] As shown in Figure 1 , compared with the negative control group, targeted binding proteins 2, 3, 4, and 5 were significantly bound to the KK-LC-1 protein.

[0044] The specific protein sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, and SEQ ID No.5 are as follows:

[0045] SEQ ID No. 1:

[0046] DLGKKLLEAARAGQDDEVRILMANGAPFTADAAGATPLHLAAAAGHLEIVEVLLKYGADVNAADAAGATPLHLAAAAGHLEIVEVLLKYGADVNAADAAGATPLHLAAAAGHLEIVEVLLKYGADVNAQDKSGKTSADLAADAGHEDIAEVLQKAA

[0047] SEQ ID No. 2:

[0048] DLGKKLLEAARAGQDDEVRILMANGAPFTFDLFGLTLHLAAQWGHLEIVEVLLKYGADVNADDDWGDTPLHLAAQDGHLEIVEVLLKYGADVNAWDMFGITPLHLAATLGHLEIVEVLLKYGADVNAQDKYGKTPADMAADAGHEDIAEVLQKAA

[0049] SEQ ID No.3:

[0050] DLGKKLLEAARAGQDDEVRILMANGAPFTEDAYETPLHLAAYWGHLEIVEVLLKYGADVNARDFWGFTPLHLAAYLGHLEIVEVLLKYGADVNAWDSFGITPLHLAAAQGHLEIVEVLLKYGADVNAQDKSGKTPADLAADAGHEDIAEVLQKAA

[0051] SEQ ID No.4:

[0052] DLGKKLLEAARAGQDDEVRILMANGAPFTEDWVDTPMHLAAFSGHLEIVEVLLKYGADVNAYDAEGITPLHLAARAGHLEIVEVLLKYGADVNADDMLGFTPLHLAAIDGHLEIVEVLLKYGADVNAHDKSGKTPADLAADAGHEYIAEVLQKAA

[0053] SEQ ID No.5:

[0054] DLGKKLLEAARAGQDDEVRILMANGAPFTDDIKGVTPLHLAAIMGHLEIVEVLLKYGADVNAMDLLGHTPLHLAALQGHLEIVEVLLKYGADVNADDWQGKTPLHLAAVMGHLEIVEVLLKYGADVNAQDKSGKTSADLAADAGHEDIAEVLQKAA

[0055] Example 2: Verification of the binding of the targeting binding protein to the cell surface:

[0056] 1. KK-LC-1-expressing cell line MKN45 and negative cell line SK-HEP-1 were cultured in the laboratory and allowed to adhere. Plated onto confocal microplates. After blocking, the cells were incubated with targeted binding protein 1 for 1 hour. Unbound proteins were then washed away, and diluted anti-His secondary antibody was added and incubated for another 1 hour. After washing, cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI).

[0057] 2. Observe the fluorescence of the cell plate under a confocal microscope.

[0058] As shown in FIG2 , compared with negative cells, the targeted binding protein has significant binding to MKN45 cells.

[0059] Example 3: Targeted validation of targeted binding proteins in mice

[0060] 1. Incubate the targeted binding protein 1 with the fluorescent dye cy5 to obtain the fluorescent targeted binding protein 1.

[0061] 2. Cultivate MKN45 cells until they adhere to the wall, select 5-week-old nude mice, and inoculate them subcutaneously in the groin for 10 6 The above cells were cultured and observed for several days until the tumor grew into a mass.

[0062] 3. Inject fluorescent targeting proteins into tumor-bearing mice via tail vein injection, and use near-infrared in vivo imaging.

[0063] As shown in FIG3 , targeted binding proteins can be targeted and enriched in tumor tissues.

[0064] Example 4: Preparation and Application of KK-LC-1 CBA Detection Kit

[0065] 1. Add EDC and NHS to APC fluorescent magnetic beads, mix well, separate the magnetic beads with a magnetic rack after activation, add KK-LC-1 targeting binding protein to the magnetic beads, separate the magnetic beads with a magnetic rack after the labeling reaction, add blocking agent, mix well and block, separate the magnetic rack after blocking, and obtain the fluorescent magnetic bead-labeled targeting KK-LC-1 targeting binding protein, which is stored at 4°C.

[0066] 2. Add KK-LC-1 targeting binding protein to biotin solution (feed ratio is 30:1), mix well, and after the labeling reaction is completed, purify and separate using PD-10 column to obtain biotin-labeled KK-LC-1 targeting binding protein, and store at 4°C.

[0067] 3. All detection steps must be performed in the dark. Dilute the fluorescent magnetic bead-labeled targeted KK-LC-1 binding protein at a ratio of 1:500 and the biotin-labeled KK-LC-1 binding protein at a ratio of 1:1000. Mix the KK-LC-1 protein standard, diluted biotin-labeled KK-LC-1 binding protein, and diluted fluorescent magnetic bead-labeled KK-LC-1 binding protein at a volume ratio of 1:1:1 (50 μl each), shake well, and react at room temperature for 1 hour. After the reaction, perform magnetic separation. Add SA-PE antibody, shake well, and react at room temperature for 30 minutes to 1 hour. After the reaction, perform magnetic separation. Detect the fluorescence intensity by upflow cytometry, and draw a standard curve based on the fluorescence intensity.

[0068] The standard curve is shown in Figure 4. 2 The value was 0.99, which proved that the kit could quantitatively detect different concentrations of KK-LC-1 protein.

[0069] 4. All testing steps must be performed in the dark. Serum samples from 10 cancer patients were collected (2 patients had pathologically positive KK-LC-1 expression, and 8 patients had negative expression). The serum or plasma sample to be tested, biotin-labeled KK-LC-1 targeting binding protein, and fluorescent magnetic bead-labeled KK-LC-1 targeting binding protein were mixed in a 1:1:1 volume ratio (50 μl each). The mixture was shaken to mix thoroughly and incubated at room temperature for 1 hour. After completion of the reaction, magnetic separation was performed. SA-PE antibody was added, shaken to mix thoroughly, and incubated at room temperature for 30 minutes to 1 hour. After completion of the reaction, magnetic separation was performed. Fluorescence intensity was measured by upflow cytometry, and the serum protein content was calculated based on the standard curve.

[0070] The KK-LC-1 levels in the serum of 10 patients are shown in FIG5 . The serum protein levels of pathology-positive patients were significantly higher than those of pathology-negative patients, indicating that the kit can detect the expression level of KK-LC-1 in the patient's serum.

[0071] The KK-LC-1 targeting binding protein can be used in any serum (plasma) detection kit, such as a CBA kit, a chemiluminescence kit, etc. This embodiment uses the CBA kit as an example for elaboration.

[0072] The method for quantitatively detecting KK-LC-1 by the CBA kit comprises the following steps: adding a serum or plasma sample to be tested and a biotin-labeled KK-LC-1 targeting binding protein to a KK-LC-1 targeting binding protein labeled with fluorescent magnetic beads, and shaking and mixing; performing magnetic separation after the reaction; adding an SA-PE antibody, shaking and mixing, performing magnetic separation after the reaction, detecting the fluorescence intensity, drawing a standard curve, and calculating the concentration of KK-LC-1 in the serum or plasma sample to be tested.

[0073] The detection principle of the CBA kit for quantitative detection of KK-LC-1 described herein is as follows: The invention utilizes a double-antibody sandwich chemiluminescent immunoassay technique. A biotin-labeled KK-LC-1 target binding protein and a human serum or plasma sample to be tested are added to a KK-LC-1 target binding protein solution labeled with APC fluorescent magnetic beads. The KK-LC-1 protein in the sample fully binds to the two target binding proteins, forming an antibody-antigen-antibody APC magnetic particle complex. After magnetic separation and washing of the magnetic particles, SA-PE fluorescent antibody is added to form a PE fluorescent-antibody-antigen-antibody-magnetic particle complex. Following magnetic separation and washing, the PE fluorescence intensity of the magnetic beads is detected by flow cytometry. The KK-LC-1 concentration in the sample is proportional to the PE fluorescence, and the KK-LC-1 concentration in the sample can be calculated by plotting a standard curve.

[0074] Example 5: Application of a KK-LC-1 targeting binding protein, wherein the KK-LC-1 targeting binding protein is used to prepare a KK-LC-1 positive tumor targeting drug.

[0075] The KK-LC-1 positive tumor targeting drug includes:

[0076] Protein-coupled cytotoxic drugs, such as MMAE, PE, maytansine, camptothecin compounds, calicheamicin compounds, etc.;

[0077] Fusion immune cytokines such as IL-2, IL-7, IL-15, IL-21, TNF-α, etc.;

[0078] Immune cell binding proteins such as fused CD3 binding peptides, proteins or antibodies, CD28 binding peptides, proteins or antibodies, CD16A binding peptides, proteins or antibodies, NKp46 binding peptides, proteins or antibodies;

[0079] As well as genetically modified immune cells containing KK-LC-1 targeting binding protein genes and KK-LC-1 targeting binding protein sequences, such as CAR-T, CAR-NK, etc.

[0080] The derivatives of the KK-LC-1 targeting binding protein and various derivative products containing the KK-LC-1 targeting binding sequence are used in the preparation of KK-LC-1 positive tumor targeting drugs.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A KK-LC-1 targeting binding protein, characterized in that The KK-LC-1 targeting binding protein is a protein that can specifically bind to the KK-LC-1 protein. The protein sequence of the KK-LC-1 targeting binding protein is shown in one of SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, and SEQ ID No.

5.

2. The derivative of KK-LC-1 targeting binding protein according to claim 1, characterized in that: Including bacteriophage, fluorescent dye coupling products, chelates, and radionuclide complex coupling products.

3. The kit for the KK-LC-1 targeting binding protein according to claim 1, characterized in that: The kit for the KK-LC-1 targeting binding protein is a CBA kit.

4. The kit for the KK-LC-1 targeting binding protein according to claim 3, characterized in that: The CBA kit comprises a fluorescent magnetic bead-labeled KK-LC-1 targeting binding protein and a biotin-labeled KK-LC-1 targeting binding protein.

5. The kit for the KK-LC-1 targeting binding protein according to claim 4, characterized in that: The preparation method of the fluorescent magnetic bead-labeled targeted KK-LC-1 targeting binding protein is as follows: EDC and NHS are added to the chemiluminescent magnetic beads, mixed, and the magnetic beads are separated by a magnetic frame after activation, and the KK-LC-1 targeting binding protein is added to the magnetic beads, mixed, and the magnetic beads are separated by a magnetic frame after the labeling reaction is completed, and a blocking agent is added, and the magnetic frame is separated after blocking to obtain the final product; The preparation method of the biotin-labeled KK-LC-1 targeting binding protein is as follows: adding the KK-LC-1 targeting binding protein to a biotin solution, mixing, and after the labeling reaction is completed, purifying and separating using a PD-10 column to obtain a final product.

6. The use of the KK-LC-1 targeting binding protein according to claim 4, characterized in that: The KK-LC-1 targeting binding protein is used to prepare a KK-LC-1 targeting binding protein kit, and the KK-LC-1 targeting binding protein kit is used for quantitative detection of KK-LC-1.

7. The use of the KK-LC-1 targeting binding protein according to claim 3, characterized in that: The method for quantitatively detecting KK-LC-1 by the CBA kit comprises the following steps: adding the serum or plasma sample to be detected and the biotin-labeled KK-LC-1 targeting binding protein to the KK-LC-1 targeting binding protein labeled with fluorescent magnetic beads, and shaking and mixing; magnetic separation after the reaction is completed; adding SA-PE antibody, shaking and mixing, magnetic separation after the reaction is completed, detecting the fluorescence intensity, drawing a standard curve, and calculating the concentration of KK-LC-1 in the serum or plasma sample to be detected.

8. The use of the KK-LC-1 targeting binding protein according to claim 2, characterized in that: The KK-LC-1 targeting binding protein is used for preparing KK-LC-1 positive tumor targeting drugs.

9. The use of the KK-LC-1 targeting binding protein according to claim 8, characterized in that: The KK-LC-1 positive tumor targeting drug includes: protein-coupled cytotoxic drugs, fused immune cytokines, immune cell binding proteins, and genetically modified immune cells containing KK-LC-1 targeting binding protein genes and KK-LC-1 targeting binding protein sequences.

10. The use of the KK-LC-1 targeting binding protein according to claim 2, characterized in that: The derivatives of the KK-LC-1 targeting binding protein and various derivative products containing the KK-LC-1 targeting binding sequence are used in the preparation of KK-LC-1 positive tumor targeting drugs.

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