A kk-lc-1 targeted binding protein, derivatives, kits and uses thereof

Abstract

This invention discloses a KK-LC-1 targeting binding protein and its derivatives, a kit, and applications. 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. The KK-LC-1 targeting binding protein of this invention has a molecular weight of only about 17 kDa, approximately one-tenth that of an antibody, exhibiting good tissue penetration. Furthermore, its production process is simple and yields high quantities. The targeting binding protein of this invention demonstrates good targeting activity against KK-LC-1-positive tumors both in vivo and in vitro, and can be used for targeted tumor drug delivery, representing a potential drug for tumor immunotherapy. Simultaneously, a KK-LC-1 serum detection kit was developed using the targeting binding protein, enabling quantitative detection of KK-LC-1 levels in the human body.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a KK-LC-1 targeted binding protein, its derivatives, a reagent kit, and their applications. Background Technology

[0002] KK-LC-1 (Kita-Kyushu lung cancer antigen-1, also known as CT83 or cxorf61), located on chromosome Xq22, is a 556 bp protein belonging to the cancer-testis antigen family. Its protein molecular weight is 12.784 kDa, and it is sublocalized in 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. Furthermore, small molecule compounds targeting KK-LC-1 can downregulate ALDH1 expression, thus achieving tumor regression. This suggests that KK-LC-1 may play an important regulatory role in tumorigenesis and development.

[0003] Under physiological conditions, KK-LC-1 has an extremely narrow expression profile, being expressed only in testicular tissue and not in other healthy tissues. However, in tumor tissues, it has been detected with high expression rates in various cancer types, including lung cancer, gastric cancer, breast cancer, and liver cancer, making it a potential target for targeted therapy. For example, in gastric cancer, KK-LC-1 is expressed in nearly 80% of gastric cancer tissues but not in normal gastric tissues. Furthermore, gastric cancer patients with high KK-LC-1 expression have a poorer prognosis, suggesting that KK-LC-1 is a potential target for treating gastric cancer. Current research has demonstrated that when a peptide-conjugated drug targeting KK-LC-1 is infused into tumor-bearing mice via the tail vein, subcutaneous inoculation with gastric cancer cells expressing high KK-LC-1 significantly inhibits tumor growth and prolongs survival.

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

[0005] In summary, KK-LC-1 is a potential target for anti-tumor drug development. However, current antibodies against KK-LC-1 often suffer from problems such as large molecular weight, poor penetration into solid tumors, strong immunogenicity requiring humanization, complex production processes, poor stability, high requirements for transportation and storage, and high research and development and usage costs. Therefore, it is crucial to develop low molecular weight targeted binding proteins with high affinity and high penetration.

[0006] Meanwhile, existing literature has confirmed a positive correlation between serum KK-LC-1 expression levels and tumor burden in lung adenocarcinoma patients, suggesting that it may serve as a good serum biomarker for tumors. However, there are currently no in vitro diagnostic reagents for KK-LC-1 available for clinical use. Therefore, developing a rapid, accurate, and highly sensitive detection kit for KK-LC-1 levels in the human body for the prediction, diagnosis, and prognostic monitoring of KK-LC-1 positive tumors has significant clinical application value. Summary of the Invention

[0007] To address the problems in the prior art, this invention provides a KK-LC-1 targeting binding protein that can specifically bind to the KK-LC-1 protein and can efficiently target KK-LC-1 positive tumors in both in vivo and in vitro experiments. Based on this, this invention provides a derivative of the KK-LC-1 targeting binding protein, a CBA kit for quantitative detection of KK-LC-1, and applications of the KK-LC-1 targeting binding protein.

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

[0009] A KK-LC-1 targeting binding protein, wherein the KK-LC-1 targeting binding protein is a protein that specifically binds 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, or SEQ ID No. 5.

[0010] Preferably, the amino acid sequence of the KK-LC-1 target-binding protein is 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 are any amino acids.

[0011] A derivative of a KK-LC-1 targeting and binding protein, including bacteriophages, fluorescent dye conjugates, chelates, and radionuclide complex conjugates.

[0012] A kit for targeting and binding KK-LC-1 protein, the kit being the CBA kit.

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

[0014] Furthermore, the preparation method of the fluorescent magnetic bead-labeled 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 rack. 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 rack. A blocking agent is added, and after blocking, the magnetic beads are separated using a magnetic rack to obtain the final product.

[0015] Furthermore, the preparation method of the biotin-labeled KK-LC-1 targeting binding protein is as follows: add the KK-LC-1 targeting binding protein to a biotin solution, mix well, and after the labeling reaction is completed, purify and separate 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 the quantitative detection of KK-LC-1.

[0017] Furthermore, the method for quantitative detection of KK-LC-1 using the CBA kit includes the following steps: adding the serum or plasma sample to be tested, along with biotin-labeled KK-LC-1 targeting and binding protein, to fluorescently labeled KK-LC-1 targeting and binding protein, and vortexing to mix; magnetic separation after the reaction; adding SA-PE antibody, vortexing to mix, magnetic separation after the reaction, detecting fluorescence intensity, plotting a standard curve, and calculating the concentration of KK-LC-1 in the serum or plasma sample to be tested.

[0018] Application of a KK-LC-1 targeting protein, wherein the KK-LC-1 targeting protein is used to prepare a KK-LC-1 positive tumor-targeting drug.

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

[0020] Protein-conjugated cytotoxic drugs, such as MMAE, PE, maytansine, camptothecin compounds, cazithromycin compounds, etc.

[0021] It incorporates immune cytokines such as IL-2, IL-7, IL-15, IL-21, and TNF-α.

[0022] Immune cell binding proteins such as fusion CD3 binding peptides, proteins or antibodies, CD28 binding peptides, proteins or antibodies, CD16A binding peptides, proteins or antibodies, and NKp46 binding peptides, proteins or antibodies.

[0023] And transgenic modified immune cells containing the KK-LC-1 targeting protein gene and the KK-LC-1 targeting protein sequence, such as CAR-T and CAR-NK.

[0024] Furthermore, the application of the derivatives of the KK-LC-1 targeting binding protein and various derivatives containing the KK-LC-1 targeting binding sequence 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] This invention provides a novel, high-affinity KK-LC-1 specific targeting protein with a molecular weight of only about 17 kDa, approximately one-tenth that of an antibody. It exhibits excellent tissue penetration and is produced in a simple and high-yield manner. The targeting protein of this invention demonstrates good targeting activity against KK-LC-1-positive tumors both in vivo and in vitro, making it suitable for targeted drug delivery to tumors and a potential agent for tumor immunotherapy. Simultaneously, a KK-LC-1 serum detection kit was developed using this targeting protein, enabling quantitative detection of KK-LC-1 levels in the human body. Attached Figure Description

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

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

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

[0030] Figure 4 This is the standard curve graph in Example 4;

[0031] Figure 5 This is a graph showing the KK-LC-1 content in the serum of 10 patients in Example 4. Detailed Implementation

[0032] The present invention will be further described below with reference to embodiments.

[0033] The KK-LC-1 specific target-binding protein involved in this invention is synthesized through an E. coli expression system, which is simple to synthesize, has high yield, and is inexpensive. Example 1

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

[0035] 1. Using an E. coli expression system, target-binding proteins 2, 3, 4, and 5 were synthesized, and negative control target-binding protein 1 was expressed. The proteins were purified using a nickel column, and endotoxins were removed to <0.1 EU / ug using a GenScript endotoxin removal kit.

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

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

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

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

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

[0041] 2. Coat KK-LC-1 protein onto a flat-bottomed 96-well plate, block it, and incubate with the same concentration of target-binding proteins 1, 2, 3, 4, and 5 for 2 hours. Wash away the target-binding proteins with PBS containing 0.5% Tween 20 (0.5% PBST), then add diluted anti-His secondary antibody and continue incubation for 1 hour. Wash away 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 changes from colorless to blue. Stop the reaction by adding 1M dilute hydrochloric acid. The solution changes from blue to yellow.

[0042] 3. Use an ELISA reader to measure absorbance at OD450;

[0043] like Figure 1 As shown, compared with the negative control group, the target-binding proteins 2, 3, 4, and 5 showed significant binding 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 Example 1

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

[0056] 1. KK-LC-1 positive cell line MKN45 and KK-LC-1 negative cell line SK-HEP-1 were cultured and adhered in the laboratory, then plated onto confocal microscopy dishes. After blocking, the cells were incubated with target-binding protein 1 for 1 hour. Unbound protein was then washed away, and diluted anti-His secondary antibody was added, followed by incubation for another 1 hour. After washing, the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) dye.

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

[0058] like Figure 2 As shown, compared with negative cells, the target-binding protein significantly binds to MKN45 cells. Example 3

[0059] Targeting and validation of the binding protein in mice

[0060] 1. Mix and incubate the target-binding protein 1 with the fluorescent dye cy5 to obtain fluorescent target-binding protein 1.

[0061] 2. Culture the MKN45 cell line until it adheres to the culture vessel, then select 5-week-old nude mice and subcutaneously inoculate them with 10 saturates in the groin area. 6 The cells were examined and observed for several days until the tumor grew into a mass.

[0062] 3. Fluorescent targeting-binding proteins were injected into tumor-bearing mice via tail vein injection, and near-infrared in vivo imaging was performed.

[0063] like Figure 3 As shown, the target-binding protein can be targeted and enriched in tumor tissue. Example 4

[0064] Preparation and application of KK-LC-1 detection kit for CBA

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

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

[0067] 3. All detection steps must be performed in the dark. Dilute the fluorescent magnetic bead-labeled KK-LC-1 targeting protein 1:500 and the biotin-labeled KK-LC-1 targeting protein 1:1000. Mix the KK-LC-1 protein standard, diluted biotin-labeled KK-LC-1 targeting protein, and diluted fluorescent magnetic bead-labeled KK-LC-1 targeting protein at a volume ratio of 1:1:1 (50 μL each), vortex to mix, and react at room temperature for 1 hour. After the reaction, perform magnetic separation. Add SA-PE antibody, vortex to mix, and react at room temperature for 30 min to 1 h. After the reaction, perform magnetic separation and detect the fluorescence intensity using a flow cytometer. Plot a standard curve based on the fluorescence intensity.

[0068] Standard curve such as Figure 4 As shown, R 2 The value was 0.99, demonstrating that the kit can quantitatively detect different concentrations of KK-LC-1 protein.

[0069] 4. All detection steps must be performed under light-protected conditions. Serum samples from 10 cancer patients were selected (2 patients showed positive KK-LC-1 expression in pathological results, and 8 patients showed 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 at a volume ratio of 1:1:1 (50 μL each), vortexed, and reacted at room temperature for 1 hour. After the reaction, magnetic separation was performed. SA-PE antibody was added, vortexed, and reacted at room temperature for 30 min to 1 h. After the reaction, magnetic separation was performed, and the fluorescence intensity was detected by flow cytometry. The protein content in the serum was calculated according to the standard curve.

[0070] The KK-LC-1 levels in the serum of 10 patients were as follows: Figure 5 As shown, the serum protein content of patients with positive pathological results was significantly higher than that of patients with negative results, suggesting that the kit can detect the expression level of KK-LC-1 in the patient's serum.

[0071] The KK-LC-1 targeting protein can be used in any serum (plasma) assay kit, such as the CBA kit and chemiluminescence assay kit. This example focuses on the CBA kit.

[0072] The method for quantitative detection of KK-LC-1 using the CBA kit includes the following steps: adding the serum or plasma sample to be tested, along with biotin-labeled KK-LC-1 targeting and binding protein, to fluorescently labeled KK-LC-1 targeting and binding protein, and vortexing to mix; magnetic separation after the reaction; adding SA-PE antibody, vortexing to mix, magnetic separation after the reaction, detecting fluorescence intensity, plotting 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 of the present invention is as follows: The present invention adopts chemiluminescent immunoassay technology based on double antibody sandwich method. Biotin-labeled KK-LC-1 targeting binding protein and serum or plasma sample of the person to be tested are added to a solution of KK-LC-1 targeting binding protein labeled with APC fluorescent magnetic beads. The KK-LC-1 protein in the sample will fully bind to the two targeting binding proteins to form an antibody-antigen-antibody APC magnetic microparticle complex. After magnetic separation and washing of the magnetic microparticles, SA-PE fluorescent antibody is added to form a PE fluorescence-antibody-antigen-antibody-magnetic microparticle complex. After magnetic separation and washing, the PE fluorescence intensity in the magnetic beads is detected by flow cytometry. The concentration of KK-LC-1 in the sample is directly proportional to the PE fluorescence. The concentration of KK-LC-1 in the sample can be calculated by plotting a standard curve. Example 5

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

[0075] The KK-LC-1 positive tumor-targeting drugs include:

[0076] Protein-conjugated cytotoxic drugs, such as MMAE, PE, maytansine, camptothecin compounds, cazithromycin compounds, etc.

[0077] It incorporates immune cytokines such as IL-2, IL-7, IL-15, IL-21, and TNF-α.

[0078] Immune cell binding proteins such as fusion CD3 binding peptides, proteins or antibodies, CD28 binding peptides, proteins or antibodies, CD16A binding peptides, proteins or antibodies, and NKp46 binding peptides, proteins or antibodies.

[0079] And transgenic modified immune cells containing the KK-LC-1 targeting protein gene and the KK-LC-1 targeting protein sequence, such as CAR-T and CAR-NK.

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

[0081] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A KK-LC-l targeted binding protein, characterized in that, KK-LC-1 targeting binding protein is a protein that specifically binds to KK-LC-1 protein. The KK-LC-1 targeting binding protein is used to prepare KK-LC-1 positive tumor-targeting drugs. The protein sequence of KK-LC-1 targeting binding protein is shown in SEQ ID No. 3.

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