Vκ4-1-IGLC polypeptide and its use

Abstract

The present invention discloses an immunoglobulin kappa light chain (Vκ4-1-IgLC) polypeptide having a unique Vκ4-1 and its use. The amino acid sequence of the Vκ4-1-IgLC polypeptide is shown in SEQ ID NO.1, and the polypeptide can be used as a target or marker for preparing pharmaceuticals for the diagnosis and / or treatment of tumors or inflammatory diseases, and can also be used for preparing inhibitors of the integrin-FAK signaling pathway.

Description

Technical Field

[0001] The present invention relates to the technical fields of immunology and cancer diagnosis and treatment, and particularly relates to an immunoglobulin kappa light chain (Vκ4-1-IgLC) polypeptide having a specific Vκ4-1 and its use.

Background Art

[0002] According to classical immunological theory, the basic structure of immunoglobulin (Ig) is composed of four peptide chains, that is, two identical heavy chains and two identical light chains are linked by disulfide bonds to form a complete Ig molecule. In addition to Ig molecules having a classical tetrapeptide chain structure, several light chains that do not bind to Ig heavy chains generally exist in normal human peripheral blood and various body fluids. These light chains are called free light chains (FLC), and exist in the form of monomers (molecular weight 22-27 kDa), covalently or non-covalently bound dimers (44-55 kDa), or polymers such as Bence Jones protein (BJP) in urine.

[0003] Conventionally, FLC has been considered an excessive byproduct produced by B cells and having no function under physiological conditions. However, there is increasing evidence indicating that FLC is significantly associated with the progression and severity of inflammatory diseases such as autoimmune diseases, diabetes, and central nervous system inflammation. In particular, two intractable diseases, amyloid protein immunoglobulin light chain (AL) and light chain deposition disease (LCDD), have been found to be mediated by abnormally folded insoluble FLC. Both AL and LCDD are intractable diseases caused by the irreversible deposition of free κ (kappa) chains or λ (lambda) chains in the extracellular space, characterized by misfolding of free light chains and deposition in tissues and organs such as the heart, kidney, liver, and lung, leading to tissue structure damage, organ dysfunction, and progression of progressive diseases. AL-related FLC shows a specific filamentous structure in tissues, and LCDD-related FLC shows amorphous deposition in tissues. Both AL and LCDD exhibit common monoclonal characteristics but lack diversity. Furthermore, the κ chains of AL- or LCDD-related FLC usually show the same Vκ4-1 / Jκ3 rearrangement. To date, AL- and LCDD-related FLC have been thought to be associated with B cell abnormalities. However, the number of cases without abnormal proliferation of B cells or plasma cells is increasing, suggesting that there may be other causes of FLC.

[0004] There are two types of immunoglobulin light chains, κ and λ, and the κ chain is more frequently used in the process of encoding Ig in B lymphocytes. Also, similar to the heavy chain, the κ chain also has diversity within the Ig molecule. The κ chain gene has 40 V segments, and these segments are randomly recombined with 5 J segments to form the complete coding sequence of the κ chain variable region. Therefore, the κ chains expressed by different B lymphocytes show different combinations of Vκ and VJ. Similarly, the hypervariable regions (CDR3 regions that mainly determine the specificity of antibodies) encoded by some Vκ segments and some VJ segments are also different. However, according to our research results (CN1940069A), it has been revealed that different types of tumor cells have the same or very similar light chain variable region sequences, that is, they show a specific Vк4-1 / J3 combination sequence. Also, the CDR3 sequence in the combination of Vк4-1 / J3 is highly conserved in the Ig light chain variable region derived from tumor cells, is specific compared to the CDR3 derived from B lymphocytes, and shows the difference between tumor cells and B lymphocytes. Research has shown that its CDR3 sequence can be used as a target for tumor diagnosis or treatment.

[0005] However, new discoveries were made in subsequent research, leading to the present invention.

Summary of the Invention

[0006] Our research has revealed that immunoglobulin κ-type light chains with unique Vκ4-1 are widely expressed in cancer cells of various lineages and are expressed at low levels in non-cancer cells. Furthermore, the sequence of a fragment of free light chain Vκ4-1 that can be used to label free Ig light chains having Vκ4-1 was obtained. Based on this, the technical solution of the present invention will be described in detail below.

[0007] In a first aspect, the present invention provides

Chemical

[0008] In a second aspect, the present invention provides the use of the above polypeptide in the preparation of a pharmaceutical for the diagnosis and / or treatment of malignant tumors and / or inflammation.

[0009] Alternatively, preferably, in the above use, the malignant tumor includes at least one of glioma, medulloblastoma, large cell lung cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, skin cancer, oral cancer, seminoma, osteosarcoma, leiomyosarcoma, angiosarcoma, liposarcoma, synovial sarcoma, rhabdomyosarcoma, B-cell lymphoma, T-cell lymphoma, leukemia and myeloma.

[0010] Alternatively, preferably, in the above use, the inflammatory disease includes at least one of systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, inflammatory nephropathy, systemic amyloidosis, Alzheimer's disease (early-onset dementia) and Parkinson's disease.

[0011] In a third aspect, the present invention provides an antibody pharmaceutical prepared by using the above polypeptide as an antigen, which is a recognition antibody or a blocking antibody.

[0012] Alternatively, preferably, the above antibody pharmaceutical is a monoclonal antibody, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:3 or SEQ ID NO:4.

[0013] Alternatively, preferably, the amino acid sequence of the light chain variable region of the above antibody pharmaceutical is shown in SEQ ID NO:5.

[0014] In a fourth aspect, the present invention also provides a small molecule pharmaceutical prepared by targeting the gene sequence or mRNA sequence of the above polypeptide. Examples of the small molecule pharmaceutical include, but are not limited to, gRNA and siRNA, which can specifically target the gene sequence or mRNA sequence of the above polypeptide and block its expression.

[0015] In a fifth aspect, the present invention also provides the use of the polypeptide in the preparation of an inhibitor of the integrin-FAK signaling pathway.

[0016] The present invention has the following beneficial effects.

[0017] Based on the sequence analysis of the immunoglobulin kappa light chain having a unique Vκ4-1, the present invention obtains a functional polypeptide that can specifically label a free Ig light chain sequence located in the conserved region of Vκ4-1 and containing the unique Vκ4-1. An antibody prepared based on this polypeptide, or a polypeptide or small molecule that can specifically bind to this target, specifically recognizes and binds to the immunoglobulin kappa light chain containing Vκ4-1 in malignant tumors and / or inflammatory disease tissues, and inhibits its function, thereby significantly inhibiting the growth of cancer cells in vitro and in vivo or suppressing inflammation. Further molecular studies have revealed that an antibody based on this polypeptide can also inhibit the activation of the integrin-FAK signaling pathway and can be used as an inhibitor of the integrin-FAK signaling pathway.

Brief Description of the Drawings

[0018]

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[0019] The technical solution of the present invention will be described in detail below with reference to preferred examples so that those skilled in the art can better understand and implement the technical solution.

Example

[0020] Example 1. Discovery of a light chain having a unique Vκ4-1 rearrangement pattern

[0021] Previous studies on tumor epithelial cells such as breast cancer, colorectal cancer, and lung squamous cell carcinoma have shown that Vκ4-1 is present in all variable regions of the Igκ light chain, and these patterns have been published (see GenBank: AY505537 - AY505541). Furthermore, Vκ4-1 has a high homology with hydrophobic FLC, and it has been found that Vκ4-1 is present in LCDD or AL.

[0022] Next, tissue samples including cancer tissues, peritumoral tissues, and distal normal tissues were selected from 5 cases of colorectal cancer. The samples were obtained from Peking University People's Hospital with written consent. This study was conducted in accordance with a protocol approved by the Institutional Review Board and approved by the Clinical Research Ethics Committee of Peking University People's Hospital (2015PHB212 - 01). The obtained cancer tissues were sorted into EpCAM + cancer cells by flow cytometry, and the sorting method was as follows.

[0023] First, the tissue was cut into small pieces (about 1 mm 3 ) and washed with 1×PBS. The epithelial cells were incubated in 1×PBS containing 5 mmol / L EDTA and 5 mmol / L DTT at 37°C for 1 hour and separated from the tissue by shaking. The digested epithelial cells were detached from the gentleMACS Dissociator (Miltenyi Biotec) and filtered through a nylon mesh. Next, the cells were washed 3 times with 1×PBS containing 2% fetal bovine serum (FBS) (10099141, Gibco), blocked in 1×PBS containing 5% fetal bovine serum at 4°C for 30 minutes, and stained with anti - human CD19 (11 - 0199 - 41, eBioscience) and anti - human EpCAM (12 - 9326 - 42, eBioscience) at 4°C for 30 minutes. Next, EpCAM + cells were subjected to fluorescence - activated cell sorting (FACS) using FACSAria II (BD Biosciences), and EpCAM + cancer cells were obtained.

[0024] Next, EpCAM +Cancer cells were used as the research subject, and the transcription products of the Igκ light chain variable region were studied by multiplex RT-PCR amplification and IR-Seq sequencing technology.

[0025] IR-Seq Sample Preparation and Sequencing: Sorted EpCAM + Total RNA was extracted from cancer cells using Trizol reagent (15596018, Life Technology). Two rounds of PCR were performed under the reaction conditions specified in the kit instructions using the iRepertoire (registered trademark) commercial kit and the Igκ (iRepertoire) primer set. In the first round, reverse transcription was completed, and tag and sequencing primers were introduced into the PCR product using nested gene-specific primers complementary to the V gene and C gene. In the second round of PCR, common (sequencing) primers were used for exponential amplification. The DNA concentration of the eluted PCR product was detected, and 100 ng of DNA was recovered for sequencing. Subsequent quality control and sequencing were performed by Novogene.

[0026] In 5 patients, the predominance of the light chain with a unique Vκ4-1 pattern was observed.

[0027] According to the IR-Seq sequencing results, the full-length sequence of the Igκ chain with a unique Vκ4-1 variable region was obtained as follows:

Chemical Formula

[0028] Here, 28 amino acids at positions 5-32 (SEQ ID NO: 1, all within the Vκ4-1 region) function as a specific target and can label the free immunoglobulin κ chain with a unique Vκ4-1 variable region.

[0029] Example 2: Preparation of an antibody targeting an Ig light chain with a unique Vκ4-1 pattern

[0030] To understand the function of these non-B cell-derived Igκ light chain variable regions having the Vκ4-1 pattern, monoclonal antibodies were prepared using the sequence shown in SEQ ID NO:1 (artificially synthesized) as an antigen.

[0031] Preparation of monoclonal antibodies: Six- to eight-week-old female BALB / c mice (purchased from Beijing Weike Experimental Animal Technology Co., Ltd.) were immunized with the above 28-amino acid polypeptide antigen conjugated to human albumin. After immunization, spleen cells were collected, fused with myeloma cells, and positive clones of hybridoma cell lines were screened using the above 28-amino acid polypeptide. Positive clones of the hybridoma cells obtained after the final screening were named 5D3 and 6G5, respectively.

[0032] Ten-week-old female BALB / c prolific mice were intraperitoneally injected with 500 μL / mouse of incomplete Freund's adjuvant, and one week later, hybridoma cells were intraperitoneally injected. 5D3 and 6G5 hybridoma cells were separately cultured in RPMI 1640 + 10% FBS + 1% PS medium at 37 °C in a 5% CO 2 incubator. The expression of antibodies in the culture supernatant was detected. After confirmation of expression, the hybridoma cells cultured until the logarithmic growth phase were collected, centrifuged, and the supernatant was discarded. The cells were resuspended in pre-warmed serum-free 1640 medium and washed twice. The cells were dispensed at 2.0×10 6 hybridoma cells / 500 μL for intraperitoneal injection into each mouse. The mice received intraperitoneal injections separately. When the mice showed significant production of ascites (after about 7 to 10 days), the ascites was collected with a 9-gauge injection needle under sterile conditions and centrifuged at 4 °C and 1200 rpm for 10 minutes to remove the hybridoma cells in the ascites. 30% glycerol was added to the ascites supernatant and mixed well, and then the solution was dispensed and stored at -20 °C for later use.

[0033] An appropriate amount of Protein G column packing was filled into a chromatography column, and the column was washed and equilibrated with 10 - 20 times the column volume of cold PBS (pH 7.4). The ascites containing the monoclonal antibody was centrifuged at 4°C and 10,000 rpm for 5 minutes to remove visible impurities. This liquid was diluted at a ratio of 3:1 with PBS, incubated with the Protein G column packing, and rotated overnight at 4°C. The next day, the liquid that passed through the column was collected, repeatedly loaded into the column 10 - 20 times, and then allowed to flow down naturally. Next, the column was washed with 10 - 20 times the column volume of cold PBS (pH 7.4) to remove non-specifically bound protein impurities. Then, the bound antibody was eluted with 0.1 mol / L glycine-HCl buffer (pH 3.0) and immediately neutralized to pH 7.4 using 1 mol / L Tris-HCl (pH 11.0). After elution, the purification column was washed with 10 - 20 times the column volume of PBS (pH 7.4) and stored in 20% ethanol. The eluted antibody was ultrafiltered with PBS, quantified, added with 30% - 50% glycerol, and stored at -20°C for later use. The entire operation procedure was carried out in a 4°C cold room or on ice. The purity of the antibody was detected by SDS-PAGE. The antibodies corresponding to the obtained hybridoma cell lines were named 5D3 and 6G5, respectively.

[0034] Evaluation of the specificity of the monoclonal antibody: Two 293T cell lysates transfected with Vκ1-5 / Jκ3-LC and Vκ4-1 / Jκ3-LC plasmids (both tagged with Myc) were used as experimental subjects. After SDS-PAGE separation, the cell lysates were incubated with 5D3 and 6G5 respectively, and an anti-Myc tag antibody was used as a control to detect the ability of the monoclonal antibody to recognize the Vκ1-5 pattern and Vκ4-1 pattern (LC: light chain).

[0035] The results are shown in Figure 2. As a result of electrophoresis, both 6G5 and 5D3 specifically recognized Vκ4-1 / Jκ3-LC, but did not recognize Vκ1-5 / Jκ3-LC, indicating that they can specifically recognize the amino acid sequence at positions 5-32 of Vκ4-1. Anti-Myc in the figure represents an anti-Myc tag antibody.

[0036] The sample was subjected to sequencing. For monoclonal antibody 5D3, the amino acid sequence of the heavy chain variable region was as shown in SEQ ID NO:4, its coding gene was as shown in SEQ ID NO:7, the amino acid sequence of the light chain variable region was as shown in SEQ ID NO:5, and its coding gene was as shown in SEQ ID NO:8. For monoclonal antibody 6G5, the amino acid sequence of the heavy chain variable region was as shown in SEQ ID NO:3, and its coding gene was as shown in SEQ ID NO:6. The DNA sequence and amino acid sequence alignment of the heavy chain variable regions between the two monoclonal antibodies were as shown in Figure 3.

[0037] Example 3: Detection of immunoglobulin κ light chains with unique Vκ4-1 in different cancer samples by monoclonal antibodies

[0038] In tissue samples of endometrial cancer, colorectal cancer (colon cancer), breast cancer, and esophageal cancer, a light chain Igκ with a unique Vκ4-1 pattern named Vκ4-1 / Jκ-Igκ was detected by immunohistochemistry using monoclonal antibody 5D3, and normal tissue was used as a negative control.

[0039] Immunohistochemical scoring criteria: For scoring cytoplasmic staining, four intensity grades (0, none; 1, weak; 2, moderate; 3, strong) and the percentage of positive cells (0% - 100%) were used. The final score was the product of the intensity grade and the percentage of positive cells (range: 0 - 300). If the final score exceeded 100, it was considered strong positive staining, and if the final score was 100 or less, it was considered weak positive staining.

[0040] The results were as shown in Figure 4. The immunoglobulin kappa light chain (immunoglobulin kappa chain) with unique Vκ4-1 was highly expressed in all four cancer tissues and was lowly expressed in non-malignant tumor cells (the staining of normal tissues in the normal group was very weak).

[0041] Furthermore, more tumor tissues including glioma, medulloblastoma, large cell lung cancer, esophageal cancer, gastric cancer, colorectal cancer (colon cancer), breast cancer, renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, skin cancer, seminoma, osteosarcoma, uterine leiomyosarcoma (leiomyosarcoma), rhabdomyosarcoma, liposarcoma, angiosarcoma, synovial sarcoma, Hodgkin lymphoma, B cell lymphoma (B lymphoma), and T cell lymphoma (T lymphoma) were selected, and immunohistochemical detection of Vκ4-1 / Jκ-Igκ was performed using monoclonal antibody 6G5 with normal tissues as a control.

[0042] The statistics of the detection results were as shown in Figure 5. Vκ4-1 / Jκ-Igκ recognized by monoclonal antibody 6G5 was highly expressed in various malignant tumor cells and was lowly expressed in non-malignant tumor cells (normal tissues).

[0043] To further verify whether Vκ4-1 / Jκ-Igκ was overexpressed in cancer cells, the expression frequencies of Vκ4-1 / Jκ-Igκ in cancer cells and normal epithelial cells of the same individual with colorectal cancer were compared.

[0044] Tissues were collected from 10 patients with colorectal cancer. Tissues from the surgical incision site (distal tissue, normal tissue), tissues around the cancer, and cancer tissues were collected from each patient, and immunohistochemical staining was performed using 6G5 and 5D3, respectively. The results are as shown in Fig. 6, presenting the results of two cases. No positive staining was observed in normal tissues, weak positive staining was observed in tissues around the cancer, and strong staining was observed in cancer tissues, and the results were consistent with both monoclonal antibodies. The results indicated that the expression frequency of Vκ4-1 / Jκ-Igκ in colorectal cancer tissues was significantly higher than that in normal tissues, and there was also a significant difference in the expression frequency of Vκ4-1 / Jκ-Igκ between colorectal cancer tissues and tissues around the cancer. These results suggested that Vκ4-1 / Jκ-Igκ was widely expressed only in cancer cells.

[0045] Example 4. Research on the characteristics of Vκ4-1 / Jκ-Igκ

[0046] Using the monoclonal antibody 5D3 as a detection tool, cell lines MDA-MB-231 (breast cancer cells), NCI-H520 (lung squamous cell carcinoma cells), HT-29 (colorectal cancer cells), and U2OS (osteosarcoma cells) (obtained from the American Type Culture Collection (ATCC) and maintained by the Peking University Human Disease Genomics Center) were used as experimental subjects, and subcellular localization analysis of Vκ4-1 / Jκ-Igκ was performed by immunofluorescence method to detect whether Vκ4-1 / Jκ-Igκ was hydrophobic and free in both cancer cell lines.

[0047] Immunofluorescence method: The cell line was quickly washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 minutes. The fixed cells were blocked with 5% BSA and treated with 0.02% Triton X-100 at room temperature for 30 minutes to create pores in the cell membrane (to allow the antibody to enter the cell). Next, the cells were immunostained with 5D3 (10 μg / mL) diluted in blocking buffer at room temperature for 3 hours and washed three times with PBS for 5 minutes each. The cells were incubated with Alexa594 anti-mouse secondary antibody and Alexa488 anti-rabbit secondary antibody (Molecular Probes) at room temperature for 1 hour and then washed. Images were taken with a confocal microscope using a Leica STED imager (Leica Microsystems).

[0048] Immunofluorescence was as shown in Figure 7, and the results indicated that Vκ4-1 / Jκ-Igκ could be secreted and deposited in the extracellular matrix.

[0049] Next, the SW480 cell line and HT-29 cell line (both colorectal cancer cell lines) were used as research subjects for Western blot detection. The samples in the four lanes of the reducing electrophoresis were culture supernatant, negative control (medium control), cell lysate, and ECM extract, respectively. The samples for non-reducing electrophoresis were serum, SW480 cells, and HT-29 cells, respectively. The antibodies used for non-reducing electrophoresis were anti-human IgG antibody (anti-IgG Fc), anti-human free Igκ antibody (anti-free κ light chain), and 6G5. 6G5 was used for reducing electrophoresis.

[0050] The results are as shown in Fig. 8. The left panel shows the results of reducing electrophoresis, and the right panel shows the results of non-reducing electrophoresis. The Igκ light chain with the Vκ4-1 / Jκ sequence mainly existed as free multimers in the cytoplasm, culture supernatant, and extracellular matrix. In both reducing electrophoresis (left) and non-reducing electrophoresis (right), it was shown that the Igκ light chain with the Vκ4-1 / Jκ sequence was in the free form and mainly existed in the form of multimers. When the Igκ light chain with the Vκ4-1 / Jκ sequence was detected using an anti-human IgG antibody (anti-IgG Fc) in electrophoresis under non-reducing conditions, it did not form a 4-peptide chain structure with the IgG heavy chain. However, when the Igκ light chain with the Vκ4-1 / Jκ sequence was detected using an anti-human free Igκ antibody (anti-κ free light chain), it was in the free form consistent with the signal of 6G5.

[0051] These results indicate that Vκ4-1 / Jκ-Igκ exhibits the property of being free in both reducing and non-reducing states and takes various forms including monomers, dimers, and polymers in the culture supernatant and ECM extracts. This suggests the possibility that non-B cell-derived Vκ4-1 is an ECM protein. The light chain Igκ with the unique Vκ4-1 in tissues, namely Vκ4-1 / Jκ (Vκ4-1 rearranged in combination with other Jκ genes), is actually a type of free Ig light chain and will be referred to as Vκ4-1 / Jκ-FLC hereinafter.

[0052] Example 5. Promotion of tumor invasion and metastasis by Vκ4-1 / Jκ-FLC

[0053] To investigate the effect of Vκ4-1 / Jκ-FLC on cancer development and progression, Chinese hamster ovary cells (CHO) were used as a eukaryotic expression platform, and the biologically active recombinant protein r-Vκ4-1 / Jκ-FLC was prepared.

[0054] The full-length gene sequence containing Vκ4-1 (SEQ ID NO: 2, including the C region and V region) was cloned into the pGEX-4T-2 vector, and further subcloned into the pcDNA3.1 myc-His(-)B vector using the same restriction enzyme cleavage sites. The recombinant protein r-Vκ4-1 / Jκ-FLC containing a His tag at the C-terminus was expressed in CHO cells and purified by Capto (商標) L affinity chromatography (GE Healthcare, USA).

[0055] Purification and identification of eukaryotic recombinant proteins (CHO expression system): The samples were centrifuged at 3000 rpm for 15 minutes at 4 °C, and the culture supernatants were collected. Capto L specifically binds to the variable region of the κ light chain and can be used for the purification of Igκ light chain eukaryotic recombinant proteins. An appropriate amount of Capto L was packed into the chromatography column and pre-equilibrated with a cold equilibration solution (20 mmol / L sodium dihydrogen phosphate, 150 mmol / L NaCl, pH 7.2) 10 times the column volume. The culture supernatant (input) was added to the column, which was sealed and incubated with rotation overnight at 4 °C. Next, the liquid in the column was allowed to flow through the column (flow-through), washed with the cold equilibration solution to remove unbound and non-specifically bound proteins, and then eluted with a cold elution solution (0.1 mol / L sodium citrate, pH 2.0 - 3.5). The eluate was neutralized to pH 7.4 with a neutralization solution (1 mmol / L Tris-HCl, pH 11.0), concentrated by ultrafiltration, quantified by BCA, and stored at -80 °C for later use. The purified protein was identified by SDS-PAGE and Western blot.

[0056] After successfully establishing a cell culture and expression system, it was discovered that r-Vκ4-1 / Jκ-FLC expressed by CHO cells was secreted into the cell culture supernatant (Figure 9A). The collected culture supernatant was subjected to affinity chromatography using a CaptoL affinity column (specifically binding to the variable region of the Igκ type light chain), and the purified recombinant protein was verified using SDS-PAGE, Western blot, and a Superdex200 column. It was confirmed that a highly pure recombinant protein, r-Vκ4-1 / Jκ-FLC, was obtained (Figures 9B-D).

[0057] SW480 cells and HT-29 cells were used as the research subjects, and r-Vκ4-1 / Jκ-FLC was added for intervention culture. The experimental results are shown in Figures 10 and 11. Figure 10A shows the cell proliferation and self-renewal ability of SW480 cells treated with 1 μg / mL of r-Vκ4-1 / Jκ-FLC detected by a colony formation assay. Figure 10B shows the cell proliferation and self-renewal ability of HT-29 cells treated with 1 μg / mL of r-Vκ4-1 / Jκ-FLC detected by a colony formation assay. In Figure 11, A and B show the effects of r-Vκ4-1 / Jκ-FLC on the migration and invasion ability of SW480 cells, which were treated with 1 μg / mL of r-Vκ4-1 / Jκ-FLC and detected by a Transwell assay (A) and a Matrigel Transwell assay (B), respectively. C and D show the effects of r-Vκ4-1 / Jκ-FLC on the migration and invasion ability of HT-29 cells, which were treated with 1 μg / mL of r-Vκ4-1 / Jκ-FLC and detected by a Transwell assay (C) and a Matrigel Transwell assay (D), respectively. The results indicate that treatment with r-Vκ4-1 / Jκ-FLC increases the survival / proliferation and migration ability of SW480 cells and HT-29 cells compared to the negative control.

[0058] Next, three gRNAs targeting the constant region of Igκ were used to knockout Igκ in SW480 cells and HT-29 cells.

[0059]

Table 1

[0060] The results showed that after knocking out Igκ, the migration ability of both cells decreased. As can be seen from Figure 12, A shows the cell migration ability detected by the Transwell assay after knocking out Igκ in SW480 cells, and B shows the cell migration ability detected by the Transwell assay after knocking out Igκ in HT-29 cells. After knocking out Igκ with three gRNAs, the migration ability of the cells decreased significantly compared with the control group of the empty vector (vector).

[0061] To further demonstrate that Vκ4-1 / Jκ-FLC exerts its tumorigenesis-promoting effect mainly through the secreted form rather than intracellular Igκ, first, intracellular Igκ was knocked out, and then the recombinant protein r-Vκ4-1 / Jκ-FLC was added to the culture system. The results of the Transwell assay showed that the exogenous addition of the recombinant protein r-Vκ4-1 / Jκ-FLC could partially reverse the inhibition of cell migration ability caused by Igκ knockout (Figure 13). These results demonstrated that Vκ4-1 / Jκ-FLC mainly exerts its tumorigenesis-promoting effect in the secreted form.

[0062] Furthermore, to demonstrate that Vκ4-1 / Jκ-FLC exerts its tumor-promoting effect mainly through the secreted form, a 5D3-specific antibody was added to the culture system to block secreted Vκ4-1 / Jκ-FLC (Igκ secreted from the cells themselves). The results showed that adding 5D3 to block the secreted Vκ4-1 / Jκ-FLC significantly inhibited cell migration. The results were as shown in Fig. 14. A shows the experimental results of cell migration ability detected by Transwell after adding 50 μg / mL of 5D3 to the SW480 cell culture supernatant, and B shows the experimental results of cell migration ability detected by Transwell after adding 50 μg / mL of 5D3 to the HT-29 cell culture supernatant. The results showed that the addition of 5D3 significantly reduced the migration ability of both cells compared to the negative control PBS and mIgG.

[0063] Next, 50 μg / mL of 5D3 was added to the SW480 cell culture supernatant and the HT-29 cell culture supernatant respectively, and at the same time 10 μg / mL of r-Vκ4-1 / Jκ-FLC was added to the culture system respectively, and the cell migration ability was detected by Transwell assay. The experimental results were as shown in Fig. 15. Adding exogenous recombinant protein r-Vκ4-1 / Jκ-FLC could reverse the inhibitory effect of the 5D3 antibody on cell migration after blocking with the antibody.

[0064] Furthermore, a subcutaneous xenograft tumor model of nude mice was established using SW480 cells, and recombinant protein r-Vκ4-1 / Jκ-FLC was injected around the tumors at different doses to detect the tumor growth-promoting ability of r-Vκ4-1 / Jκ-FLC. The results showed that recombinant protein r-Vκ4-1 / Jκ-FLC promoted tumor growth in a dose-dependent manner, and both the tumor volume and weight were higher than those of the control group. As can be seen from Fig. 16, A is a photo of the three tumor groups at the end of the experiment and the growth curve of the tumor volume, and B is a statistical chart of the tumor volume and tumor weight of the three groups at the end of the experiment.

[0065] In conclusion, in vivo and in vitro experiments confirmed that Vκ4-1 / Jκ-FLC secreted from tumor cells plays an important role in the growth and metastasis of colorectal cancer.

[0066] Example 6. Inhibition of tumor growth by monoclonal antibodies 5D3 and 6G5

[0067] In Example 5, it was confirmed that monoclonal antibody 5D3 can specifically block the tumorigenic promotion properties of Vκ4-1 / Jκ-FLC in vitro. Next, the effects of monoclonal antibodies 6G5 and 5D3 on the growth of colorectal cancer xenografts in SCID mice were investigated.

[0068] The animal models were classified into two groups. One model group was established by subcutaneous injection of SW480 cells into SCID mice (CDX), and the other model group was established by subcutaneous injection of tumor cells from colorectal cancer patients into SCID mice (PDX). After tumor formation, intravenous administration (5 mg / kg) was performed every 4 days, and mouse IgG (mIgG) was used as a control to dynamically observe the tumor growth rate and dynamically monitor the body weight changes of the mice. At the end of the experiment, the differences in tumor volume and tumor weight between 6G5 and the control group, as well as the differences in tumor volume and tumor weight between 5D3 and the control group, were compared.

[0069] The experimental results of the two groups are shown in Figures 17-18 (6G5) and Figures 19-20 (5D3).

[0070] The results showed that both monoclonal antibodies 6G5 and 5D3 had antitumor effects and significantly inhibited the increase in tumor volume and weight compared with the control group, but had no significant effect on the body weight of the mice and were equivalent to the control group.

[0071] Example 7. Inhibition of integrin-FAK signaling pathway by monoclonal antibodies 5D3 and 6G5

[0072] Control antibody mIgG (50 μg / ml) or antibodies 5D3 and 6G5 were added to the supernatants of SW480 and HT-29 cell cultures. The cells were collected after 24 hours, and the phosphorylation levels of the major molecules involved in the integrin-FAK signaling pathway were detected by Western blotting to detect the effect of the antibodies on the activation of the integrin-FAK pathway. The results are as shown in Figure 21, and it was shown that both 6G5 and 5D3 can reduce the phosphorylation levels of FAK, Src, Erk, and Akt, which are major molecules downstream of the integrin signaling pathway, thereby inhibiting the activation of the integrin-FAK signaling pathway.

[0073] The procedure for the in vivo experiment was the same as in Example 6, and at the end of the experiment, the difference in the activation of the integrin-FAK signaling pathway between the 6G5, 5D3, and control groups was compared. The results are as shown in Figures 22 and 23, and basically they were consistent with the results detected at the cellular level in vitro.

[0074] Example 8. Inhibition of osteosarcoma growth in vivo by monoclonal antibody 5D3

[0075] Next, the effect of monoclonal antibody 5D3 on the growth of osteosarcoma in nude mice was investigated in vivo.

[0076] The animal model was established by subcutaneous injection of the human osteosarcoma cell line 143B into nude mice (CDX). After tumor formation, the animals were divided into two groups of six each. One model group was intravenously administered monoclonal antibody 5D3 at a dose of 5 mg / kg every four days, and the other group was administered mouse IgG (mIgG) as a control, with the same administration schedule and dose as those of 5D3. The growth rate of the tumors in vivo in the two mouse groups was dynamically observed, and the change in the body weight of the mice was also dynamically monitored. At the end of the experiment, the differences in tumor volume and tumor weight between the 5D3-administered group and the control group were compared.

[0077] The experimental results are as shown in Fig. 24 (5D3), and the tumor growth rate of the mice in the experimental group was significantly slower than that in the control group. From the comparison of the volumes of the excised tumors, it can also be seen that the overall tumor volume of the experimental group was significantly smaller than that of the mIgG control group.

[0078] The results showed that the monoclonal antibody 5D3 had an antitumor effect and significantly inhibited the increase in tumor volume and weight compared with the control group.

[0079] Example 9. Vκ4-1-IgLC polypeptide for use in assisting the diagnosis of multiple sclerosis

[0080] Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system. Multiple inflammatory demyelinating plaques are observed in the white matter of the central nervous system during the acute active phase of the disease, and are frequently seen in the optic nerve, spinal cord and brainstem. In the field of neurology, this disease is a common intractable disease. In addition, high levels of Igκ free light chains are also found in the sera and cerebrospinal fluids of patients with this disease, which is an important indicator for identifying MS. However, Igκ free light chains have always been considered to be produced by B cells. We performed a detection assay using an antibody that can specifically recognize a 28-amino acid peptide of Vκ4-1 (amino acids at positions 5-32 of the full-length sequence shown in SEQ ID NO: 1).

[0081] Serum samples from 10 patients with multiple sclerosis (MS) were selected, and sera from 8 healthy individuals were used as controls. 1 μL of the cryopreserved samples was collected, diluted with 10 μL of PBS, and subjected to denaturing SDS-PAGE electrophoresis. Next, the proteins were electrotransferred onto a nitrocellulose membrane and detected using a rabbit-specific antibody obtained by immunization with a 28-amino acid peptide of Vκ4-1 / Jκ (SEQ ID NO: 1).

[0082] The results were as shown in Fig. 25. Antibodies specific for the 28-amino acid peptide of Vκ4-1 specifically recognized free Vκ4-1 / Jκ light chain tetramers in the sera of MS patients, but tetramers were not detected in healthy individuals (healthy controls). The results indicated that a unique Vκ4-1 rearrangement occurred in Igκ free Ig light chains in the sera of MS patients, suggesting that the 28-amino acid peptide might be used as an auxiliary diagnostic marker for MS.

[0083] Example 10. Vκ4-1-IgLC polypeptide for use in assisting the diagnosis of Alzheimer's disease

[0084] In our previous studies, it was found that Ig free light chains were widely present in non-B cells and highly expressed in tumor cells, proliferating cells, and inflammatory cells. It has been shown that this is a cytoskeleton-related protein, which may exist in the cytoplasm and form fibrous structures, or may deposit in the extracellular matrix due to its hydrophobic properties. Although there has been no evidence suggesting an association with amyloidosis in Alzheimer's disease (AD) until now, new findings were obtained when pathological sections of 10 AD patients were stained with 6G5.

[0085] Pathological sections from 10 AD patients were collected, deparaffinized in xylene as usual, and the xylene was removed with an alcohol gradient. Then, the sections were boiled in sodium citrate buffer for 5 minutes and allowed to cool naturally. 1% hydrogen peroxide was added to inhibit peroxidase (for 10 minutes at room temperature). 1% hydrogen peroxide was added to inhibit peroxidase (for 10 minutes at room temperature). The sections were washed with PBS, blocked with 10% horse serum (for 10 minutes at room temperature), and then incubated with 6G5 (overnight at 4°C). The sections were washed with PBS, stained with HRP-labeled anti-mouse IgG, and observed under a microscope. The results were as shown in Fig. 26.

[0086] The results showed that in all 10 patients, different forms of strong dendritic staining (Figure 26A) or local and plaque (senile plaque) staining (Figure 26B) were observed in the cerebral cortex. Slight deposition of the light chain was seen at sites distal to the cortex, and the deposition was mainly in a diffuse state (Figure 26C). On the other hand, no positive reaction was seen in normal brain tissue (Figure 26D).

[0087] Example 11. Vκ4-1-IgLC polypeptide for use in assisting the diagnosis of systemic amyloidosis

[0088] Systemic amyloidosis is a refractory disease characterized by irreversible organ failure caused by the deposition of free Ig light chains in various tissues. This pathogenic free Ig light chain has long been thought to be produced by B cells. However, previous studies (such as protein extraction, amino acid and nucleotide sequence alignment, etc.) have shown that Ig free light chains with a Vκ4-1 variable region produced by non-B cells have high homology with the free Ig light chains that cause systemic amyloidosis. Subsequently, a detection experiment was conducted using 6G5, a monoclonal antibody specific for the 28-amino acid peptide (positions 5-32) of Vκ4-1.

[0089] Serum samples from 3 patients with cardiac amyloidosis were selected, and the sera of 2 healthy individuals were used as controls. 1 μL of the serum sample was collected, diluted with 10 μL of PBS, and subjected to denaturing SDS-PAGE electrophoresis. Next, the protein was electrotransferred onto a nitrocellulose membrane and detected using 6G5. The results were as shown in Figure 27.

[0090] The results showed that the Vκ4-1 specific antibody can specifically recognize the free Igκ light chain in the serum of patients with systemic amyloidosis, but cannot recognize normal human Igκ, indicating that the 28-amino acid target polypeptide can be used as an auxiliary diagnostic marker for patients with systemic amyloidosis.

[0091] Example 12. Vκ4-1-IgLC polypeptide for use in assisting the diagnosis of inflammatory kidney disease

[0092] Approximately 90% of kidney diseases are associated with pathogenic Igs. For example, IgA nephropathy is caused by the deposition of IgA in the mesangial region of the glomerulus, membranous nephropathy is caused by the deposition of IgG between glomerular podocytes and the basement membrane, and renal amyloidosis is caused by the deposition of free light chains in the glomerular basement membrane. However, although it has long been believed in the art that these Igs are derived from B cells, the inventors have discovered that these pathogenic Igs are mainly produced by local non-immune cells.

[0093] 1% hydrogen peroxide was added to frozen pathological sections collected from 5 cases of nodular nephrosis to inhibit peroxidase (for 10 minutes at room temperature). The sections were washed with PBS and blocked with 10% horse serum (for 10 minutes at room temperature). Next, the sections were incubated with monoclonal antibody 6G5 against the polypeptide having Vκ4-1 (overnight at 4°C), and mouse IgG was set as a control. The sections were washed with PBS, stained with FITC-labeled anti-mouse IgG, and observed under a microscope. The results were as shown in Figure 28. The results showed that the glomeruli of all 5 patients exhibited strong fluorescence staining on the basement membrane (A), but no positive staining was observed with irrelevant mouse IgG (B), indicating that 6G5 can specifically recognize the deposition of Vκ4-1 / Jκ-FLC in the glomerular basement membrane of human nodular nephrosis patients.

[0094] Example 13. Vκ4-1-IgLC polypeptide for use in assisting the diagnosis of inflammatory gastrointestinal diseases

[0095] Ulcerative colitis and Crohn's disease are intractable chronic inflammations commonly seen in clinical practice and often lead to changes from inflammation to cancer. Chronic gastritis often progresses to gastric cancer. The inventors have discovered high levels of free Ig light chains (Vκ4-1 / Jκ-Igκ) that may promote the inflammatory process in the glandular epithelial cells of the tissues of the above two diseases.

[0096] Pathological sections were taken from inflammatory colorectal polyps, ulcerative colitis, and chronic gastritis, and were routinely deparaffinized with xylene and the xylene was removed with a gradient of alcohol. The sections were boiled in sodium citrate buffer for 5 minutes and cooled naturally. 1% hydrogen peroxide was added to inhibit peroxidase (for 10 minutes at room temperature). 1% hydrogen peroxide was added to inhibit peroxidase (for 10 minutes at room temperature). The sections were washed with PBS, blocked with 10% horse serum (for 10 minutes at room temperature), and then incubated with 5D3 (overnight at 4°C). The sections were washed with PBS, stained with HRP-labeled anti-mouse IgG, and observed under a microscope. As can be seen from Figure 29, the results showed that the inflammatory colorectal polyps (A), ulcerative colitis (B), and chronic gastritis epithelial cells (C) were all positively stained (brown), while the normal colorectal tissue (D) was negatively stained (blue).

[0097] In this specification, the concept of the present invention has been described in detail with reference to specific examples, but the above examples are only described for understanding the central concept of the present invention. It should be noted that for those skilled in the art, obvious changes, equivalent alternatives, or other improvements made without departing from the concept of the present invention should be included in the protection scope of the present invention.

Claims

1. An amino acid sequence is 【Chemical 1】 a Vκ4-1-IgLC polypeptide, characterized in that it is as shown in .

2. Use of the polypeptide according to claim 1 in the preparation of a medicament for the diagnosis and / or treatment of a malignant tumor or an inflammatory disease.

3. The use according to claim 2, characterized in that the malignant tumor includes at least one of glioma, medulloblastoma, large cell lung cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, skin cancer, oral cancer, seminoma, osteosarcoma, leiomyosarcoma, angiosarcoma, liposarcoma, synovial sarcoma, rhabdomyosarcoma, B-cell lymphoma, T-cell lymphoma, leukemia and myeloma.

4. The use according to claim 2, characterized in that the inflammatory disease includes at least one of systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, inflammatory nephropathy, systemic amyloidosis, Alzheimer's disease and Parkinson's disease.

5. An antibody medicament, characterized in that the polypeptide according to claim 1 is used as an antigen for preparing the antibody medicament, and the antibody medicament is a recognition antibody or a blocking antibody.

6. The antibody medicament according to claim 5, which is a monoclonal antibody, and the amino acid sequence of its heavy chain variable region is as shown in SEQ ID NO: 3 or SEQ ID NO:

4.

7. The antibody medicament according to claim 6, characterized in that the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

5.

8. A small molecule medicament, characterized in that the gene sequence or mRNA sequence corresponding to the polypeptide according to claim 1 is used as a target for preparing the small molecule medicament.

9. Use of the polypeptide according to claim 1 in the preparation of an inhibitor of the integrin-FAK signaling pathway.

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