Single-domain antibody targeting human IGFL1 and application of single-domain antibody

Abstract

The invention relates to the technical field of antibody engineering, in particular to a single-domain antibody targeting human IGFL1 and application of the single-domain antibody, and particularly relates to application of the single-domain antibody in preparation of drugs for treating breast cancer. According to the single-domain antibody targeting the human IGFL1, a heavy chain variable region comprises three complementary determining regions, namely CDR1, CDR2 and CDR3, and the amino acid sequence of the CDR1 is shown as SEQ ID No.10; the amino acid sequence of the CDR2 is as shown in SEQ ID No. 11; the amino acid sequence of the CDR3 is as shown in SEQ ID No. 1 or SEQ ID No. 2. According to the invention, a single-domain antibody artificial synthesis library is successfully constructed, and two single-domain antibody sequences with good anti-IGFL1 effects are screened out. The single-domain antibody provided by the invention can be used for preparing drugs for treating IGFL1 positive tumors, especially triple negative breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of antibody engineering technology, and particularly relates to a single-domain antibody targeting human IGFL1 and its applications, especially its application in the preparation of drugs for treating breast cancer. Background Art

[0002] IGFL1 (insulin-like growth factor family-related protein 1) is associated with certain skin diseases, inflammatory diseases and cancers, and plays an oncogenic function of driving tumor proliferation, migration and invasion. Therefore, it has become a potential target for tumor treatment. The expression level of IGFL1 in various diseases is closely related to disease progression, showing the potential as a biomarker. For example, in lung adenocarcinoma, the expression level of IGFL1 is closely related to the clinicopathological characteristics and prognosis of patients, and can be used as an independent risk factor for judging the prognosis of patients. In addition, the high expression of IGFL1 in thyroid eye disease also suggests its potential as a biomarker for disease diagnosis and treatment monitoring. The mechanism of action of IGFL1 in diseases such as tumors and thyroid eye disease has been gradually revealed, and it promotes cell proliferation and inflammatory responses by activating the IGF-1R signaling pathway. Preclinical research results show that IGFL1 is not only an important biomarker but also has great potential as a therapeutic target. Future research will further explore the role of IGFL1 in more diseases and develop novel treatment strategies based on IGFL1. These studies provide various potential treatment strategies for IGFL1-targeted therapy. Although there are not many successful clinical results at present, these studies provide new directions and hopes for future tumor treatment.

[0003] Breast cancer is a common malignant tumor among women in clinical practice. It is classified according to estrogen receptor (ER), progesterone receptor (PR), HER-2 receptor and Ki67 indicators, including Luminal A type, Luminal B type and triple-negative breast cancer. Among them, triple-negative breast cancer (TNBC) has a poor prognosis and relatively limited treatment methods due to the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). IGFL1 is an insulin-like growth factor family-related protein that is highly expressed in various tumors and plays an important role in the processes of tumor cell proliferation, migration and invasion. Single-domain antibody is a heavy-chain variable antibody fragment with advantages such as small molecular weight, high stability and strong tissue penetration, and shows broad application prospects in tumor targeted therapy. Therefore, a single-domain antibody targeting breast cancer with IGFL1 as the target has become a potential treatment strategy.

[0004] In the prior art, there has been no disclosure of a single-domain antibody targeting breast cancer with IGFL1 as the target. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a single-domain antibody targeting human IGFL1 and its applications. The single-domain antibody of the present invention can specifically bind to human IGFL1 and block its activity, thereby inhibiting the proliferation and stemness maintenance of triple-negative breast cancer cells, providing a new means for the treatment of IGFL1-positive tumor cells.

[0006] To solve the above technical problems, the technical solution of the present invention is as follows: In the first aspect of the present invention, a single-domain antibody targeting human IGFL1 is provided. The heavy-chain variable region of the single-domain antibody comprises three complementary determining regions, namely CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is as shown in SEQ ID No.10; The amino acid sequence of CDR2 is as shown in SEQ ID No.11; The amino acid sequence of CDR3 is as shown in SEQ ID No.1 or SEQ ID No.2.

[0007] Furthermore, the amino acid sequence of the single-domain antibody is as shown in SEQ ID No.3 or SEQ ID No.4.

[0008] The single-domain antibody of the present invention can be expressed in prokaryotic or eukaryotic expression systems through genetic engineering techniques, such as Escherichia coli, yeast, insect cells, or mammalian cells, etc., to obtain a single-domain antibody protein with biological activity.

[0009] In the second aspect of the present invention, a nucleic acid molecule encoding the single-domain antibody targeting human IGFL1 as described in the first aspect is provided.

[0010] In the third aspect of the present invention, a vector containing the nucleic acid molecule as described in the second aspect is provided.

[0011] In the fourth aspect of the present invention, a host cell containing the vector as described in the third aspect is provided.

[0012] In the fifth aspect of the present invention, the application of the single-domain antibody targeting human IGFL1 as described in the first aspect in the preparation of a human IGFL1 protein detection reagent is provided.

[0013] In the sixth aspect of the present invention, the application of the single-domain antibody targeting human IGFL1 as described in the first aspect in the preparation of a product that binds to human IGFL1 protein is provided.

[0014] In the seventh aspect of the present invention, the application of the single-domain antibody targeting human IGFL1 as described in the first aspect in the preparation of an anti-breast cancer drug is provided.

[0015] Furthermore, the breast cancer is triple-negative breast cancer.

[0016] The single-domain antibody of the present invention down-regulates the expression of IGFL1; by inhibiting the activation of the PI3K / AKT pathway to down-regulate the expression of the oncogenes C-myc and CyclinD1, thereby inhibiting the progression of tumors.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1) The present invention discovers that IGFL1 is highly expressed in breast cancer patients, especially triple-negative breast cancer patients. On this basis, the present invention successfully constructs a single-domain antibody synthetic library and screens out 7 new single-domain antibody sequences against IGFL1. After further verification of affinity and anti-tumor activity, two single-domain antibodies with the best anti-tumor effects, SdAb-IGFL1#6 and SdAb-IGFL1#8, are obtained, and their amino acid sequences are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0018] 2) The single-domain antibody provided by the present invention can specifically bind to human IGFL1 to block the activity of IGFL1, and can effectively inhibit the proliferation and stemness maintenance of human triple-negative breast cancer cells. In addition, the results of animal experiments show that the single-domain antibody of the present invention can significantly inhibit the growth of triple-negative breast cancer in situ in mice, but does not affect the body weight of mice, further confirming its potential and safety in tumor treatment. Therefore, the single-domain antibody provided by the present invention can be used to prepare drugs for treating IGFL1-positive tumors, especially triple-negative breast cancer. Brief Description of the Drawings

[0019] Figure 1 It is a result diagram for screening single-domain antibodies against IGFL1 based on the isPLA-seq technology. Among them, A is the result of sorting positive cells by flow cytometry after isPLA of 293T cells. B is the gel electrophoresis result diagram of amplifying the DNA fragment of the CDR3 region by PCR.

[0020] Figure 2 It is a result diagram for detecting the binding and affinity of the single-domain antibody against IGFL1 and the antigen. Among them, A-B is the result of detecting the binding of the antigen and antibody by GST pull-down experiment. C is the result of detecting the interaction partner of IGFL1 using the single-domain antibody against IGFL1 in the Co-IP experiment. D is the result of detecting the affinity of the single-domain antibody against IGFL1 and the IGFL1 protein by SPR.

[0021] Figure 3Results of the preparation of the single-domain antibody of IGFL1 and its entry into cells. Among them, A is a schematic diagram of the fusion expression of the single-domain antibody with the TAT cell-penetrating peptide, and the seven prepared single-domain antibodies of IGFL1. B shows the results of detecting the localization and quantity of the single-domain antibody in cells by IF experiment after adding the single-domain antibody to HCC1806 and HCC1937 cells for treatment. Scale bar: 200 μm.

[0022] Figure 4 Results of the significant inhibition of the proliferation of triple-negative breast cancer cells by the single-domain antibody of IGFL1. Among them, A shows the results of detecting the number of viable cells by CCK8 experiment after adding different concentrations of the single-domain antibody of IGFL1 to HCC1806 and MDA-MB-231 cells and treating them for 48 hours. B shows the results of detecting the tumor clone formation ability by crystal violet staining in the colony formation experiment after adding the single-domain antibody of IGFL1 to HCC1806 cells overexpressing IGFL1. C shows the results of obtaining the relative cell growth value in the cell counting experiment after adding the single-domain antibody of IGFL1 to HCC1806 cells overexpressing IGFL1. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005.

[0023] Figure 5 Results of the inhibition of tumor cell stemness by the single-domain antibody of IGFL1. Among them, A shows the results of detecting the proportion of ALDH-positive cell population by flow cytometry after treating HCC1806 cells overexpressing IGFL1 with the single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8. B shows the results of detecting the stemness of tumor cells by the mamosphere experiment after treating HCC1806 cells overexpressing IGFL1 with the single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8. Scale bar: 200 μm. C shows the results of detecting the expression of stemness markers of tumor cells by WB experiment after treating HCC1806 cells overexpressing IGFL1 with the single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8. D shows the results of detecting the expression of stemness markers by qPCR after treating HCC1806 cells overexpressing IGFL1 with the single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005.

[0024] Figure 6 Results of detecting the changes in the proteins of the IRS1 / p85 / PI3K / AKT / β-catenin signaling pathway by WB experiment after treating cells with the single-domain antibody of IGFL1.

[0025] Figure 7Results of the inhibition of the growth of murine mammary gland orthotopic carcinoma by an IGFL1 single-domain antibody. Among them, A-D show the results of inoculating the HCC1806 cell line into the mammary fat pad of mice, followed by treatment with the single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 at a dose of 10 mg / kg, and monitoring the body weight, tumor volume, and tumor weight of the mice. E-H show the results of inoculating the MDA-MB-23 cell line into the mammary fat pad of mice, followed by treatment with the single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 at a dose of 10 mg / kg, and monitoring the body weight, tumor volume, and tumor weight of the mice. I-J show the results of sectioning the MDA-MB-231 and HCC1806 cell xenografts and subsequent pathological staining with HE and IHC (Ki67 and Caspase-3). *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005. Scale bar: 100 μm. Detailed implementation mode

[0026] The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.

[0027] The cell lines involved in the embodiments of the present invention are all purchased from the ATCC cell bank. The nude mice used are purchased from Beijing Speyford Biotech Co., Ltd. The detection antibodies are purchased from CST and Abcam companies. The biochemical reagents and kits are purchased from biotechnology companies such as Beyotime and Solarbio. The molecular biology test methods not specifically described are all carried out with reference to "Molecular Cloning: A Laboratory Manual".

[0028] The specific tests for the preparation, purification, and anti-tumor activity verification of the single-domain antibody of the present invention are shown in the following embodiments.

[0029] Example 1 Screening and purification of single-domain antibody 1. Construction of a single-domain antibody synthetic library The single-domain antibodies of the synthetic library are composed of FR1, FR2, FR3, FR4, CDR1, CDR2, and CDR3. Among them, the most critical antigen-complementary determining region is CDR3. Therefore, in this synthetic library, only single-domain antibodies with diverse CDR3 regions were synthesized, and FR1, FR2, FR3, FR4, CDR1, and CDR2 were kept consistent in all single-domain antibodies. FR1, FR2, FR3, FR4, CDR1, and CDR2 are all known sequences. See the literature Yan J., Li G., Hu Y., Ou W., Wan Y. Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications. J. Transl. Med. 2014;12:1–12. doi: 10.1186 / s12967-014-0343-6. The construction method of the single-domain antibody synthetic library includes the following steps: Step 1: Construct a variable library of the CDR3 region of the single-domain antibody targeting IGFL1. Use gene synthesis technology to design and synthesize a highly diverse DNA fragment library. The core feature of this library is to introduce 20 variable amino acid sites in the CDR3 region, which are optimized and encoded using NNN codons (N = A / T / G / C), ensuring both sequence diversity and effectively reducing the probability of the appearance of stop codons. The entire CDR3 region is designed to have a fixed length of 60 bases, and the theoretical library capacity finally obtained is not less than 1×10^8 clones, ensuring that various possible amino acid sequence combinations are covered. The SdAb CDR3 DNA fragment mixture is as follows: CCA TCT ACT ACT gCg CCg CTN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNNNNN NNN NNN NNN NNN NNN NNN NNT ggg gAC AAg gAA CAC AAg T N = A / T / C / G.

[0030] Step 2: Construct the pCDH-CMV-sdAb backbone vector: Design and construct a plasmid DNA vector containing a single-domain antibody backbone. This vector should contain the framework regions (FR1, FR2, FR3, FR4, CDR1, and CDR2) of the single-domain antibody and necessary expression regulatory elements such as promoters, terminators, etc. In addition, a 3×Flag tag should be fused to the C-terminus of the vector for subsequent detection and purification. After labeling the C-terminus of the SdAb backbone with 3 x flag, it is ligated to the pCDH-CMV vector to obtain the pCDH-CMV-sdAb backbone vector.

[0031] Step 3: Connect the CDR fragment mixture to the pCDH-CMV-sdAb vector by homologous recombination to obtain a single-domain antibody synthetic library. Appropriate restriction enzymes or homologous recombination techniques can be used to ligate the CDR3 region DNA library to the corresponding position of the single-domain antibody backbone vector. Ensure that the ligated plasmid DNA vector contains the complete single-domain antibody sequence, including the FR1, FR2, FR3, FR4, CDR1, CDR2, and CDR3 regions, as well as the 3×Flag tag.

[0032] Step 4: Verify and amplify the single-domain antibody synthetic library Verify the correctness of the ligated plasmid DNA vector by methods such as sequencing and PCR amplification. Ensure that the CDR3 region DNA fragment has been correctly inserted into the single-domain antibody backbone vector without mutations or deletions. Amplify the verified plasmid DNA vector to form the final single-domain antibody synthetic library.

[0033] The above method generates a CDR3 region DNA fragment library by randomly combining bases, ensuring the diversity of single-domain antibodies, covering a large number of single-domain antibodies with different sequences, and providing a rich set of candidate sequences for screening single-domain antibodies against IGFL1. This single-domain antibody synthetic library can be used to screen single-domain antibodies against various disease-related targets, not limited to LGFL1. Through high-throughput screening techniques, single-domain antibodies with high affinity and specificity can be quickly identified and optimized, providing new tools and methods for disease diagnosis and treatment.

[0034] 2. Screening of single-domain antibodies by isPLA-seq The method for screening single-domain antibodies by isPLA-seq can specifically refer to Chinese Patent Application CN202110641192.4.

[0035] The gene fragments of the single-domain antibody synthetic library were cloned into the pCDH-CMV-sdAb vector, and the IGFL1 cDNA was cloned into the pCDNA3.1-HA-C expression vector, and then co-transfected into HEK293T cells. After 48 hours of transfection, the cells were fixed and then isPLA was performed to obtain cells with positive signals. The positive cells were sorted by flow cytometry, and the plasmids in the positive cells were amplified by PCR. The amplified DNA fragments were recovered and subjected to subsequent high-throughput next-generation sequencing, from which the DNA fragment sequence and its abundance of the CDR3 region of the single-domain antibody candidate factor that binds to IGFL1 could be obtained. The top 9 single-domain antibodies with the highest abundance were recombinant into the protein expression vector for affinity detection, and finally a single-domain antibody against IGFL1 with high affinity and specificity was obtained.

[0036] The specific process is as follows: 2.1 Cloning and co-transfection of single-domain antibody gene fragments 1) Clone the single-domain antibody gene fragments: The gene fragments of the single-domain antibody synthetic library were cloned into the expression vector with a 3×Flag tag to construct a single-domain antibody expression plasmid.

[0037] 2) Clone the IGFL1 cDNA: The IGFL1 cDNA was cloned into the expression vector with an HA tag to construct an IGFL1 expression plasmid.

[0038] 3) Co-transfect HEK293T cells: The single-domain antibody expression plasmid constructed in step 1) and the IGFL1 expression plasmid constructed in step 2) were co-transfected into HEK293T cells. After 48 hours of transfection, the cells were fixed for isPLA experiment.

[0039] 2.2 isPLA experiment and sorting of positive cells isPLA (in situ proximity ligation assay) is a highly sensitive molecular detection method for visualizing protein interactions at the single-cell level. This technique uses specific antibodies to recognize and bind to target proteins, and then a PLA probe with a segment of oligodeoxynucleotide (single-stranded DNA) recognizes and binds to the primary antibody; when two target proteins are close, the DNAs of the PLA probes of the two target proteins will pair and complement each other, and then under the action of ligase, the DNA fragments on the PLA probes are ligated together to form a circular structure, and a detectable signal is generated through rolling circle amplification (RCA).

[0040] The specific process is as follows: 1) Fixation and permeabilization: The prepared cell slides were fixed with 4% paraformaldehyde and then permeabilized with 0.2% TritonX-100.

[0041] 2) Blocking: Add the blocking solution dropwise onto the cell smear slides, ensuring that the blocking solution evenly covers the entire tissue area, and incubate at 37 °C for 1 hour.

[0042] 3) Incubating with primary antibodies: Evenly drip the diluted Flag (1:500) and HA (1:500) primary antibodies onto the blocked cell smear slides, place them in a humidified chamber, and incubate at 37 °C for 2 - 3 hours.

[0043] 4) Incubating with PLA probes: Mix the PLUS and MINUS PLA probes, dilute them according to the ratio in the kit instructions, aspirate the primary antibody solution completely, wash the slides with 1× Wash Buffer A twice, 5 minutes each time. Aspirate the excess wash buffer, then add the PLA probe solution and incubate at 37 °C for 1 hour.

[0044] 5) Ligation and amplification: Add the oligodeoxynucleotides complementary to the probes (hybridization solution) and ligase to form a closed loop. Add polymerase, using one of the probes as a template, and continuously form new closed loops through rolling circle replication.

[0045] 6) Detection: Add the fluorescein-labeled oligonucleotides (detection solution) to act on the circularized DNA to form detectable fluorescent signals.

[0046] 7) Flow cytometry sorting: Use flow cytometry to sort the cells with positive red fluorescent signals. These positive cells indicate the successful binding of the single-domain antibody to IGFL1.

[0047] 8) PCR amplification and DNA fragment recovery: Perform PCR amplification on the plasmids in the sorted positive cells and recover the amplified DNA fragments.

[0048] 2.3 High-throughput next-generation sequencing: Perform high-throughput next-generation sequencing on the recovered DNA fragments, from which the DNA fragment sequences and abundances of the CDR3 regions of the single-domain antibody candidate factors binding to IGFL1 can be obtained.

[0049] 3. Expression and purification of single-domain antibodies 3.1 Construction of expression vector: Clone the screened single-domain antibody gene sequence into the pET-28a expression vector to construct an expression vector containing the following elements: 6 histidine (His6) tags, the trans-membrane peptide TAT (amino acid sequence as shown in SEQ ID NO.16: YGRKKRRQRRR) domain, the single-domain antibody sequence recognizing IGFL1, 3×Flag tags, and the molecular weight of this expression vector is 15 kDa.

[0050] 3.2 Transformation and Expression: The constructed expression vector was transformed into Escherichia coli BL21(DE3) competent cells. Under the induction of IPTG (isopropyl-β-D-thiogalactoside), the single-domain antibody protein was expressed. The induction conditions were 0.2 mM IPTG and induction at 16°C for 16 hours.

[0051] 3.3 Purification: The expressed bacterial culture was collected, and the cells were disrupted by sonication. The supernatant was collected. Purification was carried out using a nickel bead (Ni-NTA) affinity chromatography column. Utilizing the high affinity between the His6 tag and the nickel beads, the single-domain antibody protein was specifically bound. The chromatography column was washed with a balance buffer (such as 20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0) to remove impurities. The target protein was eluted with an elution buffer (such as 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 8.0). Subsequently, the solvent in the protein solution was replaced with PBS buffer by dialysis.

[0052] 3.4 Purity Detection and Preservation: The purity of the purified single-domain antibody protein was detected by methods such as SDS-PAGE and HPLC to ensure that its purity reached over 95%. The purified single-domain antibody protein was aliquoted and stored at -80°C or freeze-dried for subsequent experimental research and drug development.

[0053] In the above method, by using the pET-28a expression vector, the single-domain antibody protein can be efficiently expressed, improving the stability and solubility of the protein. Through nickel bead affinity chromatography and elution methods, a single-domain antibody protein with high purity can be obtained, and the purity can reach over 95%. The single-domain antibody protein with a TAT transmembrane peptide domain and a 3×Flag tag at the C-terminus not only retains the specific binding ability to IGFL1 but also has good cell penetration ability and is suitable for a variety of biomedical applications.

[0054] The single-domain antibody protein obtained by the method of this example can be used in a variety of biomedical research, including cell experiments, animal experiments, and preclinical studies, providing new tools and methods for the diagnosis and treatment of IGFL1-positive tumors.

[0055] Results and Analysis: Through the above method, the present invention screened single-domain antibodies of IGFL1. The present invention constructed the SdAblibrary library and screened according to the existing isPLA-seq method, wherein the antigen complementary determining region CDR3 contains 20 amino acids. In the isPLA-seq screening, first, IGFL1-HA and the SdAbs-Flag artificial synthetic library were transiently overexpressed in HEK239T cells at the same time. The red fluorescent signal in situ in the cell was obtained by isPLA, and the positive cells were sorted out by cell flow sorting. The positive rate of the control group was 0, and the positive rate of the test group was 23.5%. The results are as follows Figure 1 A. Then, the PLA-positive cells were observed under a fluorescence microscope to emit specific red fluorescence on the organelle membrane. PCR amplification was performed by designing forward and reverse primers of CDR3. The results are shown in Figure 1 As shown in B, a 108 bp CDR3 mixture was obtained, which was recovered and subjected to second-generation sequencing to obtain the DNA and amino acid sequences of the CDR3 region. First, the top 9 single-domain antibodies SdAb-IGFL1#1-SdAb-IGFL1#9 with the highest abundance in the test group were selected for subsequent verification and experiments.

[0056] Example 2 Single domain antibody affinity verification 1. GST Pull-Down Assay In order to verify whether the nine candidate single-domain antibodies SdAb-IGFL1#1-SdAb-IGFL1#9 with the highest sequencing abundance directly bind to IGFL1, a GST Pull-Down experiment was performed.

[0057] The specific experimental process is as follows: 1) Protein extraction and purification: Clone the cDNA of IGFL1 into the PGEX-4T-1 vector to form a prokaryotic GST-IGFL1 fusion protein. Use IPTG to induce expression, collect the bacteria, lyse and remove the precipitate, then add an appropriate volume of 50% glutathione-agarose gel 4B and shake slowly on a shaker at 4°C for 30-60 minutes. Centrifuge at 4°C, 4000rpm for 5 minutes and discard the supernatant. Wash the beads with pre-cooled PBS solution and repeat this step 3 times. Aspirate the liquid on the surface of the beads, but be careful not to aspirate the beads, and you can get the agarose gel bound to GST-IGFL1.

[0058] 2) System Incubation and Pull-Down: Mix the solution containing GST-IGFL1 protein and candidate single-domain antibodies, and incubate the mixture with rotation at 4°C overnight. Centrifuge at 4000 rpm for 5 minutes at 4°C, discard the supernatant, wash with pre-cooled buffer, and repeat three times. After sucking dry the water layer above the agarose gel, add 1× protein electrophoresis loading buffer, boil the protein sample, and then aliquot and store it frozen at -80°C for subsequent detection.

[0059] Finally, 7 single-domain antibodies that bind to IGFL1 were obtained, namely IGFL1-SdAb#1, IGFL1-SdAb#4, IGFL1-SdAb#5, IGFL1-SdAb#6, IGFL1-SdAb#7, IGFL1-SdAb#8, and IGFL1-SdAb#9. The 7 single-domain antibodies all contain 3 complementarity-determining regions CDR1-3 and 4 framework regions FR1, FR2, FR3, and FR4. The amino acid sequences of CDR1-3 are shown in Tables 1-3:

[0060]

[0061]

[0062] The amino acid sequences of FR1 of the 7 single-domain antibodies are the same, all as shown in SEQ ID NO.12, and the specific amino acid sequence is MGQVQLVESGGGSVQAGGSLRLSCTAS.

[0063] The amino acid sequences of FR2 of the 7 single-domain antibodies are the same, all as shown in SEQ ID NO.13, and the specific amino acid sequence is WFRQAPGQEREAVA.

[0064] The amino acid sequences of FR3 of the 7 single-domain antibodies are the same, all as shown in SEQ ID NO.14, and the specific amino acid sequence is RFTISRDNAKNTVTLQMNNLKPEDTAIYYCAA.

[0065] The amino acid sequences of FR4 of the 7 single-domain antibodies are the same, all as shown in SEQ ID NO.15, and the specific amino acid sequence is WGQGTQVTVSS.

[0066] 2. SPR Experiment The present invention further detects the affinity of 7 candidate single-domain antibodies with sufficient sample amounts among the above 9 single-domain antibodies through surface plasmon resonance (SPR) experiment.

[0067] The specific process is as follows: 1) Experimental design: At least 8 concentration gradients, low coupling and high flow rate, affinity K D The values must fall within the concentration range. Set at least one concentration of sample replicates (completed at intervals), and set a zero-concentration sample.

[0068] 2) Kinetic analysis: Obtain kinetic ka, kd, and affinity by fitting all curves K D 。 K D =kd / ka.

[0069] 3) Measure the response value at steady state, high ligand coupling level (high ligand concentration, coupling flow rate, coupling loading time).

[0070] Results and analysis: The specific binding of antibodies to antigens is the key mechanism for their biological functions. Single-domain antibodies often specifically bind to antigens through their CDR3 regions. First, to verify whether 9 candidate single-domain antibodies directly bind to IGFL1, GST pull-down experiments were performed. The results showed that 7 of the single-domain antibodies directly bound to IGFL1 (the results are shown in Figure 2 A and Figure 2 B), including SdAb-IGFL1#1, SdAb-IGFL1#4, SdAb-IGFL1#5, SdAb-IGFL1#6, SdAb-IGFL1#7, SdAb-IGFL1#8, SdAb-IGFL1#9. In addition, SdAb-IGFL1#4 was also able to co-precipitate the known interacting protein p53 from 293T cells (the results are shown in Figure 2 C).

[0071] Furthermore, the affinities of these 7 candidate single-domain antibodies were detected by SPR experiments. Their dissociation constants (K D ) were 150.2 nM, 5.925 μM, 9.5 μM, 170.6 nM, 186.7 nM, 31.83 nM, and 1.068 μM, respectively. However, SdAb-IGFL1#1 had non-specific binding. Therefore, two candidate antibodies, SdAb-IGFL1#6 and SdAb-IGFL1#8, were selected according to the affinity order (the results are shown in Figure 2 D). The above results indicate that the single-domain antibodies of IGFL1 specifically bind to IGFL1 and have strong affinities. In summary, 7 candidate single-domain antibodies that specifically bind to IGFL1 were obtained through isPLA-seq and subsequent experimental techniques.

[0072] Example 3 Preparation of single-domain antibodies and treatment of cells IGFL1 is a secreted protein that plays a role in promoting tumors by being secreted extracellularly. The present invention hypothesizes that the single-domain antibody of IGFL1 exerts its effect by inhibiting the maturation and secretion process of intracellular IGFL1 protein, or by inhibiting extracellularly secreted IGFL1. Therefore, in the present invention, a single-domain antibody fused with the TAT transmembrane peptide at the N-terminus was prepared, and it was found that it could enter cells and exert its effect with the highest efficiency.

[0073] 1. Construction of recombinant plasmid containing TAT The DNA sequence of the TAT transmembrane peptide was ligated with the DNA sequence of the candidate nanobody using (G4S)3 and constructed into the pET-28a vector for fusion expression.

[0074] The construction process is as follows: 1.1 Design and synthesis of fusion gene fragments (1)Sequence design: The sequence of the TAT transmembrane peptide (such as: 5'TACGGGCGTAAAAAACGTCGTCAACGTCGTCGT3') is SEQ ID NO.17 (G4S)3 flexible linker (5'GGTGGTGGTTCTGGTGGTGGTTCTGGTGGTGGTTCT3') sequence SEQ ID NO.18 Candidate single-domain antibody sequence (such as VHH fragment) NdeI and XhoI restriction sites (CATATG&CTCGAG) were introduced at the 5' and 3' ends respectively.

[0075] (2)Synthesis method: The complete fusion fragment (TAT(G4S)3 single-domain antibody) was directly synthesized by Tsingke Gene Synthesis Company, or synthesized in segments and spliced by overlap PCR.

[0076] 1.2 PCR amplification of fusion gene 1) Overlap PCR: The TAT, (G4S)3, and single-domain antibody fragments were amplified separately, and primers for the overlapping region were designed.

[0077] First-round PCR: Each fragment was amplified separately.

[0078] Second-round PCR: Using the equimolar ratio of mixed fragments as the template, the complete fusion gene was amplified with the outer primers.

[0079] 2) PCR conditions: Pre-denaturation: 98°C, 30 sec 30 cycles: 98°C for 10 sec, 55°C for 15 sec, 72°C for 30 sec / kb Final extension: 72°C, 5 min.

[0080] 3) Gel electrophoresis verification: Detect the size of the PCR product by 1% agarose gel electrophoresis, and cut and recover the target band.

[0081] 1.3 Double digestion of vector and inserted fragment 1) Digestion of pET28a vector: Reaction system (20 μL): pET28a 1 μg NdeI 1 μL XhoI 1 μL 10× Buffer 2 μL ddH2O to make up to 20 μL React at 37°C for 2 hours and inactivate at 65°C for 10 minutes.

[0082] 2) Digestion of fusion gene fragment: Same conditions as vector digestion.

[0083] 3) Purify the digested products: Use a gel recovery kit to purify the linearized vector and inserted fragment.

[0084] 1.4 Ligation reaction Ligation system (10 μL): ① Linearized pET28a 50 ng ② Fusion gene fragment (3:1 molar ratio in excess) ③ T4 DNA ligase 1 μL ④ 10× Ligase Buffer 1 μL ⑤ ddH2O to make up to 10 μL Ligate at 16°C for 2 hours or at room temperature for 1 hour.

[0085] 1.5 Transformation and screening of positive clones 1) Transform DH5α competent cells: ① Take 5 μL of the ligation product and add it to 50 μL of DH5α competent cells, and incubate on ice for 30 minutes.

[0086] ② Heat shock at 42°C for 45 seconds and incubate on ice for 2 minutes.

[0087] ③ Add 500 μL of LB (without antibiotics) and resuscitate at 37°C for 1 hour.

[0088] ④ Spread on an LB plate containing kanamycin (50 μg / mL) and incubate overnight at 37°C.

[0089] 2) Colony PCR verification: ① Pick a single colony and verify it by PCR using T7 universal primers or gene-specific primers.

[0090] ② Send the positive clone for sequencing to confirm the sequence correctness.

[0091] 1.6 Plasmid extraction and transformation into expression strains 1) Extract the plasmid of the positive clone: Use a plasmid miniprep kit to extract the recombinant plasmid (pET-28a TAT(G4S)3 single-domain antibody).

[0092] 2) Transform BL21(DE3) competent cells: Follow the above transformation steps to obtain the expression strain.

[0093] 2. Treat cells with the single-domain antibody Add the prepared single-domain antibody to the cell culture supernatants of triple-negative breast cancer cell lines HCC1806 and HCC1937 at a concentration of 2 μg / mL. After 48 hours of treatment, detect the amount of the single-domain antibody and its localization in the cells using an Anti-VHH (488) fluorescent secondary antibody.

[0094] Taking the addition of the single-domain antibody to HCC1806 cells as an example, the specific experimental steps are as follows: 2.1 Cell seeding and culture Seed HCC1806 cells at an appropriate density (such as 5×10 4 / well) into 24-well plates (containing sterile coverslips) or confocal dishes. Incubate at 37°C and 5% CO2 until the cell density reaches 60 - 70% (24 hours).

[0095] 2.2 Treatment with the single-domain antibody Dilute the purified single-domain antibody with pre-warmed complete medium to a final concentration of 2 μg / mL.

[0096] Set up control groups: 1) Negative control: Only medium (without antibody).

[0097] 2) Isotype control: Nonspecific isotype nanobody (such as AntiRFP VHH).

[0098] Treat the cells: 1) Aspirate the original medium and add fresh medium containing the single-domain antibody (500 μL / well).

[0099] 2) Incubate at 37°C and 5% CO2 for 48 hours.

[0100] 2.3 Cell Fixation and Permeabilization 1) Fixation: Aspirate the culture medium and gently wash the HCC1806 cells 3 times with pre-cooled PBS. Add 4% PFA (500 μL / well) and fix at room temperature for 15 minutes. Wash 3 times with PBS, 5 minutes each time.

[0101] 2) Permeabilization: Add 0.1% Triton X-100 (prepared with PBS) and permeabilize at room temperature for 10 minutes. Wash 3 times with PBS, 5 minutes each time.

[0102] 2.4 Blocking and Prevention of Non-specific Binding Blocking: Add 1% BSA (prepared with PBS) and block at room temperature for 30 minutes. Aspirate the blocking solution without washing.

[0103] 2.5 Incubation with Fluorescent Secondary Antibody Antibody Incubation: Dilute the AntiVHH(488) secondary antibody with 1% BSA (at the ratio specified in the instruction manual, such as 1:500). Add the secondary antibody solution (200 μL / well), incubate in the dark at room temperature for 1 hour (or overnight at 4°C). Wash 3 times with PBS, 5 minutes each time (operate in the dark).

[0104] 2.6 Nuclear Staining and Mounting 1) DAPI Staining: Add 1 μg / mL DAPI (prepared with PBS) and incubate in the dark for 5 minutes. Wash 3 times with PBS, 5 minutes each time.

[0105] 2) Mounting: Use forceps to take out the coverslip, invert it on the glass slide, and add the anti-fluorescence quenching mounting medium (ProLongGold). After drying in the dark, store at 4°C for detection.

[0106] Replace the HCC1806 cells with HCC1937 cells, and the experimental method of adding the single-domain antibody to HCC1937 cells is the same as above.

[0107] Results and Analysis: A single-domain antibody with a purity greater than 95% was obtained through prokaryotic expression, enabling it to enter the cells and exert its function with maximum efficiency (the results are as shown in Figure 3 Figure A). At the same time, the single-domain antibody of IGFL1 can indeed enter the triple-negative breast cancer cell lines HCC1806 and HCC1937 under the action of the TAT cell-penetrating peptide (the results are as shown in Figure 3 Figure B).

[0108] Example 4 Activity Detection of Single-Domain Antibody Verify the activity of the single-domain antibody through three cell experiments: CCK8 assay, colony formation assay, and cell stemness detection assay.

[0109] 1.1 CCK8 assay To detect whether the IGFL1 single-domain antibody has cytotoxic activity against tumor cells, the present invention detected the proliferation of HCC1806 and MDA-MB-231 cells under the treatment of the single-domain antibody through the CCK8 assay.

[0110] The specific method is as follows: 1) Cell culture: HCC1806 and MDA-MB-231 cells were respectively cultured in DMEM medium containing 10% fetal bovine serum in an incubator at 37°C and 5% CO2. When the cells reached 70-80% confluence, experimental treatment was carried out.

[0111] 2) Single-domain antibody treatment: The cells were seeded into 96-well plates at a density of 5000-10000 cells / well, with 100 μL of medium in each well. After the cells adhered, different concentrations of the IGFL1 single-domain antibody (0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM) were added respectively, and three replicates were set for each concentration. The control group was added with an equal volume of PBS.

[0112] 3) CCK8 detection: After 48 hours of treatment, 10 μL of CCK8 reagent was added to each well and cultured for another 1-2 hours, and the specific time was optimized according to cell types and experimental conditions. The absorbance (OD value) was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0113] 4) Data analysis: Calculate the ratio of the OD value of the single-domain antibody treatment group to the OD value of the control group at each concentration, and draw a concentration-response curve. Use nonlinear regression analysis to calculate the half-maximal inhibitory concentration (IC50).

[0114] 1.2 Colony formation assay To identify the single-domain antibodies with high affinity and the best anti-tumor activity effect, the present invention selected SdAb-IGFL1#6 and SdAb-IGFL1#8 single-domain antibodies for in-depth study according to their dissociation constants and IC50.

[0115] The specific method is as follows: 1) Cell culture: HCC1806 cells were cultured in DMEM medium containing 10% fetal bovine serum in an incubator at 37°C and 5% CO2. When the cells reached 70-80% confluence, experimental treatment was carried out.

[0116] 2) Single-domain antibody treatment: Cells were seeded into a 6-well plate at a density of 500 cells / well, with 2 mL of medium per well. After the cells adhered, single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 were added respectively.

[0117] 3) Colony formation assay: After treatment, the cells were cultured for 10 - 14 days until visible colonies formed. The cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The colonies were observed and counted under a microscope, and a colony was defined as a cluster of at least 50 cells.

[0118] 4) Data analysis: Calculate the ratio of the number of colonies in the single-domain antibody treatment group to the number of colonies in the control group.

[0119] 1.3 Cell stemness detection experiments, including ALDH, WB, and mamosphere detection experiments One of the most important roles of IGFL1 in promoting the progression of triple-negative breast cancer is to maintain the stemness of tumor cells. Therefore, developing single-domain antibodies that can inhibit the stemness of tumor cells is particularly important for the research and development of tumor therapeutic drugs. This invention detected the effect of IGFL1 single-domain antibodies on the stemness of tumor cells.

[0120] 1.3.1 ALDH detection This invention detected the effect of SdAb-IGFL1#6 and SdAb-IGFL1#8 on the proportion of ALDH + cells in HCC1806 cells with overexpressed IGFL1. The results showed that these two single-domain antibodies reduced the proportion of ALDH + cells, and the reduced proportion was about 20 - 30%.

[0121] The specific method is as follows: 1) Cell staining The cells were prepared into a single-cell suspension, and activated BAAA was added for staining. At the same time, a DEAB control group was set. Incubate at 37 °C for 30 - 60 minutes to allow ALDH to fully react with the substrate.

[0122] 2) Flow cytometry detection Use flow cytometry to detect the fluorescence intensity of the cells, and analyze the ALDH activity through the FL1 channel. Distinguish ALDH-high activity cells (ALDHbr) and low-activity cells according to the fluorescence intensity.

[0123] 3) Data analysis Calculate the percentage of ALDHbr cells, and analyze the differences in ALDH activity under different samples or treatment conditions. Combine cell phenotype analysis to explore the relationship between ALDH activity and cell function.

[0124] 1.3.2 WB experiment The specific method is as follows: 1) Protein separation Perform SDS-PAGE and select an appropriate gel concentration according to the molecular weight of the target protein. Electrophoretically separate the proteins to ensure clear bands.

[0125] 2) Membrane transfer Transfer the separated proteins onto a PVDF or nitrocellulose membrane to ensure the membrane transfer efficiency. Use the wet transfer or semi-dry transfer method, and control the membrane transfer time and voltage to be 2 hours and 120V respectively.

[0126] 3) Antibody incubation Block the non-specific binding sites on the membrane with 5% skim milk powder. Add the primary antibody and incubate overnight at 4°C; add the secondary antibody and incubate for 2 hours at room temperature.

[0127] 4) Color development and imaging Use a chemiluminescence or fluorescence imaging system to detect the target protein bands. Adjust the exposure time to ensure clear visibility of the bands.

[0128] 1.3.3 qPCR experiment 1) RNA extraction Add 500 μl of Trizol reagent to the cell sample, fully lyse the cells, let it stand for 5 minutes, then add 100 μl of chloroform, shake well and let it stand for 5 minutes. Centrifuge at 12000 rpm and 4°C for 10 minutes, take the supernatant and add an equal volume of isopropanol, let it stand for 10 minutes and then centrifuge again, discard the supernatant. Wash the RNA precipitate with 1 ml of 75% ethanol, centrifuge at 4°C and 7000 rpm for 5 minutes, discard the supernatant and dry at room temperature for 5 - 10 minutes. Add 25 μL of DEPC water to dissolve the RNA and store it at -80°C for later use.

[0129] 2) RNA concentration and purity determination Use a nucleic acid and protein detector to measure the RNA concentration and purity (A260 / A280 ratio) to ensure that the RNA concentration is within an appropriate range and the purity meets the requirements.

[0130] 3) Reverse transcription Prepare the reverse transcription reaction system according to the kit instructions, which usually includes reverse transcriptase, reaction buffer, primers, etc., and add the RNA template to the reaction system. Carry out the reverse transcription reaction at an appropriate temperature, usually 42°C for 15 - 30 minutes, and then terminate the reaction at 70°C to obtain the cDNA template.

[0131] 4) Primer design According to the gene sequence of the stemness marker, primers were designed using primer design software (such as Primer Premier). The primer length was 20 - 25 bp, the Tm value was about 60 °C, and the amplified fragment length was 150 - 250 bp. Avoid the formation of secondary structures within the primer itself or between primers to ensure primer specificity.

[0132] 5) Primer verification Use conventional PCR amplification to verify primer specificity and observe whether a single target band is amplified without primer dimers.

[0133] Further verify primer amplification efficiency and specificity through qPCR amplification curves and melting curves to ensure that the primers are suitable for qPCR experiments.

[0134] 6) Configuration of qPCR reaction system Configure the qPCR reaction according to the following system: 10 μL of 2×qPCR Mix, 1 μL of 2 μM primer F, 1 μL of 2 μM primer R, 1 μL of cDNA template, and make up to 20 μL with ultrapure water. Set 3 technical replicates for each sample, and at the same time set a no-template control (NTC) and an internal reference gene control.

[0135] 7) Amplification program Pre-denature at 95 °C for 2 minutes; 95 °C for 15 seconds, 60 °C for 30 seconds, for 40 cycles; after amplification, perform melting curve analysis, from 65 °C to 95 °C, staying for 5 seconds at every 0.5 °C. The amplification program can be appropriately adjusted according to different instruments and reagents.

[0136] 8) Data analysis Collect fluorescence signals through the qPCR instrument software, calculate the Ct value, and analyze the relative expression level of the target gene using the 2^-ΔΔCt method. Compare the Ct values of different treatment groups with the control group and calculate the change in relative expression level.

[0137] 9) Result interpretation If the Ct value of the target gene is low and the difference from the Ct value of the internal reference gene is small, it indicates that the expression level of this gene is high; otherwise, the expression level is low.

[0138] 1.3.4 Mamosphere detection This invention detected the effects of SdAb-IGFL1#6 and SdAb-IGFL1#8 on the formation of stem cell tumor spheres in HCC1806 cells.

[0139] The specific method is as follows: 1) Cell seeding Prepare the cells into a single-cell suspension and seed them into a low-attachment culture dish at an appropriate density. Add an appropriate amount of serum-free medium to each culture dish.

[0140] 2) Mamosphere formation Culture under the conditions of 37 °C and 5% CO2, and change the culture medium regularly. Observe the formation process of mamospheres, and record their size and quantity.

[0141] 3) Data analysis Calculate the mamosphere formation efficiency (MFE), and analyze the differences in stem cell activity under different treatment conditions. Combine cell phenotype analysis to explore the relationship between mamospheres and tumorigenesis, drug resistance, etc.

[0142] Results and analysis: 2.1 Results of the CCK8 assay To detect whether the IGFL1 single-domain antibody has cytotoxic activity against tumor cells, the proliferation of HCC1806 and MDA-MB-231 cells treated with the single-domain antibody for 48 h was detected by the CCK8 assay, as Figure 4 shown. The results showed that seven IGFL1 single-domain antibodies (SdAb-IGFL1#1, SdAb-IGFL1#4, SdAb-IGFL1#5, SdAb-IGFL1#6, SdAb-IGFL1#7, SdAb-IGFL1#8, and SdAb-IGFL1#9) all exhibited strong cytotoxicity and killed tumor cells in a concentration-dependent manner. Their IC50 values in HCC1806 cells were: SdAb-IGFL1#1: 134.26 nM, SdAb-IGFL1#4: 237.63 nM, SdAb-IGFL1#5: 160.89 nM, SdAb-IGFL1#6: 212.21 nM, SdAb-IGFL1#7: 21.90 nM, SdAb-IGFL1#8: 83.32 nM, and SdAb-IGFL1#9: 76.68 nM. Their IC50 values in MDA-MB-231 cells were: SdAb-IGFL1#1: 374.79 nM, SdAb-IGFL1#4: 206.95 nM, SdAb-IGFL1#5: 680.53 nM, SdAb-IGFL1#6: 461.21 nM, SdAb-IGFL1#7: 533.68 nM, SdAb-IGFL1#8: 383.63 nM, and SdAb-IGFL1#9: 785.26 nM ( Figure 4A). The results showed that 7 single-domain antibodies had strong cytotoxicity and killed tumor cells in a concentration-dependent manner. Among them, SdAb-IGFL1#6 and SdAb-IGFL1#8 showed high anti-tumor activity in both cell lines, with low IC50 values and high affinity. These single-domain antibodies provide new candidate drugs for the treatment of IGFL1-positive tumors.

[0143] 2.2 Results of colony formation assay How the IGFL1 single-domain antibody inhibits the activity of tumor cells is one of the cores of the present invention. To identify the single-domain antibody with the best anti-tumor activity effect, the IGFL1 single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 were selected for in-depth study according to their dissociation constants. First, the results of the colony formation assay showed that overexpression of IGFL1 significantly enhanced the colony formation ability of HCC1806 cells, and both SdAb-IGFL1#6 and SdAb-IGFL1#8 single-domain antibodies could inhibit the colony formation caused by IGFL1 (as shown in Figure 4 Figure B). Second, the results of the cell counting assay showed that overexpression of IGFL1 significantly promoted the proliferation of HCC1806 cells, and both SdAb-IGFL1#6 and SdAb-IGFL1#8 single-domain antibodies could significantly inhibit IGFL1-mediated cell proliferation (as shown in Figure 4 Figure C). In summary, the IGFL1 single-domain antibody has strong cytotoxicity against triple-negative breast cancer cells.

[0144] Conclusion: These results indicate that SdAb-IGFL1#6 and SdAb-IGFL1#8 can significantly inhibit the colony formation of triple-negative breast cancer cells HCC1806 by blocking the activity of IGFL1, indicating that they have strong anti-tumor activity. This further confirms that these two single-domain antibodies have significant effects in inhibiting tumor cell proliferation and colony formation, providing strong evidence for subsequent mechanism research and preclinical experiments.

[0145] 2.3 Results of cell stemness detection experiments (including ALDH, WB, mamosphere detection experiments, qPCR) One of the most important roles of IGFL1 in promoting the progression of triple-negative breast cancer is to maintain the stemness of tumor cells. Therefore, developing single-domain antibodies that can inhibit the stemness of tumor cells is particularly important for the research and development of anti-tumor drugs. The present invention detected the effect of the IGFL1 single-domain antibody on the stemness of tumor cells. First, overexpression of IGFL1 increased the ALDH of HCC1806 + cells, and the treatment with SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly reduced the ALDH +The proportion of cells, and the reduced proportion is about 20 - 30% (as Figure 5 shown in A). Secondly, SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly inhibited the formation of tumor spheres with stem cell characteristics in HCC1806 cells overexpressing IGFL1 (as Figure 5 shown in B). In terms of molecular mechanism, SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly reduced the expression of IGFL1 and stem cell markers in tumor cells overexpressing IGFL1, including SOX2, SOX9, Nanog, and OCT4, etc. (as Figure 5 shown in C). The qPCR results showed that SdAb-IGFL1#6 and SdAb-IGFL1#8 reduced the mRNA expression of IGFL1 and stem cell markers (as Figure 5 shown in D). In summary, the IGFL1 single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly inhibited the maintenance of stem cell characteristics in triple-negative breast cancer cells by blocking the activity of IGFL1.

[0146] To explore the molecular mechanism by which IGFL1 single-domain antibodies inhibit tumors, the present invention treated breast cancer tumor cells with SdAb-IGFL1#6 and SdAb-IGFL1#8. The WB experimental results showed that in the cell line overexpressing IGFL1, both SdAb-IGFL1#6 and SdAb-IGFL1#8 reduced the expression of IGFL1. Secondly, SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly inhibited the phosphorylation of β-catenin, IRS-1, PI3K, and AKT mediated by IGFL1. In addition, SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly inhibited the expression of downstream genes of the PI3K / AKT pathway, including C-myc, CyclinD1, etc. (as Figure 6 shown). The above results indicate that the IGFL1 single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 : 1) inhibit tumor progression by downregulating the expression of IGFL1; 2) inhibit the activation of the PI3K / AKT pathway to downregulate the expression of the pro-oncogenes C-myc and CyclinD1.

[0147] Example 5 Verification of antitumor effect in vivo Verifying anti-tumor activity through animal experiments: Establish a subcutaneous xenograft tumor model of triple-negative breast cancer in nude mice (the method for model establishment can refer to the literature: Wang H, Shi Y, Chen CH, Wen Y, Zhou Z, Yang C, Sun J, Du G, Wu J, Mao X, Liu R, Chen C. KLF5-induced lncRNA IGFL2-AS1 promotes basal-like breast cancer cell growth and survival by upregulating the expression of IGFL1. Cancer Lett. 2021 Sep 1;515:49-62. doi: 10.1016 / j.canlet.2021.04.016. Epub 2021 May 27. MID: 34052325.), and administer the single-domain antibody by intraperitoneal injection. Regularly measure the tumor volume, and the results show that the single-domain antibody can significantly inhibit tumor growth.

[0148] Establish an orthotopic tumor model with HCC1806 cells, and the experimental method for detecting the in vivo tumor suppression effect of the single-domain antibody is as follows: 1) Cell line preparation: Culture HCC1806 cells in DMEM medium containing 10% fetal bovine serum, and place them in an incubator at 37°C and 5% CO2. When the cells reach 70-80% confluence, perform experimental treatment.

[0149] 2) Establishment of orthotopic tumor model: Inject the HCC1806 cell suspension (1×10^6 cells / 100 μL) into the mammary fat pad of mice to establish an orthotopic tumor model.

[0150] 3) Start single-domain antibody treatment 6 days after the tumor begins to grow. Administer SdAb-IGFL1#6 and SdAb-IGFL1#8 by intraperitoneal injection respectively. Inject 10 mg / kg 10 times, and administer the drug on the 6th day after random grouping, once every other day.

[0151] 4) Evaluation of treatment effect: Every other day, measure the long and short diameters of the tumor with calipers, and calculate the tumor volume (V = 0.5 × long diameter × short diameter 2 ). After the treatment is over, collect the tumor tissue and weigh the tumor weight. The results are as Figure 7 shown.

[0152] Replace the HCC1806 cells with MDA-MB-231 cells, and re-establish the orthotopic tumor model. The experimental method is the same as above.

[0153] Results and analysis: Based on the inhibitory effects of the single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 against IGFL1 in triple-negative breast cancer, an in vivo animal experiment of orthotopic mammary tumors in mice was conducted. Consistent with the in vitro test results, the single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly reduced the volume and weight of the orthotopic tumors formed by HCC1806 and MDA-MB-231 cells in the mammary glands of mice ( Figure 7 A-C and Figure 7 E-G), and SdAb-IGFL1#6 and SdAb-IGFL1#8 had no significant effect on the body weight of mice ( Figure 7 D and Figure 7 H). In addition, immunohistochemistry experiments showed that in the tumors of mice, the treatment with the single-domain antibodies against IGFL1 significantly promoted the expression of the apoptosis-related marker Caspase-3, and conversely, inhibited the expression of the proliferation marker Ki67 ( Figure 7 I and Figure 7 J). It shows that in vivo, the single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 can inhibit IGFL1, thereby promoting tumor apoptosis and inhibiting tumor proliferation, and ultimately achieving an effective therapeutic effect on triple-negative breast cancer.

[0154] The above results indicate that the single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 can achieve an effective therapeutic effect on triple-negative breast cancer by inhibiting IGFL1 in vivo and have no significant effect on the body weight of mice, indicating their good safety and therapeutic effect. This further confirms that single-domain antibodies have a significant effect in inhibiting the proliferation of tumor cells and provides strong evidence for subsequent mechanism research and preclinical experiments.

[0155] The above are only embodiments of the present invention and do not limit the patent scope of the present invention accordingly. Any equivalent transformation made using the content of the specification of the present invention, directly or indirectly applied in other related technical fields, shall be similarly included within the patent protection scope of the present invention.

Claims

1. A single-domain antibody targeting human IGFL1, characterized in that, The variable region of the heavy chain of the single-domain antibody comprises three complementarity-determining regions, namely CDR1, CDR2 and CDR3, The amino acid sequence of the said CDR1 is as shown in SEQ ID No.10; The amino acid sequence of the said CDR2 is as shown in SEQ ID No.11; The amino acid sequence of the said CDR3 is as shown in SEQ ID No.1 or SEQ ID No.

2.

2. The single-domain antibody targeting human IGFL1 according to claim 1, characterized in that, The amino acid sequence of the variable region of the heavy chain of the single-domain antibody is as shown in SEQ ID No.3 or SEQ ID No.

4.

3. A nucleic acid molecule encoding the single-domain antibody targeting human IGFL1 as claimed in claim 1 or 2.

4. A vector containing the nucleic acid molecule as claimed in claim 3.

5. A host cell containing the vector as claimed in claim 4.

6. Use of the single-domain antibody targeting human IGFL1 as claimed in claim 1 or 2 in the preparation of a reagent for detecting human IGFL1 protein.

7. Use of the single-domain antibody targeting human IGFL1 as claimed in claim 1 or 2 in the preparation of a product that binds to human IGFL1 protein.

8. Use of the single-domain antibody targeting human IGFL1 as claimed in claim 1 or 2 in the preparation of a drug for treating breast cancer.

9. The application according to claim 8, wherein The breast cancer is triple-negative breast cancer.

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