CN120309727B - Single-domain antibody targeting human IGFL1 and application thereof - Google Patents

Abstract

The invention relates to the technical field of antibody engineering, in particular to a single domain antibody targeting human IGFL <1 > and application thereof, and particularly relates to application thereof in preparation of a medicament for treating breast cancer. The heavy chain variable region of the single domain antibody targeting human IGFL1 comprises three complementarity determining regions, namely CDR1, CDR2 and CDR3, wherein the amino acid sequence of the CDR1 is shown as SEQ ID No.10, the amino acid sequence of the CDR2 is shown as SEQ ID No.11, and the amino acid sequence of the CDR3 is shown as SEQ ID No.1 or SEQ ID No. 2. The invention successfully constructs a single-domain antibody artificial synthesis library and screens out two single-domain antibody sequences with better anti-IGFL 1 effect. The single domain antibody provided by the invention can be used for preparing medicines for treating IGFL positive tumors, in particular to triple negative breast cancer.

Description

A single-domain antibody targeting human IGFL1 and its application

Technical Field

The present invention relates to the field of antibody engineering technology, and in particular to a single-domain antibody targeting human IGFL1 and applications thereof, and in particular to applications thereof in preparing drugs for treating breast cancer.

Background Art

IGFL1 (insulin-like growth factor family-related protein 1) is associated with certain skin diseases, inflammatory disorders, and cancers, acting as an oncogene that drives tumor proliferation, migration, and invasion, making it a potential target for cancer therapy. IGFL1 expression levels in various diseases are closely correlated with disease progression, demonstrating its potential as a biomarker. For example, in lung adenocarcinoma, IGFL1 expression levels are closely associated with patient clinicopathological features and prognosis, making it an independent risk factor for prognosis. Furthermore, high IGFL1 expression in thyroid eye disease suggests its potential as a biomarker for disease diagnosis and treatment monitoring. The mechanisms of action of IGFL1 in diseases such as cancer and thyroid eye disease are gradually being elucidated. IGFL1 promotes cell proliferation and inflammatory responses by activating the IGF-1R signaling pathway. Preclinical studies have demonstrated that IGFL1 is not only an important biomarker but also holds great potential as a therapeutic target. Future studies will further explore the role of IGFL1 in additional diseases and develop novel IGFL1-based therapeutic strategies. These studies provide a variety of potential therapeutic strategies for IGFL1 targeted therapy. Although there are not many successful clinical results at present, these studies provide new directions and hope for future tumor treatment.

Breast cancer is a common malignancy in women. It is classified based on the expression of estrogen receptors (ER), progesterone receptors (PR), HER-2 receptors, and Ki67 markers, including luminal A, luminal B, and triple-negative breast cancer. Triple-negative breast cancer (TNBC) lacks expression of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2), resulting in a poor prognosis and limited treatment options. IGFL1, a protein associated with the insulin-like growth factor family, is highly expressed in various tumors and plays a crucial role in tumor cell proliferation, migration, and invasion. Single-domain antibodies (SDOs), heavy-chain variable antibody fragments, offer advantages such as small molecular weight, high stability, and strong tissue penetration, showing broad application prospects in targeted tumor therapy. Therefore, SDOs targeting IGFL1 have become a potential therapeutic strategy for breast cancer.

In the prior art, there is no disclosed single-domain antibody targeting IGFL1 for the treatment of breast cancer.

Summary of the Invention

To address the shortcomings of existing technologies, the present invention provides a single-domain antibody targeting human IGFL1 and its application. 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 approach for the treatment of IGFL1-positive tumor cells.

In order to solve the above technical problems, the technical solutions of the present invention are as follows:

In a first aspect of the present invention, a single-domain antibody targeting human IGFL1 is provided, wherein the heavy chain variable region of the single-domain antibody comprises three complementarity determining regions, namely CDR1, CDR2 and CDR3.

The amino acid sequence of the CDR1 is shown in SEQ ID No. 10;

The amino acid sequence of the CDR2 is shown in SEQ ID No. 11;

The amino acid sequence of the CDR3 is shown in SEQ ID No. 1 or SEQ ID No. 2.

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

The single-domain antibody of the present invention can be expressed in a prokaryotic or eukaryotic expression system, such as Escherichia coli, yeast, insect cells or mammalian cells, by genetic engineering technology to obtain a single-domain antibody protein with biological activity.

In a 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.

In the third aspect of the present invention, a vector containing the nucleic acid molecule according to the second aspect is provided.

In the fourth aspect of the present invention, a host cell containing the vector according to the third aspect is provided.

In a fifth aspect of the present invention, there is provided use of the single-domain antibody targeting human IGFL1 as described in the first aspect in preparing a human IGFL1 protein detection reagent.

In a sixth aspect of the present invention, there is provided use of the single-domain antibody targeting human IGFL1 as described in the first aspect in preparing a product that binds to human IGFL1 protein.

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

Furthermore, the breast cancer is triple-negative breast cancer.

The single-domain antibody of the present invention downregulates the expression of IGFL1 and inhibits the activation of the PI3K/AKT pathway to downregulate the expression of oncogenes C-myc and CyclinD1, thereby inhibiting the progression of tumors.

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

1) The present invention discovered that IGFL1 is highly expressed in breast cancer patients, particularly those with triple-negative breast cancer. Based on this discovery, the present invention successfully constructed a synthetic library of single-domain antibodies and screened seven novel anti-IGFL1 single-domain antibody sequences. Further validation of affinity and anti-tumor activity yielded two single-domain antibodies (SdAb-IGFL1#6 and SdAb-IGFL1#8) with the highest anti-tumor efficacy. Their amino acid sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

2) The single-domain antibody provided by the present invention can specifically bind to human IGFL1 to block IGFL1 activity, effectively inhibiting the proliferation and stemness maintenance of human triple-negative breast cancer cells. Furthermore, animal experimental results showed that the single-domain antibody of the present invention can significantly inhibit the growth of triple-negative breast carcinoma in situ in mice without affecting the mice's body weight, 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 the treatment of IGFL1-positive tumors, particularly triple-negative breast cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of screening for IGFL1 single-domain antibodies using isPLA-seq technology. A shows the results of isPLA-seq followed by flow cytometry sorting of positive cells from 293T cells. B shows the gel electrophoresis results of DNA fragments amplified from the CDR3 region using PCR.

Figure 2 shows the results of antigen binding and affinity testing of IGFL1 single-domain antibodies. Panels A-B show the results of antigen-antibody binding assays using GST pull-down assays. Panel C shows the results of a Co-IP assay using the IGFL1 single-domain antibody to detect IGFL1's interacting partners. Panel D shows the affinity of the IGFL1 single-domain antibody to the IGFL1 protein using SPR.

Figure 3 shows the preparation and intracellular delivery of IGFL1 single-domain antibodies. A: Schematic diagram of single-domain antibody expression fused with TAT transmembrane peptide, and seven prepared IGFL1 single-domain antibodies. B: Results of intracellular localization and quantity of single-domain antibodies detected by IF assay after treatment of HCC1806 and HCC1937 cells with single-domain antibodies. Scale bar: 200 μm.

Figure 4 shows the significant inhibitory effect of IGFL1 single-domain antibody on triple-negative breast cancer cell proliferation. A shows the results of a CCK8 assay to measure viable cell counts after adding different concentrations of IGFL1 single-domain antibody to HCC1806 and MDA-MB-231 cells for 48 hours. B shows the results of a crystal violet staining assay to measure tumor colony formation ability after adding IGFL1 single-domain antibody to HCC1806 cells overexpressing IGFL1. C shows the results of a cell counting assay to measure relative cell growth after adding IGFL1 single-domain antibody to HCC1806 cells overexpressing IGFL1. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005.

Figure 5 shows the results of IGFL1 single-domain antibodies inhibiting tumor cell stemness. A shows the results of flow cytometry analysis of the proportion of ALDH-positive cells in HCC1806 cells overexpressing IGFL1 treated with SdAb-IGFL1#6 and SdAb-IGFL1#8 single-domain antibodies. B shows the results of mamosphere analysis of tumor cell stemness in HCC1806 cells overexpressing IGFL1 treated with SdAb-IGFL1#6 and SdAb-IGFL1#8 single-domain antibodies. Scale bar, 200 μm. C shows the results of Western blotting of stemness markers in HCC1806 cells overexpressing IGFL1 treated with SdAb-IGFL1#6 and SdAb-IGFL1#8 single-domain antibodies. D shows the results of qPCR analysis of stemness markers in HCC1806 cells overexpressing IGFL1 treated with SdAb-IGFL1#6 and SdAb-IGFL1#8 single-domain antibodies. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005.

Figure 6 shows the results of WB experiments to detect changes in IRS1/p85/PI3K/AKT/β-catenin signaling pathway proteins after cells were treated with IGFL1 single domain antibody.

Figure 7 shows the results of IGFL1 single-domain antibody inhibition of mouse breast carcinoma in situ growth. A-D show the results of HCC1806 cells inoculated into the mammary fat pad of mice, followed by treatment with 10 mg/kg of the single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8. Mouse body weight, tumor volume, and tumor weight were monitored. E-H show the results of MDA-MB-23 cells inoculated into the mammary fat pad of mice, followed by treatment with 10 mg/kg of the single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8. Mouse body weight, tumor volume, and tumor weight were monitored. I-J show the results of subsequent HE and IHC (Ki67 and Caspase-3) staining of MDA-MB-231 and HCC1806 cell xenograft tumor sections, respectively. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005. Scale bar 100 μm.

DETAILED DESCRIPTION

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

The cell lines used in the examples of the present invention were purchased from the ATCC cell bank, the nude mice used were purchased from Beijing Sibeifu Biological Co., Ltd., the detection antibodies were purchased from CST and Abcam, and the biochemical reagents and kits were purchased from Beyotime Biotechnology Co., Ltd. and Solebio Biotechnology Co., Ltd. Molecular biology experimental methods not specifically described were performed in accordance with the Molecular Cloning Experiment Guide.

Specific experiments for the preparation, purification and anti-tumor activity verification of the single domain antibody of the present invention are detailed in the following examples.

Example 1 Screening and purification of single domain antibodies

1. Construction of a synthetic library of single-domain antibodies

The synthetic single-domain antibodies (SDAs) in this library are composed of FR1, FR2, FR3, FR4, and CDR1, CDR2, and CDR3. CDR3 is the most critical complementarity-determining region, so only SDAs with diverse CDR3 regions were synthesized in this synthetic library. FR1, FR2, FR3, FR4, and CDR1 and CDR2 sequences remain consistent across all SDAs. The sequences of FR1, FR2, FR3, FR4, and CDR1 and CDR2 are known, as described in 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 method for constructing a single domain antibody artificial synthesis library comprises the following steps:

Step 1: Construct a variable CDR3 library of single-domain antibodies targeting IGFL1. A highly diverse library of DNA fragments was designed and synthesized using gene synthesis. A key feature of this library is the introduction of 20 variable amino acid sites in the CDR3 region, optimized using NNN codons (N = A/T/G/C). This ensures sequence diversity while effectively reducing the occurrence of stop codons. The entire CDR3 region was designed to have a fixed length of 60 bases. The resulting library had a theoretical capacity of at least 1×10^8 clones, ensuring coverage of a wide range of possible amino acid sequence combinations. The sdAb CDR3 DNA fragment mixture was as follows: CCA TCT ACT ACT gCg CCg CTN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNT ggg gAC AAg gAA CAC AAg T N = A/T/C/G.

Step 2: Construct the pCDH-CMV-sdAb backbone vector: Design and construct a plasmid DNA vector containing the single-domain antibody backbone. This vector should contain the single-domain antibody framework regions (FR1, FR2, FR3, FR4, CDR1, and CDR2) and necessary expression regulatory elements, such as a promoter and terminator. In addition, a 3× Flag tag should be fused to the C-terminus of the vector to facilitate subsequent detection and purification. The sdAb backbone is tagged with a 3× Flag tag at the C-terminus and then ligated into the pCDH-CMV vector to obtain the pCDH-CMV-sdAb backbone vector.

Step 3: Connect the CDR fragment mixture to the pCDH-CMV-sdAb vector by homologous recombination to obtain a single-domain antibody synthetic library. The CDR3 region DNA library can be connected to the corresponding position of the single-domain antibody backbone vector using appropriate restriction enzymes or homologous recombination technology. Ensure that the connected plasmid DNA vector contains the complete single-domain antibody sequence, including FR1, FR2, FR3, FR4, CDR1, CDR2 and CDR3 regions, as well as 3×Flag tags.

Step 4: Validation and amplification of the single domain antibody synthetic library

Verify the correctness of the ligated plasmid DNA vector through sequencing and PCR amplification. Ensure that the CDR3 region DNA fragment has been correctly inserted into the single-domain antibody backbone vector and has no mutations or deletions. Amplify the verified plasmid DNA vector to form the final single-domain antibody synthetic library.

This method, through random base combination, generates a library of CDR3 region DNA fragments, ensuring the diversity of single-domain antibodies. This library encompasses a wide range of single-domain antibodies with diverse sequences, providing a rich set of candidate sequences for screening single-domain antibodies against IGFL1. This synthetic single-domain antibody library can be used to screen for single-domain antibodies against a variety of disease-related targets, not just LGFL1. High-throughput screening techniques can rapidly identify and optimize single-domain antibodies with high affinity and specificity, providing new tools and approaches for disease diagnosis and treatment.

2. isPLA-seq screening of single domain antibodies

For details about the isPLA-seq method for screening single-domain antibodies, please refer to Chinese patent application CN202110641192.4.

Gene fragments from the synthetic single-domain antibody library were cloned into the pCDH-CMV-sdAb vector, and the IGFL1 cDNA was cloned into the pCDNA3.1-HA-C expression vector. These fragments were co-transfected into HEK293T cells. 48 hours after transfection, the cells were fixed and subjected to isPLA to isolate cells with positive signals. Positive cells were sorted by flow cytometry, and plasmids from these cells were amplified by PCR. The amplified DNA fragments were recovered and subsequently subjected to high-throughput next-generation sequencing. This revealed the CDR3 region DNA sequences and abundance levels of candidate single-domain antibodies that bind to IGFL1. The top nine most abundant single-domain antibodies were recombined into protein expression vectors and affinity tested, ultimately yielding single-domain antibodies with high affinity and specificity against IGFL1.

The specific process is as follows:

2.1 Cloning and co-transfection of single-domain antibody gene fragments

1) Cloning of single-domain antibody gene fragments: Clone the gene fragments of the single-domain antibody synthetic library into an expression vector with a 3×Flag tag to construct a single-domain antibody expression plasmid.

2) Cloning of IGFL1 cDNA: Clone IGFL1 cDNA into an expression vector with an HA tag to construct an IGFL1 expression plasmid.

3) Co-transfection of HEK293T cells: Co-transfect HEK293T cells with the single-domain antibody expression plasmid constructed in step 1) and the IGFL1 expression plasmid constructed in step 2). 48 hours after transfection, fix the cells and perform the isPLA assay.

2.2 isPLA experiment and positive cell sorting

isPLA (in situ proximity ligation assay) is a highly sensitive molecular detection method used to visualize protein interactions at the single-cell level. This technique uses specific antibodies to recognize and bind to target proteins. A PLA probe containing a stretch of oligodeoxynucleotide (single-stranded DNA) then recognizes and binds to the primary antibody. When two target proteins come into proximity, the DNA fragments of the PLA probes pair and complement each other. Ligase then ligates the DNA fragments on the PLA probes, forming a circular structure that generates a detectable signal through rolling circle amplification (RCA).

The specific process is as follows:

1) Fixation and permeabilization: Fix the cell slides with 4% paraformaldehyde and then permeabilize with 0.2% TritonX-100.

2) Blocking: Add blocking solution dropwise to the cell slide, ensuring that the blocking solution evenly covers the entire tissue area. Incubate at 37°C for 1 hour.

3) Incubation with primary antibodies: Evenly add diluted Flag (1:500) and HA (1:500) primary antibodies onto the blocked cell slides, place in a humidified chamber, and incubate at 37°C for 2-3 hours.

4) PLA Probe Incubation: Mix the PLUS and MINUS PLA probes and dilute according to the kit instructions. Aspirate the primary antibody solution and wash the slides twice with 1x Wash Buffer A for 5 minutes each. Aspirate the excess wash buffer, then add the PLA probe solution dropwise and incubate at 37°C for 1 hour.

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

6) Detection: Add fluorescein-labeled oligonucleotides (detection solution) to react with the circularized DNA to form a detectable fluorescent signal.

7) Flow cytometry sorting: Cells with positive red fluorescent signals are sorted using flow cytometry. These positive cells indicate that the single-domain antibody successfully binds to IGFL1.

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

2.3 High-throughput next-generation sequencing: The recovered DNA fragments are subjected to high-throughput next-generation sequencing, thereby obtaining the CDR3 region DNA fragment sequences and abundance of the single-domain antibody candidate factors that bind to IGFL1.

3. Single Domain Antibody Expression and Purification

3.1 Construction of expression vector: The screened single-domain antibody gene sequence was cloned into the pET-28a expression vector to construct an expression vector containing the following elements: a 6-histidine (His6) tag, a transmembrane peptide TAT domain (amino acid sequence as shown in SEQ ID NO. 16: YGRKKRRQRRR), a single-domain antibody sequence that recognizes IGFL1, and a 3×Flag tag. The molecular weight of the expression vector is 15 kDa.

3.2 Transformation and Expression: The constructed expression vector was transformed into competent E. coli BL21 (DE3) cells. The single-domain antibody protein was expressed under induction with IPTG (isopropyl-β-D-thiogalactopyranoside). The induction conditions were 0.2 mM IPTG and 16°C for 16 hours.

3.3 Purification: Harvest the expressed bacterial culture, disrupt the cells by sonication, and collect the supernatant. Purify the protein using a nickel-bead (Ni-NTA) affinity chromatography column. The His6 tag binds specifically to the nickel beads with high affinity, allowing the single-domain antibody protein to bind specifically. Wash the column with equilibration buffer (e.g., 20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0) to remove impurities. Elute the target protein with elution buffer (e.g., 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 8.0). Then, exchange the solvent in the protein solution with PBS buffer by dialysis.

3.4 Purity Testing and Storage: The purity of the purified single-domain antibody protein should be tested by SDS-PAGE and HPLC to ensure that its purity reaches 95% or above. The purified single-domain antibody protein should be aliquoted and stored at -80°C or lyophilized for subsequent experimental research and drug development.

In this method, the use of the pET-28a expression vector enables efficient expression of single-domain antibody proteins, improving protein stability and solubility. Nickel-bead affinity chromatography and elution methods can yield highly purified single-domain antibody proteins, with purity exceeding 95%. Single-domain antibody proteins with a C-terminal TAT transmembrane peptide domain and a 3×Flag tag not only retain specific binding to IGFL1 but also exhibit excellent cell penetration, making them suitable for a variety of biomedical applications.

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.

Results and Analysis:

Using the above method, the present invention screened for single-domain antibodies against IGFL1. The present invention constructed an SdAb library and performed screening according to the existing isPLA-seq method, wherein the antigen complementarity determining region (CDR3) comprises 20 amino acids. In the isPLA-seq screening, IGFL1-HA and the synthetic SdAbs-Flag library were first transiently overexpressed in HEK239T cells. In situ red fluorescent signals were obtained using isPLA, and positive cells were isolated by flow cytometry. The positive rate in the control group was 0, while the positive rate in the experimental group was 23.5%, as shown in Figure 1A. Subsequently, the specific red fluorescence emitted from the organelle membranes of the sorted PLA-positive cells was observed under a fluorescence microscope. PCR amplification was performed by designing forward and reverse primers for CDR3. The results are shown in Figure 1B, and a 108 bp CDR3 mixture was obtained. After recovery, it was 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.

Example 2 Verification of single domain antibody affinity

1. GST Pull-Down Experiment

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.

The specific experimental process is as follows:

1) Protein Extraction and Purification: Clone the IGFL1 cDNA into the PGEX-4T-1 vector to form a GST-IGFL1 fusion protein for prokaryotic expression. Induce expression with IPTG, harvest the cells, lyse, and remove the precipitate. Add an appropriate volume of 50% glutathione-Sepharose 4B and gently shake on a shaker at 4°C for 30-60 minutes. Centrifuge at 4000 rpm for 5 minutes at 4°C and discard the supernatant. Wash the beads with pre-chilled PBS and repeat this step three times. Aspirate the liquid from the beads, but be careful not to remove the beads themselves. This will yield the GST-IGFL1-bound agarose gel.

2) System Incubation and Pull-Down: Mix the solution containing GST-IGFL1 protein and the candidate single-domain antibody, rotate and incubate the mixture overnight at 4°C. Centrifuge at 4000 rpm for 5 minutes at 4°C, discard the supernatant, and wash with pre-chilled buffer three times. Aspirate the aqueous layer above the agarose gel and add 1× protein electrophoresis loading buffer. Boil the protein sample, aliquot, and store frozen at -80°C for subsequent testing.

Finally, seven 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 seven single-domain antibodies all include three complementary determining regions (CDR1-3) and four framework regions (FR1, FR2, FR3, and FR4). The amino acid sequences of CDR1-3 are shown in Tables 1-3:

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

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

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

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

2. SPR Experiment

The present invention continues to test the affinity of 7 candidate single-domain antibodies with sufficient sample size among the above 9 single-domain antibodies through surface plasmon resonance (SPR) experiments.

The specific process is as follows:

1) Experimental Design: At least eight concentration gradients, low coupling, high flow rate, and _the affinity KD value must fall within the concentration range. Set up at least one sample concentration for replicates (completed at intervals), and set up a zero concentration sample.

2) Kinetic analysis: By fitting all curves, _the kinetic ka, kd and affinity KD were obtained. KD = _kd /ka.

3) Measure the response when steady state is reached, with high ligand coupling levels (high ligand concentration, coupling flow rate, coupling loading time).

Results and Analysis:

Specific binding of antibodies to antigens is a key mechanism for their biological function. Single-domain antibodies (SDAbs) often bind specifically to antigens through their CDR3 regions. First, to verify whether nine candidate SDAbs directly bind to IGFL1, GST pull-down experiments were performed. Results demonstrated that seven of these SDAbs, including SdAb-IGFL1#1, SdAb-IGFL1#4, SdAb-IGFL1#5, SdAb-IGFL1#6, SdAb-IGFL1#7, SdAb-IGFL1#8, and SdAb-IGFL1#9, directly bound to IGFL1 (Figures 2A and 2B). Furthermore, SdAb-IGFL1#4 coprecipitated p53, a known interacting protein, from 293T cells (Figure 2C).

Furthermore, the affinity of these seven candidate single-domain antibodies was tested using SPR assays, with dissociation constants ( _KD ) of 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 exhibited nonspecific binding. Therefore, two candidate antibodies, SdAb-IGFL1#6 and SdAb-IGFL1#8, were selected based on their affinity (results shown in Figure 2D). These results demonstrate that these IGFL1 single-domain antibodies specifically bind to IGFL1 with strong affinity. In summary, seven candidate single-domain antibodies that specifically bind to IGFL1 were identified using isPLA-seq and subsequent experimental techniques.

Example 3 Preparation of single domain antibodies and treatment of cells

IGFL1 is a secreted protein that promotes tumor growth through extracellular secretion. The present invention hypothesizes that IGFL1 single-domain antibodies exert their effects by inhibiting the maturation and secretion of intracellular IGFL1 protein, or by inhibiting extracellular secreted IGFL1. Therefore, the present invention prepared a single-domain antibody with a TAT transmembrane peptide fused to its N-terminus, and found that it can enter cells with maximum efficiency to exert its effects.

1. Construction of plasmid containing TAT recombinant protein

The DNA sequence of TAT transmembrane peptide was connected to the candidate nanobody DNA sequence using (G4S) ₃ and constructed into the pET-28a vector for fusion expression.

The build process is as follows:

1.1 Design and synthesis of fusion gene fragments

(1) Sequence design:

TAT cell-penetrating peptide (e.g., 5'TACGGGCGTAAAAAACGTCGTCAACGTCGTCGT3') sequence SEQ ID NO. 17

(G4S) ₃ flexible linker (5'GGTGGTGGTTCTGGTGGTGGTTCTGGTGGTGGTTCT3') Sequence SEQ ID NO.18

Candidate single domain antibody sequences (e.g. VHH fragments)

NdeI and XhoI restriction sites (CATATG & CTCGAG) were introduced at the 5' and 3' ends, respectively.

(2) Synthesis method:

The complete fusion fragment (TAT(G4S) ₃ single domain antibody) was directly synthesized by Qingke Gene Synthesis Co., Ltd., or synthesized in segments and then spliced by overlapping PCR.

1.2 PCR amplification of fusion genes

1) Overlap PCR:

TAT, (G4S) ₃ and single domain antibody fragments were amplified respectively, and primers for overlapping regions were designed.

First round PCR: amplify each fragment separately.

Second round of PCR: Using the mixed fragments in equal molar ratio as templates, the outer primers were used to amplify the complete fusion gene.

2) PCR conditions:

Initial denaturation: 98°C, 30 sec

30 cycles: 98°C 10 sec, 55°C 15 sec, 72°C 30 sec/kb

Final extension: 72°C, 5 min.

3) Gel electrophoresis verification:

1% agarose gel electrophoresis was used to check whether the PCR product size was correct, and the gel was cut to recover the target band.

1.3 Double enzyme digestion of vector and insert

1) pET28a vector digestion:

Reaction system (20 μL):

pET28a 1 μg

NdeI 1 μL

XhoI 1 μL

10× Buffer 2 μL

Add ddH ₂ O to 20 μL

Incubate at 37°C for 2 hours and inactivate at 65°C for 10 minutes.

2) Enzyme digestion of fusion gene fragments:

Same enzyme digestion conditions as the vector.

3) Purification of enzyme digestion products:

Purify the linearized vector and insert using a gel recovery kit.

1.4 Ligation reaction

Ligation system (10 μL):

① Linearized pET28a 50 ng

② Fusion gene fragment (3:1 molar excess)

③ T4 DNA ligase 1 μL

④ 10× Ligase Buffer 1 μL

⑤ Add ddH ₂ O to 10 μL

Ligate for 2 hours at 16°C or 1 hour at room temperature.

1.5 Transformation and positive clone screening

1) Transformation of DH5α competent cells:

① Take 5 μL of ligation product and add 50 μL of DH5α competent cells and incubate on ice for 30 minutes.

② Heat shock at 42°C for 45 seconds, followed by an ice bath for 2 minutes.

③ Add 500 μL LB (without antibodies) and incubate at 37°C for 1 hour.

④ Spread the plate onto LB plates containing kanamycin (50 μg/mL) and culture at 37°C overnight.

2) Colony PCR verification:

① Pick a single colony and verify it by PCR using T7 universal primers or gene-specific primers.

② The positive clones were sent for sequencing to confirm the sequence correctness.

1.6 Plasmid extraction and transformation into expression strains

1) Extraction of positive clone plasmids:

The recombinant plasmid (pET-28a TAT(G4S) ₃ single domain antibody) was extracted using a plasmid extraction kit.

2) Transformation into BL21(DE3) competent cells:

The expression strain was obtained by the same transformation steps as above.

2. Treatment of cells with single domain antibodies

The prepared single-domain antibodies were added to the culture supernatant of triple-negative breast cancer cell lines HCC1806 and HCC1937 at a concentration of 2 μg/mL. After 48 hours of treatment, the amount of single-domain antibodies and their localization in the cells were detected by Anti-VHH (488) fluorescent secondary antibody.

Taking the addition of single-domain antibodies to HCC1806 cells as an example, the specific experimental steps are as follows:

2.1 Cell plating and culture

Seed HCC1806 cells at an appropriate density (e.g., 5 × 10 ⁴ /well) in a 24-well plate (containing sterile slides) or confocal culture dish. Incubate at 37°C, 5% CO ₂ until the cell density reaches 60-70% (24 hours).

2.2 Single domain antibody treatment

Purified single-domain antibodies were diluted with prewarmed complete medium to a final concentration of 2 μg/mL.

Set up a control group:

1) Negative control: culture medium only (without antibody).

2) Isotype control: non-specific isotype nanobody (such as AntiRFP VHH).

Treat cells:

1) Aspirate the original culture medium and add fresh culture medium containing the single domain antibody (500 μL/well).

2) Incubate at 37°C, 5% _CO₂ for 48 hours.

2.3 Cell fixation and permeabilization

1) Fixation: Aspirate the culture medium and gently wash HCC1806 cells three times with pre-chilled PBS. Add 4% PFA (500 μL/well) and fix for 15 minutes at room temperature. Wash three times with PBS for 5 minutes each.

2) Permeabilization: Add 0.1% Triton X100 (in PBS) and permeabilize for 10 minutes at room temperature. Wash three times with PBS, 5 minutes each time.

2.4 Blocking and nonspecific binding

Blocking: Add 1% BSA (in PBS) and block for 30 minutes at room temperature. Aspirate the blocking solution and do not wash.

2.5 Fluorescent secondary antibody incubation

Antibody incubation: Dilute AntiVHH (488) secondary antibody with 1% BSA (according to the ratio in the instructions, e.g., 1:500). Add secondary antibody solution (200 μL/well) and incubate at room temperature for 1 hour (or overnight at 4°C) in the dark. Wash three times with PBS for 5 minutes each (in the dark).

2.6 Nuclear staining and sealing

1) DAPI staining: Add 1 μg/mL DAPI (in PBS) and incubate for 5 minutes in the dark. Wash three times with PBS, 5 minutes each time.

2) Mounting: Remove the slide with tweezers, place it upside down on a glass slide, and add a drop of anti-fluorescence quenching mounting medium (ProLongGold). After drying in the dark, store at 4°C until assayed.

HCC1806 cells were replaced with HCC1937 cells, and the experimental method of adding single-domain antibodies to HCC1937 cells was the same as above.

Results and Analysis:

Through prokaryotic expression, a single-domain antibody with a purity greater than 95% was obtained, enabling it to enter cells with maximum efficiency and exert its effect (results shown in Figure 3A). Furthermore, the IGFL1 single-domain antibody was indeed able to enter the triple-negative breast cancer cell lines HCC1806 and HCC1937 under the action of the TAT transmembrane peptide (results shown in Figure 3B).

Example 4 Activity detection of single domain antibodies

The activity of the single-domain antibody was verified through three cell experiments: CCK8 assay, clone formation assay, and cell stemness assay.

1.1 CCK8 assay

In order to detect whether the IGFL1 single-domain antibody has killing activity on tumor cells, the present invention detected the proliferation of HCC1806 and MDA-MB-231 cells under the condition of single-domain antibody treatment by CCK8 assay.

The specific method is as follows:

1) Cell Culture: HCC1806 and MDA-MB-231 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum in a 37°C, 5% _CO2 incubator. Experimental treatments were performed when cells reached 70-80% confluence.

2) Single-domain antibody treatment: Cells were seeded at a density of 5,000–10,000 cells/well in a 96-well plate with 100 μL of culture medium per well. After the cells adhered, different concentrations of IGFL1 single-domain antibody (0.1 nM, 1 nM, 10 nM, 100 nM, and 1000 nM) were added, with triplicate wells for each concentration. An equal volume of PBS was added to the control group.

3) CCK8 Assay: After 48 hours of treatment, add 10 μL of CCK8 reagent to each well and continue incubation for 1-2 hours (the specific time will be optimized based on the cell type and experimental conditions). Measure the absorbance (OD) at 450 nm using a microplate reader.

4) Data Analysis: Calculate the ratio of the OD value of the single-domain antibody-treated group to the OD value of the control group at each concentration and plot a concentration-response curve. Calculate the half-maximal inhibitory concentration (IC50) using nonlinear regression analysis.

1.2 Clone formation assay

In order to identify single-domain antibodies with high affinity and optimal anti-tumor activity, the present invention selected SdAb-IGFL1#6 and SdAb-IGFL1#8 single-domain antibodies for in-depth research based on their dissociation constants and IC50.

The specific method is as follows:

1) Cell Culture: HCC1806 cells were cultured in DMEM supplemented with 10% fetal bovine serum in a 37°C, 5% _CO2 incubator. Experimental treatments were performed when cells reached 70-80% confluence.

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

3) Colony formation assay: After treatment, culture cells for 10–14 days until visible colonies form. Fix cells with 4% paraformaldehyde and stain with 0.1% crystal violet. Observe and count colonies under a microscope. A colony is defined as a colony of at least 50 cells.

4) Data analysis: Calculate the ratio of the number of clones in the single-domain antibody-treated group to the number of clones in the control group.

1.3 Cell stemness detection experiments, including ALDH, WB, and mamosphere detection experiments

One of the most important roles of IGFL1 in promoting triple-negative breast cancer progression is maintaining the stemness of tumor cells. Therefore, developing single-domain antibodies (SDAs) that can inhibit tumor cell stemness is crucial for the development of therapeutics for cancer. This study examined the effects of IGFL1 SDAs on tumor cell stemness.

1.3.1 ALDH detection

The present invention tested the effects of SdAb-IGFL1#6 and SdAb-IGFL1#8 on the proportion of ALDH ⁺ cells in HCC1806 cells overexpressing IGFL1. The results showed that these two single-domain antibodies reduced the proportion of ALDH ⁺ cells by approximately 20-30%.

The specific method is as follows:

1) Cell staining

Prepare cells into a single-cell suspension and add activated BAAA for staining. Simultaneously set up a DEAB control group and incubate at 37°C for 30-60 minutes to allow ALDH to fully react with the substrate.

2) Flow cytometry

Flow cytometry was used to measure cell fluorescence intensity and analyze ALDH activity using the FL1 channel. ALDH-high (ALDHbr) and low-activity cells were distinguished based on fluorescence intensity.

3) Data Analysis

Calculate the percentage of ALDHbr cells and analyze differences in ALDH activity among different samples or treatment conditions. Combined with cell phenotypic analysis, explore the relationship between ALDH activity and cell function.

1.3.2 WB experiment

The specific method is as follows:

1) Protein separation

Perform SDS-PAGE and select an appropriate gel concentration based on the molecular weight of the target protein. Separate the proteins by electrophoresis to ensure clear bands.

2) Transfer

Transfer the separated proteins to a PVDF or nitrocellulose membrane to ensure efficient transfer. Use a wet or semi-dry transfer method, with a transfer time of 2 hours and a voltage of 120V.

3) Antibody incubation

Nonspecific binding sites on the membrane were blocked with 5% skim milk powder. Primary antibody was added and incubated at 4°C overnight; secondary antibody was added and incubated at room temperature for 2 hours.

4) Color development and imaging

Use a chemiluminescent or fluorescent imaging system to detect target protein bands. Adjust the exposure time to ensure clear bands.

1.3.3 qPCR experiments

1) RNA extraction

Add 500 μl of Trizol reagent to the cell sample to fully lyse the cells. Let stand for 5 minutes, then add 100 μl of chloroform, vortex to mix, and let stand for 5 minutes. Centrifuge at 12,000 rpm and 4°C for 10 minutes. Remove the supernatant and add an equal volume of isopropanol. Let stand for 10 minutes, then centrifuge again and discard the supernatant. Wash the RNA pellet with 1 ml of 75% ethanol and centrifuge at 7,000 rpm for 5 minutes at 4°C. Discard the supernatant and dry at room temperature for 5-10 minutes. Dissolve the RNA in 25 μl of DEPC water and store at -80°C until needed.

2) RNA concentration and purity determination

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

3) Reverse transcription

Prepare the reverse transcription reaction system according to the kit instructions, which typically includes reverse transcriptase, reaction buffer, primers, etc. Add the RNA template to the reaction system. Perform the reverse transcription reaction at an appropriate temperature, typically 42°C for 15-30 minutes, and terminate the reaction at 70°C to obtain the cDNA template.

4) Primer design

Based on the gene sequence of the stemness marker, use primer design software (such as Primer Premier) to design primers with a length of 20-25 bp, a Tm of approximately 60°C, and an amplified fragment length of 150-250 bp. Avoid forming secondary structures within or between primers to ensure primer specificity.

5) Primer verification

Use conventional PCR amplification to verify primer specificity and observe whether a single target band is amplified without primer dimers.

The primer amplification efficiency and specificity were further verified by qPCR amplification curves and melting curves to ensure that the primers were suitable for qPCR experiments.

6) qPCR reaction system configuration

Prepare the qPCR reaction as follows: 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. Perform three technical replicates for each sample, along with a no-template control (NTC) and an internal reference gene control.

7) Amplification procedure

Denaturation was performed at 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds. Melting curve analysis was performed after amplification, with a dwell time of 5 seconds at 0.5°C increments from 65°C to 95°C. The amplification procedure may be adjusted depending on the instrument and reagents.

8) Data Analysis

Fluorescence signals were collected using qPCR instrument software, and Ct values were calculated. The 2-ΔΔCt method was used to analyze the relative expression of target genes. The Ct values of the different treatment groups were compared with those of the control group to calculate the changes in relative expression.

9) Interpretation of results

If the Ct value of the target gene is low and the difference with the Ct value of the reference gene is small, it means that the expression level of the gene is high; otherwise, the expression level is low.

1.3.4 Mamosphere Detection

The present invention detected the effects of SdAb-IGFL1#6 and SdAb-IGFL1#8 on the formation of stem tumor spheres of HCC1806 cells.

The specific method is as follows:

1) Cell seeding

Prepare cells into a single-cell suspension and seed them into low-attachment culture dishes at an appropriate density. Add an appropriate amount of serum-free culture medium to each dish.

2) Mamosphere formation

Culture at 37°C, 5% CO ₂ , and change the medium regularly. Observe the formation of mamospheres and record their size and number.

3) Data Analysis

Calculate mamosphere formation efficiency (MFE) and analyze differences in stem cell activity under different treatment conditions. Combined with cell phenotypic analysis, explore the relationship between mamospheres and tumorigenesis, drug resistance, and other issues.

Results and Analysis:

2.1 CCK8 experimental results

To test whether IGFL1 single-domain antibodies have cytotoxic activity against tumor cells, the CCK8 assay was used to measure the proliferation of HCC1806 and MDA-MB-231 cells after 48 h of single-domain antibody treatment, as shown in Figure 4. The results showed that all 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) exhibited strong cytotoxicity and killed tumor cells in a concentration-dependent manner. The IC50 values of SdAb-IGFL1#1 in HCC1806 cells were 134.26 nM, 237.63 nM, 160.89 nM, 212.21 nM, 21.90 nM, 83.32 nM and 76.68 nM, respectively. The 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 the seven single-domain antibodies exhibited potent cytotoxicity and killed tumor cells in a concentration-dependent manner. Among them, SdAb-IGFL1#6 and SdAb-IGFL1#8 exhibited high antitumor activity in both cell lines, with low IC50 values and high affinity. These single-domain antibodies provide new drug candidates for the treatment of IGFL1-positive tumors.

2.2 Results of clone formation experiments

The key question of how IGFL1 single-domain antibodies inhibit tumor cell activity is central to this invention. To identify the single-domain antibodies with the best anti-tumor activity, IGFL1 single-domain antibodies (SdAb-IGFL1#6 and SdAb-IGFL1#8) were selected for in-depth study based on their dissociation constants. First, clonogenic assays demonstrated that IGFL1 overexpression significantly enhanced the clonogenicity of HCC1806 cells. Both SdAb-IGFL1#6 and SdAb-IGFL1#8 inhibited IGFL1-induced clonogenicity (Figure 4B). Second, cell counting assays demonstrated that IGFL1 overexpression significantly promoted HCC1806 cell proliferation. Both SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly inhibited IGFL1-mediated cell proliferation (Figure 4C). In summary, IGFL1 single-domain antibodies exhibit potent cytotoxicity against triple-negative breast cancer cells.

Conclusion: These results demonstrate that SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly inhibit the colony formation of triple-negative breast cancer cells HCC1806 by blocking IGFL1 activity, indicating their potent anti-tumor activity. This further confirms the significant efficacy of these two single-domain antibodies in inhibiting tumor cell proliferation and colony formation, providing strong evidence for subsequent mechanistic studies and preclinical experiments.

2.3 Cell Stemness Test Results (including ALDH, WB, mamosphere assay, qPCR)

One of the most important roles of IGFL1 in promoting triple-negative breast cancer progression is maintaining tumor cell stemness. Therefore, developing single-domain antibodies (SDAbs) that can inhibit tumor cell stemness is crucial for the development of cancer therapeutics. The present invention examined the effects of IGFL1 SDAbs on tumor cell stemness. First, IGFL1 overexpression increased the proportion of ALDH ⁺ cells in HCC1806 cells. Simultaneous treatment with SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly reduced the proportion of ALDH ⁺ cells by approximately 20-30% (Figure 5A). Second, SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly inhibited the formation of stem-like tumor spheres in IGFL1-overexpressing HCC1806 cells (Figure 5B). Molecularly, SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly reduced the expression of IGFL1 and stemness markers, including SOX2, SOX9, Nanog, and OCT4, in IGFL1-overexpressing tumor cells (Figure 5C). qPCR results showed that SdAb-IGFL1#6 and SdAb-IGFL1#8 reduced the expression of IGFL1 and stemness marker mRNA (Figure 5D). In summary, IGFL1 single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly inhibited the maintenance of stemness characteristics in triple-negative breast cancer cells by blocking IGFL1 activity.

To investigate the molecular mechanism of tumor inhibition by IGFL1 single-domain antibodies, the present invention treated breast cancer cells with SdAb-IGFL1#6 and SdAb-IGFL1#8. Western blot analysis showed that both SdAb-IGFL1#6 and SdAb-IGFL1#8 reduced IGFL1 expression in IGFL1-overexpressing cell lines. Furthermore, SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly inhibited IGFL1-mediated phosphorylation of β-catenin, IRS-1, PI3K, and AKT. Furthermore, SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly suppressed the expression of downstream genes in the PI3K/AKT pathway, including C-myc and CyclinD1 (as shown in Figure 6). The above results show that IGFL1 single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8: 1) downregulate the expression of IGFL1; 2) inhibit the activation of the PI3K/AKT pathway to downregulate the expression of oncogenes C-myc and CyclinD1, thereby inhibiting tumor progression.

Example 5 Verification of tumor inhibition effect in vivo

Anti-tumor activity was verified through animal experiments: A subcutaneous xenograft tumor model of triple-negative breast cancer in nude mice was established (model establishment methods can be found in the reference: 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.). The single-domain antibody was administered via intraperitoneal injection. Tumor volume was measured regularly, and the results showed that the single-domain antibody significantly inhibited tumor growth.

The orthotopic tumor model was established using HCC1806 cells, and the in vivo tumor inhibition effect of the single-domain antibody was tested using the following experimental methods:

1) Cell line preparation: HCC1806 cells were cultured in DMEM supplemented with 10% fetal bovine serum in a 37°C, 5% _CO2 incubator. Experimental treatments were performed when the cells reached 70-80% confluence.

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

3) Six days after tumor growth began, single-domain antibody treatment was initiated. SdAb-IGFL1#6 and SdAb-IGFL1#8 were administered intraperitoneally at a dose of 10 mg/kg for 10 doses, starting on day 6 after randomization and continuing every other day.

4) Treatment Effect Assessment: Every other day, the long and short diameters of the tumors were measured using calipers, and tumor volume was calculated (V = 0.5 × long diameter × short ^diameter² ). After treatment, tumor tissue was collected and weighed. The results are shown in Figure 7.

HCC1806 cells were replaced with MDA-MB-231 cells to re-establish the orthotopic tumor model, and the experimental method was the same as above.

Results and Analysis:

This example demonstrates the inhibitory effects of IGFL1 single-domain antibodies (SdAb-IGFL1#6 and SdAb-IGFL1#8) on triple-negative breast cancer by conducting mouse mammary orthotopic tumor studies. Consistent with in vitro results, SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly reduced the volume and weight of orthotopic tumors formed by HCC1806 and MDA-MB-231 cells in the mouse mammary gland (Figures 7A-C and 7E-G). Furthermore, SdAb-IGFL1#6 and SdAb-IGFL1#8 had no significant effect on mouse body weight (Figures 7D and 7H). Furthermore, immunohistochemistry revealed that treatment with the IGFL1 single-domain antibody significantly promoted the expression of the apoptosis marker Caspase-3 in mouse tumors, while suppressing the expression of the proliferation marker Ki67 (Figures 7I and 7J). This indicates that in vivo, single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 can inhibit IGFL1, thereby promoting tumor apoptosis and inhibiting tumor proliferation, ultimately achieving an effective therapeutic effect on triple-negative breast cancer.

These results demonstrate that the single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 effectively treat triple-negative breast cancer in vivo by inhibiting IGFL1, without significantly affecting mouse body weight, demonstrating their favorable safety profile and therapeutic efficacy. This further confirms the significant efficacy of single-domain antibodies in inhibiting tumor cell proliferation, providing strong evidence for subsequent mechanistic studies and preclinical experiments.

The above are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or any direct or indirect application in other related technical fields, are also included in the patent protection scope of the present invention.

Claims (8)

1. A single domain antibody targeting human IGFL1, wherein the heavy chain variable region of said single domain antibody comprises three complementarity determining regions, namely CDR1, CDR2 and CDR3,

The amino acid sequence of the CDR1 is shown as SEQ ID No. 10;

the amino acid sequence of the CDR2 is shown as SEQ ID No. 11;

the amino acid sequence of the CDR3 is shown as SEQ ID No.1 or SEQ ID No. 2.

2. The single domain antibody targeting human IGFL1 according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the single domain antibody is shown as SEQ ID No.3 or SEQ ID No. 4.

3. A nucleic acid molecule encoding the single domain antibody of claim 1 or 2 that targets human IGFL 1.

4. A vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the vector of claim 4.

6. Use of a single domain antibody targeting human IGFL as defined in claim 1 or 2 in the preparation of a human IGFL protein detection reagent.

7. Use of a single domain antibody targeting human IGFL1 according to claim 1 or 2 in the manufacture of an anti-breast cancer medicament.

8. The use according to claim 7, wherein the breast cancer is a triple negative breast cancer.