CN120309727A - A single domain antibody targeting human IGFL1 and its application - Google Patents

A single domain antibody targeting human IGFL1 and its application

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CN120309727A
CN120309727A CN202510789845.1A CN202510789845A CN120309727A CN 120309727 A CN120309727 A CN 120309727A CN 202510789845 A CN202510789845 A CN 202510789845A CN 120309727 A CN120309727 A CN 120309727A
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igfl1
single domain
domain antibody
sdab
cells
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CN120309727B (en
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陈策实
严飞
刘蓉
陈雪纯
朱申美
马文珊
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Kunming Medical University
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Kunming Medical University
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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

Single-domain antibody targeting human IGFL1 and application thereof
Technical Field
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.
Background
IGFL1 (insulin-like growth factor family related protein 1) is associated with certain skin diseases, inflammatory diseases and cancers, and functions as oncogenes driving tumor proliferation, migration and invasion, and thus becomes a potential target for tumor therapy. IGFL1 expression levels in various diseases are closely related to disease progression, showing potential as biomarkers. For example, in lung adenocarcinoma, IGFL1 expression levels are closely related to the clinical pathology and prognosis of a patient and can be an independent risk factor for determining patient prognosis. In addition, the high expression of IGFL < 1 > in thyropathy also suggests that IGFL < 1 > can be used as a potential marker for disease diagnosis and treatment monitoring. IGFL1 has been revealed to be effective in tumor and thyroid eye diseases, which promote cell proliferation and inflammatory response by activating IGF-1R signaling pathway. Preclinical studies indicate that IGFL1 is not only an important biomarker, but also has great potential as a therapeutic target. Future studies will further explore the role of IGFL1 in more diseases and develop new therapeutic strategies based on IGFL 1. These studies provide a number of potential therapeutic strategies for IGFL targeted therapies, and although the current successful clinical outcomes are not numerous, they provide new directions and promise for future tumor therapies.
Breast cancer is a relatively common malignancy among women in clinic, and is classified according to Estrogen Receptor (ER), progestin Receptor (PR), HER-2 receptor and Ki67 index, including Luminal A, luminal B, and triple negative breast cancer. Wherein Triple Negative Breast Cancer (TNBC) has relatively limited therapeutic approaches due to the lack of expression of Estrogen Receptor (ER), progestogen Receptor (PR) and human epidermal growth factor receptor 2 (HER 2), with poor prognosis. IGFL1 is an insulin-like growth factor family related protein, is highly expressed in various tumors, and plays an important role in the processes of proliferation, migration, invasion and the like of tumor cells. The single domain antibody is a heavy chain variable antibody fragment, has the advantages of small molecular weight, high stability, strong tissue penetrating power and the like, and has wide application prospect in tumor targeted therapy. Therefore, a single domain antibody directed against IGFL's 1 targeted for the treatment of breast cancer is a potential therapeutic strategy.
In the prior art, no single domain antibody for IGFL's targeted treatment of breast cancer has been disclosed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a single domain antibody targeting human IGFL < 1 > and application thereof. The single domain antibody of the invention can specifically bind to human IGFL and block the activity thereof, thereby inhibiting proliferation and stem maintenance of triple negative breast cancer cells and providing a novel means for treating IGFL1 positive tumor cells.
In order to solve the technical problems, the technical scheme of the invention is as follows:
in a first aspect of the invention there is provided a single domain antibody targeting human IGFL1, the heavy chain variable region of which 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.
Further, 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 invention can be expressed in a prokaryotic or eukaryotic expression system, such as escherichia coli, saccharomycete, insect cells or mammalian cells, etc., by genetic engineering technology, and the single domain antibody protein with biological activity is obtained.
In a second aspect of the invention there is provided a nucleic acid molecule encoding a single domain antibody as described in the first aspect which targets human IGFL.
In a third aspect of the invention there is provided a vector comprising a nucleic acid molecule as described in the second aspect.
In a fourth aspect of the invention there is provided a host cell comprising a vector as described in the third aspect.
In a fifth aspect of the invention there is provided the use of a single domain antibody targeting human IGFL1 as described in the first aspect in the preparation of a human IGFL1 protein detection reagent.
In a sixth aspect of the invention there is provided the use of a single domain antibody targeting human IGFL1 as described in the first aspect in the preparation of a product that binds to human IGFL1 protein.
In a seventh aspect of the invention there is provided the use of a single domain antibody targeting human IGFL1 as described in the first aspect in the manufacture of an anti-breast cancer medicament.
Further, the breast cancer is triple negative breast cancer.
The single domain antibody of the invention can inhibit the progress of tumor by down regulating IGFL expression and down regulating the expression of oncogene C-myc, cyclinD1 by inhibiting PI3K/AKT pathway activation.
Compared with the prior art, the invention has the following beneficial effects:
1) The invention discovers IGFL that the antibody is highly expressed in the body of a breast cancer patient, in particular to a triple negative breast cancer patient, on the basis, the invention successfully constructs a single domain antibody artificial synthesis library, and 7 new single domain antibody sequences resisting IGFL1 are screened out. Through further verification of affinity and anti-tumor activity, two single domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 with optimal anti-tumor effects are obtained, and the amino acid sequences of the single domain antibodies are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.
2) The single domain antibody provided by the invention can be specifically combined with human IGFL to block the activity of IGFL1, and can effectively inhibit proliferation and dryness maintenance of human triple negative breast cancer cells. In addition, animal experiment results show that the single-domain antibody can obviously inhibit the growth of mouse triple-negative breast in-situ cancer, but does not influence the weight of the mouse, and further proves the potential and safety of the single-domain antibody in tumor treatment. Therefore, 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 cancers.
Drawings
FIG. 1 is a graph showing the results of screening IGFL1 single domain antibodies based on isPLA-seq technology. Wherein a is the result of sorting positive cells by flow cytometry after isPLA of 293T cells. B is a graph showing the result of gel electrophoresis performed by amplifying a DNA fragment of the CDR3 region by PCR.
FIG. 2 is a graph showing the results of binding and affinity detection of IGFL single domain antibodies to antigens. Wherein A-B is the binding result of the antigen-antibody detected by GST pull-down experiment. C is the result of detection of IGFL1 interaction partner in Co-IP experiments using IGFL a1 single domain antibody. D is the affinity result of SPR assay IGFL single domain antibody to IGFL1 protein.
FIG. 3 is a graph showing the results of IGFL single domain antibody production and intracellular entry. Wherein, A, TAT penetrating peptide fusion expresses schematic diagram of single domain antibody, and 7 IGFL single domain antibodies are prepared. B results of measuring the localization and amount of intracellular single domain antibodies using IF experiments after treatment of single domain antibodies into HCC1806 and HCC1937 cells were scaled to 200. Mu.m.
Fig. 4 is a graph showing that IGFL single domain antibodies significantly inhibit proliferation of triple negative breast cancer cells. Wherein A is the result of detecting the number of living cells using CCK8 assay after 48 hours of treatment with IGFL single domain antibodies at different concentrations into HCC1806 and MDA-MB-231 cells. B is the result of cloning experiments performed on cells in HCC1806 cells overexpressing IGFL1 by adding IGFL single domain antibody, and detecting the clonogenic capacity of the tumor by crystal violet staining. C is the result of adding IGFL single domain antibody to HCC1806 cells overexpressing IGFL1, and counting the cells to obtain relative cell growth values. * P <0.05, < P <0.005, < P <0.0005, < P <0.00005.
FIG. 5 is a graph showing the results of IGFL single domain antibodies inhibiting tumor cell stem activity. Wherein. A is the result of detecting the proportion of ALDH-positive cell populations by flow cytometry using SdAb-IGFL1#6 and SdAb-IGFL1#8 single domain antibodies to treat HCC1806 cells overexpressing IGFL 1. B is the result of detecting the dryness of tumor cells by mamosphere experiments, scale bar, 200 μm, by treating HCC1806 cells overexpressing IGFL1 with SdAb-IGFL1#6 and SdAb-IGFL1#8 single domain antibodies. The single domain antibodies with the C of SdAb-IGFL # 1#6 and SdAb-IGFL # 1#8 treat HCC1806 cells which overexpress IGFL1, and the expression result of the stem markers of tumor cells is detected by a WB experiment. The single domain antibodies of SdAb-IGFL1#6 and SdAb-IGFL1#8 were used to treat HCC1806 cells overexpressing IGFL1 and the expression results of the dry markers were detected by qPCR. * P <0.05, < P <0.005, < P <0.0005, < P <0.00005.
FIG. 6 shows the results of WB experiments to detect changes in IRS1/p85/PI 3K/AKT/beta-catenin signaling pathway protein after IGFL single domain antibody treatment of cells.
FIG. 7 is a graph showing the results of IGFL A single domain antibodies inhibiting the growth of breast carcinoma in situ in mice. Wherein. A-D was the result of HCC1806 cell line inoculation into mouse mammary fat pad, followed by single domain antibody SdAb-IGFL1#6 and SdAb-IGFL1#8 , mg/kg treatment, monitoring mouse body weight, tumor volume and tumor weight. E-H is the result of MDA-MB-23 cell line inoculation into mouse mammary fat pad, followed by administration of single domain antibodies SdAb-IGFL # 16 and SdAb-IGFL #8 10 mg/kg treatment, monitoring mouse body weight, tumor volume, and tumor weight. I-J are graphs of the results of the sections of MDA-MB-231 and HCC1806 cell transplants and subsequent pathological staining of HE and IHC (Ki 67 and Caspase-3), respectively. * P <0.05, < P <0.005, < P <0.0005, < P <0.00005. Scale bar 100 μm.
Detailed Description
The following describes the technical scheme of the present invention in further detail with reference to the accompanying drawings and specific examples, but the present invention is not limited to the following technical scheme.
The cell lines involved in the examples of the present invention were all purchased from ATCC cell banks, nude mice were purchased from bio-limited company of beijing Bei Fu, detection antibodies were purchased from CST and Abcam, and biochemical reagents and kits were purchased from biotech limited companies such as bi yun tian, soribao, etc. Molecular biology test methods which are not specifically described are all carried out by referring to the molecular cloning experiment guidelines.
The preparation, purification and anti-tumor activity verification of the single domain antibody of the invention are shown in the following examples.
Example 1 screening and purification of Single-Domain antibodies
1. Construction of Single-domain antibody synthetic library
In the artificial library, only single domain antibodies with various CDR3 regions are synthesized, and the FR1, FR2, FR3, FR4, CDR1 and CDR2 are consistent in all single domain antibodies because the single domain antibodies are composed of the FR1, FR2, FR3, FR4, CDR1, CDR2 and CDR3, wherein the most critical antigen complementarity determining region is CDR 3. FR1, FR2, FR3, FR4 and CDR1, CDR2 are all known sequences, see 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 artificial synthesis library comprises the following steps:
The first step is to construct a variable library of CDR3 regions of a single domain antibody targeting IGFL1, and design and synthesize a highly diversified DNA fragment library by using a gene synthesis technology. The library is characterized in that 20 variable amino acid sites are introduced into a CDR3 region, NNN codons (N=A/T/G/C) are adopted for optimized coding, so that sequence diversity is ensured, and the occurrence probability of a stop codon is effectively reduced. The whole CDR3 region is designed to be of a fixed length of 60 bases, and the theoretical library capacity of the finally obtained library is not lower than 1X 10-8 clones, so that a plurality of possible amino acid sequence combinations are ensured to be covered. SdAb CDR3 DNA fragment mixtures are 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.
And constructing a pCDH-CMV-sdAb skeleton vector, namely designing and constructing a plasmid DNA vector containing a single-domain antibody skeleton. The vector should contain the framework regions (FR 1, FR2, FR3, FR4, CDR1 and CDR 2) and the necessary expression regulatory elements of the single domain antibody, such as promoters, terminators, etc. In addition, the C-terminal of the vector should be fused with a3 XFlag tag to facilitate subsequent detection and purification. The SdAb backbone C-terminal tag 3 x flag was then ligated to pCDH-CMV vector to obtain pCDH-CMV-sdAb backbone vector.
Thirdly, connecting the CDR fragment mixture to the pCDH-CMV-sdAb vector by a homologous recombination method to obtain the single domain antibody artificial library. The CDR3 region DNA library can be ligated to the corresponding positions of the single domain antibody backbone vector using appropriate restriction enzymes or homologous recombination techniques. The plasmid DNA vector after ligation was ensured to contain the complete single domain antibody sequences including FR1, FR2, FR3, FR4, CDR1, CDR2 and CDR3 regions, and a 3×flag tag.
Fourth step, verifying and amplifying the artificial synthetic library of single domain antibody
And verifying the correctness of the connected plasmid DNA vector by methods such as sequencing, PCR amplification and the like. Ensuring that the CDR3 region DNA fragment has been properly inserted into the single domain antibody backbone vector and that there is no mutation or deletion. Amplifying the verified plasmid DNA vector to form the final single domain antibody artificial synthesis library.
The method ensures the diversity of single-domain antibodies by randomly combining bases to generate a CDR3 region DNA fragment library, covers a large number of single-domain antibodies with different sequences, and provides abundant candidate sequences for screening the single-domain antibodies of IGFL. The single domain antibody synthetic library can be used for screening single domain antibodies aiming at various disease related targets, and is not limited to LGFL < 1 >. Through high throughput screening technology, single domain antibodies with high affinity and specificity can be rapidly identified and optimized, providing new tools and methods for diagnosis and treatment of diseases.
2. IsPLA-seq screening of Single-Domain antibodies
The isPLA-seq screening method for single domain antibodies can be specifically referred to the Chinese patent application CN202110641192.4.
The gene fragment of the single domain antibody artificial synthesis library is cloned into a pCDH-CMV-sdAb vector, IGFL cDNA is cloned into a pCDNA3.1-HA-C expression vector, and HEK293T cells are co-transfected. Cells were fixed 48 hours after transfection and isPLA were performed to obtain cells with positive signals. Positive cells are sorted by a flow cytometer, the plasmids in the positive cells are subjected to PCR amplification, and the amplified DNA fragments are recovered and subjected to subsequent high-throughput second-generation sequencing, so that the CDR3 DNA fragment sequence of the single-domain antibody candidate factor combined with IGFL and the abundance thereof can be obtained. And recombining the first 9 single domain antibodies with highest abundance onto a protein expression vector, and carrying out affinity detection to finally obtain the single domain antibody of the IGFL antibody with high affinity and specificity.
The specific process is as follows:
2.1 Cloning and Co-transfection of Single-Domain antibody Gene fragments
1) Cloning single-domain antibody gene fragment, namely cloning the gene fragment of the single-domain antibody artificial synthesis library into an expression vector with a3 xFlag tag, and constructing a single-domain antibody expression plasmid.
2) Cloning IGFL1 cDNA IGFL1 cDNA was cloned into an expression vector carrying an HA tag to construct a IGFL expression plasmid.
3) Co-transfecting HEK293T cells, namely co-transfecting the single domain antibody expression plasmid constructed in the step 1) and the IGFL expression plasmid constructed in the step 2) into HEK293T cells. Cells were fixed 48 hours after transfection and isPLA experiments were performed.
2.2 IsPLA experiment and sorting of Positive cells
IsPLA (in situ proximity ligation assay) is a highly sensitive molecular assay for protein interaction studies visualized at the single cell level. The technology uses specific antibodies to identify and bind target proteins, then uses PLA probes with a segment of oligodeoxynucleotide (single-stranded DNA) to identify and bind primary antibodies, when two target proteins are close, the DNA of the PLA probes of the two target proteins are paired and complemented, and then under the action of ligase, DNA fragments on the PLA probes are connected together to form a ring structure, and a detectable signal is generated through Rolling Circle Amplification (RCA).
The specific process is as follows:
1) Fixing and permeabilizing, namely fixing the treated cell climbing tablet by 4% paraformaldehyde and permeabilizing by 0.2% TritonX-100.
2) Sealing, namely dripping the sealing liquid on a cell climbing sheet, ensuring that the sealing liquid uniformly covers the whole tissue area, and incubating for 1 hour at 37 ℃.
3) Incubation of primary antibody diluted Flag (1:500) and HA (1:500) primary antibody were evenly dropped onto the blocked cell slide, placed in a wet box and incubated at 37℃for 2-3 hours.
4) Incubating PLA probe, mixing PLUS and MINUS PLA probe, diluting according to the instruction ratio of the kit, sucking to dry the primary antibody solution, washing the glass slide with 1 Xwashing buffer A for 2 times and 5 minutes each time. Excess wash buffer was aspirated and PLA probe solution was then added dropwise and incubated for 1 hour at 37 ℃.
5) Ligation and amplification by addition of oligodeoxynucleotides (hybridization solution) complementary to the probes and ligase to form a closed loop. Adding polymerase, using one of the probes as a template, and the rolling circle replication continuously forms a new closed loop.
6) Detection by addition of fluorescein-labeled oligonucleotides (detection solutions) to the circularized DNA to form a detectable fluorescent signal.
7) Flow cytometry sorting cells with positive red fluorescent signal were sorted using a flow cytometer. These positive cells indicate successful binding of the single domain antibody to IGFL a 1.
8) PCR amplification and DNA fragment recovery, namely, carrying out PCR amplification on plasmids in the selected positive cells, and recovering the amplified DNA fragments.
2.3 High throughput second generation sequencing, high throughput second generation sequencing is performed on the recovered DNA fragment, so that CDR3 region DNA fragment sequence and abundance of the single domain antibody candidate factor combined with IGFL can be obtained.
3. Single domain antibody expression and purification
3.1 The construction of an expression vector, namely cloning a single domain antibody gene sequence obtained by screening into a pET-28a expression vector, and constructing the expression vector which comprises 6 histidine (His 6) tags, a penetrating peptide TAT (amino acid sequence is shown as SEQ ID NO. 16: YGRKRRQRRR) structural domain, a single domain antibody sequence of IGFL1 is identified, and a 3 xFlag tag, wherein the molecular weight of the expression vector is 15 kDa.
3.2 Transformation and expression the constructed expression vector is transformed into competent cells of escherichia coli BL21 (DE 3). Under the induction of IPTG (isopropyl-beta-D-thiogalactoside), the single domain antibody protein is expressed. The induction conditions were 0.2mM IPTG and 16℃for 16 hours.
3.3 Purification by collecting the expressed bacterial culture, disrupting the cells by sonication, and collecting the supernatant. Purification was performed using a nickel bead (Ni-NTA) affinity column, utilizing the high affinity of His6 tag to nickel beads, specifically binding to single domain antibody proteins. The column is washed with equilibration buffer (e.g., 20mM Tris-HCl, 500 mM NaCl, 20mM imidazole, pH 8.0) to remove impurities. The target protein is eluted with an elution buffer (e.g., 20mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 8.0). The solvent in the protein solution was then replaced by PBS buffer by dialysis.
3.4 Purity detection and preservation, namely detecting the purity of the purified single-domain antibody protein by SDS-PAGE, HPLC and other methods, and ensuring that the purity of the single-domain antibody protein reaches more than 95 percent. And subpackaging the purified single domain antibody protein, and storing at-80 ℃ or freeze-drying for later experimental study and drug development.
In the method, the single-domain antibody protein can be efficiently expressed by using the pET-28a expression vector, so that the stability and the solubility of the protein are improved. Through nickel bead affinity chromatography and elution, high purity single domain antibody protein can be obtained, and the purity can reach more than 95%. The single domain antibody protein with TAT penetrating peptide structural domain and 3 xFlag label at the C end not only maintains the specific binding capacity with IGFL < 1 >, but also has good cell penetrating capacity, and is suitable for various biomedical applications.
The single domain antibody protein obtained by the method of the embodiment can be used for various biomedical researches, including cell experiments, animal experiments and preclinical researches, and provides a new tool and method for diagnosing and treating IGFL positive tumors.
Results and analysis:
By the above method, the present invention performs screening of IGFL single domain antibodies. The present invention performed SdAb library library construction and screening according to the existing isPLA-seq method, wherein the CDR3 of the antigen complementarity determining region comprises 20 amino acids. In isPLA-seq selection, first, IGFL-HA was transiently overexpressed simultaneously with SdAbs-Flag artificial libraries in HEK239T cells. Red fluorescent signal in situ in the cells was obtained by isPLA, positive cells were sorted out by cell flow sorting, the positive rate of control group was 0, the positive rate of test group was 23.5%, and the results are shown in fig. 1A. And then, observing the separated PLA positive cell organelle membrane under a fluorescence microscope to emit specific red fluorescence, performing PCR amplification by designing forward and reverse primers of CDR3, obtaining a CDR3 mixture of 108 bp as shown in figure 1B, recovering the mixture, performing second generation sequencing to obtain DNA and amino acid sequences of a CDR3 region, and firstly selecting the first 9 single domain antibodies SdAb-IGFL1#1-SdAb-IGFL #9 with highest abundance in a detection group, and performing subsequent verification and experiment.
Example 2 Single domain antibody affinity validation
1. GST Pull-Down experiment
To verify whether the highest-sequenced abundance 9 candidate single domain antibodies SdAb-IGFL #1-SdAb-IGFL # 1#9 bind directly to IGFL1, a GST Pull-Down experiment was performed.
The specific experimental process is as follows:
1) Protein extraction and purification, cloning IGFL < 1 > cDNA onto PGEX-4T-1 carrier to form prokaryotic expression GST-IGFL < 1 > fusion protein, inducing expression with IPTG, collecting thallus, cracking and eliminating precipitate, adding proper amount of 50% glutathione-agarose gel 4B, and shaking slowly for 30-60 min at 4 deg.C. Centrifuge at 4000rpm for 5min at 4℃and discard the supernatant. The beads were washed with pre-chilled PBS solution and this step was repeated 3 times. The liquid on the surface of the beads was aspirated, but attention was paid not to the fact that the GST-IGFL-bound agarose gel was obtained.
2) System incubation with Pull-Down mixing a solution containing GST-IGFL protein and a candidate single domain antibody and incubating the mixture at 4℃with spin mixing overnight. Centrifugation at 4000rpm for 5 min at 4℃and washing with pre-chilled buffer was repeated three times after discarding the supernatant. After the water layer above the agarose gel is absorbed, 1 x protein electrophoresis loading buffer is added, and after the protein sample is boiled, the protein sample is split-packaged and frozen in a-80 ℃ refrigerator for subsequent detection.
Finally, 7 single domain antibodies binding to IGFL1 were obtained, IGFL-SdAb #1, IGFL1-SdAb #4, IGFL1-SdAb #5, IGFL1-SdAb #6, IGFL1-SdAb #7, IGFL1-SdAb #8 and IGFL1-SdAb #9, respectively. 7 single domain antibodies, each comprising 3 complementarity determining regions CDR1-3 and 4 framework regions FR1, FR2, FR3 and FR4, CDR1-3 amino acid sequences see tables 1-3:
the FR1 amino acid sequences of the 7 single domain antibodies are the same, and are shown in SEQ ID NO.12, and the specific amino acid sequence is MGQVQLVESGGGSVQAGGSLRLSCTAS.
The FR2 of the 7 single-domain antibodies have the same amino acid sequence as shown in SEQ ID NO.13, and the specific amino acid sequence is WFRQAPGQEREAVA.
The FR3 of the 7 single-domain antibodies have the same amino acid sequence as shown in SEQ ID NO.14, and the specific amino acid sequence is RFTISRDNAKNTVTLQMNNLKPEDTAIYYCAA.
The FR4 amino acid sequences of the 7 single domain antibodies are the same as those of the single domain antibody shown in SEQ ID NO.15, and the specific amino acid sequence is WGQGTQVTVSS.
2. SPR experiment
The present invention continues to detect the affinity of 7 candidate single domain antibodies of the 9 single domain antibodies described above, which obtained a sufficient sample size, by Surface Plasmon Resonance (SPR) experiments.
The specific process is as follows:
1) The experimental design is that at least 8 concentration gradients, low coupling and high flow rate, and the value of the affinity K D must fall within the concentration range. Sample repetition (interval completed) with at least one concentration set, zero concentration samples set.
2) Kinetic analysis by fitting all curves, the kinetics ka, kd and affinity K D.KD =kd/ka were obtained.
3) The response at steady state was measured for 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, and single domain antibodies tend to specifically bind to antigens through their CDR3 regions. First, to verify whether 9 candidate single domain antibodies bind directly to IGFL1, a GST pull-down experiment was performed, and the results demonstrated that 7 single domain antibodies bind directly to IGFL1 (results shown in FIGS. 2A and 2B), including SdAb-IGFL1#1, sdAb-IGFL1#4, sdAb-IGFL1#5, sdAb-IGFL1#6, sdAb-IGFL1#7, sdAb-IGFL1#8, sdAb-IGFL1#9. Furthermore SdAb-IGFL #4 also was able to co-precipitate the known interacting protein p53 from 293T cells (results are shown in FIG. 2C).
Further, the affinity of these 7 candidate single domain antibodies was examined by SPR experiments with dissociation constants (K D) of 150.2 nM, 5.925. Mu.M, 9.5. Mu.M, 170.6 nM,186.7 nM,31.83 nM,1.068. Mu.M, respectively. However SdAb-IGFL #1 had non-specific binding, so two candidate antibodies SdAb-IGFL # 16 and SdAb-IGFL #8 were selected according to affinity order (results are shown in fig. 2D). The result shows that IGFL1 single domain antibody is specifically combined with IGFL1 and has stronger affinity. In summary, 7 candidate single domain antibodies that specifically bind IGFL1 were obtained by isPLA-seq and subsequent experimental techniques.
EXAMPLE 3 preparation and treatment of Single-Domain antibodies cells
IGFL1 is a secreted protein which acts to promote tumors by secretion outside the cell. The present invention assumes that the IGFL1 single domain antibody functions by inhibiting the maturation and secretion process of the intracellular IGFL1 protein, or by inhibiting extracellular secreted IGFL 1. Therefore, the single domain antibody with the N-terminal fused TAT penetrating peptide is prepared, and can enter cells to play a role in maximum efficiency.
1. Construction of recombinant protein plasmid containing TAT
And (3) connecting the DNA sequence of the TAT transmembrane peptide with the DNA sequence of the candidate nano antibody by using (G4S) 3, and constructing into a pET-28a vector for fusion expression.
The construction process is as follows:
1.1 Design and Synthesis of fusion Gene fragments
(1) Sequence design:
TAT transmembrane peptide (e.g. 5 'TACGGGCGTAAAACGTCGTCAAACGTCGTCGT3') sequence SEQ ID NO.17
(G4S) 3 flexible linker sequence SEQ ID NO.18
Candidate single domain antibody sequences (e.g., VHH fragments)
NdeI and XhoI cleavage sites (CATATG & CTCGAG) were introduced at the 5 'and 3' ends, respectively.
(2) The synthesis method comprises the following steps:
The complete fusion fragment (TAT (G4S) 3 single domain antibody) is synthesized directly by the Optimago gene synthesis company, or spliced by overlapping PCR after segmental synthesis.
1.2 PCR amplification of fusion genes
1) Overlapping PCR:
and respectively amplifying TAT, (G4S) 3 and single domain antibody fragments, and designing an overlapping region primer.
The first round of PCR was performed to amplify each fragment separately.
And in the second round of PCR, the mixed fragments with equal molar ratio are used as templates, and the outside primers are used for amplifying the complete fusion genes.
2) PCR conditions:
Pre-denaturation at 98 ℃,30 sec
30 Cycles of 98℃10 sec, 55℃15 sec, 72℃30 sec/kb
Final extension at 72 ℃,5 min.
3) Gel electrophoresis verification:
And detecting whether the size of the PCR product is correct or not by 1% agarose gel electrophoresis, and cutting the gel to recover the target band.
1.3 Double cleavage of vector and insert
1) Cleavage of pET28a vector:
Reaction system (20 μl):
pET28a 1 μg
NdeI 1 μL
XhoI 1 μL
10× Buffer 2 μL
ddH 2 O was added to 20. Mu.L
The reaction was carried out at 37℃for 2 hours and inactivated at 65℃for 10 minutes.
2) Cleavage of the fusion gene fragment:
and (3) enzyme cutting conditions with a carrier.
3) Purifying the enzyme cutting product:
Linearized vectors and inserts were purified using a gel recovery kit.
1.4 Ligation reaction
Ligation system (10 μl):
① Linearization pET28a 50 ng
② Fusion gene fragment (3:1 molar excess)
③ T4 DNA ligase 1. Mu.L
④ 10× Ligase Buffer 1 μL
⑤ ddH2 O is added to 10 mu L
The connection was carried out at 16℃for 2 hours or at room temperature for 1 hour.
1.5 Transformation and positive clone selection
1) Transformation of DH 5. Alpha. Competent cells:
① mu.L of ligation product was added to 50. Mu.L of DH 5. Alpha. Competent cells, and ice-incubated for 30 minutes.
② Heat shock at 42 ℃ for 45 seconds and ice bath for 2 minutes.
③ Mu.L LB (no antibody) was added and resuscitated at 37℃for 1 hour.
④ Plated on LB plates containing kanamycin (50. Mu.g/mL) and incubated overnight at 37 ℃.
2) Colony PCR verification:
① Single colonies were picked and verified by PCR with T7 universal primers or gene specific primers.
② Positive clones were sequenced to confirm sequence correctness.
1.6 Plasmid extraction and transformation of expression strains
1) Extracting positive cloning plasmids:
the recombinant plasmid (pET-28 a TAT (G4S) 3 single domain antibody) was extracted using the plasmid miniprep kit.
2) Transformation of BL21 (DE 3) competent cells:
The above transformation procedure was followed to obtain the expression strain.
2. Single domain antibody-treated cells
The prepared single domain antibodies were added to the triple negative breast cancer cell lines HCC1806 and HCC1937 cell culture supernatants at a concentration of 2 μg/mL, respectively, and after 48 hours of treatment, the amount of single domain antibodies was detected by Anti-VHH (488) fluorescence, as well as localization in the cells.
Taking the example of single domain antibody addition to HCC1806 cells, the specific experimental procedure is as follows:
2.1 Cell plating and culture
HCC1806 cells are seeded at appropriate density (e.g., 5x 10 4/well) in 24-well plates (containing sterile slide) or confocal dishes. Culturing at 37deg.C with 5% CO 2 until the cell density reaches 60-70% (24 hr).
2.2 Single domain antibody treatment
Purified single domain antibodies were diluted with pre-warmed complete medium to a final concentration of 2 μg/mL.
Setting a control group:
1) Negative control, medium only (no antibody added).
2) Isotype control, non-specific isotype nanobody (e.g., ANTIRFP VHH).
Treating cells:
1) The original medium was aspirated and fresh medium (500. Mu.L/well) containing the single domain antibody was added.
2) Incubation was performed at 37 ℃ for 48 hours with 5% CO 2.
2.3 Cell fixation and permeabilization
1) Fixation, media was aspirated and HCC1806 cells were gently washed 3 times with pre-chilled PBS. 4% PFA (500. Mu.L/well) was added and the mixture was allowed to stand at room temperature for 15 minutes. The PBS was washed 3 times for 5 minutes each.
2) Permeabilization by adding 0.1% Triton X100 (PBS formulation) and permeabilization at room temperature for 10 min. The PBS was washed 3 times for 5 minutes each.
2.4 Blocking and non-specific binding blockade
Blocking 1% BSA (PBS formulation) was added and blocked for 30 minutes at room temperature. The blocking solution is sucked and discarded without washing.
2.5 Incubation of fluorescent secondary antibody
Antibody incubation AntiVHH (488) secondary antibody (in the stated ratio, e.g., 1:500) was diluted with 1% BSA. The secondary antibody solution (200. Mu.L/well) was added and incubated at room temperature for 1 hour (or 4℃overnight) in the absence of light. The cells were washed 3 times with PBS for 5 minutes (light-protected operation).
2.6 Nuclear dyeing and sealing sheet
1) DAPI staining 1. Mu.g/mL DAPI (PBS formulation) was added and incubated for 5 min in the dark. The PBS was washed 3 times for 5 minutes each.
2) And sealing the slide, namely taking out the climbing slide by using tweezers, reversely buckling the climbing slide on the slide, and dripping the anti-fluorescence quenching sealing tablet (ProLong Gold). After drying in the dark, the sample is stored at 4 ℃ for detection.
The experimental procedure for the addition of single domain antibodies to HCC1937 cells was as described above with the replacement of HCC1806 cells with HCC1937 cells.
Results and analysis:
Single domain antibodies with purities greater than 95% were obtained by prokaryotic expression and allowed to function with maximum efficiency into cells (results are shown in figure 3A). Meanwhile, IGFL1 single domain antibody was indeed able to enter the triple negative breast cancer cell lines HCC1806 and HCC1937 under the action of 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 by three cell experiments, CCK8, clonogenic and cell stem assay.
1.1 CCK8 experiment
In order to detect whether IGFL1 single domain antibodies have killing activity on tumor cells, the proliferation of HCC1806 and MDA-MB-231 cells under the single domain antibody treatment condition is detected through a CCK8 experiment.
The specific method comprises the following steps:
1) Cell culture HCC1806 and MDA-MB-231 cells were cultured in DMEM medium containing 10% fetal bovine serum, respectively, and cultured in an incubator at 37℃with 5% CO 2 . When the cells reach 70-80% confluence, experimental treatments are performed.
2) Single domain antibody treatment cells were seeded into 96-well plates at a density of 5000-10000 cells/well, 100 μl of medium per well. After cell attachment, IGFL single domain antibodies (0.1 nM, 1 nM, 10nM, 100 nM, 1000 nM) were added at different concentrations, each concentration being provided with 3 multiplex wells. An equal volume of PBS was added to the control group.
3) CCK8 detection after 48 hours of treatment, 10. Mu.L of CCK8 reagent was added to each well and incubation was continued for 1-2 hours, with specific times optimized according to cell type and experimental conditions. Absorbance (OD value) was measured at a wavelength of 450 nm using a microplate reader.
4) Data analysis, namely calculating 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 drawing a concentration-response curve. The median inhibitory concentration (IC 50) was calculated using nonlinear regression analysis.
1.2 Cloning formation experiments
In order to identify the single domain antibodies with high affinity and optimal anti-tumor activity effect, the present invention selected SdAb-IGFL1#6 and SdAb-IGFL # 1#8 single domain antibodies based on their dissociation constants and IC50 for intensive studies.
The specific method comprises the following steps:
1) Cell culture HCC1806 cells were cultured in DMEM medium containing 10% fetal bovine serum in an incubator at 37 ℃ with 5% CO 2. When the cells reach 70-80% confluence, experimental treatments are performed.
2) Single domain antibody treatment cells were seeded into 6-well plates at a density of 500 cells/well, 2 mL medium per well. After cell attachment, sdAb-IGFL # 1#6 and SdAb-IGFL #8 single domain antibodies were added, respectively.
3) Cloning experiments following treatment, cells were cultured for 10-14 days until visible clones were formed. Cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The number of clones, defined as colonies of at least 50 cells, was observed and counted microscopically.
4) And (3) data analysis, namely calculating the ratio of the clone number of the single domain antibody treatment group to the clone number of the control group.
1.3 Cell stem assay comprising ALDH, WB, mamosphere assay
IGFL1 one of the most important effects of promoting the progression of triple negative breast cancer is to maintain the stem nature of tumor cells. Therefore, the development of single domain antibodies capable of inhibiting tumor cell stem property is particularly important for the development of tumor therapeutic drugs. The invention detects the influence of IGFL1 single domain antibody on the stem property of tumor cells.
1.3.1 ALDH detection
The present invention examined the effect of SdAb-IGFL1#6 and SdAb-IGFL1#8 on the proportion of ALDH + cells in HCC1806 cells overexpressing IGFL 1. The results indicate that these two single domain antibodies reduced the proportion of ALDH + cells by about 20-30%.
The specific method comprises the following steps:
1) Cell staining
Cells were prepared as single cell suspensions, stained with activated BAAA, and DEAB control was set. Incubation is carried out at 37℃for 30-60 minutes to allow the ALDH to react well with the substrate.
2) Flow cytometer detection
Cell fluorescence intensity was measured using a flow cytometer, and ALDH activity was analyzed by FL1 channel. ALDH high-activity cells (ALDHbr) and low-activity cells are distinguished according to fluorescence intensity.
3) Data analysis
The percentage of ALDHbr cells was calculated and the difference in ALDH activity was analyzed for different samples or treatment conditions. In conjunction with cellular phenotyping, the relationship between ALDH activity and cellular function was investigated.
1.3.2 WB experiment
The specific method comprises the following steps:
1) Protein separation
SDS-PAGE was performed to select appropriate gel concentrations based on the molecular weight of the target protein. The protein is separated by electrophoresis, so that the band is clear.
2) Transfer film
The separated proteins were transferred to PVDF or nitrocellulose membranes to ensure membrane transfer efficiency. The wet or semi-dry transfer method was used to control the transfer time and voltage to 2 hours and 120V, respectively.
3) Antibody incubation
The nonspecific binding sites on the membrane were blocked with 5% nonfat dry milk. Adding primary antibody for incubation at 4 ℃ overnight, and adding secondary antibody for incubation at room temperature for 2 hours.
4) Color development and imaging
Target protein bands are detected using chemiluminescent or fluorescent imaging systems. The exposure time was adjusted to ensure that the stripes were clearly visible.
1.3.3 QPCR experiment
1) RNA extraction
To the cell sample, 500. Mu.l Trizol reagent was added, the cells were thoroughly lysed, and after 5 minutes of standing, 100. Mu.l chloroform was added, and after shaking and mixing, the cells were left standing for 5 minutes. 12000 Centrifuging at 4 ℃ for 10 minutes at rpm, adding equal volume of isopropanol into the supernatant, standing for 10 minutes, centrifuging again, and discarding the supernatant. The RNA pellet was washed with 1 ml of 75% ethanol, centrifuged at 4℃and 7000 rpm min, the supernatant was discarded and dried at room temperature for 5-10 min. 25. Mu.L of DEPC water was added to dissolve RNA and stored at-80℃until use.
2) RNA concentration and purity determination
The concentration and purity (A260/A280 ratio) of the RNA are measured by using a nucleic acid protein detector, so that the concentration of the RNA is ensured to be in a proper range, and the purity meets the requirement.
3) Reverse transcription
The reverse transcription reaction system is prepared according to the instruction of the kit, and generally comprises reverse transcriptase, reaction buffer, primers and the like, and an RNA template is added into the reaction system. The reverse transcription reaction, typically 42℃for 15-30 minutes, is carried out at an appropriate temperature, followed by 70℃termination to obtain the cDNA template.
4) Primer design
According to the gene sequence of the dry marker, primer design software (such as PRIMER PREMIER) is used for designing primers, the length of the primers is 20-25 bp, the Tm value is about 60 ℃, and the length of amplified fragments is 150-250 bp. The secondary structure formed between the primers is avoided, and the specificity of the primers is ensured.
5) Primer verification
Primer specificity was verified using conventional PCR amplification, and whether a single target band was amplified was observed without primer dimer.
The amplification efficiency and the specificity of the primer are further verified through a qPCR amplification curve and a dissolution curve, so that the primer is ensured to be suitable for qPCR experiments.
6) QPCR reaction system configuration
The qPCR reaction was carried out in the following manner, 10. Mu.L of 2 XqPCR Mix, 1. Mu.L of 2. Mu.M primer F, 1. Mu.L of 2. Mu.M primer R, 1. Mu.L of cDNA template, and 20. Mu.L of ultrapure water were supplemented. Each sample was set up with 3 technical replicates, while No Template Control (NTC) and reference gene control were set up.
7) Amplification procedure
Pre-denaturation at 95 ℃ for 2 min, 15 sec at 95 ℃, 30 sec at 60 ℃ for 40 cycles, dissolution profile analysis after amplification was performed, 5 sec at 65 ℃ to 95 ℃ per 0.5 ℃. The amplification procedure can be appropriately adjusted depending on the instrument and the reagent.
8) Data analysis
Fluorescent signals are collected through qPCR instrument software, ct values are calculated, and the relative expression quantity of the target genes is analyzed by adopting a 2-delta Ct method. And comparing Ct values of different treatment groups with those of a control group, and calculating the change of the relative expression quantity.
9) Interpretation of results
If the Ct value of the target gene is lower and the difference between the Ct value of the target gene and the Ct value of the reference gene is smaller, the expression quantity of the target gene is higher, otherwise, the expression quantity is lower.
1.3.4 Mamosphere detection
The present invention examined the effect of SdAb-IGFL1#6 and SdAb-IGFL1#8 on HCC1806 cell stem tumor sphere formation.
The specific method comprises the following steps:
1) Cell seeding
Cells were prepared as single cell suspensions and inoculated into low adhesion dishes at appropriate densities. An appropriate amount of serum-free medium was added to each dish.
2) Mamosphere formation of
Culturing at 37deg.C under 5% CO 2, and periodically replacing culture medium. The formation process of mamosphere was observed and its size and number were recorded.
3) Data analysis
The efficiency of formation (MFE) was calculated mamosphere and the differences in stem cell activity under different treatment conditions were analysed. In combination with cell phenotype analysis, mamosphere was examined for its relationship with tumorigenesis, drug resistance, and the like.
Results and analysis:
2.1 CCK8 experimental results
To examine whether IGFL1 single domain antibodies had killing activity against tumor cells, proliferation of HCC1806 and MDA-MB-231 cells under the condition of single domain antibody treatment 48 h was examined by CCK8 assay, as shown in FIG. 4. The results show that 7 IGFL1 single domain antibodies (SdAb-IGFL 1#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. Its IC50 in HCC1806 cells was ：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, respectively. IC50 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, respectively (FIG. 4A). The results show that 7 single domain antibodies have strong cytotoxicity and kill tumor cells in a concentration-dependent manner. Of these, sdAb-IGFL1#6 and SdAb-IGFL1#8, which exhibit higher antitumor activity in both cell lines, have lower IC50 values and higher affinities. These single domain antibodies provide new drug candidates for the treatment of IGFL positive tumors.
2.2 Results of cloning experiments
It is one of the cores of the present invention how IGFL single domain antibodies inhibit the activity of tumor cells. To identify the best single domain antibodies with anti-tumor activity effects, IGFL1 single domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 were selected for intensive studies based on their dissociation constants. First, the results of the clonogenic experiments showed that IGFL1 overexpression significantly enhanced the clonogenic capacity of HCC1806 cells, and that both single domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 could inhibit IGFL 1-induced clonogenic (as shown in fig. 4B). Second, cell count experiments showed that IGFL1 overexpression significantly promoted proliferation of HCC1806 cells, and that both single domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly inhibited IGFL 1-mediated cell proliferation (as shown in fig. 4C). In conclusion, IGFL1 single domain antibodies have strong cytotoxicity to triple negative breast cancer cells.
These results indicate that SdAb-IGFL # 1#6 and SdAb-IGFL # 1#8 can significantly inhibit the clone formation of triple negative breast cancer cells HCC1806 by blocking IGFL1 activity, indicating that they have stronger anti-tumor activity. This further demonstrates that these two single domain antibodies have a significant effect in inhibiting tumor cell proliferation and clonogenic formation, providing strong evidence for subsequent mechanistic studies and preclinical experiments.
2.3 Cell dryness test results (including ALDH, WB, mamosphere test, qPCR)
IGFL1 one of the most important effects of promoting the progression of triple negative breast cancer is to maintain the stem nature of tumor cells. Therefore, the development of single domain antibodies capable of inhibiting tumor cell stem property is particularly important for the development of tumor therapeutic drugs. The invention detects the influence of IGFL1 single domain antibody on the stem property of tumor cells. First, IGFL1 overexpression increased HCC1806ALDH + cells, while the addition of SdAb-IGFL1#6 and SdAb-IGFL1#8 treatments significantly reduced the proportion of ALDH + cells by about 20-30% (as shown in FIG. 5A). Second, sdAb-IGFL1#6 and SdAb-IGFL1#8 significantly inhibited the formation of IGFL1 overexpressed HCC1806 cell stem tumor balls (as shown in fig. 5B). On molecular mechanisms SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly reduced expression of IGFL1 and dry markers, including SOX2, SOX9, nanog, OCT4, etc., in tumor cells overexpressing IGFL1 (as shown in fig. 5C). qPCR results showed that SdAb-IGFL1#6 and SdAb-IGFL1#8 reduced IGFL1 and expression of dry marker mRNA (as shown in FIG. 5D). Taken together, IGFL1 single domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly inhibited the maintenance of triple negative breast cancer cell stem characteristics by blocking the activity of IGFL 1.
In order to explore the molecular mechanism of IGFL1 single domain antibodies for inhibiting tumors, the invention utilizes SdAb-IGFL1#6 and SdAb-IGFL1#8 to treat breast cancer tumor cells, and WB experiment results show that in a cell line which overexpresses IGFL1, both SdAb-IGFL1#6 and SdAb-IGFL1#8 reduce the expression of IGFL 1. Second, sdAb-IGFL1#6 and SdAb-IGFL1#8 significantly inhibit IGFL 1-mediated phosphorylation of β -catenin, IRS-1, P3K and AKT. In addition, sdAb-IGFL1#6 and SdAb-IGFL1#8 significantly inhibit the expression of genes downstream of the PI3K/AKT pathway, including C-myc, cyclinD1, etc. (as shown in FIG. 6). The above results indicate that IGFL1 single domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 :1) down-regulate expression of the oncogene C-myc, cyclinD1 by down-regulating IGFL1 expression, 2) by inhibiting PI3K/AKT pathway activation, thereby inhibiting tumor progression.
EXAMPLE 5 verification of in vivo tumor inhibiting Effect
The anti-tumor activity is verified by animal experiments, a model of subcutaneous transplantation tumor of a triple negative breast cancer nude mouse is established (the model establishment method can refer to ：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.）, for administration of single domain antibodies by intraperitoneal injection, and the tumor volume is measured regularly, so that the result shows that the single domain antibodies can obviously inhibit the growth of tumors.
An in-situ tumor model is established by using HCC1806 cells, and an in-vivo tumor inhibition experimental method for detecting the single domain antibody is as follows:
1) Cell lines were prepared by culturing HCC1806 cells in DMEM medium containing 10% fetal bovine serum at 37deg.C in an incubator containing 5% CO 2. When the cells reach 70-80% confluence, experimental treatments are performed.
2) In situ tumor model establishment HCC1806 cell suspension (1X 10≡6 cells/100. Mu.L) was injected into mouse mammary fat pad to establish in situ tumor model.
3) After 6 days of tumor growth, single domain antibody treatment was started. Abdominal cavity injections were administered by SdAb-IGFL # 16 and SdAb-IGFL # 1, respectively. 10 mg/kg was injected 10 times, given on day 6 after randomization, once every other day.
4) Treatment effect evaluation every other day, the long and short diameters of the tumor were measured by calipers, and the tumor volume was calculated (v=0.5×long diameter×short diameter 2). After the treatment was completed, tumor tissues were collected, and the tumor weights were weighed, and the results are shown in fig. 7.
HCC1806 cells are replaced by MDA-MB-231 cells, an in-situ tumor model is built again, and the experimental method is the same as that described above.
Results and analysis:
This example conducted a mouse mammary carcinoma in situ animal experiment based on the inhibitory effect of IGFL1 single domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 in triple negative breast cancer. Consistent with the in vitro assay results, the single domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 significantly reduced the volume and weight of in situ tumors formed by HCC1806 and MDA-MB-231 cells in the mouse mammary gland (fig. 7A-C and 7E-G), and SdAb-IGFL1#6 and SdAb-IGFL1#8 had no significant effect on mouse body weight (fig. 7D and 7H). In addition, immunohistochemical experiments showed that IGFL single domain antibody treatment significantly promoted expression of apoptosis-related marker Caspase-3 in mouse tumors, in contrast, inhibited expression of proliferation marker Ki67 (fig. 7I and 7J). It is demonstrated that in vivo, the single domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 can promote tumor apoptosis and inhibit tumor proliferation by inhibiting IGFL1, and finally have effective treatment effects on triple-negative breast cancer.
The results show that the single-domain antibodies SdAb-IGFL1#6 and SdAb-IGFL1#8 can have an effective treatment effect on triple negative breast cancer in vivo by inhibiting IGFL1, have no obvious influence on the weight of mice, and have good safety and treatment effect. This further demonstrates that the single domain antibodies have a significant effect in inhibiting tumor cell proliferation, providing strong evidence for subsequent mechanism studies and preclinical experiments.
The foregoing is merely exemplary embodiments of the present invention and are not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention or direct or indirect application in other related technical fields are included in the scope of the present invention.

Claims (9)

1.一种靶向人IGFL1的单域抗体，其特征在于，所述单域抗体的重链可变区包括三个互补决定区，即CDR1、CDR2和CDR3，1. A single domain antibody targeting human IGFL1, characterized in that the heavy chain variable region of the single domain antibody includes three complementary determining regions, namely CDR1, CDR2 and CDR3, 所述CDR1的氨基酸序列如SEQ ID No.10所示；The amino acid sequence of the CDR1 is shown in SEQ ID No.10; 所述CDR2的氨基酸序列如SEQ ID No.11所示；The amino acid sequence of the CDR2 is shown in SEQ ID No.11; 所述CDR3的氨基酸序列如SEQ ID No.1或SEQ ID No.2所示。The amino acid sequence of the CDR3 is shown as SEQ ID No.1 or SEQ ID No.2. 2.根据权利要求1所述的靶向人IGFL1的单域抗体，其特征在于，所述单域抗体重链可变区的氨基酸序列如SEQ ID No.3或SEQ ID No.4所示。2. The single-domain antibody targeting human IGFL1 according to claim 1, characterized in that the amino acid sequence of the heavy chain variable region of the single-domain antibody is shown in SEQ ID No.3 or SEQ ID No.4. 3.编码如权利要求1或2所述的靶向人IGFL1的单域抗体的核酸分子。3 . A nucleic acid molecule encoding the single-domain antibody targeting human IGFL1 as claimed in claim 1 . 4.含有如权利要求3所述核酸分子的载体。4. A vector containing the nucleic acid molecule according to claim 3. 5.含有如权利要求4所述载体的宿主细胞。5. A host cell containing the vector according to claim 4. 6.如权利要求1或2所述靶向人IGFL1的单域抗体在制备人IGFL1蛋白检测试剂中的应用。6. Use of the single-domain antibody targeting human IGFL1 as claimed in claim 1 or 2 in the preparation of a human IGFL1 protein detection reagent. 7.如权利要求1或2所述靶向人IGFL1的单域抗体在制备与人IGFL1蛋白结合的产品中的应用。7. Use of the single-domain antibody targeting human IGFL1 as claimed in claim 1 or 2 in the preparation of a product binding to human IGFL1 protein. 8.如权利要求1或2所述靶向人IGFL1的单域抗体在制备抗乳腺癌药物中的应用。8. Use of the single-domain antibody targeting human IGFL1 as claimed in claim 1 or 2 in the preparation of anti-breast cancer drugs. 9.根据权利要求8所述的应用，其特征在于，所述乳腺癌为三阴性乳腺癌。9. The use according to claim 8, characterized in that the breast cancer is triple-negative breast cancer.
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