Screening and application of nano antibody of DDR1 antigen

Abstract

The invention relates to screening and application of a nano antibody of a DDR1 antigen. Specifically, the invention relates to a nano antibody aiming at DDR1 or an extracellular domain thereof or an antigen binding fragment thereof, and the amino acid sequence of the nano antibody comprises CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2 and CDR3 as shown in SEQ ID NO: 3; the CDR1 is shown as SEQ ID NO: 1, or the CDR1 is shown as SEQ ID NO: 4, the CDR2 is shown as SEQ ID NO: 5, and the CDR3 is shown as SEQ ID NO: 6.

Description

Technical Field

[0001] This invention belongs to the field of nanobodies, specifically relating to nanobodies targeting DDR1 ECD. Background Technology

[0002] Cancer is a leading cause of death worldwide, with nearly 10 million people (or almost one-sixth of all cancer deaths) dying from it in 2020. Over the past decade, immune checkpoint inhibitors in T-cell targeted immunotherapy have revitalized the powerful anti-tumor efficacy of endogenous T cells, revolutionizing treatment options for patients with solid tumors. However, the tumor microenvironment (TME) of large solid tumors creates physical barriers and prevents immune cell infiltration, leading to tumor progression and maintenance. Therefore, tumor-centric cancer treatment must be TME-centered, and collagen is the most abundant TME protein. DDR1 is a receptor-type tyrosine kinase, a single-span type I transmembrane protein with six distinct protein domains. Its N-terminal DS domain binds to collagen, and upon binding, specific tyrosine residues in the activation loop of the C-terminal tyrosine kinase domain in the cytoplasm are phosphorylated, and the phosphorylation signal is then transmitted downstream. DDR1 is expressed at low levels in healthy organs and controls cell proliferation, survival, differentiation, aggregation, adhesion, migration and invasion, collagen fiber formation, and ECM remodeling; however, overactivation of DDR1 may lead to the progression of various human diseases, including inflammation, cancer, and fibrotic diseases. Abnormal DDR1 signaling is thought to contribute to the malignant transformation of various cancers, including solid tumors and hematologic malignancies.

[0003] In a recent study, Sun et al. identified a novel mechanism by which collagen fiber remodeling hinders immune cell infiltration in solid tumors. They discovered that the extracellular domain (ECD) of DDR1 is a key component responsible for the arrangement of isotropic collagen fibers. Upon binding to collagen, it establishes a physical barrier around the tumor, thereby preventing T cell invasion and resulting immune rejection. Monoclonal antibodies targeting the human DDR1-ECD induce collagen fiber rearrangement, allowing immune cells to migrate in vitro and enhancing T cell infiltration. This suggests that the application of antibodies is promising for the treatment of DDR1-related solid tumors. Summary of the Invention

[0004] Discidin domain receptor 1 (DDR1) is a tyrosine kinase of collagen receptors that mediates various signal transductions to regulate cell polarity, tissue morphogenesis, and cell differentiation to maintain cellular and tissue homeostasis. In cancer, DDR1 regulates the interaction between tumor cells and their microenvironment, disrupting normal cell-matrix communication. The binding of DDR1-ECD to collagen strengthens aligned collagen fibers, promotes collagen fiber alignment and physical barrier formation, and inhibits immune infiltration. Antibodies targeting DDR1-ECD can disrupt collagen fiber alignment and increase T cell infiltration in tumors. Traditional monoclonal antibodies and nanobodies, as the smallest naturally occurring antigen-specific binding functional fragments, possess relatively long complementarity-determining regions that can penetrate into cracks on protein surfaces or at domain interfaces. They can bind to antigenic epitopes that traditional antibodies cannot reach, exhibiting excellent tissue permeability and reaching dense tissues such as solid tumors. Here we screened and obtained two high-affinity nanobodies targeting DDR1-ECD, which can competitively inhibit the binding of collagen to DDR1-ECD and inhibit the phosphorylation level of DDR1, laying the foundation for further research and application of anti-DDR1-ECD nanobody drugs.

[0005] Specifically, the present invention relates to the following technical solutions:

[0006] 1. Nanobodies or antigen-binding fragments targeting DDR1 or its extracellular domain, wherein the amino acid sequence comprises:

[0007] CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3; or

[0008] CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6.

[0009] 2. The nanobody or its antigen-binding fragment as described in Project 1, with an amino acid sequence as shown in SEQ ID NO:7 or 8.

[0010] 3. The nanobody or its antigen-binding fragment according to Project 1 or 2 is linked to an Fc tag; preferably, the Fc is IgG-Fc, more preferably IgG1-Fc, and most preferably human IgG1-Fc.

[0011] In this invention, any Fc sequence can be used, as long as it can contribute to protein expression, such as IgG-Fc, preferably IgG1-Fc, and more preferably human IgG1-Fc.

[0012] 4. The nanobody or its antigen-binding fragment according to Project 3, wherein the C-terminus of the nanobody or its antigen-binding fragment is connected to the N-terminus of the Fc.

[0013] 5. Nucleic acid molecules that encode nanobodies or antigen-binding fragments thereof as described in any one of items 1-4.

[0014] 6. An expression vector comprising the nucleic acid molecule described in item 5.

[0015] 7. A pharmaceutical composition comprising, as an active ingredient, any one of items 1-4, a nanobody or an antigen-binding fragment thereof.

[0016] 8. Use of any nanobody or antigen-binding fragment thereof described in any one of items 1-4 in the preparation of products, drugs or kits for the treatment of cancer.

[0017] 9. According to the use described in Item 8, the cancer is a DDR1-positive cancer, such as a DDR1-positive glioma, non-small cell lung cancer, head and neck cancer, hepatocellular carcinoma, and breast cancer.

[0018] Technical effects:

[0019] This study constructed a nanobody synthesis library and obtained high-affinity nanobodies targeting DDR1-ECD using yeast surface display technology and magnetic bead sorting. ELISA and SPR results showed that the nanobodies exhibited nM-level high affinity for DDR1-ECD and effectively blocked the interaction between DDR1-ECD and collagen. Phosphorylation inhibition experiments demonstrated that at the cellular level, the intervention of Nb-Fc effectively blocked the interaction between DDR1-ECD and collagen, thereby inhibiting intracellular phosphorylation of DDR1, suppressing downstream transmission of proliferation signaling pathways, slowing cancer cell proliferation, and inhibiting tumor growth. This provides a theoretical basis for the development of small antibody drugs. Attached Figure Description

[0020] Figure 1 The design and construction of the synthetic nanobody library are shown; A shows the CDR regions of the nanobody library (marked with different colors). As shown, partial randomization was achieved using degenerate bases to ensure library diversity. B shows a schematic diagram of nanobodies with N-terminal Flag and C-terminal v5 labels (nanobody PDB ID: 7KKJ) displayed on yeast cells. C is a schematic diagram of the nanobody magnetic screening strategy. Biotinylated antigens are shown in red. Nanobodies bound to the antigen were separated using the magnetic bead sorting (MACS) technique, and the enriched cells were amplified for the next round of screening.

[0021] Figure 2Yeast display and isolation of high-affinity DDR1 nanobodies; A shows a flowchart of the screening strategy for anti-DDR1 nanobodies. B shows flow cytometry results of yeast cells labeled with Alexa Fluor647 and bound to DDR1 after the first and fourth rounds of screening. C shows that nanobodies 9 (Nb9) and 20 (Nb20) exhibit efficient binding to 5 nM DDR1-ECD.

[0022] Figure 3 Nb9-Fc and Nb20-Fc exhibit high affinity for DDR1-ECD and act as collagen antagonists. A shows the sequence analysis of Nb9 and Nb20. CDR1, CDR2, and CDR3 regions are indicated in purple, brown, and tan, respectively. B shows the molecular design and purification of Nb-Fc. C shows the binding affinity of the nanobody to human, mouse, and monkey DDR1-ECD. D shows the SPR assay for the affinity of the nanobody for human DDR-ECD. E shows a competitive ELISA comparing the inhibitory efficacy of Nb9-Fc and Nb20-Fc on collagen. The IC50 values ​​for inhibiting collagen binding to DDR1-ECD by Nb9-Fc and Nb20-Fc are 47 nM and 85 nM, respectively.

[0023] Figure 4 Functional validation of Nb9-Fc and Nb20-Fc is shown; A shows the His pulldown experiment of Nb-Fc binding to hDDR1. SDS-PAGE results show that hDDR1 co-precipitates with Nb9-Fc and Nb20-Fc in the nickel column, indicating that hDDR1 interacts with Nb9-Fc and Nb20-Fc; B shows the flow cytometry detection of the binding of the two Nb-Fcs to HEK293T cells transfected with stable DDR1; C shows the confocal imaging detection of the binding of Nb9-Fc and Nb20-Fc to HEK293T cells transfected with transient DDR1.

[0024] Figure 5 The diagram illustrates the immune rejection mechanism of DDR1 and the inhibitory effect of Nb-Fc on DDR1 phosphorylation. A represents the mechanism of application of DDR1-related tumor antibodies. BC represents the detection of pDDR1 and beta-actin after treatment with different concentrations of Nb9-Fc and Nb20-Fc. DDR1-transfected HEK293T cells were pre-incubated with different concentrations of Nb-Fc and treated with 10 μg / ml collagen, followed by Western blotting to assess pDDR1 levels. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0026] Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents used are commercially available reagents.

[0027] Materials Methods

[0028] 1. DNA plasmid construction

[0029] For protein expression, cDNA encoding the extracellular domain (ECD) of Human-DDR1 (hDDR1, residues 19-416 of UniPort entry Q 08345), Mouse-DDR1 (residues 20-414 of UniPort entry Q 03146), and Monkey-DDR1 (residues 45-442 of UniPort entry F7BW77) were cloned into the pTT5 vector (Anhui General Biotechnology Co., Ltd.), and an HRV3C protease cleavage site and an 8×His tag were fused to the C-terminus. The C-terminus of the DDR1-ECD was then conjugated with a human IgG1-Fc sequence to generate human DDR1-Fc (hDDR1-Fc). In immunofluorescence and flow cytometry experiments, the full-length human DDR1b cDNA (1-913aa, residues 19-416 of UniPort entry Q 08345) was fused with a c-terminal His-tag or eGFP. All protein expression constructs were subcloned into the pTT5 vector (Anhui General Biotechnology Co., Ltd.). The full-length DDR1-b was subcloned into both the pTT5 and pLVX-puro vectors (Anhui General Biotechnology Co., Ltd.).

[0030] 2. Expression and purification of recombinant proteins

[0031] Both His-labeled and Fc-labeled hDDR1 were expressed in HEK293f cells (Wuhan Pronosei Biotechnology Co., Ltd.). Cells were grown in SMM293-TII medium containing 1% penicillin and streptomycin, and transfected at cell density with 1 μg / ml DNA and 2 μg / ml polyethyleneimine (PEI) to a concentration of 2.5–3 × 10⁶. After 96 hours of transfection, the culture supernatant was collected. His-labeled hDDR1 was purified using a Ni-NTA affinity column and eluted in a buffer containing 50 mM hpeps, 400 mM sodium chloride, and 500 mM imidazole. The His tag was further removed using HRV3C protease. Fc-labeled hDDR1 was incubated with Protein A resin, eluted with 50 mM citrate and 50 mM sodium citrate (pH 3.0) buffer, and rapidly neutralized in 1 M Tris (pH 9.0) buffer. All proteins were further purified on a Superdex 200 Increase 10 / 300GL column in PBS (pH 7.4). hDDR1 or hDDR1-Fc was mixed with EZ-Link NHS-PEG4-Biotin reagent (21330, Thermo Fisher Scientific Inc.) at a molecular ratio of 1:10. Biotinylated proteins were further purified on a Superdex 200 Increase 10 / 300GL column to remove excess biotin.

[0032] 3. Nanobody Library Construction

[0033] Nanobody synthetic libraries were constructed according to existing methods (e.g., see McMahon, C., et al., Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nat Struct Mol Biol, 2018, 25(3): p. 289-296), and the design and construction process is illustrated in the following diagram. Figure 1 As shown in the diagram. In short, the library was generated using 12 different primers via a two-step overlapping extension PCR process to produce insert fragments (the primer sequences are shown in Table 1). DNA fragments containing different lengths of the CDR3 region sequence (i.e., DNA fragments containing three different lengths of nanobody sequences obtained after overlapping extension PCR) were mixed at a molar ratio of 1:2:1 and co-electroporated with pYD-C digestion vector (Beijing Solarbio Science & Technology Co., Ltd.) at a molar ratio of 1:3 into EBY100 yeast competent cells (Beijing Solarbio Science & Technology Co., Ltd.). The library size was assessed by counting clones on SDCAA plate dilutions.

[0034] Table 1

[0035]

[0036] 4. Screening and sequencing of nanobodies

[0037] 2.5 x 10e9 EBY100 yeast cells, after electroporation, were centrifuged at 2000g for 5 min and induced in SDCAA medium at 20°C for 60 h. In the first round of screening, 2 μM biotinylated hDDR1 was incubated with AlexFlour647-labeled streptavidin for 30 min, followed by resuspending the yeast cells in the mixture and incubating at 4°C for 3 h. Cells were then incubated with magnetic beads bound to anti-AlexaFlour647 monoclonal antibody and concentrated on an LS column (Miltenyi). Finally, the cells adsorbed on the column were eluted and cultured overnight in SDCAA medium. In the second round of screening, the antigen concentration was reduced to 1 μM, and 2 x 10e8 cells were induced, following the same steps as the first round. In the following selection, 2 x 10e8 cells were incubated with gradually decreasing concentrations of antigen for MACS. 29 yeast cells were randomly selected from the final screening sample for induction. The binding signal with low concentrations of hDDR1 was detected by FCM. Clones 9 (Nb9) and 20 (Nb20) with the best binding signal were amplified and plasmids were extracted for sequencing.

[0038] 5. Expression and purification of Nb-Fc

[0039] Fc tags were coupled to the C-terminus of Nb9 and Nb20 cDNA, and His tags were fused to the N-terminus to obtain Nb9-Fc and Nb20-Fc. The DNA from Nb9-Fc and Nb20-Fc was then cloned into the pTT5 vector (Anhui General Biotechnology Co., Ltd.) to obtain Nb-Fc plasmids (i.e., Nb9-Fc plasmid and Nb20-Fc plasmid). The Nb-Fc plasmids were introduced into Hek293F cells (Wuhan Pronosei Biotechnology Co., Ltd.) for protein expression. The protein was incubated with protein A resin, eluted with 50 mM citric acid and 50 mM sodium citrate (pH 3.0) buffer, and rapidly neutralized in 1 Mtris (pH 9.0) buffer.

[0040] 6. ELISA

[0041] 1 μg / ml hDDR1 / mDDR1 / monDDR1 (i.e., human / mouse / monkey DDR1-ECD) was immobilized on a 96-well microplate and incubated with a series of different concentrations of Nb9-Fc or Nb20-Fc. The plates were washed three times with PBST buffer and cultured with anti-human IgG HRP-linked antibody (purchased from Cell Signaling Technology) at a 1:5000 dilution. Finally, TMB was used for color development for 15 min, and the reaction was terminated with 2M hydrochloric acid. The absorbance at 450 nm was measured using a microplate reader. In the inhibition assay, 10 μg / ml rat tail collagen (purchased from Sigma-Aldrich, C7661) was immobilized on a 96-well plate. Biotinylated hDDR1-Fc protein was incubated with serially diluted Nb-Fc antibody at room temperature for 30 min, then added to the 96-well plate and incubated at 37°C for 90 min. The bound DDR1-Fc was detected using HPR-labeled streptavidin (diluted 1:5000 in PBS). The reaction was terminated with 2M hydrochloric acid and the binding absorbance at 450 nm was measured using an ELISA reader.

[0042] 7. Surface Plasmon Resonance

[0043] SPR experiments for kinetic analysis were performed at 25°C using a BIAcore8K system (Cytiva). hDDR1 was diluted to 10 μg / mL with sodium acetate (pH 4.5) and immobilized on an activated CM5 chip. Serially diluted 1:1 Nb-Fc protein at concentrations ranging from 400 nM to 50 nM were passed through the sensor chip using a single-cycle kinetic method. The data were fitted using BIAcore evaluation software with a 1:1 binding model selected.

[0044] 8. Pulldown test

[0045] The molar ratio of hDDR1 to Nb-Fc was 1.5:1, and the mixture was incubated at 4°C for 4 h, followed by incubation with a 1 ml nickel column for 30 min. The protein complex was eluted with PBS buffer containing 500 mM imidazole. The experimental results were further evaluated by SDS-PAGE.

[0046] 9. Transient transfection and immunofluorescence

[0047] Hek293T cells (Wuhan Pronosei Biotechnology Co., Ltd.) were grown in DMEM containing 10% fetal bovine serum and 1% penicillin and streptomycin (P / S). The pTT5 vector (Anhui General Biotechnology Co., Ltd.) containing full-length hDDR1 cDNA with eGFP was transiently transfected into the cells using PEI reagent. Forty-eight hours after transfection, the cells were incubated with 50 μg / ml Nb-Fc at 37°C for 2 hours. The cells were then washed with PBS and confocal imaging was performed using PE-conjugated anti-human IgG staining.

[0048] 10. Culture and flow cytometry analysis of stable DDR1 cell lines

[0049] Hek293T cells (Wuhan Pronosei Life Sciences Co., Ltd.) were grown in DMEM containing 10% fetal bovine serum and 1% penicillin and streptomycin (P / S). The pLVX-puro vector containing full-length hDDR1 cDNA (prepared in section "1. DNA Plasmid Construction") was transiently transfected into the cells using PEI reagent. 72 h post-transfection, the medium was replaced with puromycin-containing medium, and stable transfected cells were selected. Stable HEK293T-DDR1 cells were collected and resuspended in PBS buffer containing 200 nM Nb-Fc for 90 min at 4°C. Finally, the cells were stained with PE-conjugated anti-human IgG antibody (i.e., Anti-human IgG HRP-linked Antibody: 32935, purchased from Cell Signaling Technology) for 30 min, and Nb binding signals were detected by flow cytometry.

[0050] 11. DDR1l phosphorylation inhibition experiment

[0051] Stable 293T-DDR1 cells (i.e., the stable DDR1 cell line obtained above) were stimulated at 37°C for 90 min with 10 μg / ml rat tail type I collagen (purchased from Sigma-Aldrich, C7661). In the DDR1 inhibition assay, the stable 293T-DDR1 cells were pre-incubated with Nb9-Fc and Nb20-Fc antibodies at concentrations of 10, 20, and 40 μg / ml, and then rat tail type I collagen was added to the mixture to a final concentration of 10 μg / ml. Cell extracts were prepared after 90 min of incubation. The tyrosine phosphorylation status of DDR1 cells after treatment was detected by Western blotting using an anti-tyrosine phosphorylation epitope primary antibody (Phospho-Tyrosine (4G10) Mouse mAb: 96125 (Cell Signaling Technology)).

[0052] Experimental results

[0053] 1. Construction of nanobody synthesis library and screening of magnetic beads

[0054] The fully synthesized yeast library was prepared according to publicly available methods (e.g., see McMahon, C., et al., Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nat Struct Mol Biol, 2018. 25(3): p. 289-296). To isolate candidate nanobodies, we used DDR1-ECD as the antigen to screen synthetic antibody libraries assembled in yeast. First, we prepared biotin-labeled DDR1-ECD protein, and then performed iterative staining and magnetic bead enrichment based on the biotin-streptavidin system. Figure 2 As shown in Figure A, four rounds of magnetic bead enrichment were performed using different screening strategies and reduced concentrations of biotinylated hDDR1-ECD protein as bait. In the first two rounds of screening, the biotinylated hDDR1-ECD antigen was increased to form a tetramer with fluorophore-labeled streptavidin. Since the proportion of binding antibody clones in the library was very low, the initial screening needed to emphasize screening effectiveness. Antibodies binding to the antigen were immobilized on fluorophore-labeled magnetic beads and then eluted in a non-magnetic state. The next round of screening continued with amplification induction, during which the concentration of biotinylated antigen was gradually reduced to obtain high-affinity binders. After magnetic bead enrichment, according to flow cytometry results, the cell positivity rate increased from 4.13% in the first round of screening to 30.8% in the fourth round. Figure 2 B).

[0055] 2. Screening for high-affinity antibodies

[0056] After four rounds of screening, high-affinity yeast colonies were isolated, and several individual colonies were randomly selected to bind to the biotinylated hDDR1 antigen. Flow cytometry results showed that the positive binding rates of 11 clones to the 10 nM antigen were all greater than 35%. Clones with positive rates higher than 35% were then subjected to flow cytometry detection with 5 nM hDDR1 antigen. The binding signals of Nb9 and Nb20 monoclonal antibodies to the 5 nM antigen were 72.1% and 55.3%, respectively. Figure 2 As shown in C, plasmids encoding Nb9 and Nb20 DNA were finally extracted and sequenced. The sequences are as follows: Figure 3 As shown in Figure A.

[0057] 3. Affinity detection of Nb9-Fc and Nb20-Fc and antagonists of effective collagen-DDR1 interaction.

[0058] DDR1-ECD exhibits a strong interaction with collagen (approximately 7.85 ± 2.54 nM). This strong affinity results in a tight binding, while the dense physical barrier prevents monoclonal antibodies with larger antigen-binding regions from easily penetrating. To effectively block this interaction, nanobodies with higher affinity and more precise epitope recognition are needed. In this study, ELISA results showed that Nb9-Fc had an affinity of 1.15 nM for hDDR, and Nb20-Fc had an affinity of 0.38 nM for hDDR. Further SPR testing was used to assess the binding characteristics of the two Nb-Fc proteins to the hDDR1-ECD domain; the KD values ​​for Nb9-Fc and Nb20-Fc were 0.99 and 1.99 nM, respectively. Both NB-Fc9 and NB-Fc20 bind to mDDR1 and monDDR1 proteins. Nb20-Fc exhibits higher affinity for mDDR1 and monDDR1 proteins than Nb9-Fc, at 2.28 nM and 3.43 nM respectively, while NB-Fc9 shows affinity of 7.09 nM and 9.46 nM. In a competitive inhibition assay against rat tail type I collagen, ELISA results showed that both NB-Fc9 and NB-Fc20 can block collagen binding to hDDR1-Fc in a dose-dependent manner, with IC50 values ​​of 47 nM and 85 nM respectively.

[0059] (see Figure 3 C- Figure 3 E).

[0060] 4. Functional characterization of Nb-Fc

[0061] Two Nb-Fc antibodies and hDDR1 protein were incubated at a molar ratio of 1:1.5, respectively. A nickel column pull-down experiment was then performed. Figure 4 As shown in Figure A, the SDS-PAGE assay revealed a band of hDDR1 and dimerized Nb-Fc in the elution product. Pull-down assays confirmed the specific binding of both Nb-Fc antibodies to DDR1-ECD. In addition to protein-level validation, we also performed cellular-level antibody function assays.

[0062] HEK293T-DDR1 stable transgenic cells (preparation method see "10. Culture and Flow Cytometry Detection of Stable DDR1 Cell Lines" above) were interacted with Nb-Fc and fluoresced with anti-human-Fc-PE dye (Goat anti-Human IgG Fc Secondary Antibody PE: 12-4998-82 (Thermo Fisher Scientific)). Flow cytometry analysis was then performed. Figure 4As shown in Figure B, approximately 70% of HEK293T-DDR1 cells exhibited positive PE signals, indicating that both Nb-Fc molecules interact with DDR1 on the cell surface. Simultaneously, in the immunofluorescence experiment, HEK293-DDR1 transiently transfected cells coated on confocal dishes (preparation method described in "9. Transient Transfection and Immunofluorescence" above) were treated with antibody dilution and stained, and finally analyzed using a confocal microscope. The results are as follows: Figure 4 As shown in C, the green fluorescence represents DDR1 expressed on the cell membrane surface. We observed red PE fluorescence on the cell membrane surface, which is a signal of antibody binding. This indicates that the two anti-DDR1 Nb-Fc antibodies can bind to the natural cell surface DDR1 protein.

[0063] 5. Nb-Fc inhibits DDR1 phosphorylation

[0064] To assess whether antibodies induce DDR1 phosphorylation, cells were incubated with antibodies in the absence of collagen, and DDR1 tyrosine phosphorylation was evaluated. The results showed that adding antibodies NB-Fc9 or NB-Fc20 in the absence of collagen did not induce DDR1 phosphorylation, demonstrating that neither antibody exhibited agonist properties in HEK293 cells. Since collagen leads to intracellular lysine phosphorylation of DDR1, HEK293T cells pretreated with Nb9-Fc and Nb20-Fc showed significantly reduced DDR1 intracellular domain phosphorylation levels in the presence of collagen. This indicates that Nb9-Fc and Nb20-Fc can effectively block the high-affinity interaction between collagen and DDR1 under physiological conditions. HEK293T cells treated with 40 μg / ml Nb9-Fc and 40 μg / ml Nb20-Fc showed the lowest DDR1 phosphorylation levels, which may provide a basis for the practical application of nanobodies (see [link to relevant documentation]). Figure 5 (BC). In summary, antibodies Nb9-Fc and Nb20-Fc act as antagonists of collagen-induced DDR1 phosphorylation and inhibit DDR1 phosphorylation in a dose-dependent manner.

[0065] Results Discussion

[0066] DDR1, a receptor-type tyrosine kinase, participates in tumor growth and metastasis, including tumor dormancy, and its role in cancer progression may be tumor- and environment-dependent. In triple-negative breast cancer, DDR1 binding to collagen fibers promotes tumorigenesis and development by mediating immune rejection through the inhibition of T-cell infiltration. In luminal breast cancer, the binding of collagen type III to DDR1 activates tumor dormancy and inhibits tumor growth. In the MMTV-PyMT luminal BCa model, DDR1 deficiency transforms tumors into a more aggressive basal phenotype, leading to increased lung metastasis through upregulation of fibrosis and hypoxia. Recent studies have found that MMP-mediated COL-cleaved COLI (cCOLI) and intact COLI (iCOLI) differentially regulate PDAC bioenergetics, growth, and metastatic spread by influencing DDR1 activity and expression. DDR1 is overexpressed relative to normal tissue in head and neck squamous cell carcinoma (HNSCC) and promotes cancer cell growth and metastasis through COLVIII and XI, but COLIII maintains tumor dormancy through DDR1-mediated STAT1 signaling. In fibrosis-associated hepatocellular carcinoma (HCC), high DDR1 levels are associated with poor prognosis, and ECM containing cCOLI (but not iCOLI) activates DDR1 and downstream pro-tumor effects. As a cancer target for kinases, many small molecule drugs have been developed targeting the DDR1 kinase domain, but currently, no highly selective DDR1 inhibitors have entered clinical application. Non-selective tyrosine kinase inhibitors, such as imatinib and nilotinib, have shown resistance, toxicity, and side effects in clinical use. Antibody drugs offer higher target selectivity than kinase inhibitors, and several DDR1-related monoclonal antibodies have entered clinical trials.

[0067] This study selected a yeast surface display system to screen nanobodies. Based on a previously described method for constructing a fully synthetic nanobody library, a yeast surface-displayed nanobody library was built, eliminating the need for alpaca immunization and reducing time and cost. After four rounds of screening, two nanobodies with high affinity for the HuDDR1 antigen were obtained. ELISA, SPR, and Western blotting results showed that the two Nb-FC antibodies exhibited high affinity for the antigen and could also compete with collagen, achieving an inhibitory effect on DDR1 phosphorylation at the cellular level. Anti-DDR1 antibodies could be used for glioma, non-small cell lung cancer, hepatocellular carcinoma, and breast cancer.

[0068] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0069] Sequence Listing

[0070] Nb9: CDR1: GYIFFPSFM (SEQ ID NO:1)

[0071] CDR2: EFVAGIGRGGTTYY (SEQ ID NO:2)

[0072] CDR3: TAVYYCAVYCGVLGIYLCHLN (SEQ ID NO:3)

[0073] Nb20: CDR1: GYIFSSTCM (SEQ ID NO:4)

[0074] CDR2: EFVATISSGAITYY (SEQ ID NO:5)

[0075] CDR3: TAVYYCAVSTYCLFRFI (SEQ ID NO:6)

[0076] Nb9 ---- QVQLQESGGGLVQAGGSLRLSCAASGYIFFPSFMGWYRQA PGKEREFVAGIGRGGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPE DTAVYYCAVYCGVLGIYLCHLNYWGQGTQVTVSS (SEQ ID NO:7)

[0077] Nb20 ----- QVQLQESGGGLVQAGGSLRLSCAASGYIFSSTCMGWYR QAPGKEREFVATISSGAITYYADSVKGRFTISRDNAKNTVYLQMNSLKP EDTAVYYCAVSTYCLFRFIYWGQGTPVTVSS (SEQ ID NO:8)

Claims

1. Nanobodies or antigen-binding fragments targeting DDR1 or its extracellular domain, wherein the amino acid sequence comprises: CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3; or CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:

6.

2. The nanobody or its antigen-binding fragment according to claim 1, wherein the amino acid sequence is as shown in SEQ ID NO:7 or 8.

3. The nanobody or its antigen-binding fragment according to claim 1 or 2, wherein it is linked to an Fc tag; preferably, the Fc is IgG-Fc, more preferably IgG1-Fc, and most preferably human IgG1-Fc.

4. The nanobody or its antigen-binding fragment according to claim 3, wherein, The C-terminus of the nanobody or its antigen-binding fragment is connected to the N-terminus of the Fc.

5. A nucleic acid molecule encoding a nanobody or an antigen-binding fragment thereof as described in any one of claims 1-4.

6. An expression vector comprising the nucleic acid molecule of claim 5.

7. A pharmaceutical composition comprising, as an active ingredient, any one of claims 1-4, a nanobody or an antigen-binding fragment thereof.

8. Use of the nanobody or antigen-binding fragment thereof as described in any one of claims 1-4 in the preparation of a product, drug or kit for treating cancer.

9. The use according to claim 8, wherein the cancer is a DDR1-positive cancer, such as a DDR1-positive glioma, non-small cell lung cancer, head and neck cancer, hepatocellular carcinoma, and breast cancer.

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