Nanometer antibody as well as sequence and application thereof

Abstract

The invention relates to the field of biological medicine, and discloses a nano antibody aiming at DDR1, and a sequence and application thereof. The system disclosed by the invention reveals a DDR1-mediated immune escape network, and completes complete development work from a single-target antibody to a multifunctional fusion protein. The research and development of the DDR1nb-Fc and the derivative fusion protein DDR1nb-Fc-mCD80 of the DDR1nb-Fc and the derivative fusion protein DDR1nb-Fc-mCD80 of the DDR1nb-Fc provide a novel treatment strategy with high efficiency and safety for intractable tumors with abundant collagen matrixes.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to nanobodies targeting DDR1, their sequences, and applications. Background Technology

[0002] Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase (RTK) that plays a crucial role in tissue repair, fibrosis, and tumor progression by binding to collagen and regulating the homeostasis of the extracellular matrix (ECM). DDR1 is overexpressed in various malignant tumors, including breast cancer, lung cancer, ovarian cancer, and melanoma. Aberrant expression and high activity of DDR1 promote tumor growth, and it plays a vital role in the progression and metastasis of various solid tumors. It has been shown to regulate cancer cell survival, adhesion, proliferation, motility, and invasion in different environments. Previous studies have also found that DDR1 is significantly overexpressed in colorectal cancer, gastric cancer, and other malignant tumors, and is closely associated with poor patient prognosis and reduced immune cell infiltration, suggesting that DDR1 plays an important role in tumor progression.

[0003] The molecular structure of DDR1 comprises three parts: an extracellular domain (DDR1-ECD), a transmembrane region, and an intracellular kinase domain. DDR1's immune rejection function depends on its DDR1-ECD, not its internal kinase domain. The DDR1-ECD possesses a collagen-binding domain, which can be detached by matrix metalloproteinases and bind to collagen, promoting collagen fiber arrangement, remodeling the ECM, and enhancing the tumor microenvironment's rejection of immune cells, thereby reducing the number of tumor-infiltrating lymphocytes (TILs). Antitumor therapy targeting DDR1 can relieve immunosuppression in the tumor microenvironment by inhibiting collagen fiber arrangement. In triple-negative breast cancer (TNBC), DDR1 expression is negatively correlated with the intratumoral abundance of antitumor T cells. Generally, the response to immunotherapy depends on the presence and activation of effector T cells in the tumor microenvironment (TME). Knocking out DDR1 or blocking DDR1 function with antibodies can promote intratumoral CD8... + T cell infiltration and inhibition of tumor growth in a mouse model of breast cancer. Binding of the DDR1 monoclonal antibody to DDR1-ECD leads to collagen fiber disorganization, thereby enhancing immune cell infiltration and increasing intratumoral infiltration of CD8.+ T and CD4 + The total number of T cells and the promotion of IFNγ production lead to complete tumor regression in an immunocompetent TNBC mouse model. DDR1 also plays a role in tumor growth by controlling the interaction between tumor cells and the surrounding collagen matrix.

[0004] Inhibiting DDR1 is an effective strategy for improving immunotherapy. Targeting and blocking the collagen-binding function of DDR1 will help reduce the rejection of immune cells by the tumor microenvironment (TME), promote immune cell infiltration, thereby improving the tumor immune microenvironment and slowing tumor progression. Given the important role of DDR1 in tumors, developing specific therapeutic strategies is of great significance. Due to its promising anti-tumor prospects, DDR1 inhibitors have been launched in rapid succession. However, most reported DDR1 / 2 inhibitors have broad inhibitory effects on several kinases, poor selectivity, and limited efficacy. Since most kinase inhibitors bind to the ATP-binding region of DDR1, they cannot directly inhibit the interaction between DDR1-ECD and collagen, thus losing the function of directly inhibiting collagen fiber arrangement and enhancing TME immune infiltration. Regarding antibody development targeting DDR1, only a few monoclonal antibodies have been reported so far. Unlike traditional antibodies with a molecular size of approximately 150 kDa, nanobodies are typically about 15 kDa and can be recombinantly expressed in bacteria. Nanobodies have several advantages, including low expression cost, small size, good solubility, strong permeability, low immunogenicity, and can effectively diffuse within the TME. Nanobodies, due to their unique advantages such as high stability and good tissue penetration, have shown great potential in targeted cancer therapy. There is an urgent practical need to screen and identify nanobodies with high affinity and high specificity against DDR1. Summary of the Invention

[0005] The present invention aims to at least partially solve one of the technical problems existing in the prior art. To this end, the present invention provides the following nanobodies, their sequences and applications.

[0006] Nanobody

[0007] In one aspect, the present invention provides an anti-DDRI nanobody comprising a complementarity-determining region (CDR) of a VHH chain, wherein the CDR of the VHH chain comprises: CDR1 with the amino acid sequence shown in SEQ ID NO:1; CDR2 with the amino acid sequence shown in SEQ ID NO:2; CDR3 with the amino acid sequence shown in SEQ ID NO:3; or CDR1 with the amino acid sequence shown in SEQ ID NO:4; CDR2 with the amino acid sequence shown in SEQ ID NO:2; CDR3 with the amino acid sequence shown in SEQ ID NO:3; or CDR1 with the amino acid sequence shown in SEQ ID NO:5; CDR2 with the amino acid sequence shown in SEQ ID NO:6; CDR3 with the amino acid sequence shown in SEQ ID NO:7; or CDR1 with the amino acid sequence shown in SEQ ID NO:1; CDR2 with the amino acid sequence shown in SEQ ID NO:2; CDR3 with the amino acid sequence shown in SEQ ID NO:8.

[0008] The nanobodies described in this invention typically comprise a VHH consisting of four framework regions (FRs) and three complementarity-determining regions (CDRs), referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The antigen-binding fragment contains at least a portion of the nanobody, sufficient to confer the ability of the fragment to specifically bind to DDR1. In some embodiments, the nanobodies described in this invention may be truncated at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to omit one or both of those framework regions, as long as they substantially maintain antigen binding and specificity.

[0009] In some implementations, the CDR1, CDR2, and CDR3 included in the VHH are determined using the Kabat numbering system.

[0010] In some embodiments, the nanobody or its antigen-binding fragment further comprises a heavy chain framework region derived from human immunoglobulins (e.g., a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain germline antibody gene), the heavy chain framework region optionally comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) reversion mutations from human residues to camel residues.

[0011] In some embodiments, the nanobody or its antigen-binding fragment further comprises a framework region derived from a camel-derived heavy chain antibody.

[0012] In some embodiments, the nanobody or its antigen-binding fragment comprises FR1, FR2, FR3, and FR4 sequences, wherein FR1 comprises the amino acid sequence X0X1QLVESGGGX 10 VX 12 X 13 GGSLX 18 LSCX b X 22 X 23 S, where: X0 is: missing, or selected from D or Q; X1 is selected from: V, L, or D; X 10 Selected from: L or S; X 12 Selected from: H, Q, R, or A; X 13 Selected from: P or A; X 18 Selected from: R or T; X b For: missing, or A; X 22 Selected from: A, V, or T; X 23 Selected from: V or A; and the length of the sequence is 24 to 26 amino acids.

[0013] FR2 contains the amino acid sequence Y1Y2WY4RQAPGKEREY 14 Y 15 Y 16 Where: Y1 is selected from: I or M; Y2 is selected from: G or R; Y4 is selected from: F or Y; Y 14 Selected from: G or L; Y 15 Selected from: V or A; Y 16 Selected from: C or H; and the length of the sequence is 16 amino acids.

[0014] FR3 contains the amino acid sequence (Z1Z2)ADSVKGRFTISRZ 15 NZ 17 Z 18 Z 19 TZ 21 Z 22 LQMNSLKPEDTAVYZ 37 C, where: Z1Z2 is either jointly missing or NY; Z 15 Selected from: M or D; Z 17 Selected from: I or A; Z 18 Selected from: R or K; Z 19 Selected from: K or N; Z 21 Selected from: L or V; Z 22 Selected from: Y or S; Z 37 Selected from: F or Y; and the length of the sequence is 36 or 38 amino acids.

[0015] FR4 contains the amino acid sequence WGW3GW5QVTW9SS, wherein: W3 is selected from Q or P; W5 is selected from T or A; W9 is selected from V or I; and the sequence is 11 amino acids in length.

[0016] In this invention, the nanobodies or antigen-binding fragments thereof may include variants that differ from their derived nanobodies or antigen-binding fragments only in one or more (e.g., up to 20, 15, 10, or 5 conserved substitutions of amino acid residues) conserved substitutions, or have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with their derived antibodies or antigen-binding fragments, and substantially retain the biological function of their derived nanobodies or antigen-binding fragments (e.g., specific binding to DDR1, neutralizing the biological activity of DDR1).

[0017] Bispecific or multispecific antibodies

[0018] On the other hand, the present invention provides a bispecific or multispecific antibody comprising the nanobody of the present invention or its antigen-binding fragment. To generate the bispecific or multispecific antibody, the nanobody of the present invention or its antigen-binding fragment may be linked (e.g., by chemical coupling, gene fusion, non-covalent association, or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimics).

[0019] In some implementations, the bispecific or multispecific antibody specifically binds to DDR1 and additionally specifically binds to one or more other targets.

[0020] In some embodiments, the bispecific or multispecific antibody further comprises at least one second antibody having a second binding specificity against a second target.

[0021] Antibody preparation

[0022] The antibodies of the present invention can be prepared by various methods known in the art, such as through genetic engineering recombination techniques. For example, DNA molecules encoding the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification, the resulting DNA molecules can be inserted into an expression vector, and then transfected into host cells. The transfected host cells can then be cultured under specific conditions to express the antibodies of the present invention. The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules.

[0023] On the other hand, the present invention provides an isolated nucleic acid molecule that encodes the nanobody of the present invention or its antigen-binding fragment or the bispecific or multispecific antibody of the present invention.

[0024] On the other hand, the present invention provides a vector (e.g., a cloning vector or an expression vector) containing the isolated nucleic acid molecules of the present invention. In some embodiments, the vector of the present invention is, for example, a plasmid, a granule, a bacteriophage, etc.

[0025] On the other hand, the present invention provides a host cell comprising the isolated nucleic acid molecules or carriers as described above. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

[0026] On the other hand, a method for preparing the nanobodies of the present invention or their antigen-binding fragments or bispecific or multispecific antibodies is provided, comprising culturing host cells as described above under conditions that allow protein expression, and recovering the antibody or its antigen-binding fragments or bispecific or multispecific antibodies from the cultured host cell culture.

[0027] Conjugate

[0028] On the other hand, the present invention also provides conjugates, wherein the nanobody of the present invention or its antigen-binding fragment or bispecific or multispecific antibody and the conjugated portion thereof.

[0029] In some embodiments, the nanobody of the present invention or its antigen-binding fragment may optionally be conjugated to the coupling portion via a linker.

[0030] In some embodiments, the conjugation portion is selected from protein tags. Such protein tags are well known in the art, and examples include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and those skilled in the art know how to select a suitable protein tag according to the desired purpose (e.g., purification, detection, or tracing). In some exemplary embodiments, the C-terminus of the nanobody of the present invention or its antigen-binding fragment is attached with a His tag (e.g., 6×His).

[0031] In some embodiments, the coupling portion is selected from detectable markers, such as enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), or biotin. The detectable markers described in this invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances, such as acridinium esters), magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assays, radioimmunoassays, fluorescence immunoassays, chemiluminescent immunoassays, etc.). In some embodiments, the detectable markers described above can be linked to the nanobodies or antigen-binding fragments of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0032] In some embodiments, the coupling portion is selected from therapeutic agents, such as anti-inflammatory drugs or immunosuppressants.

[0033] In some embodiments, the coupling moiety is selected from other bioactive peptides.

[0034] Pharmaceutical Composition

[0035] On the other hand, the present invention provides a pharmaceutical composition comprising the nanobody or antigen-binding fragment thereof described in the present invention, bispecific or multispecific antibodies, isolated nucleic acid molecules, a carrier, a host cell, or a conjugate, and pharmaceutically acceptable carriers and / or excipients.

[0036] In some embodiments, the pharmaceutical composition may also contain additional pharmaceutically active agents.

[0037] In some implementations, the additional pharmaceutically active agent is an anti-inflammatory drug or an immunosuppressant.

[0038] In some embodiments, the nanobody or antigen-binding fragment thereof, bispecific or multispecific antibody, isolated nucleic acid molecule, carrier, host cell, or conjugate of the present invention, along with the additional pharmaceutically active agent, may be provided as separate components or as mixed components in the pharmaceutical composition. Therefore, the nanobody or antigen-binding fragment thereof, bispecific or multispecific antibody, isolated nucleic acid molecule, carrier, host cell, or conjugate of the present invention, along with the additional pharmaceutically active agent, may be administered simultaneously, separately, or sequentially.

[0039] In some embodiments, the pharmaceutically acceptable carrier and / or excipient may comprise a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such a sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0040] The pharmaceutical compositions of the present invention may include, in a “therapeutic effective amount” or “preventative effective amount”, the nanobody or antigen-binding fragment thereof described herein, a bispecific or multispecific antibody, an isolated nucleic acid molecule, a carrier, a host cell, or a conjugate. A “preventative effective amount” refers to an amount sufficient to prevent, stop, or delay the onset of a disease. A “therapeutic effective amount” refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Therapeutic effective amounts may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient’s own immune system, the patient’s general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc.

[0041] Therapeutic applications

[0042] On the other hand, the present invention provides a method for preventing and / or treating DDR1-related diseases in subjects, comprising administering to a subject in need of the nanobody or antigen-binding fragment thereof, a bispecific or multispecific antibody, an isolated nucleic acid molecule, a carrier, a host cell, or a conjugate, or a pharmaceutical composition of the present invention. The present invention also relates to the use of said nanobody or antigen-binding fragment thereof, bispecific or multispecific antibody, isolated nucleic acid molecule, carrier, host cell, or conjugate, or pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of DDR1-related diseases in subjects.

[0043] In some embodiments, the DDR1-related disease is characterized by elevated DDR1 expression and / or excessive DDR1 activity.

[0044] In some implementations, the subject is a mammal, such as a human.

[0045] In some embodiments, the nanobody or its antigen-binding fragment, bispecific or multispecific antibody, isolated nucleic acid molecule, carrier, host cell, or pharmaceutical composition may be used alone or in combination with other pharmaceutically active agents (e.g., anti-inflammatory drugs or immunosuppressants).

[0046] The nanobodies or antigen-binding fragments thereof, bispecific or multispecific antibodies, isolated nucleic acid molecules, carriers, host cells, or conjugates of the present invention, or the pharmaceutical compositions of the present invention, can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use.

[0047] A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the antibody or antigen-binding fragment of the present invention into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0048] The nanobodies or antigen-binding fragments thereof, bispecific or multispecific antibodies, isolated nucleic acid molecules, carriers, host cells, or conjugates of the present invention, or the pharmaceutical compositions of the present invention, may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intrabladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration is parenteral (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route of administration and / or method will vary depending on the intended purpose. In some embodiments, the nanobodies or antigen-binding fragments thereof, bispecific or multispecific antibodies, isolated nucleic acid molecules, carriers, host cells, or conjugates of the present invention, or the pharmaceutical compositions of the present invention, are administered by intravenous injection or bolus.

[0049] Detection Application

[0050] On the other hand, the present invention provides a method for detecting the presence or amount of DDR1 in a sample, which includes using the nanobody or antigen-binding fragment or conjugate of the present invention.

[0051] In some embodiments, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0052] In some embodiments, the conjugate used in the method comprises the nanobody of the present invention or an antigen-binding fragment thereof, and a detectable label.

[0053] In some embodiments, the nanobody or its antigen-binding fragment used in the method carries a detectable label.

[0054] In some embodiments, the nanobodies or antigen-binding fragments thereof used in the method do not carry a detectable label. Therefore, the method may also include using other reagents (such as a second antibody) carrying a detectable label to detect the nanobodies or antigen-binding fragments of the present invention.

[0055] In some implementations, the method includes the following steps:

[0056] (1) Contact the sample with the nanobody or its antigen-binding fragment or conjugate of the present invention;

[0057] (2) Detect the formation of a complex between the nanobody or its antigen-binding fragment or conjugate and DDR1 or detect the amount of the complex.

[0058] The formation of the complex indicates the presence of DDR1 or cells expressing DDR1.

[0059] The method can be used for diagnostic purposes or non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

[0060] In some embodiments, the method is used to diagnose whether a subject has a disease related to DDR1. In such embodiments, the method may further include the step of comparing the amount of DDR1 in a sample from the subject with a reference value. The reference value may be the level of DDR1 in a sample from a subject known not to have a disease related to DDR1 (e.g., a healthy control) (also referred to as a "negative reference value"). For example, if the amount of DDR1 in a sample from the subject is elevated relative to a negative reference value, it indicates that the subject has a disease related to DDR1.

[0061] In some embodiments, the DDR1-related diseases are characterized by elevated DDR1 expression and / or excessive DDR1 activity. In some embodiments, the DDR1-related diseases are tumors selected from the group consisting of: gastric cancer, liver cancer, kidney tumors, lung cancer, small bowel cancer, bone cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, cervical cancer, adrenal tumors, pancreatic cancer, bladder tumors, or combinations thereof.

[0062] In some embodiments, the sample may be selected from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissue (e.g., surgically removed tumor tissue, biopsy sections, or fine-needle aspiration tissue), histological preparations, etc.

[0063] On the other hand, the present invention provides the use of nanobodies or their antigen-binding fragments or conjugates in the preparation of diagnostic reagents for detecting the presence or level of DDR1 in a sample or for diagnosing whether a subject has a disease related to DDR1.

[0064] In some embodiments, the conjugates used to prepare the detection reagents comprise the nanobodies of the present invention or antigen-binding fragments thereof, and a detectable label.

[0065] In some embodiments, the nanobodies or antigen-binding fragments thereof used to prepare the detection reagents are labeled with detectable tags.

[0066] In some embodiments, the nanobodies or antigen-binding fragments thereof used to prepare the detection reagents do not carry a detectable label. In such embodiments, the detection reagents may further comprise other reagents (such as a second antibody) capable of detecting the nanobodies or antigen-binding fragments thereof of the present invention.

[0067] This invention systematically reveals the DDR1-mediated immune escape network and completes the development process from single-target antibodies to multifunctional fusion proteins. The development of DDR1nb-Fc and its derived fusion protein DDR1nb-Fc-mCD80 provides a novel therapeutic strategy that is both highly effective and safe for refractory tumors rich in collagen matrix. Attached Figure Description

[0068] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0069] Figure 1 This is a diagram showing the amplification results of the VHH fragment displayed by phage according to an embodiment of the present invention. (A) Representative bands of the first round of amplification of the VHH fragment displayed by phage. (B) Representative bands of the second round of amplification of the VHH fragment displayed by phage (products amplified by new primers: the first 9, and products amplified by old primers: the last 7).

[0070] Figure 2 This is a graph showing the in vivo antitumor effects of the top ten candidate antibodies with the highest affinity according to embodiments of the present invention. (A) Images of mouse tumors after treatment with the 10 candidate antibodies. (B) Quantitative analysis of tumor tissue weight after treatment. (C) Tumor growth curves of tumor-bearing mice in each group. (n=3 independent experiments; statistical method: one-way ANOVA, *P<0.05).

[0071] Figure 3 This is a graph showing the in vivo antitumor activity results of the four candidate antibodies with the best efficacy provided according to the embodiments of the present invention. (A) Curves of weight change in mice in each group. (B) Tumor growth curves of tumor-bearing mice in each group (n=3 independent experiments; statistical method: one-way ANOVA, ****P<0.0001).

[0072] Figure 4 The image shows the results of ELISA detection of the binding of four candidate antibodies to mouse DDR1-Fc and human DDR1-his proteins (n=3 independent experiments) according to the embodiment of the present invention.

[0073] Figure 5 The graph shows the affinity results of four candidate antibodies provided in the embodiments of the present invention with mouse DDR1 and human DDR1 proteins, respectively.

[0074] Figure 6The graph shows the affinity test results of four candidate antibodies provided in the embodiments of the present invention with mouse E0771 cells that highly express DDR1 (n=3 independent experiments; statistical method: one-way ANOVA, *P<0.05, **P<0.01, ***P<0.001).

[0075] Figure 7 This is a graph showing the affinity test results between the candidate antibody and tumor tissue provided in an embodiment of the present invention.

[0076] Figure 8 This is a graph showing the in vitro antitumor activity results of the candidate antibody provided according to an embodiment of the present invention (n=4 independent experiments).

[0077] Figure 9 This is a graph showing the in vivo tumor-targeting effect of the candidate antibody provided according to an embodiment of the present invention. (A) In vivo fluorescence imaging detection of mice at 12, 24, 36, 48 and 72 hours after administration. (B) The ratio of the fluorescence intensity of tumor tissue (area in blue ellipse box) to the total fluorescence intensity of the mouse body at different time points (n=3 independent experiments; statistical method: two-way ANOVA, *P<0.05, **P<0.01).

[0078] Figure 10 The figures show the antitumor effects of four candidate DDR1 antibodies provided in the embodiments of the present invention in a mouse triple-negative breast cancer subcutaneous tumor model. (A) Mouse tumor growth curve. (B) Mouse body weight change curve. (C) Tumor images of mice in each group at the study endpoint (day 21 after tumor inoculation). (D) Violin plot of tumor weight statistics of mice in each group at the study endpoint (day 21 after tumor inoculation) (n=7 biological replicates; statistical method: two-way ANOVA, **P<0.01, ***P<0.001, ****P<0.0001).

[0079] Figure 11 The images shown are representative H&E staining images of tumor tissue from the E0771 subcutaneous tumor model provided in this embodiment of the invention: tubular structures in the central cavity (black arrow), pathological mitotic figures (green arrow), hemorrhage (red arrow), lymphocytes (blue arrow), and neutrophils (yellow arrow).

[0080] Figure 12 The Masson staining results provided in this embodiment of the invention indicate that four candidate antibodies disrupt the orderly arrangement of collagen fibers in tumor tissue.

[0081] Figure 13 These are representative images of second harmonic imaging of frozen sections of E0771 mouse tumor tissue provided in an embodiment of the present invention.

[0082] Figure 14This is a graph showing the effects of four candidate DDR1 antibodies provided according to embodiments of the present invention on T cells in the E0771 subcutaneous tumor model, both within the tumor and in the spleen. (A) Tumor infiltration CD45 + Flow cytometry analysis of immune cells. (B) CD4 in tumors and spleen. + and CD8 + Flow cytometry analysis of T cells. (C) Response of four candidate antibodies to intratumorally infiltrated CD45 in the E0771 subcutaneous tumor model. + CD3 + CD4 + and CD8 + The effect of immune cell percentage, and on CD4 in the spleen + and CD8 + The effect of T cell percentage (n=7 biological replicates; statistical method: one-way ANOVA, *P<0.05, **P<0.01, ***P<0.001).

[0083] Figure 15 This is a graph showing the expression detection results of CD45, CD8 and NK1.1 in the E0771 subcutaneous tumor model provided by the embodiment of the present invention (n=7 biological replicates; statistical method: one-way ANOVA, *P<0.05, **P<0.01, ***P<0.001).

[0084] Figure 16 This is a graph showing the effect of the candidate antibody provided according to embodiments of the present invention on neutrophils in the tumor and spleen in the E0771 subcutaneous tumor model. (A) CD45 in the tumor and spleen + CD11B + Ly6G + Flow cytometry analysis of neutrophils. (B)CD45 + CD11B + Ly6G + The percentage of neutrophils in tumors. (C)CD45 + CD11B + Ly6G + neutrophils in tumors CD45 + The percentage of immune cells. (D)CD45 + CD11B + Ly6G + The percentage of neutrophils in spleen cells. (E)CD45 + CD11B + Ly6G + Neutrophils in spleen CD45 +Percentage of immune cells (n=7 biological replicates; statistical method: one-way ANOVA, *P<0.05, **P<0.01).

[0085] Figure 17 The figures show the antitumor effects of four candidate antibodies provided in the KPC subcutaneous tumor model according to embodiments of the present invention. (A) Tumor growth curve in mice. (B) Body weight change curve in mice. (C) Tumor images of mice in each group at the study endpoint (day 26 after tumor inoculation) (n=7 biological replicates; statistical method: two-way ANOVA, **P<0.01, ***P<0.001, ****P<0.0001).

[0086] Figure 18 This is an image showing the H&E staining results of tumor tissue from a KPC pancreatic ductal carcinoma subcutaneous tumor model provided in an embodiment of the present invention.

[0087] Figure 19 This is a graph showing the in vivo anti-gastric cancer tumor function detection results of DDR1nb-3-Fc provided according to an embodiment of the present invention.

[0088] Figure 20 This is a graph showing the results of DDR1nb-Fc treatment for non-small cell lung cancer (NSCLC) according to an embodiment of the present invention.

[0089] Figure 21 These are schematic diagrams of fusion protein expression plasmids provided according to embodiments of the present invention. (A) Schematic diagram of Fc-mCD80 fusion protein expression plasmid. (B) Schematic diagram of DDR1nb-3-Fc-mCD80 fusion protein expression plasmid.

[0090] Figure 22 The figures show the affinity test results of the DDR1nb-3-Fc-mCD80 fusion protein with human and mouse DDR1 proteins according to embodiments of the present invention. (A) Affinity test of DDR1nb-3-Fc-mCD80 fusion protein with human DDR1-ECD-his by BLI method. (B) Affinity test of DDR1nb-3-Fc-mCD80 fusion protein with mouse DDR1-ECD-his protein by BLI method. (C) Affinity test of DDR1nb-3-Fc-mCD80 fusion protein with human DDR1-ECD-his by ELISA method. (D) Affinity test of DDR1nb-3-Fc-mCD80 fusion protein with mouse DDR1-ECD-his protein by ELISA method.

[0091] Figure 23 This is a graph showing the affinity analysis results of the DDR1nb-3-Fc-mCD80 fusion protein provided in the embodiments of the present invention with mouse CD28, CTLA-4 and PD-L1 proteins.

[0092] Figure 24 This is a CCK8 assay result of tumor cell survival rate after co-culturing mouse spleen lymphocytes according to an embodiment of the present invention. Data are expressed as mean ± standard deviation (n=4); statistical analysis was performed using one-way ANOVA (**P<0.01, ***P<0.001, ****P<0.0001).

[0093] Figure 25 This describes the apoptosis and necrosis of tumor cells after co-incubation with mouse primary spleen lymphocytes according to embodiments of the present invention. Data are expressed as mean ± standard deviation (n=6); statistical analysis was performed using one-way ANOVA (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0094] Figure 26 This is an in vivo tumor targeting result diagram of DDR1nb-3-Fc-mCD80 provided according to an embodiment of the present invention. (A) In vivo fluorescence imaging detection of tumor-bearing mice at 12, 24, 36 and 48 hours after administration. (B) The ratio of fluorescence intensity of tumor tissue (area within the blue ellipse box) to the total fluorescence intensity of the mouse body at different time points. Data are expressed as mean ± standard deviation (n=3); statistical analysis was performed using one-way ANOVA (*P<0.05).

[0095] Figure 27 These are representative H&E staining images of the major organs of mice treated with the fusion protein according to embodiments of the present invention.

[0096] Figure 28 This is a graph showing the in vivo antitumor function detection results of the Fc-mCD80 and DDR1nb-3-Fc-mCD80 fusion protein provided according to embodiments of the present invention. Detailed Implementation

[0097] The following examples are provided to help those skilled in the art better understand the present invention. It should be noted that the following examples are not intended to limit the scope of protection claimed by the present invention, but are merely illustrative. Unless otherwise specified, the raw materials, reagents, or devices mentioned in the following examples are commercially available or obtained through known existing methods.

[0098] When the present invention uses the terms “for example,” “such as,” “like,” “including,” “comprising,” or variations thereof, these terms will not be considered limiting terms, but will be interpreted as meaning “but not limited to” or “not limited to.”

[0099] Unless otherwise specified by the invention or obviously contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0100] As used herein, the term "camel-derived antibody" refers to an antibody against an antigen produced by camel-dwelling animals (including camels, alpacas, and llamas) after immunization or antigen invasion. Those skilled in the art know that among the antibodies produced by camel-dwelling animals are "camelid heavy-chain antibodies" (HCAbs) lacking the light chain. These antibodies contain only one variable domain of the heavy chain (VHH) and two conventional CH2 and CH3 regions. The VHH region, when cloned and expressed separately, exhibits excellent structural stability and antigen-binding activity. The VHH is currently the smallest known unit capable of binding to a target antigen.

[0101] As used herein, the term "nanobody" has the meaning commonly understood by those skilled in the art, referring to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camel antibody or a shark antibody). Typically, a nanobody consists of four framework regions and three complementarity-determining regions, having a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to lack one or both of those framework regions, as long as they substantially maintain antigen binding and specificity. Nanobodies are also called single-domain antibodies (sdAbs), and the two terms are used interchangeably.

[0102] As used herein, the term "antigen-binding fragment" of a nanobody refers to a polypeptide containing a fragment of a nanobody that retains the ability to specifically bind to the same antigen bound by the nanobody and / or competes with the nanobody for specific binding to the antigen; it is also referred to as the "antigen-binding moiety." See Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. The antigen-binding fragment of the antibodies of this invention can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the nanobody of this invention. In some embodiments, the "antigen-binding fragment" of the nanobody may be truncated at the N-terminus or C-terminus compared to a full-length nanobody to contain only a portion of FR1 and / or FR4, or lack one or both of those backbone regions, as long as it substantially retains antigen binding and specificity.

[0103] Antigen-binding fragments of nanobodies can be obtained from a given nanobody (e.g., the nanobody provided by the present invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of nanobodies can be specifically screened in the same manner as for intact nanobodies.

[0104] In this invention, unless the context clearly indicates otherwise, when referring to the term "nanobody," it includes not only the complete nanobody but also the antigen-binding fragment of the nanobody.

[0105] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residue in the variable region of an antibody responsible for antigen binding. The nanobody contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given nanobody, those skilled in the art will readily identify the CDR defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0106] As used in this invention, the term "framework region" or "FR" residue refers to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0107] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) of that interaction. In this invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0108] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of antigen binding site / antigen complex formation and dissociation. The "binding rate constant" (k...) a or k on ) and "dissociation rate constant" (k dis or k offBoth can be calculated from concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). dis / k on The ratio is equal to the dissociation constant K. D (See Davies et al., Annual Rev Biochem, 1990; 59:439-473). K can be measured by any effective method. D k on and k dis The dissociation constant can be measured in Biacore using surface plasmon resonance (SPR) in some implementations. Alternatively, it can be measured using bioluminescence interferometry or Kinexa.

[0109] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0110] As used in this invention, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as *Escherichia coli* or *Bacillus subtilis*, fungal cells such as yeast cells or *Aspergillus*, insect cells such as S2 *Drosophila* cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells. Host cells may include single cells or populations of cells.

[0111] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be performed using, for example, a computer program such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a 12-bit nick length penalty, and a 4-bit nick penalty. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoIBiol. 48:444-453 (1970)) algorithm in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blos sum 62 matrix or a PAM250 matrix, along with 16, 14, 12, 10, 8, 6, or 4 nick weights and 1, 2, 3, 4, 5, or 6 length weights.

[0112] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved substitutions of amino acids are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0113] The twenty common amino acids involved in this invention are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0114] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences, Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing). Company (1995), and including but not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, delayed absorption agents, and preservatives. For example, pH adjusters include but are not limited to phosphate buffers. Surfactants include but are not limited to cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include but are not limited to sodium chloride. Preservatives include but are not limited to various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include but are not limited to sugars, NaCl, and their analogues. Delayed absorption agents include but are not limited to monostearates and gelatin. Diluents include but are not limited to water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), etc. Preservatives include but are not limited to various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art as being able to stabilize the desired activity of an active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). In some exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w / v) NaCl), glucose solutions (e.g., 5% glucose), solutions containing surfactants (e.g., 0.01% polysorbate 20), pH buffer solutions (e.g., phosphate buffer solutions), Ringer's solutions, and any combination thereof.

[0115] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., a disease related to DDR1) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to the expected survival (if no treatment was received).

[0116] As used in this invention, the term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) suffers from a disease related to DDR1.

[0117] As described in this invention, "fusion protein of this invention" refers to a bifunctional fusion protein that has both the anti-DDR1 nanobody described in the first aspect of this invention and an immunomodulatory molecule portion.

[0118] In this invention, a fusion protein is provided, wherein the nanoantibody fusion protein has a structure from the N-terminus to the C-terminus as shown in Formula I:

[0119] Z1-Z2-L-Z3 (Formula I)

[0120] In the formula, Z1 is the VHH chain of the anti-DDR1 nanobody as described in the second aspect of the present invention; Z2 is the Fc segment of the immunoglobulin; L is the adapter sequence; and Z3 is the immunomodulatory molecular part. The complementarity-determining region (CDR) of the VHH chain of the anti-DDR1 nanobody consists of the following: CDR1 with the amino acid sequence shown in SEQ ID NO:1; CDR2 with the amino acid sequence shown in SEQ ID NO:2; and CDR3 with the amino acid sequence shown in SEQ ID NO:8. The framework region (FR) of the nanobody includes the sequences FR1, FR2, FR3, and FR4, wherein FR1 contains the amino acid sequence VQLVESG GGLVQPGGSLRLSCVAS; FR2 contains the amino acid sequence IGWFRQAPGKERE GVSC; FR3 contains the amino acid sequence NYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFC; and FR4 contains the amino acid sequence WGQGTQVTVSS. The immunomodulatory molecular part is the CD80 protein. The adapter sequence is GGGGSGGGGSGGGGS. The CD80 protein sequence is VDEQLSKSVKDKVLLPCRYNSPHEDESEDRIYWQKHDKVVLSVIAGKLKVWPEYKNRTLYDNTTYSLIILGLVLSDRGTYSCVVQKKERGTYEVKHLALV KLSIKADFSTPNITESGNPSADTKRITCFASGGFPKPRFSWLENGRELPGINTTISQDPESELYTISSQLDFNTTRNHTIKCLIKYGDAHVSEDFTWEKPPEDPPDSK. The VHH chain sequence is QVQLVESGGGLVQPGGSLRLS CVASGFTANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTI SRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYFSNDCDLNNYDHWGQGTQVTVSS.

[0121] In this invention, a polynucleotide is provided that encodes a protein selected from the group consisting of: anti-DDRI nanobody as described above, VHH chain of anti-DDRI nanobody as described above, or anti-DDRI nanobody as described above.

[0122] In this invention, an expression vector is provided, which contains the polynucleotides described above.

[0123] In this invention, a host cell is provided, which contains the expression vector as described above, or whose genome integrates the polynucleotides as described above.

[0124] In this invention, a method for generating anti-DDRI nanobodies is provided, comprising the steps of: (a) culturing host cells as described above under conditions suitable for nanobodies to generate, thereby obtaining a culture containing the anti-DDRI nanobodies; and (b) separating or recovering the anti-DDRI nanobodies from the culture.

[0125] In this invention, an immunoconjugate is provided comprising: (a) a VHH chain of an anti-DDRI nanobody as described above, an anti-DDRI nanobody as described above, or a nanobody fusion protein as described above; and (b) a conjugation portion selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0126] In this invention, the use of the anti-DDRI nanobody or the nanobody fusion protein as described above is provided for the preparation of (a) a reagent for detecting DDRI molecules; (b) a drug for treating tumors; wherein the tumor is selected from the group consisting of: gastric cancer, liver cancer, kidney tumors, lung cancer, small intestinal cancer, bone cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, cervical cancer, adrenal tumors, pancreatic cancer, bladder tumors, or combinations thereof.

[0127] In this invention, a pharmaceutical composition is provided, comprising: (i) an anti-DDRI nanobody as described above, a VHH chain of an anti-DDRI nanobody as described above, or an anti-DDRI nanobody as described above, a nanobody fusion protein as described above, or an immunoconjugate as described above; and (ii) a pharmaceutically acceptable carrier.

[0128] The above-mentioned pharmaceutical composition is used to prepare a drug for treating tumors, wherein the tumors are selected from the group consisting of: gastric cancer, liver cancer, kidney tumors, lung cancer, small intestine cancer, bone cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, cervical cancer, adrenal tumors, pancreatic cancer, bladder tumors, or combinations thereof.

[0129] In this invention, a recombinant protein is provided, the recombinant protein having: (i) the sequence of the VHH chain of the anti-DDRI nanobody as described above, or the sequence of the nanobody as described above, or the sequence of the nanobody fusion protein as described above; and (ii) an optional tag sequence to assist in expression and / or purification.

[0130] The VHH chain of the anti-DDRI nanobody described above, the nanobody described above, the nanobody fusion protein described above, or the immunoconjugate described above are used to prepare pharmaceuticals, reagents, detection plates, or kits; wherein the reagents, detection plates, or kits are used to detect DDRI protein in a sample; wherein the pharmaceuticals are used to treat or prevent DDRI-positive tumors.

[0131] In this invention, a non-disease diagnostic method for detecting DDRI protein in a sample is provided, comprising the steps of: (1) contacting the sample with a nanobody or a nanobody fusion protein as described above; and (2) detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of DDRI protein in the sample.

[0132] Example 1: Screening and Identification of DDR1 Nanobodies

[0133] This invention obtains a monoclonal nanobody library against DDR1 by immunizing alpacas, and then screens the antibodies using phage display and yeast display technologies to select DDR1 nanobodies with high affinity and specificity. The selected candidate antibodies are expressed, purified, and functionally evaluated, including affinity, selectivity, and antitumor function tests. These experiments verify the binding properties of the candidate antibodies and their application value in tumor models.

[0134] 1. Alpaca Immunity

[0135] Alpacas were immunized with a mixture of recombinant human DDR1-ECD-his and mouse DDR1-ECD-his proteins, alternatingly with the adjuvant GERBU in a 1:1 ratio. The immunizations were administered via subcutaneous injection at multiple sites for a total of five immunizations, with an interval of 7 days between each immunization. Peripheral blood was collected 10 days after the last immunization, and serum was separated and the immunization effect was detected by ELISA.

[0136] For the alpaca immunization experiment, 250 μg of protein was administered weekly. The first three immunizations were against human DDR1-ECD protein, followed by two shock immunizations against mouse DDR1-ECD protein, for a total of five immunizations. Through cross-immunization, alpacas were successfully induced to produce immune responses against both human and mouse DDR1-ECD proteins. Starting from the third immunization, 1 ml of blood was collected from alpacas after each immunization, and serum was separated by centrifugation at 300 × g for 5 min. The serum was limitedly diluted, and ELISA experiments were performed separately in 96-well high-affinity polystyrene microplates pre-coated with 100 μl of 1 μg / ml human and mouse DDR1-ECD protein to detect the titer of antibodies against human and mouse DDR1-ECD in alpaca serum.

[0137] The results showed that with increasing immunization frequency, the antibody titers against human and mouse DDR1-ECD antigens in alpacas significantly increased. This indicates that the alpaca's immune system gradually recognized and enhanced its response to these antigens, demonstrating the "memory effect" and progressively increasing characteristics of the immune response. After the fifth immunization, the alpaca's immunotiter reached 64K, indicating a high immunotiter level that met the criteria for library construction. Therefore, 150 ml of alpaca peripheral blood was collected, and PBMCs were isolated for subsequent experiments.

[0138] 2. Construction and screening of phage display libraries

[0139] Total RNA was extracted from isolated alpaca peripheral blood PBMCs, reverse transcribed into cDNA, and then amplified by two rounds of PCR using single-domain antibody amplification primers. Figure 1 This refers to the PCR product results from agarose gel electrophoresis. The first round of PCR yielded bands of approximately 1000 bp and 700 bp, respectively. The 700 bp fragment was excised and recovered as the template for the second round of PCR. The second round of PCR yielded a band of approximately 400 bp, which is the target band, representing the Variable Domain of Heavy Chain (VHH) antibody fragment composed of the variable region of the heavy chain.

[0140] SS320 electroporation competent *E. coli* cells were prepared, and the quality of the prepared competent cells was verified by QC. The obtained VHH fragment was cloned into the pCANTab5f vector. The prepared pCANTab5f-VHH plasmid was electroporated into SS320 electroporation competent *E. coli* cells to obtain the original *E. coli* library. 1×10⁻⁶ cells of the original bacterial culture were used. -6 The plating was spread on LB agar plates containing ampicillin, incubated upside down overnight, and colony counts were performed, yielding a total of 258 colonies. The calculated initial library volume was 2.5 × 10⁻⁶. 8 Thirty-two single clones were randomly selected from the plate and transferred to 2YT medium containing ampicillin. The culture was incubated at 37°C with shaking for approximately 6–8 hours, and then sent for sequencing (using universal sequencing primer M13R). The sequencing results showed that the empty vector rate and antibody repetition rate of the phage display library were no higher than 10%, indicating that the constructed original library had high quality.

[0141] Solid-phase screening of phages that cross-binded with human and mouse antigens was performed using gradually decreasing concentrations of human and mouse DDR1-ECD protein, for a total of four rounds of screening. After each round of screening, the selected (output) phages were used to infect ER2738 *E. coli*. All infected *E. coli* were then plated on LB-AMP plates. The output titer of the phages was determined by counting colonies and eluting. The phages obtained from each round of screening were amplified and precipitated with PEG to become the input phages for the next round of screening.

[0142] After four rounds of screening, phages that cross-bind with human and mouse antigens were successfully enriched. Single clones were picked from LB agar plates containing *E. coli* infected with the output phages from the fourth round of screening. The binding of the phages to human and mouse DDR1-ECD in the selected single clones was detected by ELISA. Positive clones were sent for next-generation sequencing, resulting in 11 DDR1 antibody sequences. These included one human-mouse cross-binding antibody sequence, one antibody sequence binding only to m-DDR1-his, and nine antibody sequences binding only to h-DDR1-his.

[0143] 3. Construction and screening of yeast display libraries

[0144] The VHH fragment was amplified and enriched by three rounds of PCR, and the mixture of the fragment and a vector containing the V5 tag expression label was electroporated into freshly prepared EBY100 Saccharomyces cerevisiae competent cells. The resulting yeast culture was used as the original yeast display library for the DDR1 nanobody. The original library was plated on SDCAA plates, and single colonies were counted. The calculated volume of the original DDR1 yeast display library was 2 × 10⁻⁶ cells / year. 9 .

[0145] Protein expression was induced in the original library obtained by electroporation using SGCAA medium. The positive level of expression tag V5 tag was detected by flow cytometry. The display rate of the original library expression tag V5 tag in unsorted yeast was 20.1%.

[0146] Next, the yeast display library was subjected to the first round of magnetic sorting using biotinylated human DDR1-ECD protein to screen for yeast strains displaying anti-human DDR-ECD antibodies. The strains obtained from the first round of magnetic sorting were amplified and cultured in SDCAA medium for 1 day, followed by induction culture in SDCAA medium for 2 days. The display positivity rate of the yeast display library after the first round of sorting was detected by flow cytometry. The cells were incubated with 100 μl of biotinylated h-DDR1-ECD protein (10 μg / ml) at a concentration of 1×10⁻⁶ cells / ml. 6 Yeast cells were incubated for 1 hour, followed by incubation with FITC-V5 Tag Monoclonal Antibody and PE-streptavidin for 30 minutes before analysis. After one round of magnetic sorting, the V5 tag display rate of the yeast display library significantly increased from 20.1% to 50.88%. Simultaneously, 41.24% of the yeast cells displayed anti-human DDR-ECD antibodies.

[0147] We screened yeast cells expressing anti-human DDR1-ECD nanobodies through one round of magnetic sorting. The bacterial culture enriched by the first round of magnetic sorting was expanded and cultured with SDCAA for 1 day, and then induced to express the cells with SGCAA for 2 days.

[0148] To screen for nanobodies that can cross-bind with human and mouse DDR1-ECD proteins, a yeast display library after one round of magnetic sorting was subjected to a two-round flow sorting using biotinylated mouse DDR1-ECD protein. Specifically, yeast cells were incubated with 100 μl of biotinylated mouse DDR1-ECD protein (10 μg / ml) for 1 hour, followed by incubation with FITC-V5 Tag Monoclonal Antibody and APC-streptavidin for 1 hour. Double-positive yeast cells simultaneously displaying the V5 tag and anti-mouse DDR1-ECD nanobodies were isolated from P3.

[0149] Nanobodies that cross-bind human and mouse DDR1-ECD proteins were successfully screened from a yeast display library using a combination of magnetic sorting and flow cytometry. Next, yeast culture that had undergone two rounds of screening was plated on SDCAA plates, and single clones were picked. A total of 1000 single clones were picked and amplified using SDCAA medium. Each single clone was then induced to display the target antibody using SDCAA medium. The binding of each single clone to human and mouse DDR1-ECD proteins was detected by high-throughput flow cytometry. Based on the flow cytometry results, clones with a positive antibody display rate of >15% for both human and mouse DDR1-ECD were selected for amplification. The candidate clones underwent culture PCR to amplify the target antibody sequences, followed by next-generation sequencing. Finally, 79 positive DDR1 nanobody sequences were screened.

[0150] A total of 90 candidate positive antibody sequences were screened from bacteriophage and yeast displays, and were numbered sequentially from DDR1nb-1 to DDR1nb-90.

[0151] Table 1. Top Ten Antibody Sequences by Affinity Detection

[0152] Antibody number CDR1-IMGT CDR2-IMGT CDR3-IMGT DDR1nb-2 GFTANYYD ISSSGSGT AAKRRDYSSNDCDLNNYDH DDR1nb-3 GFTANYYD ISSSGSGT AAKRRDYFSNDCDLNNYDH DDR1nb-4 GFTANYYD ISSSGSGT AAKRRDYSSNDCDLNNYDH DDR1nb-8 GFTANYYD ISSSGSGT AAKRRDYSSNDCDLNNYDH DDR1nb-9 GFTANYYD ISSSGSGT AAKRRDYSSNDCDLNNYDH DDR1nb-11 GFAANYYD ISSSGSGT AAKRRDYSSNDCDLNNYDH DDR1nb-12 GFTFSMYS IASGSGST WGAGY DDR1nb-13 GFTANYYD ISSSGSGT AAKRRDYSSNDCDLNNYDH DDR1nb-19 GFTANYYD ISSSGSGT AAKRRDYSSNDCDLNNYDH DDR1nb-31 GFTANYYD ISSSGSGT AAKRRDYSSNDCDLNNYDH

[0153] Table 2. Top 10 Antibody Sequences by Affinity Detection

[0154]

[0155]

[0156] Table 3. Top Ten Antibody Sequences by Affinity Detection

[0157] Antibody number FR3-IMGT FR4-IMGT DDR1nb-2 ADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFC WGQGTQVTVSS DDR1nb-3 NYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFC WGQGTQVTVSS DDR1nb-4 NYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFC WGQGTQVTISS DDR1nb-8 NYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFC WGQGAQVTVSS DDR1nb-9 NYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFC WGQGTQVTVSS DDR1nb-11 NYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFC WGQGTQVTVSS DDR1nb-12 NYADSVKGRFTISRDNAKNTVSLQMNSLKPEDTAVYYC WGPGTQVTVSS DDR1nb-13 NYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFC WGQGTQVTVSS DDR1nb-19 NYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFC WGQGTQVTVSS DDR1nb-31 NYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFC WGQGTQVTVSS

[0158] In the above sequences, the complementarity-determining region (CDR) of the VHH chain consists of the following: CDR1 with an amino acid sequence as shown in SEQ ID NO:1; CDR2 with an amino acid sequence as shown in SEQ ID NO:2; CDR3 with an amino acid sequence as shown in SEQ ID NO:3; SEQ ID NO:1: GFTANYYD; SEQ ID NO:2: G ISSSGSGT; SEQ ID NO:3: AAKRRDYSSNDCDLNNYDH.

[0159] Or the amino acid sequence as shown in SEQ ID NO:4: CDR1; the amino acid sequence as shown in SEQ ID NO:2: CDR2; the amino acid sequence as shown in SEQ ID NO:3: CDR3; SEQ ID NO:4: GFAANYYD.

[0160] Or the amino acid sequence as shown in SEQ ID NO:5 for CDR1; the amino acid sequence as shown in SEQ ID NO:6 for CDR2; the amino acid sequence as shown in SEQ ID NO:7 for CDR3; SEQ ID NO:5: GFTFSMYS; SEQ ID NO:6: IASGSGST; SEQ ID NO:7: WGAGY.

[0161] Or the amino acid sequence is as shown in SEQ ID NO:1 for CDR1; the amino acid sequence is as shown in SEQ ID NO:2 for CDR2; the amino acid sequence is as shown in SEQ ID NO:8 for CDR3; SEQ ID NO:8: AAKRRDYFSNDCDLNNYDH.

[0162] The amino acid sequence of the VHH chain of the anti-DDRI nanobody is shown in SEQ ID NO:11-20 (sequence summary in the table).

[0163] DDR1nb-2SEQ ID NO:9

[0164] VQLVESGGGLVHPGGSLRLSCAVSGFTANYYDIGWFRQAPGKEREGVSCISSSGSGTADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGTQVTVSS

[0165] DDR1nb-3SEQ ID NO:10

[0166] VQLVESGGGLVQPGGSLRLSCVASGFTANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYFSNDCDLNNYDHWGQGTQVTVSS

[0167] DDR1nb-4SEQ ID NO:11

[0168] VQLVESGGGLVHPGGSLRLSCAVSGFTANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGTQVTISS

[0169] DDR1nb-8SEQ ID NO:12

[0170] LQLVESGGGLVHPGGSLRLSCAVSGFTANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGAQVTVSS

[0171] DDR1nb-9SEQ ID NO:13

[0172] LQLVESGGGSVQAGGSLTLSCTASGFTANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGTQVTVSS

[0173] DDR1nb-11SEQ ID NO:14

[0174] VQLVESGGGLVHPGGSLRLSCAVSGFAANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGTQVTVSS

[0175] DDR1nb-12SEQ ID NO:15

[0176] QVQLVESGGGLVRPGGSLRLSCAASGFTFSMYSMRWYRQAPGKERELVAHIASGSGSTNYADSVKGRFTISRDNAKNTVSLQMNSLKPEDTAVYYCWGAGYWGPGTQVTVSS

[0177] DDR1nb-13SEQ ID NO:16

[0178] DVQLVESGGGSVQAGGSLRLSCAVSGFTANYYDIGWVRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGTQVTVSS

[0179] DDR1nb-19SEQ ID NO:17

[0180] DVQLVESGGGLVHPGGSLRLSCAVSGFTANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGTQVTVSS

[0181] DDR1nb-31SEQ ID NO:18

[0182] DVQLVESGGGLVHPGGSLRLSCAVSGFTANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGTQVTVSS

[0183] For positive sequences, his-tag pcDNA3.4 expression plasmids were constructed. Ninety plasmids and GFP lentivirus were transfected into 293F cells for low-level expression. Cell transfection supernatants were collected on day 5 post-transfection, and affinity tests were performed on the 90 transfection supernatants and negative transfection supernatants using ELISA microplates coated with human DDR1-ECD and mouse DDR1-ECD proteins, respectively. ELISA screening identified 19 transfection supernatants with affinity for both human and mouse DDR1-ECD. The corresponding plasmids were transfected into Top10 competent *E. coli* cells, and amplification and plasmid extraction were performed to obtain 19 candidate sequence expression plasmids. These 19 plasmids were transfected into 293F cells and purified using a Ni column.

[0184] Affinity was measured for the 19 candidate DDR1nbs expressing the his tag using ELISA microplates coated with mouse DDR1-ECD and human DDR1-ECD proteins, respectively, and the candidate antibodies were ranked by affinity. The top ten antibodies in terms of affinity were: DDR1nb-2-his, DDR1nb-3-his, DDR1nb-4-his, DDR1nb-8-his, DDR1nb-9-his, DDR1nb-11-his, DDR1nb-12-his, DDR1nb-13-his, DDR1nb-19-his, and DDR1nb-31-his.

[0185] This invention selects active candidate antibodies directly through in vivo antitumor activity screening. To ensure the antibodies can realistically interact with DDR1-ECD in the tumor microenvironment in vivo, thereby more accurately assessing antibody efficacy, and considering the short in vivo half-life of nanobodies, expression plasmids fused with the human IgG1 Fc fragment at the C-terminus of each of the ten sequences were constructed to express the DDR1nbs-Fc fusion protein. The fused Fc fragment extends the antibody half-life. The electrophoretic bands of the 10 antibodies all ranged from 35 to 55 kDa.

[0186] DDR1nb-2-Fc sequence: SEQ ID NO:19

[0187] MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVHPGGSLRLSCAVSGFTANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGTQVTVSSAHHSEDPSSAAASGHEGQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0188] DDR1nb-3-Fc sequence: SEQ ID NO:20

[0189] MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVQPGGSLRLSCVASGFTANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYFSNDCDLNNYDHWGQGTQVTVSSEPKTPKPGHEGQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0190] DDR1nb-4-Fc sequence: SEQ ID NO:21

[0191] MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVHPGGSLRLSCAVSGFTANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGTQVTISSEPKTPKPGHEGQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0192] DDR1nb-8-Fc sequence: SEQ ID NO:22

[0193] MGWSCIILFLVATATGVHSKAQPAMAQLQLVESGGGLVHPGGSLRLSCAVSGFTANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGAQVTVSSAHHSEDPSSAAASGHEGQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0194] DDR1nb-9-Fc sequence: SEQ ID NO:23

[0195] MGWSCIILFLVATATGVHSKAQPAMAQLQLVESGGGSVQAGGSLTLSCTASGFTANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGTQVTVSSAHHSEDPGHEGQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0196] DDR1nb-11-Fc sequence: SEQ ID NO:24

[0197] MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVHPGGSLRLSCAVSGFAANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGTQVTVSSAHHSEDPSSAAASGHEGQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0198] DDR1nb-12-Fc sequence: SEQ ID NO:25

[0199] MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVRPGGSLRLSCAASGFTFSMYSMRWYRQAPGKERELVAHIASGSGSTNYADSVKGRFTISRDNAKNTVSLQMNSLKPEDTAVYYCWGAGYWGPGTQVTVSSEPKTPKPGHEGQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0200] DDR1nb-13-Fc sequence: SEQ ID NO:26

[0201] MGWSCIILFLVATATGVHSKAQPAMADVQLVESGGGSVQAGGSLRLSCAVSGFTANYYDIGWVRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGTQVTVSSAHHSEDPGHEGQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0202] DDR1nb-19-Fc sequence: SEQ ID NO:27

[0203] MGWSCIILFLVATATGVHSKAQPAMADVQLVESGGGLVHPGGSLRLSCAVSGFTANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGTQVTVSSAHHSEDPGHEGQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0204] DDR1nb-31-Fc sequence: SEQ ID NO:28

[0205] MGWSCIILFLVATATGVHSKAQPAMADVQLVESGGGLVHPGGSLRLSCAVSGFTANYYDIGWFRQAPGKEREGVSCISSSGSGTNYADSVKGRFTISRMNIRKTLYLQMNSLKPEDTAVYFCAAKRRDYSSNDCDLNNYDHWGQGTQVTVSSAHHSEDPSSAAASGHEGQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0206] E0771 is a murine TNBC cell line that highly expresses DDR1 and exhibits tumorigenicity in C57BL / 6 mice. Studies have shown that DDR1 knockout significantly inhibits tumor growth in an E0771 mouse breast cancer model. This model is currently used for screening existing DDR1 monoclonal antibodies. Similarly, we constructed an E0771 mouse TNBC model for in vivo screening of candidate DDR1 antibodies. Tumor-bearing mice were treated twice weekly with intraperitoneal injections of the candidate DDR1nbs-Fc (10 mg / kg). The in vivo tumor-suppressive effects of 10 candidate antibodies (n=3) were then evaluated. The results are as follows: Figure 2 As shown, among the ten candidate antibodies, DDR1nb-3-Fc, DDR1nb-8-Fc, DDR1nb-13-Fc, and DDR1nb-31-Fc exhibited potential anti-tumor effects. Tumor growth curves for mice in the antibody-treated groups are detailed below. Figure 3 Furthermore, none of these four antibodies caused a significant decrease in the body weight of the mice that were treated.

[0207] The affinity of four candidate antibodies for mouse DDR1-Fc and human DDR1-his was detected by ELISA. Using two different labeled antigens for detection helped rule out non-specific binding between the candidate antibodies and the labels. Results are shown below. Figure 4 All four antibodies can bind to mouse DDR1-Fc and human DDR1-his, respectively. Among them, the EC50 of DDR1nb-3-Fc... 50 EC for mouse DDR1-Fc (7.975 ng / ml) and human DDR1-his (14.84 ng / ml); DDR1nb-8-Fc 50 EC for mouse DDR1-Fc (1118 ng / ml) and human DDR1-his (2070 ng / ml); DDR1nb-13-Fc 50 ECGs of mouse DDR1-Fc (177.7 ng / ml) and human DDR1-his (167.6 ng / ml) and DDR1nb-31-Fc were measured. 50 The results were compared with mouse DDR1-Fc (383.2 ng / ml) and human DDR1-his (158.5 ng / ml).

[0208] Bio-Layer Interferometry (BLI) was used. Candidate antibodies DDR1nb-3-Fc, DDR1nb-8-Fc, DDR1nb-13-Fc, and DDR1nb-31-Fc were immobilized using an AHC sensor at a concentration of 5 μg / ml for 80 s. The buffer was 0.02% PBST (PBS + 0.02% Tween 20). Human DDR1-His and mouse DDR1-His were diluted to 50, 25, 12.5, 6.25, 3.13, and 0 nM. Affinity was assessed: equilibration for 60 s, binding for 180 s, dissociation for 300 s, and detection at 25 °C. Results are shown below. Figure 5 DDR1nb-3-Fc, DDR1nb-8-Fc, DDR1nb-3-Fc, and DDR1nb-31-Fc all exhibit high affinity for human / mouse DDR1-ECD. Among them, DDR1nb-3-Fc shows the highest affinity for human / mouse DDR1-ECD, with its protein binding rate constant (Kb) being the highest. on The value is 2.593 × 10 5 M -1 s -1 Dissociation rate constant (K) off The value is 3.988 × 10 -5 s -1 Equilibrium dissociation constant (K) D The value is 1.538 × 10 -10 M (Table 4).

[0209] Table 4. Affinity kinetic constants of the four candidate DDR1 antibodies with human / mouse DDR1-ECD.

[0210]

[0211] Multiple studies have demonstrated that DDR1-ECD detaches from the surface of tumor cells, thereby binding to collagen fibers in the tumor microenvironment and promoting collagen fiber arrangement. Because DDR1-ECD easily detaches from the cell surface, detecting the binding of DDR1 antibodies to cells expressing DDR1 is challenging. In this experiment, single-cell suspensions of mouse E0771 adherent cells highly expressing DDR1 were obtained using three methods: trypsin digestion, Accutase cell digestion solution digestion, and cell scraping physical method.

[0212] Compared to isotype control (human IgG1-Fc), all four candidate antibodies significantly bound to single cells obtained by the three methods. Figure 6This indicates that all four candidate antibodies can bind to residual DDR1-ECD on the cell membrane. Among the three methods, trypsin digestion resulted in the greatest DDR1-ECD detachment, followed by Accutase cell digestion solution. Accutase is a cell digestion solution containing both proteolytic and collagenase activities; compared to trypsin digestion, it is gentler and retains more cell surface DDR1-ECD. The cell scraping method, which directly scrapes adherent cells, yields single cells with the highest retention of surface DDR1-ECD, demonstrating the strongest ability to retain cell surface antigens.

[0213] Immunohistochemical staining was used to detect the binding of isotype control (human IgG1-Fc) and DDR1nb-3-Fc, DDR1nb-8-Fc, DDR1nb-13-Fc, and DDR1nb-31-Fc to mouse E0771 TNBC tumor tissue. The secondary antibody was anti-human IgG1-hrp. The results are as follows: Figure 7 As shown, compared to the isotype control, all four candidate antibodies were able to bind to mouse tumor tissue.

[0214] The affinity of four candidate antibodies for human / mouse DDR1-ECD-his (1 μg / ml) was detected by ELISA. Results are as follows: Figure 7 As shown, all four candidate antibodies exhibited high affinity for human / mouse DDR1-ECD-his protein, but weak affinity for human / mouse DDR2-ECD-his protein, indicating that all four candidate antibodies have excellent selectivity.

[0215] Example 2: DDR1 nanobody exerts anti-tumor effect by inhibiting collagen fiber arrangement.

[0216] This embodiment further explores the antitumor effects and mechanisms of four candidate DDR1 antibodies in TNBC and PAAD mouse models. Triple-negative breast cancer (TNBC), pancreatic cancer (PAAD), non-small cell lung cancer (NSCLC), and gastric cancer (CA) are among the most challenging tumor types to treat clinically. Their high invasiveness and resistance to traditional therapies make them significant challenges for tumor immunotherapy. Therefore, exploring novel targeted therapies is crucial for improving patient outcomes. This embodiment will analyze in detail how candidate antibodies can disrupt the collagen fiber arrangement in the tumor microenvironment, break down the tumor collagen barrier, and regulate immune cell infiltration, thereby improving antitumor immune responses and providing new strategies for tumor immunotherapy.

[0217] 1. DDR1 nanobodies do not directly kill tumor cells.

[0218] The cytotoxicity of four candidate antibodies, DDR1nb-3-Fc, DDR1nb-8-Fc, DDR1nb-13-Fc, and DDR1nb-31-Fc, was detected in human MDAMB468 triple-negative breast cancer cells, human MCF7 Luminal A breast cancer cells, and mouse MC38 colon cancer cells, respectively.

[0219] The results are as follows Figure 8 As shown, none of the four candidate antibodies significantly inhibited the proliferation of the three types of tumor cells after 48 hours of treatment. Even at a high concentration of 100 μg / ml, no significant cytotoxicity was observed. Therefore, it can be inferred that the candidate antibodies do not directly kill or inhibit the proliferation of tumor cells in vitro.

[0220] 2. In vivo tumor targeting of candidate antibodies

[0221] Subcutaneous tumor-bearing nude mice were intraperitoneally injected with 10 μg / ml of CY7-labeled human IgG1 Fc (is otypecontrol), DDR1nb-3-Fc, DDR1nb-8-Fc, DDR1nb-13-Fc, and DDR1nb-31-Fc, respectively. In vivo imaging was performed at 12, 24, 36, 48, and 72 hours post-administration to detect fluorescence distribution. Figure 9 A) and calculate the ratio of the total fluorescence intensity in the tumor region (blue box) to the total fluorescence intensity of the entire body of the same mouse. Figure 9 B). For example Figure 9 As shown, among the four candidate antibodies, DDR1nb-3-Fc and DD R1nb-31-Fc have the most significant tumor targeting. They are significantly enriched in the tumor area 24 hours after intraperitoneal administration, and the fluorescence is still mainly concentrated in the tumor area 72 hours after administration.

[0222] 3. Study on the efficacy, safety and mechanism of candidate antibodies in TNBC subcutaneous tumor model

[0223] 3.1 The candidate antibody exerted significant anti-tumor effects in a mouse TNBC subcutaneous tumor model.

[0224] The in vivo antitumor efficacy of four candidate antibodies was tested in a mouse E0771 TNBC subcutaneous tumor model. The results showed that all four candidate antibodies could effectively inhibit tumor growth in mice. Figure 10 A), and does not cause an abnormal decrease in body weight in tumor-bearing mice. Figure 10 B). Tumors were isolated on day 21 after inoculation, and compared with the control group, the tumors in all four candidate antibody treatment groups showed significant shrinkage. Figure 10 C), weighing the tumors revealed that the total weight of the tumor masses in mice treated with the four candidate DDR1 antibodies was significantly reduced. Figure 10 D).

[0225] like Figure 11 As shown, in the tumor tissue of the control group, a large number of tumor cells were densely packed, with significantly irregular cell morphology and tubular structures in the central lumen of the tumor cell clusters (black arrows). The tumor cells had abundant, eosinophilic cytoplasm, with nuclei of varying sizes, high atypia, and obvious pathological mitotic figures (green arrows). The interstitial blood vessels were slightly dilated, congested, and hemorrhagic (red arrows). Scattered tumor necrosis was observed near the tumor cells. No obvious infiltration of lymphocytes or neutrophils was observed. In contrast, in the groups treated with the four candidate DDR1 antibodies, fewer tumor cells were densely packed, with scattered pathological mitotic figures (green arrows). The interstitial blood vessels were highly dilated, congested, and hemorrhagic (red arrows). There was extensive tumor necrosis near the tumor cells, with a large number of lymphocytes (blue arrows) and neutrophils (yellow arrows) infiltrating. These results indicate that all four candidate DDR1 antibodies can significantly promote the intratumoral infiltration of immune cells and lead to tumor cell necrosis.

[0226] 3.2 Candidate antibodies inhibit the normal arrangement of collagen fibers in TNBC tumors

[0227] Masson's trichrome staining was performed on the tumor tissues of each group of tumor-bearing mice. Collagen fibers stained blue, while cytoplasm and muscle appeared red. Figure 12 As shown, in the control group, orderly arranged blue collagen fibers were visible within the tumor. In the tumor tissues of the four candidate DDR1 antibody administration groups, obvious disordered collagen fiber arrangement and abnormal disordered collagen fiber proliferation were observed, which is speculated to be compensatory hyperplasia.

[0228] Collagen fibers are among the most abundant structural proteins in living organisms, exhibiting a non-centrosymmetric molecular arrangement. This structure allows collagen fibers to effectively generate second harmonic generation (SHG) signals, while other tissue components (such as cells and fats) typically do not produce SHG signals or produce extremely weak signals. The generated SHG signal is usually located at half the excitation wavelength; therefore, by selectively detecting the signal at this wavelength using appropriate filter equipment, imaging information of collagen fibers can be obtained. The intensity and pattern of this signal can reflect the alignment, density, and structural characteristics of collagen fibers. SHG signals from frozen sections of tumor tissue were collected using multiphoton confocal microscopy to image the boundaries of tumor sections. Figure 13As shown, the generated SHG signal appears as red fluorescence, while the cell nuclei, stained with DCS1 nuclear dye, appear as blue fluorescence. The results showed that in the control group, the tumor tissue boundary exhibited a very clear SHG signal, with neatly arranged collagen fibers forming a distinct barrier. However, in the DDR1nb-3-Fc and DDR1nb-31-Fc treatment groups, the SHG signal at the tumor tissue boundary was relatively weak, indicating disruption of the collagen fiber barrier at the tumor tissue boundary.

[0229] 3.3 Candidate antibodies promote the infiltration of immune cells in TNBC tumors

[0230] The separated tumor tissues were dissected and found that the tumors in the four candidate antibody treatment groups were hollow, with a large amount of yellow pus flowing out upon dissection. In contrast, each tumor in the control group was homogeneous, solid, and firm, with no pus discharge. Therefore, it is speculated that the tumor tissues in the treatment groups contained a large number of infiltrating immune cells. Single-cell suspensions were obtained by grinding each tumor fragment, stained, and analyzed by flow cytometry. Results are shown below. Figure 14 and Figure 15 .

[0231] Depend on Figure 14 It can be seen that among the candidate DDR1nb-Fc, DDR1nb-3-Fc, DDR1nb-8-Fc, and DDR1nb-13-Fc all significantly promote CD45. + Immune cells and CD3 + Intratumoral infiltration of T cells. Due to the limited sample size and significant individual variability, although not statistically significant, all four candidate antibodies promoted intratumoral CD4 infiltration to some extent. + T cell infiltration was observed. DDR1nb-3-Fc, to some extent, promoted intratumoral CD8 cell infiltration. + T cell infiltration. CD4+ in the spleen of the DDR1nb-31-Fc treatment group. + and CD8 + The increased proportion of T cells, which was not observed in the other three candidate antibody dosing groups, suggests that DDR1nb-31-Fc may have a broad systemic immune activation effect.

[0232] To further explore the effect of candidate DDR1 antibodies on intratumoral immune cell infiltration, the expression of CD45, CD8, and NK1.1 in tumor tissue sections was detected by immunohistochemical staining. Results are shown below. Figure 15 Significant upregulation of CD45, CD8, and NK1.1 was detected in the tumors of mice treated with DDR1nb-3-Fc, DDR1nb-8-Fc, and DDR1nb-31-Fc. This indicates a significant increase in the total number of infiltrating immune cells, CD8+ T cells, and NK and NKT cells within the tumor.

[0233] Neutrophils are the most abundant immune cells. In the tumor microenvironment (TME), neutrophil metabolism, chemokines, and matrix are altered, and they perform multiple functions, including secreting pro-inflammatory mediators and regulating immune activation. However, the role and mechanism of neutrophils in the TME are not yet thoroughly understood. Some researchers believe that neutrophil infiltration within tumor cells is detrimental to antitumor immune responses. Therefore, this section further examines flow cytometry analysis of neutrophils infiltrating tumor cells.

[0234] The results are as follows Figure 16 As shown, surprisingly, in tumor tissues that showed significant growth inhibition after treatment with the candidate DDR1 antibodies DDR1nb-3-Fc, DDR1nb-13-Fc, and DDR1nb-31-Fc, total CD45 was present. + CD11b + Ly6G + The proportion of neutrophils, however, increased significantly. In mice treated with DDR1nb-31-Fc, neutrophils accounted for a smaller percentage of total CD45 cells. + The proportion of TILs increased significantly. However, similar results as in tumor TME were not observed in the spleen; CD45 levels were significantly increased in the spleens of mice treated with the four candidate antibodies. + CD11b + Ly6G + The proportion of neutrophils was not statistically different compared to the control group. This further illustrates that CD45 + CD11b + Ly6G + Neutrophil infiltration is a characteristic increase in tumor tissue following DDR1 antibody treatment. This result appears to contradict current understanding of the function of neutrophils in TME. This example also found that, compared to the control group, all four candidate DDR1 antibodies significantly promoted intratumoral Ly6G... + CD62L + Neutrophil infiltration within the tumor, with these neutrophils being enriched in areas of tumor necrosis.

[0235] 3.4 The candidate antibody has good safety profile.

[0236] Whole blood was collected from E0771 tumor-bearing mice in different treatment groups at the study endpoint. Serum was separated, and the concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were detected by ELISA. The results showed that in the E0771 TNBC model, compared with the control group, administration of the four candidate antibodies did not cause abnormal upregulation of AST and ALT concentrations in mouse serum.

[0237] H&E staining was performed on the major organs (heart, liver, spleen, lung, and kidney) of mice collected at the study endpoint. The results showed that, compared with the control group, administration of the four candidate DDR1 antibodies did not cause significant damage to the heart, liver, spleen, lung, and kidney tissues of tumor-bearing mice. This result further demonstrates that the candidate antibodies all have good safety profiles.

[0238] 4. The candidate antibody exhibited significant anti-tumor activity in the PAAD subcutaneous tumor model.

[0239] A KPC PAAD subcutaneous tumor-bearing mouse model was constructed using KPC pancreatic ductal carcinoma cells. The antitumor effects of four candidate DDR1nbs were explored in this model. Results showed that DDR1nb-3-Fc, DDR1nb-8-Fc, and DDR1nb-31-Fc all effectively inhibited tumor growth compared to the control group. DDR1nb-3-Fc exhibited the strongest antitumor effect. DDR1nb-13-Fc inhibited tumor growth to some extent. Figure 17 ).

[0240] H&E staining was performed on tumor tissues from each group of mice, and the results are shown in the figure. Figure 18In the control group, the cells were disordered, with densely packed tumor cells and a clearly malignant tumor mesenchyme (TME). Significant nuclear atypia was observed, with large, deeply stained tumor cell nuclei and an increased nucleoplasm-to-cytoplasm ratio, indicating active tumor proliferation. Numerous mitotic figures further reflected rapid tumor proliferation. While some inflammatory cell infiltration was present in the control group, it was not pronounced. Compared to the control group, the tumor tissue in the DDR1nb-3-Fc treatment group showed decreased cell density and reduced nuclear atypia, suggesting partial inhibition of tumor cell proliferation. Increased necrotic areas were observed, along with typical apoptotic features, including condensed micronuclear fragments and tissue vacuolation. The tumor cell density in the DDR1nb-8-Fc treatment group was slightly higher, indicating continued active tumor proliferation, but with reduced atypia compared to the control group. Decreased mitotic figures suggested suppressed tumor cell proliferation. The tumor cells were more regularly arranged, with some areas showing lightly stained nuclei and abundant cytoplasm, possibly indicating increased cell differentiation. In the DDR1nb-13-Fc treatment group, tumor cell density decreased slightly, nuclear atypia in some cells was reduced, staining was lighter, and a small number of necrotic areas were observed, suggesting that the tumor may be affected locally by the drug. Compared with the control group, the DDR1nb-31-Fc treatment group showed significantly reduced cell density, extensive necrosis, and significantly reduced nuclear atypia, with lighter nuclear staining and a decreased nucleocytoplasmic ratio. Overall, H&E staining structures showed that all four candidate DDR1 antibodies exhibited certain antitumor effects, with DDR1nb-3-Fc showing the best antitumor efficacy.

[0241] In the results of this study, it was found that all four candidate DDR1 antibodies showed no significant toxicity to the major tissues and organs of TNBC subcutaneous tumor-bearing mice. The safety of the candidate antibodies in KPC subcutaneous tumor model mice was further investigated. Similarly, administration of any of the four candidate antibodies did not cause a significant decrease in the body weight of the experimental mice. At the study endpoint, the major organs (heart, liver, spleen, lung, and kidney) of the tumor-bearing mice in each group were harvested and weighed. The results showed that the weights of the aforementioned major organs in the mice treated with the four candidate DDR1 antibodies were not significantly different from those in the control group. This result further demonstrates that all four candidate DDR1 antibodies have good safety profiles.

[0242] Compared with the control group, the spleen tissue structure of mice in the four candidate DDR1 antibody treatment groups was normal, with clear differentiation between the splenic corpuscles, red pulp, and white pulp regions. No significant immune response or cellular damage was observed, and the immune function of the spleen tissue remained normal. No significant pathological changes were observed in the heart tissue in any treatment group, and the liver tissue showed a similar structure and arrangement to the control group. The kidney tissue of mice in all treatment groups (DDR1nb-3-Fc, DDR1nb-8-Fc, DDR1nb-13-Fc, DDR1nb-31-Fc) showed a structure similar to the control group, with no significant kidney damage. Although slight cellular infiltration was observed in the kidney tissue, it did not reach a significant level. The lung tissue structure was normal in all treatment groups, with no significant pathological changes, and no abnormal immune cell infiltration or inflammatory response was observed. H&E staining results showed no significant pathological changes or tissue damage in any of the major organs (heart, liver, kidney, lung, and spleen) in the four candidate DDR1nbs-Fc and isotype control antibody treatment groups, indicating that these antibodies have good safety.

[0243] 5. DDR1nb-Fc Treatment for Gastric Cancer MFC

[0244] 5.1 Construction of a mouse subcutaneous gastric cancer model:

[0245] Construction of a subcutaneous mouse gastric cancer MFC model: Mouse gastric cancer cells (MFC) were cultured in 10 cm cell culture dishes. After reaching the logarithmic growth phase, the cells were digested and collected by centrifugation at 1500 rpm / min. Six-week-old male 615 mice (19-23 g) were subcutaneously injected with 1 x 102 cells into the right rib area. 5 Cells per 0.1 ml of suspension; tumor volume reached 200 mm² after 1 week. 3 The subcutaneous tumor model of gastric cancer was considered to have been successfully constructed.

[0246] 5.2 Animal experimental treatment regimen: After the tumor-bearing animal model was successfully established, the mice were randomly divided into two groups: Group 1: control group (administered with PBS); Group 2: experimental group (intraperitoneal injection of DDR1nb-3-Fc at a dose of 10 mg / kg).

[0247] Mice were administered medication on days 1, 3, 6, and 9 of treatment, with a treatment cycle of 12 days. Mouse body weight, tumor length and width were measured on days 1, 3, 6, 9, and 12 from the start of administration, and tumor volume was calculated. After 12 days, mice were dissected, and tumors and major organs were removed for sample fixation and analysis. Tumor tissue was fixed in 4% paraformaldehyde for 24 hours, then embedded in paraffin, and cut into 4μm sections. This experiment aimed to observe the effects of DDR1nb-3-Fc on angiogenesis in gastric cancer, thereby assessing its therapeutic effect. Angiogenesis-related factors (CD31, VEGFA, HIF-1α) were stained onto the gastric cancer tissue sections. Experimental results are as follows: Figure 19As shown. Figure 19 A represents the average tumor volume in mice during the treatment period. Figure 19 B represents the weight change curve of mice during the treatment period. Figure 19 C is an image of the tumor dissected from the mouse at the end of the experiment. Figure 19 D represents the immunohistochemical staining of CD31 in tumor tissue. Figure 19 E represents the immunohistochemical staining of VEGFA in tumor tissue. Experimental results showed that the DDR1nb-3-Fc antibody, after intraperitoneal injection into a gastric cancer model mouse, effectively reduced tumor volume and inhibited angiogenesis within the tumor tissue, demonstrating good anti-tumor efficacy and therapeutic safety.

[0248] 6. DDR1nb-Fc for the treatment of non-small cell lung cancer (NSCLC)

[0249] Six-week-old male C57 mice were purchased and housed in the SPF-grade animal facility of the School of Pharmacy, Fudan University, with normal diet and water. After acclimatization to standard conditions for one week, they were used to construct a mouse subcutaneous tumor model. Lung cancer cells (LLC) were cultured in 10cm sterile cell culture dishes using DMEM (Dulbecco's Modified Eagle's Medium) complete medium containing 10% FBS (fetalbovine serum). Once the cells reached the logarithmic growth phase, they were digested and collected by centrifugation at 1500 rpm / min. Seven-week-old male C57 mice (18-22g) were subcutaneously injected with 5*10g of the drug into the right rib area. 5 Cells per 0.1 mL suspension; tumor volume reached 100 mm² after 1 week. 3 The subcutaneous tumor model of non-small cell lung cancer was considered successfully constructed. The tumor volume measurement formula is V = 0.52 * a * b. 2 (Where a represents the long axis of the tumor, and b represents the short axis of the tumor) After the tumor-bearing animal model was successfully established, the mice were randomly divided into 6 groups according to their body weight and tumor formation status:

[0250] Group 1. Isotype Control: Intraperitoneal injection of 10 mpk;

[0251] Group 2. DDR1nb-3-Fc intraperitoneal injection of 10 MPa;

[0252] Group 3. DDR1nb-8-Fc intraperitoneal injection of 10 MPk;

[0253] Group 4. DDR1nb-13-Fc intraperitoneal injection of 10 MPa;

[0254] Group 5. DDR1nb-31-Fc intraperitoneal injection of 10 MPa;

[0255] Group 6. Atezolizumab 10mpk intraperitoneal injection.

[0256] Treatment was administered on days 1, 4, 8, 11, 15, and 18 after model establishment, with a total treatment period of 18 days. Mouse body weight, tumor long axis, and short axis were measured on days 1, 4, 8, 11, 15, and 18 after model establishment, and tumor volume was calculated (tumor volume formula V = 0.52 * a * b). 2 Twenty-one days after model establishment, the mice were dissected, and tumors and major organs were removed for sample fixation and analysis. The experimental results are as follows: Figure 20 As shown. Figure 20 A represents the average tumor volume in mice during the treatment period. Figure 20 B is an image of the tumor dissected from the mouse at the end of the experiment. Figure 20 C represents the weight change curve of mice during treatment. Figure 20 D represents a comparison of the weights of the major organs obtained from the samples taken from the mice. The experimental results showed that in a C57 mouse model of LLC non-small cell lung cancer, intraperitoneal injection of DDR1nb-8-Fc and DDR1nb-13-Fc 10mpk effectively slowed tumor growth, demonstrating good anti-tumor efficacy and therapeutic safety.

[0257] Example 3: Construction and Antitumor Effect Study of DDR1nb-3-Fc-mCD80 Fusion Protein

[0258] Previous examples demonstrated that the candidate DDR1 antibody exhibited significant therapeutic effects in various cancer models. Its mechanism of action involves key aspects of TME regulation. By specifically inhibiting the DDR1 signaling pathway, it effectively disrupts the orderly arrangement of pathological collagen fibers in tumor tissue, thereby remodeling the immunosuppressive TME and promoting the infiltration of anti-tumor immune cells such as cytotoxic T lymphocytes (CTLs).

[0259] CD80, as a key co-stimulatory molecule, plays a dual regulatory role in T-cell immune responses. On the one hand, it binds to the CD28 receptor on the T cell surface through its N-terminal domain, transmitting co-stimulatory signals to promote T-cell activation and proliferation. On the other hand, it mediates the termination of immunosuppressive signals by binding to CTLA-4 through its C-terminal domain. Recent studies have also found that CD80 can competitively block the binding of PD-L1 and PD-1, forming a unique "triple regulatory" mechanism—that is, simultaneously activating the CD28 co-stimulatory pathway and relieving the dual immune checkpoint inhibition of CTLA-4 and PD-L1. This multi-target regulatory characteristic makes it a highly promising immune agonist; however, systemic use of CD80 agonists may lead to excessive immune activation and systemic toxicity.

[0260] The inventors previously discovered that the Fc-mCD80 fusion protein, constructed by placing the extracellular region of CD80 at the C-terminus of IgG1 Fc, has significant advantages over the traditional N-terminal linked CD80-Fc. Alphafold2 structural prediction showed that the CD80 domain of Fc-mCD80 exhibits a more extended conformation, with its key functional domains (including the CD28 / CTLA-4 binding site and the PD-L1 competitive binding region) fully exposed. Combined with ELISA results, Fc-mCD80 showed 3.2-fold, 4.7-fold, and 5.8-fold increased affinity for CD28, CTLA-4, and PD-L1 compared to CD80-Fc, respectively, demonstrating superior multi-target binding properties and excellent anti-tumor activity in mouse models. However, animal experiments indicated that systemic administration of Fc-mCD80 still carries the risk of immune-related adverse reactions, suggesting the need to develop tumor-targeted delivery strategies to improve treatment safety.

[0261] Fc-mCD80 sequence: SEQ ID NO:29

[0262] MGWSCIILFLVATATGVHSEPKSCDKTHTCPPCPAPELLGGPSVFLF

[0263] PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

[0264] EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ

[0265] PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK

[0266] TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS

[0267] LSPGKGGGGSGGGGSGGGGSVDEQLSKSVKDKVLLPCRYNSPHEDESEDR

[0268] IYWQKHDKVVLSVIAGKLKVWPEYKNRTLYDNTTYSLIILGLVLSDRGTY

[0269] SCVVQKKERGTYEVKHLALVKLSIKADFSTPNITESGNPSADTKRITCFASG

[0270] GFPKPRFSWLENGRELPGINTTISQDPESELYTISSQLDFNTTRNHTIKCLIK

[0271] YGDAHVSEDFTWEKPPEDPPDSK

[0272] Based on the above, this embodiment proposes an innovative fusion modification scheme. A bifunctional fusion protein of anti-DDR1 nanobody and CD80 (DDR1nb-3-Fc-mCD80) is constructed. This design integrates a dual mechanism of action: (1) the DDR1 antibody component (DDR1nb-3) breaks the physical barrier and reprograms the immunosuppressive microenvironment by interfering with the pathological remodeling of collagen fibers in the tumor matrix; (2) after the optimized conformation of the CD80 component is precisely delivered to the tumor site, it achieves multidimensional T cell activation by activating the CD28 co-stimulatory signal and blocking the CTLA-4 / PD-L1 dual checkpoint. It is particularly noteworthy that the DDR1 antibody component can enrich CD80 in the tumor tissue by means of its targeting, thereby improving the local immune activation effect while effectively avoiding the potential risks brought about by systemic immune activation.

[0273] DDR1nb-3-Fc-mCD80 sequence: SEQ ID NO:30

[0274] Plasmid extraction

[0275] Take 100 μl of glycerol bacteria frozen at -80℃ and transfer it to LB medium containing 150 ml of ampicillin. Incubate overnight at 37℃ and 220 rpm. Collect OD. 600 Centrifuge the bacterial culture at 0.8–0.9 μL at 8000 rpm for 3 min and discard the supernatant. Resuspend the bacterial cells in 2 ml of sterile water and combine them in a 50 ml centrifuge tube. Centrifuge at 8000 rpm for 3 min and discard the supernatant. Perform plasmid extraction according to the plasmid extraction kit protocol, using one reagent (i.e., 8 ml of P1, P2, and P4 reagents) per 150 ml of bacterial culture. Filter the obtained plasmid solution through a 0.22 μm filter membrane. Quantify 2 μl using Nanodrop and aliquot into sterile 1.5 ml EP tubes and store at -20 °C.

[0276] 2.293F protein expression

[0277] 293F cells were cultured at 36–38°C with 80%–85% CO2 and shaking at a speed of 115–125 rpm (shaking amplitude 50 mm). The cell density was 3.0 × 10⁻⁶ cells / year. 6 When cells / ml and cell viability ≥95%, prepare for transfection. Add an appropriate amount of plasmid to 293F Hi-exp medium, mix gently, and the plasmid dilution volume should be 5% of the total volume. Add an appropriate amount of transfection reagent to 293F Hi-exp medium, mix gently, and the transfection reagent dilution volume should be 5% of the total volume. Add the transfection reagent dilution to the plasmid dilution, mix gently, and incubate at room temperature for 10–15 min to allow the plasmid-transfection reagent complex to fully react and form. Slowly add the incubated plasmid-transfection reagent complex to the cultured cells to be transfected, gently shaking the flask while adding. 16–22 h after transfection, add 5% (v / v) 293F Hi-exp feed to the flask, gently shaking the flask while adding. Return the flask to the incubator for further culture. During transient expression, sample the supernatant daily to check the glucose concentration, maintaining a glucose concentration above 4 g / L. On day 7 after transfection, collect the cell supernatant for protein purification.

[0278] 3. Protein purification (protein A column)

[0279] All containers used for solution preparation were thoroughly rinsed and then washed with pure water. Buffer A: Prepare a 1×PBS solution by dissolving PBS powder in pure water, filter the solution through a 0.22 μm aqueous membrane, and sonicate to remove air bubbles. Buffer B: Prepare a 0.1 M glycine solution, adjust the pH to 3.2 with concentrated hydrochloric acid, filter the solution through a 0.22 μm aqueous membrane, and sonicate to remove air bubbles. Filter pure water through a 0.22 μm aqueous membrane and sonicate to remove air bubbles. Dilute anhydrous ethanol (pure) with pure water to a 20% ethanol solution and sonicate to remove air bubbles. Prepare a 1×TBS solution, filter the solution through a 0.22 μm aqueous membrane, and sonicate to remove air bubbles. Inject the protein solution into a mobile phase of PBS, elute with a 0.1 M pH 3.2 glycine-hydrochloric acid buffer, and collect the purified protein solution. The obtained protein solution was adjusted to pH 7 with pH 8.8 Tris-HCl solution, concentrated using an ultrafiltration tube with a molecular weight of 30 kD, and repeatedly concentrated with PBS to replace the solvent.

[0280] 4. ELISA Affinity Test

[0281] Prepare CBS carbonate coating buffer (Na₂CO₃ 1.59g, NaHCO₃ 2.93g, add sterile water to 1L; pH 9.6). Dilute the required antigen to a final concentration of 1μg / ml using CBS coating buffer and coat the MaxiSorp-treated transparent ELISA plates overnight at 4°C (100μl / well). The next day, remove the antigen coating buffer and wash 5 times with PBST (containing 0.05% Tween 20). Add 200μl / well of 3% PBSA and block at 37°C for 2h. Remove the blocking buffer and wash 5 times with PBST. Add the assay protein and incubate at 37°C for 1h. The control wells contain PBS (these wells are also coated with antigen; use PBS instead of the supernatant for incubation). Repeat the above steps, remove the liquid from the wells, and wash 5 times with PBST. Add 100 μl of Rabbit Anti-Camelid VHH Antibody [HRP] and mAb secondary antibody (1:10000 dilution) and incubate at room temperature for 45 min. Repeat the above procedure, removing the liquid from the wells and washing 5 times with PBST. Add 100 μl / well of TMB chromogenic buffer and incubate at 37°C for 10 min. Add 50 μl / well of stop buffer (2M HCl) and read the OD values ​​from the wells using a microplate reader. 450 value.

[0282] 5. Detection of immune cell killing ability

[0283] E0771 tumor cells in logarithmic growth phase were digested with trypsin and their cell density was adjusted to 4 × 10⁻⁶. 5 Cells were seeded at a density of 100 μl / ml in 96-well plates. After 24 hours, spleen tissue from 8-week-old C57BL / 6 mice was collected, homogenized into single cells, and passed through a 70 μm cell sieve. The mouse spleen single-cell suspension was washed twice with PBS, and then 5 times the volume of 1× erythrocyte lysis buffer was added. The mixture was pipetted and incubated for 3 min at room temperature. The cells were then centrifuged at 350 g, 4 °C for 5 min. The cell pellet was collected, and the cell concentration was adjusted to 2×10⁻⁶ cells / ml. 6 100 μl of spleen lymphocyte suspension was added to each well of a 96-well plate containing E0771 cells. DDR1nb-3-Fc, Fc-mCD80, or DDR1nb-3-Fc-mCD80 was then added to each well, with a final concentration of 10 μg / ml. After co-culturing at 37°C for 48 h, the supernatant and the suspended lymphocytes in the supernatant were discarded. 100 μl of complete culture medium and 10 μl of CCK8 assay reagent were added to each well, and the plate was incubated at 37°C for 4 h. The absorbance was measured at 450 nm.

[0284] 6. Apoptosis detection

[0285] Expand the experimental system to 6-well plates. E0771 tumor cells in logarithmic growth phase were seeded into each well (2 ml). After 24 h of culture, a suspension of cleaved spleen lymphocytes was added to each well, followed by the addition of DDR1nb-3-Fc, Fc-mCD80, or DDR1nb-3-Fc-mCD80, to a final concentration of 10 μg / ml. After co-culturing at 37℃ for 48 h, the supernatant and the suspended lymphocytes were discarded. Cells were washed twice with pre-chilled PBS buffer (4℃). Cells were digested with 0.3 ml of EDTA-free trypsin solution in each well, and the cell suspension was collected. The cells were resuspended in 1× binding buffer to adjust the cell concentration to 1×10⁻⁶ cells / well. 7 Cells / ml. Take 100 μl of cell suspension, add 5 μl of FITC Annexin V and 5 μl of PI, gently vortex to mix, and incubate at room temperature in the dark for 15 min. Add 200 μl of 1× binding buffer to each tube and perform flow cytometry analysis.

[0286] 7. Detection of immune cell migration ability

[0287] Collect the culture supernatant of E0771 cells after 48 h of culture, centrifuge at 350×g for 5 min, and the supernatant is the conditioned medium for E0771 tumor cells. Incubate this conditioned medium with DDR1nb-3-Fc, Fc-mCD80, or DDR1nb-3-Fc-mCD80 at a final concentration of 10 μg / ml at 37℃ for 1 h. After incubation, place the conditioned medium in the lower chamber of an 8.0 μm pore size transwell chamber, and add 5×10⁻⁶ ppm of the supernatant to the upper chamber. 6 Single-cell suspensions of spleen cells from C57BL / 6 mice after erythropoiesis were cultured at 37°C for 2 hours, and the number of spleen lymphocytes migrating in the lower chamber was quantitatively counted by flow cytometry.

[0288] 8. Preparation of tumor single-cell suspension

[0289] 15 ml of 10× Collagenase IV (10 mg / ml) and 22.5 ml of DNase I solution (1 mg / ml) were added to 112.5 ml of RPMI-1640 medium and vortexed to prepare 1× single-cell digestion solution. 0.6 mg of tumor tissue was excised into 5 ml centrifuge tubes and thoroughly minced to <2 mm. 4 ml of 1× single-cell digestion solution was added to each tube for single-cell separation. The tubes were incubated in a 37°C water bath for 20–30 min, shaking occasionally every 5 min. After digestion, complete culture medium was added to each tube to stop the digestion. The tissue was passed through a 70 μm sieve and ground using the rubber end of a syringe. The tissue was then transferred to a 50 ml centrifuge tube and rinsed with 1 ml of RPMI-1640 medium (+5% FBS) (when grinding the tumor, the cell filter was rinsed multiple times with culture medium; grinding continued until maximum dissociation of the tumor tissue was achieved). Centrifuge at 350×g for 5 min, and resuspend the cell pellet in complete culture medium. Add 1× red blood cell lysis buffer to resuspend the cell pellet, incubate at room temperature for 3 min, centrifuge at 350×g for 10 min, remove the supernatant, and the cell pellet is obtained. Add complete culture medium to each well to resuspend the cell pellet.

[0290] 9. Experimental Results

[0291] 9.1 Construction and characterization of the DDR1nb-3-Fc-mCD80 fusion protein

[0292] In previous research, the inventors successfully constructed an Fc-mCD80 fusion protein expression system. This molecule is formed by fusing the human IgG1 Fc fragment with the extracellular region of mouse CD80 through gene recombination technology. The expression vector uses the pcDNA3.4 eukaryotic expression plasmid, which has a strong CMV promoter and an hIgG1 signal peptide sequence. Optimization of the Kozak sequence ensured efficient secretory expression in 293F suspension cells. After purification by Protein A affinity chromatography, the obtained Fc-mCD80 protein was verified to effectively bind to the T cell co-stimulatory receptor CD28 and the immune checkpoint molecules PD-L1 / CTLA-4, exhibiting significant anti-tumor activity. Based on previous research, this study further developed a novel DDR1nb-3-Fc-mCD80 fusion protein. This molecule introduces the DDR1nb-3 nanobody, targeting the tumor microenvironment, into the N-terminus of Fc-mCD80. While maintaining the original pcDNA3.4 vector backbone, a codon-optimized DDR1nb-3-Fc sequence was inserted at the multiple cloning site. Both vectors were expressed in 293F cells via PEI MAX-mediated transient transfection, utilizing the protein folding and glycosylation modification capabilities unique to mammalian expression systems to ensure the functional integrity of the fusion protein. Figure 21 This is a schematic diagram of the fusion protein expression plasmid.

[0293] The predicted molecular weight of the Fc-mCD80 fusion protein single strand is 50.7 kDa. Under reducing electrophoresis (SDS-PAGE) conditions, this protein migrates to the 66-90 kDa region due to glycosylation. The predicted molecular weight of the DDR1nb-3-Fc-mCD80 fusion protein single strand is 66.7 kDa. This protein migrates to the 70-100 kDa region due to glycosylation.

[0294] The affinity of the DDR1nb-3-Fc-mCD80 fusion protein for human and mouse DDR1 proteins was detected using the BLI biomembrane interferometry method. DDR1nb-3-Fc-mCD80 was immobilized using an AHC sensor at a concentration of 5 μg / ml for 80 s. The buffer was 0.02% PBST (PBS + 0.02% Tween 20). Human DDR1-ECD-his and mouse DDR1-ECD-his were diluted to 50, 25, 12.5, 6.25, 3.13, and 0 nM. Affinity was measured at equilibration (60 s), binding (180 s), and dissociation (300 s) at 25 °C. The results showed that DDR1nb-3-Fc-mCD80 has a high affinity for human DDR1-ECD protein. on 3.799×10 5 M-1S-1, K off It is 5.495×10 -4 S-1, K D It is 1.446×10 -9 M( Figure 22 A). DDR1nb-3-Fc-mCD80 and the K of mouse DDR1-ECD protein on 3.987×10 5 M-1S-1, K off It is 5.912×10 -4 S-1, K D It is 1.483×10 -9 M( Figure 22 B). ELISA affinity assays further confirmed that DDR1nb-3-Fc-mCD80 can specifically bind to human DDR1-ECD and mouse DDR1-ECD. Coating with 1 μg / ml of human DDR1-ECD-his or mouse DDR1-ECD-his and incubating with different concentrations of DDR1nb-3-Fc-mCD80, with anti-hIgG1-hrp as the secondary antibody, yielded its EC50. 50 The values ​​were 2.303 μg / ml ( Figure 22 C) and 2.655 μg / ml ( Figure 22 D).

[0295] Our team has previously verified the affinity of Fc-mCD80 for CD28, CTLA-4, and PD-L1 proteins, respectively. Building on this, we further used ELISA to detect the affinity of the DDR1nb-3-Fc-mCD80 fusion protein for Fc-tagged mouse CD28 (EC1). 50 =2.024μg / ml), his-labeled mouse CTLA-4 (EC) 50 =77.86 ng / ml) and Fc-tagged mouse PD-L1 protein (EC) 50 Affinity (=670.9 ng / ml) Figure 23 The results showed that the DDR1nb-3-Fc-mCD80 fusion protein retained the affinity of CD80 for CD28, CTLA-4, and PD-L1.

[0296] Adherent E0771 tumor cells were co-incubated with a suspension of lymphocytes extracted from the spleen of C57BL / 6 mice. PBS (solvent control), DDR1nb-3-Fc, Fc-mCD80, or DDR1nb-3-Fc-mCD80 were added to a final concentration of 10 μg / ml. After co-culturing at 37℃ for 48 h, the viability of the adherent tumor cells was assessed using the CCK8 assay. Results are as follows: Figure 5-7 As shown, DDR1nb-3-Fc, Fc-mCD80, and DDR1nb-3-Fc-mCD80 all promoted the killing effect of primary mouse spleen lymphocytes on tumor cells. This result further validates the immune-activating function of CD80 and also indicates that DDR1nb-3-Fc possesses a certain immune-activating function.

[0297] Following the above method, apoptosis detection kits and flow cytometry were used to detect the apoptosis and necrosis of tumor cells after co-incubation with primary mouse spleen lymphocytes for 30 hours. The results showed that DDR1nb-3-Fc, Fc-mCD80, and DDR1nb-3-Fc-mCD80 all significantly inhibited the survival rate of tumor cells after co-incubation with spleen lymphocytes. Among them, co-incubation with Fc-mCD80 and DDR1nb-3-Fc-mCD80 enhanced the inhibitory effect of spleen lymphocytes on tumor cells compared to co-incubation with DDR1nb-3-Fc, indicating a stronger necrosis-inducing function. This also demonstrates that Fc-mCD80 has a stronger anti-tumor immunomodulatory effect. The DDR1nb-3-Fc-mCD80 fusion protein constructed in this study retained the potent immune-activating effect of Fc-mCD80. Co-incubation with DDR1nb-3-Fc, Fc-mCD80, and DDR1nb-3-Fc-mCD80 failed to promote tumor cell apoptosis, and all three groups showed a reduced proportion of early apoptosis compared to the control group. This further indicates that the inhibitory effect of splenic lymphocytes promoted by DDR1nb-3-Fc, Fc-mCD80, and DDR1nb-3-Fc-mCD80 on E0771 tumor cells is not achieved through an apoptosis-promoting pathway.

[0298] Studies have reported that the DDR1-ECD monoclonal antibody promotes the migration of immune cells. Therefore, this study further analyzed whether the fusion protein also promotes the migration of immune cells. Conditioned culture medium from E0771 tumor cells was collected. This conditioned culture medium was then incubated with PBS (solvent control) and DDR1nb-3-Fc, Fc-mCD80, or DDR1nb-3-Fc-mCD80 at a final concentration of 10 μg / ml at 37°C for 1 h. After incubation, the conditioned culture medium was placed in the lower chamber of an 8.0 μm pore size transwell chamber, and a suspension of mouse spleen lymphocytes was added to the upper chamber. The chambers were incubated at 37°C for 2 h, and the number of migrating spleen lymphocytes in the lower chamber was quantitatively counted by flow cytometry. The results showed that DDR1nb-3-Fc, Fc-mCD80, and DDR1nb-3-Fc-mCD80 all significantly promoted the migration of mouse spleen lymphocytes into the tumor conditioned culture medium.

[0299] 9.2 In vivo targeting assay of DDR1nb-3-Fc-mCD80 fusion protein

[0300] To further investigate the in vivo tumor-targeting activity of DDR1nb-3-Fc-mCD80, Fc-mCD80 and DDR1nb-3-Fc-mCD80 were conjugated to CY7 fluorescence and intraperitoneally injected into tumor-bearing nude mice. Fluorescence distribution was observed at 12, 24, 36, and 48 hours post-administration, and the proportion of total fluorescence intensity within the tumor region to the total fluorescence intensity throughout the mouse's body was calculated. The results showed that Fc-mCD80 lacked effective tumor-targeting function. In contrast, DDR1nb-3-Fc-mCD80 significantly accumulated in the tumor region at 24 and 36 hours post-administration, significantly enhancing the tumor-targeting activity of the CD80 fusion protein.

[0301] 9.3 Detection of in vivo antitumor activity of DDR1nb-3-Fc-mCD80

[0302] A mouse model of KPC pancreatic cancer subcutaneous tumor was established, and mice were intraperitoneally injected twice weekly with equimolar amounts of hIgG1Fc (control, 1.15 × 10⁻⁶). -7 mol / kg), DDR1nb-3-Fc (10mg / kg,1.15×10 -7 mol / kg), Fc-mCD80 (1.15×10 -7 mol / kg) and DDR1nb-3-Fc-mCD80 (1.15×10) -7 (mol / kg). Mouse tumor growth curves are shown in [reference needed]. Figure 26 DDR1nb-3-Fc, Fc-mCD80, and DDR1nb-3-Fc-mCD80 all significantly inhibited tumor growth. Among them, DDR1nb-3-Fc-mCD80 had the strongest inhibitory effect on tumor growth, slightly better than DDR1nb-3-Fc and Fc-mCD80.

[0303] H&E staining was performed on mouse tumor tissues. In the control group, tumor cells exhibited irregular morphology, larger nuclei, and increased nuclear-cytoplasmic ratio, showing strong atypia. Cells were densely packed, with significant cell proliferation and relatively frequent cell division. Few immune cells were observed, and the tumor tissue lacked a significant immune response, suggesting the presence of immune escape or an immunosuppressive environment. The stroma in the control group was relatively dense, with collagen fibers arranged regularly among tumor cells, potentially limiting immune cell penetration and leading to immune suppression. In the DDR1nb-3-Fc treatment group, tumor cells were loosely packed, with cell death in some areas, uneven cell morphology, and milder nuclear atypia. Tumor cell differentiation was observed in some areas, possibly indicating that some cells had ceased proliferation. Compared to the control group, immune cell infiltration was significantly increased, especially in areas of tumor necrosis, with more pronounced infiltration of lymphocytes and neutrophils, possibly related to the immune activation response induced by treatment. The looser stroma surrounding tumor cells may facilitate immune cell penetration and enhance the anti-tumor immune response. In the Fc-mCD80 treatment group, the tumor tissue showed a looser structure and decreased tumor cell density. Fader nuclear staining suggested potentially higher levels of apoptosis or necrosis. Significant inflammatory cell infiltration was observed, possibly indicating immune activation. In the DDR1nb-3-Fc-mCD80 dual-target fusion protein treatment group, the tumor tissue showed the most pronounced tissue damage, with large areas of necrosis, loose intercellular matrix, and abundant inflammatory cell infiltration, suggesting an enhanced immune response. More pronounced apoptotic features such as nuclear fragmentation and pyknosis indicated that DDR1nb-3-Fc-mCD80 treatment induced the most significant tumor cell death.

[0304] In summary, DDR1nb-3-Fc alone had a weak direct effect on tumor tissue, showing only mild inflammatory infiltration. Fc-mCD80 treatment induced a decrease in tumor cell density, accompanied by a certain degree of inflammatory infiltration, suggesting a possible immune-activating effect. The combination therapy with DDR1nb-3-Fc-mCD80 showed the strongest effect, with tumor tissue exhibiting significant necrosis and inflammatory infiltration, suggesting that DDR1nb-3-Fc-mCD80 may inhibit tumor growth by enhancing the immune response.

[0305] 9.4 Safety analysis of the DDR1nb-3-Fc-mCD80 fusion protein

[0306] Based on H&E staining results ( Figure 27As can be seen, in the DDR1nb-3-Fc treatment group, the cardiac muscle cells of mice were arranged normally, with no obvious pathological changes. Liver tissue cells were arranged regularly, and the liver lobule structure was not significantly affected. The spleen and kidney structures were not significantly affected, and the alveolar structure was relatively intact. However, in the Fc-mCD80 treatment group, the alveolar structure in the lung tissue was significantly damaged, the alveolar walls were thickened, and the gaps between some alveoli were wide. Some alveolar areas showed signs of local damage or necrosis. Significant cellular edema and immune cell infiltration indicated that there might be a strong inflammatory response or treatment-induced damage in this area. In contrast, in the DDR1nb-3-Fc-mCD80 treatment group, compared to the Fc-mCD80 treatment group, lung tissue damage was significantly reduced, showing only a mild inflammatory response.

[0307] In vivo antitumor function assay of 9.5Fc-mCD80 and DDR1nb-3-Fc-mCD80 fusion protein

[0308] (1) Construction of a subcutaneous mouse model of gastric cancer:

[0309] Construction of a subcutaneous mouse gastric cancer MFC model: Mouse gastric cancer cells (MFC) were cultured in 10 cm cell culture dishes. After reaching the logarithmic growth phase, the cells were digested and collected by centrifugation at 1500 rpm / min. Six-week-old male 615 mice (19-23 g) were subcutaneously injected with 1 x 102 cells into the right rib area. 5 Cells per 0.1 ml of suspension; tumor volume reached 200 mm² after 1 week. 3 The subcutaneous tumor model of gastric cancer was considered to have been successfully constructed.

[0310] (2) Animal experimental treatment regimen: After the tumor-bearing animal model was successfully established, the mice were randomly divided into: Group 1: control group (administered with PBS); Group 2: experimental group (intraperitoneal injection of Fc-mCD80 at a dose of 12.2 mg / kg); Group 3: experimental group (intraperitoneal injection of DDR1nb-3-Fc-mCD80 at a dose of 15.3 mg / kg); the drugs were administered on the 1st, 3rd, 6th and 9th days of treatment, and the treatment cycle was 12 days.

[0311] (3) Animal experimental data recording and collection:

[0312] Mice body weight, tumor length and width were measured on days 1, 3, 6, 9, and 12 after drug administration, and tumor volume was calculated. Twelve days later, the mice were dissected, and tumors and major organs were removed for sample fixation and analysis. Figure 28 A is an image of the tumor dissected from the mouse at the end of the experiment; Figure 28 B represents the average tumor volume in mice during the treatment period; Figure 28C represents the body weight change curve of mice during treatment. The results showed that, compared with Fc-mCD80, the DDR1nb-3-Fc-mCD80 fusion protein effectively reduced tumor volume and inhibited angiogenesis in tumor tissue after intraperitoneal injection in gastric cancer model mice, demonstrating good anti-tumor effects and therapeutic safety.

[0313] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An anti-DDRI nanobody, characterized in that, Complementarity determining regions CDRs of a VHH chain, characterized in that the complementarity determining regions CDRs of the VHH chain consist of: a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 1 ; a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 2; a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 3; or a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 1 ; a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 2; a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 3; or a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 1 ; a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 2; a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 3; or a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 1 ; a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 2; a CDR3 having an amino acid sequence as set forth in SEQ ID NO:

3.

2. The nanobody of claim 1, wherein The framework regions FR of the Nanobody comprise FR1, FR2, FR3 and FR4 sequences, wherein the FR1 comprises the amino acid sequence X0X1QLVESGGGX 10 VX 12 X 13 GGSLX 18 LSCX b X 22 X 23 S, wherein: X0 is: absent, or selected from D or Q; X1 is selected from: V, L or D; X 10 is selected from: L or S; X 12 is selected from: H, Q, R or A; X 13 selected from: P or A; X 18 selected from: R or T; X b is: a deletion, or is A; X 22 is selected from: A, V or T; X 23 is selected from: V or A; and the length of the sequence is 24 to 26 amino acids.

3. The Nanobody of claim 2, wherein The FR2 comprises the amino acid sequence Y1Y2WY4RQAPGKEREY 14 Y 15 Y 16 wherein: Y1 is selected from: I or M; Y2 is selected from: G or R; Y4 is selected from: F or Y; Y 14 selected from: G or L; Y 15 selected from: V or A; Y 16 is selected from: C or H; and the length of the sequence is 16 amino acids.

4. The nanobody of claim 3, wherein the FR3 comprises an amino acid sequence (Z1Z2)ADSVKGRFTISRZ 15 NZ 17 Z 18 Z 19 TZ 21 Z 22 LQMNSLKPEDTAVYZ 37 C, wherein: Z1Z2 is: collectively absent, or is NY; Z 15 selected from: M or D; Z 17 selected from: I or A; Z 18 selected from: R or K; Z 19 selected from: K or N; Z 21 selected from: L or V; Z 22 selected from: Y or S; Z 37 selected from: F or Y; and the sequence has a length of 36 or 38 amino acids.

5. The nanobody of claim 4, wherein the FR4 comprises an amino acid sequence WGW3GW5QVTW9SS, wherein: W3 is selected from: Q or P; W5 is selected from: T or A; W9 is selected from: V or I; and the sequence has a length of 11 amino acids.

6. A VHH chain of an anti-DDRI nanobody, characterized in that, the VHH chain of the Nanobody comprises a CDR1, a CDR2 and a CDR3 as defined in claim 1.

7. The VHH chain of the anti-DDRI nanobody of claim 6, wherein, the amino acid sequence of the VHH chain of the anti-DDRI Nanobody is as set forth in SEQ ID NOs: 9-18.

8. An anti-DDRI nanobody, characterized in that, the anti-DDRI Nanobody is a Nanobody against a DDRI epitope, and has a VHH chain of an anti-DDRI Nanobody as defined in claim 6.

9. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of claim 1. the polynucleotide encodes a protein selected from the group consisting of an anti-DDRI Nanobody as defined in claim 1, a VHH chain of an anti-DDRI Nanobody as defined in claim 6, or an anti-DDRI Nanobody as defined in claim 8.

10. An expression vector, characterized in that, the expression vector contains a polynucleotide as defined in claim 9.

11. A host cell, characterized in that, the host cell contains an expression vector as defined in claim 10, or has integrated into its genome a polynucleotide as defined in claim 9.

12. A method of producing an anti-DDRI nanobody, characterized in that, comprising the steps of: (a) culturing the host cell of claim 11 under conditions suitable for production of a Nanobody, thereby obtaining a culture containing the anti-DDRI Nanobody; and (b) isolating or recovering the anti-DDRI Nanobody from the culture.

13. A nanobody fusion protein, characterized in that, the Nanobody fusion protein has a structure from N-terminus to C-terminus as set forth in Formula I: Z1-Z2-L-Z3 (Formula I) wherein Z1 is a VHH chain of an anti-DDRI Nanobody as defined in claim 6; Z2 is an Fc fragment of an immunoglobulin; L is a linker sequence; and Z3 is an immunomodulatory molecule moiety.

14. An immunoconjugate, comprising, The immunoconjugate comprises: (a) a VHH chain of an anti-DDRI Nanobody according to claim 6, an anti-DDRI Nanobody according to claim 8, or a Nanobody fusion protein according to claim 13; and (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

15. Use of an anti-DDRI nanobody according to claim 8 or a nanobody fusion protein according to claim 13, characterized in that, For the preparation of (a) a reagent for detecting a DDRI molecule; (b) a medicament for treating a tumor; wherein the tumor is selected from the group consisting of gastric cancer, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, colorectal cancer, breast cancer, large intestine cancer, prostate cancer, cervical cancer, adrenal gland tumor, pancreatic cancer, bladder tumor, or a combination thereof.

16. A pharmaceutical composition comprising, Comprising: (i) an anti-DDRI Nanobody according to claim 1, a VHH chain of an anti-DDRI Nanobody according to claim 6, or an anti-DDRI Nanobody according to claim 8, a Nanobody fusion protein according to claim 13, or an immunoconjugate according to claim 14; and, (ii) a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 14, wherein For the preparation of a medicament for treating a tumor selected from the group consisting of gastric cancer, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, colorectal cancer, breast cancer, large intestine cancer, prostate cancer, cervical cancer, adrenal gland tumor, pancreatic cancer, bladder tumor, or a combination thereof.

18. A recombinant protein, characterized in that, The recombinant protein has: (i) the sequence of a VHH chain of an anti-DDRI Nanobody according to claim 6, or the sequence of a Nanobody according to claim 8, or the sequence of a Nanobody fusion protein according to claim 13; and (ii) optionally a tag sequence to assist expression and / or purification.

19. Use of a VHH chain of an anti-DDRI nanobody according to claim 6, a nanobody according to claim 8, a nanobody fusion protein according to claim 13, or an immunoconjugate according to claim 14, characterized in that, For use in the preparation of a medicament, a reagent, an assay plate, or a kit; wherein the reagent, assay plate, or kit is for detecting a DDRI protein in a sample; wherein the medicament is for treating or preventing a tumor that is positive for DDRI.

20. A non-disease diagnostic method of detecting DDRI protein in a sample, comprising, Comprising the steps of: (1) contacting a sample with a Nanobody according to claim 8 or a Nanobody fusion protein according to claim 13; (2) detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of a DDRI protein in the sample.

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