Anti-DDR2 nanobody and its use

Abstract

The present invention relates to nanobodies that bind to DDR2 and various derivatives of the nanobodies, as well as pharmaceutical compositions containing these antibodies and derivatives, and their pharmaceutical uses. The anti-DDR2 nanobodies have high binding activity and can be used for the diagnosis, prevention, and / or treatment of diseases mediated by the abnormal expression of DDR2 (e.g., cancer and inflammatory diseases), and can also be used for imaging of cells expressing DDR2, for example, for biopsy navigation and intraoperative navigation.

Description

[Technical Field]

[0001] This invention relates to anti-DDR2 antibodies. More specifically, this invention relates to nanobodies that bind to DDR2 and various derivatives of said nanobodies. This invention also relates to pharmaceutical compositions comprising these antibodies and derivatives, and their pharmaceutical applications. [Background technology]

[0002] The discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase (RTK) that utilizes extracellular matrix collagen as its ligand. In addition to its kinase function, DDR2 activates β1-integrin to promote cell adhesion. DDR2's unique functions include mediating signal transduction from the extracellular matrix to the cell, ensuring a balanced regulation of the extracellular matrix, and participating in the regulation of cell growth, differentiation, and metabolism. Activation of DDR2 by extracellular matrix collagen is essential for normal development and tissue homeostasis; however, abnormal activation of these receptors after damage or disease is detrimental.

[0003] The role of DDR2 in regulating the extracellular matrix suggests that dysregulation of its signaling can lead to tumorigenesis, including invasion and metastasis. DDR2 has been reported to be highly expressed at the anterior margin of many invasive breast tumor samples. In-situ hybridization in adjacent sections of human ovarian or lung cancer has shown the detection of DDR2 in peritumoral stromal cells. Therefore, DDR2 is considered to be crucial for metastasis in many cancer cells (such as breast cancer cells) and is also thought to be an important target in many other cancers (such as ovarian cancer, lung cancer, head and neck cancer, and pancreatic cancer).

[0004] Furthermore, DDR2 is considered an important target in inflammation (e.g., arthritis such as osteoarthritis and rheumatoid arthritis) and fibrosis (such as pulmonary fibrosis, liver cirrhosis, renal fibrosis, or cutaneous fibrosis). DDR2 is primarily expressed in interstitial cells such as fibroblasts, myofibroblasts, and smooth muscle cells in the kidneys, skin, lungs, heart, and connective tissue. Numerous studies have shown that abnormal expression of DDR2 is associated with the progression of various diseases, including inflammation, liver fibrosis, renal fibrosis, pulmonary fibrosis, skin scarring, and atherosclerosis. Studies using mouse inflammation models have revealed elevated DDR2 expression in the knee joints of aged mice. Studies using rat models of rheumatoid arthritis have also shown elevated DDR2 expression in their synovial cells.

[0005] WO2019243431A1 discloses that in a xenograft model derived from melanoma cells, imatinib-mediated DDR2 targeting enhances the antitumor effect of BRAF inhibitors, delays the development of acquired drug resistance, and resolves targeted therapy-induced tumor fibrosis.

[0006] Therefore, developing high-affinity antibodies that target DDR2 is considered to have broad practical significance for imaging, diagnosis, prevention, and treatment of cancer, inflammation, fibrotic diseases, and other conditions. [Overview of the project]

[0007] In a first embodiment, the present invention provides a nanobody that binds to DDR2, the nanobody comprising CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 comprise or are selected from CDR1, CDR2, and CDR3 of any nanobody having the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, and SEQ ID NO: 20, or equivalent variants thereof.

[0008] In some embodiments, CDR1, CDR1 and CDR3 are defined by one of the following definition schemes: IMGT, Kabat, Chothia, Contact, or Martin.

[0009] In some embodiments, the nanobody includes CDR1, CDR2, and CDR3, (a) CDR1 contains or is the sequence shown in SEQ ID NO: 1 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 2 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 3 or its equivalent variant. (b) CDR1 contains or is the sequence shown in SEQ ID NO: 5 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 6 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 7 or its equivalent variant. (c) CDR1 contains or is the sequence shown in SEQ ID NO: 9 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 10 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 11 or its equivalent variant. (d) CDR1 includes or is the sequence shown in SEQ ID NO: 13 or its equivalent variant, CDR2 includes or is the sequence shown in SEQ ID NO: 14 or its equivalent variant, CDR3 includes or is the sequence shown in SEQ ID NO: 15 or its equivalent variant, (e) CDR1 contains or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 contains or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 contains or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0010] In some embodiments, the nanobody comprises or is selected from any of the sequences of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20 and their respective equivalent variants.

[0011] In another embodiment, the present invention provides a polynucleotide comprising a polynucleotide encoding any of the above-described antibodies. In another embodiment, the present invention also provides a vector comprising the polynucleotide. In another embodiment, the present invention also provides a non-human host cell comprising the vector. The present invention also provides a cell line that produces the antibodies of the present invention, and a method for producing antibodies by culturing the antibody-producing cell line.

[0012] In a second embodiment, the present invention provides a chimeric antigen receptor comprising an extracellular domain capable of binding to an antigen, a transmembrane domain, and an intracellular domain, wherein the extracellular domain capable of binding to the antigen comprises any nanobody described in the first embodiment. In another embodiment, the present invention provides a polynucleotide comprising a polynucleotide encoding the chimeric antigen receptor. In another embodiment, the present invention provides a vector comprising the above polynucleotide. In another embodiment, the present invention provides modified immune cells expressing the chimeric antigen receptor.

[0013] In a third aspect, the present invention provides a bispecific antibody comprising a first binding portion that binds to a first antigen and a second binding portion that binds to a second antigen, wherein the first binding portion comprises any nanobody described in the first aspect.

[0014] In a fourth aspect, the present invention provides an antibody-drug conjugate comprising an antibody targeting DDR2, a drug, and a linker connecting the nanobody and the drug, wherein the antibody targeting DDR2 comprises or is any of the nanobody described in the first aspect.

[0015] In a fifth aspect, the present invention provides an siRNA conjugate for targeting DDR2-positive cells, comprising (i) an antibody targeting DDR2 which comprises or is a nanobody as described in the first aspect, (ii) an siRNA that inhibits the expression of survival genes in DDR2-positive cells, and (iii) a linker located between the nanobody and the siRNA.

[0016] In a sixth aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any of the above-mentioned antibodies, polynucleotides, vectors, host cells, immune cells and / or complexes, and a pharmaceutically acceptable carrier.

[0017] In a seventh embodiment, the present invention provides the use of any of the above antibodies, polynucleotides, vectors, host cells, immune cells, or complexes in the manufacture of a drug for treating and / or preventing diseases mediated by abnormal expression of DDR2.

[0018] In the eighth embodiment, the present invention provides any of the above-mentioned antibodies, polynucleotides, vectors, host cells, immune cells, or complexes for treating and / or preventing diseases mediated by abnormal expression of DDR2.

[0019] In a ninth embodiment, the present invention provides a method for treating and / or preventing a disease mediated by abnormal expression of DDR2, comprising administering an effective amount of any of the above-mentioned antibodies, polynucleotides, vectors, host cells, immune cells, or complexes to a target.

[0020] In a tenth aspect, the present invention provides an imaging and / or diagnostic reagent comprising any nanobody described in the first aspect and a detectable label linked to the nanobody.

[0021] In an eleventh aspect, the present invention provides the use of an imaging and / or diagnostic reagent according to the tenth aspect in the manufacture of a kit for diagnosing a disease mediated by abnormal expression of DDR2 in a subject.

[0022] In a twelfth embodiment, the present invention provides an imaging and / or diagnostic reagent according to the tenth embodiment for diagnosing a disease mediated by abnormal expression of DDR2 in a subject.

[0023] In a thirteenth embodiment, the present invention provides a method for diagnosing a disease mediated by abnormal expression of DDR2 in a subject, comprising administering an effective amount of the imaging and / or diagnostic reagent described in the tenth embodiment to the subject. In some embodiments, the method comprises administering an effective amount of the imaging and / or diagnostic reagent described in the tenth embodiment to the subject, detecting and reading a signal generated by the imaging and / or diagnostic reagent, and determining from the intensity of the signal that the subject is suffering from or at risk of suffering from a disease mediated by abnormal expression of DDR2.

[0024] In a fourteenth embodiment, the present invention provides a method for treating a disease mediated by abnormal expression of DDR2 in a subject, the method comprising: (a) administering an effective amount of any of the above imaging and / or diagnostic reagents to the subject; (b) detecting and reading a signal generated by the imaging and / or diagnostic reagent; (c) determining whether the signal exceeds a predetermined threshold, and if the signal exceeds the predetermined threshold, determining that the subject is suffering from a disease mediated by abnormal expression of DDR2, preferably the threshold being the median level derived from a subject not suffering from the disease; and (e) administering to the subject determined to be suffering from the disease a therapy that reduces the activity and / or expression of DDR2, for example, an anti-cancer therapy or an anti-inflammatory therapy (for example, an anti-fibrotic therapy). In a preferred embodiment, the anti-fibrotic therapy is pirfenidone and / or nintedanib.

[0025] In the relevant embodiments described above, the diseases mediated by the abnormal expression of DDR2 include cancer and inflammatory diseases. In some embodiments, the cancers include, but are not limited to, melanoma, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, uterine cancer, cervical cancer, bladder cancer, stomach cancer, and skin cancer. In some embodiments, the inflammatory diseases include, but are not limited to, rheumatoid arthritis, osteoarthritis, skin scarring, atherosclerosis, retinal vascular disease, and fibrotic diseases. The fibrotic diseases include, but are not limited to, pulmonary fibrosis (especially idiopathic pulmonary fibrosis), hepatic fibrosis, cirrhosis, cutaneous fibrosis, renal fibrosis, pancreatic fibrosis, systemic sclerosis, cardiac fibrosis, and macular degeneration.

[0026] The anti-DDR2 nanobodies of the present invention have high binding activity and can be used in the diagnosis, prevention and / or treatment of diseases mediated by abnormal expression of DDR2 (e.g., cancer and inflammatory diseases), and can also be used for imaging of DDR2-expressing cells, such as biopsy navigation and intraoperative navigation.

[0027] Other aspects and advantages of the present invention are expected to become apparent from the following detailed description relating to the present invention. [Brief explanation of the drawing]

[0028] [Figure 1] Figure 1 shows the SDS-PAGE results for the expression and purification of DDR2 nanobodies. [Figure 2] Figure 2 shows the ELISA validation of the DDR2 nanobody. [Figure 3] Figure 3 shows the flow cytometry results for DDR2 nanobodies. [Figure 4] Figure 4 shows the immunofluorescence results of DDR2 nanobodies. [Figure 5] Figure 5 shows the affinity constants of DDR2 nanobodies to the extracellular segment of the DDR2 antigen. [Figure 6] Figure 6 shows in vitro imaging results of DDR2 nanobodies in mouse lung tissue. [Figure 7] Figure 7 shows the in vitro imaging results of DDR2 nanobodies in human lung tissue. [Figure 8] Figure 8 shows in vitro imaging results of DDR2 nanobodies in pig and rabbit lung tissue. [Figure 9] Figure 9 shows the in vitro imaging results of DDR2 nanobodies in the retina. [Figure 10] Figure 10 shows the in vivo imaging results of the DDR2 nanobody. [Figure 11] Figure 11 shows a Western blot (WB) of the inhibition of the DDR2 protein phosphorylation induction by collagen I by nanobody 1A12. [Figure 12] Figure 12 shows the statistics of inhibition by nanobody 1A12 against the induction of DDR2 protein phosphorylation by collagen I. [Figure 13] Figure 13 shows DNA electrophoresis for enzymatic cleavage identification of plasmids expressing anti-DDR2 nanobodies. [Figure 14] Figure 14 shows the SDS-PAGE electrophoresis staining of purified anti-DDR2 nanobodies. [Figure 15] Figure 15 shows a Western blot of the purified anti-DDR2 nanobodies. [Figure 16] Figure 16 shows flow cytometry measurements of the activity of anti-DDR2 nanobodies. [Figure 17] Figure 17 is a Western blot showing the inhibition of collagen-induced phosphorylation of DDR2 protein by anti-DDR2 nanobodies. [Figure 18] Figure 18 is a schematic diagram of a CAR molecule constructed with a DDR2 nanobody 1A12 sequence. [Figure 19] Figure 19 shows the DDR2-positive 293T phagocytosis rate of adenovirus-infected mouse macrophages packaged with CAR molecules constructed in 1A12. [Figure 20] Figure 20 shows the localization of CAR-M constructed in 1A12. [Figure 21] Figure 21 shows targeted phagocytosis of CAR-M constructed with 1A12 against 293T-DDR2. [Figure 22] Figure 22 shows the in vivo therapeutic effect of CAR-M constructed with 1A12. [Figure 23] Figure 23 shows 68Ga PET-CT imaging of a BLM-modeled mouse at day 15. [Modes for carrying out the invention]

[0029] (definition) In this invention, “approximately” means that the numerical value is within the allowable error range of a specific value measured by a person skilled in the art, and that the numerical value depends to some extent on how it was measured or measured (i.e., the limitations of the measurement system). For example, in each implementation in the art, “approximately” may mean a standard deviation of 1 or more than 1. Or, “approximately” or “substantially including” may mean a range of up to 20%. In particular for biological systems or processes, the term may mean up to one digit or up to five times the numerical value. Unless otherwise specified, whereever a specific value appears in this specification and the claims, “approximately” or “substantially including” should be understood to mean that it is within the allowable error range of that specific value.

[0030] In this specification, compositions or methods described as "including" one or more elements or steps are open-ended, meaning that the elements or steps are essential, but other elements or steps may be added to the scope of the composition or method. Any composition or method described as "including" one or more elements or steps is also described as a corresponding, more limited composition or method "substantially consisting of the elements or steps," meaning that the composition or method includes the essential elements or steps, and may include additional elements or steps that are basic and novel in nature but do not have an essential effect on the composition or method.

[0031] As used herein, the term “antibody” refers to any form of antibody that exhibits a desired physiological activity (e.g., by inhibiting the binding of a ligand to its receptor or by inhibiting ligand-induced receptor signaling). “Antibody fragment” and “antigen-binding fragment” generally refer to antigen-binding fragments and antibody analogs of an antibody that include at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. In some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.

[0032] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, wherein, with the exception of naturally occurring variants that may exist in small amounts, each antibody constituting the population is identical. Monoclonal antibodies possess very high specificity and can target a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations which generally contain multiple different antibodies targeting multiple different determinants (epitopes), each monoclonal antibody targets only a single determinant on the antigen. The modifier "monoclonal" describes the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be understood as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies used in this invention may be produced by hybridoma or recombinant DNA methods.

[0033] Monoclonal antibodies may include "chimeric" antibodies, humanized antibodies, or fully human antibodies. In some embodiments, the antibody constitutes part of a larger biomolecule, such as a fusion protein or antibody-drug conjugate. The antibody fragment retains at least some of the binding specificity of the parent antibody. Generally, when activity is expressed in moles, the antibody fragment retains at least 10% of the parent's binding activity. Preferably, the antibody fragment retains at least 20%, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, or more of the parent antibody's binding affinity to the target.

[0034] As used herein, the term “heavy chain antibody” refers to an antibody consisting solely of a heavy chain, lacking a light chain, and comprising two constant regions (CH2 and CH3), one hinge region, and one heavy chain variable region (VHH). Examples include, but are not limited to, naturally occurring heavy chain antibodies, naturally occurring light chain-deficient antibodies, heavy chain antibodies derived from conventional four-chain antibodies, and artificial antibodies. Heavy chain antibodies may originate from species of the Camelidae family, such as antibodies produced in camels, llamas, dromedaries, alpacas, and draft horses. Other species outside the Camelidae family can also naturally produce light chain-deficient heavy chain antibodies, and such heavy chain antibodies are within the scope of this invention.

[0035] As used herein, the term "nanobody" refers to a single-domain antibody consisting solely of the variable domain of a heavy chain antibody, obtained by cloning the variable domain of a heavy chain antibody. It is also called a VHH (Variable domain of heavy chain antibody) or monodomain antibody, and is the smallest functional antigen-binding fragment. Nanobodies recognize antigens with high specificity and affinity similar to IgG antibodies, but their smaller size (approximately 15 kD) allows them to penetrate tumor tissue more effectively. Furthermore, nanobodies are resistant to harsh pH levels, thermal denaturation, protein hydrolysis, solvents, and detergents. They can be expressed and produced with high yield and high solubility.

[0036] As used herein, the term "humanized antibody" refers to an antibody that includes a CDR derived from an antibody of a non-human mammal, along with the framework region (FR) and constant region of a human antibody.

[0037] An "equivalent variant" of an antibody or polypeptide refers to an antibody or polypeptide that has a certain degree of homology or sequence identity with respect to the amino acid sequence of the antibody or polypeptide. In some embodiments, the sequence identity is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%. In some embodiments, the equivalent variant has one, two, three, four, or five additions, deletions, substitutions, or combinations thereof compared to the reference antibody or polypeptide. In some embodiments, the equivalent variant of an antibody or polypeptide retains the activity (e.g., epitope binding) or structure (e.g., salt bridges) of the reference sequence.

[0038] As used herein, the term "variant" of a sequence refers to a sequence that differs from the target sequence by one or more amino acid residues but retains the physiological activity of the target molecule.

[0039] As used herein, the term "% identity" between two sequences refers to a function of the number of identical positions in those sequences (i.e., % homology = number of identical positions / total number of positions × 100), where the number of gaps and the length of each gap are taken into consideration, and the gaps are introduced to optimally align the two sequences. Sequence comparison and determination of % identity between two sequences may be performed by mathematical algorithms.

[0040] "Conservative substitution" refers to amino acid substitutions known to those skilled in the art, and such substitutions generally do not alter the physiological activity of the target molecule. Generally, it is common knowledge among those skilled in the art that a single amino acid substitution in a non-essential region of a polypeptide substantially does not alter its physiological activity or does not essentially alter it. "Does not alter or does not essentially alter" means that, when measured by the same or similar method, one or more aspects differ from the comparison subject by about 20% or less, about 15% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less.

[0041] CDR3 is generally considered to play a more important role than other CDRs in antigen recognition; therefore, when substitution is performed, it is preferable in the present invention to perform conservative substitutions for CDRs other than CDR3. In some embodiments, CDR3 is not substituted. Preferred amino acid substitutions include, but are not limited to, (1) substitutions that reduce the hydrolysis sensitivity of the protein, (2) substitutions that reduce the oxidation sensitivity, (3) substitutions that alter the binding affinity of the formed protein complex, and (4) substitutions that provide or modify other physical and chemical or functional properties of these analogs. The analogs may include a variety of sequence mutations other than naturally occurring peptide sequences. For example, one or more amino acid substitutions (preferably conservative amino acid substitutions) may be performed in naturally occurring sequences (preferably in polypeptide portions other than regions that form intermolecular contacts). Conservative amino acid substitutions should not essentially alter the structural features of the parent sequence (for example, it is preferable that the amino acid substitutions are not of a type that tends to disrupt helices present in the parent sequence or that has been characterized to disrupt other secondary structures of the parent sequence).

[0042] The binding domain of the antibody or its antigen-binding fragment of the present invention can generally be expected to have a signal peptide consisting of 15 to 30 amino acids located at the N-terminus of the secreted protein. When the signal peptide sequence is synthesized, it is recognized by a signal recognition particle (SRP), and protein synthesis is temporarily paused or relaxed. When the signal recognition particle carries the ribosome to the endoplasmic reticulum, protein synthesis resumes. Guided by the signal peptide, the newly synthesized protein enters the endoplasmic reticulum lumen, but the signal peptide sequence is cleaved by the signal peptidase. Furthermore, if a transport termination sequence is present at the C-terminus of the nascent peptide chain, such as in ovalbum which contains an internal signal peptide, it may not be cleaved by the signal peptidase. Neither the precursor nor the mature form undergoes a process of cleavage by the signal peptidase.

[0043] According to the present invention, the term “binding” preferably relates to specific binding. Where ligand-receptor, antibody-antigen, or other binding pairs are mentioned, “specific” binding refers to determining whether there is a binding reaction of the protein to a heterogeneous population of proteins and / or other biochemical reagents. Thus, under given conditions, a particular ligand or antigen binds to a particular receptor or antibody and does not bind in significant amounts to other proteins present in the sample. “Specific binding” means that the monoclonal antibody or its antigen-binding fragment of the present invention can specifically interact with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or more amino acids of each human target molecule. The “specific binding” of an antibody is mainly evaluated by two parameters: a qualitative parameter (binding epitope or antibody binding site) and a quantitative parameter (binding affinity or binding strength). The binding epitope of an antibody may be measured by FACS, epitope mapping, mass spectrometry, or peptide ELISA. The Biacore method and / or ELISA method can measure the binding strength of an antibody to a specific epitope. Generally, the signal-to-noise ratio is used as a representative method for calculating binding specificity. In such a signal-to-noise ratio, the signal represents the binding strength of the antibody to the target epitope, and the noise represents the binding strength of the antibody to other non-target epitopes. Preferably, if the signal-to-noise ratio for the target epitope is about 50, the antibody being evaluated can be considered to bind to the target epitope in a specific manner, i.e., to "specifically bind". If an antigen-binding protein (including an antibody) binds to the antigen with a high binding affinity determined from the value of its affinity constant (KD), then the antigen-binding protein (including an antibody) "specifically binds" to the antigen. In some embodiments, the affinity constant KD is 10 -9 Less than M. As used herein, the term "KD" refers to the affinity constant of a particular antibody-antigen interaction.

[0044] As used herein, the terms “patient” or “subject” refer to any organism that is administered, or may be administered, an antibody, a derivative thereof, or a pharmaceutical composition provided for experimental, diagnostic, preventive, cosmetic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the subject is human. In some embodiments, the subject has or is susceptible to one or more diseases or conditions. The patient may exhibit one or more symptoms of a disease or condition, or may be diagnosed with already having one or more diseases or conditions. In some embodiments, the patient has received, or has received, a specific therapy for diagnosing and / or treating this type of disease, disease, or condition.

[0045] In this invention, the term "treatment" refers to therapies and preventive measures that prevent or mitigate undesirable physiological changes or the onset or progression of a disease in a subject. Favorable or expected clinical effects include, but are not limited to, symptom reduction, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or mitigation of disease progression, reduction or mitigation of the disease state, and partial or complete cure of the disease. Furthermore, "treatment" may also refer to an extension of survival time compared to not receiving treatment. Subjects to be treated include subjects suffering from the disease or disease, subjects at risk of suffering from the disease or disease, or subjects to be prevented from suffering from the disease or disease.

[0046] As used herein, the term “prevention” includes preventing or mitigating the onset of clinically significant disease progression, or preventing or mitigating the onset of a preclinically significant disease stage in an at-risk individual. This includes preventive treatment for human individuals at risk of disease progression.

[0047] When “administration,” “treatment,” or “prevention” is used in reference to animals, humans, experimental subjects, cells, tissues, organs, or biofluids, it refers to the contact of exogenous drugs, therapeutic agents, diagnostic agents, or compositions with animals, humans, subjects, cells, tissues, organs, or biofluids. “Administration” and “treatment” may also refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, or experimental methods. Cell therapy includes contacting reagents with cells, or contacting reagents with fluids, wherein the fluids come into contact with cells. “Administration” and “treatment” also mean in vitro and ex vivo treatments performed on cells, for example, with reagents, diagnostic agents, conjugate compositions, or with other cells.

[0048] As used herein, the terms “therapeutic dose” or “effective dose” refer to the amount of the antibody or derivative thereof of the present invention administered alone or in combination with another therapeutic agent to cells, tissues, or a person to be treated that effectively prevents or alleviates the disease or condition to be treated. The therapeutic dose further refers to an amount sufficient to alleviate the symptoms, and the alleviation of symptoms means, for example, treating, curing, preventing or alleviating the relevant medical condition, or improving the treatment rate, cure rate, prevention rate or alleviation rate for the condition. When an active ingredient is administered alone to an individual, the therapeutic dose refers to that single ingredient. When administered in combination, the therapeutic dose refers to the amount of the combined active ingredients that produce a therapeutic effect, regardless of whether it is administered concomitantly, sequentially, or simultaneously. The reduction of symptoms by the therapeutic dose is generally at least 10%, generally at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

[0049] As used herein, the term “pharmaceutically acceptable carrier” includes materials that, when combined with the active ingredient of a composition, preserve the physiological activity of the ingredient and do not cause a destructive reaction against the target immune system. These carriers may include stabilizers, preservatives, salts, or sugar complexes or crystals. “pharmaceutically acceptable” means molecules and components that do not cause an allergic reaction or similar undesirable reaction when administered to the human body.

[0050] As used herein, the term “chimeric antigen receptor (CAR)” refers to a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from the polypeptide of the induced extracellular domain, and at least one intracellular domain. “Chimeric antigen receptors (CARs)” may also be called “chimeric receptors,” “T-body,” or “chimeric immune receptors (CIRs).” “Antigen-binding extracellular domain” refers to any oligopeptide or polypeptide capable of binding to a specific antigen. “Intracellular domain” refers to any oligopeptide or polypeptide known to function as a domain that transmits signals in a cell, leading to the activation or inhibition of biological processes.

[0051] As used herein, the terms “antibody-drug conjugate” or “ADC” refer to an antibody covalently bound to a therapeutic active substance or active pharmaceutical ingredient, thereby enabling the therapeutic active substance or active pharmaceutical ingredient to exert its pharmacological function by targeting the antibody’s binding target. The therapeutic active substance or active pharmaceutical ingredient may also be a cytotoxin capable of killing the cells targeted by the ADC. Covalent binding of the therapeutic active substance, active pharmaceutical ingredient, or cytotoxin may be carried out in a non-site-specific manner by a standard chemical linker that binds the payload to a lysine or cysteine ​​residue, or, preferably, in a site-specific manner by a complex that allows for complete control of the complex site and the drug-antibody ratio (DAR) of the resulting ADC.

[0052] The term "siRNA" refers to a double-stranded RNA molecule with 20–25 nucleotides in length, containing two substantially complementary, antiparallel nucleic acid strands. These two strands are named the sense strand and antisense strand in relation to the target RNA. The siRNA moiety triggers the degradation of the target RNA (e.g., mRNA) through a mechanism called post-transcriptional gene silencing (referred to here as RNA interference or RNAi). Generally, the nucleotides in each strand of the siRNA moiety are mostly ribonucleotides, but each or both of the two strands may further contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. The siRNA moiety may also contain chemically modified ribonucleotides or essential modifications at multiple nucleotides. These modifications may include all types of modifications disclosed herein or known in the art. Any such modifications are intended to fall within the definition of "siRNA".

[0053] The terms "survival genes" and "essential genes" are interchangeable to refer to genes that are essential for the survival or proliferation of a particular cell. Even a single functional deletion in these genes can cause the cell to exhibit a lethal phenotype, preventing it from surviving or proliferating. Typical survival genes are genes that encode important proteases in the transcription, replication, and expression processes of genetic material, and include, but are not limited to, POLR2A (DNA-directed RNA polymerase II subunit RPB1, the largest subunit of RNA polymerase II, capable of synthesizing mRNA precursors and many functional non-coding RNAs, and forming the polymerase active site with the second largest subunit), POLR2B (DNA-directed RNA polymerase II subunit RPB2, the second largest subunit of RNA polymerase II, forming the polymerase active site with the largest subunit), DKC1 (H / ACA ribonucleoprotein complex subunit DKC1, a ribonucleoprotein complex subunit), CENPE (Centromere-associated protein E), and eIF-3b (eukaryotic translation initiation factor 3 subunit b). Furthermore, the term "survival genes in DDR2-positive cells" refers to genes essential for the survival or proliferation of DDR2-positive cells.Methods for determining whether a particular gene in human cells is an essential gene are known in this field; see, for example, Wang T et al., Identification and characterization of essential genes in the human genome. Science 27 Nov 2015: Vol.350, Issue 6264, pp.1096-1101, or Vincent A. Blomen et al., Gene essentiality and synthetic lethality in haploid human cells. Science 27 Nov 2015: Vol.350, Issue 6264, pp.1092-1096.

[0054] (Anti-DDR2 nanobody) In this invention, camel-derived heavy chain antibodies are obtained by immunizing alpacas with a human DDR2 extracellular segment (its amino acid sequence is UniProtKB / Swiss-Prot:Q16832.2 aa22~aa399), and then the corresponding nanobodies are obtained.

[0055] In one aspect of the present invention, the present invention provides a nanobody that binds to DDR2, the nanobody comprising CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 comprise or are selected from CDR1, CDR2, and CDR3 of any nanobody having the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, and SEQ ID NO: 20, or equivalent variants thereof.

[0056] In some embodiments, CDR1, CDR1 and CDR3 are defined by one of the following definition schemes: IMGT, Kabat, Chothia, Contact, or Martin. In some embodiments, CDR1, CDR1 and CDR3 are defined by the IMGT definition scheme.

[0057] In some embodiments, the nanobody includes CDR1, CDR2, and CDR3, (a) CDR1 contains or is the sequence shown in SEQ ID NO: 1 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 2 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 3 or its equivalent variant. (b) CDR1 contains or is the sequence shown in SEQ ID NO: 5 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 6 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 7 or its equivalent variant. (c) CDR1 contains or is the sequence shown in SEQ ID NO: 9 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 10 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 11 or its equivalent variant. (d) CDR1 includes or is the sequence shown in SEQ ID NO: 13 or its equivalent variant, CDR2 includes or is the sequence shown in SEQ ID NO: 14 or its equivalent variant, CDR3 includes or is the sequence shown in SEQ ID NO: 15 or its equivalent variant, (e) CDR1 contains or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 contains or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 contains or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0058] In some embodiments, the nanobody comprises or is selected from any of the sequences of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20 and their respective equivalent variants.

[0059] In some embodiments, the equivalent variants of CDR1, CDR2, and CDR3 refer to those having a single amino acid substitution, deletion, or insertion compared to the reference sequence.

[0060] In some embodiments, each equivalent variant of the nanobody refers to one that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to any of the sequences of SEQ ID NOs. 4, SEQ ID NOs. 8, SEQ ID NOs. 12, SEQ ID NOs. 16, and SEQ ID NOs. 20, and has the same or equivalent CDR1, CDR2, and CDR3.

[0061] In some embodiments, the nanobody includes CDR1, CDR2, and CDR3, (a) CDR1 contains the sequence shown in SEQ ID NO: 1, CDR2 contains the sequence shown in SEQ ID NO: 2, CDR3 contains the sequence shown in SEQ ID NO: 3, (b) CDR1 contains the sequence shown in SEQ ID NO: 5, CDR2 contains the sequence shown in SEQ ID NO: 6, and CDR3 contains the sequence shown in SEQ ID NO: 7. (c) CDR1 contains the sequence shown in SEQ ID NO: 9, CDR2 contains the sequence shown in SEQ ID NO: 10, and CDR3 contains the sequence shown in SEQ ID NO: 11. (d) CDR1 contains the sequence shown in SEQ ID NO: 13, CDR2 contains the sequence shown in SEQ ID NO: 14, CDR3 contains the sequence shown in SEQ ID NO: 15, or (e) CDR1 contains the sequence shown in SEQ ID NO: 17, CDR2 contains the sequence shown in SEQ ID NO: 18, and CDR3 contains the sequence shown in SEQ ID NO: 19.

[0062] In some embodiments, the nanobody includes any sequence selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, and SEQ ID NO: 20.

[0063] In some embodiments, the nanobody includes CDR1, CDR2, and CDR3, (a) CDR1 is the sequence shown in sequence number 1, CDR2 is the sequence shown in sequence number 2, and CDR3 is the sequence shown in sequence number 3. (b) CDR1 is the sequence shown in sequence number 5, CDR2 is the sequence shown in sequence number 6, and CDR3 is the sequence shown in sequence number 7. (c) CDR1 is the sequence shown in sequence number 9, CDR2 is the sequence shown in sequence number 10, and CDR3 is the sequence shown in sequence number 11. (d) CDR1 is the sequence shown in SEQ ID NO: 13, CDR2 is the sequence shown in SEQ ID NO: 14, CDR3 is the sequence shown in SEQ ID NO: 15, or (e) CDR1 is the sequence shown in sequence number 17, CDR2 is the sequence shown in sequence number 18, and CDR3 is the sequence shown in sequence number 19.

[0064] In some embodiments, the nanobody is any sequence selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, and SEQ ID NO: 20.

[0065] In some embodiments, the substitutions described herein are conservative substitutions.

[0066] A "conservative (amino acid) substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid having a similar side chain. The families of amino acid residues having similar side chains, as defined in this field, include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferable that non-essential amino acid residues in immunoglobulin polypeptides be substituted with other amino acid residues from the same side chain family. In another embodiment, the amino acid string may be replaced with a string of a side-chain family member that has a similar structure but a different order and / or configuration.

[0067] Furthermore, it will be understood by those skilled in the art that the antibodies disclosed herein may be modified such that their amino acid sequences differ from those of the naturally occurring conjugated polypeptides from which they are derived. For example, the polypeptide or amino acid sequence derived from a given protein may be similar to the start sequence, for example, having a certain percentage of identity, such as being identical to the start sequence by 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or any two of these values.

[0068] In some embodiments, the antibody includes an amino acid sequence or one or more parts that are not generally related to the antibody. Exemplary modifications are described in more detail here. For example, the antibodies disclosed herein may include a flexible linker sequence or may be modified to add a functional moiety (e.g., polyethylene glycol (PEG), a drug, a toxin, or a label).

[0069] The antibodies, their variants, or derivatives of the present invention include modified derivatives, i.e., those in which the antibody is covalently bonded to any type of molecule, such that the covalent bond does not inhibit the binding of the antibody to the epitope. Antibodies may be modified, but are not limited to, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein cleavage by hydrolysis, or linkage with cell ligands or other proteins. Any of the various chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical lysis, acetylation, formylation, or metabolic synthesis of tunicamycin. Furthermore, antibodies may contain one or more non-classical amino acids.

[0070] In some embodiments, the antibody may be conjugated with a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, immunomodulator, drug, or PEG.

[0071] Antibodies may be combined with or fused with therapeutic agents, which may include detectable labels (e.g., radioactive labels), immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic agents or diagnostic agents, cytotoxic agents (which may be drugs or toxins), ultrasound enhancers, non-radioactive labels, combinations thereof, and other reagents of this kind known in the art.

[0072] Antibodies may be labeled detectably by binding to a chemiluminescent compound. The presence of a chemiluminescently labeled antigen-binding polypeptide is then determined by detecting whether luminescence occurs during the chemical reaction. Particularly useful examples of chemiluminescent labeling compounds include luminol, isoluminol, heat-stable acridinium esters, imidazole, acridine salts, and oxalic acid esters.

[0073] Also, fluorescent metals (for example, 152 The antibody may be detectably labeled with Eu or other lanthanide elements. These metals may be linked to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various parts of the antibody are well-known.

[0074] (Humanized antibody) In any embodiment of the present invention, the nanobody bound to DDR2 is preferably a humanized antibody.

[0075] Humanized antibodies are antibody molecules derived from non-human species antibodies that bind to a desired antigen having one or more complementarity-determining regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule. Generally, framework residues in the human framework region are substituted with corresponding residues from the antibody derived from the CDR donor to alter, and preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interaction between CDRs and framework residues to identify framework residues important for antigen binding and sequence comparison, and further to identify unnatural framework residues at specific locations. Antibodies may be humanized using various techniques known in the art, such as CDR transplantation, veneering or resurfacing, and chain shuffling.

[0076] Fully human antibodies are particularly ideal for treating human patients. Human antibodies may be manufactured using various methods known in this field, such as phage display technology using antibody libraries derived from human immunoglobulin sequences.

[0077] Furthermore, mice may be modified to produce human antibodies by using genes that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy-chain and light-chain immunoglobulin gene complexes may be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy-chain and light-chain genes, human variable regions, constant regions, and diversity regions may be introduced into mouse embryonic stem cells. By introducing human immunoglobulin gene loci by homologous recombination, the mouse heavy-chain and light-chain immunoglobulin genes can be made to lose function individually or simultaneously. In particular, homozygous deletion of the JH region inhibits the production of endogenous antibodies. Chimeric mice are obtained by amplifying the modified embryonic stem cells and microinjecting them into blastulae. Next, the chimeric mice are raised to obtain homozygous offspring that express human antibodies. The genetically modified mice are immunized with a selected antigen using a conventional method, the antigen being, for example, all or part of a desired target polypeptide. Monoclonal antibodies against the antigen may be obtained from the immunized genetically modified mice using conventional hybridoma technology. The human immunoglobulin genes present in genetically modified mice are rearranged during the B-cell differentiation process, followed by class switching and somatic mutations. Therefore, it is possible to use this technique to produce IgG, IgA, IgM, and IgE antibodies that are useful for therapy.

[0078] Fully human antibodies that recognize the selected epitopes may be generated using a technique called "guided selection." In this method, non-human monoclonal antibodies, such as mouse antibodies, are selected to guide the selection of fully human antibodies that recognize the same epitopes.

[0079] The DNA encoding the desired monoclonal antibody may be easily isolated and sequenced using standard procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the mouse antibody heavy and light chains). Isolated and subcloned hybridoma cells are used as a preferred source of this type of DNA. After isolation, the DNA may be placed into an expression vector and then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or non-immunoglobulin-producing myeloma cells. More specifically, the isolated DNA may be used to clone constant and variable region sequences for antibody production. Essentially, this requires extracting RNA from selected cells, converting it to cDNA, and PCR amplification using Ig-specific primers. As described herein, transformed cells expressing the desired antibody can be grown in relatively large quantities, thus enabling the clinical and commercial supply of immunoglobulins.

[0080] Furthermore, one or more CDRs of the antigen-binding polypeptides of this disclosure can be inserted into the framework region using conventional recombinant DNA technology, for example, by inserting a human framework region to humanize a non-human antibody. The framework region may be a naturally occurring or common framework region, and is preferably a human framework region. Preferably, the polynucleotide produced by the combination of the framework region and the CDR encodes an antibody that specifically binds to at least one epitope of a desired polypeptide (e.g., LIGHT). Preferably, one or more amino acid substitutions may be made within the framework region, and preferably, the amino acid substitutions improve the binding of the antibody to its antigen. Such methods may also be used to produce antibody molecules lacking one or more intrachain disulfide bonds by substituting or deleting cysteine ​​residues in one or more variable regions involved in intrachain disulfide bonds. Other modifications to the polynucleotide are included in this disclosure and are within the scope of the art.

[0081] With respect to camelid antibodies, particularly heavy chain antibodies and nanobodies (VHHs), according to the present invention, humanization of a polypeptide involves replacing one or more camelid amino acids with their human counterparts found in human consensus sequences without causing the polypeptide to lose its typical characteristics; in other words, such humanization does not significantly affect the antigen-binding ability of the resulting polypeptide. Such methods are well known to those skilled in the art.

[0082] In one embodiment, "humanization" of VHH refers to replacing one or more amino acid residues in the amino acid sequence (and more specifically, the framework sequence) of a naturally occurring VHH sequence with one or more amino acid residues at the corresponding positions in a VH domain derived from a normal human four-chain antibody. This may be done using humanization techniques known in the art. In some embodiments, possible humanization substitutions or combinations of humanization substitutions may be determined by methods known in the art, for example, by comparing the sequence of VHH with the sequence of a naturally occurring human VH domain. In some embodiments, the humanization substitutions are selected such that the resulting humanized VHH still retains advantageous functional properties. Generally, as a result of humanization, the VHH of the present invention becomes more "human-like" than the corresponding naturally occurring VHH domain, while still retaining advantageous properties, such as reduced immunogenicity. In each embodiment, the humanized VHH of the present invention may be obtained by any suitable method known in the art, and is not strictly limited to polypeptides obtained using polypeptides containing naturally occurring VHH domains as starting materials.

[0083] In each embodiment, "humanization" and "camelization" may be performed in the following manner: A nucleotide sequence encoding a naturally occurring VHH domain or VH domain is provided, and then one or more codons of the nucleotide sequence are modified in a manner known in the art so that the new nucleotide sequence encodes a "humanized" or "camelized" VHH, respectively. Next, such nucleic acids may be expressed in a manner known in the art to provide the specified VHH of the present application. Optionally, an amino acid sequence of the specified humanized or camelized VHH of the present application may be designed from an amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized from scratch using a peptide synthesis technique known in the art. Alternatively, a nucleotide sequence encoding a specified humanized or camelized VHH may be designed from an amino acid sequence or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized from scratch using a nucleic acid synthesis technique known in the art, and then the nucleic acid thus obtained may be expressed in a manner known in the art to provide the specified VHH of the present application. Other suitable methods and techniques for obtaining the VHH and / or the nucleic acid encoding it from naturally occurring VH sequences or VHH sequences as starting materials are known in the art, and to provide the VHH or the nucleotide sequence or nucleic acid encoding it, it may be appropriate to combine, for example, one or more parts of one or more naturally occurring VH sequences (such as one or more FR sequences and / or CDR sequences), one or more parts of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences.

[0084] Furthermore, the present invention provides a nanobody that binds to humanized DDR2, the nanobody comprising CDR1, CDR2, and CDR3, (a) CDR1 contains or is the sequence shown in SEQ ID NO: 1 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 2 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 3 or its equivalent variant. (b) CDR1 contains or is the sequence shown in SEQ ID NO: 5 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 6 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 7 or its equivalent variant. (c) CDR1 contains or is the sequence shown in SEQ ID NO: 9 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 10 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 11 or its equivalent variant. (d) CDR1 includes or is the sequence shown in SEQ ID NO: 13 or its equivalent variant, CDR2 includes or is the sequence shown in SEQ ID NO: 14 or its equivalent variant, CDR3 includes or is the sequence shown in SEQ ID NO: 15 or its equivalent variant, (e) CDR1 contains or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 contains or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 contains or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0085] In another aspect of the present invention, the present invention provides a nucleotide sequence comprising, or comprising, a polynucleotide encoding an antibody of any of the above embodiments. In some embodiments, the polynucleotide comprises, or is selected from, any sequence of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and their respective equivalent variants.

[0086] (Chimera antigen receptor (CAR)) Chimeric Antigen Receptor (CAR) T cells are T cells that express chimeric receptors that can recognize specific antigens on their surface and transmit signals. CAR T cells play a crucial role in antitumor activity by expressing chimeric antigen receptor (CAR) molecules. CAR molecules generally consist of an extracellular segment, a transmembrane region, and an intracellular segment. The extracellular segment is typically a single-chain variable region (ScFv) formed by linking the antibody heavy and light chain variable regions with a single peptide segment. The intracellular segment is a chimeric form of the intracellular segments of various signaling molecules, and the transmembrane region originates from the transmembrane region of other molecules. The genes for the single-chain variable segment are, for example, isolated from hybridomas that produce monoclonal antibodies that recognize target antigens. Because CAR-expressing T cells are independent of the expression of major histocompatibility antigen type I on tumor cells, they can directly recognize tumor cell surface antigens and simultaneously activate T cells, thereby effectively killing tumor cells. Simply put, CAR T cells recognize specific molecules on the surface of tumor cells through antigen-antibody recognition, and are then activated by intracellular signaling, proliferate, and exert cell-killing functions.

[0087] Furthermore, in another aspect of the present invention, the present invention provides a chimeric antigen receptor comprising an extracellular domain capable of binding to an antigen, a transmembrane domain, and an intracellular domain, wherein the extracellular domain capable of binding to the antigen comprises any of the above-described nanobodies.

[0088] In some embodiments, the CAR of the present invention may include, for example, a transmembrane domain such as a T cell receptor α or β chain, CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, Tlr4, FcγR I, FcγR II, FcγR III, FCER1, or CD36. In some embodiments, the transmembrane domain includes CD8α or CD28. In some embodiments, the transmembrane domain includes a Tlr4 transmembrane region.

[0089] In some embodiments, the CAR of the present invention may include, for example, an intracellular signaling domain such as CD3ζ, CD28, 4-1BB (or CD137), CD27, and OX40 (or CD134). In some embodiments, the intracellular domain includes one of CD3ζ and CD28, 4-1BB (or CD137), CD27, and OX40 (or CD134). In some embodiments, the intracellular domain includes two of CD3ζ and CD28, 4-1BB (or CD137), CD27, and OX40 (or CD134). In some embodiments, the intracellular domain includes one or more of the CD86 intracellular functional region, the Tlr4 CSD region (i.e., the Tlr4 intracellular Toll-interleukin-1 receptor (TIR) ​​domain), and the FcγRI intracellular functional region. In some embodiments, the intracellular domain includes the Tlr4 CSD region and the FcγRI intracellular functional region.

[0090] Furthermore, the present invention provides a chimeric antigen receptor comprising an extracellular domain capable of binding to an antigen, a transmembrane domain, and an intracellular domain, wherein the extracellular domain capable of binding to the antigen comprises any of the above nanobodies, the transmembrane domain comprises a Tlr4 transmembrane region, and the intracellular domain comprises a Tlr4 CSD region and an FcγR I intracellular functional region.

[0091] Furthermore, the present invention also provides modified immune cells that express any of the above-mentioned chimeric antigen receptors.

[0092] In some embodiments, the immune cells are selected from T cells, NK cells, macrophages, and neutrophils. In some embodiments, the T cells are cytotoxic T lymphocytes, NKT cells, helper T cells, or suppressive and / or regulatory T cells. In some embodiments, the macrophages are M1 macrophages or M2 macrophages.

[0093] In some embodiments, the present invention provides CAR-T cells expressing a chimeric antigen receptor, wherein the extracellular domain of the chimeric antigen receptor comprises any nanobody described herein.

[0094] In some embodiments, the present invention provides CAR-T cells expressing a chimeric antigen receptor, wherein the extracellular domain of the chimeric antigen receptor comprises any of the nanobodies described herein, the nanobodies comprising CDR1, CDR2, and CDR3, and (a) CDR1 contains or is the sequence shown in SEQ ID NO: 1 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 2 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 3 or its equivalent variant. (b) CDR1 contains or is the sequence shown in SEQ ID NO: 5 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 6 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 7 or its equivalent variant. (c) CDR1 contains or is the sequence shown in SEQ ID NO: 9 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 10 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 11 or its equivalent variant. (d) CDR1 includes or is the sequence shown in SEQ ID NO: 13 or its equivalent variant, CDR2 includes or is the sequence shown in SEQ ID NO: 14 or its equivalent variant, CDR3 includes or is the sequence shown in SEQ ID NO: 15 or its equivalent variant, (e) CDR1 contains or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 contains or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 contains or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0095] In some embodiments, the present invention provides CAR-M cells (macrophages) expressing a chimeric antigen receptor, wherein the extracellular domain of the chimeric antigen receptor comprises any of the nanobodies described herein.

[0096] In some embodiments, the present invention provides CAR-M cells expressing a chimeric antigen receptor, wherein the extracellular domain of the chimeric antigen receptor comprises any of the nanobodies described herein, the nanobodies comprising CDR1, CDR2, and CDR3, and (a) CDR1 contains or is the sequence shown in SEQ ID NO: 1 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 2 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 3 or its equivalent variant. (b) CDR1 contains or is the sequence shown in SEQ ID NO: 5 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 6 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 7 or its equivalent variant. (c) CDR1 contains or is the sequence shown in SEQ ID NO: 9 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 10 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 11 or its equivalent variant. (d) CDR1 includes or is the sequence shown in SEQ ID NO: 13 or its equivalent variant, CDR2 includes or is the sequence shown in SEQ ID NO: 14 or its equivalent variant, CDR3 includes or is the sequence shown in SEQ ID NO: 15 or its equivalent variant, (e) CDR1 contains or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 contains or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 contains or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0097] In some embodiments, the nanobody comprises or is selected from any of the sequences of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20 and their respective equivalent variants.

[0098] (bispecific antibody) Bispecific antibodies (BsAbs) contain two artificial antibodies that can specifically recognize and bind to different antigens or different antigenic sites. If these two antigens are located on different cell surfaces, such bispecific antibodies can bridge the gap between these two antigen molecules, forming intercellular crosslinks and mediating the development of targeted effector function in cells. Bispecific antibodies used in immunotherapy are artificial antibodies containing two specific cell receptor antigen-binding sites that can bridge the gap between diseased cells (target cells) and functional cells (immune cells) to induce a targeted immune response. Mediating immune cells (T cells, NK cells, etc.) to kill tumor cells with BsAbs is currently a hot spot in applied immunotherapy research. The mechanism of action is that BsAbs can simultaneously bind to tumor-associated antigens and target molecules on immune effector cells, activating immune cells while directly guiding the targeted killing of tumor cells by immune effector cells.

[0099] Furthermore, in another aspect of the present invention, the present invention provides a bispecific antibody comprising a first binding portion that binds to a first antigen and a second binding portion that binds to a second antigen, wherein the first binding portion comprises any of the above-described nanobodies.

[0100] In some embodiments, the second antigen targeted by the bispecific antibody is a specific antigen on the surface of an immune cell. In some embodiments, the second antigen is an antigen or epitope of a B cell, T cell, myeloid cell, plasma cell, or mast cell.

[0101] In some embodiments, the second antigen is selected from CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD89, CD123, TCRγ / δ, NKp46, and KIR.

[0102] In some embodiments, the second antigen is CD16a. CD16a(FcγRIIIa) is a low-affinity receptor for the IgG Fc domain, which is involved in antibody-dependent cell-mediated cytotoxicity (ADCC) and in triggering natural killer (NK) lysis of target cells.

[0103] In some embodiments, the second antigen is CD89. CD89 is a glycoprotein, also known as the immunoglobulin α Fc receptor (Fc α RI), and is an effective cytotoxicity-inducing molecule expressed on the surface of neutrophils, monocytes, macrophages, and eosinophils.

[0104] In some embodiments, the second antigen is CD3. CD3 is present only on the surface of T cells, consists of six peptide chains, and is always tightly bound to the T cell receptor (TCR) to form a TCR-CD3 complex containing eight peptide chains, which is involved in both T cell recognition of antigens and signal transduction.

[0105] In some embodiments, the present invention provides a bispecific antibody comprising a first binding portion that binds to a first antigen and a second binding portion that binds to a second antigen, wherein the first antigen is DDR2, the first binding portion comprises nanobodies, and the second antigen is an antigen or epitope of a B cell, T cell, myeloid cell, plasma cell, or mast cell, preferably a T cell surface antigen, preferably CD3, and the nanobodies comprise CDR1, CDR2, and CDR3, and (a) CDR1 contains or is the sequence shown in SEQ ID NO: 1 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 2 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 3 or its equivalent variant. (b) CDR1 contains or is the sequence shown in SEQ ID NO: 5 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 6 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 7 or its equivalent variant. (c) CDR1 contains or is the sequence shown in SEQ ID NO: 9 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 10 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 11 or its equivalent variant. (d) CDR1 includes or is the sequence shown in SEQ ID NO: 13 or its equivalent variant, CDR2 includes or is the sequence shown in SEQ ID NO: 14 or its equivalent variant, CDR3 includes or is the sequence shown in SEQ ID NO: 15 or its equivalent variant, (e) CDR1 contains or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 contains or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 contains or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0106] In some embodiments, the present invention provides a bispecific antibody comprising a first binding portion that binds to a first antigen and a second binding portion that binds to a second antigen, wherein the first antigen is DDR2, and the first binding portion comprises nanobodies, the nanobodies comprising CDR1, CDR2, and CDR3, and (a) CDR1 contains or is the sequence shown in SEQ ID NO: 1 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 2 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 3 or its equivalent variant. (b) CDR1 contains or is the sequence shown in SEQ ID NO: 5 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 6 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 7 or its equivalent variant. (c) CDR1 contains or is the sequence shown in SEQ ID NO: 9 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 10 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 11 or its equivalent variant. (d) CDR1 includes or is the sequence shown in SEQ ID NO: 13 or its equivalent variant, CDR2 includes or is the sequence shown in SEQ ID NO: 14 or its equivalent variant, CDR3 includes or is the sequence shown in SEQ ID NO: 15 or its equivalent variant, (e) CDR1 contains or is the sequence shown in SEQ ID NO: 17 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 18 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 19 or its equivalent variant. Furthermore, the second antigen is DDR2, and the second binding portion includes a nanobody, and the nanobody includes CDR1, CDR2, and CDR3, (a) CDR1 contains or is the sequence shown in SEQ ID NO: 1 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 2 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 3 or its equivalent variant. (b) CDR1 contains or is the sequence shown in SEQ ID NO: 5 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 6 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 7 or its equivalent variant. (c) CDR1 contains or is the sequence shown in SEQ ID NO: 9 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 10 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 11 or its equivalent variant. (d) CDR1 includes or is the sequence shown in SEQ ID NO: 13 or its equivalent variant, CDR2 includes or is the sequence shown in SEQ ID NO: 14 or its equivalent variant, CDR3 includes or is the sequence shown in SEQ ID NO: 15 or its equivalent variant, (e) CDR1 contains or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 contains or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 contains or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0107] In some embodiments, the nanobody comprises or is selected from any of the sequences of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20 and their respective equivalent variants.

[0108] In some embodiments, the nanobodies included in the first bonding portion and the nanobodies included in the second bonding portion are different.

[0109] In some embodiments, the present invention provides a bispecific antibody comprising a first binding portion that binds to a first antigen and a second binding portion that binds to a second antigen, wherein the first binding portion comprises a nanobody, the nanobody comprising or being the sequence of SEQ ID NO: 4, and the second binding portion comprises a nanobody, the nanobody comprising or being the sequence of SEQ ID NO: 12.

[0110] In some embodiments, the first and second bonding portions are linked by a linker. The linker is preferably flexible, thereby preventing the effector molecule or polypeptide from being limited to a single undesirable conformation. The linker is preferably composed mainly of amino acids having small side chains, such as glycine, alanine, and serine, thereby providing the flexibility. In some embodiments, the linker comprises a polymer of glycine and serine, (GS) n (GGS) n (GGGS) n This includes, but is not limited to, (SEQ ID NO: 26) and its combinations, where n is an integer of 1 or more. In some embodiments, the linker sequence is (GGGS)3 (SEQ ID NO: 27). Other different linkers may also be used, such as various flexible linker designs used to link different antibody variable regions. The size and sequence configuration of the linkers may be determined by computer modeling and conventional techniques.

[0111] (Antibody-drug conjugate (ADC)) Antibody-drug conjugates (ADCs) are novel, efficient biopharmaceuticals designed for targeted therapy, particularly in the treatment of cancer. ADCs are complex molecules composed of an antibody (a complete mAb or antibody fragment) linked to a biologically active drug or cytotoxic compound by a stable chemical linker that may result in unstable binding. By combining the antibody's inherent targeting ability with the cytotoxic drug's ability to kill cells, antibody-drug conjugates can achieve highly sensitive differentiation between healthy and diseased tissues based on antibody and antigen expression. This means that, compared to conventional chemotherapeutic agents, antibody-drug conjugates actively target and attack antigen-expressing cells, resulting in very little impact on healthy cells that express only small amounts of the antigen or do not express it at all.

[0112] Furthermore, in another aspect of the present invention, the present invention provides an antibody-drug conjugate comprising an antibody targeting DDR2, a drug, and a linker for linking the antibody and the drug, wherein the antibody targeting DDR2 comprises or is any of the nanobodies described herein.

[0113] In some embodiments, the drug to be conjugated with the antibody of the present invention is a cytotoxic agent, a chemotherapeutic agent, or a therapeutic radioisotope.

[0114] In some embodiments, the cytotoxic agent is a low molecular weight toxin, a peptide toxin, or a protein toxin. In some embodiments, the toxin is selected from lysine, lysine A chain, ethidium bromide, colchicine, dihydroxyanthracin dione, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, abrin, abrin A chain, volkensin A chain, α-sarsina, mitogellin, restrictosin, curcin, and crotin.

[0115] In some embodiments, the chemotherapeutic agent is selected from alkylating agents, anthracycline antibiotics, antimetabolites, microtubule / mitosis inhibitors, histone deacetylase inhibitors, kinase inhibitors, peptide antibiotics, platinum-based antitumor agents, topoisomerase inhibitors, DNA crosslinking agents, and cytotoxic antibiotics.

[0116] In some embodiments, the therapeutic radioisotopes are selected from Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra-223, Ru-106, Na-24, Sr-89, Tb-149, Th-227, Xe-133, Yb-169, and Yb-177.

[0117] In some embodiments, the present invention provides an antibody-drug conjugate comprising an antibody targeting DDR2, a drug (preferably MMAE, calicheamicin, IR700, SG3199, DM1, DXd, MMAF, PE-38, SN-38, or yttrium-90), and a linker that links the antibody and the drug, wherein the antibody comprises or is composed of nanobodies, and the nanobodies comprise CDR1, CDR2, and CDR3, (a) CDR1 contains or is the sequence shown in SEQ ID NO: 1 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 2 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 3 or its equivalent variant. (b) CDR1 contains or is the sequence shown in SEQ ID NO: 5 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 6 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 7 or its equivalent variant. (c) CDR1 contains or is the sequence shown in SEQ ID NO: 9 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 10 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 11 or its equivalent variant. (d) CDR1 includes or is the sequence shown in SEQ ID NO: 13 or its equivalent variant, CDR2 includes or is the sequence shown in SEQ ID NO: 14 or its equivalent variant, CDR3 includes or is the sequence shown in SEQ ID NO: 15 or its equivalent variant, (e) CDR1 contains or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 contains or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 contains or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0118] (siRNA complex) In another aspect of the present invention, the present invention provides an siRNA complex targeting DDR2-positive cells, comprising (i) an antibody targeting DDR2, (ii) an siRNA that inhibits the expression of survival genes in DDR2-positive cells, and (iii) a linker located between the nanobody and the siRNA, wherein the antibody targeting DDR2 comprises or is any of the nanobody described herein.

[0119] The pharmacological portion of the siRNA complex disclosed herein, the "siRNA portion that inhibits the expression of survival genes in DDR2-positive cells," is a single double-stranded RNA molecule that, upon being taken up by target cells, inhibits the expression of survival genes in DDR2-positive cells by specifically targeting the degradation of the mRNA of specific survival genes via an RNAi mechanism.

[0120] Designing siRNAs for target genes is a common technique in this field. For example, there are several siRNA design sites that offer online design services (e.g., DSIR: http: / / biodev.extra.cea.fr / DSIR / DSIR.html, or siDirect version 2.0: http: / / sidirect2.rnai.jp / ). These design sites allow for the simultaneous acquisition of multiple representative siRNA sequences for different fragments of a target gene from the target gene's mRNA. These representative siRNAs may be sorted using several options (e.g., thermodynamics, higher-order structure). Candidate siRNA sequences may be further screened (manually or automatically) to exclude sequences containing SNP sites or binding to non-open reading frame fragments. In some embodiments, overlapping results from different online design sites are selected as candidate siRNA sequences. Candidate siRNA sequences may also be analyzed for their high likelihood of being off-target using off-target prediction software (e.g., http: / / rnai.cs.unm.edu / rnai / off-target).

[0121] In some embodiments, the siRNA sequence of the siRNA portion targets survival genes in DDR2-positive cells. For example, the siRNA sequence targets one or more fragments of one or more of the polr2a, polr2b, dkc1, cenpe, and eif-3b genes. In some embodiments, upon delivery of the siRNA sequence to the cell, it binds to the target gene via the RNAi pathway, thereby inhibiting the expression of the target gene. In some embodiments, inhibition of target gene expression causes apoptosis, death, and / or inhibition of proliferation in DDR2-positive cells. In some embodiments, apoptosis, death, and / or inhibition of proliferation in DDR2-positive cells mediates the treatment or cure of the disease. Suitable survival genes include, but are not limited to, one or more of the polr2a, polr2b, dkc1, cenpe, eif-3b, and survivin genes. siRNA sequences targeting survival genes may be obtained and tested by the methods described above.

[0122] In some embodiments, the present invention provides an siRNA complex targeting DDR2-positive cells, comprising (i) an antibody targeting DDR2, (ii) an siRNA that inhibits the expression of survival genes in DDR2-positive cells, and (iii) a linker located between the antibody and the siRNA, wherein the antibody targeting DDR2 comprises or is a nanobody, and the nanobody comprises CDR1, CDR2, and CDR3, (a) CDR1 contains or is the sequence shown in SEQ ID NO: 1 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 2 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 3 or its equivalent variant. (b) CDR1 contains or is the sequence shown in SEQ ID NO: 5 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 6 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 7 or its equivalent variant. (c) CDR1 contains or is the sequence shown in SEQ ID NO: 9 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 10 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 11 or its equivalent variant. (d) CDR1 includes or is the sequence shown in SEQ ID NO: 13 or its equivalent variant, CDR2 includes or is the sequence shown in SEQ ID NO: 14 or its equivalent variant, CDR3 includes or is the sequence shown in SEQ ID NO: 15 or its equivalent variant, (e) CDR1 contains or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 contains or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 contains or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0123] In some embodiments, an antibody comprising any of the nanobodies described herein may also comprise two complete heavy chains, each of which nanobodies constitutes a variable region of the respective complete heavy chain, and each complete heavy chain further comprises a heavy chain constant region (e.g., a human heavy chain constant region). In some embodiments, an antibody comprising any of the nanobodies described herein may also comprise a divalent nanobody.

[0124] In some embodiments, the Fc region of the heavy chain constant region is mutated. In some embodiments, the Fc region is mutated to reduce antibody-mediated cytotoxicity (ADCC). Exemplary Fc region mutations may be L234A, L235A, G237A, and P329G, where the positional numbers are based on the EU index.

[0125] (Pharmaceutical composition) Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any of the above-mentioned antibodies, nucleotide sequences, vectors, host cells, immune cells, or complexes, and a pharmaceutically acceptable carrier.

[0126] In any of the above embodiments, the pharmaceutical composition is used to treat and / or prevent diseases mediated by abnormal expression of DDR2, such as cancer or inflammatory diseases (e.g., pulmonary fibrosis, retinal vascular disease).

[0127] To manufacture a pharmaceutical composition or a sterile composition, a drug is mixed with a pharmaceutically acceptable carrier or excipient. By mixing with a physiologically acceptable carrier, excipient, or stabilizer, a formulation may be manufactured, for example, in the form of a lyophilized powder, suspension, aqueous solution, or suspending agent. Pharmaceutically acceptable carriers are well known in the art. Methods for manufacturing aqueous compositions containing active ingredients are known in the art. Generally, these compositions may be manufactured as injectable or spray preparations, such as liquids that are solutions or suspensions, or in solid form suitable for preparation as a solution or suspension before injection or spraying.

[0128] (Reagents for imaging and / or diagnostics) In another aspect of the present invention, the present invention provides an imaging and / or diagnostic reagent comprising any of the above-described nanobodies and a detectable label.

[0129] Furthermore, the present invention provides the use of any of the above nanobodies in the manufacture of reagents for imaging and / or diagnostics.

[0130] Furthermore, the present invention provides any of the above-mentioned nanobodies for use in imaging and / or diagnostic applications.

[0131] The above-mentioned imaging and / or diagnostic reagents may be used for disease diagnosis or lesion imaging, such as pulmonary fibrosis imaging or retinal neovascularization imaging. These reagents may also be used as developing reagents for intraoperative image navigation, for example, by providing navigation to guide the surgeon through optical imaging of the lesion, thereby enabling accurate resection of the lesion.

[0132] The detectable labels included in the imaging and / or diagnostic reagents of the present invention may be, for example, fluorescent labels, chemiluminescent labels, paramagnetic labels, radioisotope labels, or enzyme labels.

[0133] The choice of label depends on the detection method. For example, fluorescent labels (e.g., indocyanine green (ICG), rare earth chelates (e.g., europium chelates)), fluorescein-type labels (e.g., fluorescein, fluorescein isothiocyanate, 5-carboxyfluorescein, 6-carboxyfluorescein, dichlorotriazinylamine fluorescein), rhodamine-type labels (e.g., ALEXA568 (Invitrogen), or dansyl chloride), VIVOTAG 680 XLFLUOROCHROMETM (Perkin Elmer), phycoerythrin, 7-hydroxycoumarin, lissamine, cyanine, phycoerythrin, Texas Red, BODIPY (Invitrogen), or analogs thereof are suitable for optical detection.

[0134] Chemiluminescent labels (e.g., luminol, luciferase, luciferin, aequorin) may be used. Such diagnosis and detection may be carried out by linking the nanobodies of the present invention to a detectable substance, the detectable substance including, but not limited to, various enzymes, the enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase, or by linking them to prosthetic group complexes, the prosthetic group complexes including, but not limited to, streptavidin-biotin and avidin-biotin.

[0135] Paramagnetic labels and radioisotope labels may be employed, and those detectable by positron emission tomography (PET) or single-photon emission computed tomography (SPECT) are preferred. The radioisotope labels include bismuth ( 213 Bi), carbon ( 11 C, 13 C, 14 C), chromium ( 51 Cr), cobalt ( 57 Co, 60 Co), copper ( 64 Cu), dysprosium ( 165 Dy), erbium ( 169 Er), fluorine ( 18 F), gadolinium ( 153 Gd, 159 Gd), gallium ( 68 Ga, 67 Ga), germanium ( 68 Ge), gold ( 198 Au), holmium ( 166 Ho), hydrogen ( 3 H), indium ( 111 In, 112 In, 113 In, 115 In), iodine ( 121 I, 123 I, 125 I, 131 I), iridium ( 192 Ir), iron ( 59 Fe), krypton ( 81m Kr), lanthanum ( 140 La), lutetium ( 177 Lu), manganese ( 54 Mn), molybdenum ( 99 Mo), nitrogen ( 13 N, 15 N), oxygen ( 15 O), palladium ( 103 Pd), phosphorus ( 32 P), potassium ( 42 K), praseodymium ( 142 Pr), promethium ( 149 Pm), rhenium ( 186Re, 188 Re), Rhodium ( 105 Rh), Rubidium ( 81 Rb, 82 Rb), Ruthenium ( 82 Ru, 97 Ru), Samarium ( 153 Sm), scandium ( 47 Sc), Selenium ( 75 Se), sodium ( 24 Na), strontium ( 85 Sr, 89 Sr, 92 Sr), Sulfur ( 35 S), Technetium ( 99 Tc), thallium ( 201 Tl), Suzu ( 113 Sn, 117 Sn), xenon ( 133 Xe), Ytterbium ( 169 Yb, 175 Yb, 177 Yb), Yttrium ( 90 Y), zinc ( 65 This includes, but is not limited to, various positron-emitting metals and non-radioactive paramagnetic metal ions for positron emission tomography, such as paramagnetic aluminum (Al) ions, barium (Ba) ions, calcium (Ca) ions, cerium (Ce) ions, dysprosium (Dy) ions, erbium (Er) ions, europium (Eu) ions, gadolinium (Gd) ions, holmium (Ho) ions, iridium (Ir) ions, lithium (Li) ions, magnesium (Mg) ions, and manufactured metals. Cancer (Mn) ions, molybdenum (M) ions, neodymium (Nd) ions, osmium (Os) ions, oxygen (O) ions, palladium (Pd) ions, platinum (Pt) ions, rhodium (Rh) ions, ruthenium (Ru) ions, samarium (Sm) ions, sodium (Na) ions, strontium (Sr) ions, terbium (Tb) ions, thulium (Tm) ions, tin (Sn) ions, titanium (Ti) ions, tungsten (W) ions, zirconium (Zi) ions, especially Co +2 CR +2 , Cr +3 Cu +2 Fe+2 Fe +3 , Ga +3 Mn +3 Ni +2 Ti +3 , V + , V +4 Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art. For example, radioisotopes may be compounded by the chloramine-T method.

[0136] In some embodiments, the present invention provides an imaging and / or diagnostic reagent comprising any of the above-described nanobodies and a detectable label linked to the nanobodies, wherein the detectable label is a fluorescent label. In some embodiments, the fluorescent label is indocyanine green (ICG).

[0137] In some embodiments, the present invention provides an imaging and / or diagnostic reagent comprising any of the above nanobodies and a detectable label linked to the nanobodies, wherein the detectable label is a fluorescent label, preferably indocyanine green (ICG), and the nanobodies comprise CDR1, CDR2, and CDR3, (a) CDR1 contains or is the sequence shown in SEQ ID NO: 1 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 2 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 3 or its equivalent variant. (b) CDR1 contains or is the sequence shown in SEQ ID NO: 5 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 6 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 7 or its equivalent variant. (c) CDR1 contains or is the sequence shown in SEQ ID NO: 9 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 10 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 11 or its equivalent variant. (d) CDR1 includes or is the sequence shown in SEQ ID NO: 13 or its equivalent variant, CDR2 includes or is the sequence shown in SEQ ID NO: 14 or its equivalent variant, CDR3 includes or is the sequence shown in SEQ ID NO: 15 or its equivalent variant, (e) CDR1 contains or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 contains or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 contains or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0138] In some embodiments, the present invention provides an imaging and / or diagnostic reagent comprising any of the above-described nanobodies and a detectable label linked to the nanobodies, wherein the detectable label is a radioisotope. In some embodiments, the radioisotope is 68 Ga or 64 It is Cu.

[0139] In some embodiments, the present invention provides an imaging and / or diagnostic reagent comprising any of the above-described nanobodies and a detectable label linked to the nanobodies, wherein the detectable label is a radioisotope, preferably 68 Ga or 64 It is made of Cu, and the nanobody includes CDR1, CDR2, and CDR3, (a) CDR1 contains or is the sequence shown in SEQ ID NO: 1 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 2 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 3 or its equivalent variant. (b) CDR1 contains or is the sequence shown in SEQ ID NO: 5 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 6 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 7 or its equivalent variant. (c) CDR1 contains or is the sequence shown in SEQ ID NO: 9 or its equivalent variant, CDR2 contains or is the sequence shown in SEQ ID NO: 10 or its equivalent variant, and CDR3 contains or is the sequence shown in SEQ ID NO: 11 or its equivalent variant. (d) CDR1 includes or is the sequence shown in SEQ ID NO: 13 or its equivalent variant, CDR2 includes or is the sequence shown in SEQ ID NO: 14 or its equivalent variant, CDR3 includes or is the sequence shown in SEQ ID NO: 15 or its equivalent variant, (e) CDR1 contains or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 contains or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 contains or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0140] In some embodiments, the nanobody comprises or is selected from any of the sequences of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20 and their respective equivalent variants.

[0141] (Method and Use) One aspect of the present invention provides a method for treating and / or preventing diseases mediated by abnormal expression of DDR2, the method comprising administering an effective amount of any of the above antibodies, polynucleotides, vectors, host cells, immune cells, or complexes to a target.

[0142] Furthermore, another aspect of the present invention provides the use of any of the above antibodies, polynucleotides, vectors, host cells, immune cells, or complexes in the manufacture of a drug for treating and / or preventing a disease mediated by abnormal expression of DDR2.

[0143] Furthermore, another aspect of the present invention provides any of the above-mentioned antibodies, polynucleotides, vectors, host cells, immune cells, or complexes for treating and / or preventing diseases mediated by abnormal expression of DDR2.

[0144] Another aspect of the present invention provides a method for diagnosing a disease mediated by abnormal expression of DDR2 in a subject, the method comprising administering an effective amount of any of the above imaging and / or diagnostic reagents to the subject. In some embodiments, the method comprises administering an effective amount of any of the above imaging and / or diagnostic reagents to the subject, detecting and reading a signal generated by the imaging and / or diagnostic reagent, and determining from the intensity of the signal that the subject has or is at risk of having a disease mediated by abnormal expression of DDR2. In some embodiments, it is determined whether the signal exceeds a predetermined threshold, and if the signal exceeds the predetermined threshold, it is determined that the subject has a disease mediated by abnormal expression of DDR2. In some embodiments, the threshold is the median level derived from subjects that do not have the disease.

[0145] Furthermore, another aspect of the present invention provides the use of any of the above-mentioned imaging and / or diagnostic reagents in the manufacture of a kit for diagnosing diseases mediated by abnormal expression of DDR2 in a subject.

[0146] Furthermore, another aspect of the present invention provides any of the above-described imaging and / or diagnostic reagents for diagnosing diseases mediated by abnormal expression of DDR2 in a subject.

[0147] In some embodiments, the present invention provides a method for treating a disease mediated by abnormal expression of DDR2 in a subject, the method comprising: (a) administering an effective amount of any of the above imaging and / or diagnostic reagents to the subject; (b) detecting and reading a signal generated by the imaging and / or diagnostic reagent; (c) determining whether the signal exceeds a predetermined threshold, and if the signal exceeds the predetermined threshold, determining that the subject is suffering from a disease mediated by abnormal expression of DDR2, preferably the threshold being the median level derived from a subject not suffering from the disease; and (e) administering to the subject determined to be suffering from the disease a therapy that reduces the activity and / or expression of DDR2, such as an anti-cancer therapy or an anti-inflammatory therapy (e.g., an anti-fibrotic therapy). In a preferred embodiment, the disease mediated by abnormal expression of DDR2 is pulmonary fibrosis, particularly idiopathic pulmonary fibrosis. In a preferred embodiment, the anti-fibrotic therapy is pirfenidone and / or nintedanib.

[0148] Appropriate routes of administration include parenteral administration (e.g., intramuscular, intravenous, or subcutaneous) and oral administration. Other common methods of administration include via endotracheal intubation, oral, inhalation, topical, or percutaneous, subcutaneous, intraperitoneal, and intra-arterial injection.

[0149] The appropriate dose is determined by the clinician based on parameters or factors known in this field or thought to influence or expected to influence treatment. Generally, the starting dose is slightly lower than the optimal dose, and is then gradually increased until the necessary or optimal effect is achieved against any adverse events. Important monitoring indicators include, for example, measuring inflammatory symptoms or the levels of inflammatory cytokines produced. [Table 1-1] [Table 1-2] [Table 1-3]

[0150] (Examples) Example 1: Antibody Screening Anti-DDR2 heavy-chain antibodies were screened from alpacas immunized with human DDR2 extracellular segments (UniProtKB / Swiss-Prot:Q16832.2 aa22~aa399). Next, the antibodies were sequenced, their VHH portions were identified, and five nanobodies were obtained, named 1A1, 1A5, 1A12, 1B2, and 1B3, respectively. Their sequences are shown in the table above, and these will be used for further characterization and experiments.

[0151] Example 2: Expression and purification of five screened DDR2 nanobodies The experimental steps were as follows: 1. Transformation 100 μL of BL21(DE3) competent cells were obtained from one tube and placed on ice. 1 μL of plasmid (expression vector for five screened DDR2 nanobody cells) was added, the bottom of the tube was gently tapped, and the tube was left on ice for 25-30 minutes. After a 45-second water bath at 42°C, the tube was immediately returned to ice and held for 2 minutes. 500 μL of antibiotic-free LB medium was added, and the mixture was treated at 37°C and 250 rpm for 1 hour. The mixture was centrifuged at 3000 rpm for 1 minute, 100 μL of supernatant was retained, and the supernatant was pipetted to ensure uniformity. The mixture was then spread onto ampicillin-containing LB agar plates (LBA+) and incubated overnight at 37°C.

[0152] 2. Preservation and amplification of bacterial strains (1) Save Single clones were picked from the plates and added to 5-10 mL of LBA+ liquid medium, then processed overnight at 37°C and 250 rpm. The following day, 750 μL of bacterial suspension was obtained and added to 250 μL of 80% LB glycerol. The fragment, vector, strain name, and date were labeled, and the mixture was homogenized before storage at -20°C / -80°C. (After storing the strain in glycerol for 3-4 months, it is necessary to re-plate it to activate it and protect the strain.)

[0153] (2) Amplification On day 1, 10 μL of the glycerol-containing strain was obtained and added to 10 mL of LBA+ liquid medium (in 50 mL centrifuge tubes), for a total of 15 tubes. These were processed overnight at 37°C and 250 rpm. 2 L of LBA+ liquid medium was prepared and dispensed into eight 1 L Erlenmeyer flasks, each containing 250 mL, and processed at 121°C for 16 minutes.

[0154] On the second day, the bacterial suspension amplified overnight was inoculated into a 1 L Erlenmeyer flask at a 6% concentration. Simultaneously, 250 μL of 100 mg / mL Amponol was added, and the mixture was treated at 37°C and 250 rpm for 2-3 hours (OD value was 0.8-1). A final concentration of 1 mM IPTG was added to induce cell growth, and the mixture was treated at 32°C and 200 rpm for 5-6 hours. The bacterial cells were collected (10000 rpm, 5 minutes) and stored frozen at -20°C.

[0155] 3. Protein extraction and purification (1) Protein extraction Bacterial cells were obtained from a -20°C environment, thawed at room temperature, resuspended in bacterial lysate (500 mL of cells: 50 mL of lysate), disrupted under ultrasound for 5 minutes (450-500 W, 7 seconds on, 3 seconds off), treated in a homogenizer at 1000 Pa for 10 minutes, and then treated at 4°C and 12000 rpm for 30 minutes, with the supernatant remaining.

[0156] (2) Purification of proteins 200 μL of magnetic beads (Genscript Biotech Corporation, L00776) were aspirated and added to a 1.5 mL EP tube. The tube was washed 5-6 times with wash buffer (1 mL of wash buffer each time), the wash buffer was discarded, and the magnetic beads were added to the bacterial suspension supernatant. The tube was mixed uniformly for 1 hour at 4°C, turning it over as needed. The centrifuge tube was placed on a magnetic stand, and after the supernatant became clear, it was transferred to a new centrifuge tube. 200 μL of new magnetic beads were added, and the tube was mixed uniformly for 1 hour at 4°C, turning it over as needed. The old magnetic beads were washed 5-6 times with wash buffer, and then eluted three times with elution buffer, each time using 1 mL for 5 minutes, turning the tube over to mix uniformly. Finally, all eluates were collected and stored frozen at -20°C.

[0157] 4. Protein concentration and medium exchange (1) Pour 20% alcohol from the ultracentrifuge filter, rinse three times with distilled water, add 15 mL of distilled water, process at 4°C and 4000 rpm for 10-12 minutes, pour out the distilled water from the inner and outer tubes, add the eluate (the amount added should not exceed the mark on the inner tube), and concentrate until it reaches approximately 500 μL.

[0158] (2) Add 4.5 mL of filtered PBS and process at 4°C and 4000 rpm for 30 minutes. Repeat the above steps three times. Finally, aspirate approximately 500-700 μL of the sample from the PBS and place it in a 1.5 mL EP tube. Wash the ultracentrifuge filter and immerse the inner tube in 20% ethanol.

[0159] (3) The protein concentration was measured using the BCA method.

[0160] The results analysis was as follows: The SDS-PAGE results for the five expressed and purified DDR2 nanobodies (1A1, 1A5, 1A12, 1B2, 1B3) are shown in Figure 1, and the size of the antibody protein (indicated by circles) was approximately 15 KD, which was correct and as expected.

[0161] Example 3: Preliminary identification of binding between DDR2 nanobodies and DDR2 antigen extracellular segments by ELISA. The experimental steps were as follows: Flag tags were fused to nanobody gene coding sequences to express five purified DDR2 antibodies containing Flag tags: 1A1, 1A5, 1A12, 1B2, and 1B3. These antibodies were then coated with 100 ng / well of DDR2 antigen extracellular segment, blocked overnight with goat serum, rinsed three times with PBS, coated with 200 ng / well of anti-DDR2 (five purified DDR2 antibodies, all containing Flag tags) at 37°C for 1 hour, treated with anti-Flag (Sigma, F1804-200UG, 1 mg / mL) at 1:2500 at 37°C for 1 hour, treated with HRP (G&M) at 1:5000 at 37°C for 30 minutes (signal amplification with horseradish peroxidase-labeled anti-Flag antibody, TMB color development), and then stopped with 1M HCl. A blank control was simultaneously established.

[0162] The results analysis was as follows: ELISA validation was performed against five purified and expressed nanobodies and DDR2 antigen extracellular segments that had been confirmed as correct in the previous step, as well as against the negative control protein, bovine serum albumin (BSA).

[0163] The results of the binding analysis to the extracellular segment of the DDR2 antigen are shown in Figure 2A, and it was found that all five nanobodies, namely 1A1, 1A5, 1A12, 1B2, and 1B3, can exhibit binding activity to the DDR2 antigen.

[0164] The results of the binding analysis with bovine serum albumin, a negative control protein, are shown in Figure 2B. It was found that none of the five nanobodies, namely 1A1, 1A5, 1A12, 1B2, and 1B3, showed binding activity to the BSA protein.

[0165] Therefore, it was confirmed that the five nanobodies, namely 1A1, 1A5, 1A12, 1B2, and 1B3, have binding activity to the DDR2 antigen and do not bind to the negative control protein BSA.

[0166] Example 4: Flow cytometry identification of binding between DDR2 nanobodies and DDR2 antigens The experimental steps were as follows: A549 cells were infected with an adenovirus overexpressing the DDR2 antigen. DDR2 nanobodies (five purified DDR2 nanobodies expressing the His tag, fused to the nanobodies gene coding sequence) were incubated at 4°C for 1 hour, washed once with PBS, incubated with anti-His-AF488 as a secondary antibody at 4°C for 30 minutes, washed once with PBS, and collected by centrifugation at 300g for 5 minutes. The cells were then resuspended in 200 μL. Flow cytometry measurements were performed using the ACEA NovoCyte next-generation intelligent flow cytometer.

[0167] The results of the analysis were as follows: The results of the binding analysis with the DDR2 antigen are shown in Figure 3, and it was found that all five nanobodies, namely 1A1, 1A5, 1A12, 1B2, and 1B3, were able to exhibit binding activity to the DDR2 antigen.

[0168] Example 5: Immunofluorescence identification of binding between DDR2 nanobodies and DDR2 antigens The experimental steps were as follows: Cells overexpressing the DDR2 antigen were seeded → fixed overnight with 4% paraformaldehyde → blocked with 5% skim milk for 30 minutes → DDR2 nanobodies were incubated at room temperature for 2 hours → anti-His AF488 secondary antibody was applied → DAPI staining and imaging with a fluorescence microscope.

[0169] The results of the analysis were as follows: The results of the analysis of binding to the DDR2 antigen are shown in Figure 4, and it was found that all five nanobodies, namely 1A1, 1A5, 1A12, 1B2, and 1B3, were able to exhibit binding activity to the DDR2 antigen.

[0170] Example 6: Affinity of DDR2 nanobodies to the extracellular segment of the DDR2 antigen The experimental steps were as follows: For five purified DDR2 nanobodies, namely 1A1, 1A5, 1A12, 1B2, and 1B3, the affinity of antibodies to the antigens was measured using biolayer interferometry (BLI) in the ForteBio molecular interaction analysis system, and the affinity of the measured nanobodies to the DDR2 antigen extracellular segment protein was analyzed.

[0171] The experimental materials were as follows: The antibody was dissolved in PBS (pH 7.4), the antigen in PBS (pH 7.4), the sensor was Ni-NTA, the kinetics buffer was PBST (PBS + 0.02% Tween-20, pH 7.4), the regeneration buffer was 10 mM glycine-HCl (pH 1.7), and the re-charged buffer was 10 mM NiCl in water.

[0172] The operating steps were as follows: a. The probe was pre-moistened in kinetics buffer for 10 minutes. b. Establish baseline 1. A baseline was established for the biosensor using the kinetics buffer for 180 seconds. c. Fixation. The His-tagged DDR2 antigen extracellular segment was diluted to 20 μg / mL with kinetics buffer and captured with a sensor until it reached 4 nM (300 seconds). d. Establish Baseline 2. A 60-second baseline was established for the biosensor using the kinetics buffer. e. Binding. The antibody solution was diluted to a certain concentration with kinetics buffer (2-fold dilution from 100 nM to 3.125 nM), and the sensor was placed in the antibody solution to bind (600 seconds). f. Dissociation. The sensor was dissociated in the kinetics buffer (600 seconds). The g sensor was regenerated. It was tested with 10 mM glycine-HCl (pH 1.7) for 5 seconds. h. Neutralize. After the sensor was regenerated, it was neutralized in a kinetics buffer for 5 seconds. i. Repeat the regeneration step g and neutralization step h. This was done a total of 3 times (30 seconds). j. Establish baseline 3. After the sensor was regenerated, 10 mM NiCl was added and held for 60 seconds.

[0173] Pharmacokinetic curves were created, and each relevant parameter was calculated. Multiple binding-dissociation curves with appropriate concentration gradients were selected, and a 1:1 binding model was used to fit all curves. The three curves with the best fit were selected, graphed, and analyzed. Finally, important parameters such as affinity, binding constant, and dissociation constant were obtained.

[0174] The results of the analysis were as follows: The results of the binding of the five purified DDR2 nanobodies, namely 1A1, 1A5, 1A12, 1B2, and 1B3, to the DDR2 antigen are shown in Figure 5. [Table 2]

[0175] Example 7: In vitro imaging results of DDR2 nanobodies The experimental steps were as follows: 1. Labeling of DDR2 nanobody with indocyanine green (ICG) Nb-DDR2 was dissolved in PBS at a concentration of 2 mg / mL and vortexed to homogeneously mix the contents.

[0176] ICG-NHS was dissolved in DMSO at a concentration of 2 mM and vortexed to homogeneously mix it.

[0177] 500 μL of 2 mg / mL Nb-DDR2 solution was transferred to a 1.5 mL centrifuge tube, and 18 μL of 2 mM ICG-NHS solution was added to the Nb-DDR2 solution in nine separate additions of 2 μL each time. After each addition, the mixture was vortexed for several seconds to ensure uniform mixing.

[0178] The pH of the mixed solution was measured, and the pH was adjusted to 8.5-9 with a 2M NaOH solution.

[0179] The centrifuge tube was placed in a shaker and reacted at 60 rpm at room temperature for 2 hours.

[0180] The solution was centrifuged multiple times at 14,000 g for 10 minutes using a 0.5 mL ultracentrifuge filter to remove unreacted ICG-NHS. The solution was then replaced with 0.9% NaCl, filtered through a 0.22 μm membrane filter, and stored at 4°C.

[0181] 2. Imaging of mouse lung tissue sections Fresh lung tissue from normal mice was sliced ​​to a thickness of 300 μm. One slice served as a control group (no treatment), another was induced with bleomycin, and yet another was induced with mTGF-β cytokine. All treatments took 72 hours (both treatment groups were able to promote DDR2 expression). Next, each group was stained with ICG-labeled DDR2 nanobody 1A12 (1 μg / sample) for 30 minutes, and then washed multiple times with PBS to remove nonspecific staining. Imaging was then performed using a bioimaging system, and the imaging results are shown in Figure 6.

[0182] The results of the analysis were as follows: Purified DDR2 nanobodies labeled with ICG can specifically bind to mouse DDR2-highly expressing tissues, making them suitable for accurate in vitro imaging.

[0183] 3. Imaging of lung tissue sections in patients with idiopathic pulmonary fibrosis (IPF) Using lung tissue from normal donors and patients with idiopathic pulmonary fibrosis (IPF), the lung tissue from IPF patients expressed high levels of DDR2, while the lung tissue from normal donors expressed little to no DDR2. The lung tissue was sliced ​​in vitro to a thickness of 600 μm, with the normal group used as the control and the lung tissue sections from IPF patients used as the experimental group. After staining with ICG-labeled DDR2 nanobody 1A12, in vitro bioimaging was performed, and the results are shown in Figure 7.

[0184] The results analysis was as follows: Purified DDR2 nanobodies labeled with ICG can specifically bind to human DDR2-highly expressing tissues, making them suitable for accurate in vitro imaging.

[0185] 4. Imaging of lung tissue sections from pigs and rabbits Fresh lung tissue from normal pigs and rabbits was sliced ​​to a thickness of 600 μm, and DDR2 expression in the lung tissue was induced by adding bleomycin at concentrations of 1 μg / mL, 2 μg / mL, and 4 μg / mL. Lung tissue without bleomycin was used as a blank control. After induction for 72 hours, 2 μg of ICG-labeled DDR2 nanobody 1A12 was added, and the tissue was incubated at 37°C for 1 hour. The tissue sections were washed five times with PBS to remove unbound probes, and near-infrared imaging was performed using an in vivo imaging device for small animals. The results are shown in Figure 8.

[0186] The results analysis was as follows: ICG-labeled DDR2 nanobodies can specifically recognize DDR2 highly expressed in porcine and rabbit lung tissue, and therefore can be used for accurate in vitro imaging.

[0187] 5. Retinal neovascularization imaging Vascular smooth muscle cells are the main component of the vascular wall and can express DDR2. Retinas were collected from normal mice and oxygen-induced lesion mice, spread on glass slides, and 2 μg of ICG-labeled DDR2 nanobody 1A12 was added. The cells were incubated at room temperature for 1 hour, washed multiple times with PBS to remove unbound probes, and near-infrared imaging was performed using an in vivo imaging device for small animals. The results are shown in Figure 9.

[0188] The results analysis was as follows: ICG-labeled DDR2 nanobodies can specifically recognize DDR2 in the mouse retina and indicate the vascular distribution in the retina and the state of retinal vascular disease caused by oxygen induction.

[0189] Example 8: In vivo imaging results of DDR2 nanobodies The experimental steps were as follows: A mouse pulmonary fibrosis model was constructed by administering bleomycin at a dose of 1.7 mg / kg to the lungs of C57 mice via endotracheal intubation. In the mouse pulmonary fibrosis model, DDR2 was highly expressed in the lung tissue. After modeling for one week, 75 μg of ICG-labeled 1A12 nanobodies were injected via the tail vein, and in vivo imaging was performed. Mice 1 and 2 were pulmonary fibrosis models, while mouse 3 was a normal mouse that was not modeled. The site of pulmonary fibrosis in the mice was identified by imaging using a small animal in vivo imaging device.

[0190] As is clear from the results, the anti-DDR2 nanobodies provided by the present invention, as shown in Figure 10, can all enable accurate imaging of pulmonary fibrosis lesions in the body of mice, and can therefore be used for accurate in vivo diagnosis of early pulmonary fibrosis in the future.

[0191] Example 9: Inhibition of collagen-induced phosphorylation of DDR2 protein by nanobody 1A12 (functional verification) The experimental steps were as follows: 293T cells overexpressing the DDR2 antigen were seeded in 6-well plates. After the cells had grown to 70-80%, the medium was changed to 0.75% FBS to starve the cells. After 12 hours, the medium was changed again to fresh 0.75% FBS, and different amounts of DDR2 antibody 1A12 were added and incubated. After 2 hours, collagen was added to stimulate the cells, and after 12 hours, the cells were lysed, proteins were collected, and Western blotting was performed.

[0192] The experimental results are shown in Figures 11 and 12. As is clear from the results, 1A12 can inhibit collagen-induced phosphorylation of the DDR2 protein, and moreover, it exhibits a dose-dependent effect.

[0193] Example 10: Identification of anti-DDR2 heterodimer nanobodies 1A1-1A12 by plasmid enzymatic cleavage In this example, heterodimeric nanobodies 1A1 to 1A12 were constructed, including the nanobodies 1A1 and 1A12 described above. The linking method used was GGGSGGGSGGGS (Sequence ID 27), with the linking site located between the N-terminus of sequence 1A1 and the C-terminus of sequence 1A12. Characterization and experiments were performed on the antibody.

[0194] The experimental steps were as follows: 1. Glycerol-containing bacterial strains expressing DDR2 nanobodies 1A1-1A12 were gently shaken overnight. The following day, a small volume of plasmid extraction was performed. To 500 ng of plasmid, 0.5 μL of NcoI and XhoI rapid restriction enzymes and 1 μL of 10× buffer were added, and secondary distilled water was added to make a 10 μL system. This was incubated at 37°C for 30 minutes.

[0195] 2. Weigh 0.3 g of agarose into an Erlenmeyer flask, add 30 mL of TAE buffer, heat the Erlenmeyer flask in a microwave oven until the agarose dissolves, cool the liquid agarose gel to about 65°C, add ethidium bromide, mix thoroughly until homogeneous, fix the mold, set the comb, pour in the agarose solution, and allow it to solidify.

[0196] 3. Samples and DNA markers were added to the wells, and electrophoresis was performed at 120 V for 20 minutes to obtain an agarose gel, which was then exposed and developed.

[0197] The results were as shown in Figure 13. As was clear from the results, since the enzyme cleavage size of the plasmid was correct, it was the target plasmid.

[0198] Example 11: Coomassie Brilliant Blue staining of the purified proteins of anti-DDR2 heterodimeric nanobodies 1A1 to 1A12 The experimental steps were as follows. For 2 μg of the purified anti-DDR2 nanobodies 1A1 to 1A12, 5× protein loading buffer was added, boiled for denaturation, loaded onto an SDS-PAGE gel, and electrophoresed at 220 V for 1 hour. After electrophoresis, the SDS-PAGE gel was placed in an appropriate amount of Coomassie Brilliant Blue staining solution, stained for 30 minutes on a shaker at a rotation speed of 60 rpm, the staining solution was discarded, replaced with double-distilled water, and washed 2 to 3 times, and photographed under white light.

[0199] The results were as shown in Figure 14. As was clear from the results, the purified heterodimeric nanobodies 1A1 to 1A12 had the correct size and no other impurity bands.

[0200] Example 12: WB identification of anti-DDR2 heterodimeric nanobodies 1A1 to 1A12 The experimental steps were as follows. For 2 μg of the purified anti-DDR2 nanobodies 1A1 to 1A12, 5× protein loading buffer was added, boiled for denaturation, loaded onto an SDS-PAGE gel, and electrophoresed at 220 V for 1 hour. After electrophoresis, the SDS-PAGE gel was obtained and transferred, the transfer conditions were 400 mA for 35 minutes, blocked with 5% skim milk for 1 hour, incubated with anti-His-HRP at room temperature for 1 hour, washed 3 times with TBST, and developed after loading.

[0201] The results are shown in Figure 15. As is clear from the results, the purified heterodimer nanobodies 1A1 to 1A12 were of the correct size.

[0202] Example 13: Flow cytometry identification of the binding activity of anti-DDR2 heterodimer nanobodies 1A1-1A12 to the DDR2 antigen. The experimental steps were as follows: DDR2 293T and 293T cells were digested, transferred to a 1.5 mL centrifuge tube, 1 μg of anti-DDR2 nanobody 1A1-1A12 was added, and the tubes were incubated for 1 hour. The tubes were then centrifuged at 300 g for 5 minutes, the supernatant was discarded, PBS was added and the tubes were washed three times, anti-His-488 secondary antibody was added and the tubes were incubated in the dark for 1 hour, washed three times with PBS, and loaded into a flow cytometer for measurement.

[0203] The results are shown in Figure 16. As is clear from the results, the purified heterodimeric nanobodies 1A1-1A12 have the ability to bind to the corresponding DDR2 antigen.

[0204] Example 14: Inhibition of collagen-induced phosphorylation of DDR2 protein by anti-DDR2 heterodimer nanobodies 1A1-1A12 (functional verification) The experimental steps were as follows: 1. Seed DDR2-293T cells in a 6-well plate, with each group in one well, and wait until the cell density reaches 85%. 2. The cells were starved for 12 hours by replacing the medium with 0.75% fetal bovine serum, and antibody solutions 1A1 to 1A12 at different concentrations were prepared in the same starvation medium. 3. Discard the supernatant from the starvation medium and replace it with antibody medium of a different concentration, then incubate for 2 hours. 4. Collagen was added to stimulate the cells, resulting in a collagen concentration of 30 μg / mL. After 12 hours of stimulation, the cells were lysed and proteins were extracted. 5. Phosphorylation levels of the DDR2 protein in cells from each group were measured using WB experiments.

[0205] The results are shown in Figure 17. As is clear from the results, the heterodimeric nanobodies 1A1-1A12 had an inhibitory effect when they bound to the DDR2 antigen.

[0206] Example 15: Chimeric antigen receptor CAR molecular vectors were constructed using the DDR2 nanobody 1A12 sequence (see Figure 18), and adenovirus was packaged in them. By infecting primary mouse macrophages with these vectors, αDDR2 CAR-M cells were obtained and used in the following experiments.

[0207] Empty-M (uninfected macrophages) and αDDR2 CAR-M were co-cultured with 293T-DDR2 cells in a 1:1 ratio. The FITC and APC double-positive rates were statistically analyzed as the phagocytosis rate of 293T-DDR2 cells, and the results are shown as mean ± standard error (Mean ± SEM). The results are shown in Figure 19, and there was a statistically significant difference between Empty-M and CAR-M. It was found that CAR-M cells constructed with 1A12 phagocytosed DDR2-positive 293T cells at a significantly higher rate.

[0208] Empty-M, αDDR2 CAR-M each 5×10 5 Individual cells were injected into the tail vein of mice modeling the left lung using bleomycin. On day 15, the mice were euthanized, the left lung was removed, and CD68 immunohistochemistry was performed on normal mice that had not been injected with macrophages as controls. The results are shown in Figure 20. The results of CD68 immunohistochemistry show the following: Brown fine granules were observed in lung sections of mice from each group. In the NC group and the BLM+Empty-M group, weak positive expression of CD68 was observed in the lung tissue sections, mainly distributed around the airways and in the alveolar septa. In the BLM+CAR-M group, positive expression of CD68 was clearly enhanced and uniformly distributed. From these results, it is thought that adoptive αDDR2 CAR-M cells can be localized to regions that highly express DDR2, i.e., the left lung of mice. CAR-M cells constructed with 1A12 were concentrated in DDR2-highly expressing tissues of mice and localized there.

[0209] We investigated the targeted phagocytosis of αDDR2 CAR-M cells onto 293T-DDR2 cells using a lattice light sheet super-resolution microscope. αDDR2 CAR-M cells exhibit green fluorescence, and 293T-DDR2 cells were stained red with Dil (an orange-red fluorescent stain). The two were mixed in a 1:1 ratio, loaded, and continuously scanned for 16 hours, with images taken every 3 minutes. The results are shown in Figure 21. Green (CAR-M) and red (293T-DDR2) cells were observed. The yellow cells are thought to have formed after the green CAR-M cells sufficiently phagocytosed the red 293T-DDR2 cells, and the red staining solution on the 293T-DDR2 cells entered the interior of the CAR-M cells. This demonstrated that CAR-M cells constructed with 1A12 can target and effectively phagocytose DDR2-positive 293T cells.

[0210] In each group of mice, 40 μL of 0.75 mg / mL bleomycin (BLM) was administered intratracheally. On day 8, micro-CT scans were performed to screen for mice that successfully modeled the left lung, and these mice were divided into groups according to the degree of lesion. On day 10, PBS, Empty-M, and αDDR2 CAR-M were injected via the tail vein, respectively. On day 26, micro-CT scans were performed again to evaluate the therapeutic effect, and on day 28, the mice were euthanized, and their left lungs were removed and fixed.

[0211] As shown in Figure 22, A is the experimental procedure, B is a weight change graph of mice whose weight was measured every three days after BLM modeling, C is a survival curve graph of the mice in each group from the day of BLM modeling to day 28 after modeling, and D is a CT 3D image obtained by micro-CT scanning of the lungs of the mice in each group on day 8 and day 26 after modeling, respectively.

[0212] When looking at body weight, in the CAR-M group, the body weight of the mice tended to decrease and then increase, and almost recovered to the original weight level on the 28th day. In the other two groups, although weight recovery was observed, they could not recover to the original weight. When looking at the survival curves, in the CAR-M group, there were no dead mice, while in the other two groups, dead mice appeared, and the PBS group was more serious. When looking at the CT, in the CAR-M group, the lung lesions of the mice had mostly recovered compared to those on the 8th day, while in the other two groups, the recovery was not obvious or the condition deteriorated. In short, compared with the control, the mice injected with αDDR2 CAR-M had significantly higher body weight, significantly increased survival rate (no deaths occurred until the 28th day), the degree of lesions was controlled, and there was a significant therapeutic effect.

[0213] Example 16: Procedure for labeling nanobody 1A12 with radionuclides ( 64 Cu, 68 ]>[Ga and other common radionuclides) 1. Binding with NOTA (1) Centrifuge the antibody solution at 12,000 g to obtain the supernatant. Replace the solvent of the antibody solution with 0.1 M ammonium acetate (pH = \alpha). After replacement, the concentration of nanobody 1A12-cys becomes 1.33 mg / mL. Measure the concentration before and after replacement and comply with the concentration after replacement.

[0214] (2) Use 50 mM Mal-NOTA (dissolved in DMSO), set the molar ratio of the antibody to Mal-NOTA to 1:5, dilute Mal-NOTA with DMSO until it is less than 10% of the reaction system volume (for example, if the reaction system is 1 mL, dilute Mal-NOTA to 80 - 90 μL, shake and mix uniformly to fully dissolve Mal-NOTA in DMSO). Add the Mal-NOTA solution to the antibody solution in 10 μL aliquots several times. After each addition, shake for dozens of seconds to mix uniformly, and adjust the pH of the solution to 7 (the reaction solvent is 0.1 M ammonium acetate, and the pH hardly needs to be adjusted, just measure and confirm). Place it on a shaker and react at room temperature for 2 hours. The rotation speed of the shaker was 150 rpm.

[0215] It should be noted that there is an unclear value "\alpha" in the original text for the pH value in step (1) of item . The translation has been done as accurately as possible based on the existing content. (3) After the reaction was completed, the antibody solution was centrifuged at 12,000 g, and the supernatant was obtained. The antibody solution was purified and concentrated with 0.1 M ammonium acetate, concentrated to 50 - 80 μL per 100 μg of antibody, sealed with a sealing film, and stored in a refrigerator at -80 °C.

[0216] 2. 68 Labeling with common nuclides such as Ga 64 and Cu (1) 68 Ga 64 Cu or other nuclides were added to the precursor solution ( 64 In the case of Cu, the concentration was made as high as possible. The total radioactivity input for 100 μg of the precursor was about 1.5 mCi, and the final reaction volume was 150 μL or less), and they were uniformly mixed, and the pH was adjusted to 4 - 5 (Generally, even without adjustment, the pH was 4 - 5 after uniform mixing).

[0217] (2) If there is a vibrating thermostat, place it in the vibrating thermostat and react at 37 °C for 2 hours. If not, heat at 37 °C and react for 2 hours (using a water bath, oven, etc.).

[0218] (3) Spot the sample on filter paper for paper chromatography, develop with a developing solution, and measure the labeling rate by iTLC (the developing solution is sodium citrate).

[0219] Example 17: Nuclide 68 Imaging of Nanobody 1A12 Labeled with Ga According to the procedure described in Example 16, 68 Ga-labeled Nanobody 1A12 was prepared.

[0220] On the 15th day of modeling with bleomycin (BLM) in mice (BLM-15d) and control normal mice (Ctrl), respectively, at 200 μCi / mouse 68Ga-labeled nanobody 1A12 was administered via tail vein, and PET-CT imaging was performed. The imaging results are shown in Figure 23. The results indicate that the BLM-modeled mice clearly took up more of the nanobody into their lungs compared to normal mice. This suggests that nanobodies can be used in PET-CT imaging when radionuclides are attached to them.

[0221] While the present invention is specifically disclosed in preferred embodiments and optional features, those skilled in the art should understand that modifications, improvements, and changes may be made to the invention disclosed herein, and such modifications, improvements, and changes will be deemed to be within the scope of the invention. The materials, methods, and examples provided herein are representative and illustrative of preferred embodiments and do not limit the scope of the invention.

Claims

1. A nanobody that binds to DDR2, Including CDR1, CDR2, and CDR3, The CDR1, CDR2, and CDR3 are nanobodies comprising or being CDR1, CDR2, and CDR3, whose amino acid sequence is Sequence ID No.

4.

2. The nanobody according to claim 1, wherein CDR1, CDR2, and CDR3 are defined by any of the following definition methods: IMGT, Kabat, Chothia, Contact, or Martin.

3. The nanobody according to claim 1, wherein CDR1 comprises or is the sequence shown in SEQ ID NO: 1, CDR2 comprises or is the sequence shown in SEQ ID NO: 2, and CDR3 comprises or is the sequence shown in SEQ ID NO:

3.

4. The nanobody according to claim 1, wherein the nanobody is a humanized nanobody.

5. The nanobody according to claim 1, wherein the nanobody includes or is an equivalent variant having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the sequence of sequence 4.

6. A polynucleotide comprising a polynucleotide encoding the nanobody described in claim 1.

7. The polynucleotide according to claim 6, wherein the polynucleotide includes, or is, the sequence of SEQ ID NO: 21 or an equivalent variant having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:

21.

8. A vector comprising the polynucleotide described in claim 6.

9. A non-human host cell comprising the vector according to claim 8.

10. A chimeric antigen receptor comprising an extracellular domain capable of binding to an antigen, a transmembrane domain, and an intracellular domain, wherein the extracellular domain capable of binding to the antigen includes the nanobody described in claim 1.

11. The chimeric antigen receptor according to claim 10, wherein the transmembrane domain comprises a Tlr4 transmembrane region, and the intracellular domain comprises a Tlr4 CSD region and an FcγRI intracellular functional region.

12. Modified immune cells expressing the chimeric antigen receptor described in claim 10.

13. The immune cell according to claim 12, which is a macrophage.

14. A bispecific antibody comprising a first binding portion that binds to a first antigen and a second binding portion that binds to a second antigen, wherein the first binding portion includes the nanobody described in claim 1.

15. The bispecific antibody according to claim 14, wherein the second binding portion includes the nanobody described in claim 1.

16. The bispecific antibody according to claim 15, wherein the nanobody contained in the first binding portion and the nanobody contained in the second binding portion are different.

17. An antibody-drug conjugate comprising an antibody targeting DDR2, a drug, and a linker for linking the antibody and the drug, wherein the antibody targeting DDR2 includes or is a nanobody as described in claim 1.

18. (i) an antibody targeting DDR2, which includes or is a nanobody as described in claim 1; (ii) an siRNA that inhibits the expression of survival genes in DDR2-positive cells; and (iii) a linker located between the nanobody and the siRNA, wherein the siRNA complex targets DDR2-positive cells.

19. A reagent for imaging and / or diagnostics comprising a nanobody according to claim 1 and a detectable label linked to the nanobody.

20. The reagent according to claim 19, wherein the detectable label is a fluorescent label or a radioisotope.

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