Anti-DDR2 nanobodies and uses thereof

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] The present invention relates to anti-DDR2 antibodies. More specifically, the present invention relates to nanobodies that bind to DDR2 and various derivatives of said nanobodies. The present invention also relates to pharmaceutical compositions containing these antibodies and derivatives and their pharmaceutical uses. [Background technology]

[0002] Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase (RTK) that utilizes extracellular matrix collagens as its ligands. In addition to its kinase function, DDR2 activates β1-integrins to promote cell adhesion. The unique function of DDR2 is to mediate signaling from the extracellular matrix to the cytoplasm, thereby balancing extracellular matrix regulation, and to participate in the regulation of cell growth, differentiation, and metabolism. Activation of DDR2 by extracellular matrix collagens is essential for normal development and tissue homeostasis, but aberrant activation of these receptors after injury or disease is detrimental.

[0003] The role of DDR2 in regulating the extracellular matrix suggests that dysregulation of its signaling may lead to tumorigenesis, including invasion and metastasis. DDR2 has been reported to be highly expressed at the leading edge of many invasive breast tumor samples. In situ hybridization of adjacent sections of human ovarian or lung cancers has demonstrated that DDR2 is detected in stromal cells surrounding the tumor. Therefore, DDR2 is thought to be crucial for the metastasis of many cancer cells (e.g., breast cancer) and is also an important target for many other cancers (e.g., ovarian cancer, lung cancer, head and neck cancer, and pancreatic cancer).

[0004] DDR2 is also considered an important target for inflammation (e.g., arthritis, including osteoarthritis and rheumatoid arthritis) and fibrosis (e.g., pulmonary fibrosis, liver cirrhosis, renal fibrosis, and skin fibrosis). DDR2 is primarily expressed in interstitial cells, such as fibroblasts, myofibroblasts, and smooth muscle cells, in the kidney, skin, lung, heart, and connective tissue. A growing body of evidence suggests that abnormal DDR2 expression is involved in 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 aging mice. Studies using a rat model of rheumatoid arthritis have also revealed elevated DDR2 expression in the synovial cells.

[0005] WO2019243431A1 discloses that in a melanoma cell-derived xenograft model, imatinib-mediated targeting of DDR2 can enhance the antitumor effect of BRAF inhibitors, postpone the development of acquired drug resistance, and resolve targeted therapy-induced tumor fibrosis.

[0006] Therefore, the development of high-affinity antibodies targeting DDR2 is likely to have broad practical implications for imaging, diagnosis, prevention, and treatment of cancer, inflammation, fibrotic diseases, and other conditions. Summary of the Invention

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

[0008] In some embodiments, the CDR1, CDR1, and CDR3 are defined according to any of the IMGT, Kabat, Chothia, Contact, or Martin definitions.

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

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

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

[0012] In a second aspect, 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 nanobodies described in the first aspect. In another aspect, the present invention provides a polynucleotide comprising a polynucleotide encoding the chimeric antigen receptor. In another aspect, the present invention provides a vector comprising the polynucleotide described above. In another aspect, the present invention provides a modified immune cell expressing the chimeric antigen receptor.

[0013] In a third aspect, the present invention provides a bispecific antibody comprising a first binding moiety that binds to a first antigen and a second binding moiety that binds to a second antigen, wherein said first binding moiety comprises any of the nanobodies according to the first aspect.

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

[0015] In a fifth aspect, the present invention provides an siRNA complex targeting DDR2-positive cells, comprising (i) an antibody that targets DDR2, which comprises or is any of the Nanobodies according to the first aspect, (ii) an siRNA that inhibits expression of a survival gene 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 antibodies, polynucleotides, vectors, host cells, immune cells and / or complexes described above, and a pharmaceutically acceptable carrier.

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

[0018] In an eighth aspect, 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 aspect, the present invention provides a method for treating and / or preventing a disease mediated by abnormal expression of DDR2, comprising administering to a subject an effective amount of any of the above-mentioned antibodies, polynucleotides, vectors, host cells, immune cells or complexes.

[0020] In a tenth aspect, the present invention provides an imaging and / or diagnostic reagent comprising any of the Nanobodies according to the first aspect and a detectable label linked to said 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 aberrant expression of DDR2 in a subject.

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

[0023] In a thirteenth aspect, the present invention provides a method for diagnosing a disease mediated by aberrant expression of DDR2 in a subject, comprising administering to the subject an effective amount of an imaging and / or diagnostic reagent according to aspect 10. In some embodiments, the method comprises administering to the subject an effective amount of an imaging and / or diagnostic reagent according to aspect 10, detecting and reading a signal produced 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 aberrant expression of DDR2.

[0024] In a fourteenth aspect, the present invention provides a method for treating a disease mediated by aberrant expression of DDR2 in a subject, the method comprising: (a) administering to the subject an effective amount of any of the imaging and / or diagnostic reagents described above; (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 afflicted with a disease mediated by aberrant expression of DDR2, preferably the threshold being a median level derived from subjects not afflicted with the disease; and (e) administering to the subject determined to be afflicted with the disease a therapy that reduces DDR2 activity and / or expression, for example, an anti-cancer therapy or an anti-inflammatory therapy (e.g., an anti-fibrotic therapy). In a preferred embodiment, the anti-fibrotic therapy is pirfenidone and / or nintedanib.

[0025] In related aspects of the above, the diseases mediated by aberrant expression of DDR2 include cancer and inflammatory diseases. In some embodiments, the cancer includes, but is 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, gastric cancer, and skin cancer. In some embodiments, the inflammatory disease includes, but is 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 (particularly idiopathic pulmonary fibrosis), liver fibrosis, liver cirrhosis, skin 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 for 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 cells expressing DDR2, for example, biopsy navigation and intraoperative navigation.

[0027] Other aspects and advantages of the present invention will become apparent from the following detailed description of the invention. [Brief explanation of the drawings]

[0028] [Figure 1] Figure 1 shows the SDS-PAGE results of the expression and purification of DDR2 nanobodies. [Figure 2] Figure 2. ELISA validation of DDR2 nanobodies. [Figure 3] Figure 3 shows the flow cytometry results of DDR2 nanobodies. [Figure 4] FIG. 4 shows the immunofluorescence results of DDR2 nanobodies. [Figure 5] FIG. 5. Affinity constants of DDR2 nanobodies for the extracellular segment of the DDR2 antigen. [Figure 6] Figure 6 shows the results of in vitro imaging of DDR2 nanobodies in mouse lung tissue. [Figure 7] Figure 7 shows in vitro imaging results of DDR2 nanobodies in human lung tissue. [Figure 8] FIG. 8 shows in vitro imaging results of DDR2 nanobodies in pig and rabbit lung tissue. [Figure 9] FIG. 9 shows in vitro imaging results of DDR2 nanobody in the retina. [Figure 10] FIG. 10 shows in vivo imaging results of DDR2 nanobodies. [Figure 11] FIG. 11 is a Western blot (WB) showing the inhibition of DDR2 protein phosphorylation induced by Collagen 1 by nanobody 1A12. [Figure 12] FIG. 12 shows the statistics of the inhibition of DDR2 protein phosphorylation induced by Collagen 1 by nanobody 1A12. [Figure 13] FIG. 13 shows DNA electrophoresis of enzyme cleavage identification for a plasmid expressing an anti-DDR2 nanobody. [Figure 14] FIG. 14 is an SDS-PAGE electrophoresis staining of purified anti-DDR2 nanobodies. [Figure 15] Figure 15 is a western blot of purified anti-DDR2 nanobodies. [Figure 16] FIG. 16: Flow cytometry measurement of the activity of anti-DDR2 nanobodies. [Figure 17] FIG. 17 is a western blot of anti-DDR2 nanobody inhibition of collagen-induced DDR2 protein phosphorylation. [Figure 18] FIG. 18 is a schematic diagram of a CAR molecule constructed with the DDR2 nanobody 1A12 sequence. [Figure 19] Figure 19 shows the phagocytosis rate of DDR2-positive 293T cells by mouse macrophages infected with adenovirus packaged with a CAR molecule constructed by 1A12. [Figure 20] Figure 20 shows the localization of CAR-M constructed with 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] FIG. 23 shows 68Ga PET-CT imaging of a mouse modeled with BLM on day 15. DETAILED DESCRIPTION OF THE INVENTION

[0029] (definition) In the present invention, "about" refers to a numerical value that is within an acceptable error range of the specific value as determined by one of ordinary skill in the art, and the numerical value will depend to some extent on how it is measured or determined (i.e., the limitations of the measurement system). For example, in each practice in the art, "about" may mean within or more than 1 standard deviation. Alternatively, "about" or "substantially comprising" may mean a range of up to 20%. Particularly for biological systems or processes, the term may mean up to an order of magnitude or up to 5 times the numerical value. Unless otherwise specified, when a specific value appears in the specification and claims, "about" or "substantially comprising" should be interpreted as meaning within an acceptable error range of the specific value.

[0030] References herein to compositions or methods "comprising" one or more elements or steps are open-ended, meaning that those elements or steps are essential, but that other elements or steps may be added within the scope of the composition or method. Note that any composition or method described as "comprising" one or more elements or steps describes a corresponding, more limited composition or method "consisting essentially of those elements or steps," meaning that the composition or method includes those essential elements or steps, but may also include additional elements or steps of basic and novel characteristics that do not essentially affect the composition or method.

[0031] As used herein, the term "antibody" refers to any form of antibody that exhibits a desired biological activity (e.g., through inhibiting binding of a ligand to its receptor or inhibiting ligand-induced receptor signaling). "Antibody fragment" and "antigen-binding fragment" generally refer to antigen-binding fragments of antibodies and antibody analogs that contain at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody. In some embodiments, the antibody is a monoclonal antibody. In some 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, each antibody in the population being identical except for naturally occurring variants that may be present in minor amounts. Monoclonal antibodies have high specificity, being able to target a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations which typically include several different antibodies directed against several different determinants (epitopes), each monoclonal antibody targets only a single determinant on the antigen. The modifier "monoclonal" refers to the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in the present invention may be produced by hybridoma or recombinant DNA technology.

[0033] Monoclonal antibodies may include "chimeric," humanized, or fully human antibodies. In some embodiments, an antibody is part of a larger biological molecule, such as a fusion protein or antibody-drug conjugate. Antibody fragments retain at least some of the binding specificity of the parent antibody. Generally, antibody fragments retain at least 10% of the binding activity of the parent, when activity is expressed in molar terms. Preferably, antibody fragments retain 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 binding affinity of the parent antibody for the target.

[0034] As used herein, the term "heavy chain antibody" refers to an antibody that lacks a light chain and consists of only a heavy chain, including two constant regions (CH2 and CH3), a hinge region, and a heavy chain variable region (VHH). Examples include, but are not limited to, naturally occurring heavy chain antibodies, antibodies naturally lacking a light chain, heavy chain antibodies derived from conventional four-chain antibodies, and artificial antibodies. Heavy chain antibodies may be derived from species of the Camelidae family, such as antibodies produced in camels, llamas, dromedaries, alpacas, and draft horses. Species other than Camelidae can also produce heavy chain antibodies naturally lacking a light chain, and such heavy chain antibodies are within the scope of the present invention.

[0035] As used herein, the term "nanobody" refers to a single-domain antibody consisting only of the heavy chain variable region, obtained by cloning the variable region of a heavy chain antibody. Also known as a VHH (variable domain of heavy chain antibody) or single-domain antibody, this is the smallest functional antigen-binding fragment. Nanobodies recognize antigens with high specificity and affinity similar to IgG antibodies, but their small size (approximately 15 kDa) allows them to penetrate tumor tissue more easily. Nanobodies are also resistant to harsh pH, ​​heat denaturation, protein hydrolysis, solvents, and detergents. They can be expressed and produced with high yield and solubility.

[0036] As used herein, the term "humanized antibody" refers to an antibody comprising CDRs derived from an antibody of a non-human mammal and framework regions (FRs) and constant regions of a human antibody.

[0037] An "equivalent variant" of an antibody or polypeptide refers to an antibody or polypeptide having a degree of homology or sequence identity 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, an 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 subject sequence in one or more amino acid residues but retains the biological activity of the molecule of interest.

[0039] As used herein, the term "% identity" between two sequences refers to a function of the number of identical positions the sequences have (i.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps and the length of each gap, which are introduced during optimal alignment of the two sequences. The comparison of sequences and determination of % identity between two sequences may be accomplished by a mathematical algorithm.

[0040] "Conservative substitutions" refer to amino acid substitutions known to those skilled in the art, which generally do not alter the biological activity of a molecule of interest. It is generally recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter or essentially alter the biological activity. "Unaltered or essentially unchanged" refers to a difference in one or more aspects of the polypeptide that is less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% compared to a comparable molecule, when measured using the same or similar methods.

[0041] CDR3 is generally considered to play a more important role in antigen recognition than other CDRs. Therefore, when substitutions are made, conservative substitutions for CDRs other than CDR3 are preferred in the present invention. In some embodiments, CDR3 is not substituted. Preferred amino acid substitutions include, but are not limited to, (1) substitutions that reduce the hydrolysis susceptibility of the protein, (2) substitutions that reduce the oxidative susceptibility, (3) substitutions that change the binding affinity of the formed protein complex, and (4) substitutions that provide or modify other physical, chemical, or functional properties of these analogs. Analogs may contain various sequence variations other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally occurring sequence (preferably in the polypeptide portion other than the region forming intermolecular contacts). Conservative amino acid substitutions should not essentially alter the structural features of the parent sequence (e.g., amino acid substitutions that tend to disrupt helices present in the parent sequence or that have been characterized as disrupting other secondary structures of the parent sequence).

[0042] The binding domain of the antibody or antigen-binding fragment thereof of the present invention may generally be expected to have a signal peptide consisting of 15 to 30 amino acids located at the N-terminus of a secretory protein. When the signal peptide sequence is synthesized, it is recognized by the signal recognition particle (SRP), pausing or relaxing protein synthesis. Protein synthesis resumes when the SRP directs the ribosome to the endoplasmic reticulum. Guided by the signal peptide, the newly synthesized protein enters the endoplasmic reticulum lumen, but the signal peptide sequence is cleaved off by a signal peptidase. Furthermore, when a stop transport sequence is present at the C-terminus of a nascent peptide chain, as in the case of ovalbumin, it may not be cleaved off by a signal peptidase. Neither the precursor nor the mature form is subject to cleavage by a signal peptidase.

[0043] According to the present invention, the term "binding" preferably refers to specific binding. When referring to a ligand-receptor, antibody-antigen, or other binding pair, "specific" binding refers to determining whether or not there is a binding reaction between the proteins in a heterogeneous population of proteins and / or other biochemical reagents. Thus, under given conditions, a specific ligand or antigen binds to a specific receptor or antibody and does not bind in significant amounts to other proteins present in a sample. "Specific binding" refers to the ability of a monoclonal antibody or antigen-binding fragment thereof of the present invention to 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 primarily 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. Biacore and / or ELISA 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 measuring and calculating binding specificity. In such a signal-to-noise ratio, the signal represents the binding strength of the antibody to the epitope of interest, and the noise represents the binding strength of the antibody to other non-target epitopes. Preferably, when the signal-to-noise ratio for the epitope of interest is about 50, the antibody being evaluated can be considered to bind to the epitope of interest in a specific manner, i.e., "specifically bind." An antigen-binding protein (including an antibody) "specifically binds" to an antigen if it binds to the antigen with high binding affinity as determined by the affinity constant (KD) value. In some embodiments, the affinity constant KD is 10 -9 is 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 term "patient" or "subject" refers to any organism that receives or may receive the provided antibodies, derivatives thereof, or pharmaceutical compositions for experimental, diagnostic, prophylactic, 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 is suffering from or susceptible to one or more diseases or conditions. The patient may exhibit one or more symptoms of the disease or condition, or may already be diagnosed with one or more diseases or conditions. In some embodiments, the patient is undergoing or has undergone a particular therapy to diagnose and / or treat such diseases, diseases, or conditions.

[0045] As used herein, the term "treatment" refers to therapeutic and preventative measures that prevent or alleviate the onset or progression of undesirable physiological changes or disease conditions in a subject. Beneficial or expected clinical effects include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delaying or slowing the progression of disease, alleviation or mitigation of the disease state, and partial or total cure of disease, whether or not such effects are detectable. "Treatment" may also refer to prolonging survival compared to not receiving treatment. Subjects to be treated include those suffering from the disease or condition, as well as those at risk of developing the disease or condition, or those in whom the disease or condition is to be prevented.

[0046] As used herein, the term "prevention" includes preventing or alleviating the onset of clinically significant disease progression, or preventing or alleviating the onset of a pre-clinically significant disease stage in an at-risk individual, including prophylactic treatment of human individuals at risk for disease progression.

[0047] "Administration," "treatment," or "prevention," when used with reference to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" can refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cell therapy includes contacting a reagent with a cell or a reagent with a fluid, where the fluid contacts the cell. "Administration" and "treatment" also refer to in vitro and ex vivo treatments of cells, for example, with a reagent, diagnostic agent, binding composition, or by other cells.

[0048] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of the antibody or derivative thereof of the present invention, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, that effectively prevents or alleviates the disease or condition being treated. A therapeutically effective dose also refers to an amount sufficient to alleviate symptoms, such as treating, curing, preventing, or alleviating the associated medical condition, or improving the treatment, cure, prevention, or alleviation rate for the condition. When administered to an individual as a single active ingredient, a therapeutically effective amount refers to that ingredient alone. When administered as a combination, a therapeutically effective amount refers to the amount of the combined active ingredients that produces a therapeutic effect, regardless of whether they are administered concomitantly, sequentially, or simultaneously. A therapeutically effective amount generally alleviates symptoms by 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 a material that, when combined with an active ingredient of a composition, allows the ingredient to retain its biological activity and does not induce a destructive reaction in the subject's immune system. These carriers may include stabilizers, preservatives, salt or sugar complexes or crystals, etc. "Pharmaceutically acceptable" refers to molecules and components that do not induce allergic or similar undesirable reactions 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 derived extracellular domain, and at least one intracellular domain. A "chimeric antigen receptor (CAR)" is also sometimes referred to as a "chimeric receptor," "T-body," or "chimeric immune receptor (CIR)." An "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to a specific antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that transduces a signal in a cell, leading to the activation or inhibition of a biological process.

[0051] As used herein, the term "antibody drug conjugate" or "ADC" refers to an antibody covalently attached to a therapeutically active agent or active pharmaceutical ingredient, such that the therapeutically active agent or active pharmaceutical ingredient can target the antibody's binding target and exhibit its pharmacological function. The therapeutically active agent or active pharmaceutical ingredient may be a cytotoxin capable of killing cells targeted by the ADC. Covalent attachment of the therapeutically active agent, active pharmaceutical ingredient, or cytotoxin may be achieved in a non-site-specific manner using standard chemical linkers that attach the payload to lysine or cysteine ​​residues, or, preferably, in a site-specific manner of conjugation, which allows complete control of the conjugation site and the drug-antibody ratio (DAR) of the resulting ADC.

[0052] The term "siRNA" refers to double-stranded RNA 20-25 nucleotides in length, comprising two substantially complementary, antiparallel nucleic acid strands. These strands are designated the sense and antisense strands relative to the target RNA. The siRNA portion triggers degradation of the target RNA (e.g., mRNA) through a mechanism known as post-transcriptional gene silencing (herein referred to as RNA interference or RNAi). Generally, the nucleotides in each strand of the siRNA portion are mostly ribonucleotides, but each or both of the two strands may further comprise one or more non-ribonucleotides, e.g., deoxyribonucleotides and / or modified nucleotides. The siRNA portion may also comprise ribonucleotides with chemical modifications or may contain inherent 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 be included within the definition of "siRNA."

[0053] The terms "survival genes" and "essential genes" are used interchangeably to refer to genes that are essential for the survival or proliferation of a particular cell; functional deletion of a single one of these genes results in a lethal phenotype in that cell, rendering it unable to survive or proliferate. Typical survival genes are genes encoding proteases important in the processes of transcription, replication, and expression of genetic material, including, but not limited to, POLR2A (DNA-directed RNA polymerase II subunit RPB1, the largest subunit of RNA polymerase II, capable of synthesizing pre-mRNA and many functional non-coding RNAs, and forming the polymerase active center 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 center with the largest subunit), DKC1 (H / ACA ribonucleoprotein complex subunit DKC1), CENPE (Centromere-associated protein E), and eIF-3b (eukaryotic translation initiation factor 3 subunit b). Furthermore, the term "survival gene in DDR2-positive cells" refers to a gene that is essential for the survival or proliferation of DDR2-positive cells.Methods for determining whether a particular gene in a human cell is an essential gene are known in the art, 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 the present invention, camel-derived heavy chain antibodies are obtained by immunizing alpacas with the human DDR2 extracellular segment (the amino acid sequence of which is UniProtKB / Swiss-Prot:Q16832.2 aa22 to aa399), and the corresponding nanobodies are then obtained.

[0055] In one aspect of the invention, the invention provides a Nanobody that binds to DDR2, wherein the Nanobody comprises CDR1, CDR2 and CDR3, which CDR1, CDR2 and CDR3 comprise or are selected from the CDR1, CDR2 and CDR3 of any of the Nanobodies having the amino acid sequences SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16 and SEQ ID NO: 20, or their respective equivalent variants.

[0056] In some embodiments, the CDR1, CDR1, and CDR3 are defined according to any of the IMGT, Kabat, Chothia, Contact, or Martin definitions. In some embodiments, the CDR1, CDR1, and CDR3 are defined according to the IMGT definitions.

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

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

[0059] In some embodiments, the equivalent variants of each of the 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, equivalent variants of each of the Nanobodies refer to those that have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, and SEQ ID NO:20, and have the same or equivalent CDR1, CDR2, and CDR3.

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

[0062] In some embodiments, the Nanobody comprises 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 comprises CDR1, CDR2, and CDR3, and (a) CDR1 is the sequence shown in SEQ ID NO: 1, CDR2 is the sequence shown in SEQ ID NO: 2, and CDR3 is the sequence shown in SEQ ID NO: 3; (b) CDR1 is the sequence shown in SEQ ID NO: 5, CDR2 is the sequence shown in SEQ ID NO: 6, and CDR3 is the sequence shown in SEQ ID NO: 7; (c) CDR1 is the sequence shown in SEQ ID NO: 9, CDR2 is the sequence shown in SEQ ID NO: 10, and CDR3 is the sequence shown in SEQ ID NO: 11; (d) CDR1 is the sequence set forth in SEQ ID NO: 13, CDR2 is the sequence set forth in SEQ ID NO: 14, and CDR3 is the sequence set forth in SEQ ID NO: 15, or (e) CDR1 is the sequence shown in SEQ ID NO: 17, CDR2 is the sequence shown in SEQ ID NO: 18, and CDR3 is the sequence shown in SEQ ID NO: 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. Amino acid residue families with similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar 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). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, the amino acid string may be replaced with a string of structurally similar side chain family members that differ in order and / or composition.

[0067] It will also be understood by those skilled in the art that the antibodies disclosed herein may be modified such that their amino acid sequences differ from the naturally occurring binding polypeptide from which they are derived. For example, a polypeptide or amino acid sequence derived from a designated protein may resemble the starting sequence, e.g., have a certain percentage identity, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or a range between any two of these values, the same as the starting sequence.

[0068] In some embodiments, an antibody comprises one or more amino acid sequences or moieties not generally associated with antibodies. Exemplary modifications are described in more detail herein. For example, the antibodies disclosed herein may comprise flexible linker sequences or may be modified to attach a functional moiety (e.g., polyethylene glycol (PEG), a drug, a toxin, or a label).

[0069] The antibodies, variants, or derivatives of the present invention include modified derivatives, i.e., those that involve the covalent attachment of any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from binding to the epitope. Antibodies may be modified, for example, but not limited to, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, hydrolytic protein cleavage, or linkage to a cellular ligand or other protein. Any of a variety of chemical modifications may be performed using known techniques, including, but not limited to, specific chemical lysis, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Antibodies may also contain one or more non-classical amino acids.

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

[0071] The antibody may be conjugated or fused to a therapeutic agent, which may include a detectable label (e.g., a radioactive label), an immunomodulatory agent, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent (which may be a drug or a toxin), an ultrasound-enhancing agent, a non-radioactive label, combinations thereof, or other reagents of this type known in the art.

[0072] The antibody may be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescently labeled antigen-binding polypeptide is then determined by detecting the appearance of luminescence during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, heat-stable acridinium esters, imidazole, acridine salts, and oxalate esters.

[0073] Also, fluorescent metals (e.g., 152 Antibodies may be detectably labeled with metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to antibodies are well known.

[0074] (humanized antibody) In any aspect of the invention, the Nanobody that binds 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 and have one or more complementarity-determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Generally, framework residues in the human framework regions are replaced 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, such as modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding and sequence comparison, as well as identifying unnatural framework residues at specific positions. Antibodies may be humanized using a variety of techniques known in the art, such as CDR grafting, veneered or resurfacing, and chain shuffling.

[0076] Completely human antibodies are particularly ideal for treating human patients. Human antibodies can be produced by a variety of methods known in the art, such as phage display technology using antibody libraries derived from human immunoglobulin sequences.

[0077] Alternatively, mice that are unable to express functional endogenous immunoglobulins but are capable of expressing human immunoglobulin genes can be engineered to produce human antibodies. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. Introducing human immunoglobulin loci by homologous recombination can render mouse heavy and light chain immunoglobulin genes functional, either individually or simultaneously. In particular, homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastula embryos to generate chimeric mice. The chimeric mice are then bred to obtain homozygous offspring that express human antibodies. The genetically engineered mice are immunized with a selected antigen using conventional methods, such as the entire or partial target polypeptide of interest. Monoclonal antibodies against the antigen can be obtained from the immunized genetically engineered mice using conventional hybridoma technology. The human immunoglobulin genes harbored in genetically engineered mice are rearranged during B cell differentiation, followed by class switching and somatic mutation, making it possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies using this technology.

[0078] Fully human antibodies that recognize a chosen epitope may be generated by a technique called "guided selection," in which a non-human monoclonal antibody, such as a murine antibody, is selected to guide the selection of a fully human antibody that recognizes the same epitope.

[0079] DNA encoding the desired monoclonal antibody can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding mouse antibody heavy and light chains). Isolated and subcloned hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which may then be transfected into prokaryotic or eukaryotic host cells, such as Escherichia 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 the selected cells, converting it to cDNA, and PCR amplifying it using Ig-specific primers. As described herein, transformed cells expressing the desired antibody can be grown in relatively large quantities, enabling clinical and commercial supplies of immunoglobulins.

[0080] Alternatively, one or more CDRs of an antigen-binding polypeptide of the present disclosure can be inserted into framework regions using conventional recombinant DNA techniques, e.g., inserting human framework regions to humanize a non-human antibody. The framework regions may be naturally occurring or consensus framework regions, and preferably are human framework regions. Preferably, the polynucleotide produced by combining the framework regions and CDRs 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 can be made within the framework regions, and preferably the amino acid substitutions improve binding of the antibody to its antigen. Such methods can also be used to produce antibody molecules lacking one or more intrachain disulfide bonds by amino acid substitution or deletion of one or more variable region cysteine ​​residues involved in intrachain disulfide bonds. Other modifications to the polynucleotide are encompassed by this disclosure and within the skill of the art.

[0081] For camelid antibodies, in particular heavy chain antibodies and nanobodies (VHH), humanizing a polypeptide according to the present invention comprises replacing one or more camelid amino acids with their human counterparts found in the human consensus sequence without causing the polypeptide to lose its typical characteristics, i.e., the humanization does not significantly affect the antigen-binding capacity of the resulting polypeptide. Such methods are well known to those skilled in the art.

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

[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, respectively, is provided, and one or more codons in said nucleotide sequence are then altered in a manner known in the art so that the new nucleotide sequence encodes a "humanized" or "camelized" VHH, respectively. Such nucleic acids may then be expressed in a manner known in the art to provide the desired VHH of the present application. Optionally, the amino acid sequence of a desired humanized or camelized VHH of the present application may be designed from the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using peptide synthesis techniques known in the art. Alternatively, a nucleotide sequence encoding a desired humanized or camelized VHH of the present application may be designed from the amino acid or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using nucleic acid synthesis techniques known in the art, and then the resulting nucleic acid may be expressed in a manner known in the art to provide the desired VHH of the present application. Other suitable methods and techniques for obtaining the VHHs of the present application and / or nucleic acids encoding same starting from naturally occurring VH or VHH sequences are known in the art and may include, for example, combining in an appropriate manner one or more parts of one or more naturally occurring VH sequences (such as one or more FR and / or CDR sequences), one or more parts of one or more naturally occurring VHH sequences (such as one or more FR or CDR sequences) and / or one or more synthetic or semi-synthetic sequences to provide the VHHs of the present application or nucleotide sequences or nucleic acids encoding them.

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

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

[0086] (Chimeric Antigen Receptor (CAR)) Chimeric antigen receptor (CAR) T cells are T cells that express chimeric receptors capable of recognizing specific antigens on their surface and transmitting signals. CAR T cells play an important role in anti-tumor therapy by expressing chimeric antigen receptor (CAR) molecules. CAR molecules generally comprise an extracellular segment, a transmembrane region, and an intracellular segment. The extracellular segment is typically a single-chain variable fragment (ScFv) formed by linking antibody heavy and light chain variable regions via a single peptide segment. The intracellular segment is a chimera of intracellular segments from various signaling molecules, and the transmembrane region is derived from the transmembrane region of another molecule. Genes for the single-chain variable segment are isolated, for example, from hybridomas producing monoclonal antibodies that recognize target antigens. Because CAR molecule-expressing T cells are independent of the expression of major histocompatibility complex type I on tumor cells, they can directly recognize tumor cell surface antigens and simultaneously activate T cells, thereby effectively killing tumor cells. In simple terms, CAR T cells recognize specific molecules on the surface of tumor cells through antigen-antibody recognition, and then are activated by intracellular signaling, proliferate, and exert their cell-killing function.

[0087] Furthermore, in another aspect, 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 nanobodies described above.

[0088] In some embodiments, a CAR of the invention may comprise a transmembrane domain comprising, for example, 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, FcγRI, FcγRII, FcγRII, FcγRII, FCER1, or CD36. In some embodiments, the transmembrane domain comprises CD8α or CD28. In some embodiments, the transmembrane domain comprises a Tlr4 transmembrane region.

[0089] In some embodiments, a CAR of the present invention may comprise intracellular signaling domains consisting of, for example, CD3ζ, CD28, 4-1BB (or CD137), CD27, and OX40 (or CD134). In some embodiments, the intracellular domain comprises one of CD3ζ and CD28, 4-1BB (or CD137), CD27, and OX40 (or CD134). In some embodiments, the intracellular domain comprises two of CD3ζ and CD28, 4-1BB (or CD137), CD27, and OX40 (or CD134). In some embodiments, the intracellular domain comprises one or more of a CD86 intracellular functional region, a Tlr4 CSD region (i.e., a Tlr4 intracellular Toll-Interleukin 1 Receptor (TIR) ​​domain), and an FcγR I intracellular functional region. In some embodiments, the intracellular domain comprises a Tlr4 CSD region and an FcγR I 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 nanobodies described above, the transmembrane domain comprises a Tlr4 transmembrane region, and the intracellular domain comprises a Tlr4 CSD region and an FcγRI intracellular functional region.

[0091] Furthermore, the present invention also provides modified immune cells that express any of the above 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 suppressor and / or regulatory T cells. In some embodiments, the macrophages are M1 macrophages or M2 macrophages.

[0093] In some embodiments, the present invention provides a CAR-T cell that expresses a chimeric antigen receptor, wherein the extracellular domain of said chimeric antigen receptor comprises any of the nanobodies described herein.

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

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

[0096] In some embodiments, the 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, wherein the Nanobody comprises CDR1, CDR2, and CDR3, and (a) CDR1 comprises or is the sequence shown in SEQ ID NO: 1 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 2 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 3 or an equivalent variant thereof; (b) CDR1 comprises or is the sequence set forth in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 7 or an equivalent variant thereof; (c) CDR1 comprises or is the sequence set forth in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 11 or an equivalent variant thereof; (d) CDR1 comprises or is the sequence set forth in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 15 or an equivalent variant thereof; or (e) CDR1 comprises or is the sequence set forth in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 19 or an equivalent variant thereof.

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

[0098] (bispecific antibody) Bispecific antibodies (BsAbs) contain two artificial antibodies that can specifically recognize and bind to different antigens or antigenic sites. If the two antigens are located on different cell surfaces, such bispecific antibodies can bridge the two antigen molecules, forming an intercellular bridge and mediating the cells to produce directed effector functions. Bispecific antibodies used in immunotherapy are artificial antibodies that contain two specific cell receptor antigen-binding sites, bridging the gap between diseased cells (target cells) and functional cells (immune cells) to initiate directed immune responses. BsAbs mediating immune cell (e.g., T cells, NK cells) killing tumor cells is currently a hot topic in applied immunotherapy research. Their mechanism of action is that BsAbs can simultaneously bind to tumor-associated antigens and target molecules on immune effector cells, activating immune cells and directly guiding the immune effector cells to specifically kill tumor cells.

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

[0100] In some embodiments, the second antigen targeted by the bispecific antibody is a specific antigen on the surface of an immune cell, hi some embodiments, the second antigen is an antigen or epitope of a B cell, a T cell, a myeloid cell, a plasma cell, or a 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, TCR gamma / delta, NKp46, and KIR.

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

[0103] In some embodiments, the second antigen is CD89, a glycoprotein also known as immunoglobulin alpha Fc receptor (Fc alpha RI), which 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, which is present only on the surface of T cells, consists of six peptide chains, and always tightly binds to the T cell receptor (TCR) to form a TCR-CD3 complex containing eight peptide chains, and is involved in both T cell recognition of antigens and signal transduction.

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

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

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

[0108] In some embodiments, the nanobody contained in the first binding moiety and the nanobody contained in the second binding moiety are different.

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

[0110] In some embodiments, the first and second binding moieties are connected by a linker. The linker is preferably a flexible linker so that the effector molecule or polypeptide is not restricted to a single undesired conformation. The linker is preferably composed primarily of amino acids with small side chains, such as glycine, alanine, and serine, thereby providing said flexibility. In some embodiments, the linker comprises a polymer of glycine and serine, such as (GS) n , (GGS) n , (GGGS) n (SEQ ID NO: 26) and combinations thereof, where n is an integer greater than or equal to 1. 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 that have been used to link different antibody variable regions. Linker size and sequence configuration may be determined by computer modeling and routine techniques.

[0111] (Antibody-drug conjugates (ADCs)) Antibody-drug conjugates (ADCs) are novel and efficient biopharmaceuticals designed for targeted therapy, particularly in the treatment of cancer. ADCs are complex molecules composed of an antibody (intact mAb or antibody fragment) linked to a biologically active drug or cytotoxic compound via a stable chemical linker, which may result in an unstable bond. Combining the unique targeting ability of antibodies with the cell-killing potential of cytotoxic drugs, ADCs can achieve highly sensitive differentiation of healthy and diseased tissues depending on the expression of the antibody and antigen. This means that, compared to conventional chemotherapeutic agents, ADCs actively target and attack antigen-expressing cells, with minimal impact on healthy cells that only weakly or do not express the antigen.

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

[0113] In some embodiments, the drug conjugated to the antibody of the 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, hi some embodiments, the toxin is selected from ricin, ricin A chain, ethidium bromide, colchicine, dihydroxyanthracin dione, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, abrin, abrin A chain, volkensin A chain, α-sarcina, mitogellin, restrictocin, 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 cross-linking agents, and cytotoxic antibiotics.

[0116] In some embodiments, the therapeutic radioisotope is 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 connecting the antibody and the drug, wherein the antibody comprises or is a nanobody, the nanobody comprising CDR1, CDR2, and CDR3, and (a) CDR1 comprises or is the sequence shown in SEQ ID NO: 1 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 2 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 3 or an equivalent variant thereof; (b) CDR1 comprises or is the sequence set forth in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 7 or an equivalent variant thereof; (c) CDR1 comprises or is the sequence set forth in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 11 or an equivalent variant thereof; (d) CDR1 comprises or is the sequence set forth in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 15 or an equivalent variant thereof; or (e) CDR1 comprises or is the sequence set forth in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 19 or an equivalent variant thereof.

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

[0119] The pharmacological portion of the siRNA complex of the present disclosure, the "siRNA portion that inhibits expression of survival genes in DDR2-positive cells," is a single double-stranded RNA molecule that, when taken up by target cells, inhibits expression of survival genes in DDR2-positive cells by specifically targeting the degradation of the mRNA of a particular survival gene via the RNAi mechanism.

[0120] Methods for designing siRNAs for target genes are common in the art. For example, there are several siRNA design sites that provide 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 simultaneous generation of multiple representative siRNA sequences for different fragments of a target gene from the mRNA of the target gene. These representative siRNAs may be sorted by multiple options (e.g., thermodynamics, higher-order structure, etc.). Candidate siRNA sequences may be further screened (manually or automatically) to eliminate sequences containing SNP sites or binding to non-open reading frame fragments. In some embodiments, overlapping results from different online design sites may be selected as candidate siRNA sequences. Candidate siRNA sequences may also be analyzed for siRNA sequences with high off-target potential 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 a survival gene in DDR2-positive cells. For example, the siRNA sequence targets one or more fragments of one or more of the following genes: polr2a, polr2b, dkc1, cenpe, and eif-3b. In some embodiments, when the siRNA sequence is delivered to a cell, it binds to the target gene via the RNAi pathway, thereby inhibiting expression of the target gene. In some embodiments, inhibition of expression of the target gene causes apoptosis, death, and / or growth inhibition of the DDR2-positive cells. In some embodiments, apoptosis, death, and / or growth inhibition of the DDR2-positive cells mediates treatment or cure of the disease. Suitable survival genes include, but are not limited to, one or more of the following genes: polr2a, polr2b, dkc1, cenpe, eif-3b, and survivin. siRNA sequences targeting survival genes may be obtained and tested using 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 expression of a survival gene in DDR2-positive cells; and (iii) a linker positioned between the antibody and the siRNA, wherein the antibody targeting DDR2 comprises or is a nanobody, and the nanobody comprises CDR1, CDR2, and CDR3, and (a) CDR1 comprises or is the sequence shown in SEQ ID NO: 1 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 2 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 3 or an equivalent variant thereof; (b) CDR1 comprises or is the sequence set forth in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 7 or an equivalent variant thereof; (c) CDR1 comprises or is the sequence set forth in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 11 or an equivalent variant thereof; (d) CDR1 comprises or is the sequence set forth in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 15 or an equivalent variant thereof; or (e) CDR1 comprises or is the sequence set forth in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 19 or an equivalent variant thereof.

[0123] In some embodiments, an antibody comprising any of the Nanobodies described herein may be an antibody comprising two complete heavy chains, each comprising the variable region of a complete heavy chain, and each complete heavy chain further comprising 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 comprise a bivalent 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 cell-mediated cytotoxicity (ADCC). Exemplary Fc region mutations may be L234A, L235A, G237A, and P329G, where position numbers are according to the EU index.

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

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

[0127] To prepare 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 prepared in the form of, for example, a lyophilized powder, a suspension, an aqueous solution, or a suspension. Pharmaceutically acceptable carriers are well known in the art. Methods for preparing aqueous compositions containing active ingredients are known in the art. Generally, these compositions are prepared as injections or sprays, such as liquids that are solutions or suspensions, or may be prepared in solid form suitable for preparation as a solution or suspension before injection or spray.

[0128] (Imaging and / or diagnostic reagents) In another aspect, the invention provides an imaging and / or diagnostic reagent comprising any of the above-mentioned Nanobodies and a detectable label.

[0129] Furthermore, the present invention provides the use of any of the above-mentioned Nanobodies in the manufacture of an imaging and / or diagnostic reagent.

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

[0131] The imaging and / or diagnostic reagents described above may be used for diagnosing diseases or imaging lesions, such as, for example, pulmonary fibrosis imaging, retinal neovascularization imaging, etc. The reagents may also be used as developing reagents for intraoperative image navigation, for example, optical imaging of lesions to guide a surgeon through surgery and thereby enable precise resection of the lesion.

[0132] The detectable label contained in the imaging and / or diagnostic reagent of the present invention may be, for example, a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label or an enzyme label.

[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 also be employed. Such diagnosis and detection may be achieved by linking the Nanobody of the invention to a detectable substance, including, but not limited to, various enzymes, including, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase, or by linking it to prosthetic group complexes, such as, but not limited to, streptavidin-biotin and avidin-biotin.

[0135] Paramagnetic and radioisotope labels may be employed, preferably those detected by Positron Emission Tomography (PET) or Single-Photon Emission Computed Tomography (SPECT). Radiolabels 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), tin ( 113 Sn, 117 Sn), xenon ( 133 Xe), Ytterbium ( 169 Yb, 175 Yb, 177 Yb), yttrium ( 90 Y), zinc ( 65 Zn), various positron-emitting metals for positron emission tomography and non-radioactive paramagnetic metal ions, 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, manganese (Man) ions, and the like. Gunn (Mn) ion, molybdenum (M) ion, neodymium (Nd) ion, osmium (Os) ion, oxygen (O) ion, palladium (Pd) ion, platinum (Pt) ion, rhodium (Rh) ion, ruthenium (Ru) ion, samarium (Sm) ion, sodium (Na) ion, strontium (Sr) ion, terbium (Tb) ion, thulium (Tm) ion, tin (Sn) ion, titanium (Ti) ion, tungsten (W) ion, zirconium (Zi) ion, especially Co +2 , C.R. +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 conjugated by the chloramine-T method.

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

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

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

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

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

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

[0142] Furthermore, another aspect of the present invention provides use of any of the above-mentioned antibodies, polynucleotides, vectors, host cells, immune cells or complexes in the manufacture of a medicament 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 aberrant expression of DDR2 in a subject, the method comprising administering to the subject an effective amount of any of the imaging and / or diagnostic reagents described above. In some embodiments, the method comprises administering to the subject an effective amount of any of the imaging and / or diagnostic reagents described above, detecting and reading a signal produced by the imaging and / or diagnostic reagent, and determining from the intensity of the signal that the subject has or is at risk for a disease mediated by aberrant expression of DDR2. In some embodiments, determining whether the signal exceeds a predetermined threshold, and determining that the subject has a disease mediated by aberrant expression of DDR2 if the signal exceeds the predetermined threshold, in some embodiments, the threshold is a median level from subjects without the disease.

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

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

[0147] In some embodiments, the present invention provides a method for treating a disease mediated by aberrant expression of DDR2 in a subject, the method comprising: (a) administering to the subject an effective amount of any of the imaging and / or diagnostic reagents described above; (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 afflicted with a disease mediated by aberrant expression of DDR2, preferably the threshold being a median level derived from subjects not afflicted with the disease; and (e) administering to the subject determined to be afflicted with the disease a therapy that reduces DDR2 activity and / or expression, e.g., an anti-cancer therapy or an anti-inflammatory therapy (e.g., an anti-fibrotic therapy). In a preferred embodiment, the disease mediated by aberrant expression of DDR2 is pulmonary fibrosis, particularly idiopathic pulmonary fibrosis. In a preferred embodiment, the anti-fibrotic therapy is pirfenidone and / or nintedanib.

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

[0149] The appropriate dose is determined by the clinician based on parameters or factors known in the art or believed or assumed to affect treatment. Generally, the starting dose is slightly lower than the optimal dose, and is then increased by small increments until the desired or optimal effect is achieved, regardless of any adverse events. Important monitoring parameters include, for example, measuring inflammatory symptoms or the levels of inflammatory cytokines produced. [Table 1-1] [Table 1-2] [Table 1-3]

[0150] (Example) Example 1: Antibody screening Anti-DDR2 heavy chain antibodies were screened from alpacas immunized with the human DDR2 extracellular segment (UniProtKB / Swiss-Prot: Q16832.2 aa22-aa399). The antibodies were then sequenced to confirm their VHH moieties. Five nanobodies, designated 1A1, 1A5, 1A12, 1B2, and 1B3, whose sequences are shown in the table above, were obtained for further characterization and experiments.

[0151] Example 2: Expression and purification of the five screened DDR2 nanobodies The experimental steps were as follows: 1. Transformation A 100 μL tube of BL21(DE3) competent cells was placed on ice, 1 μL of plasmid (expression vector for one of the five DDR2 nanobodies screened) was added, and the tube was gently tapped. The tube was then placed on ice for 25–30 minutes, then placed in a water bath at 42°C for 45 seconds, and immediately returned to ice for 2 minutes. 500 μL of antibiotic-free LB medium was added and incubated at 37°C and 250 rpm for 1 hour. The cells were then centrifuged at 3000 rpm for 1 minute, and 100 μL of the supernatant was removed. The mixture was mixed uniformly by pipetting and plated on an ampicillin-containing LB agar plate (LBA+) and incubated overnight at 37°C.

[0152] 2. Strain storage and amplification (1) Preservation A single clone was picked from the plate and added to 5-10 mL of LBA+ liquid medium, incubated overnight at 37°C and 250 rpm. The next day, 750 μL of the bacterial suspension was added to 250 μL of 80% LB glycerol, labeled with the fragment, vector, strain name, and date, mixed uniformly, and stored at -20°C / -80°C. (After storing the strain in glycerol for 3-4 months, it is necessary to re-plate it to activate and protect the strain.)

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

[0154] On the second day, the overnight amplified bacterial solution was inoculated into a 1 L Erlenmeyer flask at a ratio of 6%, and 250 μL of 100 mg / mL Amp was added at the same time. The mixture was treated at 37°C and 250 rpm for 2-3 hours (OD value reached 0.8-1). IPTG was added to a final concentration of 1 mM for induction, and the mixture was treated at 32°C and 200 rpm for 5-6 hours. The bacterial cells were collected (10,000 rpm, 5 minutes) and frozen and stored at -20°C.

[0155] 3. Protein Extraction and Purification (1) Protein extraction The bacterial cells were retrieved from a -20°C environment, thawed at room temperature, resuspended in bacterial lysis solution (500 mL of bacterial cells: 50 mL of lysis solution), disrupted by 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, and the supernatant was retained.

[0156] (2) Protein purification 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 each time). The wash buffer was discarded, and the magnetic beads were added to the bacterial supernatant and mixed end-over-end at 4°C for 1 hour. The centrifuge tube was placed on a magnetic stand. After the supernatant became clear, the tube was transferred to a new centrifuge tube, and 200 μL of new magnetic beads were added. The beads were mixed end-over-end at 4°C for 1 hour. The old magnetic beads were washed 5–6 times with wash buffer, and then eluted three times with elution buffer (1 mL each time for 5 minutes). Finally, all eluates were collected and stored frozen at −20°C.

[0157] 4. Protein Concentration and Medium Exchange (1) Pour off the 20% alcohol from the ultracentrifugal filter, rinse three times with secondary distilled water, add 15 mL of secondary distilled water, and process at 4°C and 4000 rpm for 10 to 12 minutes. Pour off the secondary distilled water from the inner and outer tubes, add the eluate (the amount added should not exceed the scale on the inner tube), and concentrate until the volume is 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, approximately 500-700 μL of the sample in PBS was aspirated and placed in a 1.5 mL EP tube. The ultracentrifugal filter was washed and the inner tube was immersed in 20% ethanol.

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

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

[0161] Example 3: Preliminary identification of binding between DDR2 nanobodies and the extracellular segment of the DDR2 antigen by ELISA The experimental steps were as follows: Five purified DDR2 antibodies bearing a Flag tag, i.e., 1A1, 1A5, 1A12, 1B2, and 1B3, were expressed by fusing a Flag tag to the nanobody gene coding sequence. Then, the cells were coated with the extracellular segment of the DDR2 antigen at 100 ng / well, blocked overnight with goat serum, rinsed three times with PBS, and incubated with anti-DDR2 (five purified DDR2 antibodies, all containing a Flag tag) at 200 ng / well for 1 hour at 37°C, anti-Flag (Sigma, F1804-200UG, 1 mg / mL) at 1:2500 for 1 hour at 37°C, HRP (G&M) at 1:5000 for 30 minutes at 37°C (horseradish peroxidase-conjugated anti-Flag antibody for signal amplification and TMB development), developed for 30 minutes at 37°C, and stopped with 1M HCl. A blank control was also run.

[0162] The results were analyzed as follows: ELISA validation was performed on the five purified and expressed nanobodies and the extracellular segment of the DDR2 antigen that were confirmed to be correct in the previous step, as well as the negative control protein bovine serum albumin (BSA).

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

[0164] The results of the binding analysis with the negative control protein bovine serum albumin are shown in Figure 2B, which showed that all five nanobodies, i.e., 1A1, 1A5, 1A12, 1B2, and 1B3, had no binding activity with BSA protein.

[0165] Therefore, it was confirmed that 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 of DDR2 nanobodies to DDR2 antigen The experimental steps were as follows: A549 cells were infected with adenovirus overexpressing the DDR2 antigen and DDR2 nanobodies (His-tags were fused to the nanobody gene coding sequence to express five purified DDR2 nanobodies with His-tags) for 1 h at 4 °C, washed once with PBS, incubated with anti-His-AF488 as a secondary antibody for 30 min at 4 °C, washed once with PBS, collected by centrifugation at 300 g for 5 min, and resuspended in 200 μL of PBS. Flow cytometry measurements were performed using an ACEA NovoCyte new generation intelligent flow cytometer.

[0167] The results were as follows: The analysis results of binding to the DDR2 antigen are shown in Figure 3, which shows that all five nanobodies, i.e., 1A1, 1A5, 1A12, 1B2, and 1B3, exhibited binding activity to the DDR2 antigen.

[0168] Example 5: Immunofluorescence identification of binding of DDR2 nanobodies to DDR2 antigen 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, incubated with the DDR2 nanobody at room temperature for 2 hours, and then stained with anti-His AF488 secondary antibody. The cells were then stained with DAPI and photographed under a fluorescent microscope.

[0169] The results were analyzed as follows: The analysis results of binding to the DDR2 antigen are shown in Figure 4, which showed that all five nanobodies, i.e., 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: The five purified DDR2 nanobodies, i.e., 1A1, 1A5, 1A12, 1B2, and 1B3, were analyzed for their affinity to the antigen using biolayer interferometry (BLI) in the ForteBio molecular interaction analysis system, and the affinity of the nanobodies tested for 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 was dissolved 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 operation steps were as follows: a. The probe was pre-wetted in kinetics buffer for 10 minutes. b. Set baseline 1. The biosensor was baselined for 180 seconds in kinetics buffer. c. Immobilization: The His-tagged DDR2 antigen extracellular segment was diluted to 20 μg / mL in kinetics buffer and captured by the sensor until it reached 4 nM (300 seconds). d. Set baseline 2. The biosensor was baselined for 60 seconds in kinetics buffer. e. Binding: The antibody solution was diluted to a certain concentration with kinetics buffer (diluted two-fold from 100 nM to 3.125 nM), and the sensor was placed in the antibody solution to allow binding (600 seconds). f. Dissociation: The sensor was dissociated in kinetics buffer (600 seconds). g. Regeneration of the sensor: 10 mM glycine-HCl (pH 1.7) for 5 seconds. h. Neutralization After the sensors were regenerated, they were neutralized in kinetics buffer for 5 seconds. i. Repeat regeneration step g and neutralization step h for a total of three times (30 seconds). j. Set baseline 3. After the sensor was regenerated, 10 mM NiCl was added and held for 60 seconds.

[0173] Pharmacokinetic curves were generated and relevant parameters were calculated. Multiple binding / dissociation curves with appropriate concentration gradients were selected, and all curves were fitted using a 1:1 binding model. The three curves with the best fit were selected and graphed for analysis. Finally, important parameters such as affinity values, binding constants, and dissociation constants were obtained.

[0174] The results were analyzed as follows: The binding analysis results 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. DDR2 nanobody labeling with indocyanine green (ICG) Nb-DDR2 was dissolved in PBS at a concentration of 2 mg / mL and vortexed to mix uniformly.

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

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

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

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

[0180] Unreacted ICG-NHS was removed by multiple centrifugations at 14,000 g for 10 min using a 0.5 mL ultracentrifugal filter, the solution was replaced with 0.9% NaCl, and the protein solution was filtered through a 0.22 μm membrane filter and then stored at 4°C.

[0181] 2. Mouse Lung Tissue Section Imaging Fresh lung tissue from normal mice was sliced ​​at 300 μm thickness. One was an untreated control, one was induced with bleomycin, and the other was induced with mTGF-β cytokine for 72 hours (both treatment groups can promote DDR2 expression). Next, each group was stained with ICG-labeled DDR2 nanobody 1A12 (1 μg / sample) for 30 minutes, followed by multiple rinses with PBS to remove nonspecific staining. Next, the slices were imaged using a bioimaging system. The imaging results are shown in Figure 6.

[0182] The results were analyzed as follows: ICG-labeled purified DDR2 nanobody can specifically bind to DDR2-high-expressing tissues in mice, and can be used for accurate in vitro imaging.

[0183] 3. Lung tissue section imaging in patients with idiopathic pulmonary fibrosis (IPF) Lung tissues from normal donors and patients with idiopathic pulmonary fibrosis (IPF) were used. The lung tissues from IPF patients highly expressed DDR2, while the lungs from normal donors expressed little or no DDR2. They were sliced ​​in vitro at a thickness of 600 μm. The normal group (Normal) served as the control, and the lung tissue slices from IPF patients served as the experimental group. After staining with ICG-labeled DDR2 nanobody 1A12, in vitro bioimaging was performed. The results are shown in Figure 7.

[0184] The results were analyzed as follows: ICG-labeled purified DDR2 nanobody can specifically bind to human DDR2-high-expressing tissues and can be used for accurate in vitro imaging.

[0185] 4. Pig and Rabbit Lung Tissue Section Imaging Fresh lung tissue from normal pigs and rabbits was sliced ​​to a thickness of 600 μm, and bleomycin was added at concentrations of 1 μg / mL, 2 μg / mL, and 4 μg / mL to induce DDR2 expression in the lung tissue. Lung tissue without bleomycin served as a blank control. After 72 hours of induction, 2 μg of ICG-labeled DDR2 nanobody 1A12 was added and incubated at 37°C for 1 hour. The tissue slices were then rinsed five times with PBS to remove unbound probes, and near-infrared imaging was performed using a small animal in vivo imaging system. The results are shown in Figure 8.

[0186] The results were analyzed as follows: ICG-labeled DDR2 nanobody can specifically recognize DDR2, which is highly expressed in pig and rabbit lung tissues, and can be used for accurate in vitro imaging.

[0187] 5. Retinal Neovascularization Imaging Vascular smooth muscle cells, the main component of the vascular wall, express DDR2. Retinas from normal mice and mice with oxygen-induced lesions were collected and applied to glass slides. 2 μg of ICG-labeled DDR2 nanobody 1A12 was added and incubated at room temperature for 1 hour. The samples were then rinsed multiple times with PBS to remove unbound probes. Near-infrared imaging was performed using a small animal in vivo imaging system. The results are shown in Figure 9.

[0188] The results were analyzed as follows: ICG-labeled DDR2 nanobody can specifically recognize DDR2 in the mouse retina, and indicate the status of retinal vascular distribution and oxygen-induced retinal vascular disease.

[0189] Example 8: In vivo imaging results of DDR2 nanobodies The experimental steps were as follows: A mouse pulmonary fibrosis model was established by administering bleomycin at a dose of 1.7 mg / kg via intratracheal intubation to the lungs of C57 mice. In this mouse pulmonary fibrosis model, DDR2 expression was high in the lung tissue. After one week of modeling, 75 μg of ICG-labeled 1A12 nanobody was injected via the tail vein and bioimaging was performed. Mice #1 and #2 were pulmonary fibrosis models, while mouse #3 was a normal mouse. The location of pulmonary fibrosis in the mice was identified by imaging using a small animal bioimaging device.

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

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

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

[0193] Example 10: Identification of anti-DDR2 heterodimeric nanobodies 1A1 to 1A12 by enzymatic cleavage of plasmids In this example, heterodimeric nanobodies 1A1 to 1A12 were constructed by combining the above nanobodies 1A1 and 1A12. The linker sequence was GGGSGGGSGGGS (SEQ ID NO: 27), and the linking site was between the N-terminus of the 1A1 sequence and the C-terminus of 1A12. These antibodies were then characterized and subjected to experiments.

[0194] The experimental steps were as follows: 1. Glycerol-containing strains expressing DDR2 nanobodies 1A1 to 1A12 were gently shaken overnight. The next day, a small volume of plasmid was extracted. 500 ng of plasmid was mixed with 0.5 μL of NcoI and XhoI rapid restriction enzymes, 1 μL of 10x buffer, and supplemented with distilled water to make a 10 μL solution. The mixture was then incubated at 37°C for 30 minutes.

[0195] 2. Weigh out 0.3 g of agarose and place it in an Erlenmeyer flask. Add 30 mL of TAE buffer, place the Erlenmeyer flask in a microwave oven, and heat until the agarose dissolves. Cool the liquid agarose gel to approximately 65°C, add ethidium bromide, and mix thoroughly until homogenous. 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 carried out at 120V for 20 minutes, and the agarose gel was obtained, exposed and developed.

[0197] The results are shown in Figure 13. As is clear from the results, the enzyme cleavage size of the plasmid was correct, and therefore it was the target plasmid.

[0198] Example 11: Coomassie brilliant blue staining of purified proteins of anti-DDR2 heterodimeric nanobodies 1A1 to 1A12 The experimental steps were as follows: 2 μg of purified anti-DDR2 nanobodies 1A1-1A12 were added to 5x protein loading buffer, boiled to denature, 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 and stained for 30 minutes at 60 rpm in a shaker. The staining solution was discarded and replaced with distilled water, followed by washing 2-3 times and photographing under white light.

[0199] The results are shown in Figure 14. As is clear from the results, the purified heterodimeric Nanobodies 1A1 to 1A12 were of the correct size and were free of other impurity bands.

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

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

[0202] Example 13: Flow cytometry identification of the binding activity of anti-DDR2 heterodimeric nanobodies 1A1 to 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, and incubated with 1 μg of anti-DDR2 nanobody 1A1-1A12 for 1 hour. The tube was then centrifuged at 300 g for 5 minutes. The supernatant was discarded, washed three times with PBS, and incubated with anti-His-488 secondary antibody in the dark for 1 hour. The tube was then washed three times with PBS and loaded onto a flow cytometer for analysis.

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

[0204] Example 14: Inhibition of collagen-induced DDR2 protein phosphorylation by anti-DDR2 heterodimeric nanobodies 1A1-1A12 (functional validation) The experimental steps were as follows: 1. Seed DDR2-293T cells into a 6-well plate, one well per group, 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 solutions of antibodies 1A1 to 1A12 at different concentrations were prepared in the same starvation medium. 3. The supernatant from the starvation medium was discarded and replaced with antibody medium at different concentrations and incubated for 2 hours. 4. Collagen was added for stimulation at a collagen concentration of 30 μg / mL. After stimulation for 12 hours, the cells were lysed and proteins were extracted. 5. The phosphorylation level of DDR2 protein in cells from each group was measured by WB experiment.

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

[0206] Example 15: Chimeric Antigen Receptors A CAR molecule vector was constructed using the DDR2 nanobody 1A12 sequence (see Figure 18), packaged into adenovirus, and used to infect mouse primary macrophages to obtain αDDR2 CAR-M cells, which were used in the following experiments.

[0207] Empty-M (uninfected macrophages) and αDDR2 CAR-M were co-cultured with 293T-DDR2 cells at a 1:1 ratio. The FITC and APC double-positive cells were counted as the phagocytosis rate of 293T-DDR2 cells. The results are shown as mean ± standard error (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 phagocytized DDR2-positive 293T cells at a significantly higher rate.

[0208] Empty-M, αDDR2 CAR-M 5×10 each 5 1A12 was injected intravenously into the tail vein of mice with bleomycin-induced left lung modeling. On day 15, the mice were euthanized, and the left lungs were removed. CD68 immunohistochemistry was performed on normal mice without macrophage injections as controls. The results are shown in Figure 20. The CD68 immunohistochemistry results indicated the following: fine brown granules were observed in lung sections from all mice in each group. In the NC and BLM+Empty-M groups, weak CD68 expression was observed in lung tissue sections, mainly distributed around the airways and in the alveolar septa. In the BLM+CAR-M group, CD68 expression was significantly enhanced and uniformly distributed. These results suggest that adoptively transferred αDDR2 CAR-M can localize to areas with high DDR2 expression, i.e., the left lung of mice. CAR-M cells constructed with 1A12 were shown to concentrate and localize in tissues with high DDR2 expression in mice.

[0209] We used lattice light-sheet super-resolution microscopy to verify the targeted phagocytosis of αDDR2 CAR-M against 293T-DDR2. αDDR2 CAR-M possesses green fluorescence, and 293T-DDR2 was stained red with Dil (an orange-red fluorescent dye). The two were mixed at a 1:1 ratio, loaded, and then imaged continuously 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. Note that the yellow cells were likely formed when the green CAR-M fully engulfed the red 293T-DDR2, resulting in the red dye on the 293T-DDR2 entering the CAR-M. This demonstrated that CAR-M cells constructed with 1A12 can target and effectively engulf DDR2-positive 293T cells.

[0210] Mice in each group were given 40 μL of 0.75 mg / mL bleomycin (BLM) instilled into the trachea, and on the 8th day, micro-CT scans were performed. Mice that successfully modeled the left lung were screened and divided into groups according to the severity of the lesion. On the 10th day, PBS, Empty-M, or αDDR2 CAR-M was injected via the tail vein. On the 26th day, micro-CT scans were performed again to evaluate the therapeutic effect. On the 28th day, the mice were euthanized, and the left lung was removed and fixed.

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

[0212] Body weight was measured in the CAR-M group, with weight loss followed by a tendency to increase, and by day 28, mice had almost returned to their original weight levels. The other two groups showed some weight recovery, but were unable to fully recover. Survival curves showed no deaths in the CAR-M group, while some mice died in the other two groups, with the PBS group showing even more severe disease. CT scans showed that the lung lesions in most mice in the CAR-M group had recovered by day 8, while the other two groups showed no clear recovery or worsened. In summary, compared with controls, mice injected with αDDR2 CAR-M had significantly higher body weights, significantly increased survival rates (no deaths by day 28), and controlled lesion severity, demonstrating a significant therapeutic effect.

[0213] Example 16: Nuclides ( 64 Cu, 68 Labeling procedure for nanobody 1A12 with conventional nuclides such as Ga 1. Integration with NOTA (1) The antibody solution was centrifuged at 12,000 g, and the supernatant was collected. The solvent for the antibody solution was replaced with 0.1 M ammonium acetate (pH = 7). After the replacement, the concentration of nanobody 1A12-cys was 1.33 mg / mL. The concentration was measured before and after the replacement and was based on the concentration after the replacement.

[0214] (2) 50 mM Mal-NOTA (dissolved in DMSO) was used, and the molar ratio of antibody to Mal-NOTA was 1:5. Mal-NOTA was diluted with DMSO to less than 10% of the reaction system volume (for example, if the reaction system was 1 mL, Mal-NOTA was diluted to 80-90 μL and shaken to mix uniformly, thereby dissolving Mal-NOTA sufficiently in DMSO). The Mal-NOTA solution was added to the antibody solution in several portions, 10 μL per addition, and after each addition, the solution was shaken for several tens of seconds to mix uniformly. The pH of the solution was adjusted to 7 (the reaction solvent was 0.1 M ammonium acetate, so the pH did not need to be adjusted and could be confirmed by measurement). The solution was then placed on a shaker and reacted at room temperature for 2 hours. The shaker rotation speed was 150 rpm.

[0215] (3) After the reaction was completed, the antibody solution was centrifuged at 12,000 g, and the supernatant was collected. The antibody solution was purified and concentrated with 0.1 M ammonium acetate to a volume of 50 to 80 μL per 100 μg of antibody, sealed with a sealing film, and stored in a refrigerator at -80°C.

[0216] 2. 68 Ga, 64 Labeling with conventional nuclides such as Cu (1) 68 Ga, 64 Cu or other nuclides are added to the precursor solution ( 64 In the case of Cu, the concentration was as high as possible, with the total radioactivity input for 100 μg of precursor being approximately 1.5 mCi, and the final reaction volume was 150 μL or less), and the mixture was mixed uniformly and the pH was adjusted to 4-5 (generally, the pH was 4-5 after uniform mixing without any adjustment).

[0217] (2) If you have a vibrating thermostat, place it in the vibrating thermostat and let it react at 37°C for 2 hours. If you don't have one, heat it to 37°C and let it react for 2 hours (in a water bath, oven, etc.).

[0218] (3) The sample was spotted on a filter paper for paper chromatography, developed with a developing solution, and the labeling rate was measured by iTLC (the developing solution was sodium citrate).

[0219] Example 17: Nuclides 68 Imaging of Ga-labeled nanobody 1A12 In the procedure described in Example 16, 68 Ga-labeled nanobody 1A12 was prepared.

[0220] Bleomycin (BLM)-induced model mice on day 15 (BLM-15d) and control normal mice (Ctrl) were treated with 200 μCi / mouse. 68Ga-labeled nanobody 1A12 was administered via the tail vein and PET-CT imaging was performed. The imaging results are shown in Figure 23. The results show that BLM-modeled mice showed significantly higher lung uptake than normal mice. This indicates that nanobodies conjugated with nuclides can be used for PET-CT imaging.

[0221] While the present invention has been specifically disclosed in terms of preferred embodiments and optional features, it is to be understood that those skilled in the art may make amendments, improvements, and modifications to the invention disclosed herein, and that these amendments, improvements, and modifications are considered to be within the scope of the present invention. The materials, methods, and examples provided herein are representative and illustrative of preferred embodiments and are not intended as limitations on the scope of the invention.

Claims

1. A Nanobody that binds to DDR2, comprising CDR1, CDR2 and CDR3, wherein said CDR1, CDR2 and CDR3 comprise or are selected from the CDR1, CDR2 and CDR3 of any Nanobody having the amino acid sequence 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.

2. The nanobody of claim 1 , wherein the CDR1, CDR1 and CDR3 are defined according to any of the IMGT, Kabat, Chothia, Contact or Martin definitions.

3. (a) CDR1 comprises or is the sequence set forth in SEQ ID NO: 1 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 2 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 3 or an equivalent variant thereof; (b) CDR1 comprises or is the sequence set forth in SEQ ID NO:5 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO:6 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO:7 or an equivalent variant thereof; (c) CDR1 comprises or is the sequence set forth in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 11 or an equivalent variant thereof; (d) CDR1 comprises or is the sequence set forth in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 15 or an equivalent variant thereof; or (e) CDR1 comprises or is the sequence set forth in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence set forth in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence set forth in SEQ ID NO: 19 or an equivalent variant thereof.

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

5. 2. The Nanobody of claim 1, wherein the Nanobody comprises or is selected from the sequence of any 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.

6. A polynucleotide comprising a polynucleotide encoding a Nanobody according to any one of claims 1 to 5.

7. 7. The polynucleotide of claim 6, wherein the polynucleotide comprises or is selected from any of the sequences of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25 or equivalent variants thereof.

8. A vector comprising the polynucleotide of claim 6 or 7.

9. A non-human host cell comprising the vector of 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 an antigen comprises the nanobody of any one of claims 1 to 5.

11. The chimeric antigen receptor of 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. A modified immune cell expressing the chimeric antigen receptor of claim 10 or 11.

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

14. 6. A bispecific antibody comprising a first binding moiety that binds to a first antigen and a second binding moiety that binds to a second antigen, wherein the first binding moiety comprises a nanobody according to any one of claims 1 to 5.

15. The bispecific antibody of claim 14, wherein the second binding moiety comprises a nanobody according to any one of claims 1 to 5.

16. The bispecific antibody of claim 15 , wherein the nanobody contained in the first binding moiety and the nanobody contained in the second binding moiety are different.

17. An antibody-drug conjugate comprising an antibody that targets DDR2, a drug, and a linker that links the antibody and the drug, wherein the antibody that targets DDR2 comprises or is a nanobody according to any one of claims 1 to 5.

18. 10. A siRNA complex targeting DDR2-positive cells, comprising: (i) an antibody targeting DDR2 that comprises or is a Nanobody according to any one of claims 1 to 5; (ii) an siRNA that inhibits expression of a survival gene in DDR2-positive cells; and (iii) a linker located between said Nanobody and said siRNA.

19. 6. A reagent for imaging and / or diagnosis comprising a Nanobody according to any one of claims 1 to 5 and a detectable label linked to said Nanobody.

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

Images