Anti-DDR2 nano antibody and application thereof

Abstract

Nanobodies that bind to DDR2, as well as various derivatives of the nanobodies. Pharmaceutical compositions comprising these antibodies and derivatives and pharmaceutical uses thereof. The anti-DDR2 nano antibody has strong binding activity, can be used for diagnosing, preventing and / or treating diseases (such as cancers and inflammatory diseases) mediated by abnormal expression of DDR2, and can also be used for imaging cells expressing DDR2, such as biopsy navigation and intraoperative navigation.

Description

Anti-DDR2 nanoantibodies and their applications 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 the nanobodies. The present invention also relates to pharmaceutical compositions comprising these antibodies and derivatives and their medical uses. Background Art

[0002] Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase (RTK) that utilizes the extracellular matrix protein collagen as its ligand. In addition to its kinase function, DDR2 also promotes cell adhesion by activating β1-integrin. DDR2's unique function is to mediate the transmission of extracellular matrix signals into the cell, maintaining a balanced regulation of the extracellular matrix and participating in the regulation of cell growth, differentiation, and metabolism. While activation of DDR2 by extracellular matrix collagen is essential for normal development and tissue homeostasis, aberrant activation of these receptors following injury or disease is detrimental.

[0003] The role of DDR2 in regulating the extracellular matrix suggests that dysregulation of its signaling may contribute to carcinogenesis, 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 on adjacent sections of human ovarian or lung cancer has shown that DDR2 is detected in stromal cells surrounding the tumor. Therefore, DDR2 is considered to be crucial for the metastasis of many cancer cells (such as breast cancer cells) and is also an important target for many other cancers (such as ovarian, lung, head and neck, and pancreatic cancers).

[0004] DDR2 is also considered an important target for inflammation, such as arthritis (such as osteoarthritis, rheumatoid arthritis) and fibrosis (such as pulmonary fibrosis, cirrhosis, renal fibrosis or skin fibrosis). DDR2 is mainly expressed in interstitial cells such as fibroblasts, myofibroblasts, smooth muscle cells in the kidney, skin, lung, heart and connective tissue. A lot of evidence shows that abnormal expression of DDR2 is associated with the progression of various diseases, such as inflammation, liver fibrosis, renal fibrosis, pulmonary fibrosis, skin scars and atherosclerosis. In a mouse inflammatory model study, it was found that DDR2 expression was upregulated in the knee joints of elderly mice. In a rat model study of rheumatoid arthritis, it was found that DDR2 expression was upregulated in its synovial cells.

[0005] WO2019243431A1 discloses that targeting DDR2 through imatinib can enhance the anti-tumor efficacy of BRAF inhibitors in a melanoma cell-derived xenograft model, delay the development of acquired drug resistance and offset tumor fibrosis induced by targeted therapy.

[0006] Therefore, the development of high-affinity antibodies targeting DDR2 has broad practical significance for the imaging, diagnosis, prevention and treatment of cancer, inflammation, fibrosis and other diseases.

[0007] Summary of the Invention

[0008] In a first aspect, the present invention provides a Nanobody that binds to DDR2, wherein the Nanobody comprises CDR1, CDR2, CDR3, and the CDR1, CDR2 and CDR3 comprise or are selected from the CDR1, CDR2 and CDR3 of a Nanobody having an amino acid sequence of any one of 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.

[0009] In some embodiments, the CDR1, CDR2, and CDR3 are defined based on any one of the IMGT, Kabat, Chothia, Contact, or Martin definitions.

[0010] In some embodiments, the Nanobody comprises CDR1, CDR2, CDR3, and

[0011] (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;

[0012] (b) CDR1 comprises or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof;

[0013] (c) CDR1 comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof;

[0014] (d) CDR1 comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof; or

[0015] (e) CDR1 comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0016] In some embodiments, the Nanobody comprises or is any one sequence selected from 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 thereof.

[0017] In another aspect, the present invention provides a polynucleotide comprising a polynucleotide encoding any of the above-described antibodies. In another aspect, the present invention also provides a vector comprising the aforementioned polynucleotide. In another aspect, the present invention also provides a non-human host cell comprising the aforementioned vector. Furthermore, 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.

[0018] 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 an 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 aforementioned polynucleotide. In another aspect, the present invention provides a modified immune cell expressing the chimeric antigen receptor.

[0019] 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 the first binding moiety comprises any one of the Nanobodies described in the first aspect.

[0020] In a fourth aspect, the present invention provides an antibody-drug conjugate comprising an antibody targeting DDR2, a drug, and a linker connecting the Nanobody and the drug, wherein the antibody targeting DDR2 comprises or is any Nanobody described in the first aspect.

[0021] In a fifth aspect, the present invention provides a siRNA conjugate targeting DDR2-positive cells, comprising: (i) an antibody targeting DDR2, which comprises or is any one of the nanoantibodies described in the first aspect, (ii) an siRNA that inhibits the expression of survival genes in DDR2-positive cells, and (iii) a linker located between the nanoantibody and the siRNA.

[0022] In a sixth aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the above antibodies, polynucleotides, vectors, host cells, immune cells and / or conjugates, and a pharmaceutically acceptable carrier.

[0023] In a seventh aspect, the present invention provides use of any of the above antibodies, polynucleotides, vectors, host cells, immune cells or conjugates in the preparation of a medicament for treating and / or preventing diseases mediated by abnormal DDR2 expression.

[0024] In an eighth aspect, the present invention provides any one of the above antibodies, polynucleotides, vectors, host cells, immune cells or conjugates for use in treating and / or preventing diseases mediated by abnormal DDR2 expression.

[0025] In a ninth aspect, the present invention provides a method for treating and / or preventing a disease mediated by abnormal DDR2 expression, comprising administering to a subject an effective amount of any one of the above antibodies, polynucleotides, vectors, host cells, immune cells or conjugates.

[0026] In a tenth aspect, the present invention provides an agent for imaging and / or diagnosis, comprising any one of the Nanobodies described in the first aspect and a detectable label linked to the Nanobody.

[0027] In an eleventh aspect, the present invention provides use of the reagent for imaging and / or diagnosis according to the tenth aspect in preparing a kit for diagnosing a disease mediated by abnormal DDR2 expression in a subject.

[0028] In a twelfth aspect, the present invention provides the reagent for imaging and / or diagnosis according to the tenth aspect for diagnosing a disease mediated by abnormal DDR2 expression in a subject.

[0029] In a thirteenth aspect, the present invention provides a method for diagnosing a disease mediated by abnormal DDR2 expression in a subject, the method comprising administering to the subject an effective amount of the imaging and / or diagnostic agent described in the tenth aspect. In some embodiments, the method comprises: administering to the subject an effective amount of the imaging and / or diagnostic agent described in the tenth aspect; detecting and reading a signal generated by the imaging and / or diagnostic agent; and determining, based on the intensity of the signal, that the subject has or is at risk of having the disease mediated by abnormal DDR2 expression.

[0030] In a fourteenth aspect, the present invention provides a method for treating a disease mediated by abnormal expression of DDR2 in a subject, the method comprising: (a) administering to the subject an effective amount of any of the above-mentioned imaging and / or diagnostic reagents; (b) detecting and reading a signal generated by the imaging and / or diagnostic reagent; (c) determining whether the signal exceeds a predetermined threshold, and when the signal exceeds the predetermined threshold, determining that the subject has a disease mediated by abnormal expression of DDR2, preferably wherein the threshold is the median level from subjects without the disease; and (e) administering to the subject determined to have the disease a therapy that reduces DDR2 activity and / or expression, such as an anti-cancer therapy or an anti-inflammatory therapy, such as an anti-fibrotic therapy. In a preferred embodiment, the anti-fibrotic therapy is pirfenidone and / or nintedanib.

[0031] In the above-mentioned related aspects, the diseases mediated by abnormal 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, skin cancer. In some embodiments, the inflammatory disease includes but is not limited to rheumatoid arthritis, osteoarthritis, skin scars, atherosclerosis, retinal vascular lesions, and fibrotic diseases. The fibrotic disease includes but is not limited to pulmonary fibrosis (particularly idiopathic pulmonary fibrosis), liver fibrosis, cirrhosis, skin fibrosis, renal fibrosis, pancreatic fibrosis, systemic sclerosis, cardiac fibrosis and macular degeneration.

[0032] The anti-DDR2 nanoantibodies of the present invention have strong binding activity and can be used to diagnose, prevent and / or treat diseases mediated by abnormal DDR2 expression (such as cancer and inflammatory diseases). They can also be used to image cells expressing DDR2, such as for biopsy navigation and intraoperative navigation.

[0033] Other aspects and advantages of the present invention will become apparent from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1: SDS-PAGE results of DDR2 nanobody expression and purification.

[0035] Figure 2: DDR2 nanobody ELISA validation diagram.

[0036] Figure 3: Flow cytometry results of DDR2 nanoantibody.

[0037] Figure 4: Immunofluorescence image of DDR2 nanobody.

[0038] Figure 5: Affinity constants of DDR2 nanobodies and the extracellular segment of DDR2 antigen.

[0039] Figure 6: In vitro imaging results of mouse lung tissue with DDR2 nanoantibodies.

[0040] Figure 7: In vitro imaging results of human lung tissue using DDR2 nanoantibodies.

[0041] Figure 8: In vitro imaging results of pig and rabbit lung tissues of DDR2 nanoantibodies.

[0042] Figure 9: In vitro retinal imaging results of DDR2 nanoantibodies.

[0043] Figure 10: In vivo imaging results of DDR2 nanobody.

[0044] Figure 11: Western Blot (WB) image showing that Nanobody 1A12 inhibits collagen1-induced DDR2 protein phosphorylation.

[0045] Figure 12: Statistical graph showing that Nanobody 1A12 inhibits collagen1-induced DDR2 protein phosphorylation.

[0046] Figure 13: DNA electrophoresis diagram of enzyme digestion identification of plasmid expressing anti-DDR2 nanobody.

[0047] Figure 14: SDS-PAGE electrophoresis staining of purified anti-DDR2 nanobody.

[0048] Figure 15: Western blot image of purified anti-DDR2 nanobody.

[0049] Figure 16: Flow cytometry analysis of anti-DDR2 nanobody activity.

[0050] Figure 17: Western blot image of anti-DDR2 nanobody inhibiting collagen-induced DDR2 protein phosphorylation.

[0051] Figure 18: Schematic diagram of the CAR molecule constructed using the DDR2 nanobody 1A12 sequence.

[0052] Figure 19: Graph showing the ratio of DDR2-positive 293T cells engulfed by mouse macrophages infected with adenovirus packaged with the CAR molecule constructed by 1A12.

[0053] Figure 20: Localization map of CAR-M constructed with 1A12.

[0054] FIG21 : Targeted phagocytosis of 293T-DDR2 by CAR-M constructed with 1A12.

[0055] Figure 22: In vivo therapeutic effect of CAR-M constructed with 1A12.

[0056] Figure 23: BLM model mice 15 days68 Ga PET-CT imaging. DETAILED DESCRIPTION

[0057] definition

[0058] In the present invention, "about" means that a value is within an acceptable error range for the specific value determined by one of ordinary skill in the art, which value depends in part on how the measurement or determination is made (i.e., the limitations of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation per practice in the art. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%. In addition, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise indicated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0059] Compositions or methods described herein as "comprising" one or more recited elements or steps are open-ended, meaning that the recited elements or steps are required, but other elements or steps may be added within the scope of the composition or method. It should also be understood that any composition or method described as "comprising" one or more recited elements or steps also describes a corresponding, more limited composition or method that "consists essentially of" the recited elements or steps, meaning that the composition or method includes the recited elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristics of the composition or method.

[0060] As used herein, the term "antibody" refers to any form of an antibody that exhibits a desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or by inhibiting ligand-induced receptor signal transduction). "Antibody fragment" and "antigen-binding fragment" refer to antigen-binding fragments of antibodies and antibody analogs, which typically include at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. In some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.

[0061] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homologous antibody population, i.e., the individual antibodies constituting the population are identical except for possible natural mutants that may be present in small amounts. Monoclonal antibodies are highly specific and can be directed against a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include a variety of different antibodies directed against a plurality of different determinants (epitopes), each monoclonal antibody is directed against only a single determinant on the antigen. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homologous antibody population and is not to be construed as requiring the preparation of the antibody by any particular method. For example, the monoclonal antibodies used in the present invention can be prepared by hybridoma or recombinant DNA methods.

[0062] Monoclonal antibodies can include "chimeric," humanized, or fully human antibodies. In some embodiments, antibodies form part of a larger biomolecule, such as a fusion protein or antibody-drug conjugate. Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, antibody fragments retain at least 10% of the parent binding activity. Preferably, antibody fragments retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody for the target.

[0063] As used herein, the term "heavy chain antibody" refers to an antibody that lacks a light chain and consists only of a heavy chain, comprising two constant regions (CH2 and CH3), a hinge region, and a heavy chain variable region (i.e., VHH). Examples include, but are not limited to, natural heavy chain antibodies, antibodies that are naturally free of light chains, heavy chain antibodies derived from conventional 4-chain antibodies, and engineered antibodies. Heavy chain antibodies can be from species of the Camelidae family, such as antibodies produced in camels, llamas, dromedaries, alpacas, and draft horses. Other species except the Camelidae family can produce heavy chain antibodies that naturally lack light chains; such heavy chain antibodies are within the scope of the present invention.

[0064] As used herein, the term "nanobody" refers to a single-domain antibody consisting only of the variable region of the heavy chain obtained by cloning the variable region of a heavy chain antibody, also known as VHH (Variable domain of heavy chain of heavy chain antibody) or a single-domain antibody, which is the smallest functional antigen-binding fragment. Nanobodies recognize antigens with high specificity and affinity similar to IgG antibodies, but can better penetrate tumor tissue due to their smaller size (~15kD). In addition, nanobodies are resistant to extreme pH, thermal denaturation, proteolysis, solvents, and detergents. They can be expressed and produced with high yield and high solubility.

[0065] As used herein, the term "humanized antibody" refers to an antibody comprising CDRs of an antibody derived from mammals other than humans, and framework regions (FRs) and constant regions of a human antibody.

[0066] An "equivalent variant" of an antibody or polypeptide refers to an antibody or polypeptide that has a certain degree of homology or sequence identity to the amino acid sequence of the antibody or polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In some aspects, an equivalent variant has one, two, three, four or five additions, deletions, substitutions and combinations thereof compared to a reference antibody or polypeptide. In some aspects, an equivalent variant of an antibody or polypeptide retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the reference sequence.

[0067] As used herein, a "variant" of a sequence is one that differs from the indicated sequence at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0068] As used herein, "% identity" between two sequences refers to a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and the determination of % identity between two sequences can be accomplished using a mathematical algorithm.

[0069] "Conservative substitutions" refer to amino acid substitutions known to those skilled in the art that are made without generally altering the biological activity of the resulting molecule. In general, it is recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter or substantially change the biological activity. "No alteration or no substantial alteration" means that one or more aspects of the polypeptide differ by no more than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% when compared to the subject being compared, when measured by the same or similar method.

[0070] Generally, CDR3 and CDR3 are considered to play a more important role in antigen recognition than other CDRs. Therefore, in the case of substitution, the present invention preferably performs conservative substitutions on CDRs other than CDR3. In some embodiments, CDR3 is not substituted. Preferred amino acid substitutions include, but are not limited to, substitutions that (1) reduce sensitivity to proteolysis, (2) reduce sensitivity to oxidation, (3) change the binding affinity for forming protein complexes, and (4) provide or modify other physicochemical or functional properties of these analogs. Analogs may include mutations of various sequences other than naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be performed in naturally occurring sequences (preferably in polypeptide portions outside the region forming intermolecular contacts). Conservative amino acid substitutions should not substantially change the structural characteristics of the parent sequence (e.g., amino acid substitutions should not tend to destroy the helix present in the parent sequence, or characterize other secondary structure types that destroy the parent sequence).

[0071] It is anticipated that the binding domain of the antibody or antigen-binding fragment thereof of the present invention may carry a signal peptide, which is typically located at the N-terminus of the secretory protein and generally consists of 15 to 30 amino acids. After the signal peptide sequence is synthesized, it is recognized by the signal recognition particle (SRP), protein synthesis is paused or slowed, and the signal recognition particle carries the ribosome to the endoplasmic reticulum, where protein synthesis resumes. Under the guidance of the signal peptide, the newly synthesized protein enters the endoplasmic reticulum lumen, and the signal peptide sequence is removed by the action of signal peptidase. If the termination transport sequence is present at the C-terminus of the nascent peptide chain, it may not be removed by the signal peptidase, such as ovalbumin, which contains an internal signal peptide. Neither its precursor nor its mature form is removed by the signal peptidase.

[0072] 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 the determination of the presence or absence of a binding reaction for the protein in a heterogeneous population of proteins and / or other biological reagents. Thus, under specified conditions, a specific ligand / antigen binds to a specific receptor / antibody and does not bind to other proteins present in the sample in significant amounts. "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, three, four, five, six, seven, eight or more amino acids of a respective target molecule. The "specific binding" of an antibody is primarily characterized by two parameters: a qualitative parameter (binding epitope or antibody binding site) and a quantitative parameter (binding affinity or binding strength). Antibody binding epitopes can be determined by FACS, peptide dot epitope mapping, mass spectrometry or peptide ELISA. Biacore and / or ELISA methods can determine the binding strength of an antibody to a specific epitope. Signal-to-noise ratio is typically used as a representative measurement and calculation method for binding specificity. In such a signal-to-noise ratio, the signal represents the strength of the antibody binding to the target epitope, while the noise represents the strength of the antibody binding to other non-target epitopes. Preferably, a signal-to-noise ratio of about 50 for the target epitope can be considered to indicate that the antibody being evaluated binds to the target epitope in a specific manner, i.e., "specifically binds". An antigen binding protein (including an antibody) "specifically binds" to an antigen if the antigen binding protein (including an antibody) binds to the antigen with a high binding affinity as determined by an affinity constant (KD) value. In some embodiments, the affinity constant KD is less than 10 -9 M. As used herein, the term "KD" refers to the affinity constant for a specific antibody-antigen interaction.

[0073] As used herein, the term "patient" or "subject" refers to any organism to which a provided antibody, derivative thereof, or pharmaceutical composition is or can be administered for experimental, diagnostic, preventive, cosmetic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the subject is a human. In some embodiments, the subject suffers from or is susceptible to one or more disorders or conditions. The patient may exhibit one or more symptoms of a disorder or condition, or may have been diagnosed with one or more disorders or conditions. In some embodiments, the patient is receiving or has received a therapy for the diagnosis and / or treatment of such a disease, disorder, or condition.

[0074] In the present invention, the term "treatment" refers to therapeutic and preventive measures that prevent or slow the occurrence or progression of undesirable physiological changes or symptoms in a subject. Favorable or desired clinical effects include, but are not limited to, relief of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, alleviation or alleviation of the disease state, and partial or complete cure of the disease, regardless of whether the above effects are detectable. "Treatment" may also refer to prolonged survival compared to no treatment. Subjects in need of treatment include subjects who already have the disease or condition, as well as subjects who are likely to have the disease or condition, or subjects who are to prevent the disease or condition.

[0075] As used herein, the term "prevention" includes preventing or slowing the onset of clinically significant disease development or preventing or slowing the onset of a preclinically significant disease stage in an at-risk individual. This includes prophylactic treatment of individuals at risk of developing disease.

[0076] When used in reference to an animal, human, subject, cell, tissue, organ, or biological fluid, "administering," "treating," or "preventing" means contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering" and "treating" can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating cells includes contacting an agent with cells and contacting an agent with a fluid, wherein the fluid is contacted with cells. "Administering" and "treating" also mean the in vitro and ex vivo treatment of cells, for example, by an agent, diagnostic agent, binding composition, or by other cells.

[0077] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of an antibody or derivative thereof of the present invention that is effective in preventing or alleviating the disease or condition to be treated when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject. A therapeutically effective dose further refers to an amount sufficient to cause a reduction in symptoms, such as treating, curing, preventing, or alleviating a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviating of the symptoms. When administered to an individual, the therapeutically effective amount refers to that individual ingredient. When administered in combination, the therapeutically effective amount refers to the combined amount of the active ingredients that produces the therapeutic effect, regardless of whether they are administered in combination, sequentially, or simultaneously. A therapeutically effective amount will alleviate symptoms typically by at least 10%; typically by at least 20%; preferably by at least about 30%; more preferably by at least 40% and most preferably by at least 50%.

[0078] As used herein, "pharmaceutically acceptable carriers" include materials that, when combined with the active ingredient of a composition, allow the ingredient to maintain biological activity and do not cause a destructive reaction with the subject's immune system. These carriers may include stabilizers, preservatives, salt or sugar complexes or crystals, etc. "Pharmaceutically acceptable" refers to molecules and ingredients that do not produce an allergic reaction or similar undesirable reaction when administered to a human.

[0079] As used herein, "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 from which the extracellular domain is derived, and at least one intracellular domain. "Chimeric Antigen Receptor (CAR)" is 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 that can bind to a specific antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function in a cell as a domain that transmits signals to cause activation or inhibition of a biological process.

[0080] As used herein, the term "antibody drug conjugate" or "ADC" refers to an antibody to which a therapeutically active substance or active pharmaceutical ingredient has been covalently coupled so that the therapeutically active substance or active pharmaceutical ingredient can be targeted to the binding target of the antibody to exhibit its pharmacological function. The therapeutically active substance or active pharmaceutical ingredient can be a cytotoxin that can kill the cells targeted by the ADC. Covalent attachment of the therapeutically active substance, active pharmaceutical ingredient, or cytotoxin can be performed in a non-site-specific manner using standard chemical linkers that couple payloads to lysine or cysteine ​​residues, or preferably, conjugation is performed in a site-specific manner, which allows complete control over the site of conjugation and the drug-to-antibody ratio (DAR) of the resulting ADC.

[0081] The term "siRNA" is a double-stranded RNA with a length of 20 to 25 nucleotides, comprising two antiparallel and substantially complementary nucleic acid chains. Relative to the target RNA, these two nucleic acid chains are named as the sense strand and the antisense strand. The siRNA portion triggers the degradation of the target RNA (e.g., mRNA) by a post-transcriptional gene silencing mechanism (referred to as RNA interference or RNAi herein). Typically, the nucleotides of most of the each chain of the siRNA portion are ribonucleotides, but each or both of the two chains can also include one or more non-ribonucleotides, e.g., deoxyribonucleotides and / or modified nucleotides. In addition, the siRNA portion can include ribonucleotides with chemical modifications, or can include substantial modifications at multiple nucleotide locations. These modifications can include modifications of all types disclosed herein or known in the art. Any such modification is intended to be included within the definition of "siRNA."

[0082] The term "survival genes" is used interchangeably with "essential genes" to refer to genes that are necessary for the survival or proliferation of a particular cell. Functional defects in one of these genes will cause the cell to exhibit a lethal phenotype and be unable to survive or reproduce. Typical survival genes are genes encoding key proteases in the 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, which synthesizes pre-mRNA and many functional non-coding RNAs, and together with the second largest subunit forms the polymerase active center), POLR2B (DNA-directed RNA polymerase II subunit RPB2, the second largest subunit of RNA polymerase II, which together with the first largest subunit forms the polymerase active center), DKC1 (H / ACA ribonucleoprotein complex subunit DKC1, a ribonucleoprotein complex subunit), CENPE (Centromere-associated protein E, mitosis-associated protein E), eIF-3b (eukaryotic translation initiation factor 3 subunit b, eukaryotic transcription initiation factor 3 subunit b), etc. Accordingly, the term "survival genes in DDR2-positive cells" refers to genes essential for the survival or proliferation of DDR2-positive cells. Methods for determining whether a particular gene in human cells is an essential gene are known in the art, for example, see 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.

[0083] Anti-DDR2 nanobody

[0084] The present invention obtains camel-derived heavy chain antibodies and then obtains corresponding nanobodies by immunizing alpacas with the extracellular segment of human DDR2 (whose amino acid sequence is UniProtKB / Swiss-Prot: Q16832.2 aa 22 to aa 399).

[0085] In one aspect of the invention, the invention provides a Nanobody that binds to DDR2, wherein the Nanobody comprises CDR1, CDR2, CDR3, and the CDR1, CDR2 and CDR3 comprise or are selected from the CDR1, CDR2 and CDR3 of a Nanobody having an amino acid sequence of any one of 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.

[0086] In some embodiments, the CDR1, CDR1 and CDR3 are defined based on any one of the IMGT, Kabat, Chothia, Contact or Martin definitions. In some embodiments, the CDR1, CDR1 and CDR3 are defined based on the IMGT definition.

[0087] In some embodiments, the Nanobody comprises CDR1, CDR2, CDR3, and:

[0088] (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;

[0089] (b) CDR1 comprises or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof;

[0090] (c) CDR1 comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof;

[0091] (d) CDR1 comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof; or

[0092] (e) CDR1 comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0093] In some embodiments, the Nanobody comprises or is any one sequence selected from 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 thereof.

[0094] In some embodiments, the equivalent variant of each of the CDR1, CDR2, and CDR3 refers to a single amino acid substitution, deletion, or insertion compared to the reference sequence.

[0095] In some embodiments, an equivalent variant of each of the Nanobodies refers to a sequence identity of at least 75%, 80%, 85%, 90%, 95%, 98% or 99% to any one of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16 and SEQ ID NO: 20, and having the same or equivalent CDR1, CDR2 and CDR3.

[0096] In some embodiments, the Nanobody comprises CDR1, CDR2, CDR3 and:

[0097] (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;

[0098] (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;

[0099] (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;

[0100] (d) CDR1 comprises the sequence shown in SEQ ID NO: 13, CDR2 comprises the sequence shown in SEQ ID NO: 14, and CDR3 comprises the sequence shown in SEQ ID NO: 15; or

[0101] (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.

[0102] In some embodiments, the Nanobody comprises any one sequence selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16 and SEQ ID NO:20.

[0103] In some embodiments, the Nanobody comprises CDR1, CDR2, CDR3 and:

[0104] (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;

[0105] (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;

[0106] (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;

[0107] (d) CDR1 is the sequence shown in SEQ ID NO: 13, CDR2 is the sequence shown in SEQ ID NO: 14, and CDR3 is the sequence shown in SEQ ID NO: 15; or

[0108] (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.

[0109] In some embodiments, the Nanobody is any one sequence selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16 and SEQ ID NO:20.

[0110] In some embodiments, the substitutions described herein are conservative substitutions.

[0111] "Conservative (amino acid) substitutions" refer to substitutions of amino acid residues with amino acids having similar side chains. Families of amino acid residues with similar side chains have been 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-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 substituted with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be substituted with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0112] It will also be understood by those skilled in the art that the antibodies disclosed herein can be modified so that their amino acid sequences differ from the naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a given protein can be similar to the starting sequence, such as having a certain percentage of identity, for example, it can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or a range between any two of these values ​​identical to the starting sequence.

[0113] In some embodiments, the antibodies comprise amino acid sequences or one or more moieties not typically associated with antibodies. Exemplary modifications are described in more detail herein. For example, the antibodies disclosed herein may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., polyethylene glycol (PEG), a drug, a toxin, or a label).

[0114] The antibodies, variants or derivatives thereof of the present invention include modified derivatives, i.e., 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. For example, but not limitation, the antibody can be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a variety of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Additionally, the antibody can comprise one or more non-classical amino acids.

[0115] In some embodiments, the antibody may be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

[0116] The antibodies may be conjugated or fused to therapeutic agents, which may include detectable labels (e.g., radiolabels), immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents (which may be drugs or toxins), ultrasound enhancing agents, non-radioactive labels, combinations thereof, and other such agents known in the art.

[0117] Antibodies can be detectably labeled by coupling them to a chemiluminescent compound. The presence of the chemiluminescent-labeled antigen-binding polypeptide is then determined by detecting the presence of luminescence that occurs during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, thermostable acridinium esters, imidazoles, acridinium salts, and oxalate esters.

[0118] Fluorescent emitting metals (e.g. 152 Antibodies can be detectably labeled with metals such as Eu or other metals from the lanthanide series. These metals can be attached to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to antibodies are well known.

[0119] Humanized antibodies

[0120] In any aspect of the invention, the Nanobody that binds to DDR2 is preferably a humanized antibody.

[0121] Humanized antibodies are antibody molecules derived from non-human species antibodies, which combine with the desired antigen having one or more complementary determining regions (CDRs) from non-human species and from the framework regions of human immunoglobulin molecules. Typically, the framework residues in the human framework region will be replaced by the corresponding residues from the CDR donor antibody to change, preferably improve antigen binding. These framework replacements are identified by methods well known in the art, for example, by modeling the interaction of CDR and framework residues to identify framework residues that are more important for antigen binding and sequence comparison to identify unusual framework residues at specific positions. Antibody humanization can be performed using a variety of techniques known in the art, including, for example, CDR-grafting, finishing or resurfacing, and chain shuffling.

[0122] Fully human antibodies are particularly desirable for therapeutic treatment of human patients.Human antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences.

[0123] Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells, either randomly or through homologous recombination. Alternatively, in addition to human heavy and light chain genes, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells. Mouse heavy and light chain immunoglobulin genes can be rendered functional separately or simultaneously upon introduction into the human immunoglobulin locus through homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells are amplified and microinjected into blastocysts to produce chimeric mice. Chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the normal manner with a selected antigen, such as all or part of a desired target polypeptide. Monoclonal antibodies against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice rearrange themselves during B cell differentiation, subsequently undergoing class switching and somatic mutation. Thus, using this technology, it is possible to generate therapeutically useful IgG, IgA, IgM, and IgE antibodies.

[0124] Fully human antibodies that recognize a selected epitope can also be generated using a technique called "guided selection." In this method, a non-human monoclonal antibody, such as a mouse antibody, is selected to guide the selection of a fully human antibody that recognizes the same epitope.

[0125] Conventional procedures (for example, by using oligonucleotide probes that can be specifically combined with the genes encoding mouse antibody heavy and light chains) can be used to easily separate and sequence the DNA encoding the desired monoclonal antibody. The hybridoma cells separated and subcloned are used as the preferred source of this type of DNA. After separation, DNA can be placed in an expression vector, which is then transfected into a prokaryotic or eukaryotic host cell, such as an Escherichia coli cell, ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulins. More specifically, the DNA isolated can be used to clone constant region and variable region sequences for the manufacture of antibodies. Essentially, this requires extracting RNA from selected cells, converting it into cDNA, and amplifying by PCR using Ig-specific primers. As described herein, the transformed cells expressing the desired antibody can be grown in a relatively large amount to provide clinical and commercial supply of immunoglobulins.

[0126] In addition, using conventional recombinant DNA technology, one or more CDRs of the antigen-binding polypeptides of the present disclosure can be inserted into the framework region, for example, inserting a human framework region to humanize a non-human antibody. The framework region can be a naturally occurring or shared framework region, and preferably a human framework region. Preferably, the polynucleotide encoding generated by the combination of the framework region and the CDR specifically binds to an antibody of at least one epitope of a desired polypeptide (e.g., LIGHT). Preferably, one or more amino acid substitutions can be made in the framework region, and preferably, the amino acid substitution improves the binding of the antibody to its antigen. In addition, such methods can be used to carry out amino acid substitutions or deletions of one or more variable region cysteine ​​residues that participate in intrachain disulfide bonds to produce antibody molecules lacking one or more intrachain disulfide bonds. Other changes to the polynucleotide are included in the present disclosure and are within the scope of the art.

[0127] For camelid antibodies, in particular heavy chain antibodies and nanobodies (VHH), according to the present invention, humanization of the polypeptide comprises replacing one or more camelid amino acids with their human counterparts found in the human consensus sequence, without the polypeptide losing its typical characteristics, that is, the humanization does not significantly affect the antigen binding ability of the resulting polypeptide. Such methods are well known to the skilled person.

[0128] In one embodiment, the "humanization" of 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 present at the corresponding position in the VH domain of a conventional 4-chain antibody from a human. This can be performed using humanization techniques known in the art. In some embodiments, possible humanizing substitutions or combinations of humanizing substitutions can 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 favorable functional properties. In general, as a result of humanization, the VHH of the present application can become more "human-like" compared to the corresponding naturally occurring VHH domain, while still retaining favorable properties, such as reduced immunogenicity. In various embodiments, the humanized VHH of the present application can be obtained by any suitable means known in the art, and is therefore not strictly limited to polypeptides that have been obtained using polypeptides comprising naturally occurring VHH domains as starting materials.

[0129] In various embodiments, "humanization" and "camelization" can be performed by providing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, respectively, and then changing one or more codons in the nucleotide sequence using methods known in the art, so that the new nucleotide sequence encodes a "humanized" or "camelized" VHH, respectively. This nucleic acid can then be expressed using methods known in the art to provide the desired VHH of the present application. Alternatively, based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, the amino acid sequence of the desired humanized or camelized VHH of the present application can be designed, and then synthesized de novo using peptide synthesis techniques known in the art. Furthermore, based on the amino acid sequence or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, the nucleotide sequence encoding the desired humanized or camelized VHH can be designed, and then synthesized de novo using nucleic acid synthesis techniques known in the art, after which the nucleic acid obtained can be expressed using methods known in the art to provide the desired VHH of the present application. Other suitable methods and techniques for obtaining the VHH of the present application and / or nucleic acids encoding the same using naturally occurring VH sequences or VHH sequences as starting materials are known in the art and may, for example, comprise combining one or more portions of one or more naturally occurring VH sequences (such as one or more FR sequences and / or CDR sequences), one or more portions of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences) and / or one or more synthetic or semi-synthetic sequences in a suitable manner to provide the VHH of the present application or the nucleotide sequence or nucleic acid encoding the same.

[0130] Accordingly, the present invention provides a humanized Nanobody that binds to DDR2, wherein the Nanobody comprises CDR1, CDR2, CDR3 and:

[0131] (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;

[0132] (b) CDR1 comprises or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof;

[0133] (c) CDR1 comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof;

[0134] (d) CDR1 comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof; or

[0135] (e) CDR1 comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0136] In another aspect of the present invention, the present invention provides a nucleotide sequence comprising or being a polynucleotide encoding an antibody according to any of the above aspects. In some embodiments, the polynucleotide comprises or is any sequence selected from SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and equivalent variants thereof.

[0137] Chimeric Antigen Receptor (CAR)

[0138] Chimeric Antigen Receptor (CAR) T cells are T cells that express chimeric receptors on their surface that recognize specific antigens and can transmit signals. CAR T cells play an important role in anti-tumor by expressing chimeric antigen receptor (CAR) molecules. CAR molecules usually include an extracellular segment, a transmembrane region, and an intracellular segment: the extracellular segment is usually a single-chain variable region (ScFv) formed by connecting the variable regions of the heavy chain and light chain of an antibody through a peptide segment, the intracellular segment is a chimera of the intracellular segments of various signal transduction molecules, and the transmembrane region comes from the transmembrane region of other molecules. The gene of the single-chain variable fragment is isolated from, for example, a hybridoma that produces a monoclonal antibody that recognizes the target antigen. T cells expressing CAR molecules directly recognize tumor cell surface antigens independently of the expression of major histocompatibility antigen type I on tumor cells, and activate T cells at the same time, so that T cells expressing CAR can effectively kill tumor cells. In short, CAR T cells recognize specific molecules on the surface of tumor cells through antigen-antibody recognition mode, and then activate, proliferate and exert cell-killing function through their intracellular signal transduction.

[0139] Accordingly, in another aspect of the present invention, the present invention provides a chimeric antigen receptor, which comprises 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 any one of the above-mentioned nanoantibodies.

[0140] In some embodiments, the CAR of the present invention may include, for example, the following transmembrane domains: T cell receptor α or β chain, CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, Tlr4, FcγR I, FcγR II, FcγR III, FCER1 or CD36. In some embodiments, the transmembrane domain includes CD8α or CD28. In some embodiments, the transmembrane domain includes the Tlr4 transmembrane region.

[0141] In some embodiments, the CAR of the present invention may include, for example, the following intracellular signaling domains: CD3ζ, CD28, 4-1BB (or CD137), CD27, and OX40 (or CD134). In some embodiments, the intracellular domain includes CD3ζ and one of CD28, 4-1BB (or CD137), CD27, and OX40 (or CD134). In some embodiments, the intracellular domain includes two of CD3ζ and CD28, 4-1BB (or CD137), CD27, and OX40 (or CD134). In some embodiments, the intracellular domain includes one or more of a CD86 intracellular functional region, a Tlr4CSD region (i.e., a Toll-interleukin 1 receptor (TIR) ​​domain in a Tlr4 cell), and an FcγR I intracellular functional region. In some embodiments, the intracellular domain includes a Tlr4CSD region and an FcγR I intracellular functional region.

[0142] Accordingly, the present invention provides a chimeric antigen receptor, which comprises 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 any one of the above-mentioned nanoantibodies, the transmembrane domain comprises the Tlr4 transmembrane region, and the intracellular domain comprises the Tlr4CSD region and the FcγR I intracellular functional region.

[0143] Accordingly, the present invention also provides a modified immune cell expressing any one of the above-mentioned chimeric antigen receptors.

[0144] In some embodiments, the immune cell is 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 / regulatory T cells. In some embodiments, the macrophages are M1 macrophages or M2 macrophages.

[0145] In some embodiments, the present invention provides a CAR-T cell expressing a chimeric antigen receptor, the extracellular domain of which comprises any one of the Nanobodies described herein.

[0146] In some embodiments, the present invention provides a CAR-T cell expressing a chimeric antigen receptor, the extracellular domain of which comprises any one of the Nanobodies described herein, wherein the Nanobody comprises CDR1, CDR2, CDR3 and:

[0147] (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;

[0148] (b) CDR1 comprises or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof;

[0149] (c) CDR1 comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof;

[0150] (d) CDR1 comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof; or

[0151] (e) CDR1 comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0152] In some embodiments, the present invention provides a CAR-M cell (macrophage) expressing a chimeric antigen receptor, the extracellular domain of which comprises any one of the Nanobodies described herein.

[0153] In some embodiments, the present invention provides a CAR-M cell expressing a chimeric antigen receptor, the extracellular domain of which comprises any one of the Nanobodies described herein, wherein the Nanobody comprises CDR1, CDR2, CDR3 and:

[0154] (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;

[0155] (b) CDR1 comprises or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof;

[0156] (c) CDR1 comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof;

[0157] (d) CDR1 comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof; or

[0158] (e) CDR1 comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0159] In some embodiments, the Nanobody comprises or is any one sequence selected from 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 thereof.

[0160] Bispecific antibodies

[0161] Bispecific antibodies (BsAbs) are artificial antibodies that contain two proteins that can specifically recognize and bind to different antigens or different antigenic sites. If the two antigens are located on different cell surfaces, the bispecific antibody can build a bridge between the two antigen molecules, thereby forming a cross-link between the cells and mediating the cells to produce directed effector functions. Bispecific antibodies used for immunotherapy are artificial antibodies that contain two specific cell receptor antigen binding sites. They can build a bridge between diseased cells (target cells) and functional cells (immune cells) to stimulate a directed immune response. The use of BsAbs to mediate the killing of tumor cells by immune cells (such as T cells, NK cells, etc.) is currently a hot topic in the application of immunotherapy. Its mechanism of action is that BsAbs can simultaneously bind to tumor-associated antigens and target molecules on immune effector cells, while activating immune cells and directly guiding immune effector cells to specifically kill tumor cells.

[0162] Accordingly, in another aspect of the present invention, the present invention provides a bispecific antibody comprising a first binding portion that binds to a first antigen and a second binding portion that binds to a second antigen, wherein the first binding portion comprises any one of the above-mentioned nanobodies.

[0163] In some embodiments, the second antigen targeted by the bispecific antibody is a specific antigen on the surface of an immune cell. In some embodiments, the second antigen is an antigen or epitope of a B cell, a T cell, a myeloid cell, a plasma cell, or a mast cell.

[0164] In some embodiments, the second antigen is selected from CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD89, CD123, TCRγ / δ, NKp46 and KIR.

[0165] In some embodiments, the second antigen is CD16a. CD16a (FcγRIIIa) is a low-affinity receptor for the IgG Fc domain that participates in antibody-dependent cellular cytotoxicity (ADCC) and is responsible for triggering cytolysis of target cells by natural killer (NK) cells.

[0166] In some embodiments, the second antigen is CD89. CD89, also known as immunoglobulin α Fc receptor (FcαRI), is a glycoprotein expressed on the surface of neutrophils, monocytes, macrophages, and eosinophils and is a potent cytotoxic triggering molecule.

[0167] In some embodiments, the second antigen is CD3. CD3 is present only on the surface of T cells and consists of six peptide chains. It often binds tightly to the T cell receptor (TCR) to form a TCR-CD3 complex containing eight peptide chains, which participate in T cell antigen recognition and signal transduction.

[0168] In some embodiments, 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 the first antigen is DDR2 and the first binding moiety comprises a Nanobody, and 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, preferably a T cell surface antigen, preferably CD3, wherein the Nanobody comprises CDR1, CDR2, CDR3 and:

[0169] (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;

[0170] (b) CDR1 comprises or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof;

[0171] (c) CDR1 comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof;

[0172] (d) CDR1 comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof; or

[0173] (e) CDR1 comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0174] In some embodiments, 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 the first antigen is DDR2 and the first binding moiety comprises a Nanobody comprising CDR1, CDR2, CDR3 and:

[0175] (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;

[0176] (b) CDR1 comprises or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof;

[0177] (c) CDR1 comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof;

[0178] (d) CDR1 comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof; or

[0179] (e) CDR1 comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof;

[0180] And wherein the second antigen is DDR2 and the second binding moiety comprises a Nanobody, said Nanobody comprising CDR1, CDR2, CDR3 and:

[0181] (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;

[0182] (b) CDR1 comprises or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof;

[0183] (c) CDR1 comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof;

[0184] (d) CDR1 comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof; or

[0185] (e) CDR1 comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0186] In some embodiments, the Nanobody comprises or is any one sequence selected from 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 thereof.

[0187] In some embodiments, the first binding moiety comprises a Nanobody that is different from the Nanobody comprised by the second binding moiety.

[0188] In some embodiments, 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 the first binding moiety comprises a Nanobody comprising or is the sequence of SEQ ID NO: 4, and wherein the second binding moiety comprises a Nanobody comprising or is the sequence of SEQ ID NO: 12.

[0189] In some embodiments, the first binding moiety and the second binding moiety are connected by a linker. The linker is preferably a flexible linker so that it does not restrict the effector molecule or polypeptide to a single undesirable conformation. The linker is preferably composed primarily of amino acids with small side chains, such as glycine, alanine, and serine, to provide the flexibility. In some embodiments, the linker comprises a glycine-serine polymer, including but not limited to (GS) n 、(GGS) n 、(GGGS) n (SEQ ID NO: 26) and combinations thereof, wherein n is an integer of at least 1. In some embodiments, the sequence of the linker is (GGGS) 3 (SEQ ID NO: 27). Other different linkers may also be used, including various flexible linker designs that have been successfully used to link different antibody variable regions. The size and sequence composition of the linker can be determined by conventional computer modeling and techniques.

[0190] Antibody-drug conjugates (ADCs)

[0191] Antibody drug conjugates (ADCs) are a new class of highly effective biological drugs designed for targeted therapy, especially for the treatment of cancer. ADCs are complex molecules consisting of antibodies (whole mAbs or antibody fragments) connected to bioactive drugs or cytotoxic compounds via stable chemical linkers that may have unstable bonds. By combining the unique targeting capabilities of antibodies with the cell-killing ability of cytotoxic drugs, antibody drug conjugates can achieve sensitive differentiation between healthy and diseased tissues based on the expression of antibody antigens. This means that compared to traditional chemotherapeutic agents, antibody drug conjugates actively target and attack cells expressing antigens, leaving healthy cells with little or no antigen expression less affected.

[0192] Accordingly, in another aspect of the present invention, the present invention provides an antibody-drug conjugate, which comprises an antibody targeting DDR2, a drug, and a linker connecting the antibody and the drug, wherein the antibody targeting DDR2 comprises or is any one of the nanobodies described herein.

[0193] In some embodiments, the drug for conjugation to the antibodies of the invention is a cytotoxic agent, a chemotherapeutic agent, or a therapeutic radioisotope.

[0194] In some embodiments, the cytotoxic agent is a small molecular weight toxin, a peptide toxin, or a protein toxin. In some embodiments, the toxin is selected from ricin, ricin A chain, ethidium bromide, colchicine, dihydroxy anthracin dione, diphtheria toxin, Pseudomonas exotoxin A, abrin, abrin A chain, modeccin A chain, α-sarcin, mitogellin, restrictocin, curcin, and crotonin.

[0195] In some embodiments, the chemotherapeutic agent is selected from an alkylating agent, an anthracycline, an antimetabolite, an antimicrotubule / antimitotic agent, a histone deacetylase inhibitor, a kinase inhibitor, a peptide antibiotic, a platinum-based antineoplastic drug, a topoisomerase inhibitor, a DNA cross-linking agent, and a cytotoxic antibiotic.

[0196] 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.

[0197] 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, CDR3 and:

[0198] (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;

[0199] (b) CDR1 comprises or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof;

[0200] (c) CDR1 comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof;

[0201] (d) CDR1 comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof; or

[0202] (e) CDR1 comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0203] siRNA conjugates

[0204] In another aspect of the invention, the present invention provides a siRNA conjugate targeting DDR2-positive cells, comprising: (i) an antibody targeting DDR2, (ii) an siRNA that inhibits the expression of survival genes in DDR2-positive cells, and (iii) a linker located between the Nanobody and the siRNA, wherein the antibody targeting DDR2 comprises or is any Nanobody described herein.

[0205] As the pharmacological portion of the siRNA conjugate disclosed herein, the "siRNA portion that inhibits the expression of survival genes in DDR2-positive cells" is a double-stranded RNA molecule that, after being taken up by target cells, specifically targets the degradation of the mRNA of a specific survival gene through the RNAi mechanism, thereby inhibiting the expression of the survival gene in DDR2-positive cells.

[0206] The method for designing siRNA for target gene is the routine technology of this area.For example, there are multiple siRNA design websites to provide online design service (for example DSIR:http: / / biodev.extra.cea.fr / DSIR / DSIR.html or siDirect version 2.0:http: / / sidirect2.rnai.jp / ).These design websites can obtain multiple representative siRNA sequences for the different fragments of this target gene based on the mRNA of target gene simultaneously.These representative siRNA can be sorted based on multiple options (for example thermodynamics, high-level structure etc.).Candidate siRNA sequence can be further screened (artificially or automatically), to exclude the sequence containing SNP site or in conjunction with non-open reading frame fragment.In some embodiments, the overlap or overlapping results in different online design websites are selected as candidate siRNA sequence.Candidate siRNA sequence can also analyze the siRNA sequence (as http: / / rnai.cs.unm.edu / rnai / off-target) with potential high miss-target property by miss-target (off-target) prediction software.

[0207] In some embodiments, the siRNA sequence of the siRNA portion targets a survival gene of a DDR2-positive cell. For example, the siRNA sequence targets one or more fragments of one or more of the polr2a, polr2b, dkcl, cenpe, and eif-3b genes. In some embodiments, after the siRNA sequence is delivered to the cell, it binds to the target gene via the RNAi pathway, resulting in inhibition of target gene expression. In some embodiments, inhibition of target gene expression leads to apoptosis, death, and / or proliferation inhibition of DDR2-positive cells. In some embodiments, apoptosis, death, and / or proliferation inhibition of DDR2-positive cells mediates the treatment or cure of a disease. Suitable survival genes include, but are not limited to, one or more of the polr2a, polr2b, dkcl, cenpe, eif-3b, and survivin genes. siRNA sequences targeting survival genes can be obtained and tested by the methods described above.

[0208] In some embodiments, the present invention provides a siRNA conjugate targeting DDR2-positive cells, comprising: (i) an antibody targeting DDR2, (ii) an siRNA that inhibits the expression of a survival gene in DDR2-positive cells, and (iii) a linker between the antibody and the siRNA, wherein the antibody targeting DDR2 comprises or is a nanobody, and the nanobody comprises CDR1, CDR2, CDR3 and:

[0209] (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;

[0210] (b) CDR1 comprises or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof;

[0211] (c) CDR1 comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof;

[0212] (d) CDR1 comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof; or

[0213] (e) CDR1 comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0214] In some embodiments, an antibody comprising any one of the Nanobodies described herein may be an antibody comprising two complete heavy chains, any one of the Nanobodies constituting the variable region of each complete heavy chain, each complete heavy chain further comprising a heavy chain constant region, such as a human heavy chain constant region. In some embodiments, an antibody comprising any one of the Nanobodies described herein may comprise a bivalent Nanobody.

[0215] In some embodiments, the Fc region of the heavy chain constant region is mutated. In some embodiments, the Fc region is mutated to reduce antibody-mediated cytotoxicity (ADCC). Exemplary Fc region mutations can be L234A, L235A, G237A, and P329G, wherein position numbering is according to the EU index.

[0216] Pharmaceutical composition

[0217] Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the above antibodies, nucleotide sequences, vectors, host cells, immune cells or conjugates, and a pharmaceutically acceptable carrier.

[0218] In any of the above aspects, the pharmaceutical composition is used to treat and / or prevent diseases mediated by abnormal DDR2 expression, such as cancer or inflammatory diseases, such as pulmonary fibrosis and retinal vascular disease.

[0219] To prepare a pharmaceutical composition or sterile composition, the drug is mixed with a pharmaceutically acceptable carrier or excipient. Formulations can be prepared, for example, as lyophilized powders, slurries, aqueous solutions, or suspensions by mixing with physiologically acceptable carriers, excipients, or stabilizers. Pharmaceutically acceptable carriers are well known in the art. It is also known in the art how to prepare aqueous compositions containing active ingredients. Typically, these compositions are prepared as injections or sprays, such as liquid solutions or suspensions; solid forms suitable for preparation as solutions or suspensions prior to injection or spraying can also be prepared.

[0220] Imaging and / or diagnostic agents

[0221] In another aspect of the present invention, the present invention provides an agent for imaging and / or diagnosis, comprising any one of the above-mentioned Nanobodies and a detectable label.

[0222] Accordingly, the present invention provides the use of any one of the above-mentioned Nanobodies in the preparation of reagents for imaging and / or diagnosis.

[0223] Accordingly, the invention provides any of the Nanobodies described above for use in imaging and / or diagnosis.

[0224] These imaging and / or diagnostic agents can be used for disease diagnosis or imaging of lesions, such as pulmonary fibrosis imaging and retinal neovascularization imaging. They can also be used as intraoperative imaging navigation agents, for example, to provide optical imaging of lesions, guiding surgeons during surgical navigation and achieving precise resection of the lesion.

[0225] The detectable label included in the reagent for imaging and / or diagnosis of the present invention may be, for example, a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label or an enzyme label.

[0226] The choice of label depends on the means of detection. 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 680XL FLUOROCHROME™ (Perkin Elmer), phycoerythrin, 7-hydroxycoumarin, Lissamine, cyanine, phycoerythrin, Texas Red, BODIPY (Invitrogen), or their analogs are suitable for optical detection.

[0227] Chemiluminescent labels (e.g., luminol, luciferase, luciferin, and aequorin) may also be employed. Such diagnosis and detection may also be accomplished by linking the Nanobodies of the invention to detectable substances, including but not limited to various enzymes, including but not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase, or by linking them to prosthetic group complexes, such as but not limited to streptavidin / biotin and avidin / biotin.

[0228] Paramagnetic labels and radioisotope labels may also be used, which are preferably detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT). Radiolabels include, but are not limited to, bismuth ( 213 Bi), carbon ( 11 C. 13 C. 14 C), chromium ( 51 Cr), cobalt ( 57 Co、 60 Co、Co、Copper 64 Cu), Dysprosium ( 165 Dy), erbium ( 169 Er), fluorine ( 18 F), gadolinium ( 153 Gd, 159 Gd), gallium ( 68 Ga, 67 Ga), germanium ( 68Ge), 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 ( 186 Re、 188 Re), rhodium ( 105 Rh), rubidium ( 81 Rb, 82 Rb), ruthenium ( 82 Such as 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) and zinc ( 65Zn); various positron-emitting metals and non-radioactive paramagnetic metal ions that can be used for positron emission tomography, such as paramagnetic aluminum (Al) ions, barium (Ba) ions, calcium (Ca) ions, cerium (Ce) ions, dysprosium (Dy) ions, erbium (Er) ions, europium (Eu) ions, gadolinium (Gd) ions, holmium (Ho) ions, iridium (Ir) ions, lithium (Li) ions, magnesium (Mg) ions, manganese ( Mn) ions, molybdenum (M) ions, neodymium (Nd) ions, osmium (Os) ions, oxygen (O) ions, palladium (Pd) ions, platinum (Pt) ions, rhodium (Rh) ions, ruthenium (Ru) ions, samarium (Sm) ions, sodium (Na) ions, strontium (Sr) ions, terbium (Tb) ions, thulium (Tm) ions, tin (Sn) ions, titanium (Ti) ions, tungsten (W) ions and zirconium (Zi) ions, especially Co +2 , CR +2 Cr +3 、Cu +2 、Fe +2 、Fe +3 、Ga +3 、Mn +3 、Ni +2 、Ti +3 、V + and V +4 Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art. For example, radioisotopes can be conjugated by the chloramine T method.

[0229] In some embodiments, the present invention provides a reagent for imaging and / or diagnosis, which includes any one of the above-mentioned nanobodies and a detectable label attached to the nanobody, wherein the detectable label is a fluorescent label. In some embodiments, the fluorescent label is indocyanine green (ICG).

[0230] In some embodiments, the present invention provides an agent for imaging and / or diagnosis, comprising any one of the Nanobodies described above and a detectable label attached to said Nanobody, wherein the detectable label is a fluorescent label, preferably indocyanine green (ICG), and wherein the Nanobody comprises CDR1, CDR2, CDR3 and:

[0231] (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;

[0232] (b) CDR1 comprises or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof;

[0233] (c) CDR1 comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof;

[0234] (d) CDR1 comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof; or

[0235] (e) CDR1 comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0236] In some embodiments, the present invention provides a reagent for imaging and / or diagnosis, which comprises any one of the above-mentioned nanobodies and a detectable label linked to the nanobody, wherein the detectable label is a radioisotope. In some embodiments, the radioisotope is 68 Ga or 64 Cu.

[0237] In some embodiments, the present invention provides a reagent for imaging and / or diagnosis, which comprises any one of the above-mentioned nanobodies and a detectable label linked to the nanobody, wherein the detectable label is a radioisotope, preferably 68 Ga or 64 Cu, and wherein the Nanobody comprises CDR1, CDR2, CDR3 and:

[0238] (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;

[0239] (b) CDR1 comprises or is the sequence shown in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof;

[0240] (c) CDR1 comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof;

[0241] (d) CDR1 comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof; or

[0242] (e) CDR1 comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

[0243] In some embodiments, the Nanobody comprises or is any one sequence selected from 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 thereof.

[0244] Methods and Applications

[0245] In one aspect, the present invention provides a method for treating and / or preventing diseases mediated by abnormal DDR2 expression, comprising administering to a subject an effective amount of any one of the above antibodies, polynucleotides, vectors, host cells, immune cells or conjugates.

[0246] Accordingly, another aspect of the present invention provides the use of any of the above antibodies, polynucleotides, vectors, host cells, immune cells or conjugates in the preparation of a medicament for treating and / or preventing diseases mediated by abnormal DDR2 expression.

[0247] Accordingly, another aspect of the present invention provides any one of the above antibodies, polynucleotides, vectors, host cells, immune cells or conjugates for use in treating and / or preventing diseases mediated by abnormal DDR2 expression.

[0248] Another aspect of the present invention provides a method for diagnosing a disease mediated by abnormal expression of DDR2 in a subject, the method comprising administering to the subject an effective amount of any of the above-mentioned reagents for imaging and / or diagnosis. In some embodiments, the method comprises: administering to the subject an effective amount of any of the above-mentioned reagents for imaging and / or diagnosis; detecting and reading the signal generated by the reagent for imaging and / or diagnosis; and determining that the subject has or is at risk of having a disease mediated by abnormal expression of DDR2 based on the intensity of the signal. In some embodiments, it is determined whether the signal exceeds a predetermined threshold, and when the signal exceeds the predetermined threshold, the subject is determined to have a disease mediated by abnormal expression of DDR2. In some embodiments, the threshold is the median level from subjects who do not have the disease.

[0249] Accordingly, another aspect of the present invention provides the use of any of the above-mentioned reagents for imaging and / or diagnosis in the preparation of a kit for diagnosing a disease mediated by abnormal DDR2 expression in a subject.

[0250] Accordingly, another aspect of the present invention provides any of the above-mentioned reagents for imaging and / or diagnosis for use in diagnosing a disease mediated by abnormal DDR2 expression in a subject.

[0251] In some embodiments, the present invention provides a method for treating a disease mediated by abnormal expression of DDR2 in a subject, the method comprising: (a) administering to the subject an effective amount of any of the above-mentioned imaging and / or diagnostic reagents; (b) detecting and reading the signal generated by the imaging and / or diagnostic reagent; (c) determining whether the signal exceeds a predetermined threshold, and when the signal exceeds the predetermined threshold, determining that the subject has a disease mediated by abnormal expression of DDR2, preferably wherein the threshold is the median level from subjects without the disease; and (e) administering to the subject determined to have the disease a therapy that reduces DDR2 activity and / or expression, such as an anti-cancer therapy or an anti-inflammatory therapy, such as an anti-fibrotic therapy. In a preferred embodiment, the disease mediated by abnormal expression of DDR2 is pulmonary fibrosis, particularly idiopathic pulmonary fibrosis. In a preferred embodiment, the anti-fibrotic therapy is pirfenidone and / or nintedanib.

[0252] Suitable administration routes include parenteral administration (e.g., intramuscular, intravenous or subcutaneous administration) and oral administration. Other conventional administration methods include administration via endotracheal tube, oral ingestion, inhalation, topical application or transdermal, subcutaneous, intraperitoneal, intraarterial injection.

[0253] The appropriate dosage is determined by a clinician based on parameters or factors known or suspected to affect treatment or expected to affect treatment in the art. Typically, the initial dose is slightly lower than the optimal dose and then increased in small amounts until the desired or optimal effect relative to any adverse side effects is achieved. Important monitoring indicators include measuring, for example, inflammatory symptoms or the levels of inflammatory cytokines produced.

[0254] Sequence Listing

[0255] Example

[0256] Example 1: Antibody Screening

[0257] Anti-DDR2 heavy chain antibodies were screened from alpacas immunized with the human DDR2 extracellular domain (UniProtKB / Swiss-Prot: Q16832.2 aa 22 to aa 399). The antibodies were then sequenced to confirm their VHH domains, resulting in five nanobodies, designated 1A1, 1A5, 1A12, 1B2, and 1B3. Their sequences are shown in the table above and were used for further characterization and experiments.

[0258] Example 2: Expression and purification of 5 screened DDR2 nanobodies

[0259] Experimental steps:

[0260] 1. Conversion

[0261] Place 100 μL of BL21 (DE3) competent cells on ice, add 1 μL of plasmid (expression vectors for the five selected DDR2 nanobodies), gently flick the bottom of the tube, place on ice for 25-30 minutes, place in a 42°C water bath for 45 seconds, then quickly return to the ice for two minutes. Add 500 μL of resistance-free LB medium and incubate at 37°C at 250 rpm for 1 hour. Centrifuge at 3000 rpm for 1 minute. Retain 100 μL of the supernatant and pipette to mix thoroughly. Plate the supernatant on an LB agar plate containing ampicillin (LBA+) and incubate overnight at 37°C.

[0262] 2. Strain Preservation and Amplification

[0263] 1. Storage: Pick a single colony from the plate and add it to 5-10 ml of LBA+ liquid medium and incubate at 37°C, 250 rpm, overnight. The next day, take 750 μL of the bacterial solution and add it to 250 μL of 80% LB glycerol. Write the fragment, vector, bacterial strain name, and date on the label. Mix well and store at -20°C / -80°C (Glycerol bacteria should be activated and re-incubated after 3-4 months of storage).

[0264] 2. Amplification:

[0265] Day 1: Add 10 μL of glycerol stock to 10 mL of LBA+ liquid medium (in 50 mL centrifuge tubes). Incubate 15 tubes at 37°C, 250 rpm, overnight. Prepare 2 L of LBA+ liquid medium and distribute it to eight 1 L conical flasks. Incubate at 250 mL / bottle at 121°C for 16 minutes.

[0266] The next day: inoculate the overnight amplified bacterial solution into a 1L conical flask at a ratio of 6%, and add 250μL of 100mg / ml Amp at the same time, 37℃, 250rpm for 2-3h (OD value reaches 0.8-1), add IPTG with a final concentration of 1mM to induce, 32℃, 200rpm, 5-6h, collect the bacteria (10000rpm, 5min), and freeze at -20℃.

[0267] 3. Protein Extraction and Purification

[0268] 1. Protein Extraction

[0269] The cells were taken out from -20°C and thawed at room temperature, resuspended in bacterial lysis buffer (500 ml of bacteria = 50 ml of lysis buffer), and ultrasonically disrupted for 5 min (450-500 W, working for 7 s, stopping for 3 s); homogenized at 1000 Pa for 10 min; incubated at 4°C at 12000 rpm for 30 min, and the supernatant was retained.

[0270] 2. Protein Purification

[0271] Pipette 200 μL of magnetic beads (GenScript, L00776) into a 1.5 ml EP tube and wash 5-6 times with Wash Buffer (1 ml / time). Discard the wash buffer and add the magnetic beads to the bacterial supernatant incubate at 4°C with inversion and mix for 1 hour. Place the centrifuge tube on a magnetic rack. Once the supernatant is clarified, transfer it to a new centrifuge tube and add 200 μL of new magnetic beads. Incubate at 4°C with inversion and mix for 1 hour. Wash the beads in the old tube 5-6 times with Wash Buffer and elute three times with Elution Buffer (1 ml / time, 5 minutes each time) with inversion and mix thoroughly. Collect all eluates and freeze at -20°C.

[0272] 4. Protein Concentration and Medium Exchange

[0273] 1. Pour out the 20% alcohol in the ultrafiltration tube, rinse with double distilled water three times, add 15 ml of double distilled water, 4 ℃ 4000 rpm, 10-12 min, pour out the double distilled water in the inner and outer tubes, add eluent (the amount added should not exceed the scale line of the inner tube), and concentrate to about 500 μL.

[0274] 2. Add 4.5 ml of filtered PBS and incubate at 4°C, 4000 rpm, for 30 min. Repeat the above steps three times. Finally, aspirate approximately 500-700 μL of the sample in PBS into a 1.5 ml EP tube. Rinse the ultrafiltration tube and soak the inner tube in 20% ethanol.

[0275] 3. Detect protein concentration using the BCA method.

[0276] Result analysis: The SDS-PAGE results of the five expressed and purified DDR2 nanobodies (1A1, 1A5, 1A12, 1B2, and 1B3) are shown in Figure 1. The antibody protein (circled) is approximately 15KD in size, which is in line with expectations and the size is correct.

[0277] Example 3: Preliminary identification of DDR2 nanobody binding to the extracellular segment of DDR2 antigen by ELISA

[0278] Experimental steps:

[0279] The Flag tag was fused into the nanobody gene coding sequence to express five purified DDR2 antibodies with Flag tags: 1A1, 1A5, 1A12, 1B2, and 1B3; then coated with 100 ng / well of the extracellular segment of DDR2 antigen → blocked with goat serum overnight → rinsed three times with PBS, Anti-DDR2 (five purified DDR2 antibodies, all containing Flag tags) 200 ng / well, 37°C for 1h → Anti-Flag (Sigma, F1804-200UG, 1 mg / ml) 1:2500, 37°C for 1h → G&M (HRP), 1:5000, 37°C for 30min (horseradish peroxidase-labeled anti-Flag antibody amplified the signal, TMB color development) → TMB color development at 37°C for 30min → 1M HCl termination; a blank control was also performed.

[0280] Result analysis:

[0281] The five nanobodies that were purified and expressed correctly in the previous step were verified by ELISA with the extracellular segment of DDR2 antigen and the negative control protein bovine serum albumin (BSA).

[0282] The analysis results of binding to the extracellular segment of DDR2 antigen are shown in FIG2A . It can be seen that the five nanobodies 1A1, 1A5, 1A12, 1B2, and 1B3 can all show binding activity to DDR2 antigen.

[0283] The analysis results of binding to the negative control protein bovine serum albumin are shown in Figure 2B. It can be seen that the five nanoantibodies 1A1, 1A5, 1A12, 1B2, and 1B3 bind to the BSA protein and are found to have no binding activity.

[0284] Therefore, it can be confirmed that the five Nanobodies 1A1, 1A5, 1A12, 1B2, and 1B3 have binding activity to the DDR2 antigen, but do not bind to the negative control protein BSA.

[0285] Example 4: Flow cytometry identification of DDR2 nanobody binding to DDR2 antigen

[0286] Experimental steps:

[0287] A549 cells were infected with an adenovirus overexpressing the DDR2 antigen and incubated with DDR2 nanobodies (five purified DDR2 nanobodies expressing a His-tag were fused to the nanobody gene coding sequence) at 4°C for 1 hour, washed once with PBS, and incubated with Anti-His-AF488 as a secondary antibody at 4°C for half an hour. The cells were washed once with PBS, harvested by centrifugation at 300g for 5 minutes, and resuspended in 200 μl of water. Flow cytometric analysis was performed using an ACEA NovoCyte next-generation intelligent flow cytometer.

[0288] Result analysis: The analysis results of binding to DDR2 antigen are shown in FIG3 . It can be seen that the five nanobodies 1A1, 1A5, 1A12, 1B2, and 1B3 can all show binding activity to DDR2 antigen.

[0289] Example 5: Immunofluorescence identification of DDR2 nanobody binding to DDR2 antigen

[0290] Experimental steps:

[0291] Cells overexpressing DDR2 antigen were plated → fixed with 4% paraformaldehyde overnight → blocked with 5% milk powder for 0.5 h → incubated with DDR2 nanoantibody at room temperature for 2 h → Anti-His AF488 secondary antibody → DAPI staining and photographed under a fluorescence microscope.

[0292] Result analysis: The analysis results of binding to DDR2 antigen are shown in FIG4 . It can be seen that the five nanobodies 1A1, 1A5, 1A12, 1B2, and 1B3 can all show binding activity to DDR2 antigen.

[0293] Example 6: Affinity of DDR2 Nanobodies to the Extracellular Segment of DDR2 Antigen

[0294] Experimental steps:

[0295] The five purified DDR2 nanoantibodies: 1A1, 1A5, 1A12, 1B2, and 1B3 were used to determine the affinity between the antibody and the antigen based on the ForteBio biolayer interferometry (BLI) molecular interaction analysis platform, and the affinity between the nanoantibodies and the extracellular segment protein of the DDR2 antigen was analyzed and detected.

[0296] Experimental consumables: Antibody: dissolved in PBS (pH 7.4); Antigen: dissolved in PBS (pH 7.4); Sensor: Ni-NTA; Kinetics Buffer: PBST (PBS + 0.02% Tween-20, pH 7.4); Regeneration Buffer: 10 mM Glycine-HCl, pH 1.7; Re-charged Buffer: 10 mM NiCl in H2O.

[0297] Steps:

[0298] a. Pre-wet the probe in kinetics buffer for 10 minutes;

[0299] b.Baseline 1: Baseline the Biosensors in kinetics buffer for 180s;

[0300] c. Fixation: Dilute the His-tagged antigen DDR2 extracellular segment to 20 μg / ml with kinetics buffer and capture it with the sensor; to 4 nM (300 s);

[0301] d.Baseline 2: Baseline the Biosensors in kinetics buffer for 60s;

[0302] e. Binding: The antibody solution was diluted to a certain concentration with kinetics buffer (from 100 nM, 2-fold dilution to 3.125 nM), and the sensor was inserted into the antibody solution for binding (600 s);

[0303] f. Dissociation: The sensor was dissociated in kinetics buffer (600s);

[0304] g. Sensor regeneration: 10 mM Glycine-HCl, pH 1.7 for 5 s;

[0305] h. Neutralization: After sensor regeneration, neutralize in kinetics buffer for 5 seconds;

[0306] i. Repeat regeneration step g and neutralization step h: a total of 3 times (30s);

[0307] j. Baseline 3: After sensor regeneration, add 10 mM NiCl for 60 s.

[0308] Create kinetic curves and calculate relevant parameters. Select several appropriate concentration gradient binding and dissociation curves and fit all curves using a 1:1 binding model. Select the three curves with the best fit for graphical analysis to ultimately obtain affinity values ​​and important parameters such as binding and dissociation constants.

[0309] Result analysis: The analysis results of the binding of the five purified DDR2 nanobodies: 1A1, 1A5, 1A12, 1B2, and 1B3 to the DDR2 antigen are shown in Figure 5.

[0310] Table 1: Nanobody affinity data

[0311] Example 7: In vitro imaging results of DDR2 nanobodies

[0312] Experimental steps:

[0313] (1) Indocyanine green (ICG) labeled DDR2 nanobody

[0314] Nb-DDR2 was dissolved in PBS at a concentration of 2 mg / mL and vortexed to mix.

[0315] ICG-NHS was dissolved in DMSO at a concentration of 2 mM and vortexed to mix.

[0316] Take 500 μL of 2 mg / mL Nb-DDR2 solution in a 1.5 mL centrifuge tube, add 18 μL of 2 mM ICG-NHS solution to the Nb-DDR2 solution in 9 additions, 2 μL each time, and vortex mix for a few seconds each time.

[0317] The pH of the mixed solution was measured and adjusted to 8.5-9 using 2 M NaOH solution.

[0318] The centrifuge tube was placed on a shaker at 60 rpm and reacted at room temperature for 2 h.

[0319] The unreacted ICG-NHS was removed by repeated centrifugation at 14,000 g for 10 min using a 0.5 mL ultrafiltration tube, and the solution was replaced with 0.9% NaCl. The protein solution was filtered through a 0.22 μm filter membrane and stored at 4°C.

[0320] (2) Mouse lung tissue slice imaging

[0321] Fresh lung tissue from normal mice was sliced ​​into 300 μm thick sections. One section served as an untreated control, one was induced with bleomycin, and one was induced with mTGF-β cytokine. Both sections were treated for 72 hours (both treatments promoted DDR2 expression). Each section was then stained with ICG-labeled DDR2 nanoantibody 1A12 (1 μg / sample) for 30 minutes. The samples were then rinsed multiple times with PBS to remove nonspecific staining. Images were then taken using a live imaging system. The results are shown in Figure 6.

[0322] Result analysis: The purified DDR2 nanoantibody can be labeled with ICG and specifically bind to DDR2-highly expressed tissues in mice, and can be used for precise in vitro imaging.

[0323] (3) Lung tissue imaging of patients with idiopathic pulmonary fibrosis (IPF)

[0324] Lung tissues from normal donors and idiopathic pulmonary fibrosis (IPF) patients were cut into 600 μm thickness in vitro. The lung tissues of IPF patients highly express DDR2, while the lungs of normal donors do not express or express DDR2 at very low levels. The normal group (Normal) served as the control, and the lung tissue slices of IPF patients served as the experimental group. After staining with ICG-labeled DDR2 nanoantibody 1A12, in vitro live imaging was performed. The results are shown in Figure 7.

[0325] Result analysis: The purified DDR2 nanoantibody can specifically bind to human DDR2-highly expressed tissues after being labeled with ICG, and can be used for precise in vitro imaging.

[0326] (4) Imaging of pig and rabbit lung tissue sections

[0327] Fresh lung tissues from normal pigs and rabbits were cut into 600 μm thick slices. Bleomycin was added at concentrations of 1 μg / mL, 2 μg / mL, and 4 μg / mL to induce DDR2 expression in the lung tissues. Lung tissue without bleomycin was used as a blank control. After 72 hours of induction, 2 μg of ICG-labeled DDR2 nanoantibody 1A12 was added and incubated at 37°C for 1 hour. The tissue sections were rinsed with PBS five times to remove unbound probes, and near-infrared imaging was performed using a small animal in vivo imager. The results are shown in Figure 8.

[0328] Result analysis: ICG-labeled DDR2 nanoantibody can specifically recognize DDR2 highly expressed in pig and rabbit lung tissues and can be used for precise in vitro imaging.

[0329] (5) Retinal neovascularization imaging

[0330] Vascular smooth muscle cells, the primary component of the blood vessel wall, express DDR2. Retinas from normal mice and mice with oxygen-induced lesions were mounted, then 2 μg of ICG-labeled DDR2 nanoantibody 1A12 was added and incubated at room temperature for 1 hour. Unbound probe was removed by multiple PBS rinses, and near-infrared imaging was performed using a small animal in vivo imaging system. The results are shown in Figure 9.

[0331] Analysis of results: ICG-labeled DDR2 nanoantibodies can specifically recognize DDR2 in the mouse retina, showing the vascular distribution in the retina and oxygen-induced retinal vascular lesions.

[0332] Example 8: In vivo imaging results of DDR2 nanobody

[0333] Experimental steps:

[0334] A mouse pulmonary fibrosis model was established by intubating C57 mice with bleomycin at a dose of 1.7 mg / kg. DDR2 is highly expressed in lung tissue in this mouse pulmonary fibrosis model. One week after establishment, 75 μg of ICG-labeled 1A12 nanoantibody was injected into the tail vein for in vivo imaging. Mice No. 1 and No. 2 were pulmonary fibrosis models, while mouse No. 3 was a normal, untreated mouse. The sites of pulmonary fibrosis were imaged and localized using a small animal in vivo imaging system.

[0335] The results showed that the anti-DDR2 nanoantibodies provided by the present invention can achieve accurate imaging of pulmonary fibrosis lesions in mice, and can be used for accurate in vivo diagnosis of early pulmonary fibrosis in the future, as shown in Figure 10.

[0336] Example 9: Nanobody 1A12 inhibits collagen-induced DDR2 protein phosphorylation (functional verification)

[0337] Experimental steps: 293T cells overexpressing DDR2 antigen were plated in 6-well plates. When the cells grew to 70-80%, the culture medium was changed to 0.75% FBS to starve the cells. After 12 hours, the culture medium was changed to new 0.75% FBS and different amounts of DDR2 antibody 1A12 were added for incubation. Collagen was added for stimulation 2 hours later. After 12 hours, the cells were lysed and the proteins were collected for western blotting.

[0338] The experimental results are shown in Figures 11 and 12. The results showed that 1A12 could inhibit collagen-induced DDR2 protein phosphorylation in a dose-dependent manner.

[0339] Example 10: Plasmid enzyme digestion and identification of anti-DDR2 heterodimeric nanobodies 1A1-1A12

[0340] In this example, heterodimeric Nanobodies 1A1-1A12 were constructed, comprising the aforementioned Nanobodies 1A1 and 1A12, linked by a linker sequence: GGGSGGGSGGGS (SEQ ID NO: 27), located between the N-terminus of 1A1 and the C-terminus of 1A12. These antibodies were characterized and tested.

[0341] Experimental steps:

[0342] (1) Take the glycerol bacteria expressing DDR2 nanoantibodies 1A1-1A12 and shake overnight. The next day, perform a small plasmid extraction. Take 500 ng of plasmid, add 0.5 μL of NcoI and XhoI fast endonucleases, 1 μL of 10x buffer, and adjust to 10 μL with double distilled water. Incubate at 37°C for 30 minutes.

[0343] (2) Weigh 0.3 g of agarose. Add 30 ml of TAE buffer to a flask. Place the flask in a microwave and heat until the agarose dissolves. Add ethidium bromide to the agarose gel solution that has cooled to about 65 degrees Celsius and mix thoroughly. Secure the mold, place the comb, pour in the agarose solution, and allow it to solidify.

[0344] (3) Add the sample and DNA marker to the wells, run the electrophoresis at 120 volts for 20 minutes, and take the agarose gel for exposure and development.

[0345] The results are shown in Figure 13. The results showed that the plasmid was of the correct size and was the target plasmid.

[0346] Example 11: Coomassie blue staining of purified proteins of anti-DDR2 heterodimeric nanobodies 1A1-1A12

[0347] Experimental steps: Take 2 micrograms of purified anti-DDR2 nanoantibodies 1A1-1A12, add 5x protein loading buffer and boil for denaturation, load on SDS-PAGE gel, and electrophorese at 220 volts for 1 hour. After stopping the electrophoresis, take the SDS-PAGE gel and place it in an appropriate amount of Coomassie blue dye solution. Stain on a shaker at 60 rpm for 30 minutes. Discard the dye, replace with double distilled water and wash 2-3 times, and take pictures under white light.

[0348] The results are shown in Figure 14. The results showed that the purified heterodimeric Nanobodies 1A1-1A12 were of the correct size and had no other miscellaneous bands.

[0349] Example 12: Western blot identification of anti-DDR2 heterodimeric nanobodies 1A1-1A12

[0350] Experimental steps: Take 2 micrograms of purified anti-DDR2 nanoantibodies 1A1-1A12, add 5x protein loading buffer and boil for denaturation, load on SDS-PAGE gel, electrophorese at 220 volts for 1 hour, stop electrophoresis, take SDS-PAGE gel and transfer to membrane, the transfer conditions are 400mA for 35 minutes, block with 5% skim milk powder for 1 hour, incubate with Anti-His-HRP at room temperature for 1 hour, wash 3 times with TBST, and develop on a machine.

[0351] The results are shown in Figure 15. The results showed that the purified heterodimeric Nanobodies 1A1-1A12 were of the correct size.

[0352] Example 13: Flow cytometry analysis of the anti-DDR2 heterodimeric nanobodies 1A1-1A12 to identify their binding activity to the antigen DDR2

[0353] Experimental steps: DDR2 293T and 293T cells were digested and transferred to a 1.5 ml centrifuge tube, 1 μg of anti-DDR2 nanoantibody 1A1-1A12 was added and incubated for 1 hour, centrifuged at 300 g for 5 minutes, the supernatant was discarded and PBS was added, washed three times, Anti-His-488 secondary antibody was added and incubated in the dark for 1 hour, washed three times with PBS, and detected by flow cytometry.

[0354] The results are shown in Figure 16. The results showed that the purified heterodimeric Nanobodies 1A1-1A12 had the ability to bind to the corresponding antigen DDR2.

[0355] Example 14: Anti-DDR2 heterodimeric nanobodies 1A1-1A12 inhibit collagen-induced DDR2 protein phosphorylation (functional verification)

[0356] Experimental steps:

[0357] (1) Plate DDR2-293T cells in a six-well plate, one well per group, until the cell density reaches 85%;

[0358] (2) Cells were starved for 12 h using a medium containing 0.75% fetal bovine serum, and solutions of antibodies 1A1-1A12 at different concentrations were prepared in the same starvation medium;

[0359] (3) Discard the supernatant of the starvation medium and replace it with antibody medium of different concentrations for incubation for 2 hours;

[0360] (4) Add collagen stimulation at a concentration of 30 μg / ml. After 12 hours of stimulation, cells were lysed and protein was extracted.

[0361] (5) Western blot experiments were used to detect the phosphorylation levels of DDR2 protein in each group of cells.

[0362] The results are shown in Figure 17. The results showed that the heterodimeric nanobody 1A1-1A12 had an inhibitory effect after binding to the DDR2 antigen.

[0363] Example 15: Chimeric Antigen Receptor

[0364] A CAR molecular vector constructed with the DDR2 nanobody 1A12 sequence was constructed (as shown in FIG18 ), packaged with adenovirus, and infected primary mouse macrophages to obtain αDDR2 CAR-M cells for the following experiments.

[0365] Empty-M (uninfected macrophages) and αDDR2 CAR-M were co-cultured with 293T-DDR2 cells at a 1:1 ratio. The percentage of FITC and APC double-positive cells was used as the percentage of 293T-DDR2 phagocytosis, and the results were expressed as Mean ± SEM. The results are shown in Figure 19. The difference between Empty-M and CAR-M was statistically significant. It can be seen that CAR-M cells constructed with 1A12 phagocytose DDR2-positive 293T cells at a significantly higher rate.

[0366] Empty-M and αDDR2 CAR-M 5X10 5 The αDDR2 CAR-M cells were injected into the left lung of mice induced by bleomycin. After 15 days, the mice were euthanized, and their left lungs were harvested. CD68 immunohistochemistry was performed on normal mice that had not been injected with macrophages. The results are shown in Figure 20. CD68 immunohistochemistry revealed fine, brown granules in lung sections from all groups. Weak CD68 expression was observed in lung tissue sections from the NC and BLM+Empty-M groups, primarily distributed around the airways and within the alveolar septa. CD68 expression was significantly enhanced and evenly distributed in the BLM+CAR-M group. These results suggest that the adoptively transferred αDDR2 CAR-M cells are able to localize to the left lung, an area of ​​high DDR2 expression. This indicates that the CAR-M cells constructed with 1A12 are concentrated in tissues with high DDR2 expression in mice.

[0367] Lattice light sheet super-resolution microscopy was used to verify the targeted phagocytosis of 293T-DDR2 cells by αDDR2 CAR-M. αDDR2 CAR-Ms naturally fluoresce green. 293T-DDR2 cells were stained red with Dil (an orange-red fluorescent dye). The two were mixed in a 1:1 ratio and then placed on the microscope for continuous imaging for 16 hours, with images taken every 3 minutes. The results are shown in Figure 21. Both green (CAR-M) and red (293T-DDR2) cells are visible. The yellow cells are believed to be formed after the green CAR-Ms have fully engulfed the red 293T-DDR2 cells, and the red dye carried by the 293T-DDR2 cells enters the CAR-Ms. This demonstrates that CAR-M cells constructed with 1A12 can effectively target and engulf DDR2-positive 293T cells.

[0368] Each group of mice was intratracheally instilled with 0.75 mg / ml bleomycin (BLM) in a volume of 40 μl. Micro-CT scans were taken on the 8th day to screen mice with left lung modeling and grouped according to the severity of the lesion. On the 10th day, PBS, Empty-M, and αDDR2 CAR-M were injected into the tail vein respectively. Micro-CT scans were taken again on the 26th day to evaluate the treatment effect. The mice were euthanized on the 28th day, and the left lung was fixed internally.

[0369] As shown in Figure 22, Figure A is the experimental flow chart, Figure B is a weight change chart of mice weighed every 3 days after BLM modeling, Figure C is a survival curve chart of each group of mice from the date of BLM modeling to the 28th day after modeling, and Figure D is a CT 3D image obtained by taking Micro-CT of the lungs of each group of mice on the 8th and 26th days after modeling.

[0370] In terms of body weight, mice in the CAR-M group initially decreased and then increased, essentially returning to their original weight by day 28. While weight rebounded in the other two groups, it was not able to return to its original weight. Survival curves revealed no mortality in the CAR-M group, while mortality occurred in the other two groups, with mortality being more pronounced in the PBS group. CT scans revealed that lung lesions in the CAR-M group had largely recovered compared to day 8, while those in the other two groups showed little recovery or even worsening. In summary, compared with the control group, mice injected with the αDDR2 CAR-M had significantly higher body weight, a significantly improved survival rate (no mortality until day 28), and controlled lesion severity, demonstrating a significant therapeutic effect.

[0371] Example 16: Nanobody 1A12 labeled nuclide ( 64 Cu, 68 Ga and other conventional nuclides) process

[0372] 1. Coupling NOTA

[0373] (1) The antibody solution was centrifuged at 12000 g, the supernatant was collected, and the antibody solution was solvent-displaced into 0.1 M ammonium acetate (pH = 7). After the solvent-displacement, the concentration of the nanobody 1A12-cys was 1.33 mg / mL. The concentrations were measured before and after the solvent-displacement, and the concentration after the solvent-displacement was used as the standard.

[0374] (2) Take 50mM Mal-NOTA (dissolved in DMSO) and make the molar ratio of antibody to Mal-NOTA be 1:5. Dilute Mal-NOTA with DMSO so that its volume is less than 10% of the volume of the reaction system (for example, if the reaction system is 1mL, dilute Mal-NOTA to 80-90μL, shake and mix, so that Mal-NOTA is fully dissolved in DMSO). Add Mal-NOTA solution to the antibody solution in batches, 10μL / time, shake and mix for tens of seconds after each addition, adjust the pH of the solution to 7 (the reaction solvent is 0.1M ammonium acetate, the pH basically does not need to be adjusted, just measure and confirm), place it on a shaker and react at room temperature for 2h, and the shaker speed is 150rpm.

[0375] (3) After the reaction, the antibody solution was centrifuged at 12000g, the supernatant was collected, and the antibody solution was purified and concentrated with 0.1M ammonium acetate. Every 100μg of antibody was concentrated to 50-80μL, sealed with parafilm, and stored in a -80℃ refrigerator.

[0376] 2. Marking 68 Ga, 64 Cu and other conventional nuclides

[0377] (1) Take 68 Ga or 64 Cu or other nuclides are added to the precursor solution ( 64 Cu should be as concentrated as possible, with an activity of approximately 1.5 mCi per 100 μg of precursor, and the final reaction volume should not exceed 150 μL), mix well, and adjust the pH to 4-5 (normally, no adjustment is required, the pH is 4-5 after mixing).

[0378] (2) If there is an oscillating thermostat, place it in a oscillating thermostat at 37°C for 2 hours. If not, heat it at 37°C for 2 hours (water bath, oven, etc.).

[0379] (3) Spot the sample on chromatography paper, develop with a developing agent, and detect the labeling rate using iTLC (the developing agent is sodium citrate).

[0380] Example 17: Nanobody 1A12 labeled nuclide 68 Ga imaging

[0381] According to the procedure described in Example 16, 68 Ga-labeled nanobody 1A12.

[0382] BLM-15d mice and normal control mice were injected with 200 μCi / mouse via tail vein. 68 PET-CT imaging was performed using Ga-labeled Nanobody 1A12. The imaging results are shown in Figure 23. The results showed significant lung uptake in BLM-treated mice compared to normal mice, demonstrating the potential of nanobody-conjugated radionuclides for PET-CT imaging.

[0383] It should be understood that, although the present invention has been specifically disclosed by preferred embodiments and optional features, those skilled in the art may make modifications, improvements and variations to the invention disclosed herein, and these modifications, improvements and variations are considered to be within the scope of the present invention. The materials, methods and examples provided herein are representative and exemplary of preferred embodiments and are not intended to be limiting of the scope of the present invention.

Claims

1. A Nanobody that binds to DDR2, wherein the Nanobody comprises CDR1, CDR2, CDR3, and the CDR1, CDR2, and CDR3 comprise or are selected from the CDR1, CDR2, and CDR3 of a Nanobody having an amino acid sequence of any one of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, and SEQ ID NO: 20, or equivalent variants thereof.

2. The Nanobody according to claim 1 , wherein the CDR1, CDR2 and CDR3 are defined based on any one of the definitions of IMGT, Kabat, Chothia, Contact or Martin.

3. The Nanobody according to claim 1, wherein: (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 shown in SEQ ID NO: 5 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 6 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 7 or an equivalent variant thereof; (c) CDR1 comprises or is the sequence shown in SEQ ID NO: 9 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 10 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 11 or an equivalent variant thereof; (d) CDR1 comprises or is the sequence shown in SEQ ID NO: 13 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 14 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 15 or an equivalent variant thereof; or (e) CDR1 comprises or is the sequence shown in SEQ ID NO: 17 or an equivalent variant thereof, CDR2 comprises or is the sequence shown in SEQ ID NO: 18 or an equivalent variant thereof, and CDR3 comprises or is the sequence shown in SEQ ID NO: 19 or an equivalent variant thereof.

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

5. The Nanobody according to claim 1 , comprising or being any one 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 thereof.

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

7. The polynucleotide according to claim 6, wherein the polynucleotide comprises or is any one sequence selected from SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25 or an equivalent variant of each thereof. A vector comprising the polynucleotide according to 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 according to any one of claims 1 to 5. The chimeric antigen receptor according to claim 10 , wherein the transmembrane domain comprises a Tlr4 transmembrane region, and the intracellular domain comprises a Tlr4 CSD region and an FcγR I intracellular functional region.

12. An engineered immune cell expressing the chimeric antigen receptor according to claim 10 or 11. The immune cell according to claim 12 , wherein the immune cell is a macrophage.

14. 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 the Nanobody of any one of claims 1 to 5.

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

16. The bispecific antibody according to claim 15, wherein the Nanobody comprised by the first binding moiety is different from the Nanobody comprised by the second binding moiety.

17. An antibody-drug conjugate comprising an antibody targeting DDR2, a drug, and a linker connecting the antibody and the drug, wherein the antibody targeting DDR2 comprises or is the Nanobody according to any one of claims 1 to 5.

18. An siRNA conjugate targeting DDR2-positive cells, comprising: (i) an antibody targeting DDR2, which comprises or is the Nanobody according to any one of claims 1 to 5, (ii) an siRNA that inhibits the expression of survival genes in DDR2-positive cells, and (iii) a linker between the Nanobody and the siRNA.

19. An agent for imaging and / or diagnosis, comprising the Nanobody according to any one of claims 1 to 5 and a detectable label linked to said Nanobody.

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

Images