Anti-human integrin cd103 nanobodies and uses thereof

Abstract

The application belongs to the field of biological medicine, and relates to an anti-human integrin CD103 nanobody and application. The anti-human integrin CD103 nanobody comprises three complementarity determining regions CDR1, CDR2 and CDR3; wherein the amino acid sequence of CDR1 is a sequence or a high homology sequence shown in one of SEQ ID NO: 1 to SEQ ID NO: 6, the amino acid sequence of CDR2 is a sequence or a high homology sequence shown in one of SEQ ID NO: 7 to SEQ ID NO: 12, and the amino acid sequence of CDR3 is a sequence or a high homology sequence shown in one of SEQ ID NO: 13 to SEQ ID NO: 18. 18 The F-CYNB nanobody probe can be applied to targeted imaging of cardiac myocardial fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a specific nanobody targeting integrin CD103 (integrin αEβ7) and its application, particularly in the fields of cardiac imaging, tumor imaging, evaluation of the efficacy of tumor immunotherapy and diagnosis. Background Technology

[0002] Tumor immunotherapy, such as immune checkpoint inhibitors (ICIs), has become one of the most widely used treatment methods besides surgery, radiotherapy, and chemotherapy, profoundly changing the clinical diagnosis and treatment of malignant tumors. ICIs significantly improve the survival rate of some patients by relieving the suppression of T cells and hold promise for long-term remission. However, in most solid tumors, the objective response rate of ICI monotherapy is only about 10-30%, and most patients do not benefit from it. Early assessment of treatment efficacy and prediction of tumor recurrence could allow some patients to withdraw from immunotherapy in a timely manner, saving funds or enabling timely switching of treatment regimens to avoid delays in treatment.

[0003] Radionuclide imaging enables the tracking and imaging of specific physiological or pathological processes by injecting radiolabeled molecular probes into the body. With the rise of immunotherapy, researchers increasingly hope to use radionuclide imaging to assess molecular expression in the tumor immune microenvironment, thereby determining treatment efficacy or screening patients. To achieve this goal, there is an urgent need to develop probe carriers that can specifically recognize target molecules and are suitable for in vivo imaging, which is one of the key challenges facing nanobody technology.

[0004] Currently, radionuclide imaging probes targeting tumors have the following main drawbacks:

[0005] First, it lacks specific probes targeting functional molecular targets and cannot dynamically reflect the response to immunotherapy. Most traditional molecular imaging techniques, such as... 18 F-FDG PET can only reflect the metabolic activity of the tumor to achieve the purpose of tumor imaging, but cannot identify immune cells. At the same time, it can only reflect the state of the lesion at a certain moment, and cannot continuously monitor the infiltration, activation or depletion of immune cells. It is not suitable for assessing the phenomenon unique to immunotherapy, such as "false progression" (which refers to the tumor appearing to grow larger or new lesions on images in the early stage of treatment, but in fact it is due to the large infiltration of immune cells, and the tumor may shrink or stabilize later).

[0006] Second, tumor heterogeneity and the diversity of immunotherapy approaches prevent the widespread application of probes targeting single treatments. Radionuclide probes targeting PD-1 and PD-L1 can only be used for monitoring immunotherapy in specific treatment regimens and cannot be used for other immunotherapy regimens. At the same time, the expression levels of substances such as CD70 are highly heterogeneous in different locations and at different times, and cannot represent the overall state of the lesion.

[0007] Third, there are currently no effective means for prognostic monitoring. While CD8-targeted nanobody probes can address the aforementioned issues of dynamic monitoring and multi-therapy coverage, their specificity is poor due to the presence of different populations of CD8+ T cells in the tumor immune microenvironment, including not only effector CD8+ T cells that kill tumor cells but also non-functional, exhausted CD8+ T cells. Although nanobody-based nucleoside probes targeting granzyme B, designed based on markers of effector CD8+ T cells, can specifically monitor immunotherapy, they cannot predict the subsequent evolution of the tumor microenvironment or the outcome after immunotherapy.

[0008] CD103, a member of the integrin superfamily, is a heterodimeric transmembrane receptor with a molecular weight of approximately 150-175 kDa, composed of αE and β7 subunits. CD103 binds highly specifically to E-cadherin, a cadherin ligand widely expressed on the surface of epithelial cells, dendritic cells, and specific tumor-infiltrating lymphocytes, playing a crucial biological role in the mucosal barrier and tumor microenvironment. CD103 exhibits characteristic expression in immune cell subsets: it is highly expressed in tissue-resident memory T cells (Trm) and enhances their long-term anchoring ability in epithelial tissues; it is enriched in tumor-infiltrating CD8+ T cells (TILs) and significantly positively correlated with good prognosis and immunotherapy response; its expression in regulatory T cells (Tregs) enhances the immunosuppressive function of the tumor microenvironment; simultaneously, mucosal dendritic cells, such as intestinal CD103+ DCs, also highly express CD103, inducing immune tolerance through antigen presentation. CD103, therefore, as a crucial molecule connecting epithelial homeostasis and immune response, plays a central role in mucosal immune tolerance and anti-tumor immune balance by integrating adhesion, survival, and activation signals. It has become an important biomarker for tissue-resident T cells and a significant potential target for tumor immunotherapy. In previous studies, using an AngII-induced mouse myocardial fibrosis model, the applicant found significantly elevated CD103 expression in mouse myocardial fibrosis tissue. Specifically, Figure 13 The results of an immunoblotting experiment on CD103 expression in mice with myocardial fibrosis, as previously studied at our research center, are shown. It can be seen that, using the AngII-induced mouse model of myocardial fibrosis, CD103 expression in myocardial tissue was significantly increased after 14 days compared to the control group (NaCl group). Therefore, specific nanobodies targeting CD103 hold promise for use in cardiac imaging.

[0009] However, there are currently no reports or clinical applications of specific nanobodies targeting CD103. Therefore, there is an urgent need in this field to develop novel and effective specific CD103 nanobodies to provide more accurate and reliable means for cardiac imaging, evaluation of tumor immunotherapy efficacy, tumor imaging and diagnosis. Summary of the Invention

[0010] The purpose of this invention is to provide an anti-human integrin CD103 nanobody that can bind to integrin CD103, and to provide the coding sequence of the nanobody, as well as the preparation method and application of the nanobody.

[0011] To achieve the above objectives, the present invention provides an anti-human integrin CD103 nanobody comprising three complementarity-determining regions CDR1, CDR2, and CDR3; wherein...

[0012] The amino acid sequence of CDR1 is the sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6, or is a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6 and has the same function;

[0013] The amino acid sequence of CDR2 is the sequence shown in one of SEQ ID NO: 7 to SEQ ID NO: 12, or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one of SEQ ID NO: 7 to SEQ ID NO: 12 and has the same function;

[0014] The amino acid sequence of CDR3 is the sequence shown in any one of SEQ ID NO: 13 to SEQ ID NO: 18, or is a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 13 to SEQ ID NO: 18 and has the same function.

[0015] In this invention, "having the same function" means being able to bind to human integrin CD103 protein.

[0016] The location of the CDR in the antibody or nanobody sequence can be determined by those skilled in the art using existing techniques. Typically, the CDR can be identified by sequencing the DNA of the antibody or nanobody, and the resulting sequence can then be analyzed using a dedicated database (such as the international ImMunoGeneTics database or IMGT).

[0017] In the sequence provided by this invention, CDR is drawn based on IMGT ( https: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results ).

[0018] According to a preferred embodiment of the present invention, the anti-human integrin CD103 nanobody has any of the following CDR sequence characteristics:

[0019] (1) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 13, respectively, or sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences and have the same function;

[0020] (2) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 14, respectively, or sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences and have the same function;

[0021] (3) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 3, SEQ ID NO: 9, and SEQ ID NO: 15, respectively, or sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences and have the same function;

[0022] (4) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 16, respectively, or sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences and have the same function;

[0023] (5) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 17, respectively, or sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences and have the same function;

[0024] (6) The amino acid sequences of CDR1, CDR2 and CDR3 are the sequences shown in SEQ ID NO: 6, SEQ ID NO: 12 and SEQ ID NO: 18, respectively, or sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above sequences and have the same function.

[0025] According to the present invention, in addition to the aforementioned complementarity-determining regions, the anti-human integrin CD103 nanobody further comprises four framework regions FR1, FR2, FR3, and FR4 alternately disposed with the three complementarity-determining regions, wherein,

[0026] The amino acid sequence of FR1 is shown in one of SEQ ID NO: 19 to SEQ ID NO: 24;

[0027] The amino acid sequence of FR2 is the sequence shown in one of SEQ ID NO: 25 to SEQ ID NO: 30;

[0028] The amino acid sequence of FR3 is shown in one of SEQ ID NO: 31 to SEQ ID NO: 36;

[0029] The amino acid sequence of FR4 is shown in one of SEQ ID NO: 37 to SEQ ID NO: 38.

[0030] According to a preferred embodiment of the present invention, the anti-human integrin CD103 nanobody has any of the following FR sequence characteristics:

[0031] (a) The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 31, and SEQ ID NO: 37, respectively;

[0032] (b) The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 37, respectively;

[0033] (c) The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 37, respectively;

[0034] (d) The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, and SEQ ID NO: 37, respectively;

[0035] (e) The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, and SEQ ID NO: 38, respectively;

[0036] (f) The amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36 and SEQ ID NO: 37, respectively.

[0037] This invention includes all sequences that satisfy the above sequence characteristics, and preferably, the nanobody is selected from one of the following sequences:

[0038] (i) An amino acid sequence as shown in any one of SEQ ID NO: 39 to SEQ ID NO: 44;

[0039] (ii) An amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 39 to SEQ ID NO: 44 and having the same function;

[0040] (iii) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in any one of SEQ ID NO: 39 to SEQ ID NO: 44, and retains the function of the amino acid sequence shown in any one of SEQ ID NO: 39 to SEQ ID NO: 44.

[0041] NB672-4-E-M1F2-20:

[0042] QVQLVESGGGLVQAGGSLRLSCAASGRTFRSYAMGWFRQGPGKEREFVATISASGNYTYYTDSVKGRFTISRDNAENTVYLQMSSLEPEDTAVYYCNFALYYSSDSPVGDTRYWGQGTQVTVSS (SEQ ID NO: 39).

[0043] NB672-4-E-M1F2-28:

[0044] QVQLVESGGGLVQPGGSLRLSCAASGSIDSLYIMGWYRQAPGEQRELVAIMSRAGSTQYGDSVKGRFTISRDNTKNTVYLQMNNLKPEDTAVYYCKIVHESTYWGQGTQVTVSS(SEQ ID NO:40)。

[0045] NB672-4-E-M1F2-30:

[0046] EVQLVESGGGLVQPGGSLRLSCAASGNIGSFYTMGWYRQVPGKKRELVADITNFGITNYADSMKGRFTISRDNAKMTVYLQMNSLKPEDTAVYYCNVKHNHYPYEHWGQGTQVTVSS(SEQ ID NO:41)。

[0047] NB672-4-E-M1F2-86:

[0048] EVQLVESGGGLVQAGGSLRLSCTASRRTNMAWFRRAPGKDREFIAA IMWTGVSTQYGDSVKGRATISRDNAENTAYLQMNSLKPEDTAVYFCNA DGIFEYNSYRPGSNWGQGTQVTVSS(SEQ ID NO:42)。

[0049] NB672-4-E-M1F2-98:

[0050] EVQLVESGGGLVQPGGSLRLSCAVSGSIFSDYAMGWYRQAPGKRRE FIASMTYYADSVKGRFTFSRDNAKNTVYLQMNSLKPEDTAVYYCNAQS TSWPYDFWGQGTQVTVSG(SEQ ID NO:43)。

[0051] NB672-ITGA4-Mouse ITGAE-M1F3-80:

[0052] QLQLVESGGGLVQPGGSLRLSCATSRSRFGIYNMGWYRQAPGKQRE LVATISGSSTGGDFTNYAASVKGRFTISRDNAANTMYLQMNSLKPEDTAV YYCNVDQVDTIVVDLWGQGTQVTVSS(SEQ ID NO:44)。

[0053] A second aspect of the present invention provides a nucleic acid molecule encoding the aforementioned anti-human integrin CD103 nanobody.

[0054] A third aspect of the present invention provides a carrier comprising the nucleic acid molecule described in the second aspect.

[0055] A fourth aspect of the present invention provides a host cell containing the vector described in the third aspect or the nucleic acid molecule described in the second aspect.

[0056] The host cells include, but are not limited to, bacterial cells, fungal cells, or animal cells.

[0057] The anti-human integrin CD103 nanobody of the present invention can be obtained by the following method:

[0058] (1) Yeast display library was prepared by immunizing alpacas with human and mouse CD103-related nucleic acids.

[0059] (2) Use human CD103-related proteins to affinity screen yeast display libraries;

[0060] (3) Identify positive clones;

[0061] (4) Expression and purification of anti-human integrin CD103 nanobody.

[0062] According to one specific embodiment, the present invention first immunizes alpacas with human and mouse CD103-related nucleic acids, extracts peripheral blood from alpacas to separate alpaca peripheral blood lymphocytes, and constructs a CD103-specific single-domain heavy chain antibody immune library. Then, magnetic sorting and flow cytometry are used to screen the immune library with human CD103 protein to obtain single-domain heavy chain antibodies against human CD103, thereby obtaining a highly efficient nanobody strain.

[0063] A fifth aspect of the present invention provides a method for engineering the production of anti-human integrin CD103 nanobodies, comprising the following steps:

[0064] (a) Under conditions suitable for the production of nanobodies, the host cells described above are cultured to obtain a culture containing the anti-human integrin CD103 nanobodies;

[0065] (b) Isolating and / or recovering the anti-human integrin CD103 nanobody from the culture; and optionally...

[0066] (c) Purification and / or modification of the anti-human integrin CD103 nanobody obtained in step (b).

[0067] A sixth aspect of the present invention provides an antibody-drug conjugate comprising the aforementioned anti-human integrin CD103 nanobody, a linker, and an effector; preferably, the effector comprises at least one of a radionuclide, a cytotoxic agent, a fluorescent group, an enzyme that catalyzes substrate color development, a chemiluminescent reagent, and a nanoparticle-based label. The radionuclide may be a diagnostic radionuclide or a therapeutic radionuclide, preferably a diagnostic radionuclide. 18 F, 32 P, 33 P, 45 Ti、 47 Sc、 52 Fe、 59 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 75 Sc、 77 As、 86 Y、 90 Y、 89 Sr、 89 Zr、 94 Tc, 94 Tc, 99m Tc, 99 Mo、 105 Pd, 105 Rh、 111 Ag、 111 ln、 123 I, 124 I, 125 I, 131 I, 142 Pr、 143 Pr、 149 Pm, 153 Sm、 154"1581 Gd, 161 Tb, 166 Dy、 166 Ho、 169 Er、 175 Lu、 177 Lu、 186 Re、 188 Re、 189 Re、 194 lr、 198 Au、 199 Au、 211 At、 211 Pb, 212 Bi、 212 Pb, 213 Bi、 223 Ra and 225At least one of Ac; the therapeutic radionuclide is preferably... 32 P, 47 Sc、 57 Co、 89 Sr、 90 Y、 103 Pd, 106 Ru、 124 I, 125 I, 131 I, 131 Cs、 137 Cs、 177 Lu、 192 Ir、 212 Bihe 225 At least one of Ac.

[0068] A seventh aspect of the present invention provides a pharmaceutical composition comprising the above-described anti-human integrin CD103 nanobody, or the above-described antibody-drug conjugate.

[0069] The above-mentioned pharmaceutical composition also includes one or more pharmaceutical excipients.

[0070] The eighth aspect of the present invention provides the following uses of the aforementioned anti-human integrin CD103 nanobody or antibody-drug conjugate:

[0071] (i) Prepare a reagent for detecting CD103-mediated diseases; preferably, the reagent is a kit for detecting CD103-mediated diseases;

[0072] (ii) To prepare drugs for the treatment of CD103-mediated diseases;

[0073] (iii) Prepare targeted imaging probes for myocardial fibrosis; the myocardial fibrosis is caused by heart diseases such as myocardial infarction, hypertrophic cardiomyopathy, and dilated cardiomyopathy.

[0074] The CD103-mediated diseases are preferably cancer or heart disease.

[0075] The cancers mentioned include, but are not limited to: non-small cell lung cancer, colorectal cancer, breast cancer, stomach cancer, liver cancer, etc.

[0076] The heart diseases mentioned include, but are not limited to: post-myocardial infarction myocardial fibrosis, hypertrophic cardiomyopathy, and dilated cardiomyopathy.

[0077] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0078] (1) The anti-human integrin CD103 nanobody involved in this invention can achieve high sensitivity and high specificity binding to CD103 protein, CD103 positive cell line, tissue section, etc. in vitro.

[0079] (2) The present invention relates to the anti-human integrin CD103 nanobody and its preparation. 18 F-CYNB nanobody probes can enable in vivo tumor immune imaging and predict the efficacy of tumor immunotherapy.

[0080] (3) The present invention relates to the anti-human integrin CD103 nanobody and its preparation. 18 F-CYNB nanobody probes can be used for targeted imaging of myocardial fibrosis caused by myocardial infarction, hypertrophic cardiomyopathy, dilated cardiomyopathy, etc.

[0081] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0082] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0083] Figure 1 A schematic diagram of the immunization process in alpacas is shown.

[0084] Figure 2 The flowchart for constructing the yeast display library is shown.

[0085] Figure 3 A flowchart for screening the yeast display library is shown.

[0086] Figure 4 The analysis spectrum from the NB672 flow cytometer is shown.

[0087] Figure 5 The expression and purification process of CD103 nanobody is shown.

[0088] Figure 6 The affinity (FACS) of the antibody for binding to the 293T-Human-ITGAE&ITGB7 cell line was demonstrated.

[0089] Figure 7 The affinity (FACS) of the antibody for binding to the 293T-Human-ITGAE&ITGB7 and 293T-mouse-ITGAE&ITGB7 cell lines was demonstrated.

[0090] Figure 8 The affinity (FACS) of the antibody for binding to the 293T cell line was demonstrated.

[0091] Figure 9 The affinity of the antibody for binding to the human ITGAE & ITGB7 histidine tag was demonstrated by ELISA.

[0092] Figure 10 The affinity of the antibody for binding to the human ITGA4 & ITGB7 histidine tags was demonstrated by ELISA.

[0093] Figure 11 The affinity of the antibody for binding to the mouse ITGAE & ITGB7 histidine tag was demonstrated by ELISA.

[0094] Figure 12-1 , 12-2 12-3 are the HPLC detection results of CYNB-06, CYNB-07, and CYNB-08, respectively.

[0095] Figure 13 The study showed that CD103 expression was significantly elevated in mouse myocardial fibrosis tissue.

[0096] Figure 14 for 18 Imaging results of F-CYNB-06 in a pig model of myocardial infarction (seventh day after surgery) at 0.5-2 hours.

[0097] Figure 15 for 18 Imaging comparison of F-CYNB-06 in a pig model with myocardial infarction on the seventh day after surgery and in a sham-operated control group 1 hour after injection. Detailed Implementation

[0098] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0099] definition

[0100] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art as would be understood by those skilled in the art. Furthermore, unless otherwise stated, all methods, steps, techniques, and operations not specifically detailed herein can and have been performed in a manner known per se as would be understood by those skilled in the art.

[0101] Antigen preparation materials

[0102]

[0103] Example 1: Alpaca Immunization

[0104] (1) Retrieve Human CD103 and mouse CD103 sequence information from UniProt database, subclone them into commercial vectors, construct eukaryotic expression vectors, and extract plasmids after Sanger sequencing verification;

[0105] (2) Human CD103 mRNA and mouse CD103 mRNA were transcribed. The Human CD103 mRNA sequence consists of two sequences, SEQ ID NO: 45 (Homo sapiens ITGAE) and SEQ ID NO: 46 (Homo sapiens ITGB7). The mouse CD103 mRNA sequence consists of two sequences, SEQ ID NO: 47 (Mus musculus ITAE) and SEQ ID NO: 48 (Mus musculus ITB7). An outsourced company coated the two mRNAs with LNPs to obtain Human CD103 LNP-mRNA and mouse CD103 LNP-mRNA.

[0106] (3) An outsourced company conducted alpaca immunization: Human CD103 LNP-mRNA (left) and mouse CD103 LNP-mRNA (right) were simultaneously immunized on the left and right sides of the alpaca's neck, respectively. The immunization process was as follows: Figure 1 As shown.

[0107] Example 2: Construction and Screening of Yeast Display Libraries

[0108] (1) Construction of yeast display library

[0109] like Figure 2 As shown, the procedure includes the following steps: PBMC collection, total RNA extraction, cDNA transcription, nested PCR amplification of the alpaca VHH gene, ligation of the VHH gene into a yeast display vector, EB100 cell transformation and library construction, QC PCR for positive insertions, and insertion sequence analysis. All steps were performed using standard conditions in the field, resulting in the NB672 library.

[0110] (2) Screening of yeast display library

[0111] like Figure 3 As shown, it includes the following steps:

[0112] (2-1) First round of magnetic bead screening

[0113] 1) Take 1 mL of NB672 library cells, revive them in 1 L of SD-CAA medium for 16 h, centrifuge to collect the cells, and adjust the concentration to 5 × 10⁻⁶. 6 Cell / mL, 1L SG culture based on expression at 20℃ for 24h, final expression concentration: 2.98×10⁻⁶ cells / mL, 1L SG culture. 7 cell / mL.

[0114] 2) Magnetic bead preparation: Take 300 μL of magnetic beads, wash them 3 times with PBSAT buffer on a magnetic rack, and set aside.

[0115] 3) Magnetic bead pretreatment: Human-ITGA4 & ITGB7 (human α4 & β7) were incubated with 100 μL of magnetic beads at 4℃ for 1 h (incubation volume: 100 μL), and Human-ITGAE & ITGB7 (human CD103 & β7) were incubated with 200 μL of magnetic beads at 4℃ for 1 h (incubation volume: 200 μL). Then the magnetic beads were washed 3 times with PBSAT and set aside for later use.

[0116] 4) Take 1.0 × 10 10 After washing the NB672 library expression cells with PBSAT buffer, they were incubated with pretreated target proteins Human-ITGA4 & ITGB7 negative sieve magnetic beads at 4°C for 2 hours. The magnetic beads were then removed and the suspended yeast cells were collected.

[0117] 5) Incubate yeast cells with pretreated target proteins Human-ITGAE & ITGB7-positive sieve magnetic beads at 4°C for 2 hours (incubation volume: 5 mL), wash the magnetic bead yeast complex three times with PBSAT buffer, resuspend the magnetic bead yeast complex in 1 mL of SD medium, add it to 100 mL of SD medium, and amplify.

[0118] 6) Amplify cells at 30℃ in 100mL SD medium for 15-16 hours. Remove the magnetic beads from the culture medium and store 4 vials containing 25% glycerol.

[0119] 7) The next step is to use flow cytometry to identify the magnetic bead enrichment products. The products screened in this round are named: NB672Human-ITGAE&ITGB7 M1.

[0120] 8) If the product positivity rate is high, proceed to the next step of flow cytometry separation. If the product positivity rate is low, continue magnetic bead separation until a product with a high positivity rate is obtained before proceeding to flow cytometry separation.

[0121] (2-2) Flow cytometry identification of enriched products from a single round of magnetic beads

[0122] 1) Centrifuge the cells expanded in step 5) of (2-1) and adjust the initial expression concentration to 4 × 10⁻⁶. 6 Cell / mL, 200mL, 25℃, 16h expression, final expression concentration: 1.33×10⁻⁶ cells / mL. 7 cell / mL. Divided into 9 portions (1×10⁻⁶ cells / mL each). 7 Cells, 100 μL in volume. Wash three times with PBSAT and set aside.

[0123] 2) Primary staining. Human-ITGAE & ITGB7 (100nM / 33.3nM / 11.1nM / 3.7nM / 1.23nM) were incubated with Anti-His tag (1:100 dilution) for 1 h, and Human-ITGA4 & ITGB7 (100nM) were incubated with Anti-His tag (1:100 dilution) for 1 h.

[0124] 3) Secondary staining. Cells were incubated with Human-ITGAE & ITGB7-Anti-His tag and Anti-HA-488 for 1 hour, and cells were incubated with Human-ITGA4 & ITGB7-Anti-His tag and Anti-HA-488 for 1 hour.

[0125] 4) Wash three times with ice-cold PBSAT, resuspend, identify by flow cytometry, and finally sort by a sorter.

[0126] (2-3) Two-round sorting

[0127] 1) Use a sorting machine to sort the samples from step (2-2) 4).

[0128] 2) The sorted products were amplified and cultured in 2 mL SD medium at 30℃ for 16-18 h, and 4 strains were preserved.

[0129] (2-4) Identification of products from two-stage sorting

[0130] 1) Take cells with good induction expression (NB672 Human-ITGA4 & ITGB7 M1F1, final expression concentration: 1.32 × 10⁻⁶). 7 (cells / mL; expression volume: 100mL; expression at 20℃ for 24h) in 4 EP tubes (1×10⁻⁶ cells / mL; expression volume: 100mL; expression at 20℃ for 24h) 7 The cells / tubes were washed three times with PBSAT and then set aside.

[0131] 2) Primary staining: Human-ITGAE & ITGB7 (100 nM) were incubated with Anti-His tag (1:100 dilution) for 1 h, and Human-ITGA4 & ITGB7 (100 nM) were incubated with Anti-His tag (1:100 dilution) for 1 h.

[0132] 3) Secondary staining: Cells were incubated with Human-ITGAE & ITGB7-SA-650 and Anti-HA-488 for 1 h, and cells were incubated with Human-ITGA4 & ITGB7-Anti-His tag and Anti-HA-488 for 1 h.

[0133] 4) Wash three times with ice-cold PBSAT, resuspend, and identify by flow cytometry.

[0134] (2-5) Three-round sorting

[0135] 1) Use a sorting machine to sort the samples from step 4) of (2-4).

[0136] 2) The sorted products were amplified and cultured in 2ml SD medium at 30℃ for 16-18h, and 4 strains were preserved.

[0137] (2-6) Identification of products from three-stage sorting

[0138] 1) Take cells with good induction expression (NB672 Human ITGA4-ITGAE & ITGB7 M1F2, final expression concentration: 3.22 × 10⁻⁶). 7 (cells / mL; expression volume: 100mL; expression at 20℃ for 24h) in 4 EP tubes (1×10⁻⁶ cells / mL; expression volume: 100mL; expression at 20℃ for 24h) 7 The cells / tubes were washed three times with PBSAT and then set aside.

[0139] 2) Primary staining: Human-ITGAE & ITGB7 (100 nM) were incubated with Anti-His tag (1:100 dilution) for 1 h, and Human-ITGA4 & ITGB7 (100 nM) were incubated with Anti-His tag (1:100 dilution) for 1 h.

[0140] 3) Secondary staining: Cells were incubated with Human-ITGAE & ITGB7-SA-650 and Anti-HA-488 for 1 h, and cells were incubated with Human-ITGA4 & ITGB7-Anti-His tag and Anti-HA-488 for 1 h.

[0141] 4) Wash three times with ice-cold PBSAT, resuspend, and identify by flow cytometry.

[0142] (2-7) Colony detection

[0143] 1) Coat the NB672 Human ITGA4-ITGAE&ITGB7 M1F2 product at 30℃ for 48 hours.

[0144] 2) Select single clones for colony detection and spot them on a plate.

[0145] 3) Colony PCR:

[0146]

[0147] Reaction program: 95℃ for 3 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 45 s, 30 cyc; 72℃ for 5 min.

[0148] 4) Send to bioengineering for sequencing.

[0149] (2-8) Monoclonal Identification

[0150] Single stain identification

[0151] 1) Express the monoclonal expression of (NB672 Human ITGA4-ITGAE&ITGB7 M1F2) to be validated.

[0152] 2) Primary staining. Human-ITGAE & ITGB7 (100 nM) and Anti-His tag (1:100 dilution) were incubated at 4°C for 1 h, and Human-ITGA4 & ITGB7 (100 nM) and Anti-His tag (1:100 dilution) were incubated at 4°C for 1 h.

[0153] 3) Secondary staining. Cells were incubated with Human-ITGAE & ITGB7-Anti-His tag and Anti-HA-488 at 4℃ for 1 h, and cells were incubated with Human-ITGA4 & ITGB7-Anti-His tag and Anti-HA-488 at 4℃ for 1 h.

[0154] 4) Wash three times with ice-cold PBSAT and resuspend, then identify using a flow cytometer.

[0155] 5) Data processing.

[0156] Filtering results

[0157] The flow cytometer analysis chromatogram of the products from the first round of magnetic bead screening in the NB672 library is shown below. Figure 4 The NB672 Human-ITGAE & ITGB7 M1 screening product bound to 100 nM Human-ITGAE-his, with a positive rate of 40.70% (box B-Q2), indicating successful magnetic bead screening.

[0158] The NB672 library's first-round magnetic bead screening products were stained with target proteins Human-ITGA4 & ITGB7 and analyzed by flow cytometry, resulting in the sorting of 110,000 cells.

[0159] 100,000 cells were sorted by flow cytometry after alternating sorting of products from NB672 library 2 and staining with target proteins Human-ITGAE & ITGB7.

[0160] Monoclonal sequencing analysis

[0161] The NB672 Human ITGA4-ITGAE & ITGB7 M1F2 sorted products were streaked onto plates, and single clones were selected for sequencing. A total of 3 plates were sent, yielding 288 sequences, 16 of which showed bimodal peaks, with 243 valid sequences. After obtaining the single clone sequences, they were translated into protein sequences, and unique sequences were selected for alignment and phylogenetic tree construction.

[0162] Flow cytometry for identification of monoclonal antibodies

[0163] The obtained monoclonal sequences were classified according to CDR3, and unique clones were selected for flow cytometry identification.

[0164] in conclusion

[0165] The NB672 library was screened using Human ITGAE & ITGB7 proteins. After one round of magnetic bead screening and three rounds of flow chemistry screening, the screened products met the screening requirements.

[0166] The product pools for screening and sequencing are shown in Table 1 below.

[0167] Table 1 Sequencing results of NB672 screening products

[0168]

[0169] A total of 40 monoclonal samples were selected for identification using NB672-Human-ITGA4-ITGAE-M1F2, among which 34 positive sequences of 100nM Human ITGAE & ITGB7 protein were detected; the results of monoclonal identification are shown in Table 2.

[0170] Table 2. Monoclonal Identification Results of NB672 Human ITGA4-ITGAE & ITGB7 M1F2

[0171]

[0172]

[0173]

[0174] NB672-ITGA4-Mouse ITGAE-M1F3-80 is an antibody that can target both humans and mice.

[0175] Example 4: Expression and purification of CD103 nanobodies

[0176] like Figure 5 As shown, it includes the following steps:

[0177] The selected VHH gene fragment was cloned into the pcDNA3.4-IgG1 Fc eukaryotic expression vector to construct a recombinant plasmid, which was then transformed into competent cells for amplification. High-purity plasmids were extracted using an endotoxin-free kit for transfection. Next, the recombinant plasmid was transiently transfected into HEK293 cells in logarithmic growth phase using PEI Max transfection reagent (Shanghai Qifa Experimental Reagent Co., Ltd.). Cells were cultured at 37°C and 8% CO2 for approximately 7 days, and protein expression was enhanced using a fed-batch strategy. After expression, the cell culture supernatant was collected by centrifugation and filtered for clarification. Subsequently, the supernatant containing the target protein was purified using MabSelectSureProtein A affinity chromatography packing material (Suzhou Navtech Co., Ltd.): after sample loading, the cells were first thoroughly washed with PBS buffer, then eluted with low-pH glycine buffer (pH 3.0), and immediately neutralized with Tris buffer (pH 9.0) to maintain protein activity. Finally, the purified VHH-Fc fusion protein was concentrated by ultrafiltration centrifuge tubes and replaced with PBS buffer. After Nanodrop concentration determination, SDS-PAGE and SEC-HPLC quality control, it was aliquoted and stored at -80℃ for long-term storage.

[0178] The expression levels, purity, endotoxin, molecular weight, isoelectric point, and other physicochemical properties of each clone in the NB672 project are summarized in the table below.

[0179] As shown in the table below, the expression levels of all clones in the NB672 project are very high, ranging from 419.33 to 459.33 mg / L. Meanwhile, the purity is normal, with both SDS-PAGE and SEC-HPLC purities exceeding 95%. Therefore, the antibody can be used for further analysis.

[0180]

[0181] Example 5: Assay of the binding of CD103 nanobody to CD103 antigen

[0182] I. NB672 VHH-hIgG1 Fc cell binding detection results:

[0183] (1) Wash the cells twice with PBS, resuspend the cells with PBS, and dispense the cells into PCR plates, 50 μL per well;

[0184] (2) Dilute the antibody to be tested with PBS to a starting concentration of 400 nM, dilute 1:5, take 50 μL / well and add it to the plate to make the starting concentration of antibody 200 nM, 5-fold gradient, and incubate at 4°C for 1 hour.

[0185] (3) After incubation, wash the cells three times with PBS, add 100 μL of fluorescent secondary antibody (647 Anti-HumanIgG Fc) to each well, and incubate at 4°C for 1 hour.

[0186] (4) After incubation, wash the cells once with PBS, transfer the cells to a 96-well cell plate, and analyze them by flow cytometry.

[0187] (5) Export the analysis data and analyze it using FlowJo software to obtain the MFI value;

[0188] (6) Import the data into Graphpad Prism for curve fitting.

[0189] The results are as follows Figure 6 , 7 8 and the table below (EC50 values) are shown. Figure 6 The antibody's affinity (FACS) for binding to the 293T-Human-ITGAE&ITGB7 cell line was demonstrated. Figure 7 The affinity (FACS) of the antibody for binding to the 293T-Human-ITGAE&ITGB7 and 293T-mouse-ITGAE&ITGB7 cell lines was demonstrated. Figure 8 The affinity (FACS) of the antibody for binding to the 293T cell line was demonstrated.

[0190] The test results showed that each antibody had a good binding to the 293T-Human-ITGAE&ITGB7 cell line. Among them, NB672-ITGA4-Mouse ITGAE-M1F3-80 could bind to both the 293T-Human-ITGAE&ITGB7 cell line and the 293T-Mouse-ITGAE&ITGB7 cell line.

[0191]

[0192] II. NB672 VHH-hIgG1 Fc ELISA Detection Results

[0193] (1) At a concentration of 2 μg / mL, 100 μL / well of Human-ITGAE&ITGB7 his tag, Human-ITGA4&ITGB7his tag and mouse-ITGAE&ITGB7 his tag (50 mM NaHCO3 pH=9.6) were coated and incubated overnight at 4℃.

[0194] (2) Wash three times with PBST and block with 300 μL / well of 5% milk. Incubate at 37°C for 1 h.

[0195] (3) Wash once with PBST, and then perform a 1:5 gradient dilution of the protein from 100 nM (5% milk dilution). Add 100 μL of the gradient diluted antibody protein to the wells of the ELISA plate and incubate at 37°C for 1 h.

[0196] (4) Wash 5 times with PBST and add the secondary antibody (Anti-Human IgG Fc, HRP, 1:10K) diluted with blocking buffer accordingly.

[0197] (5) Wash the microplate that has been incubated with the secondary antibody with PBST 5 times, add 100 μL of TMB single-component colorimetric solution to each well and incubate at 37°C for 7 min. Add 50 μL of 1M HCl to each well to stop the reaction and read the OD450 value.

[0198] (6) Import the data into Graphpad Prism for curve fitting.

[0199] The results are as follows Figure 9 , 10 The values ​​are shown in Table 11 and below (EC50 values). Figure 9 The affinity of the antibody for binding to the Human ITGAE & ITGB7 his tag was demonstrated by ELISA. Figure 10 The affinity of the antibody for binding to the Human ITGA4 & ITGB7 his tag was demonstrated by ELISA. Figure 11 The affinity of the antibody for binding to the mouse ITGAE & ITGB7 his tag was demonstrated by ELISA.

[0200]

[0201] The above experimental results successfully verified that each antibody has a good binding ability with human CD103.

[0202] Example 6 18 Preparation of F-CD103 nanobody-labeled precursor

[0203] Short peptide GGGGC conjugated with (±)H3 Resca-Mal:

[0204] (1) Add raw materials according to the ratio of GGGGC:Resca = 1:1.5.

[0205] (2). Add 1M Tris-HCl to adjust the pH to 7.5, and react at 37℃ for 1 hour.

[0206] (3). HPLC, C18 column, A phase water, B phase acetonitrile to collect the product, peak elution time t≈6.3 minutes, freeze-dried for storage.

[0207] NB672 VHH-hIgG1 Fc (named CYNB) conjugated with GGGGC-Resca:

[0208] (1) Add raw materials according to the ratio of CYNB:GGGGC-Resca = 1:10.

[0209] (2) Add 150 μg of Sortase A enzyme, 10% volume of 1M Tris-HCl, 50 μg of NaCl, and 100 mM CaCl2 (30 μL per 1 mL reaction system). React at 37°C for 2 hours.

[0210] (3). HPLC, + Superdex column, A-phase PBS, peak elution time t≈16. Collect in 1k ultrafiltration tube, centrifuge at 4000 rad for 30 minutes. Aliquot and store at -80℃.

[0211] Finally, three labeled precursors were obtained: CYNB-06 (prepared from NB672-4-E-M1F2-20), CYNB-07 (prepared from NB672-4-E-M1F2-30), and CYNB-08 (prepared from NB672-4-E-M1F2-98), for subsequent experiments. The HPLC detection results of CYNB-06, CYNB-07, and CYNB-08 are shown below. Figure 12-1 , 12-2 As shown in Figure 12-3.

[0212] Example 7 18 F-CYNB-06 imaging in a pig model of myocardial infarction

[0213] (1) Model establishment

[0214] An acute myocardial infarction model was established using a percutaneous coronary balloon combined with gel sponge embolization method. Specific procedures: After fasting for 12 hours, experimental pigs were induced with an intramuscular injection of 1.25 mg / kg of acetaminophen. An intravenous access was established through the marginal ear vein, and endotracheal intubation was performed, connecting the pigs to a ventilator and ECG monitor. Vital signs such as heart rate, blood pressure, pulse oximetry, and respiratory rate were monitored throughout the procedure. Isoflurane was used to maintain anesthesia. The pigs were placed supine on the experimental table, and heparin 200 μl / kg was administered for anticoagulation. The right groin was prepared, routinely disinfected, and draped. An arterial puncture was performed, and a 6F hind limb sheath was inserted. Using a left anterior oblique position (45°), under C-arm fluoroscopy, a guiding catheter was inserted through the sheath into the artery. The guiding catheter was guided to the ascending aorta using an angiography guidewire. The guidewire was withdrawn, and the guiding catheter was guided again sequentially into the openings of the left coronary sinus, left main coronary artery, and left anterior descending artery (LAD), ensuring the catheter opening was coaxial with the coronary artery opening. Coronary angiography was performed by injecting iopromide solution to locate the first diagonal branch. A guidewire was carefully inserted through a guiding catheter into the distal end of the left anterior descending coronary artery. A homemade balloon was then advanced along the guidewire to approximately 1-2 mm below the first diagonal branch. The guidewire and balloon were withdrawn, leaving the anterior portion of the homemade embolus at this location. After 5 minutes, angiography confirmed the occlusion of blood flow. The catheter and sheath were then withdrawn sequentially, and hemostasis was achieved by applying pressure to the aorta, thus ending the procedure. The sham surgery group underwent the same procedures as the acute myocardial infarction group, except that no embolus was used for blood flow occlusion.

[0215] (2) Imaging steps:

[0216] QMA column activation: First, inject 10 mL of 0.5 M sodium bicarbonate solution, then inject 10 mL of sterile water for injection, and finally inject air to dry the column.

[0217] Rinse 18 F - : 18 F - After diluting with 2 mL of pure water, pass the solution through an activated QMA column. After air drying, inject 300 μL of physiological saline. Discard the first 100 μL and collect the last 200 μL.

[0218] Labeled precursor: In 200 μL of the eluent collected 18 F - Add 10 μL of 2 mM AlCl3 (using HAC-NaOAC buffer at pH 4.5) and react at 37°C for 5 minutes. Add 200 μg of the labeled precursor, adjust the pH to 4.6-4.8 using HAC-NaOAC buffer at pH 4.5, and react at 37°C for 15 minutes.

[0219] Purification of the radionuclide-labeled probe: Activate the PD-10 column with 25 mL PBS. After adding the above reaction solution, add PBS to make a volume of 1 mL. After completely draining, add 1 mL, 1 mL, 0.5 mL, 0.5 mL, and 0.5 mL PBS respectively, collecting the contents into five 1.5 mL EP tubes, labeled 1-5. Tube 3 is the pre-labeled tube. 18 F-CYNB-06.

[0220] Tail vein injection: In vivo imaging was conducted on day 7 post-surgery in a pig model of acute myocardial infarction. (Intravenous injection) 18 F-CYNB-06, approximately 3 mCi per animal. Small animal PET imaging was performed 0.5, 1, and 2 hours later.

[0221] (3) Results and Analysis:

[0222] 18 F-CYNB-06 imaging in a pig model of myocardial infarction (seventh day post-surgery) at 0.5-2 hours: Figure 14 As shown in the figure. The results showed that there was significant specific uptake at the post-myocardial infarction fibrosis site (white dashed line in the figure), and the imaging effect was best 1 hour after probe injection.

[0223] Figure 15 for 18 Imaging comparison of F-CYNB-06 in a pig model of myocardial infarction on the seventh day after surgery and in a sham-operated control group 1 hour after injection. It can be seen that there is specific uptake of the probe in the fibrotic tissue after myocardial infarction, while no specific aggregation of the probe can be observed in the control group.

[0224] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A nanobody against human integrin CD103, characterized in that, It contains three complementary determinant regions, CDR1, CDR2, and CDR3; among them, (1) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 13, respectively; (2) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 14, respectively; (3) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 3, SEQ ID NO: 9, and SEQ ID NO: 15, respectively; (4) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 16, respectively; (5) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 17, respectively; (6) The amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 6, SEQ ID NO: 12 and SEQ ID NO: 18, respectively.

2. The anti-human integrin CD103 nanobody according to claim 1, characterized in that, It also includes four frame regions FR1, FR2, FR3, and FR4, which are alternately set with the three complementary determinant regions. The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 31, and SEQ ID NO: 37, respectively. The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 37, respectively. The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 37, respectively. The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, and SEQ ID NO: 37, respectively. The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, and SEQ ID NO: 38, respectively. The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36, and SEQ ID NO: 37, respectively.

3. The anti-human integrin CD103 nanobody according to claim 1, characterized in that, The nanobody is selected from the amino acid sequence shown in one of SEQ ID NO: 39 to SEQ ID NO:

44.

4. A nucleic acid molecule, characterized in that, Its encoding is the anti-human integrin CD103 nanobody as described in any one of claims 1-3.

5. A carrier, characterized in that, It comprises the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, It contains the carrier as described in claim 5 or the nucleic acid molecule as described in claim 4.

7. The host cell according to claim 6, characterized in that, The host cell can be a bacterial cell, a fungal cell, or an animal cell.

8. A method for generating anti-human integrin CD103 nanobodies, characterized in that, Includes the following steps: (a) Under conditions suitable for the production of nanobodies, host cells as described in claim 6 or 7 are cultured to obtain a culture containing the anti-human integrin CD103 nanobodies; (b) Isolating and / or recovering the anti-human integrin CD103 nanobody from the culture; and optionally... (c) Purification and / or modification of the anti-human integrin CD103 nanobody obtained in step (b).

9. An antibody conjugate, characterized in that, The invention includes the anti-human integrin CD103 nanobody, linker, and effector as described in any one of claims 1-3; the effector is at least one of a nuclide, a fluorescent group, an enzyme that catalyzes substrate color development, a chemiluminescent reagent, and a nanoparticle label; the nuclide is a diagnostic radionuclide.

10. The antibody conjugate according to claim 9, characterized in that, The diagnostic radionuclide is 18 F, 32 P, 33 P, 45 Ti、 47 Sc、 52 Fe、 59 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 75 Sc、 77 As、 86 Y、 90 Y、 89 Sr、 89 Zr、 94 Tc, 94 Tc, 99m Tc, 99 Mo、 105 Pd, 105 Rh、 111 Ag、 111 ln、 123 I, 124 I, 125 I, 131 I, 142 Pr、 143 Pr、 149 Pm, 153 Sm、 154"1581 Gd, 161 Tb, 166 Dy、 166 Ho、 169 Er、 175 Lu、 177 Lu、 186 Re、 188 Re、 189 Re、 194 lr、 198 Au、 199 Au、 211 At、 211 Pb, 212 Bi、 212 Pb, 213 Bi、 223 Ra and 225 At least one of Ac.

11. A pharmaceutical composition, characterized in that, Contains the anti-human integrin CD103 nanobody as described in any one of claims 1-3, or the antibody conjugate as described in claim 9 or 10.

12. The following uses of the anti-human integrin CD103 nanobody according to any one of claims 1-3, or the antibody conjugate according to claim 9 or 10: (i) Preparation of reagents for detecting CD103-mediated diseases; (ii) Preparation of targeted imaging probes for myocardial fibrosis; The CD103-mediated diseases are cancer or heart disease; the cancers are non-small cell lung cancer, colorectal cancer, breast cancer, gastric cancer, or liver cancer; the heart disease is myocardial fibrosis after myocardial infarction, hypertrophic cardiomyopathy, or dilated cardiomyopathy.

13. The use according to claim 12, characterized in that, The reagent is a kit for detecting CD103-mediated diseases.

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