Anti-Trop2 nano antibody and application thereof

Abstract

The invention relates to the technical field of nano antibodies, and discloses an anti-Trop2 nano antibody and application thereof. The nano antibody for resisting Trop2, provided by the invention, has unique variable regions CDR1, CDR2 and CDR3. The invention also provides a nucleic acid molecule for coding the nano antibody, and provides a specific molecular imaging probe containing the nano antibody. The nano antibody provided by the invention can be specifically combined with Trop2 protein, and a probe prepared from the nano antibody has excellent tumor targeting and specificity, and has a better imaging effect when being applied to tumor model mice. Through the amino acid sequence and the gene sequence of the nano antibody disclosed by the invention, a theoretical basis is provided for developing a new tumor treatment strategy based on a Trop2 target spot.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to anti-Trop2 nano antibodies and applications thereof. Background Art

[0002] Trop2, or tumor-associated antigen 2, is an important tumor-associated antigen that is widely expressed on the surface of a variety of malignant tumor cells, including breast cancer, lung cancer, gastric cancer, and colon cancer. High expression of Trop2 is closely related to tumor growth, invasion, metastasis, and prognosis, and is therefore considered a target with great potential. In the diagnosis and treatment of tumors, the therapeutic strategy of targeting Trop2 has gradually become a research hotspot, mainly including antibody-drug conjugates (ADCs), bispecific antibodies, radioligand conjugates, and chimeric antigen receptor T cell (CAR-T) therapy.

[0003] For example, gosartan (Trodelvy) is an antibody-drug conjugate (ADC) targeting Trop2 that has been clinically approved and used to treat a variety of solid tumors. Trodelvy couples chemotherapy drugs with anti-Trop2 antibodies, which can accurately deliver chemotherapy drugs directly to tumor cells, effectively improving the therapeutic effect. However, although Trodelvy has shown certain efficacy in clinical applications, it also has some side effects, such as neutropenia and diarrhea, which limits its application in certain patient groups.

[0004] As research deepens, the therapeutic strategy of Trop2 as a target is constantly expanding. As a new type of targeting molecule, Trop2 nanoantibodies have gradually become an important tool for targeted therapy in recent years due to their small molecule characteristics, high affinity and good tissue permeability. At the same time, in the field of radioactive probes, the application of Trop2 nanoantibodies has shown great potential, especially in the early diagnosis of tumors, precision radiotherapy and tumor imaging.

[0005] Although the existing Trop2 targeted therapeutic drugs such as gosartuzumab have certain efficacy, they still have certain limitations in specificity and affinity, and there is still room for improvement in efficacy. Therefore, providing nanobodies with better structural and functional properties is still of great practical significance. Summary of the invention

[0006] In view of the existing defects and problems, the present invention provides an anti-Trop2 nanobody and its application.

[0007] The solution adopted by the present invention to solve the technical problem is: the present invention provides an anti-Trop2 nanobody, the anti-Trop2 nanobody comprises a variable region,

[0008] The variable region comprises any one or more of CDR1, CDR2 or CDR3 sequences;

[0009] (a), the CDR1 has the amino acid sequence shown in SEQ ID NO.1; or the CDR2 has the amino acid sequence shown in SEQ ID NO.2; or the CDR3 has the amino acid sequence shown in SEQ ID NO.3;

[0010] or (b) an amino acid sequence after modification of the amino acid sequence described in (a) and having the same function as the amino acid sequence described in (a);

[0011] or (c) an amino acid sequence as described in (a) or (b) in which one or more amino acids are substituted, deleted or added, and which has the same function as the amino acid sequence described in (a) or (b);

[0012] or (d) an amino acid sequence having 90% or more identity with the amino acid sequence described in (a) or (b) or (c).

[0013] The above-mentioned anti-Trop2 nanobody, the variable region has:

[0014] (a), the amino acid sequence shown in SEQ ID NO.4;

[0015] or (b) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (a), and having the same function as the amino acid sequence described in (a);

[0016] or (c) an amino acid sequence having 90% or more identity with the amino acid sequence described in (a) or (b).

[0017] The present invention also provides a nucleic acid molecule encoding the above-mentioned nanobody.

[0018] The above-mentioned nucleic acid molecule, the nucleic acid molecule has:

[0019] (a), the nucleotide sequence shown in SEQ ID NO.5;

[0020] or (b), a nucleotide sequence complementary to the nucleotide sequence shown in (a);

[0021] or (c), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (a) or (b) but differing from the nucleotide sequence of (a) or (b) due to the degeneracy of the genetic code;

[0022] or (d), a nucleotide sequence obtained by substituting, deleting or adding one or two nucleotide sequences to the nucleotide sequence shown in (a), (b) or (c), and a nucleotide sequence having the same or similar functions as the nucleotide sequence shown in (a), (b) or (c);

[0023] or (e), a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of (a), (b), (c) or (d).

[0024] The application of the above-mentioned anti-Trop2 nanobody in the preparation of Trop2 molecular imaging probe.

[0025] The present invention also provides a Trop2-specific molecular imaging probe, the probe comprising a tumor targeting group and a radionuclide; the tumor targeting group is the nanobody according to claim 1 or 2; the radionuclide is 68 Ga, 124 I or 89 Any of Zr.

[0026] The above Trop2-specific molecular imaging probe, when the radionuclide is selected 68 Ga or 89 When Zr is present, the probe further comprises a chelating agent DOTA.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The anti-Trop2 nanoantibody obtained by phage display technology in the present invention can specifically bind to the Trop2 protein and can be used to detect the expression level of the Trop2 protein in biological samples.

[0029] 2. The present invention couples NOTA on the basis of nanobodies and utilizes short half-life positron nuclides 68 Ga-labeled 68 The Ga-NOTA-C3428 probe has excellent tumor targeting and specificity, and has a good imaging effect when used in tumor model mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the electrophoresis pattern of the coated antigen C3428.

[0031] Figure 2 The results of bacteriophage Elisa were verified.

[0032] Figure 3 This is the test result of Elisa positive sample.

[0033] Figure 4 This is the result of amino acid sequence analysis of C3428-1A3.

[0034] Figure 5 for 124 TLC test results of I-C3428 before and after purification.

[0035] Figure 6 for 124 I-C3428 cell blocking experiment results.

[0036] Figure 7 for 68 PET / CT imaging results of Ga-NOTA-C3428 in different model mice. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0038] Example 1: This example provides an anti-Trop2 nanobody, and its preparation method mainly includes the following contents.

[0039] 1. Phage library enrichment and antigen screening

[0040] 1.1 Preparation of phage antibody library

[0041] (1) Add 300 ml 2YT (A&G) to 30 OD glycerol stock and incubate at 37°C, 220 rpm / min until the OD value is 0.4-0.6. Add M13K07 (3×10 12 ) and shake well, let stand at 37°C for 30 min, and at 37°C, 220 rpm / min for 1 h.

[0042] (2) Centrifuge at 5000 rpm for 5 min and discard the culture medium.

[0043] (3) After centrifugation, the pellet was resuspended in an equal volume of 2YT (A&K) and incubated at 30°C, 220 rpm / min overnight.

[0044] (4) Aliquot into 35 ml / tube and centrifuge at 6000 rpm for 10 min.

[0045] (5) Transfer the supernatant to a clean tube, add 1 / 5 volume of PEG-NaCl, and incubate at 4°C for 2 h.

[0046] (6) Centrifuge at 10,000 rpm, 4°C for 10 min.

[0047] (7) Discard the supernatant, dissolve the precipitate in 10 mL of PBS, and centrifuge at 10,000 rpm, 4°C for 5 min.

[0048] (8) Transfer the supernatant to a clean tube and add 1 / 5 volume of PEG-NaCl. Incubate at 4°C for 60 min.

[0049] (9) Centrifuge at 10,000 rpm, 4°C for 10 min.

[0050] (10) Discard the supernatant, dissolve the precipitate with an appropriate amount of PBS, collect it into a 1.5 ml centrifuge tube, and store it at 4°C.

[0051] (11) Titer measurement: Dilute the collected phage to 10 10 , 10 11 , 10 12 After infecting the logarithmic phase TGI, spread it on 2YT-AG plates, culture it overnight at 37℃, and count the number of colonies.

[0052] save.

[0053] 1.2 Antigen selection and amplification

[0054] (1) Coat the immunotube with purified Trop2 antigen. The electrophoresis pattern of the coated antigen is as follows: Figure 1 As shown, the coating amount was 5-30 μg, supplemented with 0.1 M NaHCO3 to 2 mL, and allowed to stand at 37 °C for 2 h;

[0055] (2) After discarding the antigen, wash three times with PBST, add 5 mL of 4% milk PBS to block, and let stand at 37°C for 1 h.

[0056] (3) Wash three times with PBST and add 1×10 12 The antibody library phage was incubated at 37°C for 1 h.

[0057] (4) After discarding the phages, wash 10 times with PBST, add 1 mL of Gly-HCL (pH 2.0) and shake at room temperature for 8 min to elute the phages, and add 200 μL of Tris-HCL (pH 9.5) to neutralize.

[0058] (5) Take 100 μL of the eluate to measure the titer, and infect the rest with 5 mL of TG1 and let it stand at 37°C for 0.5 h.

[0059] (8) Centrifuge at 6000 rpm for 10 min, resuspend the pellet in 20 ml of 2YTAG, and shake at 220 rpm at 37°C until the OD600 is about 0.5.

[0060] (9) Add helper phage M13K07 at an MOI of 20 and incubate at 37°C for 0.5 h.

[0061] (10) Centrifuge at 6000 rpm for 10 min. Collect the precipitate and transfer to 150 mL of 2YTAK medium. Culture overnight at 28°C and 220 rpm.

[0062] (11) Collect phages and repeat the panning for three rounds.

[0063] The results of three rounds of screening using immunotubes coated with purified Trop2 protein are shown in Table 1 below.

[0064] Table 1 Results of three rounds of screening of purified protein coated immunotubes

[0065]

[0066] The results of phage amplification are shown in Table 2 below.

[0067] Table 2 Phage amplification results

[0068]

[0069] 2. Nanoantibody identification and screening

[0070] (1) The phages from the third round of panning were used to infect TG1, which was then spread on 2YT-AG plates and cultured at 37°C overnight.

[0071] (2) Pick a single clone and transfer it to a 96-deep-well plate with 200 μL of 2YT-AG medium. Incubate the plate at 37°C with shaking at 220 rpm.

[0072] (3) When the OD600 of the bacterial solution reaches about 0.5, 100 μL of the culture medium was taken for preservation. Helper phage M13K07 was added to the remaining bacterial solution, and the mixture was allowed to stand at 37°C for 0.5 h. Centrifuged at 4000 rpm for 10 min, and the supernatant was discarded. The precipitate was resuspended in 600 μL of 2YTAK, and the phage was induced at 28°C, 220 rpm overnight. The supernatant was retained after centrifugation at 4000 rpm for 10 min.

[0073] (4) Use NaHCO3 to coat the antigen on the Elisa plate at 50 ng / well and incubate at 4°C overnight.

[0074] (5) After washing three times with PBST, add 4% MPBS and block at 37°C for 1 h.

[0075] (6) After washing three times with PBST, 50 μL of phage supernatant and 4% MPBS were added and incubated at 37°C for 1 h.

[0076] (7) After washing four times with PBST, 100 μL of diluted anti-M13 antibody was added and reacted at 37°C for 1 h.

[0077] (8) After washing with PBST for 5 times, TMB colorimetric solution was added for color development. After quenching with sulfuric acid, the color was read on an ELISA reader at 450 nm. Figure 2 .

[0078] From the ELISA results, clones with signals were selected for repeated ELISA, with 100 ng / well of coating protein. They were numbered as C3428-1A3 and C3428-1H3. The ELISA results were as follows: Figure 3 , for sequencing.

[0079] The sequencing results showed that the C3428-1H3 sequence was missing and was discarded.

[0080] The base sequence of C3428-1A3 is:

[0081] GATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGAGGGGTCTTCAATATCA ATGCCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGACTTGGTCGCAACTATGTTTAATAATGGTAACACCAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACACGGCCA AGAACTATGTGTGGCTGCAAATGAACAACCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGCAAAGCGGGTATTTGGGAAAGACTATTGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGC(SEQ ID NO.5).

[0082] The amino acid sequence is shown in SEQ ID NO: 4, wherein the underlined portion is the CDR region.

[0083] DVQLQESGGGLVQPGGSLRLSCTAS GGVFNINA MGWYRQAPGKQRD LVAT MFNNGNT NYADSVKGRFTISRDTAKNYVWLQMNNLKPEDTAVYYC NAKRVFGKDY WGQGTQVTVSS (SEQ ID NO. 4)

[0084] CDR1: GGVFNINA (SEQ ID NO. 1)

[0085] CDR2: MFNNGNT (SEQ ID NO. 2)

[0086] CDR3:NAKRVFGKDY(SEQ ID NO.3)

[0087] The results of the analysis on the IMCT website are as follows: Figure 4 shown.

[0088] Example 2: This example is based on the Nanobody C3428-1A3 prepared in Example 1, using 124I labeled the nanobody C3428-1A3 to obtain 124 I-C3428 probe and test, mainly including:

[0089] (1) Ultrafiltration concentration

[0090] 1 mL, 0.2 mg / mL of Nanobody C3428-1A3 was ultrafiltered at 4000 rpm for 15 min and concentrated to a volume of 400 μL.

[0091] (2) 124 I Marking C3428-1A3

[0092] Reaction system: 150 μL 124 I (500 μCi) + 200 μL 0.1M PB + 0.2 mg C3428-1A3 + 12 μL NBS; shake gently to mix and let react at room temperature for 60 seconds, add 100 μL HSA solution to terminate the reaction, and then let stand for 60 minutes.

[0093] (3) TLC detection

[0094] The reaction solution was purified by PD-10 and connected to 5 tubes for measuring radioactivity. The products before and after purification were tested by TLC. The results were as follows: Figure 5 and as shown in Table 3 below.

[0095] Table 3 Radioactivity detection results of products before and after purification

[0096]

[0097] The results show that: 124 The labeling rate of I-C3428 was 68.4%, and the radiochemical purity was >95%.

[0098] (4) 124 I-C3428 cell experiment

[0099] 4T1-Trop2 cells and 4T1 cells were used for cell uptake experiments. The cells were digested one day before the experiment and counted at 2×10 5 Cells / well were plated in a 24-well plate and 500 μL of medium containing 10% FBS was added to each well and cultured for 24 hours. 2 hours before the experiment, the medium in the 24-well plate was removed with a pipette, and 500 μL of medium without FBS was added to each well and cultured in an incubator for 2 hours. The radioactive marker was diluted to 37 kBq / uL (0.1 μCi / μL) with physiological saline and 10 μL was added to each well. 124I-C3428, and then placed in an incubator. At the corresponding time (2h and 4h), the 24-well plate was taken out, the culture medium was removed with a pipette, and PBS was washed 3 times. 350μL 1M NaOH was added to each well. After digestion and lysis for 3min, the lysis solution was transferred to the r counting tube with a pipette. For the cell blocking experiment, 1μg cold C3428 precursor was added to each well 10min before adding the corresponding radioactive marker, and then 37kBq / uL (0.1μCi / μL) was added. 124 I-C3428, and collect the lysed cell solution at 2h and 4h, and treat in the same way as before. 124 1% of the radioactivity of I-C3428, i.e. 3.7 kBq (0.1 μCi) 124 I-C3428 was used as the reference activity. The reference activity was measured together with the collected cell lysate using a γ counter. The data were expressed as %AD / 2×105cells (percentage of total added dose per 2×105cells) after attenuation correction. The results are shown in Figure 6 .

[0100] The results show that: 124 I-C3428 has a high uptake in Trop2-transfected 4T1 cells and can be successfully blocked by C3428 nanobody, proving 124 The I-C3428 probe has a significant affinity for Trop2.

[0101] Example 3: Based on Example 1, this example constructs a radioactive tracer with nanobody C3428-1A3 as a carrier 68 Ga-NOTA-C3428 and verified its radiochemical properties, the main contents are as follows.

[0102] 1. Coupling of NOTA-C3428

[0103] (1) Ultrafiltration concentration

[0104] 2 mL, 0.2 mg / mL of Nanobody C3428-1A3 was ultrafiltered at 4000 rpm for 13 min and the volume was concentrated to 1 mL.

[0105] (2)NOTA coupling

[0106] Reaction system: 600 μL sodium bicarbonate buffer + 0.4 mg C3428-1A3 + 15 μL NOTA (10 mg / mL), at pH = 9.0, 37 ° C for 1 h

[0107] 2. Purification

[0108] The reaction solution after coupling was purified by PD-10 column.

[0109] 3. 68 Ga Marking NOTA-C3428

[0110] The purified NOTA-C3428 was used for 68 Ga labeling, labeling systems are:

[0111] Labeling system 1: 1.5mL 68 Ga+97.5μL NaAc(1M)+250μL NOTA-C3428;

[0112] Labeling system 2: 1.5mL 68 Ga+165μL NaAc(1M)+250μL NOTA-C3428;

[0113] The two systems reacted for 15 minutes at room temperature and different pH conditions. After the reaction, the labeled products were purified by PD-10 columns and the radioactivity was measured in 5 tubes. The results are shown in Table 4 below.

[0114] Table 4 Radioactivity detection results of products before and after purification with different labeling systems

[0115]

[0116] The results showed that the two different reaction systems did not affect the yield and labeling rate of radioactive labeling, and the radiochemical purity was maintained at a high level after purification.

[0117] Embodiment 4: 68 Micro-PET / CT imaging of mice with Ga-NOTA-C3428

[0118] The product obtained by labeling system 2 in Example 3 was used to perform PET / CT imaging in normal Kunming mice. Two normal mice were selected for the imaging experiment. A certain amount of 68 Ga-NOTA-C3428 probe was used for mouse Micro-PET / CT imaging, and the imaging time windows were selected as 30 min and 1 h. The results are shown in Figure 7 .

[0119] The results show that: 68 The Ga-NOTA-C3428 probe is mainly distributed in the liver, kidney, spleen and bladder in normal mice, and is less distributed in other normal tissues. 68 Ga-NOTA-C3428 can be metabolized through the liver and gallbladder and urinary metabolism, which basically conforms to the general distribution characteristics of nanoantibodies in the body.

[0120] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Anti-Trop2 nanobody, characterized in that: Contains variable regions, The variable region comprises any one or more of CDR1, CDR2 or CDR3 sequences; (a), the CDR1 has the amino acid sequence shown in SEQ ID NO.1; or the CDR2 has the amino acid sequence shown in SEQ ID NO.2; or the CDR3 has the amino acid sequence shown in SEQ ID NO.3; or (b) an amino acid sequence after modification of the amino acid sequence described in (a) and having the same function as the amino acid sequence described in (a); or (c) an amino acid sequence as described in (a) or (b) in which one or more amino acids are substituted, deleted or added, and which has the same function as the amino acid sequence described in (a) or (b); or (d) an amino acid sequence having 90% or more identity with the amino acid sequence described in (a) or (b) or (c).

2. The anti-Trop2 nanobody according to claim 1, characterized in that: The variable region has: (a), the amino acid sequence shown in SEQ ID NO.4; or (b) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as described in (a), and having the same function as the amino acid sequence as described in (a); or (c) an amino acid sequence that has more than 90% identity with the amino acid sequence as described in (a) or (b).

3. A nucleic acid molecule encoding a Nanobody according to any one of claims 1 to 2.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleic acid molecule has: (a), the nucleotide sequence shown in SEQ ID NO.5; or (b), a nucleotide sequence complementary to the nucleotide sequence shown in (a); or (c), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (a) or (b) but differing from the nucleotide sequence of (a) or (b) due to the degeneracy of the genetic code; or (d), a nucleotide sequence obtained by substituting, deleting or adding one or two nucleotide sequences to the nucleotide sequence shown in (a), (b) or (c), and a nucleotide sequence having the same or similar functions as the nucleotide sequence shown in (a), (b) or (c); or (e), a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of (a), (b), (c) or (d).

5. Use of the anti-Trop2 nanobody as claimed in claim 1 or 2 in the preparation of a Trop2 molecular imaging probe.

6. A Trop2-specific molecular imaging probe, characterized in that: The probe comprises a tumor targeting group and a radionuclide; the tumor targeting group is the nanobody according to claim 1 or 2; The radionuclide is 68 Ga, 124 I or 89 Any of Zr.

7. The Trop2-specific molecular imaging probe according to claim 6, characterized in that: When the radionuclide is selected 68 Ga or 89 When Zr is present, the probe further comprises a chelating agent DOTA.

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