Nanometer antibody targeting GPRC5D as well as preparation method and application thereof

Abstract

The invention discloses a nano antibody targeting GPRC5D as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The nano antibody is generated by mixed immunization of a GPRC5D polypeptide antigen with a sequence shown as SEQ ID NO: 1 and a GPRC5D protein antigen with a sequence shown as SEQ ID NO: 2. The preparation method comprises the following steps: mixing the GPRC5D polypeptide antigen and the GPRC5D protein antigen to immunize alpaca, sampling blood, constructing an antibody library, screening a phage display antibody library, expressing protein, and selecting the nano antibody with the highest affinity. The invention further provides a radionuclide labeled molecular probe, the nano antibody is small in molecular weight and can recognize a large number of epitopes, and the molecular probe prepared from the nano antibody can obtain high-quality images, is used for preparing products for developing multiple myeloma or detecting GPRC5D expression level and has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nanobody targeting GPRC5D, its preparation method, and its application. Background Technology

[0002] Multiple myeloma (MM) is a malignant disease characterized by the clonal proliferation of plasma cells in the bone marrow. It accounts for approximately 10% to 15% of all hematologic malignancies and is currently the second leading cause of hematologic malignancy worldwide, imposing a heavy health burden and enormous economic pressure on patients and their families. MM has an insidious onset and can affect multiple systems throughout the body, causing complex and diverse clinical manifestations. The lack of specific clinical presentations and the diversity of imaging findings easily lead to misdiagnosis and missed diagnosis of the disease.

[0003] G protein-coupled receptor family C5 member D (GPRC5D) is an orphan receptor. Studies have shown that GPRC5D is specifically highly expressed on MM cells and almost not expressed in normal tissues. Its unique expression in plasma cell lineages makes it a potentially ideal target for MM diagnosis. Therefore, GPRC5D has become a novel target for the treatment of multiple myeloma and shows great promise in targeted therapy for patients with relapsed / refractory multiple myeloma (RRMM).

[0004] GPRC5D, as a novel therapeutic target for multiple myeloma (MM), is currently available in various forms, including bispecific antibodies, antibody-drug conjugates (ADCs), CAR-T cells, and CAR-NK cells. For example, Johnson & Johnson's Talquetamab (JNJ-64407564) is a CD3 and GPRC5D bispecific antibody, which has received FDA approval and was launched on January 24, 2023. Therefore, there is an urgent need to develop a diagnostic tool targeting GPRC5D to non-invasively and visually monitor the expression of GPRC5D on the surface of MM cells, and to further develop new methods for targeting GPRC5D.

[0005] Nanobodies (Nb) were the first special antibodies discovered in camels. They consist only of a heavy chain, lacking the light chain, meaning that the only site for antigen binding is the variable region of the heavy chain; hence, they are called single-domain heavy chain antibodies (VHH). VHH molecules are very small, only 15 kDa. Due to their nanoscale size, they are the smallest naturally occurring antigen-binding fragments to date. Compared to complete antibodies, nanobodies significantly reduce molecular weight, increasing the overall metabolic rate of molecular probes, making them suitable for labeling radionuclides with shorter half-lives. 18 F or 68 Ga), and reduce non-specific binding while retaining the advantages of high antibody specificity. There are currently no reports on nanobodies targeting GPRC5D, or specific molecular imaging probes or radionuclide-labeled therapeutic probes targeting this target. SUMMARY

[0006] Based on this, the main purpose of the present application is to provide a GPRC5D-targeting nanobody, which is obtained by constructing a GPRC5D protein alpaca immune library and is easy to be clinically converted and applied.

[0007] Another purpose of the present application is to provide a radionuclide-labeled molecular probe, which is obtained by labeling the GPRC5D-targeting nanobody with a radionuclide.

[0008] Still another purpose of the present application is to provide an application of the GPRC5D-targeting nanobody in preparing a product for imaging multiple myeloma or detecting a GPRC5D expression level.

[0009] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0010] In the first aspect of the present application, a GPRC5D-targeting nanobody is provided, which is generated by mixed immunization of a GPRC5D polypeptide antigen and a GPRC5D protein antigen, wherein the amino acid sequence of the GPRC5D polypeptide antigen is shown as SEQ ID NO: 1, and the amino acid sequence of the GPRC5D protein antigen is shown as SEQ ID NO: 2.

[0011] The amino acid sequence of the GPRC5D protein in the present application is shown as SEQ ID NO: 3.

[0012] As a preference, the amino acid sequence of the GPRC5D-targeting nanobody is shown as SEQ ID NO: 4.

[0013] As a preference, the nucleotide sequence encoding the GPRC5D-targeting nanobody is shown as SEQ ID NO: 5.

[0014] In the second aspect of the present application, a preparation method of the GPRC5D-targeting nanobody is provided, which comprises the following steps: immunizing an alpaca with a GPRC5D polypeptide antigen and a GPRC5D protein antigen for 4-6 times, collecting blood, constructing an antibody library, then performing phage display antibody library screening, finally performing protein expression, and selecting a nanobody with the highest affinity, which is named as nanobody E4.

[0015] As a preference, the alpaca is a Huacaya alpaca.

[0016] In the third aspect of the present application, a radionuclide-labeled molecular probe is provided, and a preparation method thereof comprises the following steps: coupling the GPRC5D-targeting nanobody with NCS-NOTA to obtain a precursor NOTA-E4; and labeling the precursor NOTA-E4 with a radionuclide 68 Ga, and the molecular probe is named as molecular probe 68Ga-NOTA-E4.

[0017] As preferred, the preparation method of the radionuclide-labeled molecular probe comprises:

[0018] (a) solvent of the nanobody E4 is replaced with 0.1M carbonate buffer, pH 8.8-9, using a 3k dialysis bag;

[0019] (b) NCS-NOTA is dissolved in DMSO, with a final concentration of 10mg / mL;

[0020] (c) the nanobody E4 is mixed with NCS-NOTA at a molar ratio of 1:3, and after mixing, it is placed at 4℃ for 10h;

[0021] (d) purified using a 3k desalting column to obtain NOTA-E4;

[0022] (e) solvent of the NOTA-E4 is replaced with 0.25M NaOAc using a 3k dialysis bag;

[0023] (f) a germanium gallium generator is slowly eluted using 0.05M HCl, and the eluate is collected, and 1mL with the highest specific activity is collected 68 GaCl3;

[0024] (g) the NOTA-E4 is slowly mixed with 68 GaCl3 at a volume ratio of 1:1, and slowly stirred at room temperature for 10min;

[0025] (h) purified using a 3k molecular sieve to obtain the molecular probe 68 Ga-NOTA-E4.

[0026] In a fourth aspect of the present application, the nanobody targeting GPRC5D or the radionuclide-labeled molecular probe is used in the preparation of a multiple myeloma imaging product.

[0027] As preferred, the multiple myeloma imaging adopts PET imaging.

[0028] In a fifth aspect of the present application, the nanobody targeting GPRC5D or the radionuclide-labeled molecular probe is used in the preparation of a product for detecting the expression level of GPRC5D.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] 1. Easy to be clinically applied. The nanobody targeting GPRC5D in the present application has a small molecular weight and a simple structure, and can recognize a large number of antigen epitopes, and the molecular probe prepared from the nanobody can obtain high-quality images.

[0031] 2. Radionuclide 68 Ga is readily available. PET is a popular device in nuclear medicine department, and radionuclide 68 Ga is relatively easy to obtain by a germanium-gallium generator, and the labeling probe method is simple. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Figure 2 is a reduced SDS-PAGE Coomassie blue staining diagram of the final sample QC of nanobody E4 in the example; MW: protein marker nanobody E4.

[0033] Figure 2 Figure 3 is an immunofluorescence detection diagram of nanobody E4 in the example.

[0034] Figure 3 Figure 4 is a molecular probe diagram in the example. 68 Figure 5 is a Micro-PET / CT imaging diagram of Ga-NOTA-E4. DETAILED DESCRIPTION

[0035] In order to more fully understand the technical solutions, objectives and advantages of the present application, the following will further describe the technical effects of the present application in detail in combination with the drawings and specific examples. Obviously, the described examples are only some of the embodiments of the present application, not all. It should be noted that for those skilled in the art, other embodiments obtained without departing from the concept of the present application all belong to the protection scope of the present application.

[0036] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0037] Example 1

[0038] This example proposes a preparation method of a nanobody targeting GPRC5D, the steps are as follows:

[0039] (1) Obtain GPRC5D polypeptide antigen and protein antigen

[0040] The target fragment is subcloned into the pATX-GH vector by gene optimization synthesis, the cloning site is BamHI / EcoRI, and the protein obtained by expression and purification in the E. coli system is used for later immunoscreening experiment.

[0041] a) The target GPRC5D protein sequence is as follows:

[0042] MYKDCIESTGDYFLLCDAEGPWGIILESLAILGIVVTILLLLAFLFLMRKIQDCS

[0043] QWNVLPTQLLFLLSVLGLFGLAFAFIIELNQQTAPVRYFLFGVLFALCFSCLLA

[0044] HASNLVKLVRGCVSFSWTTILCIAIGCSLLQIIIATEYVTLIMTRGMMFVNMTP

[0045] CQLNVDFVVLLVYVLFLMALTFFVSKATFCGPCENWKQHGRLIFITVLFSIIIW

[0046] VVWISMLLRGNPQFQRQPQWDDPVVCIALVTNAWVFLLLYIVPELCILYRSCR

[0047] QECPLQGNACPVTAYQHSFQVENQELSRARDSDGAEEDVALTSYGTPIQPQT

[0048] VDPTQECFIPQAKLSPQQDAGGV(SEQ ID NO:3)。

[0049] GPRC5D has a total of 345 AAs, seven transmembrane helices, and strong hydrophobicity. The following polypeptide sequence is used as an antigen [polypeptide ①: GPRC5D [5-15]: CIESTGDYFLL], as shown in SEQ ID NO: 1.

[0050] b) Preparation of GPRC5D polypeptide antigen: 10 mg of naked peptide with a purity of more than 90%, of which 7 mg is coupled with KLH as an antigen, 3 mg is coupled with BSA as a screening agent, and 5 mg of polypeptide-biotin is synthesized, with a purity of more than 90%.

[0051] c) Preparation of GPRC5D protein antigen:

[0052] GST-GPRC5D-His (334 AAs, 38.02 kDa, pI = 4.93), as shown in SEQ ID NO: 2, specifically:

[0053] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEF

[0054] PNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG

[0055] VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYD

[0056] ALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQAT

[0057] FGGGDHPPKSDLEVLFQGPLGSMYKDCIESTGDYFLLCDAEGPWGIILEGGG

[0058] GSMYKDCIESTGDYFLLCDAEGPWGIILEGGGGSMYKDCIESTGDYFLLCDA

[0059] EGPWGIILEEFGGGSHHHHHH

[0060] Features:

[0061] GST tag: [1-231];

[0062] Histag: [329-335].

[0063] (2) Construction of phage immune library (library type: VHH)

[0064] a) Immunize alpaca: immunize one alpaca (Wakaya alpaca) with polypeptide-KLH mixed with GST-GPRC5D-His protein, immunize 4-6 times, with an interval of two weeks, 1 mg for the first immunization, and 500 ug for each subsequent round. Detect the immune titer of the third and fourth times, and collect blood after the titer reaches the required value. Centrifuge to obtain supernatant, and store at -20℃.

[0065] b) Construction of antibody library: extract total RNA from peripheral lymphocytes of the immunized alpaca, and obtain cDNA by reverse transcription as a template for gene amplification; use variable region fragments to amplify and clone into M13 phagemid, and transform TG1 to construct a phage library;

[0066] (3) Phage display antibody library panning (3 rounds of panning)

[0067] a) Coat the immunotubes: 50 μg / mL GPRC5D, buffer CBS, 1 mL per tube, 4°C overnight (Coat GPRC5D protein link as follows: Catalog # GPD-H52D6, https: / / www.acrobiosystems.cn / P26288human_GPRC5D%C2% A0Full_Length_Protein_FlagHis_Tag_Nanodisc.html).

[0068] b) Wash the immunotubes 3 times with 5 mL PBST.

[0069] c) Block with 5 mL 5% milk / PBST for 1 h at 30°C.

[0070] d) Wash once with 5 mL PBS.

[0071] e) Library phage panning: 1 x 10 12 Add 100 μg / mL MSP1D1 protein and 1% skim milk / PBST (or 0.2% casein / PBST) to the phage library of 1 x 10

[0072] f) Add the panned phage to the immunotubes and incubate at 30°C for 2 h.

[0073] g) Wash 4-6 times with 5 mL PBST.

[0074] h) Add 1 mL Gly-HCl (pH = 2.2) per tube to elute the phage, incubate at room temperature with shaking for about 6-8 min, and add Tris-HCl (pH = 9.6) to neutralize the solution to pH = 7.0-8.0.

[0075] i) Dilute the eluted phage and infect log-phase E. coli TG1, plate to determine titer.

[0076] (4) Polyclonal phage ELISA detection

[0077] a) Coat the immunotubes: 50 μg / mL GPRC5D, buffer CBS, 1 mL per tube, 4°C overnight (Coat GPRC5D protein link as follows: Catalog # GPD-H52D6, https: / / www.acrobiosystems.cn / P26288human_GPRC5D%C2% A0Full_Length_Protein_FlagHis_Tag_Nanodisc.html).

[0078] b) Wash 3 times with 300 μL PBST.

[0079] c) Add 300 μL 5% skim milk and block at 37°C for 2 h.

[0080] d) Dilute the phage after each round of amplification with PBS, with a 3-fold increase in dilution, and an initial concentration of 1 x 1012 pfu / mL, 100 μL diluted amplified phage was added to each well, incubated at 32°C for 1 h, and washed 3 times with 300 μL PBST.

[0081] e) 100 μL secondary antibody (anti-phage M13) dilution was added, incubated at 30°C for 1 h, and washed 3 times with 300 μL PBST.

[0082] f) 100 μL color developing solution TMB was added, color developed in the dark for 3-8 min, 50 μL 2M HCl was added to stop the reaction, and the reading was taken by an enzyme label instrument (450-620 nm).

[0083] g) According to the results of polyclonal phage ELISA, the phage specifically combined with the antigen was strongly enriched after three rounds of panning, 192 single clones were screened from the elution product of the third round, and the positive clones were sent for testing according to the results and verified twice, and finally the positive sequence was obtained, Ag: 4 μg / mL GPRC5D; NC1: 4 μg / mL MSP1D1; NC2: PBS.

[0084] (5) Obtain nanobody E4 targeting GPRC5D

[0085] The positive sequence obtained by panning was expressed by VHH recombination, and the target sequence gene was synthesized and subcloned into pATX1 vector, the subcloning site was EcoRI / HindIII, and the C-terminal had a strep tag. The target antibody was expressed by mammalian system CHO cells, and expression and purification tests were performed, and 1 mg of antibody E4 was obtained. The results of nanobody final sample (QC) are shown in Figure 1 , and it can be seen that the molecular weight of antibody E4 is about 15 kilodaltons.

[0086] The amino acid sequence of antibody E4 is 367F-R3P1-E4 (141AAS, 15.53KDa, PI 9.64), as shown in SEQ ID NO: 4, and is as follows:

[0087] MKHLWFFLLLVAAPRWVLSQVQLVESGGGTVQPGGSLRLSCAASGFTFSSYP

[0088] MSWVRQAPGKGFEWVSTIGSSGITTNYADSVKGRFTISRDNAKNTVYLQMTS LKPEDTALYQCRRGPYWGQGTQVTVSSGSWSHPQFEK.

[0089] DNA sequence: 367F-R3P1-E4, as shown in SEQ ID NO: 5, specifically as follows: gaattcgccgccaccATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCCCCTAGATGGGTGCTGTCCCAGGTGCAGCTGGTGGAAAGCGGAGGAGGAACCGTGCAGCCAGGAGGATCTCTGAGGCTGTCCTGCGCTGCTAGCGGATTCACCTTCTCCTCCTATCCTATGAGCTGGGTGAGGCAGGCTCCCGGAAAGGGATTTGAGTGGGTGAGCACCATCGGCTCCAGCGGAATCACCACCAACTATGCTGACTCCGTGAAGGGCCGGTTCACCATCAGCAGAGATAATGCTAAGAATACCGTGTATCTGCAGATGACCAGCCTGAAGCCCGAGGATACCGCCTTGTACCAGTGTAGGCGGGGCCCTTACTGGGGACAGGGAACCCAGGTTACCGTGTCCTCCGGATCCTGGAGCCACCCTCAGTTCGAGAAGtgagcggccgc.

[0090] (6) Nanobody E4 targeting GPRC5D affinity detection

[0091] The results of immunofluorescence (IF) detection of nanobody E4 are shown in Figure 2 The results show that the signal intensity of nanobody E4 for overexpressing cells is significantly higher than that of control cells.

[0092] (7) Molecular probe 68 Radiochemical synthesis of Ga-NOTA-E4

[0093] a) Nanobody E4 solvent was replaced with 0.1M carbonate buffer (pH 8.8-9) using a 3k dialysis bag.

[0094] b) NCS-NOTA was dissolved in DMSO with a final concentration of 10mg / mL.

[0095] c) Nanobody E4 was mixed with NCS-NOTA at a molar ratio of 1:3, and after mixing, it was placed at 4°C for 10h.

[0096] d) Purification was performed using a 3k desalting column to obtain NOTA-E4.

[0097] e) The solvent of NOTA-E4 was replaced with 0.25M NaOAc using a 3k dialysis bag.

[0098] f) Slowly elute the germanium gallium generator with 0.05M HC1, collect the eluate, collect 1 mL of the highest specific activity 68 GaCl3.

[0099] g) Slowly mix NOTA-E4 with GaCl3 at a volume ratio of 1:1, slowly stir at room temperature, and react for 10 min. 68 GaCl3.

[0100] h) Purify using 3k molecular sieves to obtain the molecular probe 68 Ga-NOTA-E4.

[0101] (8) Construction of a tumor animal model

[0102] a) 100 μL of AMO1 multiple myeloma cell line at a concentration of 1 x 10 9 cells / mL were inoculated into male BALB / c athymic nude mice (6-7 weeks old, 18-22 g).

[0103] b) Normally feed, and use after the tumor diameter reaches 0.5-1 cm.

[0104] (9) PET / CT imaging of the molecular probe 68 Ga-NOTA-E4

[0105] Molecular probe 68 Ga-NOTA-E4 Figure 3 The results of the 0.5 h and 1 h imaging are shown in the following table, which shows that: 68 Ga-NOTA-E4 has a high target organ / background value, and the imaging effect is good, and can be used for MM imaging and reflects the GPRC5D expression, thereby providing a basis for preclinical screening of subjects.

[0106] The above is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A GPRC5D-targeted nanobody, which is generated by immunizing a GPRC5D polypeptide antigen and a GPRC5D protein antigen, wherein the amino acid sequence of the GPRC5D polypeptide antigen is shown as SEQ ID NO: 1, and the amino acid sequence of the GPRC5D protein antigen is shown as SEQ ID NO:

2.

2. The GPRC5D-targeting nanobody of claim 1, wherein, The amino acid sequence of the GPRC5D-targeted nanobody is shown as SEQ ID NO:

4.

3. The GPRC5D-targeting nanobody of claim 2, wherein, The nucleotide sequence encoding the GPRC5D-targeted nanobody is shown as SEQ ID NO:

5.

4. Process for the preparation of a Nanobody targeting GPRC5D according to any one of claims 1 to 3, characterized in that, Comprising: A GPRC5D polypeptide antigen and a GPRC5D protein antigen are used to immunize a llama 4-6 times, blood is collected, an antibody library is constructed, then phage display antibody library screening is performed, and finally protein expression is performed, and the nanobody with the highest affinity is selected.

5. The method of claim 4, wherein the GPRC5D-targeting nanobody is prepared by, The llama is a Wacaya llama.

6. A radionuclide labeled molecular probe, characterized in that, The preparation method comprises the following steps: coupling the GPRC5D-targeted nanobody according to any one of claims 1 to 3 with NCS-NOTA to obtain a precursor NOTA-E4, and then labeling the precursor NOTA-E4 with a radionuclide 68 Ga, and the labeling is completed.

7. The radionuclide-labelled molecular probe according to claim 6, characterized in that, The preparation method comprises: (a) using a 3k dialysis bag to replace the solvent of the nanobody E4 with 0.1M carbonate buffer, pH 8.8-9; (b) dissolving NCS-NOTA in DMSO to a final concentration of 10mg / mL; (c) mixing the nanobody E4 and NCS-NOTA at a molar ratio of 1:3, uniformly mixing, and then placing in a 4℃ environment for 10h of reaction; (d) using a 3k desalting column for purification to obtain NOTA-E4; (e) using a 3k dialysis bag to replace the solvent of NOTA-E4 with 0.25M NaOAc; (f) The germanium gallium generator is slowly eluted with 0.05 M HCI and the eluate collected, the high specific activity fractions pooled 68 GaCl3; (g) NOTA-E4 was prepared by reacting NOTA-E3 with 68 GaCl3was mixed slowly in a volume ratio of 1:1, stirred slowly at room temperature, and reacted for 10 min. (h) purification using 3 k molecular sieves to obtain the molecular probe 68 Ga-NOTA-E4. 8.The GPRC5D-targeted nanobody of any one of claims 1-3, or the radionuclide-labeled molecular probe of claim 6 or 7, is used for preparing a multiple myeloma imaging product.

9. Use according to claim 8, characterized in that, The multiple myeloma imaging is performed by PET imaging. 10.The GPRC5D-targeted nanobody of any one of claims 1-3, or the radionuclide-labeled molecular probe of claim 6 or 7, is used for preparing a product for detecting the expression level of GPRC5D.

Images