A nanobody-multivalent rhamnose conjugate and its preparation method and application

Abstract

The present invention discloses a nano antibody-multivalent rhamnose conjugate and its application, wherein the nano antibody-multivalent rhamnose conjugate is obtained by modifying a nano antibody molecule with a multivalent rhamnose molecule. The present invention modifies the multivalent rhamnose molecule on the nano antibody molecule by a gentle and simple method, which not only retains the excellent affinity level of the original nano antibody, but also recruits a high level of rhamnose-specific natural endogenous antibodies to the surface of tumor cells, indirectly activates immune effector functions such as ADCC, CDC and ADCP to kill tumor cells, and forms an immune complex with the corresponding rhamnose-specific natural endogenous antibodies, significantly improving its own half-life. Compared with nano antibodies modified with monovalent rhamnose molecules and monoclonal antibodies used clinically, the nano antibody-multivalent rhamnose conjugate provided by the present invention has a more prominent anti-tumor effect in vitro and in vivo, and can be applied to related malignant tumors, including the treatment of tumors that are difficult to treat with certain monoclonal antibodies.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical engineering, and specifically relates to a nano antibody-multivalent rhamnose conjugate and a preparation method and application thereof. Background Art

[0002] Nanobodies are regarded as an important representative of the new generation of antibody drugs. Compared with traditional monoclonal antibodies, nanobodies have many obvious advantages, such as small molecular weight, low production cost, strong permeability, low immunogenicity, etc., so they have been widely used in the diagnosis and treatment of diseases such as tumors. However, nanobodies do not have the Fc end of traditional monoclonal antibodies and cannot kill tumor cells through the immune effector functions mediated by the Fc end, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC); on the other hand, nanobodies cannot bind to the neonatal FcRn receptor and enter the FcRn-mediated circulation system, and their small molecular weight leads to an extremely short half-life (about 10 minutes). These deficiencies have severely limited the widespread application of nanobodies as biological drugs.

[0003] At present, the above-mentioned defects of nano-antibodies can be effectively overcome by using antibody genetic engineering technology to fuse and express nano-antibodies with human or mouse Fc fragments. However, the disadvantage is that antibody genetic engineering technology is relatively complex, the production cost of nano-antibody-Fc fusion protein is high, the stability is poor, and it has potential immunogenicity, which affects its tumor immunotherapy effect.

[0004] In recent years, some naturally occurring antibodies have been found in human serum. These antibodies can specifically recognize and bind to some small molecule haptens such as 2,4-dinitrobenzene (DNP), α-Gal and rhamnose (Rha). Previous studies have shown that by coupling these haptens with molecules with tumor cell targeting function, the targeting molecules can anchor the haptens to the cells and recruit free anti-hapten antibodies, thereby activating the human immune system to kill the target cells. For example, by coupling the monovalent hapten DNP or Rha to the nanobody through protein site-specific modification technology, the nanobody can be endowed with the ability to mediate immune effector function to a certain extent and improve the half-life of the nanobody. However, the disadvantage is that the monovalent hapten has low binding affinity with natural endogenous antibodies, and the ability to recruit natural antibodies is insufficient, which affects the anti-tumor activity. Based on this, it is of great significance to develop new strategies to develop nano-antibody drugs with stronger anti-tumor activity. Summary of the invention

[0005] Purpose of the invention: In order to overcome the deficiencies in the prior art, the present invention provides a nanobody-multivalent rhamnose conjugate and a preparation method and application thereof, specifically a nanobody modified with a multivalent rhamnose molecule, and by optimizing the number of rhamnose in the multivalent rhamnose molecule, an optimized multivalent rhamnose molecule is provided, so that the nanobody modified with the molecule has a tumor treatment effect that is significantly superior to existing clinical monoclonal antibodies and other reported nanobodies.

[0006] Technical solution: To achieve the above purpose, the technical solution adopted by the present invention is:

[0007] One object of the present invention is to provide a nanobody-multivalent rhamnose conjugate, which is obtained by modifying a nanobody molecule with a multivalent rhamnose molecule, wherein the conjugate comprises a nanobody modified with a multivalent rhamnose molecule, wherein the multivalent rhamnose molecule is a multivalent form of a rhamnose molecule, and the multivalent rhamnose molecule can selectively recruit natural antibodies present in human serum.

[0008] Furthermore, the antibodies described in the present invention are nanobodies, preferably some mature nanobodies, including but not limited to any one of anti-EGFR nanobodies, anti-EGFRvIII nanobodies, anti-HER2 nanobodies, anti-HER3 nanobodies, anti-PSMA nanobodies, anti-VEGFR nanobodies, anti-PD-L1 nanobodies, anti-cMET nanobodies, anti-TGF-β nanobodies, anti-MUC1 nanobodies and anti-Trop-2 nanobodies.

[0009] Furthermore, the multivalent rhamnose molecule of the present invention is a molecule that can selectively recruit rhamnose-specific antibodies in human serum, that is, the multivalent rhamnose molecule is a multivalent form of rhamnose molecule.

[0010] Furthermore, the multivalent rhamnose molecule includes a divalent rhamnose molecule, a tetravalent rhamnose molecule, an octavalent rhamnose molecule, or a hexadevalent rhamnose molecule, and the structural formula is as follows:

[0011] Rhamnose molecule or

[0012] Rhamnose or

[0013] Eight-valent rhamnose or

[0014] Hexavalent rhamnose molecule

[0015] Furthermore, the synthesis path of the multivalent rhamnose molecule is:

[0016] Synthesis of divalent rhamnose molecules:

[0017]

[0018] Synthesis of tetravalent rhamnose molecules:

[0019]

[0020] Synthesis of octavalent rhamnose molecule:

[0021]

[0022] Synthesis of hexadecavalent rhamnose molecule:

[0023]

[0024] The multivalent hapten and its synthesis method, nanobody molecule, etc. involved in the present invention are as described above.

[0025] A second object of the present invention is to provide a method for preparing the above-mentioned Nanobody-multivalent rhamnose conjugate, which method comprises modifying the multivalent rhamnose molecules onto the Nanobody.

[0026] Further, the preparation process of the conjugate is as follows:

[0027]

[0028] Furthermore, the method for preparing a nanobody-multivalent rhamnose conjugate by modifying a multivalent rhamnose molecule onto a nanobody molecule comprises the following modification methods: an enzymatic modification method or a chemoenzymatic site-directed modification method.

[0029] Furthermore, the enzymatic site modification for preparing the Nanobody-multivalent rhamnose conjugate specifically comprises the following steps:

[0030] 1) Desalting the nanobody and replacing it in the enzyme reaction buffer;

[0031] 2) adding 10-50 times equivalent of multivalent rhamnose molecules and 1 / 10-1 / 2 equivalent of SrtA enzyme (or SrtA mutant) to react, and after the reaction is completed, using affinity chromatography to remove unreacted nanoantibodies, SrtA enzyme (or SrtA mutant), etc.

[0032] 3) removing excess multivalent rhamnose molecules by ultrafiltration or exclusion chromatography to obtain the corresponding nanobody-multivalent rhamnose conjugate.

[0033] The third object of the present invention is to provide the application of the nanobody-multivalent rhamnose conjugate or the method described in the field of preparing tumor immunotherapy drugs, especially in some tumors that are insensitive to the existing clinically used monoclonal antibodies or have developed drug resistance.

[0034] Furthermore, the nanobody-multivalent rhamnose conjugate is used to prepare a tumor immunotherapy drug, and the tumor immunotherapy drug is in a pharmaceutically acceptable form.

[0035] Furthermore, the preparation form of the tumor immunotherapy drug includes any one of injection, powder injection, tablet, sustained-release agent, dripping pill, granule, capsule and sustained-release micropill.

[0036] In a specific embodiment of the present invention, the selected antibody molecule is the anti-EGFR nanoantibody 7D12 (SEQID No. 1). EGFR is highly expressed in a variety of cancer cells such as breast cancer, colorectal cancer, and lung cancer, but its expression level in normal cells is very low. Studies have shown that high expression of EGFR is associated with poor prognosis and short survival of patients. Therefore, EGFR is considered to be one of the ideal targets for tumor treatment. The optimized hapten molecule is NH2-GGG-PEG2-(Rha) containing 16 rhamnose groups. 16 The selected preparation method is: desalting the nanobody 7D12 and replacing it in the enzyme reaction buffer; adding 25 times the equivalent of multivalent rhamnose molecules and 1 / 4 equivalent of enhanced SrtA enzyme to react, and after the reaction is completed, nickel ion chelated magnetic beads are used to remove unreacted 7D12 and enhanced SrtA enzyme; ultrafiltration or exclusion chromatography is used to remove excess multivalent rhamnose molecules to obtain the corresponding nanobody-multivalent rhamnose conjugate.

[0037] Beneficial effects: The advantages of the present invention over the prior art are:

[0038] (1) Compared with the existing reported antibodies, the nanobody-multivalent rhamnose conjugate of the present invention has no inhibition on the one hand in terms of the affinity level of the nanobody; on the other hand, the multivalent presentation of multivalent rhamnose can significantly increase the endogenous antibody recruitment level and the corresponding anti-tumor activity of the nanobody conjugate. Thus, the dosage of the nanobody drug can be reduced during treatment, and finally some toxic side effects can be reduced or avoided to a certain extent.

[0039] (2) The optimized multivalent rhamnose molecules provided by the present invention and the corresponding nanobody-multivalent rhamnose conjugates have more prominent anti-tumor activity than the nanobody-monovalent rhamnose conjugates modified with monovalent rhamnose molecules and the monoclonal antibodies used clinically.

[0040] (3) The nanobody-multivalent rhamnose conjugate provided by the present invention can be used to treat some tumors that are insensitive to existing clinical monoclonal antibodies or develop drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1: Schematic diagram of the preparation process of nanobody-monovalent rhamnose conjugates and nanobody-multivalent rhamnose conjugates.

[0042] Figure 2 : Nanobody 7D12, Nanobody-monovalent rhamnose conjugate 7D12-PEG2-Rha and Nanobody-multivalent rhamnose conjugate 7D12-PEG2-(Rha)2, 7D12-PEG2-(Rha)4, 7D12-PEG2-(Rha)8 and 7D12-PEG2-(Rha) 16 SDS-PAGE characterization.

[0043] Figure 3 : Characterization of flow cytometry affinity and antibody recruitment capacity. (A) Nanobody 7D12, Nanobody-monovalent rhamnose conjugate 7D12-PEG2-Rha, and Nanobody-multivalent rhamnose conjugates 7D12-PEG2-(Rha)2, 7D12-PEG2-(Rha)4, 7D12-PEG2-(Rha)8, and 7D12-PEG2-(Rha) 16 Evaluation of affinity level for target cells; (B) Nanobody 7D12, Nanobody-monovalent rhamnose conjugate 7D12-PEG2-Rha and Nanobody-multivalent rhamnose conjugate 7D12-PEG2-(Rha)2, 7D12-PEG2-(Rha)4, 7D12-PEG2-(Rha)8 and 7D12-PEG2-(Rha) 16 Evaluation of the recruitment capacity of anti-Rha antibodies.

[0044] Figure 4 : Characterization of CDC levels. (A) Nanobody 7D12, Nanobody-monovalent rhamnose conjugate 7D12-PEG2-Rha, and Nanobody-multivalent rhamnose conjugates 7D12-PEG2-(Rha)2, 7D12-PEG2-(Rha)4, 7D12-PEG2-(Rha)8, and 7D12-PEG2-(Rha) 16 Evaluation of CDC levels mediated by 7D12-PEG2-(Rha) 16 Assessment of the level of CDC mediated by

[0045] Figure 5 : Characterization of ADCP levels. Nanobody 7D12, Nanobody-monovalent rhamnose conjugate 7D12-PEG2-Rha, and Nanobody-multivalent rhamnose conjugates 7D12-PEG2-(Rha)2, 7D12-PEG2-(Rha)4, 7D12-PEG2-(Rha)8, and 7D12-PEG2-(Rha) 16 Evaluation of ADCP levels mediated by

[0046] Figure 6 :Comparative evaluation of the antitumor activity of nanobody-multivalent rhamnose conjugates and cetuximab in vitro (A) Evaluation of CDC levels mediated by different concentrations of cetuximab; (B) Nanobody-monovalent rhamnose conjugate 7D12-PEG2-Rha and nanobody-multivalent rhamnose conjugates 7D12-PEG2-(Rha)2, 7D12-PEG2-(Rha)4, 7D12-PEG2-(Rha)8 and 7D12-PEG2-(Rha) 16 Evaluation of CDC levels mediated by 7D12-PEG2-(Rha) 16 Evaluation of CDC levels mediated.

[0047] Figure 7 :Evaluation of half-life level. Nanobody 7D12 and Nanobody-multivalent rhamnose conjugate 7D12-PEG2-(Rha) 16 Half-life assessment.

[0048] Figure 8 :Nanobody 7D12, cetuximab, and nanobody-multivalent rhamnose conjugate 7D12-PEG2-(Rha) 16 Evaluation of the in vivo antitumor activity. DETAILED DESCRIPTION

[0049] The present invention discloses a nano antibody-multivalent rhamnose conjugate and a preparation method and application thereof. The nano antibody-multivalent rhamnose conjugate is obtained by modifying a nano antibody molecule with a multivalent rhamnose molecule, and the multivalent rhamnose molecule can selectively recruit rhamnose-specific natural antibodies present in human serum. The present invention modifies multivalent rhamnose on the nano antibody molecule by a mild and simple method. The obtained nano antibody-multivalent rhamnose conjugate not only retains the excellent affinity level of the original nano antibody with the target cell, but also can recruit the corresponding rhamnose-specific natural antibody, exert the immune effector function mediated by the Fc end and prolong the half-life; in addition, the present invention also optimizes the number of haptens in the multivalent rhamnose molecule to obtain a nano antibody-multivalent rhamnose conjugate with better anti-tumor effect. In particular, the nano antibody-multivalent rhamnose conjugate provided by the present invention can overcome the drug resistance of tumors to certain monoclonal antibodies (such as cetuximab).

[0050] The present invention is further described below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. All simple improvements to the preparation method of the present invention under the premise of the concept of the present invention belong to the protection scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on the known means in the art. The test materials and cell lines used in the following examples, unless otherwise specified, were purchased from conventional reagent companies.

[0051] Example

[0052] According to the method for synthesizing the nanobody-multivalent rhamnose conjugate of the present invention, the first step is to synthesize the multivalent rhamnose molecule NH2-GGG-PEG2-(Rha)n, where n=(2, 4, 8, 16). Then, NH2-GGG-PEG2-(Rha)n is modified onto the nanobody molecule to prepare the nanobody-multivalent rhamnose conjugate. At the same time, we also synthesized a monovalent rhamnose molecule (NH2-GGG-PEG2-Rha) to prepare the nanobody-monovalent rhamnose conjugate 7D12-PEG2-Rha as a control. We synthesized the nanobody-monovalent rhamnose conjugate 7D12-PEG2-Rha and four nanobody-multivalent rhamnose conjugates 7D12-PEG2-(Rha)2, 7D12-PEG2-(Rha)4, 7D12-PEG2-(Rha)8 and 7D12-PEG2-(Rha) 16 The biological activities of the two drugs were compared and evaluated, and the number of rhamnose in the polyvalent rhamnose was optimized, the interaction between the polyvalent rhamnose and the endogenous antibodies was regulated, and the nanoantibody-polyvalent rhamnose conjugate with the best anti-tumor activity was further screened.

[0053] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific multivalent hapten molecules and the results described in the examples are only used to illustrate the present invention, and should not and will not limit the technical solutions of the present invention described in detail in the claims.

[0054] Example 1: Preparation of monovalent and multivalent rhamnose molecules NH2-GGG-PEG2-(Rha)n, n=(1, 2, 4, 8, 16)

[0055] (1) The synthesis path and conditions of the monovalent rhamnose molecule NH2-GGG-PEG2-Rha are as follows:

[0056]

[0057] First, compound 1 triglycine was used as the starting material, and the amino group was protected with Cbz protecting group under the action of benzyl chloroformate to obtain compound 2, which was then condensed with tert-butyl 9-amino-4,7-dioxanononanoate under the action of condensation agent EDCI to obtain compound 3 with a yield of 74%, and then the Cbz protecting group was removed under the action of Pd / H2 to obtain the key intermediate 4. Compounds 6 and 7 were reacted with condensation agent EDCI to obtain compound 8 with a yield of 76%, and the TBDPS protecting group was removed under the action of TBAF to obtain compound 9, which was used as a sugar receptor for subsequent glycosylation reactions. Sugar donor 12 was obtained from fully acetylated rhamnose in a simple two-step reaction with a yield of 72%, and then glycosylated with sugar acceptor 9 under the catalysis of TMSOTf to obtain glycosylated product 13 with a yield of 72%. The Cbz protecting group was removed under the action of Pd / H2 to obtain free monovalent amino rhamnose derivative 14, and then amide condensed with intermediate compound 4 under the action of condensation agent EDCI to obtain compound 15. Finally, tert-butyl ester, Cbz and acetyl protecting groups were completely removed under the action of 25% TFA / DCM, Pd / H2 and MeOH / MeONa, respectively, to obtain the target compound NH2-GGG-PEG2-Rha. After testing, the NMR and mass spectrometry results of the final product were consistent with expectations, indicating that the synthesized NH2-GGG-PEG2-Rha was correct. 1 H NMR (400MHz, Methanol-d4) δ4.73 (s, 1H), 4.44 (m, 1H), 3.99 (s, 2H), 3.90 (s, 2H), 3.84-3.74 (m, 6H), 3.66-3.55 (m, 13H ), 3.41-3.34 (m, 5H), 2.55 (m, 2H), 2.33 (t, J = 7.5Hz, 2H), 2.23-2.15 (m, 1H), 2.03-1.90 (m, 1H), 1.26 (d, J = 6.2Hz, 3H). 13 C NMR (100MHz, Methanol-d4) δ173.61, 172.79, 170.46, 170.31, 167.02, 100.33, 72.62, 70.97, 70.81, 69.87, 69.76, 69.19, 69.07, 68.41, 66.78, 66 .38, 51.80, 48.06, 47.92, 47.77, 47.63, 47.49, 47.35, 47.21, 42.36, 40.35, 39.06, 38.95, 35.93, 31.81, 27.41, 16.66.MS-ESI(m / z): calculated for: 733.33; observed, 733.36, [M+Na] +

[0058] (2) The synthesis path and conditions of the divalent rhamnose molecule NH2-GGG-PEG2-(Rha)2 are as follows:

[0059]

[0060] First, compound 19 and compound 6 were reacted with EDCI as a condensing agent to obtain compound 20 with a yield of 88%. The TBDPS protecting group was removed under the action of TBAF to obtain compound 21, which was used as a sugar acceptor and glycosylated with the sugar donor fully acetylated rhamnose under the catalysis of boron trifluoride ether to obtain compound 22 with a yield of 77%. Then, the Cbz protecting group was removed under the action of Pd / H2 to obtain the key intermediate 23, which was then condensed with the intermediate 4 obtained above to obtain compound 24. Finally, the Cbz and acetyl protecting groups were removed under the action of Pd / H2 and MeOH / MeONa to obtain the final product NH2-GGG-PEG2-(Rha)2. After testing, the nuclear magnetic resonance and mass spectrometry results of the final product were consistent with expectations, indicating that the synthesized NH2-GGG-PEG2-(Rha)2 was correct. 1 H NMR (400MHz, Methanol-d4) δ 8.24-7.92 (m, 2H), 4.75 (d, J=1.6Hz, 2H), 4.38 (m, 1H), 4.02 (m, 2H), 3.92 (m, 2H), 3.85-3.74 (m, 8H), 3.69-3. 62 (m, 12H), 3.58 (m, 7H), 3.43-3.35 (m, 8H), 2.60-2.53 (m, 2H), 2.33 (m, 2H), 2.18-2.05 (m, 1H), 2.00-1.86 (m, 1H), 1.28 (d, J = 6.2Hz, 6H). 13 C NMR (100MHz, Methanol-d4) δ173.69, 172.75, 172.51, 170.33, 170.18, 166. 88, 100.36, 100.34, 72.64, 71.01, 70.99, 70.83, 69.89, 69.84, 69.77, 69.2 0, 69.14, 69.02, 68.45, 66.82, 66.36, 66.33, 52.90, 42.25, 42.09, 40.36, 3 9.10, 38.99, 36.13, 31.85, 27.97, 16.70, 16.69.MS-ESI(m / z): calculated for966.46observed,966.14,[M+Na]+

[0061] (3) The synthesis path and conditions of the tetravalent rhamnose molecule NH2-GGG-PEG2-(Rha)4 are as follows:

[0062]

[0063] Compound 27 and intermediate compound 4 were reacted with condensation agent EDCI to obtain compound 28 with a yield of 73%, and then Cbz and benzyl protecting groups were removed under the action of Pd / H2, and then the free amino group was protected with Boc protecting group to obtain key intermediate 29, which was subjected to amide condensation reaction with intermediate 23 under the action of EDCI to obtain tetravalent rhamnose molecular precursor 30, and finally Boc and acetyl protecting groups were removed under the action of 25% TFA / DCM and MeOH / MeONa, respectively, to obtain the target compound NH2-GGG-PEG2-(Rha)4. After testing, the nuclear magnetic resonance and mass spectrometry results of the final product were consistent with expectations, indicating that the synthesized NH2-GGG-PEG2-(Rha)4 was correct. 1 H NMR (400MHz, D2O) δ4.69 (s, 4H), 4.22-4.09 (m, 3H), 3.94 (s, 2H), 3.85-3.80 (m, 6H), 3.79 (s, 2H), 3.72 (M, 2H), 3.71-3.63 (m, 8H), 3.60 (M, 7H), 3.58-3.47(m, 22H), 3.38-3.25(m, 14H), 3.23(m, 3H), 2.48(m, 2H), 2.3 3-2.20 (m, 6H), 2.00 (m, 1H), 1.93-1.80 (m, 3H), 1.16 (d, J=6.2Hz, 12H). 13 C NMR (100MHz, D2O) δ174.85, 174.08, 173.58, 173.35, 174.22, 171.67, 171.23, 167.81, 163 .00, 162.76, 99.97, 72.04, 70.26, 70.09, 69.53, 69.38, 68.81, 68.77, 68.73, 68.70, 68.6 0, 66.57, 66.48, 53.37, 53.28, 48.88, 42.38, 42.33, 40.44, 39.06, 39.03, 39.00, 38.93, 3 5.60, 31.88, 31.85, 31.27, 27.28, 26.98, 20.38, 16.65.MS-MALDI-TOF (m / z): calculated for 1690.80,observed,1690.89[M+Na] +

[0064] (4) The synthesis path and conditions of the eight-valent rhamnose molecule NH2-GGG-PEG2-(Rha)8 are as follows:

[0065]

[0066] The free amino group in compound 27 was protected with a Boc protecting group to obtain compound 33, and then the benzyl protecting group was removed under the action of Pd / H2 to obtain compound 34, which was then subjected to a condensation reaction with the intermediate 23 obtained above to obtain compound 35 with a yield of 78%, which was subjected to an amide condensation reaction with the intermediate 29 synthesized above to obtain compound 37 with a yield of 67%. Finally, the Boc protecting group and the acetyl protecting group were removed under the action of 25% TFA / DCM and MeOH / MeONa to obtain the final product NH2-GGG-PEG2-(Rha)8. After testing, the nuclear magnetic resonance and mass spectrometry results of the final product were consistent with expectations, indicating that the synthesized NH2-GGG-PEG2-(Rha)8 was correct. 1 H NMR (400MHz, Methanol-d4) δ4.80-4.73(m, 8H), 4.42(m, 4H), 4.29-4.19(m, 2H), 4.04(m, 2H), 3.94(m, 2H), 3.90-3.72(m, 21H), 3.68 (m, 10H), 3.66 (m, 41H), 3.51-3.34 (m, 28H), 2.60-2.50 (m, 2H), 2.43-2.29 (m, 12H), 2.04 (m, 14H), 1.29 (d, J=6.1Hz, 24H). 13 C NMR (100MHz, Methanol-d4) δ173.86, 173.69, 173.65, 173.64, 173.59, 173.56, 173.55, 17 3.53, 173.36, 173.25, 173.13, 172.91, 172.87, 172.82, 172.46, 172.33, 170.36, 170.20, 100.33, 100.30, 72.62, 70.99, 70.96, 70.82, 69.87, 69.85, 69.28, 69.22, 69.13, 69.07, 6 8.46, 66.33, 42.13, 39.23, 39.08, 31.84, 16.79, 16.76.MS-MALDI-TOF (m / z): calculated for 3139.46, observed, 3139.67, [M+Na] +

[0067] (5) Hexavalent rhamnose NH2-GGG-PEG2-(Rha) 16 The synthetic path and conditions are as follows:

[0068]

[0069]

[0070] Compound 34 and compound 36 obtained above were reacted with EDCI to obtain compound 40 with a yield of 85%, and then intermediate 41 was obtained under the action of 25% TFA / DCM, and then amide condensed with intermediate 29 synthesized above to obtain compound 42 with a yield of 67%. Finally, the Boc protecting group and acetyl protecting group were removed under the action of 25% TFA / DCM and MeOH / MeONa to obtain the final product NH2-GGG-PEG2-(Rha) 16 The NMR and mass spectrometry results of the final product were consistent with expectations, indicating that the synthesized NH2-GGG-PEG2-(Rha) 16 correct. 1 H NMR (400MHz, Methanol-d4) δ4.74 (m, 16H), 4.47-4.27 (m, 11H), 3.86-3.82 (m, 16H), 3.79-3.75 (m, 16H), 3.69-3.60 (m, 80H), 3.60-3 .51 (m, 49H), 3.48-3.43 (m, 8H), 3.43-3.34 (m, 44H), 2.36 (m, 30H), 2.17-2.01 (m, 18H), 2.00-1.86 (m, 12H), 1.27 (d, J=6.3Hz, 48H). 13 C NMR (100MHz, Methanol-d4) δ173.69, 173.55, 173.26, 173.13, 173.09, 172.61, 1 00.32, 72.63, 71.00, 70.83, 70.12, 69.87, 69.83, 69.13, 68.47, 66.35, 62.94, 5 2.96, 48.50, 48.22, 48.07, 47.93, 47.91, 47.79, 47.65, 47.51, 47.48, 47.36, 47 .22, 39.25, 39.11, 31.87, 16.84, 16.80, 16.71.MS-MALDI-TOF(m / z): calculated for 6036.81; observed, 6036.63, [M+Na] +

[0071] Example 2: Nanobody-monovalent rhamnose conjugate 7D12-PEG2-Rha, and Nanobody-multivalent rhamnose conjugates 7D12-PEG2-(Rha)2, 7D12-PEG2-(Rha)4, 7D12-PEG2-(Rha)8 and 7D12-PEG2-(Rha) 16 Preparation

[0072] The embodiments for preparing 7D12-PEG2-Rha and 4 nanobody-multivalent rhamnose conjugates are:

[0073] 1) Desalting the nanobody and replacing it in the enzyme reaction buffer;

[0074] 2) adding 25 times the equivalent of monovalent or multivalent rhamnose molecules and 1 / 4 equivalent of enhanced SrtA enzyme to react, and after the reaction is completed, using affinity chromatography to remove unreacted nanoantibodies and enhanced SrtA enzyme.

[0075] 3) Ultrafiltration or exclusion chromatography is used to remove excess monovalent or polyvalent rhamnose molecules to obtain the corresponding product.

[0076] The process of obtaining nanobody-monovalent rhamnose and nanobody-multivalent rhamnose conjugates is as follows Figure 1 shown.

[0077] The purified 7D12-PEG2-Rha and four nanobody-multivalent rhamnose conjugates were characterized by SDS-PAGE, as shown in Figure 2 As shown, all purified products were single bands on SDS-PAGE, and relative to the unmodified nanobody 7D12, as the number of coupled rhamnose increased, the protein band gradually moved toward a higher molecular weight. These results clearly showed that monovalent or multivalent rhamnose molecules were successfully coupled to the nanobody.

[0078] Experimental Example 1: Characterization of affinity level and antibody recruitment ability of nanobody-multivalent rhamnose conjugates

[0079] The implementation plan of the flow cytometry experiment is:

[0080] 1) Take MDA-MB-468 cells (EGFR positive), A431 cells (EGFR positive) and MCF-7 cells (EGFR negative) with good growth conditions and resuspend them to 4×10 5 / mL, transfer 100 μL of cell suspension to a sterile EP tube pre-filled with 100 μL of 200 nmol / L sample (final sample concentration is 100 nmol / L), and place on ice for 30 min; wash twice with flow buffer;

[0081] 2) Add 100 μL of 1% MYC rabbit polyclonal antibody (specific affinity level assessment); or 1% rabbit serum containing anti-Rha antibody (antibody recruitment ability assessment), ice bath for 30 minutes, and wash twice with flow buffer;

[0082] 3) Add 647-coupled goat anti-rabbit IgG antibody and place on ice for 30 min; wash twice with flow cytometry buffer; and finally resuspend with 200 μL flow cytometry buffer for flow cytometry detection;

[0083] 4) Detection was performed using a flow cytometer BD FACSAricaIII, and data were analyzed using FlowJo software.

[0084] The specific affinity level evaluation results are as follows Figure 3 As shown in A, compared with the PBS blank control group, the fluorescence intensity of EGFR-positive MDA-MB-468 cells and A431 cells treated with nanoantibody 7D12, nanoantibody-monovalent rhamnose conjugate 7D12-PEG2-Rha or nanoantibody-multivalent rhamnose conjugate increased significantly, while for EGFR-negative MCF-7 cells, there was no significant change in the fluorescence intensity of the PBS blank control group compared with other groups, which indicates that the modification of monovalent or multivalent rhamnose molecules under this strategy has almost no effect on the specific binding of nanoantibodies to EGFR on the surface of tumor cells.

[0085] The results of the evaluation of the antibody recruitment ability of the nanobody-multivalent rhamnose conjugate are as follows Figure 3 As shown in B, in EGFR-positive MDA-MB-468 and A431 cells, the average fluorescence intensity of the nanobody-monovalent rhamnose conjugate 7D12-PEG2-Rha and the four nanobody-multivalent rhamnose conjugate groups increased significantly compared with the PBS and 7D12 control groups, and the average fluorescence intensity of the cells treated with the nanobody-multivalent rhamnose conjugate was significantly higher than that of 7D12-PEG2-Rha. In EGFR-negative MCF-7 cells, there was no significant change in fluorescence shift compared with the blank control group in other groups. The above results indicate that after being modified with monovalent rhamnose or multivalent rhamnose, the nanobody can selectively recruit anti-rhamnose antibodies to the surface of EGFR-positive tumor cells, and the antibody recruitment ability of the nanobody-multivalent rhamnose conjugate modified with multivalent rhamnose is significantly better than that of the nanobody-monovalent rhamnose conjugate modified with monovalent rhamnose.

[0086] Experimental Example 2: Evaluation of CDC and ADCP levels mediated by nanobody-multivalent rhamnose conjugates

[0087] The implementation scheme of the cytotoxicity test is:

[0088] CDC experiment: 1) Take MDA-MB-468 cells (EGFR positive), A431 cells (EGFR positive) or MCF-7 cells (EGFR negative) with good growth conditions and plate them in 96-well plates at 4×10 3 The cells were added into a 96-well plate at a density of 100 cells / well and cultured overnight to allow the cells to completely adhere to the wall. The old culture medium was discarded and the cells were washed twice with PBS.

[0089] 2) Add 100 μL of fresh culture medium solution containing 100 nM sample, or add different concentrations of 7D12-PEG2-(Rha) 16 (Assessment of concentration-dependent CDC levels), incubate with cells at 37°C for about 1 hour, add 50 μL of human serum containing anti-rhamnose antibodies and 50 μL of normal human complement (final concentration of human serum is 20%, final concentration of human complement is 1%), incubate with cells at 37°C for 6-8 hours, finally aspirate the old culture medium, and detect the results using the CCK8 kit.

[0090] 3) The cell viability was measured at OD450nm using a multifunctional microplate reader to calculate the cytotoxicity.

[0091] The CDC's results are as follows Figure 4 As shown in A, in EGFR-positive MDA-MB-468 and A431 cells, the cell lysis rate of the 7D12-PEG2-Rha group did not increase significantly compared with the 7D12 control group, while the cell lysis rate of the cells treated with the nanobody-multivalent rhamnose conjugate group was significantly higher than that of 7D12-PEG2-Rha. It is worth noting that the cell lysis rate increased with the increase in the number of rhamnose introduced. In contrast, in MCF-7 cells with low EGFR expression, only about 10% of the cells were lysed regardless of whether they were treated with 7D12, 7D12-PEG2-Rha or nanobody-multivalent rhamnose conjugate.

[0092] In addition, cytotoxicity was significantly correlated with the nanobody-multivalent rhamnose conjugate 7D12-PEG2-(Rha) 16 The concentration of 7D12-PEG2-(Rha) 16 As the concentration increases, the cytotoxicity gradually increases (e.g. Figure 4 B). The above data show that after being modified with multivalent rhamnose, nanobodies can effectively recruit endogenous antibodies, reconstruct the Fc-terminal function of nanobodies, stimulate CDC action to kill tumor cells, and the CDC toxicity of nanobody-multivalent rhamnose conjugate is significantly higher than that of nanobody-monovalent rhamnose conjugate 7D12-PEG2-Rha, which proves that the multivalent presentation of rhamnose can more significantly enhance the anti-tumor activity of nanobodies.

[0093] ADCP experiment:

[0094] 1) First, the target cells MDA-MB-468 and A431 cells with good growth conditions were stained with DiO dye (green cell membrane probe), and the phagocytic cells THP-1 were stained with DiI (orange-red membrane probe). 4 The cells were seeded at a density of 100 μg / well in a 24-well plate and allowed to adhere for 12 hours. The old medium was removed and the cells were washed 3 times with PBS.

[0095] 2) Add 500 μL of fresh culture medium solution containing 100 nM sample and incubate with cells at 37°C for about 1 h, then incubate with 1.5×10 5 DiI pre-stained THP-1 cells were co-cultured in the presence of 20% mixed human serum for 4 hours. Finally, the medium containing THP-1 cells was collected in a 1.5 mL EP tube, and the remaining target cells were digested with trypsin and added to the same EP tube. After the cells were collected, they were centrifuged and washed twice with flow buffer.

[0096] 3) Finally, resuspend in 200 μL flow cytometry solution and analyze by flow cytometry. The phagocytic ratio of target cells is calculated by detecting the percentage of double-positive cells to analyze the phagocytic efficiency of THP-1 cells.

[0097] The evaluation results of ADCP are as follows: Figure 5 As shown. Compared with the 7D12 control group, the target cell phagocytosis rate increased significantly after the cells were treated with the 7D12-PEG2-Rha and nanobody-multivalent rhamnose conjugate groups. It is worth noting that compared with the 7D12-PEG2-Rha group, the ADCP ratio mediated by the nanobody-multivalent rhamnose conjugate group increased significantly, and the trend was consistent with the above-mentioned CDC toxicity, which once again proved that the multivalent presentation of rhamnose can stimulate a stronger immune effect.

[0098] From the results of the above examples, it can be seen that 7D12-PEG2-(Rha) 16 The mediated CDC and ADCP levels were the highest, that is, the ADCP and CDC killing levels were significantly enhanced. This may be because the multivalent presentation of rhamnose increases the affinity of rhamnose to endogenous antibodies, enhances the efficiency of recruiting endogenous antibodies, and can provide more Fc domains to interact with complement protein C1q or FcγR of macrophages, thereby generating stronger CDC and ADCP activities. It is speculated that aggregated or multivalent Fc domains may be more conducive to effective chelation with complement C1q protein or FcγR of macrophages, resulting in stronger CDC and ADCP activities.

[0099] Experimental Example 3: Comparative evaluation of the antitumor activity of nanobody-multivalent rhamnose conjugate and cetuximab in vitro

[0100] 1) Take HT-29 cells with good growth condition (cetuximab-resistant cells) and incubate at 4×10 2 The cells were added into a 96-well plate at a density of 100 cells / well and cultured overnight to allow the cells to completely adhere to the wall. The old culture medium was discarded and the cells were washed twice with PBS.

[0101] 2) The activity evaluation experiment of the nanobody-multivalent rhamnose conjugate was the same as the above CDC experimental steps. In the cetuximab group, different concentrations of cetuximab or different concentrations of cetuximab and complement were added to the cells and incubated for 3 days. Finally, the old culture medium was aspirated and the cell viability was detected by CCK8 kit.

[0102] 3) The cell viability was measured at OD450nm using a multifunctional microplate reader to calculate the cytotoxicity.

[0103] Evaluation results such as Figure 6 As shown in Figure 2, cetuximab cannot exert its CDC function due to its low affinity for complement C1q. Figure 6 As shown in Figure A, after HT-29 cells were co-incubated with different concentrations of cetuximab and human complement, no obvious cell lysis was induced. Even when the concentration of cetuximab reached 100 μg / ml (~67 nM), only about 5% of cells were killed. However, the nanobody-multivalent rhamnose conjugate can stimulate significant CDC toxicity in a dose-dependent manner by recruiting endogenous antibodies, and as the number of introduced rhamnose increases, the CDC toxicity generated gradually becomes stronger (e.g. Figure 6 B and 6C), for example, 7D12-PEG2-(Rha) at a concentration of 4 nM 16 , can cause 34.3% cell death. In summary, the nanobody-multivalent rhamnose conjugate of the present invention (especially 7D12-PEG2-(Rha) 16 ) may provide a simple and effective method to solve the problem of cetuximab resistance.

[0104] Experimental Example 4: Pharmacokinetic evaluation of nanobody-multivalent rhamnose conjugates

[0105] 1) Obtaining mouse serum: 7D12 and 7D12-PEG2-(Rha) 16 The drug was injected into normal mice and mice pre-immunized with OVA-Rha (serum containing high titer anti-Rha antibodies) through the tail vein, and then blood samples were collected from the mice through the leg vein at 5min, 10min, 30min, 1h, 2h, 4h, 8h, 24h, 48h, 72h, and 96h. The samples were centrifuged at 5000rpm for 15min, and the supernatant was used as mouse serum for half-life determination.

[0106] 2) Enzyme-linked immunosorbent assay (ELISA) was used to determine the half-life. ①: Dilute the human recombinant EGFR to 2 μg / mL with coating buffer, add 100 μL to each well, incubate at 4°C overnight, and continue incubation at 37°C for 1 hour the next day. After coating, add 200 μL PBST to each well and wash three times; ②: After washing, add 200 μL ELISA blocking buffer and block at room temperature for 2 hours. After blocking, add 200 μL PBST to each well and wash three times; ③: Dilute the mouse serum prepared above with PBS (1:100 dilution in PBS), and then incubate at 37°C for 2 hours. For the standard curve group, different concentrations of 7D12 or 7D12-PEG2-(Rha) 16 (0.2~10nM) was added to the corresponding well plate, and then incubated at 37℃ for 2h. After incubation, 200μL PBST was added and washed 3 times; ④: HRP-coupled rabbit anti-myc tag IgG antibody (1:10,000 dilution in PBS) was added, 100μL was added to each well, and incubated at 37℃ for 1h. After incubation, 200μL PBST was added and washed 3 times; ⑤: 100μL TMB colorimetric solution was added to each well, incubated at 37℃ for 10-15min, and then 25μL of 2mol·L was added to each well. -1 The color reaction was terminated with H2SO4 solution, and then the absorbance of each well was detected at 450nm. The concentration of 7D12 or 7D12-PEG2-(Rha) at different times was calculated using the standard curve. 16 The half-life was calculated from the concentration.

[0107] like Figure 7 As shown, in normal Balb / c mice, nanobody 7D12 and nanobody-multivalent rhamnose conjugate 7D12-PEG2-(Rha) 16 The half-life of the nanobody 7D12 and the nanobody-multivalent rhamnose conjugate 7D12-PEG2-(Rha) were similar and very short, with half-lives of 11.32 minutes and 15.59 minutes, respectively. In Balb / c mice pre-immunized with Rha-OVA conjugate, the half-life of the nanobody 7D12 and the nanobody-multivalent rhamnose conjugate 7D12-PEG2-(Rha) 16 The half-life of the nanobody 7D12 is significantly different. As we expected, the half-life of the nanobody 7D12 is still very short in the presence of high titer anti-Rha antibodies, only 15.04 minutes. In contrast, the nanobody-multivalent rhamnose conjugate 7D12-PEG2-(Rha) 16 The half-life of the nanobody was significantly extended to 19.66 hours, which is 104 times that of the unmodified nanobody 7D12. This result clearly shows that the nanobody-multivalent rhamnose conjugate 7D12-PEG2-(Rha) 16Anti-Rha antibodies can be recruited to form immune complexes, greatly increasing the molecular weight and avoiding rapid filtration and clearance by the kidneys. In addition, the neonatal Fc receptor (FcRn) encoded by Fcgart can protect IgG and albumin from catabolism. This receptor can prolong the half-life of IgG and albumin, and the Fc end of IgG in the immune complex formed by the nanobody-multivalent hapten conjugate and anti-Rha antibody can bind to the neonatal Fc receptor (FcRn) and enter the FcRn-mediated circulation system, thereby prolonging the half-life of the nanobody and improving its pharmacokinetic characteristics.

[0108] Experimental Example 5: Comparative evaluation of the anti-tumor activity of nanobody-multivalent rhamnose conjugate and cetuximab in vivo

[0109] 1) Balb / c nude mice (female, 4-6 weeks old, 18-20 g) were injected subcutaneously on the left flank with 100 μL of 2×10 6 HT-29 cells (resistant to cetuximab) in PBS, and wait until the tumor volume is between 50-80mm 3 At the same time, the mice were randomly divided into 4 groups (PBS group, 7D12 group and nanobody-multivalent rhamnose conjugate 7D12-PEG2-(Rha) 16 and cetuximab group), and 5 mice in each group were subjected to parallel experiments.

[0110] 2) The drug was injected into the tail vein once every two days for 5 times. The PBS group was injected with 50 μL PBS and 50 μL mouse serum containing anti-Rha antibody each time; the 7D12 group was injected with 50 μL 7D12 (40 μM) and mouse serum containing anti-Rha antibody each time; 7D12-PEG2-(Rha) 16 Each group was injected with 50 μL 7D12-PEG2-(Rha) 16 (40 μM) and mouse serum containing anti-Rha antibody; the cetuximab group was injected with 100 μL cetuximab (1 mg) each time

[0111] 3) During treatment and ten days after stopping treatment, the tumor volume was measured with a vernier caliper every two days, and the volume was calculated using the formula: V = (length * width 2 ) / 2.

[0112] During and after treatment, changes in tumor volume were observed. Figure 8 As shown, compared with PBS or 7D12 group, the nanobody-multivalent rhamnose conjugate 7D12-PEG2-(Rha) 16The tumor volume of the mice in the PBS or 7D12 groups decreased significantly, and the growth of the tumor volume during the treatment was negligible, and it showed a slow growth after the drug was stopped. However, the tumor volume of the mice in the PBS or 7D12 groups increased to varying degrees. It is worth noting that the nanobody-multivalent rhamnose conjugate 7D12-PEG2-(Rha) 16 The in vivo anti-tumor activity of the treatment group was also significantly better than that of the cetuximab group. These results clearly show that the in vivo anti-tumor activity of the nanobody-multivalent rhamnose conjugate is greatly enhanced compared with 7D12 and cetuximab. Studies have found that the development of tumor resistance to related monoclonal antibodies often involves amino acid mutations in the target protein, resulting in a decrease or disappearance of the affinity of the monoclonal antibody drug for the target; in addition, some tumor cells can also degrade the monoclonal antibody by secreting some proteases, resulting in the inability of the monoclonal antibody drug to bind to it. Nanobodies often have smaller binding pockets and higher protease resistance, which may overcome the drug resistance of monoclonal antibodies. Therefore, the nanobody-multivalent rhamnose conjugate provided by the present invention can be used to treat certain tumors that are insensitive to monoclonal antibody drugs or develop drug resistance.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nanobody-multivalent rhamnose conjugate, characterized in that The conjugate comprises a nanobody modified with a multivalent rhamnose molecule, wherein the multivalent rhamnose molecule is a multivalent form of a rhamnose molecule, and the structural formula of the multivalent rhamnose molecule is as follows:

2. The Nanobody-multivalent rhamnose conjugate according to claim 1, characterized in that The synthesis path of the multivalent rhamnose molecule includes: Synthesis of divalent rhamnose molecules: Synthesis of tetravalent rhamnose molecules: Synthesis of octavalent rhamnose molecule: Synthesis of hexadecavalent rhamnose molecule:

3. The Nanobody-multivalent rhamnose conjugate according to claim 1, characterized in that The nanobody includes but is not limited to any one of anti-EGFR nanobody, anti-EGFRvIII nanobody, anti-HER2 nanobody, anti-HER3 nanobody, anti-PSMA nanobody, anti-VEGFR nanobody, anti-PD-L1 nanobody, anti-cMET nanobody, anti-TGF-β nanobody, anti-MUC1 nanobody and anti-Trop-2 nanobody.

4. A method for preparing the Nanobody-multivalent rhamnose conjugate according to any one of claims 1 to 3, characterized in that: Multivalent rhamnose molecules were modified onto nanobodies.

5. The method according to claim 4, characterized in that The modification method for modifying the multivalent rhamnose molecule onto the nanobody includes an enzymatic modification method or a chemical enzyme site-directed modification method.

6. The method according to claim 5, characterized in that Enzyme site-directed modifications include: 1) Desalting the nanobody and replacing it in the enzyme reaction buffer; 2) adding 10-50 times the equivalent of multivalent rhamnose molecules and 1 / 10-1 / 2 equivalent of SortaseA or SrtA mutants to react, and after the reaction is completed, removing unreacted nanobodies, SrtA enzymes or SrtA mutants by affinity chromatography; 3) removing excess multivalent rhamnose molecules by ultrafiltration or exclusion chromatography to obtain the corresponding nanobody-multivalent rhamnose conjugate.

7. Use of the nanobody-multivalent rhamnose conjugate according to any one of claims 1 to 3 in the preparation of tumor immunotherapy drugs.

8. The use according to claim 7, characterized in that: The nanobody-multivalent rhamnose conjugate is used to prepare a tumor immunotherapy drug, which is in a pharmaceutically acceptable form, and its preparation form includes any one of an injection, a tablet, a sustained-release agent, a pill, a granule, and a capsule.

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