A bivalent nanobody against the α3 region of human MICA / B and its application

Abstract

The present invention relates to the field of bioengineering technology, and discloses a bivalent nanobody against the α3 region of human MICA / B and its application. The variable region of the heavy chain of the bivalent nanobody comprises the amino acid sequence shown in SEQ ID NO:1, or comprises an amino acid sequence having similar or close biological activity to SEQ ID NO:1. The bivalent nanobody against the α3 region of human MICA / B of the present invention has the advantages of nanobodies, can stably exist in vivo for a long time, specifically binds to the α3 region of human MICA / B, effectively inhibits the hydrolysis and shedding of MICA / B on the surface of tumor cells, and further restores the immune surveillance of NK cells to achieve targeted therapy of tumors.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to a bivalent nanobody against the α3 region of human MICA / B and its application. Background Art

[0002] Colorectal cancer is one of the most common malignant tumors, and its incidence and mortality are gradually increasing in China, bringing a relatively large social and economic burden. Effective intervention and blocking of colorectal cancer metastasis are of great significance for improving the prognosis of patients and prolonging their survival expectancy. However, there is currently no ideal treatment method. Activating or modifying autologous active immune cells to perform immune surveillance and clearance on tumor cells during the "window period" of tumor metastasis is an ideal treatment method at present. However, how to effectively intervene in tumor immune escape and restore immune cell surveillance is a key issue in anti-tumor immunotherapy. MICA / B is an important ligand for various immune effector cells such as natural killer cells (NK) to recognize tumor cells. After binding to the NKG2D receptor with high affinity, it promotes the release of perforin and granzyme by NK cells to dissolve tumor cells or induce their death. Some studies have shown that highly invasive tumor cells, especially tumor cells during the "window period" of tumor metastasis, can release various proteases to hydrolyze the α3 region of MICA / B, causing MICA / B to fall off from the surface of tumor cells, resulting in NK cells being unable to effectively recognize and kill tumor cells invading the vascular system, thus enabling tumor cells to achieve "immune escape". Inhibiting the shedding of MICA / B from the surface of tumor cells can restore the killing effect of NK cells on them, which is an important link in realizing immune surveillance of tumor cells.

[0003] In the peripheral blood of camelids (such as alpacas, camels, etc.) and some cartilaginous fish (such as sharks, catfish, etc.), there is an antibody that lacks a natural light chain and only contains a single variable heavy chain region (VHH) and two conventional CH2 and CH3 regions. It is the smallest naturally occurring fragment that can bind to antigens. VHH can exist stably alone in vitro and has an independent antigen recognition ability. It is called a nanobody or camelid single-domain antibody. The crystal width of the nanobody is 2.5 mm and the length is 4 nm, and its molecular weight is only 1 / 10 of that of traditional antibodies. Compared with traditional antibodies, nanobodies have a small molecular weight and a simple structure, with higher physical and chemical stability (good solubility and good stability at different temperatures and pH values), strong binding specificity to the target, higher tissue penetration (better penetration into tissues and the ability to recognize hidden epitopes that traditional antibodies cannot bind), are suitable for large-scale industrial production (can be produced in large quantities in bacteria and yeast), are easier to modify and optimize, and are easier to humanize (can be humanized by replacing a few amino acids without significantly changing their specificity and activity). In addition, the structure of these variable domains is highly homologous to the variable domain of a subclass of human immunoglobulin IgG3 and will not cause a strong immune response in the human body. Nanobodies can be widely used in future research, clinical diagnosis, and treatment, and have broad prospects.

[0004] Developing a bivalent nanobody against the α3 region of human MICA / B to specifically bind to the α3 region of MICA / B and inhibit the hydrolysis and shedding of MICA / B from the surface of tumor cells, thereby restoring the immune surveillance of NK cells, is a possible strategy for targeted tumor therapy. Nanobodies have a small molecular weight and a simple structure, and are metabolized relatively quickly in the human body. Bivalent nanobodies improve the stability of nanobodies in the human body and extend the biological action time of the antibody in vivo. However, there is currently no relevant report on bivalent nanobodies that specifically bind to the α3 region of MICA / B. Summary of the Invention

[0005] In order to solve the technical problem in the prior art that there is no bivalent nanobody that specifically binds to the α3 region of MICA / B, the present invention provides a bivalent nanobody against the α3 region of human MICA / B and its application. The bivalent nanobody against the α3 region of human MICA / B of the present invention has the advantages of nanobodies, can stably exist in the human body for a long time, specifically binds to the α3 region of human MICA / B, effectively inhibits the hydrolysis and shedding of MICA / B protein on the surface of tumor cells, and further restores the immune surveillance of NK cells to achieve targeted tumor therapy.

[0006] The specific technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a bivalent nanobody against the α3 region of human MICA / B, comprising a heavy chain variable region; the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, or comprises an amino acid sequence having similar or comparable biological activity to SEQ ID NO: 1.

[0008] MICA / B is an important ligand for various immune effector cells such as NK cells to recognize tumor cells. Highly invasive tumor cells, especially cells in the "window period" of tumor metastasis, promote the shedding of MICA / B on the tumor cell surface by releasing various proteases to hydrolyze the α3 region of MICA / B, thereby enabling tumor cells to achieve "immune escape". The bivalent nanobody against the α3 region of human MICA / B of the present invention can specifically bind to the α3 region of human MICA / B, effectively inhibit the hydrolytic shedding of MICA / B on the tumor cell surface, and then restore the immune surveillance of NK cells, and is expected to treat tumor metastasis, recurrence, etc. to a certain extent.

[0009] Moreover, as a bivalent nanobody, the bivalent nanobody against the α3 region of human MICA / B of the present invention can stably exist in the human body for a long time, extend the biological action time of the nanobody in vivo, has stable physicochemical properties, high affinity, high tissue penetration, can be mass-produced, is easy to be applied to antibody conjugates, and can be subjected to corresponding engineering design according to the expression level of the target and the safety requirements of treatment, and has good clinical application prospects.

[0010] Optionally, the bivalent nanobody further comprises a heavy chain constant region; the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 3, or comprises an amino acid sequence having similar or comparable biological activity to SEQ ID NO: 3.

[0011] Optionally, the bivalent nanobody further comprises a light chain constant region; the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 4, or comprises an amino acid sequence having similar or comparable biological activity to SEQ ID NO: 4.

[0012] In a second aspect, the present invention provides an amino acid sequence encoding the bivalent nanobody, comprising a nucleotide sequence encoding a heavy chain variable region; the nucleotide sequence encoding the heavy chain variable region is as shown in SEQ ID NO: 2.

[0013] In a third aspect, the present invention provides a cloning or expression vector comprising the nucleic acid molecule.

[0014] The expression vector can be a phage.

[0015] In a fourth aspect, the present invention provides a host cell containing the nucleic acid molecule or the vector.

[0016] The host cell can be selected from CHO cells or Escherichia coli.

[0017] In a fifth aspect, the present invention provides the use of the bivalent nanobody or the nucleic acid molecule or the expression vector or the host cell, specifically including:

[0018] First, the use of the bivalent nanobody or the nucleic acid molecule or the expression vector or the host cell in the preparation of a drug that specifically binds to the α3 region of human MICA / B.

[0019] Second, the use of the bivalent nanobody or the nucleic acid molecule or the expression vector or the host cell in the preparation of a drug that inhibits the hydrolysis and shedding of MICA / B molecules on the surface of tumor cells.

[0020] Third, the use of the bivalent nanobody or the nucleic acid molecule or the expression vector or the host cell in the preparation of an antibody drug or an antibody-drug conjugate product for targeted tumor therapy.

[0021] Fourth, the use of the bivalent nanobody or the nucleic acid molecule or the expression vector or the host cell in the preparation of a detection reagent that binds to the MICA / B α3 region protein, and the detection reagent is an immunohistochemical reagent or an enzyme-linked immunosorbent assay (ELISA) detection reagent or a Western Blotting detection reagent.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The bivalent nanobody against the α3 region of human MICA / B of the present invention can specifically bind to the α3 region of human MICA / B, effectively inhibit the hydrolysis and shedding of MICA / B on the surface of tumor cells, and then restore the immune surveillance of NK cells to achieve targeted tumor therapy;

[0024] (2) As a bivalent nanobody, the bivalent nanobody against the α3 region of human MICA / B of the present invention has a large molecular mass, stable physicochemical properties, high affinity and high tissue penetration, can exert biological functions in vivo for a long time, and can be mass-produced, having great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a flow cytometry scatter plot of the bivalent nanobody against the α3 region of human MICA / B inhibiting the hydrolysis and shedding of MICA / B molecules on the surface of melanoma cell line A375 cells.

[0026] Figure 2 It is a schematic diagram of the bivalent nanobody against the α3 region of human MICA / B.

[0027] Figure 3 Results of the enzyme-linked immunosorbent assay (ELISA) of bivalent nanobodies against the α3 region of human MICA / B Detailed implementation manners

[0028] The present invention will be further described below in conjunction with embodiments.

[0029] The terms used in the embodiments of the present invention are for the purpose of describing specific specific implementation manners and are not intended to limit the protection scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the appended claims and any equivalents thereof are the protection scope of the present invention. The experimental methods described in the following embodiments are all conventional methods unless otherwise specified; the reagents and biological materials can be obtained from commercial sources unless otherwise specified.

[0030] Example 1: Preparation of bivalent nanobodies against the α3 region of human MICA / B

[0031] The bivalent nanobodies against the α3 region of human MICA / B are prepared through the following steps:

[0032] 1) Construction of a CHO cell line stably expressing the α3 region of human MICA / B:

[0033] In HEK293T cells, a lentivirus packaging of the α3 region of human MICA / B is constructed using a plasmid lentivirus packaging system. The obtained lentivirus supernatant is centrifuged to remove cell debris and concentrated to obtain a lentivirus concentrate. The obtained lentivirus concentrate is used to infect CHO cells, and CHO cells stably expressing the α3 region protein of human MICA / B with green fluorescent protein (GFP) are sorted by flow cytometry.

[0034] 2) Construction of a natural nanobody phage display library:

[0035] A CHO cell line stably expressing the α3 region of human MICA / B is used to immunize alpacas. After four immunizations, blood is collected every 5 - 7 days to detect the serum titer. After the titer reaches the standard, peripheral blood lymphocytes are separated for RNA extraction, reverse transcribed into cDNA and amplified. The obtained diverse antibody gene sequences are digested with a phage vector, purified and subjected to a ligation reaction to obtain a natural nanobody phage display library.

[0036] 3) Screening of nanobodies against the α3 region of human MICA / B:

[0037] Using CHO cells stably expressing human MICA / B as target cells, positive screening was performed to enrich candidate phages, and wild-type CHO cells were used as target cells for negative screening to remove non-targeted phage particles against the α3 region of human MICA / B. Screening and eluting phages were repeated for 3 rounds, and NSG sequencing was carried out to obtain a candidate nanobody DNA library that binds to the antigen of the α3 region of human MICA / B.

[0038] 4) Identification of nanobodies against the α3 region of human MICA / B by enzyme-linked immunosorbent assay (ELISA):

[0039] The obtained phages were used to infect the logarithmic-phase Escherichia coli TG1 strain and spread on plates. Monoclonal colonies were picked from the plates and inoculated into 96-deep well plates. After culturing to the logarithmic phase, they were infected with the VCSM13 helper phage to obtain monoclonal phage supernatants. 96-well plates were inoculated with CHO cells stably expressing the α3 region of human MICA / B and wild-type CHO cells. ELISA detection was carried out using the candidate monoclonal phage supernatant as the primary antibody and horseradish peroxidase-conjugated M13 as the secondary antibody. The obtained candidate positive clones were sequenced, and the gene sequence encoding the nanobody against the α3 region of human MICA / B was shown as SEQ ID NO:2, and the amino acid sequence of the nanobody against the α3 region of human MICA / B was shown as SEQ ID NO:1.

[0040] 5) Specificity detection of candidate nanobodies against the α3 region of human MICA / B:

[0041] The phage vectors of the candidate nanobodies obtained by screening were serially diluted and added to the TG1 bacterial solution. After culturing at 37 °C for 30 minutes, they were spread on 2x YT culture plates containing ampicillin and cultured with shaking at 37 °C overnight to obtain monoclonal colonies. Randomly selected single colonies were transferred to 96-well plates containing 2x YT culture medium with ampicillin and cultured with shaking at 37 °C overnight until the logarithmic growth phase. IPTG was added to a final concentration of 1 mM and cultured at 30 °C overnight. The bacterial cells were resuspended in 1×TES containing 1 mg / ml lysozyme and incubated on ice for 30 minutes, and the periplasmic extract was obtained by centrifugation. The obtained periplasmic extract was purified using Ni-NTA affinity chromatography, and the nanobodies against the α3 region of human MICA / B obtained were detected by SDS-PAGE. Using the candidate nanobodies as the primary antibody and APC-conjugated anti-Flag antibody as the secondary antibody, the binding of the candidate nanobodies to CHO cells stably expressing the α3 region of human MICA / B and wild-type CHO cells was detected by flow cytometry to obtain high-purity nanobodies against the α3 region of human MICA / B.

[0042] 6) Preparation of bivalent nanobodies against the α3 region of human MICA / B:

[0043] The obtained high-purity anti-human MICA / B α3 domain nanobody was conjugated with the heavy chain constant region amino acid sequence shown in SEQ ID NO:3, conjugated with the light chain constant region amino acid sequence shown in SEQ ID NO:4 and conjugated with the Fc segment of IgG1 to prepare a high-purity anti-human MICA / B α3 domain bivalent nanobody, as Figure 2 shown.

[0044] Example 2: Assay for inhibition of shedding of MICA / B on cell surface by anti-human MICA / B α3 domain bivalent nanobody

[0045] Flow cytometry was used to detect the inhibition of MICA / B hydrolysis on the cell surface by the anti-human MICA / B α3 domain bivalent nanobody. The melanoma cell line A375 that naturally expresses MICA / B was seeded into a 24-well plate at 3×10 5 / well, and five concentration gradients were set by 10-fold dilution and the anti-human MICA / B α3 domain bivalent nanobody (prepared in Example 1) was added, and a blank control was set, and cultured at 37 °C for 24 hours. The cells in the 24-well plate were digested with EDTA and transferred to an EP tube, and the PE-fluorescently labeled MICA / B flow antibody (PE anti-human MICA / MICB Antibody) was added, and incubated at 4 °C in the dark for 30 minutes; washed twice with 1 ml of PBS, 200 μl of PBS was added to each tube to resuspend the cell pellet, and then detected by a flow cytometer to analyze the ability of the anti-human MICA / B α3 domain bivalent nanobody to inhibit the hydrolysis of MICA / B on the surface of tumor cells.

[0046] The results were as Figure 1 shown. The anti-human MICA / B α3 domain bivalent nanobody was able to inhibit the shedding of MICA / B on the surface of A375 cells, and the inhibitory hydrolysis activity was concentration-dependent.

[0047] Example 3: Assay for binding of anti-human MICA / B α3 domain bivalent nanobody to target antigen

[0048] The ELISA plate strips were coated with 10 μg / ml of the target antigen MICA / B α3 protease protein at 37 °C for 2 hours, washed with TBST, and then 10% fetal bovine serum was added and blocked at 37 °C for 1 hour; different concentrations of the anti-human MICA / B α3 domain bivalent nanobody (prepared in Example 1) were added and reacted at 37 °C for 1 hour; after washing with TBST, the horseradish peroxidase-labeled anti-human Fc secondary antibody / goat anti-human secondary antibody was added and reacted at 37 °C for 30 minutes; the plate was washed with TBST, and the residual liquid drops were drained on absorbent paper, repeated 5 times, then 100 μl of TMB was added and reacted at 37 °C for 15 minutes, and the reaction was terminated by adding sulfuric acid termination solution, and the OD value was read at 450 nm by an enzyme-linked immunosorbent assay analyzer to analyze the binding ability of the anti-human MICA / B α3 domain bivalent nanobody to the target antigen.

[0049] The results are as Figure 3 shown, and the bivalent nanobody against the α3 region of human MICA / B binds well to the target antigen.

[0050] Unless otherwise specified, the raw materials and equipment used in the present invention are all common raw materials and equipment in the art; unless otherwise specified, the methods used in the present invention are all conventional methods in the art.

[0051] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent transformations made to the above embodiments according to the technical essence of the present invention still belong to the protection scope of the technical solution of the present invention. Sequence Listing <110> Zhejiang University <120> A bivalent nanobody against the α3 region of human MICA / B and its application <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 134 <212> PRT <213> Artificial Sequence <400> 1 His Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala 1 5 10 15 Gly Ala Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30 Ser Ser Ala Phe Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Gly Ile Gly Arg Gly Tyr Ile Asn Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Ala Pro Lys Tyr Cys Ser Gly Tyr Val Cys Thr Val Pro Ser 100 105 110 Asp Gly Met Gly Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser 115 120 125 His His His His His His 130 <210> 2 <211> 405 <212> DNA <213> Artificial Sequence <400> 2 catatggcgg tgcagctggt ggagtctggg ggaggattgg tgcaggctgg ggcctctctg 60 agtctctcct gtgcagcctc tggacgcacc ttcagcagct ctgccttcgg ctggttccgc 120 caggctccag ggaaggagcg tgagtttgta gcaggcattg gacggggtta tattaacaca 180 tactatgcag actccgtgaa gggccgcttc accatctcca gagacaacgc caagaacacg 240 gtgtatctgc aaatgaacag cctgaaacct gaggacacgg ccatgtatta ctgtgcgcca 300 aaatactgtt caggctatgt ctgtacggtg ccttcggacg gcatgggcta ctggggcaaa 360 gggacccagg tcaccgtctc ctcacaccac caccaccacc actaa 405 <210> 3 <211> 232 <212> PRT <213> Artificial Sequence <400> 3 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 4 <211> 107 <212> PRT <213> Artificial Sequence <400> 4 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105

Claims

1. A bivalent nanobody against the α3 region of human MICA / B, characterized in that, it comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:

1.

2. The bivalent nanobody according to claim 1, characterized in that, it further comprises a heavy chain constant region with the amino acid sequence shown in SEQ ID NO:

3.

3. The bivalent nanobody according to claim 1, characterized in that, it further comprises a light chain constant region with the amino acid sequence shown in SEQ ID NO:

4.

4. A nucleic acid molecule encoding the bivalent nanobody according to any one of claims 1 to 3, characterized in that, it comprises a nucleotide sequence encoding the heavy chain variable region; the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:

2.

5. A cloning vector or expression vector containing the nucleic acid molecule according to claim 4.

6. A host cell containing the nucleic acid molecule according to claim 4 or the vector according to claim 5.

7. Use of the bivalent nanobody according to any one of claims 1 to 3, or the nucleic acid molecule according to claim 4, or the vector according to claim 5, or the host cell according to claim 6 in the preparation of a reagent for in vitro binding to the target antigen MICA / B α3 region protein.

8. Use of the bivalent nanobody according to any one of claims 1 to 3, or the nucleic acid molecule according to claim 4, or the vector according to claim 5, or the host cell according to claim 6 in the preparation of a reagent for in vitro inhibiting the proteolytic shedding of MICA / B protein on the surface of tumor cells, characterized in that, the tumor is melanoma.

9. Use of the bivalent nanobody according to any one of claims 1 to 3, or the nucleic acid molecule according to claim 4, or the vector according to claim 5, or the host cell according to claim 6 in the preparation of a detection reagent for binding to MICA / B α3 region protein, characterized in that, the detection reagent is an immunohistochemical reagent or an enzyme-linked immunosorbent assay detection reagent or a Western blot detection reagent.

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