An anti-CD73 nanoantibody and its application

Abstract

The present invention discloses an anti-CD73 nanobody and its application. The anti-CD73 nanobody comprises a framework region (FR) and a complementary determining region (CDR), wherein the complementary determining region (CDR) comprises a complementary determining region (CDR1), a complementary determining region (CDR2), and a complementary determining region (CDR3). The anti-CD73 nanobody of the present invention has specific recognition and binding capabilities for CD73 and is expected to be used as a therapeutic antibody for cancer.

Description

Technical Field

[0001] The present invention relates to the field of immunology, and in particular to an anti-CD73 nanoantibody and its application. Background Art

[0002] Tumor cells can evade anti-tumor immune responses through various mechanisms, including inducing and recruiting various suppressive immune cells, secreting immunosuppressive cytokines, and producing immunosuppressive metabolites. The adenosine pathway has become a key immune checkpoint pathway for cancer treatment. In the hypoxic tumor microenvironment (TME), elevated adenosine levels are often observed due to the high expression of CD73 (ecto-5'-nucleotidase) and CD39 (nucleoside triphosphate diphosphohydrolase), which catalyze the dephosphorylation of AMP to adenosine. High concentrations of adenosine, on the one hand, impair the activation and function of T cells and natural killer (NK) cells, leading to potent immunosuppression; on the other hand, they enhance the function of regulatory T cells (Tregs) and the differentiation of M2 macrophages, which negatively impacts the recognition and activity of immune cells, thereby promoting the occurrence and development of cancer. Therefore, CD73 is the main immunosuppressive mediator of the TME. In addition to the effects of tumor cell-intrinsic CD73 on tumor cell proliferation, angiogenesis, invasion, and metastasis, CD73 expression by tumor cells and immune cells also impairs anti-tumor immunity by inhibiting the function of protective immune cells (such as effector T cells, NK cells, DCs, and B cells) while maintaining the function of regulatory immune cells (such as Tregs, MDSCs, TAMs, and CAFs). Currently, directly disrupting adenosine-mediated immunosuppression by blocking adenosine production by inhibiting the enzymatic activity of CD39 and CD73 will be a very promising therapeutic strategy. Studies have found that treatment with anti-CD73 monoclonal antibodies significantly delayed the growth of primary 4T1.2 and E0771 tumors in mice with intact immune systems and significantly inhibited the development of spontaneous 4T1.2 lung metastases. In addition, inhibition of CD73 activity, combined with targeted therapy or conventional treatment with immune checkpoint blockade (such as monoclonal antibodies against CTLA-4 or PD-1), has improved the anti-tumor effect in many preclinical cancer mouse models and has also shown good clinical activity in patients with advanced solid tumors. However, monoclonal antibodies have high target specificity but low penetration into solid tumors, and their manufacturing process is complex and the production cost is high, which limits their application in cancer treatment.

[0003] Nanobodies (Nb) are the variable regions of heavy-chain antibodies (IgG2 and IgG3) in camelids and are known as the smallest antigen-binding fragments found in nature. Compared to traditional full-length monoclonal antibodies (mAbs, approximately 150 kD), Nb have advantages such as a small molecular weight (12-15 kD), a simpler structure, low immunogenicity, high tissue permeability, high stability, high solubility, low aggregation, and ease of cloning. In addition, compared to similar mAb products, Nb production costs are significantly lower and can be used by most cancer patients. Therefore, the application of nanobody technology to develop a therapeutic anti-CD73 nanobody antibody has broad prospects. Summary of the Invention

[0004] In response to the above-mentioned deficiencies in the prior art, the present invention provides an anti-CD73 nanoantibody and its application. The nanoantibody has specific recognition and binding capabilities for CD73 and is expected to be used as a therapeutic antibody for various cancers.

[0005] The present invention provides the following technical solutions:

[0006] An anti-CD73 nanobody, the nanobody comprises a framework region FR and a complementary determining region CDR, wherein the complementary determining region CDR comprises a complementary determining region CDR1, a complementary determining region CDR2 and a complementary determining region CDR3, wherein:

[0007] The amino acid sequence of the complementarity determining region CDR1 is shown in SEQ ID NO: 2.

[0008] The amino acid sequence of the complementarity determining region CDR2 is shown in SEQ ID NO: 4.

[0009] The amino acid sequence of the complementarity determining region CDR3 is shown in SEQ ID NO: 6; or

[0010] The amino acid sequence of the complementarity determining region CDR1 is shown in SEQ ID NO: 9.

[0011] The amino acid sequence of the complementarity determining region CDR2 is shown in SEQ ID NO: 11.

[0012] The amino acid sequence of the complementarity determining region CDR3 is shown in SEQ ID NO: 13; or

[0013] The amino acid sequence of the complementarity determining region CDR1 is shown in SEQ ID NO: 16.

[0014] The amino acid sequence of the complementarity determining region CDR2 is shown in SEQ ID NO: 18.

[0015] The amino acid sequence of the complementarity determining region CDR3 is shown in SEQ ID NO: 20.

[0016] Furthermore, the frame region FR includes a frame region FR1, a frame region FR2, a frame region FR3 and a frame region FR4, wherein:

[0017] The amino acid sequence of the framework region FR1 is shown in SEQ ID NO: 1,

[0018] The amino acid sequence of the framework region FR2 is shown in SEQ ID NO: 3,

[0019] The amino acid sequence of the framework region FR3 is shown in SEQ ID NO: 5,

[0020] The amino acid sequence of the framework region FR4 is shown in SEQ ID NO: 7; or

[0021] The amino acid sequence of the framework region FR1 is shown in SEQ ID NO: 8,

[0022] The amino acid sequence of the framework region FR2 is shown in SEQ ID NO: 10,

[0023] The amino acid sequence of the framework region FR3 is shown in SEQ ID NO: 12,

[0024] The amino acid sequence of the framework region FR4 is shown in SEQ ID NO: 14; or

[0025] The amino acid sequence of the framework region FR1 is shown in SEQ ID NO: 15,

[0026] The amino acid sequence of the framework region FR2 is shown in SEQ ID NO: 17,

[0027] The amino acid sequence of the framework region FR3 is shown in SEQ ID NO: 19,

[0028] The amino acid sequence of the framework region FR4 is shown in SEQ ID NO: 21.

[0029] Furthermore, the amino acid sequence of the anti-CD73 nanobody is shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

[0030] A DNA molecule encoding the above-mentioned anti-CD73 nanobody.

[0031] An expression vector comprising the above DNA molecule.

[0032] A host cell comprising the above expression vector.

[0033] The use of the above-mentioned anti-CD73 nanoantibody in the preparation of CD73 molecular detection reagents.

[0034] The use of the above-mentioned anti-CD73 nanoantibody in the preparation of tumor treatment drugs.

[0035] The beneficial effects of the present invention are:

[0036] The anti-CD73 nanoantibody of the present invention has specific recognition and binding capabilities for CD73 and is expected to be used as a therapeutic antibody for cancer. DETAILED DESCRIPTION

[0037] The specific preparation process of the Nanobodies of the present application is described below through specific examples. However, those skilled in the art should understand that the scope of the present application should not be limited to these examples, but includes various equivalent transformations that can be made by those skilled in the art.

[0038] Example 1

[0039] (1) Animal immunization: For the first immunization, 0.5 mg CD73 antigen was mixed with Freund's complete adjuvant in a ratio of 1:1 (V:V) to obtain a mixture. 1 mL of the emulsified mixture was injected into the left and right sides of the camel's cervical lymph nodes to form multiple masses. The absorption of the subcutaneous injection masses was tracked to confirm the correct immunization. For the second immunization, 3 weeks after the first immunization, 0.25 mg CD73 antigen was mixed with Freund's incomplete adjuvant in a ratio of 1:1 (V:V) to obtain a mixture. 1 mL of the emulsified mixture was injected into the left and right sides of the camel's cervical lymph nodes to form multiple masses. The absorption of the subcutaneous injection masses was tracked to confirm the correct immunization. For the third immunization, 3 weeks after the second immunization, 0.25 mg CD73 antigen was mixed with Freund's incomplete adjuvant in a ratio of 1:1 (V:V) to obtain a mixture. 1 mL of the emulsified mixture was injected into the left and right sides of the camel's cervical lymph nodes to form multiple masses. The absorption of the subcutaneous injection masses was tracked to confirm the correct immunization. For the fourth immunization, 0.25 mg CD73 antigen was mixed with Freund's incomplete adjuvant in a 1:1 (V:V) ratio to obtain a mixture. 1 mL of the emulsified mixture was injected into the left and right sides of the camel's cervical lymph nodes. The absorption of the subcutaneous injection mass was monitored to confirm the correct immunization.

[0040] (2) Serum processing and titer detection: One week after the third and fourth immunizations, 2 mL of camel peripheral blood was collected, the serum was separated, and the human CD73 recombinant protein was coated in an ELISA 96-well plate. The antibody titer in the serum was determined by ELISA. The ELISA results showed that the titer of the camel four-immune serum was >1:64000, which met the library construction standards, indicating that high-affinity antibodies against human CD73 were present in the serum;

[0041] (3) Construction of phage display immune antibody library:

[0042] ① Collect 50 mL of peripheral blood from camels after the fourth immunization and separate PBMCs; take 2×10 7 Total RNA was extracted from PBMC using an RNA extraction kit; 5 μg of RNA was taken and cDNA was obtained using an RT-PCR reverse transcription kit;

[0043] ② The IgG2 and IgG3 heavy chain variable region sequences were obtained step by step by nested PCR. The experimental steps are as follows:

[0044] a. Design a pair of specific nested outer primers and perform the first round of PCR amplification using the cDNA as a template. The amplified region is the Leader-CH2 region of the camel heavy chain antibody gene, with product sizes of 700 bp and 900 bp. Recover the 700 bp PCR product by DNA gel electrophoresis and gel excision.

[0045] b. Design nested inner primers (5 pairs) and perform a second round of PCR amplification using the 700 bp first-round PCR product obtained in step a as a template. The amplified region is the camel heavy chain antibody variable region VHH fragment, and the product size is 400 bp; the second round 400 bp PCR product was purified and recovered using a PCR product purification kit;

[0046] c. Insert the heavy chain variable region sequence VHH obtained in step b into the linearized phagemid vector VHH-libTemplate that has been treated with enzyme digestion by enzyme digestion and ligation to obtain a recombinant vector. After purification and recovery, transform it into super-competent SS320 cells (containing helper phage M13K07); the transformed bacterial solution is resuspended in SOC medium and activated for 1 hour; the bacterial solution is diluted 10 times in a gradient, plated on LB / tet10 and LB / Carb50 culture plates, and placed in a 37°C biochemical incubator overnight; the remaining bacterial solution is transferred to 2YT / Carb50 / Kan25 liquid medium, placed in a 37°C shaker, cultured overnight, and the supernatant is harvested the next day. 1 / 4 volume of PEG / NaCl solution is added to precipitate the phage, and then resuspended in PBT solution. After dilution, the phage display immune antibody library is obtained (stored at -80°C for future use);

[0047] d. Count the number of clones on the LB / Carb50 plate obtained in step c. The library capacity of the camel antibody library Lib CD73Camel is 9.23×10 9 20 monoclonal clones were randomly picked from each plate for sequencing, and the results showed that the VHH insertion efficiency of the camel antibody library Lib CD73Camel was 90%.

[0048] (4) 5 μg / mL human CD73 recombinant protein was added to a 96-well plate (100 μL / well) and coated at 4°C overnight; NEB5αF' Escherichia coli was streaked on a 2YT / Tet10 plate and cultured overnight in an incubator at 37°C; the next day, NEB5αF' monoclonal was picked from the 2YT / Tet10 plate and added to 3 mL 2YT / Tet10 liquid culture medium, and the bacteria were grown at 37°C until OD600 = 0.8; at the same time, the antigen supernatant of the 96-well plate was removed, 200 μL of 1% BSA was added to each well for blocking, and 200 μL of 1% BSA was added to the blank well as a negative control well, and the plate was placed on a 3D rotary shaker at room temperature for 2 h; the supernatant of the protein well and the control well was removed, and 200 μL PT was used for washing, and 100 μL of the phage antibody library obtained in step (3) was added to each well, and the cells were placed on a 3D rotary shaker at room temperature for 2 h; the supernatant of the protein well and the control well was removed and washed with 200 μL of PT; 100 μL of 100 mM HCl was added to the wells and the cells were placed at room temperature for 5 min; the supernatant was aspirated and added to a 1.5 mL centrifuge tube, and 1 M Tris-HCl was used to neutralize the mixture, which was added to a centrifuge tube containing 1 mL of NEB5αF' bacteria, and the mixture was shaken at 37°C for 1 h; 20 μL of the culture medium in the centrifuge tube was taken and respectively subjected to 10 -5 , 10 -6 , 10 -7 The culture medium was diluted and plated on LB / Carb50 culture plates and placed in a 37°C biochemical incubator overnight; 1 μL of helper phage M13K07 (final concentration of 10 10 / mL), shake at 37°C, culture for 1 hour, and then transfer the culture medium to 35mL2YT / Carb50 / Kan25 culture medium, place it in a shaker, and culture it overnight at 37°C. The phages were collected to form the antibody library for each round; the above operation was repeated for 2 rounds until phage enrichment occurred, and the number of colonies in the antigen-binding wells on the LB / Carb50 culture plate was 100 times that of the negative control wells, indicating successful enrichment; the clones in the enrichment round were picked and expanded in 96 deep-well plates. After centrifugation, the supernatant was used for Phage ELISA screening, and the OD value of binding to human CD73 recombinant protein was selected: the clones with an OD value of binding to the blocking solution>2 were defined as positive clones, and sequencing and alignment were performed to obtain unique sequences, as shown in Table 1.

[0049] Table 1 Affinity ranking of nanobodies obtained by biopanning

[0050]

[0051] Experimental Example 1

[0052] Eukaryotic expression of nanobodies:

[0053] The three sequences obtained in Example 1 were expressed in eukaryotic cells using the following experimental steps:

[0054] (1) The VHH fragments of the three sequences were amplified by PCR, and the VHH fragments were inserted into the eukaryotic expression vector pFcIG containing the hFc tag using the enzyme digestion and ligation method. The VHH fragments were then electroporated into the Escherichia coli trans5α host bacteria. After screening with bleomycin, the correct recombinant plasmids were obtained by sequencing the single clones. The host bacteria containing the recombinant plasmids were then expanded and an endotoxin-free plasmid was obtained using an endotoxin removal kit.

[0055] (2) HEK293F cells were cultured in serum-free medium; the recombinant expression plasmid obtained in step (1) was transferred into HEK293F cells for expression using polyplus suspension cell transfection reagent, and feed OPM-CHOPFF05 was added 24 and 72 hours after transfection. The supernatant was collected on the 5th day, and the antibody was separated and purified using Protein A agarose purification resin, and then replaced and stored in a PBS solution. The yields of the 293F cells transiently expressed VHH-hFc recombinant antibodies of the three sequences and the SDS-PAGE electrophoresis identification results are shown in Table 2.

[0056] Table 2 Results of eukaryotic transient expression of VHH-hFc recombinant antibodies

[0057]

[0058] As shown in Table 2, the yield of 293F cells transiently expressing the three sequences of VHH-hFc recombinant antibodies was 1.98-44.55 mg / L. SDS-PAGE electrophoresis identification showed that the band size of the anti-CD73 nanobody CD73-B4 was normal and the purity was >95%, while the other antibodies had non-specific bands.

[0059] Experimental Example 2

[0060] The affinity EC50 of the anti-CD73 nanobody obtained in Example 1 to human and monkey CD73 recombinant proteins:

[0061] The affinity of the three sequences of VHH-hFc recombinant antibodies to the human CD73 extracellular region recombinant protein was determined by ELISA: human or monkey CD73 extracellular region recombinant protein (his tag) was added to an ELISA 96-well plate at 200 ng / well and coated overnight at 4°C; the VHH-hFc recombinant antibody was serially diluted (0.01 μg / mL, 1 μg / mL, 10 μg / mL) and reacted with the antigen by ELISA. HRP-labeled anti-hIgG1 Fc secondary antibody was used for color development, and the absorbance at 450 nm was measured using a microplate reader. The results are shown in Table 3.

[0062] Table 3 ELISA binding EC50 of VHH-hFc recombinant antibodies to human CD73

[0063]

[0064] As can be seen from Table 3, the affinity of the three anti-CD73 nanoantibodies (structure: VHH-hFc) to the human CD73 extracellular region recombinant protein is EC50 = 0.22-20.70 μg / mL, and only clone CD73-B4 binds to the monkey CD73 extracellular region recombinant protein with an affinity of EC50 = 0.16 μg / mL.

[0065] The following are the amino acid sequences of the framework regions FR, complementarity determining regions CDR, and the amino acid sequence of the Nanobody.

[0066] The amino acid sequence of the Nanobody is as follows: framework region FR1, complementarity determining region CDR1, framework region FR2, complementarity determining region CDR2, framework region FR3, complementarity determining region CDR3, framework region FR4.

[0067] FR1: QVQLVESGGGSVQAGGSLRLSCAASGNTTS (SEQ ID NO: 1),

[0068] CDR1: SACMA (SEQ ID NO: 2),

[0069] FR2: WFRQTPGNEREGVA (SEQ ID NO: 3),

[0070] CDR2: AIAPVGSAGNANYLDSVKG (SEQ ID NO: 4),

[0071] FR3: RFTISRDNAKNTLYLQMNSLIPGDTAMYYCAA (SEQ ID NO: 5), CDR3: RDSWWCLVSLKSEDYKY (SEQ ID NO: 6),

[0072] FR4: WGQGTQVTVSS (SEQ ID NO: 7);

[0073] FR1: QVKLVQSGGGSVQAGGSLLRLSCAASGDAFS (SEQ ID NO: 8),

[0074] CDR1: TYLMA (SEQ ID NO: 9),

[0075] FR2: WFRQAPGKEREGL (SEQ ID NO: 10),

[0076] CDR2: ASVIPGGGHTYYADSVKG (SEQ ID NO: 11),

[0077] FR3: RFALSRDAAAKTVYLQMDNLKPDDRAIYYCAA (SEQ ID NO: 12),

[0078] CDR3: RSRGGSWRFLNSNDYDY (SEQ ID NO: 13),

[0079] FR4: WGQGTQVAVSS (SEQ ID NO: 14);

[0080] FR1: QVKLVESGGGSVQAGGSLRLSCAASGYAYS (SEQ ID NO: 15),

[0081] CDR1: RYCMG (SEQ ID NO: 16),

[0082] FR2: WFRQAPGKEREGVA (SEQ ID NO: 17),

[0083] CDR2: GIYTSNGATAYAVSVTG (SEQ ID NO: 18),

[0084] FR3: RFTISQDNAILLLQMNSLKPEDTAMYYCAA (SEQ ID NO: 19), CDR3: DRRLSASWCYARLHLGLNY (SEQ ID NO: 20),

[0085] FR4: RGQGTQVTVSS (SEQ ID NO: 21).

[0086] Nanobody CD73 - B4:

[0087] QVQLVESGGGSVQAGGSLRLSCAASGNTTSSACMAWFRQTPGNERE GVAAIAPVGSAGNANYLDSVKGRFTISRDNAKNTLYLQMNSLIPGDTAM YYCAARDSWWCLVSLKSEDYKYWGQGTQVTVSS (SEQ ID NO: 22); <​​​​QVKLVQSGGGSVQAGGSLRLSCAASGDAFSTYLMAWFRQAPGKER EGLASVIPGGGHTYYADSVKGRFALSRDAAAKTVYLQMDNLKPDDRAIY YCAARSRGGSWRFLNSNDYDYWGQGTQVAVSS(SEQ ID NO: 23);

[0090] Nanobody CD73-E8:

[0091] QVKLVESGGGSVQAGGSLRLSCAASGYAYSRYCMGWFRQAPGKER EGVAGIYTSNGATAYAVSVTGRFTISQDNAILLLQMNSLKPEDTAMYYCA ADRRLSASWCYARLHLGLNYRGQGTQVTVSS(SEQ ID NO: 24).

Claims

1. An anti-CD73 nanobody, characterized in that The nanobody comprises a framework region FR and a complementarity determining region CDR, wherein the complementarity determining region CDR comprises a complementarity determining region CDR1, a complementarity determining region CDR2 and a complementarity determining region CDR3, wherein The amino acid sequence of the complementary determining region CDR1 is shown in SEQ ID NO: 9, The amino acid sequence of the complementary determining region CDR2 is shown in SEQ ID NO: 11, The amino acid sequence of the complementarity determining region CDR3 is shown in SEQ ID NO:

13.

2. The anti-CD73 nanobody according to claim 1, characterized in that The frame region FR includes a frame region FR1, a frame region FR2, a frame region FR3 and a frame region FR4, wherein: The amino acid sequence of the framework region FR1 is shown in SEQ ID NO: 8, The amino acid sequence of the framework region FR2 is shown in SEQ ID NO: 10, The amino acid sequence of the framework region FR3 is shown in SEQ ID NO: 12, The amino acid sequence of the framework region FR4 is shown in SEQ ID NO:

14.

3. The anti-CD73 nanobody according to claim 1, characterized in that The amino acid sequence of the anti-CD73 nanobody is shown in SEQ ID NO:

23.

4. A DNA molecule, characterized in that Encodes the anti-CD73 nanobody according to any one of claims 1 to 3.

5. An expression vector, characterized in that Comprising the DNA molecule according to claim 4.

6. A host cell, characterized in that The invention also comprises the expression vector according to claim 5.

7. Use of the anti-CD73 nanobody according to any one of claims 1 to 3 in the preparation of a CD73 molecule detection reagent.

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