CN113061177B

CN113061177B - CD73 enzyme activity related antigen epitope and preparation method of specific antibody aiming at epitope

Info

Publication number: CN113061177B
Application number: CN202011634625.5A
Authority: CN
Inventors: 黄建; 朱永良; 江舟; 吴敏瑾
Original assignee: Hangzhou Angkeniu Biotechnology Co ltd; Zhejiang University ZJU
Current assignee: Hangzhou Angkeniu Biotechnology Co ltd; Zhejiang University ZJU
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2022-07-01
Anticipated expiration: 2040-12-31
Also published as: CN113061177A

Abstract

The invention relates to the field of medicines, and discloses an epitope related to CD73 enzyme activity and a preparation method of a specific antibody aiming at the epitope. The epitope related to the enzyme activity of the CD73 comprises an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2, or an amino acid sequence with homology of more than 90 percent with the amino acid sequence. The amino acid sequence of the epitope has strong non-substitutability, and sufficient polypeptide can be obtained by utilizing a polypeptide synthesis method without producing a large amount of recombinant protein, so that experimental materials are saved, and the antigen preparation time is shortened; the epitope specific antibody prepared by the epitope can effectively inhibit the activity of CD73 enzyme and stimulate lymphocytes to release interferon, plays an anti-tumor role, and has wide application prospect and practical value.

Description

CD73 enzyme activity related antigen epitope and preparation method of specific antibody aiming at epitope

Technical Field

The invention relates to the field of medicines, in particular to an epitope related to CD73 enzyme activity and a preparation method of a specific antibody aiming at the epitope.

Background

A great deal of research is currently underscoring the important role of tumor microenvironment conditions in tumor progression and its important impact on tumor treatment outcome. Under the action of CD73 and CD39, ATP in tumor tissues is converted into adenosine, so that the depletion of an immunostimulating factor ATP in a tumor microenvironment and the over-expression of an immunosuppressive factor adenosine are caused, and optimal conditions are provided for the proliferation of tumor cells. It has been shown that overexpression of adenosine is associated with upregulation of CD73 on the surface of cancer cells and is dependent on upregulation of CD 73. In addition, the CD73 not only can increase the production of adenosine so as to inhibit anti-tumor reaction, but also can promote various malignant behaviors such as angiogenesis, cancer cell invasion and metastasis, and the like, so that the molecule has potential value as a cancer treatment target.

CD73, also known as extracellular-5 '-nucleotidase (5' -NT), is a cell surface glycosylated phosphatidylinositol-anchored glycoprotein consisting of 548 amino acids and having a molecular weight of about 61 kDa. The N end of the N-terminal provides a binding site for two catalytic divalent metal ions, and the C end of the N-terminal provides an AMP binding site, so that AMP generated by ATP and ADP catalyzed by CD39 can be dephosphorylated into adenosine.

In view of the importance of CD73 function, various studies have explored blocking of the anti-tumor effects of CD73 by different approaches. The small molecule compound APCP can specifically inhibit CD73, and is easy to obtain and well tolerated, so that the small molecule compound APCP is an attractive anticancer drug. However, there are some limitations to the use of APCP, such as poor blocking of CD73 enzyme activity by in vivo levels of APCP, and its half-life and biodistribution in vivo are not well understood. Therefore, it is crucial to design and develop CD73 inhibitors that are not limited by the above, and anti-CD 73 antibodies may be an effective alternative therapeutic approach.

At present, the anti-CD 73 antibody is prepared by taking a recombinant expressed CD73 full-length protein or an extracellular region as a target antigen, however, the recombinant proteins possibly have protein conformation difference with natural protein and also have a large number of nonspecific or activity-unrelated dominant antigen epitopes, so that the antibody obtained by screening may have cross reaction with other proteins, most antibodies only have binding capacity with CD73 protein but lack the capacity of blocking the enzyme activity, the possibility of drug formation is reduced, and a large number of functional experiments and epitope identification are needed to screen the anti-CD 73 antibody with therapeutic effect. The epitope specific antibody prepared according to the peptide segment of the active center of the CD73 enzyme can obviously improve the screening efficiency of the functional antibody and reduce the later stage cloning identification cost, and the prepared epitope specific antibody can effectively inhibit the activity of the CD73 enzyme and stimulate lymphocytes to release interferon, plays an anti-tumor role and has wide application prospect and practical value.

Disclosure of Invention

In order to solve the technical problems, the invention provides an epitope related to the enzyme activity of CD73 and a preparation method of a specific antibody aiming at the epitope. The epitope is used for preparing a specific antibody, the screening efficiency of a functional anti-CD 73 monoclonal antibody can be greatly improved, the cost of later-stage cloning identification is reduced, the prepared epitope specific antibody can effectively inhibit the activity of CD73 enzyme and stimulate lymphocytes to release interferon, the anti-tumor effect is achieved, and the epitope specific antibody has wide application prospect and practical value.

The specific technical scheme of the invention is as follows:

the invention discloses an epitope related to the enzyme activity of CD73, which comprises an amino acid sequence shown as SEQ ID NO. 1 or more than 90 percent of homology with the amino acid sequence.

Secondly, the invention discloses another epitope related to the enzyme activity of CD73, which comprises an amino acid sequence shown as SEQ ID NO. 2 or more than 90 percent of homology with the amino acid sequence.

The inventor finds that the amino acid sequences of SEQ ID NO 1 and SEQ ID NO 2 have strong irreplaceability, and the lack of any amino acid at the N end or the C end of the amino acid sequences can cause that the blocking degree of the amino acid sequences of the two specific antibodies (5A 4 antibody and 8-5 antibody obtained after mice are immunized by the SEQ ID NO 1 and the SEQ ID NO 2) is obviously reduced, which indicates that the amino acid sequences of the two specific antibodies respectively have homology of more than 90 percent (91.7 percent and 92.9 percent) with the SEQ ID NO 1 and the SEQ ID NO 2.

Thirdly, the invention discloses a preparation method of a specific antibody aiming at the antigen epitope, which comprises the following steps: (1) synthesizing an antigenic peptide comprising said antigenic epitope;

(2) immunizing animals by using the antigen peptide synthesized in the step (1) to obtain hybridoma cells, and collecting culture supernatant;

(3) and (3) screening, identifying and purifying the supernatant collected in the step (2) to obtain a specific antibody aiming at the epitope related to the enzyme activity of the CD 73.

Preferably, the specific process of step (1) is as follows: and synthesizing the polypeptide of the epitope by adopting a polypeptide synthesis technology, coupling the polypeptide with a carrier protein, namely Bovine Serum Albumin (BSA), by using a bifunctional coupling agent, namely EDC, and identifying the coupling efficiency by using High Performance Liquid Chromatography (HPLC) after removing free polypeptide to obtain the antigenic peptide.

Preferably, in step (2), the specific process of immunizing animals is as follows: injecting subcutaneously at multiple points at a thin and loose part of the skin, wherein the immunization comprises first injection of Freund complete adjuvant and antigen 1:1 by mixing, intensive injection of Freund incomplete adjuvant and antigen 1:1 by mixing for 3-4 times, and direct excitation injection of the last antigen.

Preferably, the immunized animal is a mouse, rat, rabbit, goat, sheep, and alpaca.

Preferably, in step (3), the screening process is as follows: coupling the synthetic polypeptide in the step (1) with a carrier protein-hemocyanin (KLH) by using a bifunctional coupling agent EDC, and performing ELISA screening by using the obtained polypeptide-KLH conjugate.

Fourthly, the invention discloses a specific antibody aiming at the epitope related to the enzyme activity of CD73, which is obtained by the preparation method.

By the method of the invention, two specific antibodies respectively aiming at the epitope SEQ ID NO. 1 and SEQ ID NO. 2 are obtained. The specific antibody aiming at the SEQ ID NO. 1 can efficiently inhibit the activity (the inhibition rate is more than or equal to 90%) of CD73 enzyme hydrolyzing AMP to generate adenosine, effectively reduce the adenosine concentration in a tumor microenvironment, and further remodel an immunosuppression microenvironment mediated by the adenosine; the specific antibody aiming at the SEQ ID NO. 2 can stimulate lymphocytes to release interferon, plays roles of immunoregulation and tumor resistance, and has wide application prospect and practical value in treating tumors with high expression of CD73 or high concentration adenosine and autoimmune diseases.

Preferably, the specific antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a nanobody or an antibody fragment derived from an animal.

Sixth, the present invention discloses a pharmaceutical composition comprising the specific antibody and a pharmaceutically acceptable carrier.

Preferably, the pharmaceutical composition further comprises oxaliplatin.

Seventh, the present invention discloses an application of the specific antibody in the preparation of a diagnostic reagent or kit for diagnosing a tumor highly expressing CD73 or high concentration of adenosine, or an autoimmune disease highly expressing CD73 or high concentration of adenosine.

Preferably, the tumor is breast cancer, colorectal cancer, gastric cancer, prostate cancer or lung adenocarcinoma positive for CD73 expression.

Eighth, the present invention discloses the use of the specific antibody or the pharmaceutical composition in the preparation of a pharmaceutical preparation, wherein the pharmaceutical preparation is used for the treatment of tumors highly expressing CD73 or high concentration of adenosine, or autoimmune diseases highly expressing CD73 or high concentration of adenosine.

Preferably, the pharmaceutical formulation comprises the specific antibody and a pharmaceutically acceptable carrier.

Preferably, the pharmaceutical formulation comprises an antibody specific for the first epitope and oxaliplatin.

The 5A4 antibody aiming at the first epitope and the chemotherapeutic drug oxaliplatin have synergistic anti-tumor effect, and the 5A4 antibody can effectively inhibit local microenvironment adenosine aggregation caused by the oxaliplatin.

Compared with the prior art, the invention has the following advantages:

(1) the CD73 enzyme activity related antigen epitope provided by the invention is a linear epitope, the amino acid sequence of the linear epitope has strong non-substitutability, and sufficient polypeptide can be obtained by utilizing a polypeptide synthesis method without producing a large amount of recombinant protein, so that experimental materials are saved, and the antigen preparation time is shortened;

(2) the antibody prepared by immunizing an animal by utilizing the CD73 enzyme activity related epitope polypeptide has equivalence in affinity with the antibody prepared by immunizing an animal by utilizing the CD73 full-length recombinant protein, but because the antibody does not contain other non-activity related dominant antigen epitopes of the CD73 protein, the specificity is stronger, and the screening efficiency is higher;

(3) the specific antibody aiming at the CD73 enzyme activity related epitope SEQ ID NO. 1 can efficiently inhibit the activity of CD73 enzyme for hydrolyzing AMP to generate adenosine (the inhibition rate is more than or equal to 90 percent), effectively reduce the concentration of the adenosine in a tumor microenvironment, and further remodel an immunosuppression microenvironment mediated by the adenosine;

(4) the specific antibody aiming at the CD73 enzyme activity related epitope SEQ ID NO. 2 can stimulate lymphocytes to release interferon, plays roles of immunoregulation and tumor resistance, and has wide application prospect and practical value in treating tumors with high expression of CD73 or high concentration adenosine and autoimmune diseases;

(5) the specific antibody aiming at the CD73 enzyme activity related epitope SEQ ID NO. 1 has synergistic and enhanced anti-tumor effect when being combined with chemotherapeutic drugs such as oxaliplatin and the like, and is a potential new clinical choice.

Drawings

FIG. 1 is a Docking Model of antibody binding to CD73 protein epitope; wherein, the figure (A) shows the combination mode of an antibody Fab segment and CD73 recognition epitope SEQ ID NO. 1; FIG. (B) is a graph showing the binding pattern of an antibody Fab fragment to the CD73 recognition epitope SEQ ID NO. 2;

FIG. 2 is a chart showing the statistics of ELISA absorbance values of different sequence polypeptides competitively blocking epitope from binding with 5A4 antibody and 8-5 antibody; wherein, the graph (A) is a statistical graph of ELISA light absorption values of different sequence polypeptides competitive blocking epitope and 5A4 antibody; FIG. B is a statistical chart of ELISA absorbance values of different sequence polypeptides competitively blocking epitope from binding with 8-5 antibody;

FIG. 3 is a peak diagram of flow binding rate of three human tumor cell lines highly expressing CD73 and 5A4 antibody;

FIG. 4 shows the flow-based assay results of the blocking of eukaryotic CD73 protein by the 5A4 antibody and the 8-5 antibody; wherein, the figure (A) is the flow detection result of the 5A4 antibody after being blocked by eukaryotic CD73 protein; FIG. B shows the flow detection results of the 8-5 antibody after blocking by eukaryotic CD73 protein;

FIG. 5 shows the binding of the 5A4 antibody to tumor tissue in tumor-bearing mice;

FIG. 6 is an evaluation of the in vitro inhibitory ability of the 5A4 antibody and the 8-5 antibody on the enzymatic activity of CD 73; wherein, the chart (A) is a chart of the HPLC detection result of MDA-MB-231 cell culture supernatant treated by 25 mug/mL 5A4 antibody, the area shown in the square box is the adenosine peak-off time point, and APCP, 8-5 antibody and 5A4 antibody are arranged in sequence from top to bottom; FIG. (B) is a statistical plot of inhibition of adenosine secretion by the 5A4 antibody, 8-5 antibody and APCP as a function of concentration gradient;

FIG. 7 is an evaluation of the in vivo inhibitory ability of the 5A4 antibody to the enzyme activity of CD 73; wherein, the graph (A) is a graph for detecting the adenosine content peak value in the tumor tissue after treatment by different dosing schedules by HPLC (upper: 5A4 antibody; middle: APCP; lower: isotype antibody); graph (B) is a statistical graph comparing the adenosine content of tumor tissue units in different treatment groups;

FIG. 8 is a statistical chart of the in vitro lymphokine interferon secretion measured by ELISA under different treatment conditions;

FIG. 9 shows CCK8 detecting MDA-MB-231 cell proliferation in vitro after incubation with 5A4 antibody and 8-5 antibody for 7 days;

FIG. 10 is a graph showing the inhibition of the growth of mouse transplantable tumors by the 5A4 antibody; wherein, after the homotypic antibody, APCP and 5A4 antibody treatment, each group of tumor-bearing mice; panel (B) is a photograph of a dissected subcutaneous graft after treatment with isotype antibody, APCP, 5A4 antibody; panel (C) is a statistical plot of growth curves of tumor volume as a function of time; panel (D) is a tumor weight statistic;

figure 11 shows that the 5a4 antibody produced a combined anti-tumor effect with oxaliplatin; wherein, the picture (A) shows that after homotype antibody, 5A4 antibody single drug, oxaliplatin single drug and 5A4 antibody are combined with oxaliplatin for treatment, subcutaneous transplantation tumor is stripped and photographed; panel (B) is a tumor weight statistic; panel (C) is a statistical plot of growth curves of tumor volume as a function of time; panel (D) is a graph of survival for four treatment groups of mice;

figure 12 is a graph of the inhibitory effect of the 5a4 antibody on adenosine production in tumor tissues following oxaliplatin treatment; wherein, the graph (A) is an adenosine HPLC peak graph (from top to bottom, homotypic antibody, oxaliplatin, 5A4 antibody and oxaliplatin +5A4 antibody are sequentially shown) of mouse tumor tissues of different treatment groups; the graph (B) is a statistical graph of adenosine content in tumor tissues per unit weight of mice of different treatment groups.

The blur of the writing in fig. 6(a), fig. 7(a), fig. 12(a) does not affect the understanding of the technical solution by those skilled in the art.

Detailed Description

The present invention will be further described with reference to the following examples.

The terminology used in the examples herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. It is intended that all such alterations and advantages be included in the invention, which occur to those skilled in the art, be considered as within the spirit and scope of the inventive concept, and that all such modifications and advantages be considered as within the scope of the appended claims and any equivalents thereof. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and biomaterials, if not specifically indicated, are commercially available.

Example 1: identification of epitope associated with CD73 enzyme activity

We simulated the spatial conformation of the binding of the epitope associated with the enzyme activity of CD73 to the antibody in a 3D environment by a method of bio-modeling Docking Model. (1) The crystal structure of NT5E (CD73) was obtained from the Program Database, and the protein IDs were 4H2F and 1CLY, respectively. (2) With the aid of Schrodingger 2015 software, the following treatments were performed on both protein structures: adding a side chain of an amino acid residue; adding a missing loop portion of the crystal structure; assignment of the protonation state of the amino acid residues and optimization of the overall protein structure under application of the OPLS2005 force field. (3) Docking of IgG Fab to NT5E was calculated on-line using ZDCK (website http:// ZDOCK. umassemed. edu /), and NT5E was selected as the receptor and 1CLY was set as the ligand during docking. As shown in FIG. 1, it was found that two amino acid sequences shown in SEQ ID NO:1 (i.e., IEFDERGNVISS) and SEQ ID NO:1 (i.e., PEDPSIKADINKWR) in NT5E were epitope-binding sites associated with the enzyme activity by conformational change.

Example 2: preparation of monoclonal antibody against epitope associated with CD73 enzyme activity

1 synthetic polypeptide-Carrier protein conjugates

Two sections of polypeptides shown as SEQ ID NO 1 and SEQ ID NO 2 are synthesized respectively by adopting a polypeptide synthesis technology, and the purity is over 95 percent by HPLC detection. Directly reacting-COOH on the synthetic polypeptide with-NH on Bovine Serum Albumin (BSA) and hemocyanin (KLH) by using a bifunctional coupling agent EDC₂Coupling according to a molar ratio of 100:1, and identifying the coupling efficiency by HPLC after removing free polypeptide to obtain the polypeptide-BSA and polypeptide-KLH conjugate.

2 polypeptide-BSA conjugate immunization of mice

5 Balb/c mice weighing 18-22g and 4 weeks old were selected. Taking the polypeptide-BSA conjugate as an antigen, uniformly mixing the polypeptide-BSA conjugate with Freund's complete adjuvant 1:1, and pumping into an injector, wherein the first immunization concentration is 1mg/mL, and the immunization dose of a mouse is 0.1 mL/mouse; the antigen concentration of the secondary immune to the quaternary immune is 0.5mg/mL, the immunization dose is 0.1 mL/mouse, and the secondary immune to the quaternary immune is uniformly mixed with Freund incomplete adjuvant 1:1 and pumped into a syringe. The mice were injected subcutaneously in multiple spots on the back of the neck with the fully emulsified antigen-adjuvant mixture, and the second immunization was performed 21 days after the first immunization, with a subsequent immunization interval of 14 days.

3 mouse serum titer detection

And (3) carrying out orbital blood collection on the immunized mice, centrifuging at 10000rpm for 5min, and taking the supernatant to carry out ELISA (enzyme-linked immuno sorbent assay) for detecting the antibody titer. The polypeptide-BSA conjugate was diluted to 20. mu.g/mL with coating buffer, 100. mu.L of the conjugate was added to each well of a 96-well plate (Nunc, USA), overnight at 4 ℃, the liquid in the wells was discarded the next day, washed 4 times with 1 XTSST buffer, and patted dry. Each well was then blocked by filling with 1% BSA and incubated at 37 ℃ for 1h before patting dry. Serum was diluted in multiples and 100. mu.L was added to each well to provide negative control wells (plus 1% BSA), incubated for 1h at 37 ℃ and washed 4 times and blotted dry. Then 100. mu.L of diluted (1:3000) goat anti-mouse secondary antibody-HRP was added to each well, incubated at 37 ℃ for 30min, washed 4 times and patted dry. 50. mu.L of TMB solution A and 50. mu.L of TMB solution B were added to each reaction well, and the reaction was carried out at room temperature for about 10min, and then 100. mu.L of 2mol/L stop solution was added to terminate the reaction. The absorbance at 450nm was read using a microplate reader (Molecular Device, USA), and the data was stored for analysis. As shown in Table 1, the serum antibody titers of 5 mice all reached 1:240000, and the mouse No. 5 with the highest titer was selected for the next fusion.

TABLE 1 serum titer test after immunization of mice

4 hybridoma cell fusion

After sacrifice by decapitation, spleens were aseptically exposed, squeezed out with forceps, blown up and collected into 50mL centrifuge tubes. The spleen cells and myeloma cells SP2/0 were then separated at 1:1 using an electrofusion apparatus (BTX ECM2001)The hybridoma cells were cultured in 2 XHAT medium and plated in 96-well cell culture plates at about 5X 10 cells per well⁵And (4) cells.

5ELISA clone screening

96-well plates (Nunc, USA) were coated with 20. mu.g/mL of the polypeptide-KLH conjugate overnight at 4 ℃, washed and blocked, hybridoma cell culture supernatants were added and incubated for 1h at 37 ℃, washed and patted dry. Goat anti-mouse secondary antibody-HRP was then added and incubated at 37 ℃ for 30min, washed and patted dry. And finally adding TMB color development solution, observing the result by naked eyes, and selecting positive clones. Hybridoma cells producing the high-affinity monoclonal antibody are obtained by 3 times of limiting dilution and ELISA screening, and are expanded and cultured after strain building. The collected cell supernatant was applied to a Protein G column (MabSelect Sure, GE), and the antibody was eluted with glycine, and immediately neutralized with 1M Tris to obtain the purified monoclonal antibody against the synthetic polypeptide. Through the screening process, two epitope specific monoclonal antibodies related to the enzyme activity of the CD73 are finally obtained: 5A4 and 8-5. The two monoclonal antibodies were sequenced and found to have the same sequence as the antibody obtained from the 2020112089878 patent by immunizing a mouse with the full-length recombinant antigen and designing the antibody for humanization.

Example 3: identification of recognition amino acid sequence of anti-CD 73 enzyme activity related epitope specific monoclonal antibody

(1) Wrapping a plate: diluting two-segment polypeptide-KLH conjugate of SEQ ID NO 1 or SEQ ID NO 2 to 2.5 mu g/mL by using a coating buffer solution, adding 100 mu L/well into an ELISA 96-well plate (Nunc company, USA), keeping overnight at 4 ℃, removing the solution in the well the next day, washing 3 times by using a 1 xTBST washing buffer solution, and patting dry;

(2) and (3) sealing: adding 100 mu L of 1% BSA to each well for blocking, incubating at 37 ℃ for 1h, and removing blocking solution;

(3) sample adding: adding 50 μ L/well of the above 5A4 or 8-5 hybridoma culture supernatant +50 μ L/well of 1% BSA or 50 μ L/well of the above 5A4 or 8-5 hybridoma culture supernatant +50 μ L/well of 1mg/mL naked peptide (amino acid sequence of different naked peptides in Table 2), setting 3 multiple wells each, incubating at 37 deg.C for 120min, patting to dry, washing 3 times with 1 × TBST washing buffer, patting to dry;

(4) adding an enzyme-labeled antibody: diluting goat anti-mouse secondary antibody-HRP (diluted with 1% BSA) at a ratio of 1:3000, adding 100 μ L/well into an ELISA plate hole, incubating at 37 ℃ for 60min, discarding the solution, washing with 1 XTSST washing buffer for 3 times, and patting to dry;

(5) adding a substrate solution for color development: adding 100 μ L of TMB substrate solution (solution A and solution B are prepared at a ratio of 1:1, and are ready for use) into each reaction hole, and reacting at 37 deg.C for 5 min;

(6) and (3) terminating the reaction: adding 100 mu L of 2mol/L sulfuric acid into each reaction hole, and stopping the reaction;

(7) reading a plate: the microplate was read in a microplate reader (Molecular Device, USA), and the data was saved and analyzed.

The results are shown in FIG. 2, PEP1-7 and PEP9-11 can block the combination of polypeptide IEFDERGNVISS-KLH conjugate and 5A4 monoclonal antibody to different degrees, and the blocking degree is gradually reduced along with the gradual reduction of the number of the amino acids at the N end and the C end of the naked peptide, which proves that the antigen recognition epitope of the 5A4 monoclonal antibody is SEQ ID NO 1; similarly, the PEP12-18 with the amino acids reduced one by one at the N end and the PEP19-23 with the amino acids reduced one by one at the C end gradually reduce the inhibition of the combination of the polypeptide PEDPSIKADINKWR-KLH conjugate and the 8-5 monoclonal antibody, and the antigen recognition epitope of the 8-5 monoclonal antibody is shown to be SEQ ID NO 2. Meanwhile, under the condition that the PEP1 and the PEP12 lack any amino acid at the N end or the C end, the blocking degree of the binding of the 5A4 monoclonal antibody and the 8-5 monoclonal antibody and the corresponding polypeptide-KLH conjugate is obviously reduced compared with that of the whole naked peptide, which indicates that the amino acid sequences recognized by the two monoclonal antibodies respectively have homology of more than 90 percent (91.7 percent and 92.9 percent) with the SEQ ID NO:1 and the SEQ ID NO: 2.

TABLE 2 amino acid sequences of different naked peptides

PEP1	IEFDERGNVISS	PEP12	PEDPSIKADINKWR
				PEP2	EFDERGNVISS	PEP13	EDPSIKADINKWR
PEP3	FDERGNVISS	PEP14	DPSIKADINKWR
				PEP4	DERGNVISS	PEP15	PSIKADINKWR
PEP5	ERGNVISS	PEP16	SIKADINKWR
				PEP6	RGNVISS	PEP17	IKADINKWR
PEP7	GNVISS	PEP18	KADINKWR
				PEP8	NVISS	PEP19	PEDPSIKADINKW
PEP9	IEFDERGNVIS	PEP20	PEDPSIKADINK
				PEP10	IEFDERGNVI	PEP21	PEDPSIKADIN
PEP11	IEFDERGNV	PEP22	PEDPSIKADI
						PEP23	PEDPSIKA

Example 4: in vitro detection of binding situation of anti-CD 73 enzyme activity related epitope specific monoclonal antibody and target protein, selecting high expression CD73 breast cancer MAD-MB-231 cell line, lung adenocarcinoma H385 cell line and colon cancer HCT116 cell line in logarithmic growth phase, adding 1mL pancreatin for digestion counting, and counting according to 5 × 10⁵One/tube was dispensed into an EP tube, centrifuged at 3500rpm for 5min and the supernatant carefully aspirated. Adding 10 mu g/mL of prepared antibody 5A4 into the experimental group according to 100 mu L/tube, re-suspending the cell precipitate, incubating for 30min at room temperature in the dark, and simultaneously setting a negative control group and an isotype control group. After incubation was complete, cells were washed 2 times with 1mL PBS per tube. Then, 100. mu.L/tube of secondary antibody diluted 1:1000 (PBS + 2% FBS solution) was added, and the cell pellet was resuspended, protected from light at room temperatureIncubate for 30 min. After completion of incubation, the supernatant was carefully aspirated after 2 PBS washes, resuspended by adding 200. mu.L of stabilizing buffer, and immunofluorescence was detected using a multicolor cytometric flow meter (Beckmann Coulter, USA). As a result, as shown in fig. 3, the 5a4 antibody was highly binding to CD73 under the cell surface steric structure and was universal.

Furthermore, we performed binding blocking experiments by incubating 5A4 antibody and 8-5 antibody with 10. mu.g/mL eukaryotic CD73 protein for 30min, and performing the above flow assay in 4 experimental groups of 5A4, 8-5, 5A4 (after incubation) and 8-5 (after incubation). As a result, as shown in FIG. 4, the binding rates of both antibodies after blocking were reduced by about 90%, demonstrating that the binding of both antibodies to CD73 was specific.

Example 5: in vivo detection of binding condition of epitope specific monoclonal antibody related to CD73 enzyme activity and target protein, 5 female Balb/c nude mice with age of 4 weeks are selected and bred in barrier for 1 week, MDA-MB-231 cells in logarithmic growth phase are digested and washed for 2 times, and then the total number of the cells is 1 × 10⁷Cell inoculum count/cell required was retained. Resuspending the cell sediment by PBS, adding equal volume of matrigel, mixing uniformly, inoculating the suspension to the left underarm fat pad of the mouse according to the dose of 200 mu L/mouse, and establishing an in-situ inoculation model of the mouse breast cancer, namely an MDA-MB-231 tumor-bearing Balb/c nude mouse.

The 5A4 antibody was fluorescently labeled using Alexa Fluor 647 antibody labeling kit (Sammer Feishel technologies, Inc., USA) and the tumor-bearing mice were injected tail vein at a dose of 150. mu.L/mouse. The mice are subjected to isoflurane inhalation anesthesia after 24h, the anesthetized mice are placed into a dark room with the tumor side facing upwards, a living body fluorescence imaging system (Saimer Feishell science and technology Co., Ltd. in the United states) is used for photographing (the excitation wavelength is 684nm, the emission wavelength is 707nm), and the anesthesia photographing is repeated every 24h until the fluorescence of the mice completely disappears.

The results are shown in fig. 5, the mouse transplanted tumor area shows high signal obviously, but no signal is shown in other tissues, which suggests that the fluorescein-conjugated 5a4 antibody specifically binds to the tumor area, i.e. the epitope-specific antibody related to the activity of CD73 enzyme can highly bind to the CD73 molecule highly expressed in human tumor tissues in vivo. Meanwhile, according to the detection condition of drug metabolism, 5 mice can be obviously combined after 24 hours, and the mice have complete drug metabolism till the 5 th day.

Example 6: biological function verification of anti-CD 73 enzyme activity related epitope specific monoclonal antibody

1 in vitro experiments

For the CD73 protein, the most important biological function is the enzymatic hydrolysis of AMP to adenosine, i.e., enzymatic activity. Therefore, we tested adenosine secretion in the culture supernatant of tumor cell lines highly expressing CD73 after treatment with 5A4 antibody and 8-5 antibody by High Performance Liquid Chromatography (HPLC). MDA-MB-231 cells in logarithmic growth phase were counted by digestion at 2X 10⁴And 24-pore plates are paved per pore. After 6h of cell attachment, the supernatant was aspirated, 5A4, 8-5, APCP and isotype IgG control in L-15 medium were added, and concentration gradients of 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.13. mu.g/mL, 1.56. mu.g/mL, 0.78. mu.g/mL and 0.39. mu.g/mL were set, respectively. After incubation for 2h at 37 ℃ the supernatant was aspirated, washed 1 time with PBS, and incubated for 30min at 37 ℃ with 200. mu.L of 500. mu. mol/L AMP in serum-free L-15. Collecting culture supernatant, centrifuging at 2000rpm for 3min, shaking 100 μ L of supernatant with methanol at 1:1, mixing, centrifuging at 15000g for 10min, sucking 200 μ L of supernatant, adding into liner tube, and detecting adenosine level by high performance liquid chromatography (Shimadzu corporation, Japan).

As shown in FIG. 6, at a concentration of 25. mu.g/mL, both the 5A4 antibody and the 8-5 antibody can significantly inhibit the secretion of adenosine, with the inhibition rates of 100% and 85%, respectively, which are significantly higher than that of the commercial CD73 small molecule inhibitor APCP. Meanwhile, the two antibodies are concentration-dependent on the inhibition of MDA-MB-231 cell adenosine secretion, which indicates that the antibodies can not only bind to CD73, but also have the function of enzyme activity inhibition.

2 in vivo experiments

The MDA-MB-231 tumor-bearing Balb/c nude mice obtained in example 5 were grouped, and the intraperitoneal injection administration was started 10 days after inoculation (subcutaneous tumor was visible). The 5A4 antibody and the antibody of the same type are administrated 8 times, 400 mg/injection is given for the 1 st time, and 200 mg/injection is given for the other 7 times respectively on the 4 th, 7 th, 10 th, 13 th, 16 th, 19 th, 22 th and 25 th days; APCP was administered 1 time daily at 400. mu.g/mouse. After the treatment is finished, the tumor is weighed and photographed, and 1mL of perchloric acid is added to be repeatedly ground until the tumor tissue is in a homogenate state. After centrifugation at 15000g for 10min, the supernatant was aspirated, filtered through a 40 μm sieve, and the content of adenosine (. mu.mol) in the filtrate was measured as a sample by the above HPLC method, and the content of adenosine (. mu.mol/g) per unit weight was calculated.

The comparison results are shown in fig. 7, compared with the antibody of the same type, the small molecule inhibitor APCP can reduce the production of adenosine in tumor tissues, while the 5a4 antibody can further significantly reduce the secretion of adenosine, which proves that the epitope-specific antibody related to the CD73 enzyme activity can inhibit the secretion of adenosine at the tissue level in vivo.

Example 7: in vitro validation of lymphocyte stimulating ability of monoclonal antibody specific for epitope associated with enzymatic activity of anti-CD 73 We examined the ability of 8-5 antibody to stimulate lymphocytes to produce interferon-gamma by means of a mixed lymphocyte reaction. PBMCs isolated from peripheral blood of two healthy persons were prepared, counted, and equal amounts of cells were mixed and plated on a U-bottom 96-well plate (20 ten thousand/100. mu.L/well). After 6h of cell clustering, medicine is added, four groups of isotype IgG, 5A4, 8-5 and 5A4+8-5 are set, the concentration gradients are respectively 50 mug/mL, 25 mug/mL, 12.5 mug/mL and 6.25 mug/mL, and each group is provided with 3 multiple holes. After incubation for 48h at 37 ℃, centrifuging at 2000rpm for 3min, carefully sucking the supernatant as a sample to be detected, and performing operation according to the specification of a human IFN-gamma ELISA detection kit (Biolegend company, USA), wherein the result is shown in FIG. 8, 8-5 antibody can obviously stimulate lymphocyte to secrete gamma-interferon compared with the same type of antibody.

Example 8: detection of Effect of anti-CD 73 enzyme Activity-related epitope-specific monoclonal antibody on tumor cell proliferation 7 time gradient sets were set in advance, and 10% of MDA-MB-231 cells in logarithmic growth phase were used⁴Concentration digestion counts per 100. mu.L/well were plated in 96-well plates. Respectively adding isovolumetric homotypic antibody, 5A4 antibody and 8-5 antibody of 10 mug/mL after the cells are attached to the wall, and CO at 37 DEG C₂And (5) incubation in an incubator. Every 24h, the following operations are carried out on the corresponding groups: mu.L of CCK-8 solution was carefully added to each well using a pipette, incubated in an incubator for 2h, and after completion, the 96-well plate was removed and mixed on a shaker for 1min, and absorbance at 450nm was measured using a multifunctional detection microplate reader (Thermo Fisher, USA).

The results are shown in FIG. 9, compared with the same type of antibody, the 5A4 antibody can obviously inhibit the proliferation of MDA-MB-231 cells, and the 8-5 antibody has a slightly lower degree of inhibition, which suggests that the epitope specific antibody related to the CD73 enzyme activity has the function of inhibiting the proliferation of tumor cells in vitro.

Example 9: detecting the effect of anti-CD 73 enzyme activity-related epitope specific monoclonal antibody on in vivo tumor growth we performed 3 times per week tumor growth monitoring on MDA-MB-231 tumor-bearing Balb/c nude mice treated with different drugs in the in vivo experiment of example 6, measured the major and minor diameters of the tumor, calculated the volume (formula: volume ═ major diameter × minor diameter/2) and recorded, and plotted the tumor volume growth curve using GraphPad Prism software. Meanwhile, the subcutaneous transplantation tumor is stripped, photographed and weighed for recording. As shown in fig. 10, from the tumor volume, both APCP and 5a4 antibodies inhibited tumor growth to some extent, and 5a4 antibody inhibited APCP more significantly than the same antibody; from the tumor weight, the 5a4 antibody exhibited better inhibitory effect than APCP, but the difference was not statistically significant. These suggest that the epitope-specific antibody related to the CD73 enzyme activity has an inhibitory effect on tumor growth in vivo.

Example 10: in order to evaluate the combined effect of the anti-CD 73 enzyme activity related epitope specific monoclonal antibody and a chemotherapeutic drug, namely oxaliplatin (high immunogenicity and capable of generating extracellular ATP), the combination of the 5A4 antibody and the chemotherapeutic drug, namely Balb/c nude mice with tumor bearing MDA-MB-231 obtained in the example 5 are grouped, four groups, namely homotypic antibody, 5A4 antibody, oxaliplatin, 5A4 antibody and oxaliplatin, are respectively arranged, and the intraperitoneal injection administration is started 10 days after inoculation (subcutaneous tumors can be touched). The 5A4 antibody and the antibody of the same type are administrated 8 times, 400 mg/injection is given for the 1 st time, and 200 mg/injection is given for the other 7 times respectively on the 4 th, 7 th, 10 th, 13 th, 16 th, 19 th, 22 th and 25 th days; oxaliplatin was co-administered 2 times, 10mg/kg on days 5 and 14, respectively. During the treatment, tumor growth was measured 3 times per week, tumor major and minor diameters were measured, and the volume (formula: volume ═ major diameter × minor diameter/2) was calculated and recorded. When the tumor volume reaches 2000mm³Or the mice are sacrificed when the tumor dies, the survival rate is calculated,tumors were weighed, photographed, and tumor volume growth and survival profiles were plotted using GraphPad Prism software.

The results are shown in figure 11, where 5a4 antibody alone inhibited tumor growth but tumor regression was less than that of oxaliplatin-treated group compared to the isotype antibody-treated group, while the combination treatment of 5a4 antibody and oxaliplatin further inhibited tumor. The same trend in survival was seen, with the 5a4 antibody in combination with oxaliplatin significantly prolonging the survival time of the mice.

Furthermore, when a large amount of chemotherapy drugs kill local tumor cells, a large amount of ATP is released after cell damage and necrosis, and then adenosine is generated by hydrolysis, and the large amount of locally accumulated adenosine can cause drug resistance of the chemotherapy drugs and inhibition of immune cell functions. We repeatedly ground the tumors exfoliated after the above different groups of treatments to a tissue homogenized state, centrifuged horizontally at 15000g for 10min, aspirated the supernatant, filtered through a 40 μm filter, the filtrate was used as a sample to detect adenosine content (. mu.mol) by the aforementioned HPLC method, and the adenosine content (. mu.mol/g) per unit weight was calculated.

The result is shown in fig. 12, compared with the control group (same type antibody), the content of adenosine in the tumor tissue of the mice treated by oxaliplatin is obviously increased, and the content of adenosine in the tissue level is obviously reduced compared with the prior art after the mice are combined with the 5A4 antibody, which indicates that the 5A4 antibody can effectively inhibit local microenvironment adenosine aggregation caused by chemotherapeutic drugs.

In conclusion, the invention provides an epitope related to the enzyme activity of CD73, and the amino acid sequence of the epitope comprises SEQ ID NO. 1 or SEQ ID NO. 2 or amino acid sequences with homology of more than 90 percent with the two. The method for preparing the antibody specific to the epitope comprises the following steps: (1) synthesizing an antigen-synthesized peptide comprising the above amino acid sequence; (2) immunizing animals by using the antigen synthetic peptide synthesized in the step (1) to obtain hybridoma cells, and collecting culture supernatant; (3) and (3) screening, identifying and purifying the supernatant collected in the step (2). The preparation method of the antibody can greatly improve the screening efficiency of the functional anti-CD 73 monoclonal antibody, reduce the cost of later-stage cloning identification, and the prepared epitope specific antibody can effectively inhibit the activity of CD73 enzyme and stimulate lymphocytes to release interferon, plays an anti-tumor role, and has wide application prospect and practical value.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art.

The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.

Sequence listing

<110> Zhejiang university

Hangzhou Angkeniu Biotechnology Co., Ltd.

<120> preparation method of CD73 enzyme activity related epitope and specific antibody aiming at epitope

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<170> SIPOSequenceListing 1.0

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<212> PRT

<213> extracellular-5 '-Nucleotidase (Ecto-5' -Nucleotidase)

<400> 1

Ile Glu Phe Asp Glu Arg Gly Asn Val Ile Ser Ser

1 5 10

<210> 2

<211> 14

<212> PRT

<213> extracellular-5 '-Nucleotidase (Ecto-5' -Nucleotidase)

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Pro Glu Asp Pro Ser Ile Lys Ala Asp Ile Asn Lys Trp Arg

1 5 10

Claims

1. An epitope peptide related to the enzyme activity of CD73 is characterized in that the amino acid sequence of the epitope peptide is shown in SEQ ID NO. 1.

2. An epitope peptide related to the enzyme activity of CD73, which is characterized in that the amino acid sequence of the epitope peptide is shown in SEQ ID NO. 2.

3. Use of the epitope peptide according to claim 1 or 2 for the preparation of a specific antibody against an epitope associated with the enzymatic activity of CD 73.

4. A preparation method of a specific antibody aiming at CD73 enzyme activity related epitope is characterized in that the amino acid sequence of the CD73 enzyme activity related epitope is shown as SEQ ID NO. 1 or SEQ ID NO. 2; the preparation method comprises the following steps:

(1) synthesizing an antigenic peptide comprising the epitope according to claim 1 or 2, and obtaining a polypeptide-BSA conjugate after conjugation with bovine serum albumin;

(2) immunizing animals by using the polypeptide-BSA conjugate synthesized in the step (1) to obtain hybridoma cells, and collecting culture supernatant;

5. Use of the epitope peptide according to claim 1 or 2 for the preparation of a pharmaceutical preparation for the treatment of tumors highly expressing CD 73.

6. The use of claim 5, wherein the pharmaceutical formulation comprises a specific antibody prepared using the epitope peptide and a pharmaceutically acceptable carrier.

7. The use of claim 5, wherein said pharmaceutical formulation comprises a specific antibody made using said epitope peptide and oxaliplatin.