CN111714642A

CN111714642A - Application of CD73 antibody-drug conjugate

Info

Publication number: CN111714642A
Application number: CN202010558833.5A
Authority: CN
Inventors: 余科; 刘亮
Original assignee: Fudan University
Current assignee: Bailisikang biomedical (Hangzhou) Co.,Ltd.; Fudan University
Priority date: 2020-06-18
Filing date: 2020-06-18
Publication date: 2020-09-29

Abstract

The invention belongs to the field of medicines, and relates to an application of a CD73 antibody-drug conjugate, in particular to an application of a CD73 antibody-drug conjugate in preparing a medicine for killing high-expression tumor cells in CD73 and activating a tumor immune microenvironment by inhibiting a CD73 adenylase pathway. Compared with the prior art, the invention proves the unique dual technical effects of the CD73-ADC, on one hand, the invention directly kills the high-expression tumor of CD73-, and on the other hand, the invention realizes the activation of immune microenvironment by inhibiting the CD73 adenylase pathway; the dual-superposition pharmacological mechanism has the characteristics of high efficiency and low toxicity, and provides a new strategy for treating refractory tumors.

Description

Application of CD73 antibody-drug conjugate

Technical Field

The invention relates to the field of medicine, in particular to application of a CD73 antibody-drug conjugate.

Background

In recent years, although targeted therapy and immunotherapy of malignant tumors have been significantly advanced, tumor therapy still faces challenges of low drug response rate and frequent secondary drug resistance. Recent studies have shown that tumor progression and drug resistance mechanisms often involve the interaction of tumors with the tumor immune microenvironment, and the adenylate enzyme CD73(NT5E) is a drug target of great interest in this field. CD73 consists of two identical 70kD subunits, anchored to the cell membrane surface by Glycosylphosphatidylinositol (GPI). High expression of CD73 has been found to be closely related to poor prognosis in a variety of tumors such as lung cancer, breast cancer, colorectal cancer, and the like. CD73 promotes tumorigenic immune escape by catalyzing AMP to produce a large amount of adenosine (Ado) that creates a tumor immunosuppressive microenvironment. The existing drug research targeting CD73 is mainly monoclonal antibody, and improves immune microenvironment by combining and interfering CD73 enzyme activity on the surface of immune cells.

However, in the course of the present application, the inventors speculate that the inhibitory effect of CD73 mab is limited when it is administered to a tumor or tumor environment where CD73 is highly expressed due to the limitations of long time intervals between mab administrations, low tissue absorption distribution rate, and reversibility of enzyme activity inhibition.

PCT patent application WO2019170131a1 discloses antibodies and antibody-drug conjugates (ADCs) targeting CD 73. The disclosed content shows that the CD73 antibody can efficiently and specifically bind to purified CD73 protein and CD73 on the surfaces of various tumor cells, blocks the catalytic activity of CD73 enzyme, has high affinity and low immunogenicity, and has a remarkable anti-tumor effect. And it is shown in the patent that CD73-ADC site-specifically coupled with monomethyyl auristatin E (MMAE) shows excellent in vitro and in vivo tumor killing effect by humanized monoclonal antibody CD 73.

However, the main strategy of the ADC research is to deliver cytotoxic agents in a targeted manner to kill tumors, and the above patent disclosure also only studies the function of the CD73 antibody-drug conjugate in directly killing tumor cells highly expressed in CD 73-. That is, in the current state of the art, the known function of CD73 antibody-drug conjugates is to directly eliminate highly expressed tumor cells of CD 73-.

Disclosure of Invention

The invention aims to provide application of a CD73 antibody-drug conjugate (hereinafter referred to as CD73-ADC) to expand the defect that the CD73 antibody-drug conjugate only has the function of directly killing highly-expressed tumor cells of CD 73-.

Theoretical research and experimental verification show that the CD73 antibody-drug conjugate can directly eliminate high-expression tumor cells in CD73-, and reduce adenosine pathways of other tumor cells, stromal cells and immune cells with abnormal expression of CD73, so that the tumor microenvironment is improved.

The research of the invention finds that the CD73 antibody-drug conjugate prepared by the site-specific coupling technology can partially or completely retain the adenylase inhibition activity exerted by the CD73 monoclonal antibody, and can directly eliminate high-expression tumor cells in CD73-, namely the research of the invention finds that the CD73 antibody-drug conjugate can realize the dual effect of overlapping the targeted cell poison and the adenylase inhibitor.

The purpose of the invention can be realized by the following technical scheme:

the invention provides an application of a CD73 antibody-drug conjugate, and firstly, the CD73 antibody-drug conjugate is applied to preparing a drug for activating a tumor immune microenvironment by inhibiting a CD73 adenylase pathway.

Further, the CD73 antibody-drug conjugate is used for preparing a drug for inhibiting the activity of free CD73 or cell membrane CD73 enzyme catalyzing the hydrolysis of adenosine phosphate (AMP) to generate adenosine (Ado).

In one embodiment of the invention, the CD73 antibody-drug conjugate is used for preparing the following drugs: the medicine activates tumor immune microenvironment by inhibiting CD73 adenylase pathway, and has no toxic effect on immune effector T lymphocyte.

In one embodiment of the invention, the CD73 antibody-drug conjugate is used for preparing the following drugs: the medicine activates a tumor immune microenvironment by inhibiting a CD73 adenylase pathway, does not show a toxic effect on immune effector T lymphocytes, and can effectively reverse the toxic and side effects of adenosine phosphohydrolysis to generate adenosine (AMP/Ado for short) on T lymphocyte proliferation and IFN-gamma secretion.

In one embodiment of the invention, the CD73 antibody-drug conjugate is used for preparing the following drugs: the medicine activates a tumor immune microenvironment by inhibiting a CD73 adenylase pathway, promotes Dendritic Cell (DC) maturation and enhances the activation effect of the DC on T lymphocytes.

In one embodiment of the invention, the CD73 antibody-drug conjugate is used for preparing the following drugs: the medicine activates a tumor immune microenvironment by inhibiting a CD73 adenylase pathway, promotes infiltration of immune effector cells, and reduces infiltration of immunosuppressive cells.

In one embodiment of the invention, the CD73 antibody-drug conjugate is used for preparing a drug for killing tumor cells highly expressed in CD73-, and activating a tumor immune microenvironment by inhibiting a CD73 adenylase pathway.

In one embodiment of the invention, the CD73 antibody-drug conjugate is used for preparing the following drugs: the medicine is a medicine which kills tumor cells highly expressed in CD 73-and simultaneously inhibits the activity of adenosine phosphohydrolysis generated by free CD73 or cell membrane CD73 enzyme.

In one embodiment of the invention, the CD73 antibody-drug conjugate is used for preparing the following drugs: the medicine can kill tumor cells highly expressed in CD73-, activate tumor immune microenvironment by inhibiting CD73 adenylase pathway, and has no toxic effect on immune effector T lymphocytes.

In one embodiment of the invention, the CD73 antibody-drug conjugate is used for preparing the following drugs: the medicine can kill tumor cells highly expressed in CD73-, activate tumor immune microenvironment by inhibiting CD73 adenylase pathway, has no toxic effect on immune T lymphocytes, and can effectively reverse the adenosine phosphohydrolysis process (AMP/Ado for short) to generate adenosine, proliferate T lymphocytes and secrete IFN-gamma.

In one embodiment of the invention, the CD73 antibody-drug conjugate is used for preparing the following drugs: the medicine can kill tumor cells highly expressed in CD73-, activate a tumor immune microenvironment by inhibiting a CD73 adenylase pathway, promote DC maturation and enhance the activation effect of dendritic cells on T lymphocytes.

In one embodiment of the invention, the CD73 antibody-drug conjugate is used for preparing the following drugs: the medicine can kill tumor cells highly expressed in CD73-, activate a tumor immune microenvironment by inhibiting a CD73 adenylase pathway, promote infiltration of immune effector cells and reduce infiltration of immunosuppressive cells.

In one embodiment of the invention, the CD73 antibody-drug conjugate is used for preparing the following drugs: the use of the CD73 antibody-drug conjugate for the preparation of a medicament comprising: the medicine can kill tumor cells highly expressed in CD73-, inhibit the activity of adenosine phosphate (AMP) catalyzed by free CD73 or cell membrane CD73 enzyme to generate adenosine (Ado), has no toxic effect on immune T lymphocytes, can effectively reverse the toxic and side effects of adenosine phosphate hydrolysis to generate adenosine on T lymphocytes proliferation and IFN-gamma secretion, simultaneously promotes DC maturation, enhances the activation effect of DC on T lymphocytes, promotes the infiltration of immune effector cells and reduces the infiltration of immune suppressor cells.

That is, the CD73 antibody-drug conjugate of the present invention may have the above-described functions.

In one embodiment of the invention, the CD73 antibody-drug conjugate is selected from the group consisting of: PCT patent application WO2019170131a1 discloses antibody-drug conjugates (ADCs).

In one embodiment of the invention, the CD73 antibody-drug conjugate is selected from the group consisting of the following defined substances in the antibody-drug conjugate (ADC) disclosed in PCT patent application WO2019170131a 1:

wherein the antibody is selected from the group consisting of: hu001c-14, Hu001c-15, Hu001c-21, Hu001c-22, Hu001c-23, Hu001c-24, Hu001c-25, Hu001c-28, Hu001c-30, Hu001c-31, Hu001c-32, Hu002c-2, Hu002c-3, Hu002c-4, Hu002c-6, Hu002c-7, Hu002c-8, Hu002c-10, Hu002c-11, Hu002c-12, Hu002c-14, Hu002c-15 and Hu002 c-16;

the drug is selected from:

(i) maytansine derivatives (DM1, DM4), auristatins and dolastatins;

(ii) monomethylyl Auristatin E (MMAE), monomethylyl auristatin F (MMAF), monomethylyl Dolastatin 10(MMAD) derivatives, or a combination thereof; and

(iii) a DNA damaging agent, preferably said DNA damaging agent comprises a duocarmycin, pyrrolo [2,1-c ] [1,4] benzodiazepine (PBD).

In one embodiment of the invention, the CD73 antibody-drug conjugate is a CD73-ADC preferably Hu001C14-BL20-MMAE (abbreviated as Hu001-MMAE in the patent) prepared by using a CD73 humanized monoclonal antibody (CD73 antibody) Hu001C-14 (abbreviated as Hu001 in the patent).

In the examples, a targeted study was conducted on Hu001-MMAE, verifying that the CD73 antibody-drug conjugate has dual anti-tumor effects and pharmacological mechanisms.

In one embodiment of the invention, the tumors are classified into negative, low, medium and high expression groups according to the comprehensive scores of the CD73 immunohistochemical staining intensity and positive rate in clinical lung cancer samples, and then the tumors derived from tumor cell lines are subjected to quantitative analysis to determine the high and low groups of the expression amount. The immunohistochemical detection and gene expression database analysis of CD73 protein in clinical tumor samples show that the expression level of CD73 in tumor is far higher than that in normal tissue, and the tumor cell line with high expression level similar to or higher than that in CD 73-is reached. This provides a good basis for the development of the clinical importance and feasibility of CD 73-ADC.

The CD73-ADC medicine can directly kill tumor cells with high expression of CD73-, and has no obvious killing effect on tumor cells with low expression.

In vitro experiments unexpectedly find that the CD73-ADC drug disclosed by the invention has consistent activity with that of a corresponding monoclonal antibody drug, and can still effectively inhibit the activity of free CD73 or cell membrane CD73 enzyme catalyzing adenosine phosphate (AMP) to be hydrolyzed to generate adenosine (Ado).

In one embodiment of the invention, the CD73-ADC drug does not exhibit a toxic effect on immune effector T lymphocytes that are under-expressed with CD 73. In vitro experiments unexpectedly find that the CD73-ADC drug not only has no toxic effect on T lymphocytes with low expression of CD73, but also can effectively reverse toxic and side effects of AMP/Ado on T lymphocyte proliferation.

In one embodiment of the invention, the CD73-ADC can significantly promote the differentiation and maturation of DC, and further enhance the activation effect of DC on T lymphocyte.

The CD73-ADC of the invention can promote the activation of DC by two aspects of action. First, the inhibitory effect of Ado on DC differentiation was reversed by inhibiting CD73 enzyme activity; second, it was unexpectedly found by in vitro experiments that CD73-ADC (MMAE conjugate) induces DC maturation more strongly and more efficiently than CD73 antibody, thereby further enhancing the activation effect of DC on T lymphocyte proliferation.

In one embodiment of the invention, the CD73-ADC drug significantly promotes infiltration of immune effector cells in tumor tissues while also reducing infiltration of immunosuppressive cells in mice. The effect of inhibiting the development of the tumor is achieved by activating the tumor immune microenvironment. In vivo experiments with multiple tumor models revealed that the CD73-ADC treated tumors showed massive infiltration and aggregation of mature DC cells, whereas no significant infiltration of mature DC was seen in the CD73 antibody treated tumors.

In one embodiment of the invention, the CD73-ADC is capable of significantly reducing infiltration of M2-type macrophages in a tumor microenvironment in vivo. Moreover, a large number of proinflammatory macrophages are infiltrated in the tumor treated by the CD73-ADC, and a large number of proinflammatory macrophages are not infiltrated in the tumor treated by the CD73 antibody.

In conclusion, the CD73-ADC researched by the invention is not only a targeted cytotoxic drug for high-expression tumors of CD 73-but also a CD73 adenylase (NT5E) inhibitor with extremely strong activity; on the one hand, targeted delivery of cytotoxic drugs leads to regression of highly expressed tumors of CD 73-while exerting a superimposed antitumor effect by binding to adenosine pathways that suppress other tumor cells aberrantly expressed in CD73 and the immune microenvironment.

Compared with the prior art, the invention proves the unique dual technical effects of the CD73-ADC, on one hand, the invention directly kills the high-expression tumor of CD73-, and on the other hand, the invention realizes the activation of immune microenvironment by inhibiting the CD73 adenylase pathway; the dual-superposition pharmacological mechanism has the characteristics of high efficiency and low toxicity, and provides a new strategy for treating refractory tumors.

The conception, specific structure and technical effects of the present invention will be further described in conjunction with the accompanying drawings, specific embodiments and examples to fully understand the objects, features and effects of the present invention.

Drawings

FIG. 1 illustrates the technical effect of the present invention. FIG. 1 shows the molecular structure of CD73-ADC and its dual anti-tumor mechanism. CD73-ADC on the one hand leads to regression of highly expressed tumors of CD 73-by targeted delivery of cytotoxic drugs, while exerting a superimposed antitumor effect by binding to the adenosine pathway that suppresses the tumor environment of other aberrant expression of CD 73.

FIG. 2 shows the expression of CD73 protein in clinical lung cancer and paracancerous tissue chip samples. The tissues are sorted according to the integrated scores of Immunohistochemistry (IHC) and divided into CD73 negative, low positive, medium positive and high positive groups, and the proportion of the two tissues in different CD73 expression groups (figure 2A) and staining pictures of representative samples (figure 2B) are calculated.

FIG. 3 is a graph showing the relative expression of CD73 mRNA in tumor cells (CCLE database) or normal human tissue (GETX database, http:// gtexport. org).

FIG. 4 shows the expression of CD73 in clinical lung cancer samples and xenograft tumors prepared from human lung cancer cell lines. Fig. 4A is a photograph of staining of clinical lung cancer samples and cell line xenograft tumors with different CD73 staining intensities. Fig. 4B is the mean gray value (left) and the relative gray value (right) for IMAGE analysis of the 200x field of view in 4A.

FIG. 5 shows the inhibitory effect of CD73-ADC on the proliferation of CD 73-low expressing tumor cell MDA-MB-453 and on the proliferation of CD 73-high expressing cell lines NCI-H441 and NCI-H292.

FIG. 6 shows the inhibitory effect of CD73 antibody and CD73-ADC on the activity of recombinant human free CD73 enzyme catalyzing the hydrolysis of adenosine phosphate (AMP) to adenosine (Ado) (IC 50).

FIG. 7 shows the inhibition of the activity of CD73 antibody and CD73-ADC on the enzymatic hydrolysis of AMP into Ado by CD73 in the cell membrane of NCI-H1299 (IC 50).

FIG. 8 is a flow cytometric fluorescence sorter (FACS) for detecting the expression level of cell membrane CD73 protein in breast cancer MDA-MB-453, lung cancer NCI-H460, NCI-H292, NCI-H441, NCI-H1299, Calu-1, human Peripheral Blood Mononuclear Cell (PBMC) and T lymphocyte.

FIG. 9 shows the effect of CD73-ADC on T lymphocyte proliferation. FIG. 9A is a T cell inhibition curve for various concentrations of ADC, and FIG. 9B is the inhibition of CD4+/CD8+ T lymphocytes by 100nM CD 73-ADC.

FIG. 10 shows that both CD73-ADC and CD73 antibodies were effective in reversing the inhibitory effect of AMP on human T-lymphocyte proliferation. The test adopts sorting to obtain CD3+ human T lymphocytes, and the cell proliferation rate is counted after 5 days of culture.

FIG. 11 is a graph showing that CD73-ADC at a concentration of 100nM is effective in reversing the inhibitory effect of AMP on IFN- γ secretion from human T lymphocytes. The test adopts sorting to obtain CD3+ human T lymphocyte, and the T lymphocyte culture supernatant is detected after the T lymphocyte is cultured for 5 days.

FIG. 12 shows that CD73-ADC can promote Dendritic Cell (DC) maturation. FIG. 12A is a FACS scatter plot of DC double-stained CD11c/CD86, and FIG. 12B is the proportion of CD11c/CD86 double-positive cells for each group.

FIG. 13 shows that CD73-ADC was effective in enhancing the effect of DC on T cell activation. Fig. 13A is a FACS scattergram of proliferation of cfse (cfse) labeled T lymphocytes, and fig. 13B is a ratio statistic of the proliferated T lymphocytes.

FIG. 14 is a graph showing that CD73 antibody and CD73-ADC were able to effectively reverse the inhibitory effect of AMP/Ado on DC differentiation; furthermore, CD73-ADC promotes DC maturation more favorably than the CD73 antibody. FIG. 14A is a FACS scattergram of double-stained CD11c/CD86, and FIG. 14B is the proportion of double-positive cells of CD11c/CD86 in each group.

FIG. 15 shows that DC treated by CD73-ADC can significantly promote the activation effect of DC on T lymphocytes in AMP/Ado environment compared with CD73 antibody. Fig. 15A is a FACS scatterplot of proliferation of CFSE labeled T cells for each group, and fig. 15B is a scale statistic of the proliferated T cells.

FIG. 16 shows the effect of CD73-ADC on the treatment of non-small cell lung carcinoma cell NCI-H441 nude mouse transplantable tumors and on the infiltration of mature DCs, M2-type and pro-inflammatory macrophages. FIG. 16A is the tumor volume at the end of the drug effect test when the tumor was taken, FIG. 16B is the fluorescence staining pattern and the double positive region signal values of mature DC markers CD11C and CD86 in tumor tissues administered with CD73 antibody (left) and CD73-ADC (right), FIG. 16C is the staining result (left) of macrophage markers F4/80 and M2 type macrophage marker CD 206C in tumor tissues administered with CD73 antibody (left) and CD73-ADC (right), and the ratio of the positive region value of F4/80 to the positive region value of CD206 in each group (right).

FIG. 17 the therapeutic effect of CD73-ADC and docetaxel on non-small cell lung carcinoma cells NCI-H292 in nude mouse transplantable tumors and the effect on mature DC, M2-type and pro-inflammatory macrophage infiltration. FIG. 17A shows the tumor volume at the end of the drug effect test, FIG. 17B shows the immunofluorescent staining pattern and the signal value of the double positive area for the mature DC markers CD11C and CD86 in tumor tissues, FIG. 17C shows the IHC staining results for CD73, macrophage marker F4/80 and macrophage marker CD206 of M2 type (left), and the ratio of the positive area value of F4/80 to the positive area value of CD206 in each group (right).

FIG. 18 is a graph of the effect of CD73-ADC and docetaxel on the treatment of non-small cell lung carcinoma cell NCI-H1299 nude mouse transplantable tumors and on mature DC, M2-type and pro-inflammatory macrophage infiltration. FIG. 18A is a graph showing the tumor volume at the time of tumor removal at the end of the drug effect test, FIG. 18B is a graph showing immunofluorescent staining patterns and signal values of double positive regions for the mature DC markers CD11C and CD86, FIG. 18C is a graph showing the staining results of CD73, macrophage marker F4/80 and macrophage marker CD206 of M2 type (left), and the ratio of the positive region value of F4/80 to the positive region value of CD206 (right) for each group.

Detailed Description

The inventor proves that the CD73 antibody-drug conjugate Hu001C14-BL20-MMAE (CD73-ADC) prepared by site-directed coupling of MMAE has a unique dual anti-tumor molecular mechanism through theoretical hypothesis, research design and extensive experimental demonstration. As shown by the technical effects summarized in figure 1, the CD73-ADC studied by the invention can directly kill tumor cells with high expression in CD73-, and has no obvious toxic or side effect on tumor cells with low expression in CD73 or normal cells; the CD73-ADC is also a very active adenylase inhibitor, can directly block the activity of catalyzing Adenosine Monophosphate (AMP) to be hydrolyzed into adenosine (Ado) by CD73, and protects and enhances the activity and the function of immune effector cells; the CD73-ADC has excellent in-vivo anti-tumor treatment effect and reduces the occurrence of immune escape. The present invention has been completed based on this finding.

Example 1 expression of CD73 in tumors at higher levels than in normal human tissue

To investigate the expression level of CD73 in tumor and normal tissues, we performed CD73 immunohistochemical staining (IHC) on clinical lung cancer and paracancer sample tissue chips (Outdo Biotech, Cat # HLugA180Su07) and nude mouse transplanted tumor sections prepared with human lung cancer NCI-H441, NCI-H292, and NCI-H1299 cell lines. Clinical lung cancer and paracarcinoma staining results were graded by a pathologist. IHC Composite Score (CS) was quantified as the intensity of staining x the percentage of positive tumor cells (cancer specimen) or lung epithelial cells (paracancerous specimen). The CS values (86 cases of non-cancer and 81 cases of paracancer) were classified into a CD 73-negative group (CS >0, n ═ 66) and a CD 73-positive group (CS >0, n ═ 101). Positive CS specimens were classified into low positive groups (median; n: 51), and the remaining 50 specimens were classified into medium positive (n: 25) and high positive (n: 25) by new median.

Staining pictures of representative clinical tumor samples from CD73 negative, low positive, medium positive and high positive groups and lung cancer cell transplants were taken, analyzed using IMAGE J software and calculated for mean and relative gray values for 3-4 independent fields (200 ×).

The relative expression level of CD73 mRNA was obtained by comparing the value of CD73 mRNA in tumor cells in CCLE database or normal human tissue in GETX database (http:// gtexport. org) with that of internal reference gene β -actin.

The results are shown in figure 2, and analysis of clinical lung cancer and paracancer samples found CD73 to be positive in 86% and 33.3% of the paracancers, with 52.35% (n ═ 45) and 6.17% (n ═ 5) moderately and highly positive. The expression level of CD73 in cancer tissues is much higher than that in paracarcinoma tissues at the protein level.

The results are shown in FIG. 3, where CD73 is expressed in tumor cell lines at the gene level much higher than in normal human tissues.

The results are shown in FIG. 4A, which is a typical staining image of representative clinical lung cancer sample groups and cell line xenograft tumors. Fig. 4B is a quantitative value of the average gray scale of fig. 4A. The results show that the expression level of CD73 in xenograft tumor models NCI-H441, NCI-H292, and NCI-H1299 was in the clinical lung cancer specimen or in the hyperactive group.

In conclusion, the results of this example show that the expression level of CD73 in tumor is much higher than that in normal human tissue, which provides the basis for further CD73-ADC research.

Example 2 killing of highly expressed tumor cells in CD73 by CD73-ADC

To investigate the killing effect of CD73-ADC on tumor cells with different CD73 expression levels, we used the CD73 low expressing cell line MDA-MB-453, the CD 73-high expressing cell lines NCI-H441 and NCI-H292 (purchased from ATCC or Chinese academy of sciences cell bank) as in vitro tumor suppression experimental models.

The cells in logarithmic growth phase were seeded at a density of 800-₂After about 5-12h of culture, CD73-ADCHu001-MMAE was added at different concentrations, 2-4 multiple wells were set for each drug concentration, and corresponding vehicle control and blank control wells, and after 5-6 days of action (enough number of cell divisions were ensured depending on the cell growth rate), the culture medium was decanted, MTS reaction solution (purchased from Promega, cat # G3581) was added at 100. mu.L/well, the reaction was carried out at 37 ℃ to the desired shade of color, the cell viability (OD490nm) of each group was determined, and the cell viability was calculated according to the formula of viability (OD dose-OD blank)/(OD control-OD blank) × 100% by analyzing the above data by GraphPad Pri 5 software, and the IC50 values of the above CD73-ADC on different cell lines were calculated, respectively.

As shown in FIG. 5, in the tested concentration range, CD73-ADC had no obvious toxic side effect (IC50>100nM) on tumor cells MDA-MB-453 with low expression of CD73, while it had strong inhibitory effect on tumor cells NCI-H441 and NCI-H292 with high expression of CD73 (IC50 is 0.023 + -0.006 nM and 0.051 + -0.007 nM, respectively).

In conclusion, the results of the example show that the CD73-ADC can kill the tumor cells with high CD73-, but has no obvious inhibition effect on the tumor cells with low expression of CD 73.

Example 3 inhibition of the Activity of CD73-ADC on free CD73 and tumor cell membrane CD73 enzymes catalyzing the hydrolysis of AMP to Ado

To investigate whether CD73-ADC has the same effect as the corresponding monoclonal antibody drug in inhibiting the activity of CD73 enzyme catalyzing the hydrolysis of adenosine phosphate (AMP) to adenosine (Ado), we performed a parallel study using CD73-ADC and CD73 antibody.

Human recombinant free CD73 enzyme (CD73 extracellular region) was diluted to 0.1. mu.g/mL with antigen diluent and plated evenly into 96-well low-adsorption plates at 25. mu.L/well. 50 μ L of CD73 antibody Hu001 and CD73-ADC Hu001-MMAE CD73 antibody diluted from 2nM to 0.0009nM in a 3-fold gradient were added to the plate, mixed well (final concentration 1nM to 0.00045nM), incubated at 37 ℃ for 1h, then 25 μ L of a mixture containing 1.2mM AMP and 0.4mM ATP was added, and incubated at 37 ℃ for 1 h. And taking out 50 mu L of the reaction solution, adding the reaction solution into another 96-hole white board, adding 50 mu L of CellTiter-Glo reagent into each hole, uniformly mixing, reacting for 3-5min in a dark place, and detecting the intensity of a fluorescence signal by using an enzyme labeling instrument.

Non-small cell lung cancer cell NCI-H1299 with high expression of CD73 is used as a target cell. After culturing 2500/well tumor cells (confirmed by preliminary experiments) in a 96-well plate at 37 ℃ for 16 hours, the cells were washed 3 times with serum-free RPMI-1640 medium, 50. mu.L of the CD73 antibody Hu001 and CD73-ADC Hu001-MMAE were added to the 96-well plate in a 3-fold gradient from 100nM to 0.045nM of the test antibody, incubated at 37 ℃ for 30min, 25. mu.L of 0.9mM AMP was added, and the plate was incubated at 37 ℃ for 3 hours at 5% CO2 (final concentration 66.6nM to 0.03 nM). 25 μ L of the culture supernatant was taken out and added to another 96-well white plate, and 25 μ L of 0.1mMATP was added and mixed well. And adding 50 mu L of CellTiter-Glo reagent into each hole, uniformly mixing, carrying out dark reaction for 3-5min, and detecting the intensity of a fluorescence signal by using an enzyme labeling instrument.

As shown in FIG. 6, both the CD73 antibody and the CD73-ADC showed significant inhibition activity of recombinant free CD73 proteolytic AMP in producing Ado, with IC50 of 0.045nM and 0.048nM, respectively.

As shown in FIG. 7, both the CD73 antibody and the CD73-ADC were able to inhibit the activity of NCI-H1299 cell membrane CD73 catalyzing the hydrolysis of AMP to produce Ado, with IC50 of 0.622nM and 0.877nM, respectively.

In conclusion, this example shows that the pharmaceutical activity of CD73-ADC is consistent with that of the corresponding CD73 antibody, and the CD73-ADC can effectively inhibit the enzymatic activity of free CD73 and cell membrane CD73 enzyme catalyzing AMP to generate Ado.

Example 4 in vitro testing of the protective Effect of CD73-ADC on immune effector T lymphocytes

Through the research results of example 2 and example 3, it is found that the CD73-ADC can not only target and kill the highly expressed cells of CD73-, but also has the function of inhibiting the CD73 enzyme from catalyzing AMP to generate Ado. Therefore, to study the effect of CD73-ADC on immune effector cells, we first examined the expression of CD73 in human Peripheral Blood Mononuclear Cells (PBMC) and T lymphocytes, and further studied the effect of CD73-ADC on T cell proliferation and the effect of reversing AMP/Ado on T lymphocyte proliferation.

Using 1x10⁵The individual cells were mixed well with CD73 antibody Ab001 (final concentration 10 μ g/mL), then incubated at 4 ℃ for 1h, the cells were washed twice with PBS to remove unbound primary antibody, then incubated with PE-labeled secondary antibody at 4 ℃ for 30min, the cells were washed twice with PBS to remove unbound secondary antibody, and finally the cells were resuspended in 200 μ L PBS and the Mean Fluorescence Intensity (MFI) was detected by flow cytofluorescence sorter (FACS).

Resuscitation, expansion and sorting of T cells: PBMC cryopreservation tubes are provided by Jiangsu Sedil Biotechnology, Inc. PBMCs were first resuscitated and cultured for 3-4 days in a medium containing 500ng/mL CD3/CD28 antibody and 100IU/mL IL-2, and then CD 3-positive T lymphocytes were sorted using a sorting kit (supplier: Stemcell, Cat # 1795).

To examine the effect of CD73-ADC on T cell proliferation, T cells were plated in 96-well plates (20000 cells/well), mixed with a vehicle (buffer) and a gradient diluted hIgG1-MMAE and Hu001-MMAE (final concentration 100nM to 0.001nM, n 4) and cultured for 4 to 5 days, then the number of cells in a fixed volume was counted by FACS, and an IC50 value was calculated as a dose graph.

In another set of experiments, T cells were plated in 96-well plates (20000 cells/well), vehicle, hIgG1-MMAE and Hu001-MMAE (100nM) were added, T lymphocytes were removed 4-5 days after culture, centrifuged, double stained with CD4+/CD8+ fluorescent antibody, fixed volume cells were read using FACS and the number of CD4+/CD8+ viable cells was counted.

In another set of experiments, the sorted T cells were fluorescently labeled, and a pre-prepared CFSE (carboxy fluoro toxin succinimidyl ester) was added to the cell suspension (final concentration 2.5. mu.M), and after labeling at 37 ℃ for 5min, the cells were washed 3 times with PBS. CFSE-labeled T cells were then plated into 96-well plates (2X 10)⁴Each cell/well), 50 μ L of each of the vehicle, hIgG1 and Hu001 (final concentration 10nM to 0.0001nM, n ═ 4) or hIgG1-MMAE and Hu001-MMAE (final concentration 100nM to 0.001nM, n ═ 4) diluted in a gradient, and 50 μ L of adenosine monophosphate (AMP, final concentration 0.25mM) were added, mixed, and the culture supernatant was collected after 4 to 5 days of culture, and fixed volumes of cells were read and counted using FACS.

Detection of T cell IFN- γ: 50 μ L/well of T cell culture supernatant was aspirated for detection of IFN-. gamma.protein concentration using an ELISA kit (Co. Biotechnology Ltd., Cat # EK180HS-48) and referring to the technical procedures provided in the kit.

As a result, as shown in FIG. 8, Ab001 (chimeric antibody corresponding to Hu001) specifically recognized and bound to CD73 on the cell membrane surface using the CD73 antibody, the expression level of CD73 on the surface of PBMC and T lymphocytes was lower than that of tumor cells highly expressed in CD 73-.

The results are shown in FIG. 9, where CD73-ADC produced no toxic side effects on T lymphocytes (IC50>100nM) and no effect on proliferation of CD4+/CD8+ T cells in the tested concentration range compared to vehicle and hIgG 1-MMAE.

As shown in FIG. 10, both the CD73 antibody and the CD73-ADC were effective in reversing the proliferation inhibition of T lymphocytes by AMP/Ado, and their EC50 values were 0.08. + -. 0.01nM and 0.09. + -. 0.01nM, respectively.

The results are shown in FIG. 11, where CD73-ADC was effective in reversing the inhibitory effect of AMP/Ado on IFN-. gamma.expression/secretion from T lymphocytes at a concentration of 100 nM.

In conclusion, the results of the present example show that CD73-ADC has no toxic effect on T lymphocytes, and can effectively reverse the toxic and side effects of AMP/Ado on proliferation of T lymphocytes and secretion of IFN-gamma.

Example 5 in vitro testing of the promoting Effect of CD73-ADC on dendritic cell maturation

Dendritic Cells (DC) are key immune effector cells for activating T cells, NK cells and the like, and the direct effect of CD73-ADC on DC and the inhibition effect of reversal AMP/Ado on DC maturation are researched in the embodiment.

Induced differentiation of DCs: firstly 1x10⁶PBMCs were seeded in 24-well plates at 1 ml/well and cells were allowed to recover for 12h in a cell culture incubator, after which GM-CSF (60ng/ml, 1000U/ml), IL-4(100ng/ml, 500U/ml) were added. After 36 hours of culture, the supernatant was aspirated, replaced with medium and containing the same concentrations of cytokines as described above, and at this point the vehicle, hIgG1-MMAE (5. mu.g/ml), Hu001-MMAE (5. mu.g/ml), was added, leaving a blank well. The incubation was continued for 4 days and LPS (0.5. mu.g/ml) was added to the blank wells. Cells were harvested after 24 hours, stained with CD11c and CD86 antibodies, and the number of fixed viable cells was read by FACS to count the proportion of double positive cells.

In another set of experiments, the procedure was the same as above, and after 36h of culture to replace medium and cytokines, vehicle, AMP (0.25mM), hIgG1 (5. mu.g/ml), Hu001 (5. mu.g/ml), hIgG1-MMAE (5. mu.g/ml), Hu001-MMAE (5. mu.g/ml) were added to the corresponding wells. After further culturing for 5 days, the cells were collected, stained with CD11c and CD86 antibodies, and the number of fixed viable cells was read by FACS to count the ratio of CD11c/CD86 double positive cells.

Co-culture of DCs with T cells: volunteer 1-derived 2x10⁵PBMCs were seeded in 96-well plates at 0.1 ml/well and allowed to recover in a cell culture incubator for 12 hours before GM-CSF (60ng/ml, 1000U/ml), IL-4(100ng/ml, 500U/ml) was added and cultured for 36h, the supernatant was aspirated off and replaced with cytokine containing the same concentration as described above, and at this point the vehicle, hIgG1-MMAE (5. mu.g/ml), Hu001-MMAE (5. mu.g/ml) was added and a blank well was left. The incubation was continued for 4 days and LPS (0.5. mu.g/ml) was added to the blank wells.After 24 hours the plates were centrifuged (1700rpm, 5min), the supernatant aspirated and washed twice with PBS, and CFSE-labeled volunteer 2-derived T lymphocytes plated into 96-well plates (2 × 10)⁴Individual cells/well), after further culturing for 3-5 days, the number of fixed living cells is read and counted by FACS, and the proliferated cells are circled according to cell groups.

In another set of experiments, the procedure was the same as above, and after 36 hours of culture to replace medium and cytokines, vehicle, AMP (0.25mM), hIgG1 (5. mu.g/ml), Hu001 (5. mu.g/ml), hIgG1-MMAE (5. mu.g/ml), Hu001-MMAE (5. mu.g/ml) were added to the corresponding wells. After further 5 days of culture, the plates were centrifuged (1700rpm, 5min), the supernatant aspirated and washed twice with PBS, CFSE-labeled volunteer 2-derived T cells were plated into 96-well plates (2 × 104 cells/well), after further 3-5 days of culture, fixed viable cell numbers were read and counted using FACS, and proliferating cells were circled according to cell clustering.

As shown in FIG. 12, CD73-ADC promotes immature DC maturation in an in vitro model system of PBMC-DC differentiation maturation.

The results are shown in FIG. 13, and in a model system of DC-T lymphocyte co-culture in vitro, CD73-ADC can effectively enhance the activation effect of DC on T lymphocytes.

The results are shown in FIG. 14, that the CD73 antibody and CD73-ADC were able to reverse the inhibitory effect of AMP/Ado on DC differentiation; compared with the CD73 antibody, the CD73-ADC has stronger effect of promoting DC maturation.

The results are shown in FIG. 15, where the CD73 antibody and CD73-ADC reversed the toxic side effects of AMP on DC differentiation-T lymphocyte activation in a model system of in vitro DC-T lymphocyte co-culture; CD73-ADC also promotes DC maturation and further enhances the T lymphocyte activation effect of DCs compared to CD73 antibody.

In conclusion, this example demonstrates that CD73-ADC can promote DC maturation more effectively and further enhance the activation effect of DC on T lymphocytes, compared to CD73 antibody.

Example 6 CD73-ADC inhibits tumor growth in NCI-H441 lung cancer in vivo, enhances DC maturation activation in the tumor microenvironment, reduces M2 type and increases infiltration of pro-inflammatory macrophages

In order to research the in-vivo tumor inhibition effect of the CD73-ADC and the influence on the tumor immune microenvironment, an NCI-H441 lung cancer cell line nude mouse transplantation tumor model is established. 100 μ L of a solution containing 5X10⁶NCI-H441 was mixed well with 100. mu.L of a cell suspension of matrigel and inoculated subcutaneously into the back of immunodeficient mice (Balb/c, nude). The volume of the tumor to be treated is 100-300 mm³Randomly grouping according to the tumor volume and the weight of the nude mice (n is 8), and adopting the dose of 3mg/kg hIgG1-MMAE and Hu001-MMAE to be administrated in tail vein once a week for 2 weeks; in another group, mice were sacrificed starting on the first day after H441 administration, i.e. every 3 days tail vein with doses of 10mg/kg hIgG1 and Hu001 (n ═ 6). After the tumor had grown to a certain volume, the tumor was removed, fixed with 4% neutral paraformaldehyde, dehydrated, paraffin embedded, sectioned and stained by immunofluorescence (CD11c and CD86) or immunohistochemistry (F4/80 and CD206), and the effect of CD73-ADC on mature DC and M2 and pro-inflammatory macrophage infiltration was observed.

The results are shown in FIG. 16, compared with hIgG1-MMAE, the tumor size of CD73-ADC Hu001-MMAE after treatment is smaller (FIG. 16A), and the effect of inhibiting the growth of in vivo transplanted tumor is good; compared to the CD73 antibody group, CD73-ADC was able to significantly promote infiltration of mature DCs in NCI-H441 transplants (fig. 16B); the CD73 antibodies Hu001 and CD73-ADC Hu001-MMAE were able to reduce infiltration of M2-type macrophages compared to the hIgG1 and hIgG1-MMAE groups (fig. 16C); compared with the CD73 antibody Hu001 group, CD73-ADC Hu001-MMAE was able to significantly increase infiltration of F4/80 positive macrophages (fig. 16C).

In conclusion, the results of this example show that CD73-ADC has good tumor inhibition effect in the NCI-H441 nude mouse transplantation tumor model; compared with the CD73 antibody, the CD73-ADC can better inhibit the tumor with higher efficiency by promoting the mature DC of immune effector cells and anti-inflammatory macrophages to gather in a tumor microenvironment, reducing infiltration of M2 type macrophages of immunosuppressive cells, and activating the immune microenvironment.

Example 7 CD73-ADC inhibits tumor growth in NCI-H292, NCI-H1299 Lung cancer in vivo, and compared to docetaxel, increases DC maturation activation in the tumor environment, decreases M2 type and increases infiltration of pro-inflammatory macrophages

In order to research the in-vivo tumor inhibition effect of the CD73-ADC and the influence on a tumor immune microenvironment, a lung cancer cell line nude mouse transplantation tumor model is established. Respectively mixing 200 μ L of the extract containing 5 × 10⁶NCI-H292 cell suspension, or 100. mu.L containing 9X10⁶NCI-H1299 was mixed well in 100. mu.L of a cell suspension of matrigel and inoculated subcutaneously into the back of immunodeficient mice (Balb/c, nude). The volume of the tumor to be treated is 100-300 mm³Randomly grouping according to the tumor volume and the weight of the nude mice (n is 8), and respectively adopting 5mg/kg hIgG1-MMAE and Hu001-MMAE dose to be administered once in tail vein once a week for 2 weeks; at the same time, 15mg/kg Docetaxel (Docetaxel) was set as a positive control. After the tumor had grown to a certain volume, the tumor was removed, fixed with 4% neutral paraformaldehyde, dehydrated, paraffin embedded, sectioned and stained by immunofluorescence (CD11c and CD86) or immunohistochemistry (F4/80 and CD206), and the effect of CD73-ADC on mature DC and M2 and pro-inflammatory macrophage infiltration was observed.

The results are shown in FIG. 17, compared with hIgG1-MMAE, NCI-H292 treated by CD73-ADC Hu001-MMAE has smaller tumor volume (FIG. 17A), and has good effect of inhibiting the growth of in vivo transplanted tumor; compared to hIgG1-MMAE and docetaxel, CD73-ADC Hu001-MMAE was able to significantly promote infiltration of mature DCs in NCI-H292 transplantable tumors (fig. 17B); compared with hIgG1-MMAE and docetaxel, CD73-ADC Hu001-MMAE can significantly reduce the expression amount of CD73 in NCI-H292 transplanted tumors, significantly reduce infiltration of M2 type macrophages, and significantly increase infiltration of F4/80 positive macrophages (FIG. 17C).

The results are shown in FIG. 18, compared with hIgG1-MMAE, the H1299 tumor treated by CD73-ADC Hu001-MMAE has smaller volume (FIG. 18A), and has good effect of inhibiting the growth of in vivo transplanted tumor; compared with hIgG1-MMAE and docetaxel, CD73-ADC Hu001-MMAE significantly promoted infiltration of mature DCs in NCI-H1299 transplantable tumors (FIG. 18B); compared with hIgG1-MMAE and docetaxel, CD73-ADC Hu001-MMAE was able to significantly reduce the expression level of CD73 in NCI-H1299 transplanted tumors, significantly reduce infiltration of M2-type macrophages, and significantly increase infiltration of F4/80-positive macrophages (FIG. 18C).

In conclusion, the results of the example show that the CD73-ADC has good tumor inhibition effect in a tumor cell line nude mouse transplantation tumor model; compared with docetaxel, the CD73-ADC can promote the mature DC of immune effector cells and anti-inflammatory macrophages to gather in a tumor microenvironment, reduce infiltration of M2 type macrophages of immunosuppressive cells, and has the effects of activating the tumor immune microenvironment and inhibiting tumors with higher efficiency.

The results are combined, so that the CD73-ADC can specifically kill high-expression tumor cells in CD73-, can block the activity of the CD73 in catalyzing AMP to hydrolyze to generate Ado, reduces the inhibition of the Ado on various immune effector cells, reduces the infiltration of immunosuppressive cells, activates a tumor microenvironment, reduces the immune escape of tumors, and has obvious antitumor activity.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims

1. The application of a CD73 antibody-drug conjugate is characterized in that the CD73 antibody-drug conjugate is used for preparing a drug for activating a tumor immune microenvironment by inhibiting a CD73 adenylase pathway.

2. The use of a CD73 antibody-drug conjugate according to claim 1, wherein the CD73 antibody-drug conjugate is used for the preparation of a medicament for inhibiting the activity of free CD73 or cell membrane CD73 enzyme catalyzing the hydrolysis of adenosine phosphate to adenosine.

3. The use of the CD73 antibody-drug conjugate of claim 1, wherein the CD73 antibody-drug conjugate is used to prepare: the medicine activates tumor immune microenvironment by inhibiting CD73 adenylase pathway, and has no toxic effect on immune effector T lymphocyte.

4. The use of the CD73 antibody-drug conjugate of claim 3, wherein the CD73 antibody-drug conjugate is used to prepare: the medicine activates a tumor immune microenvironment by inhibiting a CD73 adenylase pathway, does not show a toxic effect on immune effector T lymphocytes, and can effectively reverse toxic and side effects of adenosine phosphohydrolysis on T lymphocyte proliferation and IFN-gamma secretion in the adenosine production process.

5. The use of the CD73 antibody-drug conjugate of claim 1, wherein the CD73 antibody-drug conjugate is used to prepare: the medicine activates a tumor immune microenvironment by inhibiting a CD73 adenylase pathway, promotes dendritic cells to mature, and enhances the activation effect of the dendritic cells on T lymphocytes.

6. The use of the CD73 antibody-drug conjugate of claim 1, wherein the CD73 antibody-drug conjugate is used to prepare: the medicine activates a tumor immune microenvironment by inhibiting a CD73 adenylase pathway, promotes infiltration of immune effector cells, and reduces infiltration of immunosuppressive cells.

7. The use of the CD73 antibody-drug conjugate according to claim 1, wherein the CD73 antibody-drug conjugate is used for preparing a medicament for killing highly expressed tumor cells of CD73 "while activating tumor immune microenvironment by inhibiting CD73 adenylase pathway.

8. The use of the CD73 antibody-drug conjugate of claim 7, wherein the CD73 antibody-drug conjugate is used to prepare the following drugs: the medicine is a medicine which kills tumor cells highly expressed in CD 73-and simultaneously inhibits the activity of adenosine phosphohydrolysis generated by free CD73 or cell membrane CD73 enzyme.

9. The use of the CD73 antibody-drug conjugate of claim 7, wherein the CD73 antibody-drug conjugate is used to prepare: the medicine can kill tumor cells highly expressed in CD73-, activate tumor immune microenvironment by inhibiting CD73 adenylase pathway, and has no toxic effect on immune effector T lymphocytes.

10. The use of the CD73 antibody-drug conjugate of claim 7, wherein the CD73 antibody-drug conjugate is used to prepare: the medicine can kill tumor cells highly expressed in CD73-, activate tumor immune microenvironment by inhibiting CD73 adenylase pathway, has no toxic effect on immune effector T lymphocytes, and can effectively reverse toxic and side effects of adenosine phosphohydrolysis on T lymphocyte proliferation and IFN-gamma secretion during adenosine production.

11. The use of the CD73 antibody-drug conjugate of claim 7, wherein the CD73 antibody-drug conjugate is used to prepare: the medicine can kill tumor cells highly expressed in CD73-, activate a tumor immune microenvironment by inhibiting a CD73 adenylase pathway, promote dendritic cell maturation and enhance the activation effect of the dendritic cells on T lymphocytes.

12. The use of the CD73 antibody-drug conjugate of claim 7, wherein the CD73 antibody-drug conjugate is used to prepare: the medicine can kill tumor cells highly expressed in CD73-, activate a tumor immune microenvironment by inhibiting a CD73 adenylase pathway, promote infiltration of immune effector cells and reduce infiltration of immunosuppressive cells.

13. The use of the CD73 antibody-drug conjugate of claim 7, wherein the CD73 antibody-drug conjugate is used to prepare:

the medicine can kill tumor cells highly expressed in CD73-, inhibit the activity of adenosine phosphorylation generated by free CD73 or cell membrane CD73 enzyme catalysis, has no toxic effect on immune T lymphocytes, can effectively reverse the toxic and side effects of adenosine phosphorylation generated adenosine on T lymphocytes proliferation and IFN-gamma secretion, simultaneously promotes dendritic cell maturation, enhances the activation effect of dendritic cells on T lymphocytes, promotes the infiltration of immune effector cells, and reduces the infiltration of immune suppressor cells.