CN113956359A - Antibody and application thereof in anti-tumor - Google Patents
Antibody and application thereof in anti-tumor Download PDFInfo
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Abstract
The invention relates to an antibody and application thereof in tumor resistance. The invention provides a novel monoclonal antibody targeting ROR1, which can be combined with a target antigen human ROR1 with high affinity, has strong specificity and can effectively inhibit off-target benefits. The antibody can inhibit the growth of various hematopoietic system tumor cells and solid tumor cells, and has a strong anti-tumor effect in an animal model, thereby providing a new research basis for the development of related anti-tumor drugs.
Description
Technical Field
The invention belongs to the field of tumor immunotherapy and the field of biotechnology, and particularly provides an antibody and application thereof in tumor resistance.
Background
Because tumors have difficult healing, high mortality and low clinical manifestations after healing, the tumors become one of the most serious diseases threatening human health, and researchers try various tumor treatment means and technologies in sequence, including surgical treatment, radiotherapy, chemical drug therapy, gene therapy and the like, but no effective treatment means is found at present. With the intensive research of biotechnology and immunology, tumor immunotherapy and anti-tumor antibodies are receiving attention from the industry, and since the last 80 th century, hundreds of antibodies have been approved for tumor therapy worldwide, and the number of antibodies filed for clinical trials every year is thousands. The antibody therapy has higher targeting property and selectivity and obvious therapeutic effect, can effectively inhibit the growth and reproduction of tumor cells, can avoid adverse effects on normal tissues or cells, and ensures the normal living state of patients, so the antibody therapy has wide application in hematopoietic system tumors such as leukemia, lymphoma and myeloma, and solid tumors such as gastric cancer, colorectal cancer, breast cancer, ovarian cancer, lung cancer, melanoma, Hodgkin lymphoma and nasopharyngeal carcinoma.
Receptor tyrosine kinase-like orphan receptor1 (ROR 1) is one of the Receptor Tyrosine Kinase (RTKs) family members and has high homology with the tyrosine kinase domain of growth factor receptors. The human ROR1 molecule consists of an extracellular region including an immunoglobulin-like domain (Ig-like), two cysteine-rich frizzled domains (CRD or FZD) and a kringle (kng) domain; the intracellular domain contains a Tyrosine Kinase Domain (TKD), two serine/threonine-rich domains (Ser/ThrD), and a proline-rich domain (PRD). A great deal of research shows that ROR1 plays a key role in promoting the growth and metastasis of tumors, inducing tumor cell resistance, inhibiting apoptosis and the like, especially low-level expression in ROR1 normal tissues, but high expression in various malignant tumors or tissues, such as Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), breast cancer, ovarian cancer, melanoma, lung adenocarcinoma and the like. ROR1 is highly recognized in tumor tissues, and ROR1 is a novel tumor-specific marker and anti-tumor target based on the characteristic.
Against the ROR1 target, a variety of tumor immunotherapies have been developed, including: (1) the monoclonal antibody is an antibody which is generated by a single B cell clone, is highly uniform and only aims at a certain specific epitope, has high sensitivity, strong specificity, less cross reaction and low preparation cost, and is the most widely applied antibody medicament at present. Researchers have developed various monoclonal antibodies against ROR1 target, such as JP2021522162A, WO2021202863a1, EP3842072a1, CN112384533A, etc. disclose corresponding anti-ROR1 monoclonal antibodies; (2) the bispecific antibody contains 2 specific antigen binding sites, can bridge between target cells and functional molecules (cells), stimulates directed immune response, further enhances the targeting of antibody therapy, and EP2984107A1 discloses the bispecific antibody aiming at ROR1 and CD3 targets, and can effectively resist leukemia, diffuse large B cell lymphoma, myeloma, breast cancer, lung cancer and other tumors; (3) chimeric Antigen Receptor (CAR) T cells, CAR consists of two major domains: the T cell after genetic engineering can specifically identify the tumor cell and can utilize the activated T cell to play a strong anti-tumor role. The anti-tumor targets that can be used to construct chimeric antigen receptors are quite broad, ROR1 has also been reported to be useful for constructing CARs, e.g., WO2021202863a1, WO2020014366a1, etc. disclose CAR structures that target ROR 1; (4) an antibody-drug conjugate (ADC) is prepared by connecting a small molecular drug with bioactivity to a monoclonal antibody through a chemical link, and transporting the small molecular drug to a target cell by targeting the monoclonal antibody as a carrier. ADC drugs directed against ROR1 have also been reported, e.g., Peyman et al (Peyman B, Mozafar M, Ali Hakakian. anti-ROR1 scFv-EndoG as a novel anti-caner therapeutic drug, APJCP, 2017, 19 (1): 97-102) binding anti-ROR1 ScFv to immunotoxin can rapidly cause tumor cell apoptosis; (5) in addition to antibodies having classical structures such as monoclonal antibodies and bispecific antibodies, antibody derivatives such as single chain fragment variable antibodies (scFv) and Fab antibody fragments have been developed against ROR1, and have therapeutic effects.
Although the tumor immunotherapy taking ROR1 as a target point is widely reported, the medicines or products still have the technical problems of weak targeting, easy off-target benefit formation, strong rejection, tumor immune escape phenomenon, and the like, and the anti-tumor activity needs to be strengthened.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides a novel anti-ROR1 antibody, specifically, the antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, respectively.
Furthermore, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.
Further, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody or a single chain antibody.
A nucleic acid encoding the above antibody is provided, as well as a recombinant expression vector and a recombinant expression transformant comprising the nucleic acid.
The preparation method of the antibody comprises the following steps: culturing the recombinant expression transformant according to claim 6, and obtaining the anti-ROR1 antibody from the culture.
Provides an application of the antibody in preparing a medicament for treating tumors, wherein the tumors are ROR1 positive hematopoietic system malignant tumors or solid tumors, and specifically comprise lung cancer, breast cancer, ovarian cancer, Chronic Lymphocytic Leukemia (CLL), Acute Myelocytic Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), acute myelocytic leukemia (CML), myeloma and the like.
Advantageous effects
The anti-ROR1 antibody provided by the invention is a monoclonal antibody, has single component, high purity, obvious sequence structure, low production cost and is suitable for clinical application, and large-scale production is easy to realize by a biological fermentation mode. The antibody has strong affinity with a target antigen, can effectively identify the target antigen, has high specificity and strong targeting property, and can effectively prevent off-target benefits. The antibody can obviously inhibit the growth and reproduction of tumor cells, has killing effect on various tumor cells, and can obviously inhibit the growth of tumors, promote the secretion of immune factors, fully mobilize the immune system of an organism and exert the synergistic anti-tumor effect in animal experiments.
Drawings
FIG. 1: killing effect of anti-ROR1 antibody on hematologic tumor cells
FIG. 2: killing effect of anti-ROR1 antibody on ovarian cancer cells
FIG. 3: effect of anti-ROR1 antibodies on tumor volume in K562 mouse model
FIG. 4: anti-ROR1 antibody to K562 mouse model IFN- γ levels
FIG. 5: effect of anti-ROR1 antibodies on SKOV3 mouse model tumor volume
FIG. 6: anti-ROR1 antibody vs SKOV3 mouse model VEGF levels
Detailed Description
Example 1 screening of anti-ROR1 antibodies
In the invention, a mouse monoclonal antibody targeting ROR1 is screened and obtained by a fusion hybridoma technology.
1.1 Experimental mice immunization
The recombinant human ROR1 protein is used for immunizing mice, and the specific steps comprise: (1) selecting 20 BALB/c female mice of 6-8 weeks old, and normally breeding for 3 days to adapt to experimental environment; (2) emulsifying ROR1 protein with equal volume of Freund's complete adjuvant, resonating for 30min, mixing thoroughly, and injecting subcutaneously into mouse at multiple points; (3) after two weeks of the first immunization, carrying out the titer detection of the mice, and selecting the immune mice with high titer to carry out the intraperitoneal injection impact immunization; (4) emulsifying ROR1 protein with equal volume of Freund incomplete adjuvant, resonating for 30min, mixing thoroughly, and injecting into abdominal cavity of mouse; (5) repeating the step (4) for 3 times to strengthen the immune stress response.
1.2 cell fusion
Selecting a mouse with high anti-ROR1 antibody titer in blood, removing the neck of the mouse, killing the mouse, taking the spleen of the mouse on a sterile workbench, and washing the mouse with sterile PBS (phosphate buffer solution) for 3 times; injecting DMEM culture solution into the spleen by using an injector, and repeatedly washing to obtain spleen cells; centrifuging the obtained spleen cells at 1000rpm and 4 ℃ for 5min, and then resuspending the cells in a DMEM culture solution; mixing the obtained spleen cells with mouse myeloma cells SP2/0 in logarithmic growth phase at a ratio of 1:1, and performing cell fusion by using a PEG catalysis method; after cell fusion, the cells were seeded in a 96-well cell culture plate and cultured in a 37 ℃ incubator.
1.3 screening and preservation of Positive clones
After 10 days of cell fusion, taking cell supernatant, detecting positive clones by an ELISA method, carrying out expanded culture on the obtained positive clone hybridoma cells, collecting the cells, storing the cells in a refrigerator at the temperature of-80 ℃ and carrying out subsequent experiments.
1.4 anti-ROR1 antibody screening
Recovering and culturing positive hybridoma cells at 2 × 106Individual cells/mL hybridoma cells were seeded into dialysis-based bioreactors and antibody-containing supernatants were harvested once per week. Mouse monoclonal antibodies were purified by FPLC using Protein A (purchased from Pharmacia). Antibody concentration was determined by BCA kit or a280 absorbance, antibody purity was determined by SEC (size exclusion chromatography) and purity was checked by SDS (sodium dodecyl sulfate) gel electrophoresis and coomassie blue staining, and antibody purity was determined to be satisfactory for subsequent experiments.
The affinity of anti-ROR1 antibodies to human ROR1 was tested using a Fortebio biomacromolecule interactor (available from ehrlichio, usa). According to the invention, a plurality of alternative antibodies are obtained through a plurality of rounds of screening, wherein the result of the data of the affinity of partial antibodies with higher affinity with target antigens is shown in Table 1.
TABLE 1 affinity of anti-ROR1 antibody to human ROR1
In order to enable the anti-ROR1 antibody to be capable of carrying out efficient specific binding with a target and reduce the probability of off-target phenomenon, the #66 antibody with the highest affinity is selected for carrying out subsequent experiments.
1.5 sequencing of anti-ROR1 antibody
And culturing a hybridoma cell strain of the No. 66 antibody, extracting RNA by adopting a Trizol method, performing reverse transcription to prepare cDNA, and sequencing by using a designed and synthesized mouse IgG specific primer group. The results showed that the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, respectively; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.
Example 2 preparation of anti-ROR1 antibody
According to the sequencing result, proper primer sequences are designed, and the heavy chain and light chain variable region nucleotide sequences of the antibody are obtained through overlapping extension PCR reaction. The light and heavy chain nucleotide sequences of the antibody are constructed into eukaryotic expression vectors pcDNA3.4-G418 and pcDNA3.4-DHFR respectively through a gene recombination mode. The eukaryotic expression vector is introduced into CHO cells by an electrotransfection method, the CHO cells are cultured in a 5L bioreactor in a fed-batch mode, the cell density and the cell viability are detected every day, when the cell viability is reduced to below 75 percent or the culture period reaches 14 days, cell culture supernatant is centrifugally collected, and the antibody expression level of the cell supernatant is measured by an HPLC method. The antibody was isolated and purified from the cell culture supernatant using Protein A (purchased from Pharmacia) affinity chromatography column and the purity of the resulting product was more than 90% as confirmed by SDS-PAGE electrophoresis, and then the affinity-chromatographed product was again subjected to molecular sieve chromatography to obtain a sample with a purity of > 98%.
Example 3 killing Effect of anti-ROR1 antibodies on tumor cells
The ROR1 antibody is reported to have inhibitory effect on various hematological and solid tumors, and various hematological and solid tumor cell lines and cells were selected for examination in order to verify the physiological activity of the antibody of the present invention.
3.1 killing Effect of anti-ROR1 antibodies on hematological tumor cells
ROR1 is expressed in various hematopoietic system tumors, NB4 (acute myelogenous leukemia cell line), K562 (chronic myelogenous leukemia cell line), Raji (lymphoma cell line) and U266 (multiple myeloma cell line) are selected as research objects in the invention, and the killing effect is detected by a CCK-8 method. The specific method comprises the following steps:
(1) cells were seeded in 96-well plates at 2X 104Each group is provided with 3 multiple holes;
(2) after the cells grow to the logarithmic growth phase, replacing the fresh culture medium;
(3) adding 10 mu g/mL of anti-ROR1 antibody into each well, and incubating in a cell culture box at 37 ℃ for 12, 24 and 36 hours;
(4) adding CCK-8(Dojindo, Japan) reagent (20. mu.L/well) to each well, incubating the mixture in a cell incubator at 37 ℃ for 2 hours, and measuring the absorbance at 450 nm;
(5) according to the CCK-8 kit (Dojindo, Japan) instructions, the number of living cells was counted and the killing efficiency was estimated: the killing rate [ ((T + E) -T & E)/T ]. times.100%
Wherein T represents the number of viable target cells; e represents the number of viable effector cells; t + E equals the total number of viable target and effector cells; t & E represents the number of viable cells after killing of the target cells by effector cells.
As shown in figure 1, the antibody provided by the invention has an inhibiting effect on various blood tumor cells, wherein the killing effect on NB4, K562 and Raji is strongest, the killing rate can reach about 50% after being treated for 24 hours, and the effect is strengthened after being treated for 36 hours; however, the antibody of the present invention has strong inhibitory effect on leukemia and lymphoma cells, and has moderate inhibitory effect on myeloma cells, which indicates that the antibody can be used for treating various hematopoietic malignancies including leukemia, lymphoma and myeloma.
3.2 killing Effect of anti-ROR1 antibodies on ovarian cancer cells
According to the invention, ovarian cancer cell lines such as CAOV3, ES2, SKOV3 and Hey are selected as experimental objects, and the killing effect of the antibody provided by the invention on ovarian cancer is researched. The CCK-8 method is still adopted to detect and calculate the killing rate of the tumor cells, and the experimental steps are carried out according to section 3.1.
As shown in figure 2, the anti-ROR1 antibody provided by the invention also shows different killing effects on various ovarian cancer cells, wherein the killing effect on SKOV3 and CAOV3 is strongest, the killing rate can reach over 50% after 36 hours of action, and the killing effect on ES2 and Hey is second. Unlike the action on hematological tumor cells, the treatment time for the killing action of the antibodies provided by the present invention on solid tumors appears to be longer, for example, in the case of CAOV3 cells, the killing action is still weak at 24 hours, but is significantly enhanced at 36 hours. This result appears to be related to the physiological properties of solid tumors, which have been thought to secrete more extracellular matrix, delaying or blocking antibody molecule infiltration, and thus prolonging response time.
Example 4 in vivo inhibitory Effect of anti-ROR1 antibodies on hematological tumors
In order to further verify the anti-tumor effect of the antibody provided by the invention, the section mainly studies the anti-tumor effect of the anti-ROR1 antibody provided by the invention in a mouse body, and selects K562 cells to construct an animal model.
4.1 animal model preparation and treatment
C57BL/6 mice, 6-8 weeks old, were female and weighed 18-23 g. The experimental animals are raised in an SPF-level constant temperature and humidity room for 5 days, and are adaptive to the environment; 5% CO at 37 ℃2Culturing K562 cells in the environment, subculturing every 2-3 days, and adjusting the cells to logarithmic phase; centrifugally collecting the fine particlesCell, cell resuspended in sterile physiological saline, adjusted to 1X 107one/mL, the right flank hairs of the C57BL/6 mice were shaved off, and 100. mu.L of the cell suspension was injected subcutaneously into the right anterior flank of the mice. Tumor growth was observed daily and subsequent experiments were performed when tumor diameters reached between 3mm and 5 mm.
After successful modeling, experimental animals were randomly divided into two groups, and an anti-ROR1 antibody (10mg/kg) and an equal volume of physiological saline were injected weekly, tumor volume was measured weekly, tail vein blood was taken 4 weeks after treatment, blood samples were retained, and mice were sacrificed.
4.2 tumor volume
Tumor volume change was measured weekly after administration to experimental animals, and tumor size, tumor volume (L x W) was measured using a vernier caliper2) The/2 estimate, where L is the length or longest dimension and W is the width of the tumor.
The results are shown in fig. 3, the treatment with the anti-ROR1 antibody can effectively inhibit the growth rate of leukemia cells in mice, and significantly reduce the tumor volume, and the tumor volume of the treated group is about 50% of that of the control group after 4 weeks of treatment, which illustrates that the anti-ROR1 antibody provided by the invention can limit the in vivo proliferation process of the blood tumor.
4.3 Effect of anti-ROR1 antibodies on inflammatory factor expression in hematological tumor models
The occurrence and development of blood tumor are influenced by various factors, the antibody plays the role of anti-tumor, and is related to various factors such as cytokine secretion, immune cell activation, anoxic environment formation, tumor microenvironment change and the like, wherein in the antibody treatment process, the expression level of the immune factor is changed, a series of immune mechanisms can be mediated, and the synergistic anti-tumor effect is played. Therefore, in order to preliminarily investigate the action mechanism of the anti-ROR1 antibody, the change of IFN-gamma level in the plasma of the mice after treatment was detected.
After 4 weeks of treatment, tail vein blood was collected by centrifugation, and IFN-. gamma.concentration in plasma was measured by ELISA method, and the results are shown in FIG. 4. After the anti-ROR1 antibody is used for treatment, the expression level of IFN-gamma in blood can be obviously improved, and the IFN-gamma belongs to one of II-type interferons and has higher antiviral activity and wide immunoregulation effect, which indicates that the anti-ROR1 antibody can induce various immune ways in an organism by activating the expression of the IFN-gamma, and further plays a synergistic anti-tumor effect.
Example 5 in vivo inhibitory Effect of anti-ROR1 antibodies on ovarian cancer
In order to further verify the anti-tumor effect of the antibody provided by the invention, the section mainly studies the anti-tumor effect of the anti-ROR1 antibody provided by the invention in a mouse body, and an animal model is constructed by selecting SKOV3 cells.
5.1 animal model preparation and treatment
C57BL/6 mice, 6-8 weeks old, were female and weighed 18-23 g. The experimental animals are raised in an SPF-level constant temperature and humidity room for 5 days, and are adaptive to the environment; 5% CO at 37 ℃2Culturing SKOV3 cells in the environment, carrying out subculture every 2-3 days, and adjusting the cells to logarithmic phase; centrifuging to collect cells, resuspending the cells in sterile physiological saline, adjusting the concentration to 1 × 107one/mL, the right flank hairs of the C57BL/6 mice were shaved off, and 100. mu.L of the cell suspension was injected subcutaneously into the right anterior flank of the mice. Tumor growth was observed daily and subsequent experiments were performed when tumor diameters reached between 3mm and 5 mm.
After successful modeling, experimental animals were randomly divided into two groups, and an anti-ROR1 antibody (10mg/kg) and an equal volume of physiological saline were injected weekly, tumor volume was measured weekly, tail vein blood was taken 4 weeks after treatment, blood samples were retained, and mice were sacrificed.
5.2 tumor volume
Tumor volume change was measured weekly after administration to experimental animals, and tumor size, tumor volume (L x W) was measured using a vernier caliper2) The/2 estimate, where L is the length or longest dimension and W is the width of the tumor.
The result is shown in fig. 5, the anti-ROR1 antibody treatment can effectively inhibit the growth rate of ovarian cancer cells in mice, and obviously reduce the tumor volume, and after 4 weeks of treatment, the tumor volume of the treatment group is about 50% of that of the control group; notably, it appears that the anti-ROR1 antibody also appears to have a time of onset in vivo later than that of hematological tumors, e.g., two weeks after treatment in a hematological tumor model, the tumor growth rate in the treated group is significantly less than that in the control group, whereas in a solid tumor model, this trend is gradually evident after three weeks of treatment, consistent with the trend in the results of the cell experiments.
5.3 Effect of anti-ROR1 antibodies on inflammatory factor expression in hematological tumor models
Unlike hematopoietic tumors, which exist in a humoral setting, solid tumors often do not flow with the blood, and therefore the supply of nutrients is critical to the growth of tumor cells, it has been reported that abnormal proliferation of blood vessels is often accompanied in the development of solid tumors, during which Vascular Endothelial Growth Factor (VEGF) plays an important role. Therefore, in order to preliminarily investigate the action mechanism of the anti-ROR1 antibody, the change of VEGF level in the plasma of mice after treatment was detected.
After 4 weeks of treatment, tail vein blood was collected by centrifugation, and the VEGF concentration in the plasma was measured by ELISA, as shown in FIG. 6. After the anti-ROR1 antibody is used for treatment, the expression of VEGF can be obviously inhibited, so that the tumor tissues are difficult to obtain enough nutrients through a blood circulation system, and the growth of the tumor tissues is limited, although the mechanism of the anti-ROR1 antibody for playing the anti-tumor effect is various, we speculate that in a solid tumor model, the anti-ROR1 antibody can also play the anti-tumor effect together by regulating the level of immune factors and activating immune cells such as T cells.
While this invention has been particularly shown and described with references to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
Sequence listing
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Claims (10)
1. An anti-ROR1 antibody comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, respectively.
2. The antibody of claim 1, wherein the amino acid sequence of said heavy chain variable region is set forth in SEQ ID No.7 and the amino acid sequence of said light chain variable region is set forth in SEQ ID No. 8.
3. The antibody of claim 1, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a single chain antibody.
4. A nucleic acid encoding the antibody of any one of claims 1-3.
5. A recombinant expression vector comprising the nucleic acid of claim 4.
6. A recombinant expression transformant comprising the recombinant expression vector of claim 5.
7. A method for producing an antibody according to any one of claims 1 to 3, comprising the steps of: culturing the recombinant expression transformant according to claim 6, and obtaining the anti-ROR1 antibody from the culture.
8. Use of an antibody according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment of a tumour.
9. The use of claim 8, wherein the tumor is a ROR1 positive hematopoietic malignancy or a solid tumor.
10. The use according to claim 9, wherein the tumor is lung cancer, breast cancer, ovarian cancer, Chronic Lymphocytic Leukemia (CLL), Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), acute myelogenous leukemia (CML), myeloma.
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