WO2019079914A1 - Anti-ciz1 antibody - Google Patents

Abstract

CIZ1 exhibits cancer-specific high expression in small cell lung cancer and many other cancers. The prepared antibody can specifically bind to the antigenic determinant of the CIZ1 full-length protein and a variant and/or a fragment thereof on the cell surface, inhibit the tumor growth in vitro and in vivo, and can be applied to the treatment and detection of cancers.

Description

anti-CIZ1 antibody Technical field

The invention discloses a method for preparing and using an anti-CIZ1 antibody

Background technique

Cdkn1A-interacting zinc finger protein 1 (Ainscough, Rahman et al.) is a zinc finger protein capable of binding to p21 ^Cip1/Waf1 , which was discovered by Mitsui in 1999 (Mitsui, Matsumoto et al. 1999). CIZ1 can not only bind to CDK2 (Cyclin-dependent kinase 2) inhibitor p21 ^Cip1/Waf1 , but also to CDK2, cyclin A, cyclin E, CDC6 (Cell division cycle 6), estrogen receptor-α (estrogen receptor-α) ), as well as a variety of other protein combinations. CIZ1 plays a role in DNA replication and cell growth and is associated with diseases such as cancer, Alzheimer's disease, dystonia, and rheumatoid arthritis (Liu, Niu et al. 2016; Pauzaite, Thacker et al. 2016).

The CIZ1 full-length protein (NP_036259.2) has 898 amino acids (SEQ ID NO: 1) and can be divided into a DNA replication domain at the N-terminus and a nuclear matrix anchoring domain at the C-terminus. The human CIZ1 gene is located at 9q34 and its DNA is approximately 38 kb. Human full-length CIZ1 mRNA has 18 exons (NM_001257975.1), which undergo different cleavage to form a 2.7-3.2 kb mRNA transcript.

CIZ1 is involved in the formation of complexes and replicators prior to DNA replication. In the G1 phase of the cell cycle, CIZ1 binds to cyclin E and promotes the recruitment of Cdc6. At the same time, C1Z1 stabilizes the pre-replication complex and replicator through its nuclear matrix binding function. CIZ1 remained low in the G0/G1 phase of the cell cycle, but increased significantly in the early S phase and peaked in the late S phase. Inhibition of CIZ1 not only significantly reduced cell growth rates, but also reduced the proportion of cells entering the S phase (Coverley, Marr et al. 2005).

CIZ1 is highly expressed in a variety of malignancies, including lung cancer (Higgins, Roper et al. 2012), Ewing tumors (Rahman, Ainscough et al. 2007; Rahman, Aziz et al. 2010), colon cancer (Yin, Wang et Al.2013; Wang, Wang et al. 2014), gallbladder carcinoma (Zhang, Wang et al. 2015), prostate cancer (Liu, Ren et al. 2015), breast cancer (den Hollander and Kumar 2006; den Hollander, Rayala Et al. 2006), gastric cancer (Kim, Hahn et al. 2004), hepatocellular carcinoma (Lei, Wu et al. 2016; Wu, Lei et al. 2016), undifferentiated embryonic sarcoma of the liver (Hu, Chen et al. 2012).

Binding of CIZ1 to estrogen receptors increases the expression of downstream target genes and promotes breast cancer (den Hollander, Rayala et al. 2006). CIZ1 binds to beta-catenin T cell factor 4 (TCF4) and regulates the Int/Wingless (Wnt) signaling system (Zhang, Wang et al. 2015). Overexpression of CIZ1 increases the expression of Wnt target genes c-Myc, Snail and Cyclin D1, and promotes the growth and migration of gallbladder cancer cells.

CIZ1 is capable of acting on the downstream damage target DNA of p53 (PDRG1), which is highly expressed in colorectal cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, and uterine cancer. CIZ1 is able to induce the expression of tumor-associated genes AKT and PSA/KLK3 (Liu, Ren et al. 2015).

Overexpression of CIZ1 promotes growth, metastasis, invasion, colony formation, and promotion in animal models in a variety of cancer cells Progression of tumors (Yin, Wang et al. 2013; Liu, Ren et al. 2015; Zhang, Wang et al. 2015). Inhibition of CIZ1 expression reduced tumor formation in animal models (Zhang, Wang et al. 2015).

The CIZ1 mRNA splicing variant is associated with cancer. For example, the CIZ1 splice variant CIZ1ΔE4 (SEQ ID NO: 2) lacking the fourth exon is uniquely present in Ewing tumor cells (Rahman, Ainscough et al. 2007); The variant of the last 24 nucleotides of the 3' end of exon 14 CIZ1Δ14b (SEQ ID NO: 3) specifically appears in lung cancer (Higgins, Roper et al. 2012).

Based on the role of CIZ1 in the development of cancer, CIZ1 and its variants can serve as diagnostic criteria or therapeutic targets for cancer.

Summary of the invention

It is generally believed that CIZ1 is located in the nuclear matrix within the nucleus (Mitsui, Matsumoto et al. 1999; Ainscough, Rahman et al. 2007). However, when CIZ1 and p21 ^Cip1/Waf1 were co-transfected into cells, both translocated from the nucleus to the cytoplasm (Mitsui, Matsumoto et al. 1999). None of these results can rule out that some CIZ1 and its variants are distributed on the cell surface at some stage. Moreover, during the degradation of CIZ1 and its variants, fragments can be presented to the cell surface by the Major Histocompatibility Complex (MHC) system (Konig 2002; van Kasteren, Overkleeft et al. 2014) or by other means. CIZ1 and its variant proteins and their fragments located on the cell surface can serve as targets for the development of therapeutic or diagnostic antibodies.

In the present invention, an antibody having therapeutic and diagnostic functions is prepared by using CIZ1 full-length and variant proteins and fragments thereof on the cell surface as immunogens.

According to the present invention, tumor cells having high levels of cell surface expression of CIZ1 and its variant proteins and fragments thereof, such as small cell lung cancer NCI-H69 and NCI-H526 immunized BALB/c mice, and B cells in the spleen of immunized mice Fusion to SP20 mouse myeloma cells, antibody-secreting hybridoma cells were screened with CIZ1 and its variant proteins and fragments thereof. Hybridoma antibodies were prepared by expanding hybridization with hybridomas with high titers of CIZ1 and its variant proteins and fragments thereof.

According to the present invention, the prepared antibodies are added to different tumor cells in culture at different concentrations, and the cell activity is detected by the MTS method, thereby detecting the specific inhibitory effect of the prepared antibodies on the growth of different tumor cells.

According to the invention, the prepared antibody is incubated with the tumor cells, and the antibody bound to the surface of the tumor cells is combined with the fluorescently labeled secondary antibody, and the cells are analyzed by a fluorescence activated cell separator to determine the specific binding of the prepared antibody to the tumor. Cell surface.

According to the present invention, a tumor is established on a nude mouse by subcutaneous injection of a tumor cell, and when the tumor grows to a certain volume, the prepared antibody is injected into the tumor-bearing mouse at a certain dose. The tumor volume changes of the tumor-bearing mice were recorded, and the therapeutic function of the prepared antibody against the tumor of the experimental animals was determined. The changes in body weight of the bearing mice were also recorded, and it was confirmed that the preparation of the antibody had no serious side effects on the experimental animals.

According to the present invention, the gene sequence for preparing an antibody is obtained by RT-PCR and a specific primer, and the protein sequence of the antibody is derived. The gene sequence and protein sequence of the prepared antibody are analyzed with all antibody sequences in the database to determine the specificity of the prepared antibody sequence, and the preparation of the antibody is determined to be a new molecular entity (NME), and the CDR regions (Complementarity Determining Regions) and FR of the prepared antibody are determined. Framework Regions.

According to the present invention, the antibody produced may be a full-length antibody, a multispecific antibody, a bispecific antibody, a monovalent antibody, a multivalent antibody, an anti-idiotypic antibody ( Anti-idiotypic antibody), mini-bibody, Fab fragment, F(ab')2 fragment, Fv fragment, ScFv fragment, and other immunoreactive fragments, as well as fusion proteins. According to the present invention, the antibody produced may be IgA, IgD, IgE, IgG, IgG1, IgG2, IgG3, IgG4, IgM.

According to the invention, the antibody is prepared comprising an antibody linked to a cytotoxic substance (Antibody Drug Conjugates, ADC).

According to the present invention, the prepared antibody is a pharmaceutical composition for the treatment of cancer.

According to the present invention, the prepared antibody is a component of a diagnostic kit for diagnosis and detection of cancer.

According to the present invention, the prepared antibody can be used as a single therapeutic drug, and can be used in combination with other therapeutic drugs and therapeutic methods for cancer treatment.

According to the present invention, the prepared antibody can be used for Chimeric Antigen Receptor Cell Immunotherapy.

DRAWINGS

Figure 1 Anti-CIZ1 antibody specifically inhibits tumor cell growth

Anti-CIZ1 antibody B3A11 was added to Hela (A), A549 (B), MCF7 (C) and H69 (D) at a concentration of 1-100 μg/ml, and cell culture was continued for 72 hours. Cell viability was detected by MTS method. . The results showed that the anti-CIZ1 antibody B3A11 had no effect on the growth of human cervical cancer Hela cells, had no effect on the growth of human non-small cell lung cancer A549 cells, and had a weak inhibitory effect on human breast adenocarcinoma MCF7 cells, but on small cells. The growth of lung cancer cell H69 has a specific and significant inhibitory effect.

Figure 2 Anti-CIZ1 antibody specifically binds to tumor cell surface

Small cell lung cancer cells H69 were reacted with PBS (A), mouse IgG1 (B) and anti-CIZ1 antibody B3A11 antibody (C) for 30 min at 4 ° C, then bound to fluorescently labeled secondary antibody, and the treated cells were fluorescent. Activate cell separator analysis. As a result, it was confirmed that the anti-CIZ1 antibody B3A11 specifically binds to the cell surface of the small cell lung cancer cell H69.

Figure 3 Anti-CIZ1 antibody inhibits tumor growth in an experimental animal model

Small cell lung cancer H69 cells were transplanted subcutaneously into the right abdomen of 5-6 week old female nu/nu nude mice to establish a small cell lung cancer transplantation model. When the tumor volume grew to approximately 100 mm ³ , the tumor-bearing mice were randomly divided into two groups, one group as a control group, an equal volume of PBS, and the other group as an experimental group, each of which was injected with an anti-injection of 10 mg/kg. - CIZ1 antibody B3A11. Inject twice a week. All injections were injected intraperitoneally and the injection volume was 200 ml. Tumor volume was measured twice a week. The experimental results demonstrate that the anti-CIZ1 antibody B3A11 can significantly inhibit the growth of small cell lung cancer H69 tumors in an animal model.

Detailed ways

1. Antibody preparation

Tumor cells with high levels of cell surface expression of CIZ1 and its variant proteins and fragments thereof, such as small cell lung cancer NCI-H69 (American Type Culture Collection (ATCC), Virginia, USA), immunized BALB/c mice, spleens of immunized mice The B cells were fused with SP20 mouse myeloma cells (Chinese Academy of Sciences Cell Bank, Shanghai, China), and the antibody-secreting hybridoma cells were screened with CIZ1 and its variant proteins and fragments thereof. Hybridomas with a high titer of CIZ1 and its variant proteins and fragments thereof were selected for expansion culture, and hybridoma antibodies were prepared using the culture supernatant for antibody screening.

The cultured hybridoma cells were injected into the peritoneal cavity of Balb/c mice pre-empted with Freund's incomplete adjuvant to prepare antibody-containing ascites.

The antibody was affinity purified using Protein A gel (MabSelect, GE Healthcare, USA). The purified antibody was examined by SDS-PAGE electrophoresis with a purity greater than 90%, and the purified antibody was quantified by the Bradford method (Beijing Solabao Technology Co., Ltd., Beijing, China).

2. Hybridoma screening

The antibody-secreting hybridomas were screened by enzyme linked immunosorbent assay (ELISA). Polypeptide fragments of CIZ1 full-length protein and variant protein were synthesized and KLH was ligated (Nanjing Kingsray Biotechnology Co., Ltd., Nanjing, China). The KLH polypeptide was pre-coated into a 96-well plate, and the hybridoma culture supernatant was added to the diluted incubation and washed, and an HRP-labeled secondary antibody was added to detect binding of the antibody to a specific peptide by an ELISA method. We obtained 4 hybridomas, one of which had the highest titer of B3A11. B3A11 was selected for follow-up work.

3. Tumor cell growth inhibition experiment

The prepared B3A11 antibody was added to Hela, MCF7, A549, H69 tumor cells in culture at a concentration of 1-100 μg/ml, and cell activity was measured by MTS method (CellTiter 96 AQ _ueous Non-Radioactive Cell Proliferation Assay kit; Promega, USA). Thereby, the specific inhibition of the growth of different tumor cells by the prepared antibodies was examined.

B3A11 antibody has no effect on the growth of human cervical cancer Hela cells, has no effect on the growth of human non-small cell lung cancer A549 cells, has a weak inhibitory effect on human breast adenocarcinoma MCF7 cells, and is specific to small cell lung cancer cells H69. Strong growth inhibition (Figure 1).

4. fluorescence activated cell sorting (FACS)

The B3A11 antibody was reacted with tumor cell H69 at 4 ° C for 30 minutes. After washing, the antibody bound to the surface of the tumor cells was combined with a fluorescently labeled secondary antibody (Tianjin Sanjian Biotechnology Co., Ltd., Tianjin, China), and the cells were activated by fluorescence. The cells were analyzed by a separator (FACSAria II, BD Biosciences, USA) to confirm that the B3A11 antibody specifically binds to the cell surface of small cell lung cancer cell H69 (Fig. 2).

5. Tumor animal model

Small cell lung cancer H69 cells were transplanted into the right abdomen of 5-6 weeks old female nu/nu nude mice (Laboratory Animal Center of Chinese Academy of Medical Sciences, Beijing, China) to establish a small cell lung cancer transplantation model. When the tumor volume grew to approximately 100 mm ³ , the tumor-bearing mice were randomly divided into two groups, one group was used as a control group, an equal volume of PBS was injected, and the other group was an experimental group, and each mouse was injected with 10 mg/kg of B3A11. antibody. Inject twice a week. All injections were injected intraperitoneally and the injection volume was 200 μl. Tumor volume was measured twice a week, and the length and width of the tumor were measured with a caliper, and the tumor volume was calculated according to the formula: tumor volume = (length x width ² )/2 (Tomayko and Reynolds 1989). At the same time, the body weight of the bearing mice was measured twice a week. The B3A11 antibody was able to significantly inhibit small cell lung cancer H69 tumor growth in an animal model, and the tumor volume of the two groups of animals was statistically significantly different (Fig. 3). There was no significant difference in body weight between the two groups of animals, indicating that the B3A11 antibody did not have serious side effects.

6. Antibody sequence determination and analysis

RNA (RNeasy Micro Kit, QIAGEN, USA) was extracted from hybridoma cells and subjected to cDNA synthesis (First Strand cDNA Synthesis Kit, ThermoFisher). PCR primers (Fields, O'Connell et al. 2013) for amplifying the variable region of the light chain of the antibody and the variable region of the heavy chain were synthesized according to the literature sequence, and a PCR reaction was carried out. The PCR product was sequenced (Nanjing Kingsray Biotechnology Co., Ltd., Nanjing, China).

The anti-CIZ1 antibody B3A11 light chain variable region mRNA sequence (SEQ ID NO: 4) was obtained; the anti-CIZ1 antibody B3A11 heavy chain variable region mRNA sequence (SEQ ID NO: 6) was obtained; the protein sequence was deduced from the mRNA sequence. Obtaining anti-CIZ1 antibody B3A11 light chain variable region protein amino acid sequence (SEQ ID NO: 5); obtaining anti-CIZ1 antibody B3A11 heavy chain variable region protein amino acid sequence (SEQ ID NO: 7)

The gene sequence and protein sequence of the antibody are analyzed with all antibody sequences in the database to determine the specificity of the preparation of the antibody sequence, and the CDR region and the FR region of the anti-CIZ1 antibody B3A11 are determined (http://www.imgt.org/). The anti-CIZ1 antibody B3A11 was determined to be a new molecular entity (NME).

Sequence table

SEQ ID NO: 1 Human CIZ1 full-length protein amino acid sequence

SEQ ID NO: 2: Human CIZ1 variant CIZ1ΔE4 protein amino acid sequence

SEQ ID NO: 3: Human CIZ1 variant CIZ1-14b protein amino acid sequence

SEQ ID NO: 4: anti-CIZ1 antibody B3A11 light chain variable region mRNA sequence

SEQ ID NO: 5: Anti-CIZ1 antibody B3A11 light chain variable region protein amino acid sequence

SEQ ID NO: 6: Anti-CIZ1 antibody B3A11 heavy chain variable region mRNA sequence

SEQ ID NO: 7: Anti-CIZ1 antibody B3A11 heavy chain variable region protein amino acid sequence

references

Ainscough, JF, FARahman, et al. (2007). "C-terminal domains deliver the DNA replication factor Ciz1 to the nuclear matrix." J Cell Sci 120 (Pt 1): 115-124.

Coverley, D., J. Marr, et al. (2005). "Ciz1 promotes mammalian DNA replication." J Cell Sci 118 (Pt 1): 101-112.

Den Hollander, P. and R. Kumar (2006). "Dynein light chain 1 contributes to cell cycle progression by increasing cyclin-dependent kinase 2 activity in estrogen-stimulated cells." Cancer Res 66(11): 5941-5949.

Den Hollander, P., SK Rayala, et al. (2006). "Ciz1, a Novel DNA-binding coactivator of the estrogen receptor alpha, confers hypersensitivity to estrogen action." Cancer Res 66(22): 11021-11029.

Fields, C., DO'Connell, et al. (2013). "Creation of recombinant antigen-binding molecules derived from hybridomas secreting specific antibodies." Nat Protoc 8(6): 1125-1148.

Higgins, G., KMRoper, et al. (2012). "Variant Ciz1 is a circulating biomarker for early-stage lung cancer." Proc Natl Acad Sci U S A 109(45): E3128-3135.

Hu, X., H. Chen, et al. (2012). "Molecular cytogenetic characterization of undifferentiated embryonal sarcoma of the liver: a case report and literature review." Mol Cytogenet 5(1): 26.

Kim, NS, Y. Hahn, et al. (2004). "Gene cataloging and expression profiling in human gastric cancer cells by expressed sequence tags." Genomics 83(6): 1024-1045.

Konig, R. (2002). "Interactions between MHC molecules and co-receptors of the TCR." Curr Opin Immunol 14(1): 75-83.

Lei, L., J. Wu, et al. (2016). "CIZ1 interacts with YAP and activates its transcriptional activity in hepatocellular carcinoma cells." Tumour Biol 37(8): 11073-11079.

Liu, Q., N. Niu, et al. (2016). "The Role of Cdkn1A-Interacting Zinc Finger Protein 1 (CIZ1) in DNA Replication and Pathophysiology." Int J Mol Sci 17(2): 212.

Liu, T., X. Ren, et al. (2015). "Ciz1 promotes tumorigenicity of prostate carcinoma cells." Front Biosci (Landmark Ed) 20: 705-715.

Mitsui, K., A. Matsumoto, et al. (1999). "Cloning and characterization of a novel p21(Cip1/Wafl)-interacting zinc finger protein, ciz1." Biochem Biophys Res Commun 264(2):457-464 .

Pauzaite, T., U. Thacker, et al. (2016). "Emerging Roles for Ciz1 in Cell Cycle Regulation and as a Driver of Tumorigenesis." Biomolecules 7(1).

Rahman, F., JFAinscough, et al. (2007). "Cancer-associated missplicing of exon 4 influences the subnuclear distribution of the DNA replication factor CIZ1." Hum Mutat 28(10): 993-1004.

Rahman, FA, N. Aziz, et al. (2010). "Differential detection of alternative spliced variants of Ciz1 in normal and cancer cells using a custom exon-junction microarray." BMC Cancer 10:482.

Tomayko, MM and CP Reynolds (1989). "Determination of subcutaneous tumor size in athymic (nude) mice." Cancer Chemother Pharmacol 24(3): 148-154.

Van Kasteren, SI, H. Overkleeft, et al. (2014). "Chemical biology of antigen presentation by MHC molecules." Curr Opin Immunol 26: 21-31.

Wang, DQ, K. Wang, et al. (2014). "Ciz1 is a novel predictor of survival in human colon cancer." Exp Biol Med (Maywood) 239(7): 862-870.

Wu, J., L. Lei, et al. (2016). "CIZ1 is upregulated in hepatocellular carcinoma and promotes the growth and migration of the cancer cells." Tumour Biol 37(4): 4735-4742.

Yin, J., C. Wang, et al. (2013). "CIZ1 regulates the proliferation, cycle distribution and colony formation of RKO human colorectal cancer cells." Mol Med Rep 8(6): 1630-1634.

Zhang, D., Y. Wang, et al. (2015). "CIZ1 promoted the growth and migration of gallbladder cancer cells." Tumour Biol 36(4): 2583-2591.

Claims (10)

1. The isolated antibody is capable of specifically binding to the CIZ1 full-length protein (SEQ ID NO: 1) and variant (SEQ ID NO: 2; SEQ ID NO: 3) and/or a fragment thereof on the cell surface.
2. According to claim 1, the antibody comprises a full length antibody, a multispecific antibody, a bispecific antibody, a monovalent antibody, a multivalent antibody, an anti-idiotypic antibody ( Anti-idiotypic antibody), micro diabody, Fab fragment, F(ab') ₂ fragment, Fv fragment, ScFv fragment, and other immunoreactive fragments, as well as fusion proteins.
3. According to claim 1, the antibody is a chimeric antibody, a humanized antibody, a human antibody, a primate-derived antibody, and other animal-derived antibodies.
4. According to claim 1, the antibody is IgA, IgD, IgE, IgG, IgG1, IgG2, IgG3, IgG4, IgM.
5. According to claim 1, the light chain variable region of the antibody has the amino acid sequence listed in SEQ ID NO: 5, and the heavy chain variable region has the amino acid sequence listed in SEQ ID NO: 7.
6. According to claim 1, the antibody comprises an antibody linked to a cytotoxic substance (ADC).
7. According to claim 1, the antibody is a pharmaceutical composition for the treatment of cancer.
8. According to claim 1, the antibody is a component of a diagnostic kit for the diagnosis and detection of cancer.
9. According to claim 1, the antibody is for use in chimeric antigen receptor cellular immunotherapy.
10. According to claim 1, the antibody is used as a sole therapeutic drug as a combination therapy with other therapeutic drugs and treatment methods for cancer treatment.

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