Humanized cereblon knock-in mouse model and method for evaluating efficacy of multiple myeloma chemotherapy using same

Abstract

The present invention relates to a humanized cereblon (CRBN) knock-in mouse model and a method for evaluating the efficacy of multiple myeloma chemotherapy by using same. Specifically, the present invention relates to a humanized CRBN knock-in mouse model in which a human CRBN gene has been inserted into a CRBN knock-out mouse, and a method for evaluating the efficacy of multiple myeloma chemotherapy by using same. The humanized CRBN knock-in mouse model and the method for evaluating the efficacy of multiple myeloma chemotherapy by using same, according to the present invention, enable low-cost and high-efficiency evaluation of the efficacy of an anticancer chemotherapeutic agent according to the expression of human CRBN in multiple myeloma, in the mouse model, thus enabling research results to be effectively applied to clinical practice.

Description

Humanized cereblon knock-in mouse model and method for evaluating the efficacy of multiple myeloma chemotherapy using the same

[0001] The present invention relates to a humanized cereblon (CRBN) knock-in mouse model and a method for evaluating the effectiveness of multiple myeloma chemotherapy using the same. Specifically, the present invention relates to a humanized cereblon (CRBN) knock-in mouse model in which the human cereblon (CRBN) gene is inserted into a cereblon (CRBN) knock-out mouse, and a method for evaluating the effectiveness of multiple myeloma chemotherapy using the same.

[0002] Cereblon (CRBN) was identified as a target gene involved in mild mental retardation in humans, and its various functions have since been elucidated. CRBN directly interacts with large-conductance calcium-activated potassium channels and regulates their surface expression. CRBN was subsequently identified as a key target of thalidomide-induced teratogenicity and as a substrate receptor for the E3 ligase complex.

[0003] Meanwhile, multiple myeloma is a cancer of plasma cells in the bone marrow. Normally, plasma cells produce antibodies and play a crucial role in immune function. However, uncontrolled growth of these cells can lead to bone pain and fractures, anemia, infections, and other complications. Although the other causes of multiple myeloma remain unknown, it is the second most common hematological malignancy.

[0004] Common clinical manifestations of multiple myeloma include peripheral neuropathy, anemia, hyperviscosity, infections, and renal failure. Bone marrow stromal cells are known to contribute to chemotherapy resistance and disease progression in multiple myeloma. Disrupting the interaction between multiple myeloma cells and stromal cells is an additional target for multiple myeloma chemotherapy.

[0005] While current treatments for multiple myeloma are helping patients, the problem is that despite these treatments, the disease progresses rapidly and the risk of relapse is high. Furthermore, due to a lack of research into the treatment mechanisms of multiple myeloma, anticancer drugs are administered randomly, with subsequent treatment decisions based on observation. Therefore, there is a need to develop novel target concepts and optimal treatments through research into the mechanisms of various multiple myeloma diseases and the development of animal models.

[0006] The purpose of the present invention (the problem to be solved) is to provide a humanized cereblon knock-in mouse model in which a human cereblon gene is inserted into a cereblon knockout mouse, and a method for evaluating the efficacy of an anticancer chemotherapeutic agent in multiple myeloma using the same.

[0007] The present invention provides a humanized cereblon knock-in mouse model in which a human cereblon gene is inserted into a cereblon (CRBN) knock-out mouse.

[0008] In addition, the present invention provides a method for evaluating the efficacy of multiple myeloma chemotherapy, comprising the steps of (a) preparing a humanized cereblon knock-in mouse model in which multiple myeloma is induced; (b) administering a multiple myeloma chemotherapy agent to the mouse model; and (c) evaluating the anticancer efficacy.

[0009] As a monotherapy, the multiple myeloma anticancer chemotherapy agent is preferably an immunomodulatory imide drug (IMiD) such as thalidomide, lenalidomide, or pomalidomide, and lenalidomide is more preferably.

[0010] As combination therapy, the above multiple myeloma anticancer chemotherapy agents include ① a combination of carfilzomib [Kyprolis] and dexamethasone (Kd), ② a combination of carfilzomib [Kyprolis], lenalidomide [Revlimid] and dexamethasone (KRd), ③ a combination of elotuzumab [empliciti], lenalidomide [Revlimid] and dexamethasone (ERd), ④ a combination of ixazomib [Ninlaro], lenalidomide [Revlimid] and It is preferable to select from the group consisting of a combination of dexamethasone (IRd), ⑤ a combination of daratumumab (darzalex), lenalidomide (lenalidomide) (Revlimid) and dexamethasone (DRd).

[0011] In the combination (Kd) of carfilzomib (Kyprolis) and dexamethasone, carfilzomib (Kyprolis) is a proteasome inhibitor that inhibits proteasomes in cancer cells, thereby inducing excessive accumulation of abnormal proteins in tumor cells and causing cancer cell death, and dexamethasone is a synthetic corticosteroid with anti-inflammatory effects.

[0012] In the combination of carfilzomib (Kyprolis), lenalidomide (Revlimid), and dexamethasone (KRd), carfilzomib (Kyprolis) is a type of proteasome inhibitor that induces excessive accumulation of abnormal proteins in tumor cells by inhibiting proteasomes in cancer cells, thereby causing cancer cell death; lenalidomide (Revlimid) is a type of immunomodulatory imide drug (IMiDs) that directly inhibits cancer cell proliferation and enhances immune function; and dexamethasone is a synthetic corticosteroid with anti-inflammatory effects.

[0013] In the combination of elotuzumab (empliciti), lenalidomide (Revlimid), and dexamethasone (ERd), elotuzumab (empliciti) is a monoclonal antibody that binds to the CS1 protein, blocks CS1, and helps the immune system kill the cancer; lenalidomide (Revlimid) is a type of immunomodulatory imide drug (IMiDs) that directly suppresses cancer cell proliferation and enhances immune function; and dexamethasone is a synthetic corticosteroid with anti-inflammatory properties.

[0014] In the combination of ixazomib (Ninlaro), lenalidomide (Revlimid), and dexamethasone (IRd), ixazomib (Ninlaro) is a monoclonal antibody that acts as a proteasome (specifically, 20S proteasome) inhibitor; lenalidomide (Revlimid) is a type of immunomodulatory imide drug (IMiDs) that directly inhibits cancer cell proliferation and enhances immune function; and dexamethasone is a synthetic corticosteroid with anti-inflammatory properties.

[0015] In the combination of daratumumab (darzalex), lenalidomide (Revlimid), and dexamethasone (DRd), daratumumab (darzalex) is an anti-CD38 antibody; lenalidomide (Revlimid) is a type of immunomodulatory imide drug (IMiD) that directly inhibits cancer cell proliferation and enhances immune function; and dexamethasone is a synthetic corticosteroid with anti-inflammatory properties.

[0016] The humanized CRBN knock-in mouse model of the present invention and the method for evaluating the efficacy of multiple myeloma chemotherapy using the same can evaluate the efficacy of chemotherapy agents according to human CRBN expression in multiple myeloma at low cost and with high efficiency in a mouse model, and thus the research results can be effectively applied to clinical practice.

[0017] Figure 1 is a schematic diagram of the generation of CRBN mutant (p.V380E and p.I391V) mice using the TILD-Crispr method.

[0018] Figure 2 is a schematic diagram (F0 & F1) of the PCR primers used for mutant Crbn genotyping. Two primer pairs (indicated by the pairs of differently colored arrows and the expected sizes of the PCR amplicons) were used for genotyping. The PCR primer pairs covered the 5' region of the TILD donor DNA [primers F1, R1, and R2 (yellow), resulting in a 1246-bp product designated as "control" and a 910-bp product designated as "V380E KI"]. The 3' region of the TILD donor DNA [primers F1, R1, and R2 (green), resulting in a 1252-bp product designated as "control" and a 913-bp product designated as "I391V KI"] was used to identify individuals with the mutant Crbn KI.

[0019] Figure 3 shows a partial alignment of mouse wild type (WT) and mutant knock-in (KI) TILD-DNA.

[0020] Figure 4 shows a multiple myeloma animal model according to the present invention.

[0021] Figure 5 shows the screening results of lenalidomide using a multiple myeloma animal model according to the present invention.

[0022] Figure 6 shows the results of the combination of ① carfilzomib [Kyprolis] and dexamethasone (Kd), ② carfilzomib [Kyprolis], lenalidomide [Revlimid] and dexamethasone (KRd), ③ elotuzumab [empliciti], lenalidomide [Revlimid] and dexamethasone (ERd), ④ ixazomib [Ninlaro] using a multiple myeloma animal model according to the present invention. The results of screening for the combination of lenalidomide [Revlimid] and dexamethasone (IRd), and the combination of ⑤ daratumumab [darzalex], lenalidomide [Revlimid] and dexamethasone (DRd) are shown.

[0023] The inventors of the present invention developed a humanized CRBN knock-in mouse model in which the human CRBN gene was inserted into a CRBN knockout mouse model in order to compare the efficacy of anticancer chemotherapeutic agents of the IMiDs (immunomodulatory imide drugs) series according to the expression of CRBN in the treatment of multiple myeloma, and developed a system for evaluating the efficacy of various anticancer chemotherapeutic agents for the treatment of multiple myeloma using this model.

[0024] Accordingly, the present invention provides a humanized cereblon knock-in mouse model in which a human cereblon gene is inserted into a cereblon (CRBN) knock-out mouse.

[0025] In addition, the present invention provides a method for evaluating the efficacy of multiple myeloma chemotherapy, comprising the steps of (a) preparing a humanized cereblon knock-in mouse model in which multiple myeloma is induced; (b) administering a multiple myeloma chemotherapy agent to the mouse model; and (c) evaluating the anticancer efficacy.

[0026] As monotherapy, the multiple myeloma chemotherapy agent is preferably an immunomodulatory imide drug (IMiD), such as lenalidomide (Revlimid).

[0027] As combination therapy, the above multiple myeloma anticancer chemotherapy agents include ① a combination of carfilzomib [Kyprolis] and dexamethasone (Kd), ② a combination of carfilzomib [Kyprolis], lenalidomide [Revlimid] and dexamethasone (KRd), ③ a combination of elotuzumab [empliciti], lenalidomide [Revlimid] and dexamethasone (ERd), and ④ ixazomib [Ninlaro]. It is preferable to select from the group consisting of a combination of lenalidomide [Revlimid] and dexamethasone (IRd), ⑤ a combination of daratumumab [darzalex], lenalidomide [Revlimid] and dexamethasone (DRd).

[0028] The present invention is described in more detail below through specific examples. However, the following examples should not be construed as limiting the scope of the present invention, and those skilled in the art will be able to make various modifications or applications of the following examples.

[0029] Example

[0030] Example 1. Creation of a Cereblon knock-in mouse model

[0031] The present inventors created a humanized CRBN knock-in mouse model with the human CRBN gene inserted as follows.

[0032] C57BL / 6N female mice were treated with pregnant mare serum gonadotropin (PMSG) (10 IU) and human chorionic gonadotropin (10 IU). After 48 h, the mice were allowed to mate with C57BL / 6N male mice. The following day, female mice with vaginal plugs were euthanized, and embryos were fertilized. A mixture of sgRNA (250 ng / μL) (5'-GGGAAACCAGCTGTGCACTG-3', 5'-ACAGATCTTGCACTGGGCAA-3'), Cas9 mRNA (50 ng / μL), and cDNA donor (50 ng / μL) was microinjected into single-cell fertilized eggs, which were then cultured in vitro at 37°C for 1–2 h. Injected one-cell stage embryos were transferred into the oviducts of pseudopregnant recipient mice. F0 mice were genotyped by PCR using various primer sets (V380E and I391V) and tail samples, and the amplicons were then used for Sanger sequencing. KI-positive pups were mated with wild-type offspring to obtain F1 mice. The genotypes of F1 mice were determined by PCR using the same primer set. Mice were anesthetized with 4% isoflurane, and a 2-mm piece of tail was collected from each mouse. Genomic DNA was extracted from the tail samples using the G-DEX Genomic DNA Extraction Kit (iNtRON Biotechnology) according to the manufacturer's instructions. PCR was performed in 20 μL reaction volumes using 100 to 150 ng of extracted genomic DNA, using 2X Taq PCR smart mix2 (Solgent). Amplicons were analyzed by 2% agarose gel electrophoresis.Target amplicons were purified using the MEGAquick-spin total fragment DNA purification kit (iNtRON Biotechnology) and confirmed by Sanger sequencing. WT and KI mice were maintained under a 12-h dark / 12-h light cycle.

[0033] Example 2. Evaluation of the efficacy of lenalidomide monotherapy using a cereblon knock-in mouse model.

[0034] The present inventors evaluated the efficacy of an anticancer drug candidate as follows using a humanized Cereblon knock-in mouse model into which the human Cereblon gene of the present invention was inserted, produced in Example 1 above.

[0035] After transplanting a tumor into the CRBN KI mouse, when the transplanted tumor grew stably, the mouse was treated with 10 mg / kg of lenalidomide for 47 days, and changes in tumor size were observed using IVIS (in vivo imaging system).

[0036] As a result, the anticancer effect of lenalidomide was not observed in CRBN-deficient knockout (KO) mice, whereas the anticancer effect of lenalidomide was prominent in CRBN knock-in mice that had the human cereblon gene inserted. This suggests that the human cereblon gene plays a key role in the anticancer mechanism of action of lenalidomide. The results are shown in Figure 5.

[0037] Example 3. Evaluation of the efficacy of combination therapy using a cereblon knock-in mouse model.

[0038] The present inventors evaluated the efficacy of multiple myeloma chemotherapy using a humanized CRBN knock-in mouse model into which the human CRBN gene of the present invention was inserted, produced in Example 1, as follows.

[0039] After transplanting the tumor into the CRBN KI mouse, if the transplanted tumor grows stably, ① the combination of carfilzomib [Kyprolis] and dexamethasone (Kd), ② the combination of carfilzomib [Kyprolis], lenalidomide [Revlimid] and dexamethasone (KRd), ③ the combination of elotuzumab [empliciti], lenalidomide [Revlimid] and dexamethasone (ERd), ④ ixazomib [Ninlaro], The combination of lenalidomide (Revlimid) and dexamethasone (IRd), and the combination of daratumumab (Darzalex), lenalidomide (Revlimid), and dexamethasone (DRd) were each treated in mice at a dose of 10 mg / kg for 60 days, and changes in tumor size were observed. In addition, cell viability after drug treatment was measured according to the expression level of CRBN in tumor cells. The results are shown in Fig. 6.

[0040] In patients with high CRBN expression in multiple myeloma, lenalidomide-based drug combinations exhibited stronger anticancer effects, suggesting that the therapeutic efficacy of lenalidomide is determined by CRBN expression levels. Therefore, assessing CRBN expression levels prior to treatment planning should be considered an essential step to maximize the therapeutic efficacy of lenalidomide in patients with multiple myeloma.

Claims

1. CRBN knock-in mouse model with human cereblon (CRBN) gene inserted.

2. A method for producing a CRBN knock-in mouse model, comprising the step of inserting a human CRBN gene into a CRBN knock-out mouse. 3.(a) Step of creating a CRBN knock-in mouse model induced with multiple myeloma; (b) administering a multiple myeloma anticancer chemotherapy agent to the mouse model; and (c) A method for evaluating the effectiveness of anticancer chemotherapy for multiple myeloma, comprising a step of evaluating anticancer efficacy.

4. In paragraph 3, A method for evaluating the efficacy of multiple myeloma chemotherapy, wherein the above multiple myeloma chemotherapy agent is lenalidomide, an immunomodulatory imide drug (IMiDs).

5. In paragraph 3, The above multiple myeloma anticancer chemotherapy agents are ① a combination of carfilzomib [Kyprolis] and dexamethasone (Kd), ② a combination of carfilzomib [Kyprolis], lenalidomide [Revlimid] and dexamethasone (KRd), ③ a combination of elotuzumab [empliciti], lenalidomide [Revlimid] and dexamethasone (ERd), ④ a combination of ixazomib [Ninlaro], lenalidomide [Revlimid] and A method for evaluating the efficacy of multiple myeloma chemotherapy selected from the group consisting of a combination of dexamethasone (IRd), ⑤ a combination of daratumumab (darzalex), lenalidomide (Revlimid), and dexamethasone (DRd).

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