WO2022253280A1

WO2022253280A1 - Genetically modified non-human animal with human or chimeric bcma

Info

Publication number: WO2022253280A1
Application number: PCT/CN2022/096667
Authority: WO
Inventors: Shujin Zhang; Zan ZHANG; Yanhui NIE
Original assignee: Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
Priority date: 2021-06-01
Filing date: 2022-06-01
Publication date: 2022-12-08
Also published as: CN115010799A

Abstract

Provided are genetically modified non-human animals that express a human or chimeric (e.g., humanized) BCMA, and methods of use thereof.

Description

GENETICALLY MODIFIED NON-HUMAN ANIMAL WITH HUMAN OR CHIMERIC BCMA

CLAIM OF PRIORITY

This application claims the benefit of Chinese Patent Application App. No. CN202110608267.9, filed on June 1, 2021. The entire contents of the foregoing applications are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) BCMA, and methods of use thereof.

BACKGROUND

B-cell maturation antigen (BCMA) is a cell membrane bound tumor necrosis factor receptor family member that is expressed exclusively on late stage normal and malignant B-cells and plasma cells. Addition of two of its ligands, B-cell activating factor and a proliferation inducting ligand, to normal B-cells cause B-cell proliferation and antibody production. Serum BCMA is elevated among patients with multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) , and is a prognostic and monitoring tool for these patients. Thus, BCMA is an important target for cancer therapies.

The traditional drug research and development for these cancer therapies involve animal models. However, because of the differences between humans and animals, the test results obtained from the use of conventional experimental animals for in vivo pharmacological test may not reflect the real disease state and the interaction at the targeting sites, resulting in that the results in many clinical trials are significantly different from the animal experimental results. Therefore, the development of humanized animal models that are suitable for human antibody screening and evaluation will significantly improve the efficiency of new drug development and reduce the cost for drug research and development.

SUMMARY

This disclosure is related to an animal model with human BCMA or chimeric BCMA. The animal model can express human BCMA or chimeric BCMA (e.g., humanized BCMA) protein in its body. It can be used in the studies on the function of BCMA gene, and can be used in the screening and evaluation of anti-human BCMA antibodies. In addition, the animal models prepared by the methods described herein can be used in drug screening, pharmacodynamics studies, treatments for immune-related diseases (e.g., autoimmune diseases) , and cancer therapy for human BCMA target sites; they can also be used to facilitate the development and design of new drugs, and save time and cost. In summary, this disclosure provides a powerful tool for studying the function of BCMA protein and a platform for screening cancer drugs.

In one aspect, the disclosure is related to a genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric BCMA.

In some embodiments, the sequence encoding the human or chimeric BCMA is operably linked to an endogenous regulatory element at the endogenous BCMA gene locus in the at least one chromosome.

In some embodiments, the sequence encoding a human or chimeric BCMA comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human BCMA (NP_001183.2 (SEQ ID NO: 2) ) .

In some embodiments, the sequence encoding a human or chimeric BCMA comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 11.

In some embodiments, the sequence encoding a human or chimeric BCMA comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to amino acids 1-47 or 1-54 of SEQ ID NO: 2.

In some embodiments, the animal is a mammal, e.g., a monkey, a rodent, or a mouse.

In some embodiments, the animal is a mouse.

In some embodiments, the animal does not express endogenous BCMA.

In some embodiments, the animal has one or more cells expressing human or chimeric BCMA.

In some embodiments, the animal has one or more cells expressing human or chimeric BCMA, and a human BCMA ligand (e.g., APRIL or BAFF) can bind to the expressed human or chimeric BCMA.

In some embodiments, the animal has one or more cells expressing human or chimeric BCMA, and an endogenous BCMA ligand (e.g., APRIL or BAFF) can bind to the expressed human or chimeric BCMA.

In one aspect, the disclosure is related to a genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous BCMA with a sequence encoding a corresponding region of human BCMA at an endogenous BCMA gene locus.

In some embodiments, the sequence encoding the corresponding region of human BCMA is operably linked to an endogenous regulatory element at the endogenous BCMA locus, and one or more cells of the animal expresses a chimeric BCMA.

In some embodiments, the animal does not express endogenous BCMA.

In some embodiments, the replaced sequence encodes all or a portion of the extracellular region of endogenous BCMA.

In some embodiments, the animal has one or more cells expressing a chimeric BCMA having an extracellular region, a transmembrane region, and a cytoplasmic region, wherein the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%identical to the extracellular region of human BCMA.

In some embodiments, the extracellular region of the chimeric BCMA has a sequence that has at least 10, 20, 30, 40, or 47 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human BCMA.

In some embodiments, the sequence encoding a region of endogenous BCMA comprises exon 1 and/or exon 2, or a part thereof, of the endogenous BCMA gene.

In some embodiments, the animal is a mouse, and the sequence encoding a region of endogenous BCMA starts within exon 1 and ends within exon 2 of the endogenous mouse BCMA gene.

In some embodiments, the animal is heterozygous with respect to the replacement at the endogenous BCMA gene locus.

In some embodiments, the animal is homozygous with respect to the replacement at the endogenous BCMA gene locus.

In one aspect, the disclosure is related to a method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous BCMA gene locus, a sequence encoding a region of an endogenous BCMA with a sequence encoding a corresponding region of human BCMA.

In some embodiments, the sequence encoding the corresponding region of human BCMA comprises exon 1 and/or exon 2, or a part thereof, of a human BCMA gene.

In some embodiments, the sequence encoding the corresponding region of human BCMA starts within exon 1 and ends within exon 2 of a human BCMA gene.

In some embodiments, the sequence encoding the corresponding region of human BCMA encodes amino acids 1-47 or 1-54 of SEQ ID NO: 2.

In some embodiments, the region of an endogenous BCMA is located within the extracellular region.

In some embodiments, the animal is a mouse, and the sequence encoding a region of an endogenous BCMA starts within exon 1 and ends within exon 2 of the endogenous mouse BCMA gene.

In one aspect, the disclosure is related to a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric BCMA polypeptide, wherein the chimeric BCMA polypeptide comprises at least 25 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human BCMA, wherein the animal expresses the chimeric BCMA.

In some embodiments, the chimeric BCMA polypeptide has at least 40 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human BCMA extracellular region.

In some embodiments, the chimeric BCMA polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 1-47 or 1-54 of SEQ ID NO: 2.

In some embodiments, the nucleotide sequence is operably linked to an endogenous BCMA regulatory element of the animal.

In some embodiments, the chimeric BCMA polypeptide comprises an endogenous BCMA transmembrane region and/or an endogenous BCMA cytoplasmic region.

In some embodiments, the nucleotide sequence is integrated to an endogenous BCMA gene locus of the animal.

In some embodiments, the chimeric BCMA has at least one mouse BCMA activity and/or at least one human BCMA activity.

In one aspect, the disclosure is related to a method of making a genetically-modified non-human animal cell that expresses a chimeric BCMA, the method comprising: replacing at an endogenous BCMA gene locus, a nucleotide sequence encoding a region of endogenous BCMA with a nucleotide sequence encoding a corresponding region of human BCMA, thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the chimeric BCMA, wherein the non-human animal cell expresses the chimeric BCMA.

In some embodiments, the chimeric BCMA comprises: a humanized BCMA extracellular region; and a transmembrane and/or a cytoplasmic region of endogenous BCMA.

In some embodiments, the nucleotide sequence encoding the chimeric BCMA is operably linked to an endogenous BCMA regulatory region, e.g., promoter.

In some embodiments, the animal further comprises a sequence encoding an additional human or chimeric protein.

In some embodiments, the additional human or chimeric protein is tumor necrosis factor ligand superfamily member 13 (APRIL) , programmed cell death protein 1 (PD-1) , IL4, Colony Stimulating Factor 1 (CSF1) , IL34, C-C Motif Chemokine Receptor 2 (CCR2) , CD40, C-X-C Motif Chemokine Receptor 4 (CXCR4) , Vascular Endothelial Growth Factor (VEGF) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , Lymphocyte Activating 3 (LAG-3) , B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , CD27, CD28, CD47, CD137, CD154, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3) , Glucocorticoid-Induced TNFR- Related Protein (GITR) , Signal regulatory protein α (SIRPα) or TNF Receptor Superfamily Member 4 (OX40) .

In some embodiments, the additional human or chimeric protein is PD-1, and the animal expresses the human or chimeric PD-1.

In some embodiments, the additional human or chimeric protein is APRIL, PD-1, IL4, CSF1, IL34, CCR2, CD40, CXCR4, VEGF, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPα or OX40.

In one aspect, the disclosure is related to a method of determining effectiveness of an anti-BCMA antibody for the treatment of cancer, comprising: administering the anti-BCMA antibody to the animal described herein, wherein the animal has a cancer; and determining the inhibitory effects of the anti-BCMA antibody to the cancer.

In some embodiments, the cancer comprises one or more cells that express BCMA.

In some embodiments, the cancer comprises one or more cancer cells that are injected into the animal.

In some embodiments, determining the inhibitory effects of the anti-BCMA antibody to the cancer involves measuring tumor volume or a fluorescence level of tumor cells in the animal.

In some embodiments, the cancer is hematologic malignancies (e.g., multiple myeloma, chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL) , and Hodgkin lymphoma) , tonsil cancer, lymph node cancer, duodenum cancer, colon cancer, stomach cancer, rectum cancer, spleen cancer, salivary gland cancer, small instestine cancer, appendix cancer, thymus cancer, breast cancer, urinary bladder cancer, or gallbladder canc er.

In one aspect, the disclosure is related to a method of determining effectiveness of an anti-BCMA antibody and an additional therapeutic agent for the treatment of cancer, comprising administering the anti-BCMA antibody and the additional therapeutic agent to the animal described herein, wherein the animal has a cancer; and determining the inhibitory effects on the canc er.

In some embodiments, the animal further comprises a sequence encoding a human or chimeric programmed cell death protein 1 (PD-1) .

In some embodiments, the animal further comprises a sequence encoding a human or chimeric programmed death-ligand 1 (PD-L1) .

In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody or an anti-PD-L1 antibody.

In some embodiments, the cancer comprises one or more cancer cells that express BCMA, PD-L1, or PD-L2.

In some embodiments, the cancer is caused by injection of one or more cancer cells into the animal.

In some embodiments, determining the inhibitory effects of the treatment involves measuring tumor volume or a fluorescence level of the cancer cells in the animal.

In some embodiments, the animal has cancer is hematologic malignancies (e.g., multiple myeloma, chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL) , and Hodgkin lymphoma) , tonsil cancer, lymph node cancer, duodenum cancer, colon cancer, stomach cancer, rectum cancer, spleen cancer, salivary gland cancer, small instestine cancer, appendix cancer, thymus cancer, breast cancer, urinary bladder cancer, or gallbladder cancer.

In one aspect, the disclosure is related to a protein comprising an amino acid sequence, wherein the amino acid sequence is one of the following: an amino acid sequence set forth in SEQ ID NO: 2 or 11; an amino acid sequence that is at least 90%identical to SEQ ID NO: 2 or 11; an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 2 or 11; an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 2 or 11 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and an amino acid sequence that comprises a substitution, a deletion and /or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 2 or 11.

In one aspect, the disclosure is related to a nucleic acid comprising a nucleotide sequence, wherein the nucleotide sequence is one of the following: a sequence that encodes the protein described herein; SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 14 or 15; a sequence that is at least 90%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 14 or 15; and a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 14 or 15.

In one aspect, the disclosure is related to a cell comprising the protein described herein and/or the nucleic acid described herein.

In one aspect, the disclosure is related to an animal comprising the protein described herein and/or the nucleic acid described herein.

In one aspect, the disclosure is related to a method of determining toxicity of an anti-BCMA antibody, the method comprising administering the anti-BCMA antibody to the animal described herein; and determining weight change of the animal.

In some embodiments, the method further comprising performing a blood test (e.g., determining red blood cell count) .

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing a comparison between mouse BCMA gene locus and human BCMA gene locus.

FIG. 2 is a schematic diagram showing humanized BCMA gene locus.

FIG. 3 is a schematic diagram showing a BCMA gene targeting strategy.

FIG. 4 shows PCR identification results of F1 generation mice by primers L-GT-F/Mut-R. F1-01, F1-02, F1-03, F1-04 and F1-05 are mouse numbers. M is a marker. WT is a wild-type control. H ₂O is a water control.

FIG. 5 is a schematic diagram showing a BCMA gene targeting strategy.

FIGS. 6A-6B show sgRNA activity detection results as measured by a UCA Kit.

FIGS. 7A-7B show PCR identification results of F0 generation mice by primers L-GT-F/Mut-R and R-GT-F/R-GT-R. F0-01 and F0-02 are mouse numbers. M is a marker. WT is a wild-type control. H ₂O is a water control.

FIGS. 8A-8B show PCR identification results of F1 generation mice by primers L-GT-F/Mut-R and R-GT-F/R-GT-R. F1-27, F1-30, F1-31, F1-32, F1-34, F1-35, F1-36, F1-41, F1-42, F1-43, F1-44, F1-45 and F1-46 are mouse numbers. M is a marker. WT is a wild-type control. H ₂O is a water control.

FIG. 9 shows Southern Blot results of cells after recombination using the 5' Probe and 3' Probe. F1-27, F1-30, F1-31, F1-34, F1-35, F1-36, F1-41, F1-42, F1-43, F1-44, F1-45 and F1-46 are mouse numbers. WT is a wild-type control.

FIGS. 10A-10C show RT-PCR identification results of wild-type C57BL/6 mice (WT) or BCMA gene humanized homozygous mice (H/H) to detect expression of mouse BCMA (mBCMA) , human BCMA (hBCMA) , and GAPDH. H ₂O is a water control.

FIGS. 11A-1B shows PCR identification results of BCMA gene knockout mice. KO-12, KO-13, KO-14, KO-15, KO-16, KO-17 and KO-18 are mouse numbers. WT is a wild-type control. H ₂O is a water control.

FIG. 12 shows the alignment between human BCMA amino acid sequence (NP_001183.2; SEQ ID NO: 2) and mouse BCMA amino acid sequence (NP_035738.1; SEQ ID NO: 1) .

FIG. 13 shows the alignment between human BCMA amino acid sequence (NP_001183.2; SEQ ID NO: 2) and rat BCMA amino acid sequence (NP_001099231.1; SEQ ID NO: 52) .

DETAILED DESCRIPTION

This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) BCMA, and methods of use thereof. Experimental animal models are an indispensable research tool for studying the effects of these antibodies (e.g., BCMA antibodies) . Common experimental animals include mice, rats, guinea pigs, hamsters, rabbits, dogs, monkeys, pigs, fish and so on. However, there are many differences between human and animal genes and protein sequences, and many human proteins cannot bind to the animal's homologous proteins to produce biological activity, leading to that the results of many clinical trials do not match the results obtained from animal experiments. A large number of clinical studies are in urgent need of better animal models. With the continuous development and maturation of genetic engineering technologies, the use of human cells or genes to replace or substitute an animal's endogenous similar cells or genes to establish a biological system or disease model closer to human, and establish the humanized experimental animal models (humanized animal model) has provided an important tool for new clinical approaches or means. In this context, the genetically engineered animal model, that is, the use of genetic manipulation techniques, the use of human normal or mutant genes to replace animal homologous genes, can be used to establish the genetically modified animal models that are closer to human gene systems. The humanized animal models have various important applications. For example, due to the presence of human or humanized genes, the animals can express or express in part of the proteins with human functions, so as to greatly reduce the differences in clinical trials between humans and animals, and provide the possibility of drug screening at animal levels.

BCMA

B cell maturation antigen (BCMA) , also termed tumor necrosis factor receptor superfamily member 17 (TNFRS17) , is a type III transmembrane protein without a signal-peptide and containing cysteine-rich extracellular domains. Alignment of the human and murine BCMA protein sequences revealed a conserved motif of six cysteines in the N-terminal part, which strongly suggests that the BCMA protein belongs to the tumor necrosis factor receptor (TNFR) superfamily. BCMA, along with two related TNFR superfamily B-cell activation factor receptor (BAFF-R) and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) , critically regulate B cell proliferation and survival, as well as maturation and differentiation into plasma cells (PCs) . These three functionally related receptors support long-term survival of B cells at different stages of development by binding to B-cell activating factor (BAFF) and/or a proliferation-induced ligand (APRIL) , their cognate ligands. Specifically, BCMA is only induced in late memory B cells committed to the PC differentiation and is present on all PCs. Expression of BCMA is induced, while BAFF-R is decreased, during plasma cells (PC) differentiation from B cells. Studies from BCMA-knockdown mice further indicate that BCMA is most important for long-lived PC survival but is dispensable for overall B cell homeostasis.

B cell maturation antigen is exclusively expressed on the surface of plasmablasts and differentiated PCs, but not on memory B, naive B cells, CD34+ hematopoietic stem cells, and other normal tissue cells. BCMA mRNA and protein are more highly expressed on malignant than normal PCs, as validated by multiple gene expression profiling and immunohistochemistry (IHC) studies. cDNA copies of BCMA were detected by qPCR in several hematologic tissues including white blood cells, BM, lymph node, spleen, and tonsil. In normal tissues, low levels of BCMA cDNA copies were detected in the samples of testis, trachea and samples from gastrointestinal organs like duodenum, rectum, and stomach. When the expression was evaluated by IHC, BCMA protein expression was only detected on MM cells, lymphoid cells, or PCs from normal human organs such as duodenum, rectum, and stomach. However, BCMA protein expression was not detected on the other cell types in these organs. BCMA expression was negative on naive and memory B cells, weak on founder B cells from germinal center (GC) and Reed-Sternberg cells, positive on GC B cells, but highly positive on plasmacytoid B cells. Based on these findings, BCMA protein is highly and specifically expressed on PCs, low levels of BCMA RNA detected in these normal organs would be due to existence of PCs. These data confirm BCMA as a very promising MM antigen for targeted immunotherapy.

Both BCMA ligands APRIL (tumor necrosis factor ligand superfamily member 13) and BAFF (B-cell activating factor) , to a lesser extent, are critical BM factors supporting growth and survival of malignant PCs in MM. The levels of both ligands are significantly increased in serum samples of MM patients vs normal controls. APRIL, which does not bind to BAFF-R, preferably binds to BCMA with much higher affinity than BAFF (nM vs μM) , whereas BAFF has an approximate 100-fold selectivity for binding to BAFF receptor (BAFF-R) over BCMA. Coupled with the fact that APRIL also binds to TACI on PCs via interaction with CD138/syndecan-1, APRIL is more specific to PCs than BAFF. Importantly, APRIL directly promotes MM cell growth and survival in vivo, since APRIL knockout mice injected with human MM cell lines live longer than wild-type mice under similar conditions. These results strongly supporting targeting BCMA for novel MM treatments.

A detailed description of BCMA and its function can be found, e.g., in Sanchez, Eric, et al. "The clinical significance of B-cell maturation antigen as a therapeutic target and biomarker. " Expert Review of Molecular Diagnostics 18.4 (2018) : 319-329; Cho, Shih-Feng, Kenneth C. Anderson, and Yu-Tzu Tai. "Targeting B cell maturation antigen (BCMA) in multiple myeloma: potential uses of BCMA-based immunotherapy. " Frontiers in immunology (2018) : 1821; each of which is incorporated by reference in its entirety.

In human genomes, BCMA gene (Gene ID: 608) locus has three exons, exon 1, exon 2, and exon 3 (FIG. 1) . The BCMA protein also has an extracellular region, a transmembrane region, and a cytoplasmic region. The nucleotide sequence for human BCMA mRNA is NM_001192.3, and the amino acid sequence for human BCMA is NP_001183.2 (SEQ ID NO: 2) . The location for each exon and each region in human BCMA nucleotide sequence and amino acid sequence is listed below:

Table 1

The human BCMA gene (Gene ID: 608) is located in Chromosome 16 of the human genome, which is located from nucleotide 11,965,210 to 11,968,068 (GRCh38. p13 (GCF_000001405.39) ) . The 5'-UTR is from 11,965,210 to 11,965,324; exon 1 is from 11,965,210 to 11,965,454; the first intron is from 11,965,455 to 11,966,194; exon 2 is from 11,966,195 to 11,966,341; the second intron is from 11,966,342 to 11,967,569; exon 3 is from 11,967,570 to 11,968,068; the 3'-UTR is from 11,967,848 to 11,968,068 (base on transcript NM_001192.3) . All relevant information for human BCMA locus can be found in the NCBI website with Gene ID: 608, which is incorporated by reference herein in its entirety.

In mice, BCMA gene locus has three exons, exon 1, exon 2, and exon 3 (FIG. 1) . The mouse BCMA protein also has an extracellular region, a transmembrane region, and a cytoplasmic region. The nucleotide sequence for mouse BCMA mRNA is NM_011608.1, the amino acid sequence for mouse BCMA is NP_035738.1 (SEQ ID NO: 1) . The location for each exon and each region in the mouse BCMA nucleotide sequence and amino acid sequence is listed below:

Table 2

The mouse BCMA gene (Gene ID: 21935) is located in Chromosome 16 of the mouse genome, which is located from nucleotide 11131131 to 11137938 (GRCm39 (GCF_000001635.27) ) . The 5'-UTR is from 11,131,676 to 11,131,816, exon 1 is from 11,131,676 to 11,131,931, the first intron is from 11,131,932 to 11,133,038, exon 2 is from 11,133,039 to 11,133,197, the second intron is from 11,133,198 to 11,137,538, exon 3 is from 11,137,539 to 11,137,938, the 3'-UTR is from 11,137,823 to 11,137,938, base on transcript NM_011608.1. All relevant information for mouse BCMA locus can be found in the NCBI website with Gene ID: 21935, which is incorporated by reference herein in its entirety.

FIG. 12 shows the alignment between human BCMA amino acid sequence (NP_001183.2, SEQ ID NO: 2) and mouse BCMA amino acid sequence (NP_035738.1, SEQ ID NO: 1) . Thus, the corresponding amino acid residue or region between human and mouse BCMA can be found in FIG. 12.

BCMA genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for BCMA in Rattus norvegicus (rat) is 287034, the gene ID for BCMA in Macaca mulatta (Rhesus monkey) is 712212, the gene ID for BCMA in Canis lupusfamiliaris (dog) is 100684674, and the gene ID for BCMA in Equus caballus (horse) is 100055833. The relevant information for these genes (e.g., intron sequences, exon sequences, amino acid residues of these proteins) can be found, e.g., in NCBI database, which is incorporated by reference herein in its entirety. FIG. 13 shows the alignment between human BCMA amino acid sequence (NP_001183.2, SEQ ID NO: 2) and rat BCMA amino acid sequence (NP_001099231.1) . Thus, the corresponding amino acid residue or region between human and rat BCMA can be found in FIG. 13.

The present disclosure provides human or chimeric (e.g., humanized) BCMA nucleotide sequence and/or amino acid sequences. In some embodiments, the entire sequence of mouse exon 1, exon 2, exon 3, extracellular region, transmembrane region, and/or cytoplasmic region are replaced by the corresponding human sequence. In some embodiments, a “region” or “portion” of mouse exon 1, exon 2, exon 3, extracellular region, transmembrane region, and/or cytoplasmic region are replaced by the corresponding human sequence. The term “region” or “portion” can refer to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 500, or 555nucleotides, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or 185 amino acid residues. In some embodiments, the “region” or “portion” can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%identical to exon 1, exon 2, exon 3, extracellular region, transmembrane region, or cytoplasmic region of mouse BCMA gene; or exon 1, exon 2, exon 3, extracellular region, transmembrane region, or cytoplasmic region of human BCMA gene. In some embodiments, a region, a portion, or the entire sequence of mouse exon 1, exon 2, and/or exon 3 are replaced by human exon 1, exon 2, and/or exon 3 sequence.

In some embodiments, the genetically-modified non-human animal described herein comprises a sequence encoding a humanized BCMA protein. In some embodiments, the humanized BCMA protein comprises a humanized extracellular region. In some embodiments, the humanized BCMA protein comprises an endogenous transmembrane region. In some embodiments, the humanized BCMA protein comprises an endogenous cytoplasmic region.

In some embodiments, the genetically-modified non-human animal described herein comprises a humanized BCMA gene. In some embodiments, the humanized BCMA gene comprises 3 exons. In some embodiments, the humanized BCMA gene comprises humanized exon 1, humanized exon 2, and/or humanized exon 3.

In some embodiments, the present disclosure also provides a chimeric (e.g., humanized) BCMA nucleotide sequence and/or amino acid sequences, wherein in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%of the sequence are identical to or derived from mouse BCMA mRNA sequence, mouse BCMA amino acid sequence (e.g., SEQ ID NO: 1) , or a portion thereof; and in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%of the sequence are identical to or derived from human BCMA mRNA sequence, human BCMA amino acid sequence (e.g., SEQ ID NO: 2) , or a portion thereof.

In some embodiments, the sequence encoding amino acids 1-42 of mouse BCMA (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human BCMA (e.g., amino acids 1-47 of human BCMA (SEQ ID NO: 2) ) .

In some embodiments, the sequence encoding amino acids 1-49 of mouse BCMA (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human BCMA (e.g., amino acids 1-54 of human BCMA (SEQ ID NO: 2) ) .

In some embodiments, the sequence encoding the entirety or a portion of the extracellular region of mouse BCMA (SEQ ID NO: 1) is replaced or inactivated. In some embodiments, the sequence is replaced by a sequence encoding the entirety or a portion of the corresponding region of human BCMA (SEQ ID NO: 2) . In some embodiments, the corresponding region of human BCMA comprises the entirety or a portion of the extracellular region of human BCMA. In some embodiments, the sequence encoding amino acids 1-42 of mouse BCMA (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human BCMA (e.g., amino acids 1-47 of human BCMA (SEQ ID NO: 2) ) . In some embodiments, the sequence encoding the corresponding region of human BCMA does not include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids at the N-terminus and/or C-terminus of the extracellular region of human BCMA.

In some embodiments, the sequence encoding the extracellular domain of mouse BCMA is replaced or inactivated. In some embodiments, the sequence is replaced by a sequence encoding the extracellular domain of human BCMA. In some embodiments, the sequence encoding amino acids 1-42 of mouse BCMA (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding amino acids 1-47 of human BCMA (SEQ ID NO: 2) .

In some embodiments, the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse BCMA promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from a portion of or the entire mouse BCMA nucleotide sequence (e.g., exon 1, exon 2, exon 3, a portion thereof, or NM_011608.1) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire mouse BCMA nucleotide sequence (e.g., exon 1, exon 2, exon 3, a portion thereof, or NM_011608.1) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from a portion of or the entire human BCMA nucleotide sequence (e.g., exon 1, exon 2, exon 3, a portion thereof, or NM_001192.3) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire human BCMA nucleotide sequence (e.g., exon 1, exon 2, exon 3, a portion thereof, or NM_001192.3) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from a portion of or the entire mouse BCMA amino acid sequence (e.g., amino acids encoded by exon 1, exon 2, exon 3, a portion thereof, or NP_035738.1 (SEQ ID NO: 1) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as a portion of or the entire mouse BCMA amino acid sequence (e.g., amino acids encoded by exon 1, exon 2, exon 3, a portion thereof, or NP_035738.1 (SEQ ID NO: 1) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from a portion of or the entire human BCMA amino acid sequence (e.g., amino acids encoded by exon 1, exon 2, exon 3, a portion thereof, or NP_001183.2 (SEQ ID NO: 2) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as a portion of or the entire human BCMA amino acid sequence (e.g., amino acids encoded by exon 1, exon 2, exon 3, a portion thereof, or NP_001183.2 (SEQ ID NO: 2) ) .

The present disclosure also provides a humanized BCMA mouse amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

a) an amino acid sequence shown in SEQ ID NO: 2 or 11;

b) an amino acid sequence having a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 2 or 11;

c) an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is able to hybridize to a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 2 or 11 under a low stringency condition or a strict stringency condition;

d) an amino acid sequence having a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 2 or 11;

e) an amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 2 or 11 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

f) an amino acid sequence that comprises a substitution, a deletion and /or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 2 or 11.

The present disclosure also relates to a BCMA nucleic acid (e.g., DNA or RNA) sequence, wherein the nucleic acid sequence can be selected from the group consisting of:

a) a nucleic acid sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 14 or 15; a nucleic acid sequence encoding a homologous BCMA amino acid sequence of a humanized mouse;

b) a nucleic acid sequence that is shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 14 or 15;

c) a nucleic acid sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 14 or 15 under a low stringency condition or a strict stringency condition;

d) a nucleic acid sequence that has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the nucleotide sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 14 or 15;

e) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 2 or 11;

f) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%with, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 2 or 11;

g) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence is different from the amino acid sequence shown in SEQ ID NO: 2 or 11 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and/or

h) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence comprises a substitution, a deletion and /or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 2 or 11.

The present disclosure also relates to a BCMA protein sequence, wherein the amino acid sequence of the BCMA protein can be selected from the group consisting of:

a) all or part of the amino acid sequence shown in SEQ ID NO: 11; or amino acids 1-47 or 1-54 of SEQ ID NO: 2;

b) an amino acid sequence having a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 11; or amino acids 1-47 or 1-54 of SEQ ID NO: 2;

c) an amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 11; or amino acids 1-47 or 1-54 of SEQ ID NO: 2, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and

d) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 11; or amino acids 1-47 or of SEQ ID NO: 2.

The present disclosure also relates to a humanized BCMA gene sequence, wherein the transcribed mRNA sequence of the humanized BCMA gene can be selected from the group consisting of:

a) all or part of the nucleotide sequence shown in SEQ ID NO: 10;

b) a nucleotide sequence that at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or at least 99%identical to the nucleotide sequence shown in SEQ ID NO: 10;

c) a nucleotide sequence that is different from the nucleotide sequence shown in SEQ ID NO: 10 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; and

d) a nucleotide sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to the nucleotide sequence shown at SEQ ID NO: 10.

The present disclosure also relates to a humanized BCMA gene sequence, wherein the humanized BCMA gene can be selected from the group consisting of:

a) all or part of the nucleotide sequence shown in SEQ ID NO: 5;

b) a nucleotide sequence that at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or at least 99%identical to the nucleotide sequence shown in SEQ ID NO: 5;

c) a nucleotide sequence that is different from the nucleotide sequence shown in SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; and

d) a nucleotide sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to the nucleotide sequence shown at SEQ ID NO: 5.

The present disclosure further relates to a BCMA genomic DNA sequence of a humanized mouse. The DNA sequence is obtained by a reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence homologous to the sequence shown in SEQ ID NO: 5.

The disclosure also provides an amino acid sequence that has a homology of at least 90%with, or at least 90%identical to the sequence shown in SEQ ID NO: 2 or 11, and has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 2 or 11 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing homology is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 2 or 11 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

The disclosure also provides a nucleotide sequence that has a homology of at least 90%, or at least 90%identical to the sequence shown in SEQ ID NO: 10, and encodes a polypeptide that has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 10 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing homology is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 10 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any amino acid sequence as described herein. In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.

In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.

In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For illustration purposes, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percentage of residues conserved with similar physicochemical properties (percent homology) , e.g. leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . The homology percentage, in many cases, is higher than the identity percentage.

Cells, tissues, and animals (e.g., mouse) are also provided that comprise the nucleotide sequences as described herein, as well as cells, tissues, and animals (e.g., mouse) that express human or chimeric (e.g., humanized) BCMA from an endogenous non-human BCMA locus.

Genetically modified animals

As used herein, the term “genetically-modified non-human animal” refers to a non-human animal having exogenous DNA in at least one chromosome of the animal's genome. In some embodiments, at least one or more cells, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%of cells of the genetically-modified non-human animal have the exogenous DNA in its genome. The cell having exogenous DNA can be various kinds of cells, e.g., an endogenous cell, a somatic cell, an immune cell, a T cell, a B cell, an antigen presenting cell, a macrophage, a dendritic cell, a germ cell, a blastocyst, or an endogenous tumor cell. In some embodiments, genetically-modified non-human animals are provided that comprise a modified endogenous BCMA locus that comprises an exogenous sequence (e.g., a human sequence) , e.g., a replacement of one or more non-human sequences with one or more human sequences. The animals are generally able to pass the modification to progeny, i.e., through germline transmission.

As used herein, the term “chimeric gene” or “chimeric nucleic acid” refers to a gene or a nucleic acid, wherein two or more portions of the gene or the nucleic acid are from different species, or at least one of the sequences of the gene or the nucleic acid does not correspond to the wildtype nucleic acid in the animal. In some embodiments, the chimeric gene or chimeric nucleic acid has at least one portion of the sequence that is derived from two or more different sources, e.g., sequences encoding different proteins or sequences encoding the same (or homologous) protein of two or more different species. In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized gene or humanized nucleic acid.

As used herein, the term “chimeric protein” or “chimeric polypeptide” refers to a protein or a polypeptide, wherein two or more portions of the protein or the polypeptide are from different species, or at least one of the sequences of the protein or the polypeptide does not correspond to wild-type amino acid sequence in the animal. In some embodiments, the chimeric protein or the chimeric polypeptide has at least one portion of the sequence that is derived from two or more different sources, e.g., same (or homologous) proteins of different species. In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized protein or a humanized polypeptide.

As used herein, the term “humanized protein” or “humanized polypeptide” refers to a protein or a polypeptide, wherein at least a portion of the protein or the polypeptide is from the human protein or human polypeptide. In some embodiments, the humanized protein or polypeptide is a human protein or polypeptide.

As used herein, the term “humanized nucleic acid” refers to a nucleic acid, wherein at least a portion of the nucleic acid is from the human. In some embodiments, the entire nucleic acid of the humanized nucleic acid is from human. In some embodiments, the humanized nucleic acid is a humanized exon. A humanized exon can be e.g., a human exon or a chimeric exon.

In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized BCMA gene or a humanized BCMA nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human BCMA gene, at least one or more portions of the gene or the nucleic acid is from a non-human BCMA gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes a BCMA protein. The encoded BCMA protein is functional or has at least one activity of the human BCMA protein or the non-human BCMA protein, e.g., binding with human or non-human BCMA ligand (e.g., APRIL or BAFF) ; regulating B cell proliferation and survival, as well as maturation and differentiation into plasma cells (PCs) .

In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized BCMA protein or a humanized BCMA polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a human BCMA protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human BCMA protein. The humanized BCMA protein or the humanized BCMA polypeptide is functional or has at least one activity of the human BCMA protein or the non-human BCMA protein.

The genetically modified non-human animal can be various animals, e.g., a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo) , deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey) . For the non-human animals where suitable genetically modifiable embryonic stem (ES) cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification. Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo. These methods are known in the art, and are described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2003, which is incorporated by reference herein in its entirety.

In one aspect, the animal is a mammal, e.g., of the superfamily Dipodoidea or Muroidea. In some embodiments, the genetically modified animal is a rodent. The rodent can be selected from a mouse, a rat, and a hamster. In some embodiments, the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters) , Cricetidae (e.g., hamster, New World rats and mice, voles) , Muridae (true mice and rats, gerbils, spiny mice, crested rats) , Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice) , Platacanthomyidae (e.g., spiny dormice) , and Spalacidae (e.g., mole rates, bamboo rats, and zokors) . In some embodiments, the genetically modified rodent is selected from a true mouse or rat (family Muridae) , a gerbil, a spiny mouse, and a crested rat. In some embodiments, the non-human animal is a mouse.

In some embodiments, the animal is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In some embodiments, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm) , 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac) , 129S7, 129S8, 129T1, 129T2. These mice are described, e.g., in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999) ; Auerbach et al., Establishment and Chimera Analysis of 129/SvEv-and C57BL/6-Derived Mouse Embryonic Stem Cell Lines (2000) , both of which are incorporated herein by reference in the entirety. In some embodiments, the genetically modified mouse is a mix of the 129 strain and the C57BL/6 strain. In some embodiments, the mouse is a mix of the 129 strains, or a mix of the BL/6 strains. In some embodiments, the mouse is a BALB strain, e.g., BALB/c strain. In some embodiments, the mouse is a mix of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50%BALB/c-50%12954/Sv; or 50%C57BL/6-50%129) .

In some embodiments, the animal is a rodent. In some embodiments, the rodent is selected from BALB/c, A, A/He, A/J, A/WySN, AKR, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2. KM, NIH, ICR, CFW, FACA, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr and C57BL/Ola C57BL, C58, CBA/Br, CBA/Ca, CBA/J, CBA/st, CBA/H strains of mice and NOD, NOD/SCID, NOD-Prkdc ^scid IL-2rg ^null Background mice.

In some embodiments, the animal is a rat. The rat can be selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

The animal can have one or more other genetic modifications, and/or other modifications, that are suitable for the particular purpose for which the humanized BCMA animal is made. For example, suitable mice for maintaining a xenograft (e.g., a human cancer or tumor) , can have one or more modifications that compromise, inactivate, or destroy the immune system of the non-human animal in whole or in part. Compromise, inactivation, or destruction of the immune system of the non-human animal can include, for example, destruction of hematopoietic cells and/or immune cells by chemical means (e.g., administering a toxin) , physical means (e.g., irradiating the animal) , and/or genetic modification (e.g., knocking out one or more genes) . Non-limiting examples of such mice include, e.g., NOD-Prkdcscid IL-2rγ ^null NOD mice, NOD-Rag 1-/--IL2rg-/- (NRG) mice, Rag 2-/--IL2rg-/- (RG) mice, SCID mice, NOD/SCID mice, IL2Rγknockout mice, NOD/SCID/γc ^null mice (Ito, M. et al., NOD/SCID/γc ^null mouse: an excellent recipient mouse model for engraftment of human cells, Blood 100 (9) : 3175-3182, 2002) , nude mice, and Rag 1 and/or Rag2 knockout mice. These mice can optionally be irradiated, or otherwise treated to destroy one or more immune cell type. Thus, in various embodiments, a genetically modified mouse is provided that can include a humanization of at least a portion of an endogenous non-human BCMA locus, and further comprises a modification that compromises, inactivates, or destroys the immune system (or one or more cell types of the immune system) of the non-human animal in whole or in part. In some embodiments, modification is, e.g., selected from the group consisting of a modification that results in NOD-Prkdcscid IL-2rγ ^null NOD mice, NOD-Rag 1-/--IL2rg-/- (NRG) mice, Rag 2-/--IL2rg-/- (RG) mice, NOD mice, SCID mice, NOD/SCID mice, IL-2Rγ knockout mice, NOD/SCID/γc null mice, nude mice, Ragl and/or Rag2 knockout mice, and a combination thereof. These genetically modified animals are described, e.g., in US20150106961, which is incorporated herein by reference in its entirety.

In some embodiments, the mouse can include a replacement of all or part of mature BCMA coding sequence with human mature BCMA coding sequence.

Genetically modified non-human animals that comprise a modification of an endogenous non-human BCMA locus. In some embodiments, the modification can comprise a human nucleic acid sequence encoding at least a portion of a mature BCMA protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the mature BCMA protein sequence) . Although genetically modified cells are also provided that can comprise the modifications described herein (e.g., ES cells, somatic cells) , in many embodiments, the genetically modified non-human animals comprise the modification of the endogenous BCMA locus in the germline of the animal.

Genetically modified animals can express a human BCMA and/or a chimeric (e.g., humanized) BCMA from endogenous mouse loci, wherein the endogenous mouse BCMA gene has been replaced with a human BCMA gene and/or a nucleotide sequence that encodes a region of human BCMA sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70&, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the human BCMA sequence. In various embodiments, an endogenous non-human BCMA locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a mature BCMA protein.

In some embodiments, the genetically modified mice express the human BCMA and/or chimeric BCMA (e.g., humanized BCMA) from endogenous loci that are under control of mouse promoters and/or mouse regulatory elements. The replacement (s) at the endogenous mouse loci provide non-human animals that express human BCMA or chimeric BCMA (e.g., humanized BCMA) in appropriate cell types and in a manner that does not result in the potential pathologies observed in some other transgenic mice known in the art. The human BCMA or the chimeric BCMA (e.g., humanized BCMA) expressed in animal can maintain one or more functions of the wild-type mouse or human BCMA in the animal. For example, human or non-human BCMA receptors can bind to the expressed BCMA, upregulate or downregulate immune response, e.g., upregulate or downregulate immune response by at least 10%, 20%, 30%, 40%, or 50%. Furthermore, in some embodiments, the animal does not express endogenous BCMA. As used herein, the term “endogenous BCMA” refers to BCMA protein that is expressed from an endogenous BCMA nucleotide sequence of the non-human animal (e.g., mouse) before any genetic modification.

The genome of the animal can comprise a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human BCMA (NP_001183.2) (SEQ ID NO: 2) . In some embodiments, the genome comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 11.

The genome of the genetically modified animal can comprise a replacement at an endogenous BCMA gene locus of a sequence encoding a region of endogenous BCMA with a sequence encoding a corresponding region of human BCMA. In some embodiments, the sequence that is replaced is any sequence within the endogenous BCMA gene locus, e.g., exon 1, exon 2, exon 3, 5'-UTR, 3'-UTR, the first intron, the second intron, etc. In some embodiments, the sequence that is replaced is within the regulatory region of the endogenous BCMA gene. In some embodiments, the sequence that is replaced starts within exon 1 and ends within exon 2 of an endogenous mouse BCMA gene locus.

The genetically modified animal can have one or more cells expressing a human or chimeric BCMA (e.g., humanized BCMA) having an extracellular region and a cytoplasmic region, wherein the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the extracellular region of human BCMA. In some embodiments, the extracellular region of the humanized BCMA has a sequence that has at least 10, 20, 30, 40, or 47 amino acids (e.g., contiguously or non-contiguously) that are identical to human BCMA. Because human BCMA and non-human BCMA (e.g., mouse BCMA) sequences, in many cases, are different, antibodies that bind to human BCMA will not necessarily have the same binding affinity with non-human BCMA or have the same effects to non-human BCMA. Therefore, the genetically modified animal having a human or a humanized extracellular region can be used to better evaluate the effects of anti-human BCMA antibodies in an animal model. In some embodiments, the genome of the genetically modified animal comprises a sequence encoding an amino acid sequence that corresponds to part or the entire sequence of exons 3-6 of human BCMA, part or the entire sequence of extracellular region of human BCMA, or part or the entire sequence of amino acids 1-47 of SEQ ID NO: 2.

In some embodiments, the non-human animal can have, at an endogenous BCMA gene locus, a nucleotide sequence encoding a chimeric human/non-human BCMA polypeptide, wherein a human portion of the chimeric human/non-human BCMA polypeptide comprises a portion of human BCMA extracellular domain, and wherein the animal expresses a functional BCMA on a surface of a cell of the animal.

In some embodiments, the human portion of the chimeric human/non-human BCMA polypeptide comprises an amino acid sequence encoded by exon 1 and a portion of exon 2 of human BCMA. In some embodiments, the human portion of the chimeric human/non-human BCMA polypeptide comprises an amino acid sequence encoded by a nucleotide sequence of human BCMA exons 1-2 encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 47 amino acids. In some embodiments, the human portion of the chimeric human/non-human BCMA polypeptide does not comprise an amino acid sequence encoded by a nucleotide sequence of human BCMA exon 3 encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80 or 90 amino acids. In some embodiments, the human portion of the chimeric human/non-human BCMA polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 1-47 or 1-54 of SEQ ID NO: 2.

In some embodiments, the human portion of the chimeric human/non-human BCMA polypeptide comprises exon 1 and a portion of exon 2 of human BCMA. In some embodiments, the human portion of the chimeric human/non-human BCMA polypeptide can comprise a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to amino acids 1-47 of SEQ ID NO: 2.

In some embodiments, the human portion of the chimeric human/non-human BCMA polypeptide comprises exon 1 and a portion exon 2 of human BCMA. In some embodiments, the human portion of the chimeric human/non-human BCMA polypeptide can comprise a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 1-54 of SEQ ID NO: 2.

In some embodiments, the non-human portion of the chimeric human/non-human BCMA polypeptide comprises transmembrane and/or cytoplasmic regions of an endogenous non-human BCMA polypeptide. There may be several advantages that are associated with the transmembrane and/or cytoplasmic regions of an endogenous non-human BCMA polypeptide. For example, once a BCMA ligand or an anti-BCMA antibody binds to BCMA, they can properly transmit extracellular signals into the cells and initiate the downstream pathway. A human or humanized transmembrane and/or cytoplasmic regions may not function properly in non-human animal cells. In some embodiments, a few (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) extracellular amino acids that are close to the transmembrane region of BCMA are also derived from endogenous sequence. These amino acids can also be important for transmembrane signal transmission. In some embodiments, a few (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids at the N-terminus of the extracellular region are also derived from endogenous sequence.

Furthermore, the genetically modified animal can be heterozygous with respect to the replacement at the endogenous BCMA locus, or homozygous with respect to the replacement at the endogenous BCMA locus.

In some embodiments, the humanized BCMA locus lacks a human BCMA 5'-UTR. In some embodiments, the humanized BCMA locus comprises a rodent (e.g., mouse) 5'-UTR. In some embodiments, the humanization comprises a human 3'-UTR. In appropriate cases, it may be reasonable to presume that the mouse and human BCMA genes appear to be similarly regulated based on the similarity of their 5'-flanking sequence. As shown in the present disclosure, humanized BCMA mice that comprise a replacement at an endogenous mouse BCMA locus, which retain mouse regulatory elements but comprise a humanization of BCMA encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized BCMA are grossly normal.

The present disclosure further relates to a non-human mammal generated through the method mentioned above. In some embodiments, the genome thereof contains human gene (s) .

In some embodiments, the non-human mammal is a rodent, and preferably, the non-human mammal is a mouse.

In some embodiments, the non-human mammal expresses a protein encoded by a humanized BCMA gene.

In addition, the present disclosure also relates to a tumor bearing non-human mammal model, characterized in that the non-human mammal model is obtained through the methods as described herein. In some embodiments, the non-human mammal is a rodent (e.g., a mouse) .

The present disclosure further relates to a cell or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; and the tumor tissue derived from the non-human mammal or an offspring thereof when it bears a tumor, or the tumor bearing non-human mammal.

The present disclosure also provides non-human mammals produced by any of the methods described herein. In some embodiments, a non-human mammal is provided; and the genetically modified animal contains the DNA encoding human or humanized BCMA in the genome of the animal.

In some embodiments, the non-human mammal comprises the genetic construct as described herein (e.g., gene construct as shown in FIG. 2) . In some embodiments, a non-human mammal expressing human or humanized BCMA is provided. In some embodiments, the tissue-specific expression of human or humanized BCMA protein is provided.

In some embodiments, the expression of human or humanized BCMA in a genetically modified animal is controllable, as by the addition of a specific inducer or repressor substance.

Non-human mammals can be any non-human animal known in the art and which can be used in the methods as described herein. Preferred non-human mammals are mammals, (e.g., rodents) . In some embodiments, the non-human mammal is a mouse.

Genetic, molecular and behavioral analyses for the non-human mammals described above can performed. The present disclosure also relates to the progeny produced by the non-human mammal provided by the present disclosure mated with the same or other genotypes.

The present disclosure also provides a cell line or primary cell culture derived from the non-human mammal or a progeny thereof. A model based on cell culture can be prepared, for example, by the following methods. Cell cultures can be obtained by way of isolation from a non-human mammal, alternatively cell can be obtained from the cell culture established using the same constructs and the standard cell transfection techniques. The integration of genetic constructs containing DNA sequences encoding human BCMA protein can be detected by a variety of methods.

There are many analytical methods that can be used to detect exogenous DNA, including methods at the level of nucleic acid (including the mRNA quantification approaches using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, and in situ hybridization) and methods at the protein level (including histochemistry, immunoblot analysis and in vitro binding studies) . In addition, the expression level of the gene of interest can be quantified by ELISA techniques well known to those skilled in the art. Many standard analysis methods can be used to complete quantitative measurements. For example, transcription levels can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis (RNAdot) analysis. Immunohistochemical staining, flow cytometry, Western blot analysis can also be used to assess the presence of human or humanized BCMA protein.

Vectors

The present disclosure relates to a targeting vector, comprising: a) a DNA fragment homologous to the 5' end of a region to be altered (5' arm) , which is selected from the BCMA gene genomic DNAs in the length of 100 to 10,000 nucleotides; b) a desired/donor DNA sequence encoding a donor region; and c) a second DNA fragment homologous to the 3' end of the region to be altered (3' arm) , which is selected from the BCMA gene genomic DNAs in the length of 100 to 10,000 nucleotides.

In some embodiments, a) the DNA fragment homologous to the 5' end of a conversion region to be altered (5' arm) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000082.7; c) the DNA fragment homologous to the 3' end of the region to be altered (3' arm) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000082.7.

In some embodiments, a) the DNA fragment homologous to the 5' end of a region to be altered (5' arm) is selected from the nucleotides 11126899-11131816 of the NCBI accession number NC_000082.7; c) the DNA fragment homologous to the 3' end of the region to be altered (3' arm) is selected from the nucleotides 11133528-11138756 of the NCBI accession number NC_000082.7.

In some embodiments, a) the DNA fragment homologous to the 5' end of a region to be altered (5' arm) is selected from the nucleotides 11129848-11131816 of the NCBI accession number NC_000082.7; c) the DNA fragment homologous to the 3' end of the region to be altered (3' arm) is selected from the nucleotides 11133050-11133997 of the NCBI accession number NC_000082.7.

In some embodiments, the length of the selected genomic nucleotide sequence in the targeting vector can be about or at least 800 bp, about or at least 900 bp, about or at least 1 kB, about or at least 1.5 kb, about or at least 2 kb, about or at least 2.5 kb, about or at least 3 kb, about or at least 3.5 kb, or about or at least 4 kb.

In some embodiments, the region to be altered is exon 1, exon 2, and/or exon 3 of an endogenous BCMA gene (e.g., a sequence starting within exon 1 and ending within exon 2 of mouse BCMA gene) .

The targeting vector can further include a selected gene marker.

In some embodiments, the sequence of the 5' arm is shown in SEQ ID NO: 3; and the sequence of the 3' arm is shown in SEQ ID NO: 4. In some embodiments, the sequence of the selected genomic nucleotide sequence is shown in SEQ ID NO: 5.

In some embodiments, the sequence of the 5' arm is shown in SEQ ID NO: 14; and the sequence of the 3' arm is shown in SEQ ID NO: 15. In some embodiments, the sequence of the selected genomic nucleotide sequence is shown in SEQ ID NO: 5.

In some embodiments, the sequence is derived from human. For example, the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human BCMA, preferably a sequence starting within exon 1 and ending within exon 2 of the human BCMA. In some embodiments, the nucleotide sequence of the humanized BCMA encodes the entire or the part of human BCMA protein with the NCBI accession number NP_001183.2 (SEQ ID NO: 2) .

The disclosure also relates to a cell comprising the targeting vectors as described above.

In addition, the present disclosure further relates to a non-human mammalian cell, having any one of the foregoing targeting vectors, and one or more in vitro transcripts of the construct as described herein. In some embodiments, the cell includes Cas9 mRNA or an in vitro transcript thereof.

In some embodiments, the genes in the cell are heterozygous. In some embodiments, the genes in the cell are homozygous.

In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell.

Methods of making genetically modified animals

Genetically modified animals can be made by several techniques that are known in the art, including, e.g., nonhomologous end-joining (NHEJ) , homologous recombination (HR) , zinc finger nucleases (ZFNs) , transcription activator-like effector-based nucleases (TALEN) , and the clustered regularly interspaced short palindromic repeats (CRISPR) -Cas system. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to generate genetically modified animals. Many of these genome editing techniques are known in the art, and is described, e.g., in Yin et al., "Delivery technologies for genome editing, " Nature Reviews Drug Discovery 16.6 (2017) : 387-399, which is incorporated by reference in its entirety. Many other methods are also provided and can be used in genome editing, e.g., micro-injecting a genetically modified nucleus into an enucleated oocyte, and fusing an enucleated oocyte with another genetically modified cell.

Thus, in some embodiments, the disclosure provides replacing in at least one cell of the animal, at an endogenous BCMA gene locus, a sequence encoding a region of an endogenous BCMA with a sequence encoding a corresponding region of human or chimeric BCMA. In some embodiments, the replacement occurs in a germ cell, a somatic cell, a blastocyst, or a fibroblast, etc. The nucleus of a somatic cell or the fibroblast can be inserted into an enucleated oocyte.

FIG. 3 shows a humanization strategy for a mouse BCMA locus. In FIG. 3, the targeting strategy involves a vector comprising the 5' end homologous arm, human BCMA gene fragment, 3' homologous arm. The process can involve replacing endogenous BCMA sequence with human sequence by homologous recombination. In some embodiments, the cleavage at the upstream and the downstream of the target site (e.g., by zinc finger nucleases, TALEN or CRISPR) can result in DNA double strands break, and the homologous recombination is used to replace endogenous BCMA sequence with human BCMA sequence.

Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of replacing at an endogenous BCMA locus (or site) , a sequence encoding a region of endogenous BCMA with a sequence encoding a corresponding region of human BCMA. The sequence can include a region (e.g., a part or the entire region) of exon 1, exon 2, and/or exon 3 of a human BCMA gene. In some embodiments, the sequence encoding a corresponding region of human BCMA includes exon 1 and a portion of exon 2 of human BCMA gene (e.g., a sequence encoding amino acids 1-47 of SEQ ID NO: 2) . In some embodiments, the region is located within the extracellular region of BCMA.

In some embodiments, the methods of modifying a BCMA locus of a mouse to express a chimeric human/mouse BCMA peptide can include the steps of replacing at the endogenous mouse BCMA locus a nucleotide sequence encoding a mouse BCMA with a nucleotide sequence encoding a human BCMA, thereby generating a sequence encoding a chimeric human/mouse BCMA.

In some embodiments, the nucleotide sequences as described herein do not overlap with each other (e.g., the first nucleotide sequence, the second nucleotide sequence, and/or the third nucleotide sequence do not overlap) . In some embodiments, the amino acid sequences as described herein do not overlap with each other.

The present disclosure further provides a method for establishing a BCMA gene humanized animal model, involving the following steps:

(a) providing the cell (e.g. a fertilized egg cell) based on the methods described herein;

(b) culturing the cell in a liquid culture medium;

(c) transplanting the cultured cell to the fallopian tube or uterus of the recipient female non-human mammal, allowing the cell to develop in the uterus of the female non-human mammal;

(d) identifying the germline transmission in the offspring genetically modified humanized non-human mammal of the pregnant female in step (c) .

In some embodiments, the non-human mammal in the foregoing method is a mouse (e.g., a C57BL/6 mouse) .

In some embodiments, the non-human mammal in step (c) is a female with pseudo pregnancy (or false pregnancy) .

In some embodiments, the fertilized eggs for the methods described above are C57BL/6 fertilized eggs. Other fertilized eggs that can also be used in the methods as described herein include, but are not limited to, FVB/N fertilized eggs, BALB/c fertilized eggs, DBA/1 fertilized eggs and DBA/2 fertilized eggs.

Fertilized eggs can come from any non-human animal, e.g., any non-human animal as described herein. In some embodiments, the fertilized egg cells are derived from rodents. The genetic construct can be introduced into a fertilized egg by microinjection of DNA. For example, by way of culturing a fertilized egg after microinjection, a cultured fertilized egg can be transferred to a false pregnant non-human animal, which then gives birth of a non-human mammal, so as to generate the non-human mammal mentioned in the methods described above.

In some embodiments, the method for making the genetically modified animal comprises:

(1) providing a plasmid comprising a human BCMA gene fragment, flanked by a 5' homology arm and a 3' homology arm, wherein the 5' and 3' homology arms target an endogenous BCMA gene;

(2) providing two small guide RNAs (sgRNAs) that target the endogenous BCMA gene;

(3) modifying genome of a fertilized egg or an embryonic stem cell by using the plasmid of step (1) , the sgRNAs of step (2) , and Cas9;

(4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudopregnant female mouse or transplanting the embryonic stem cell obtained in step (3) into a blastocyst which is then transplanted into the oviduct of a pseudopregnant female mouse to produce a child mouse that functionally expresses a humanized BCMA protein; and

(5) mating the child mouse obtained in step (2) to obtain a homozygote mouse,

In some embodiments, the fertilized egg is modified by CRISPR with sgRNAs that target a 5'-terminal targeting site selected from the group consisting of SEQ ID NO: 16-20 and a 3'-terminal targeting site selected from the group consisting of SEQ ID NO: 21-26.

In some embodiments, the humanized BCMA protein comprises SEQ ID NO: 11.

In some embodiments, the 5'-terminal targeting site is SEQ ID NO: 18 and the 3'-terminal targeting site is SEQ ID NO: 24.

In some embodiments, the sequence encoding the humanized BCMA protein is operably linked to an endogenous regulatory element at the endogenous BCMA gene locus.

In some embodiments, the genetically-modified animal does not express an endogenous BCMA protein.

Methods of using genetically modified animals

Replacement of non-human genes in a non-human animal with homologous or orthologous human genes or human sequences, at the endogenous non-human locus and under control of endogenous promoters and/or regulatory elements, can result in a non-human animal with qualities and characteristics that may be substantially different from a typical knockout-plus-transgene animal. In the typical knockout-plus-transgene animal, an endogenous locus is removed or damaged and a fully human transgene is inserted into the animal′s genome and presumably integrates at random into the genome. Typically, the location of the integrated transgene is unknown; expression of the human protein is measured by transcription of the human gene and/or protein assay and/or functional assay. Inclusion in the human transgene of upstream and/or downstream human sequences are apparently presumed to be sufficient to provide suitable support for expression and/or regulation of the transgene.

In some cases, the transgene with human regulatory elements expresses in a manner that is unphysiological or otherwise unsatisfactory, and can be actually detrimental to the animal. The disclosure demonstrates that a replacement with human sequence at an endogenous locus under control of endogenous regulatory elements provides a physiologically appropriate expression pattern and level that results in a useful humanized animal whose physiology with respect to the replaced gene are meaningful and appropriate in the context of the humanized animal's physiology.

Genetically modified animals that express human or humanized BCMA protein, e.g., in a physiologically appropriate manner, provide a variety of uses that include, but are not limited to, developing therapeutics for human diseases and disorders, and assessing the toxicity and/or the efficacy of these human therapeutics in the animal models.

In various aspects, genetically modified animals are provided that express human or humanized BCMA, which are useful for testing agents that can decrease or block the interaction between BCMA and BCMA ligands (e.g., APRIL and/or BAFF) or the interaction between BCMA and anti-human BCMA antibodies, testing whether an agent can increase or decrease the immune response, and/or determining whether an agent is an BCMA agonist or antagonist. The genetically modified animals can be, e.g., an animal model of a human disease, e.g., the disease is induced genetically (a knock-in or knockout) . In various embodiments, the genetically modified non-human animals further comprise an impaired immune system, e.g., a non-human animal genetically modified to sustain or maintain a human xenograft, e.g., a human solid tumor or a blood cell tumor (e.g., a lymphocyte tumor, e.g., a B or T cell tumor) .

In some embodiments, the genetically modified animals can be used for determining effectiveness of an anti-BCMA antibody for the treatment of cancer. The methods involve administering the anti-BCMA antibody (e.g., anti-human BCMA antibody) to the animal as described herein, wherein the animal has a tumor; and determining the inhibitory effects of the anti-BCMA antibody to the tumor. The inhibitory effects that can be determined include, e.g., a decrease of tumor size or tumor volume, a decrease of tumor growth, a reduction of the increase rate of tumor volume in a subject (e.g., as compared to the rate of increase in tumor volume in the same subject prior to treatment or in another subject without such treatment) , a decrease in the risk of developing a metastasis or the risk of developing one or more additional metastasis, an increase of survival rate, and an increase of life expectancy, etc. The tumor volume in a subject can be determined by various methods, e.g., as determined by direct measurement, MRI or CT.

In some embodiments, the tumor comprises one or more cancer cells (e.g., human or mouse cancer cells) that are injected into the animal. In some embodiments, the anti-BCMA antibody prevents BCMA receptors from binding to BCMA. In some embodiments, the anti-BCMA antibody does not prevent BCMA receptors from binding to BCMA. Exemplary anti-BCMA antibodies or antibody-drug conjugates thereof include, but not limited to, Belantamab mafodotin-blmf (GSK2857916) , MEDI2228 and HDP-101.

In some embodiments, the genetically modified animals can be used for determining whether an anti-BCMA antibody is a BCMA agonist or antagonist. In some embodiments, the methods as described herein are also designed to determine the effects of the agent (e.g., anti-BCMA antibodies) on BCMA, e.g., whether the agent can stimulate immune cells or inhibit immune cells (e.g., macrophages, B cells, or DC) , whether the agent can increase or decrease the production of cytokines, whether the agent can activate or deactivate immune cells (e.g., macrophages, B cells, or DC) , whether the agent can upregulate the immune response or downregulate immune response, and/or whether the agent can induce complement mediated cytotoxicity (CMC) or antibody dependent cellular cytoxicity (ADCC) . In some embodiments, the genetically modified animals can be used for determining the effective dosage of a therapeutic agent for treating a disease in the subject, e.g., cancer, or autoimmune diseases.

The inhibitory effects on tumors can also be determined by methods known in the art, e.g., measuring the tumor volume in the animal, and/or determining tumor (volume) inhibition rate (TGI _TV) . The tumor growth inhibition rate can be calculated using the formula TGI _TV (%) =(1-TVt/TVc) x 100, where TVt and TVc are the mean tumor volume (or weight) of treated and control groups.

In some embodiments, the inhibitory effects are determined by detecting signals (e.g., fluorescence) of the tumor cells in the animal. In some embodiments, the tumor cells are labeled with fluorescent markers. In some embodiments, the tumor cells are labeled with fluorescent proteins (e.g., GFP) .

In some embodiments, the anti-BCMA antibody is designed for treating various cancers. As used herein, the term “cancer” refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term “tumor” as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the agents described herein are designed for treating or diagnosing a carcinoma in a subject. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

In some embodiments, the cancer types as described herein include, but not limited to, lymphoma, non-small cell lung cancer (NSCLC) , leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. In some embodiments, the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia. In some embodiments, the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T cell lymphoma, and Waldenstrom macroglobulinemia. In some embodiments, the sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

In some embodiments, the cancer types as described here includes cancer in tonsil, lymph node, duodenum, colon, stomach, rectum, spleen, salivary gland, small instestine, appendix, thymus, breast, urinary bladder, and gallbladder. In some embodiments, the cancer types as described here includes hematologic malignancies (e.g., multiple myeloma, chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL) , and Hodgkin lymphoma) .

In some embodiments, the antibody is designed for treating various immune disorder or immune-related diseases (e.g., psoriasis, allergic rhinitis, sinusitis, asthma, rheumatoid arthritis, atopic dermatitis, chronic obstructive pulmonary disease (COPD) , chronic bronchitis, emphysema, eczema, osteoarthritis, rheumatoid arthritis, systemic lupus erythematosus, polymyalgia rheumatica, autoimmune hemolytic anemia, systemic vasculitis, pernicious anemia, inflammatory bowel disease, ulcerative colitis, Crohn's disease, or multiple sclerosis) . Thus, the methods as described herein can be used to determine the effectiveness of an anti-BCMA antibody in inhibiting immune response.

In some embodiments, the immune disorder or immune-related diseases described here include allergy, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, primary thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, self-immune liver disease, diabetes, pain, or neurological disorders.

The present disclosure also provides methods of determining toxicity of an antibody (e.g., anti-BCMA antibody) . The methods involve administering the antibody to the animal as described herein. The animal is then evaluated for its weight change, red blood cell count, hematocrit, and/or hemoglobin.

The present disclosure also relates to the use of the animal model generated through the methods as described herein in the development of a product related to an immunization processes of human cells, the manufacturing of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.

In some embodiments, the disclosure provides the use of the animal model generated through the methods as described herein in the production and utilization of an animal experimental disease model of an immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and/or a therapeutic strategy.

The disclosure also relates to the use of the animal model generated through the methods as described herein in the screening, verifying, evaluating or studying the BCMA gene function, human BCMA antibodies, drugs for human BCMA targeting sites, the drugs or efficacies for human BCMA targeting sites, the drugs for immune-related diseases and antitumor drugs.

In some embodiments, the disclosure provides a method to verify in vivo efficacy of TCR-T, CAR-T, and/or other immunotherapies (e.g., T-cell adoptive transfer therapies) . For example, the methods include transplanting human tumor cells into the animal described herein, and applying human CAR-T to the animal with human tumor cells. Effectiveness of the CAR-T therapy can be determined and evaluated. In some embodiments, the animal is selected from the BCMA gene humanized non-human animal prepared by the methods described herein, the BCMA gene humanized non-human animal described herein, the double-or multi-humanized non-human animal generated by the methods described herein (or progeny thereof) , a non-human animal expressing the human or humanized BCMA protein, or the tumor-bearing or inflammatory animal models described herein. In some embodiments, the TCR-T, CAR-T, and/or other immunotherapies can treat the BCMA-associated diseases described herein. In some embodiments, the TCR-T, CAR-T, and/or other immunotherapies provides an evaluation method for treating the BCMA-associated diseases described herein.

Genetically modified animal model with two or more human or chimeric genes

The present disclosure further relates to methods for generating genetically modified animal model with two or more human or chimeric genes. The animal can comprise a human or chimeric BCMA gene and a sequence encoding an additional human or chimeric protein.

In some embodiments, the additional human or chimeric protein can be APRIL, programmed cell death protein 1 (PD-1) , IL4, Colony Stimulating Factor 1 (CSF1) , IL34, C-C Motif Chemokine Receptor 2 (CCR2) , CD40, C-X-C Motif Chemokine Receptor 4 (CXCR4) , Vascular Endothelial Growth Factor (VEGF) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , Lymphocyte Activating 3 (LAG-3) , B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , CD27, CD28, CD47, CD137, CD154, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , Signal regulatory protein α (SIRPα) or TNF Receptor Superfamily Member 4 (TNFRSF4 or OX40) .

The methods of generating genetically modified animal model with two or more human or chimeric genes (e.g., humanized genes) can include the following steps:

(a) using the methods of introducing human BCMA gene or chimeric BCMA gene as described herein to obtain a genetically modified non-human animal;

(b) breeding the genetically modified non-human animal with another genetically modified non-human animal, and then screening the progeny to obtain a genetically modified non-human animal with two or more human or chimeric genes.

In some embodiments, in step (b) of the method, the genetically modified animal can be mated with a genetically modified non-human animal with human or chimeric APRIL, PD-1, IL4, CSF1, IL34, CCR2, CD40, CXCR4, VEGF, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPα, or OX40. Some of these genetically modified non-human animal are described, e.g., in PCT/CN2017/090320, PCT/CN2017/099577, PCT/CN2017/099575, PCT/CN2017/099576, PCT/CN2017/099574, PCT/CN2017/106024, PCT/CN2017/110494, PCT/CN2017/110435, PCT/CN2017/120388, PCT/CN2018/081628, PCT/CN2018/081629; each of which is incorporated herein by reference in its entirety.

In some embodiments, the BCMA gene humanization is directly performed on a genetically modified animal having a human or chimeric APRIL, PD-1, IL4, CSF1, IL34, CCR2, CD40, CXCR4, VEGF, CTLA-4, BTLA, PD-L1, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPα, or OX40 gene.

As these proteins may involve different mechanisms, a combination therapy that targets two or more of these proteins thereof may be a more effective treatment. In fact, many related clinical trials are in progress and have shown a good effect. The genetically modified animal model with two or more human or humanized genes can be used for determining effectiveness of a combination therapy that targets two or more of these proteins, e.g., an anti-BCMA antibody and an additional therapeutic agent (e.g., an anti-PD-1 antibody) for the treatment of cancer. The methods include administering the anti-BCMA antibody and the additional therapeutic agent (e.g., an anti-PD-1 antibody) to the animal, wherein the animal has a tumor; and determining the inhibitory effects of the combined treatment to the tumor. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to APRIL, PD-1, IL4, CSF1, IL34, CCR2, CD40, CXCR4, VEGF, CTLA-4, BTLA, PD-L1, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPa, or OX40. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab) , an anti-PD-1 antibody (e.g., nivolumab) , or an anti-PD-L1 antibody.

In some embodiments, the animal further comprises a sequence encoding a human or humanized PD-1, a sequence encoding a human or humanized PD-L1, or a sequence encoding a human or humanized CTLA-4. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab) , an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In some embodiments, the tumor comprises one or more tumor cells that express CD80, CD86, PD-L1, and/or PD-L2.

In some embodiments, the combination treatment is designed for treating various cancer as described herein, e.g., melanoma, non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , bladder cancer, prostate cancer (e.g., metastatic hormone-refractory prostate cancer) , advanced breast cancer, advanced ovarian cancer, and/or advanced refractory solid tumor. In some embodiments, the combination treatment is designed for treating metastatic solid tumors, NSCLC, melanoma, B-cell non-Hodgkin lymphoma, colorectal cancer, and multiple myeloma. In some embodiments, the combination treatment is designed for treating melanoma, carcinomas (e.g., pancreatic carcinoma) , mesothelioma, hematological malignancies (e.g., Non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia) , or solid tumors (e.g., advanced solid tumors) . In some embodiments, the combination treatment is designed for treating liver cancer, pancreatic cancer, osteosarcoma, breast cancer, ovarian cancer, endometrial cancer, oral squamous cell carcinoma, cervical cancer, renal cancer, head and neck cancer, or brain cancer.

In some embodiments, the methods described herein can be used to evaluate the combination treatment with some other methods. The methods of treating a cancer that can be used alone or in combination with methods described herein, include, e.g., treating the subject with chemotherapy, e.g., campothecin, doxorubicin, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, adriamycin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, bleomycin, plicomycin, mitomycin, etoposide, verampil, podophyllotoxin, tamoxifen, taxol, transplatinum, 5-flurouracil, vincristin, vinblastin, and/or methotrexate. Alternatively or in addition, the methods can include performing surgery on the subject to remove at least a portion of the cancer, e.g., to remove a portion of or all of a tumor (s) , from the patient.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Materials and Methods

The following materials were used in the following examples.

BbsI, EcoRI, BamHI, BglII, SpeI enzymes were purchased from NEB, and the catalog numbers are R0539L, R0101M, R0136M, R0144M, R0133M respectively;

C57BL/6 mice were purchased from the National Rodent Laboratory Animal Seed Center, China National Academy of Food and Drug Control;

Ambion In Vitro Transcription Kit was purchased from Ambion, Cat. No. AM1354;

Cas9mRNA was obtained from SIGMA, Cat. No. CAS9MRNA-1EA;

The UCA kit was obtained from Biositu, Cat. No. BCG-DX-001.

Example 1: BCMA gene humanized mice

Mouse BCMA gene (NCBI Gene ID: 21935, Primary source: MGI: 1343050, UniProt: O88472, located at positions 11131131 to 11137938 of chromosome 16 NC_000082.7, with transcript NM_011608.1 and its encoded protein NP_035738.1 (SEQ ID NO: 1) ) and human BCMA gene (NCBI Gene ID: 608, Primary source: HGNC: 11913, UniProt ID: Q02223, located at positions 11965210 to 11968068 of chromosome 16 NC_000016.10, with transcript NM_001192.3 and its encoded protein NP_001183.2 (SEQ ID NO: 2) ) were used. A schematic diagram of the comparison between mouse BCMA gene and human BCMA gene is shown in FIG. 1.

A nucleotide sequence encoding a human BCMA protein (human BCMA sequence) can be introduced into the endogenous BCMA locus of a mouse, so that the mouse expresses a human or humanized BCMA protein. Specifically, using gene editing technology, under the control of the mouse BCMA gene regulatory element, the corresponding mouse sequence was replaced with human BCMA sequence, and a schematic diagram of the humanized BCMA locus is shown in FIG. 2.

A schematic diagram of a targeting strategy is shown in FIG. 3. As shown, the targeting vect or V1 contains the upstream and downstream homology arm sequences, and the A fragment cont aining the nucleotide sequence encoding the human BCMA protein. The upstream homology arm sequence (5′ homology arm, SEQ ID NO: 3) is identical to the nucleotide sequence from nucleot ide 11126899 to 11131816 of the NCBI accession number NC_000082.7. The downstream homo logy arm sequence (3′ homology arm, SEQ ID NO: 4) is identical to the nucleotide sequence fro m nucleotide 11133528 to 11138756 of the NCBI accession number NC_000082.7. The human B CMA sequence (SEQ ID NO: 5) contained on the A fragment is identical to the nucleotide seque nce from nucleotide 11965325 to 11966205 of the NCBI accession number NC_000016. The jun ction sequence upstream of the human BCMA sequence is designed as

5'-ctgtccactcttcccgtttctttcagtgatccagt ccctc

gcagatggctgggcagtgctcccaaaatgaatatt-3' (SEQ ID NO: 6) , where the last "c" of the sequence "ccctc" is the last nucleotide of mouse sequence, a nd the "a" in the sequence

is the first nucleotide of human sequence. The junction sequenc e downstream of human BCMA sequence is designed as 5'-attatctgtctgatgttcttttcataaaggtgtga ccaa t

gaaagggacgtacacggtgctctggatcttcttgg-3' (SEQ ID NO: 7) , wherein the "t" in the sequence " c caat" is the last nucleotide of human sequence, and the first "t" in the sequence " tcagt" is the first nucleotide in mouse sequence.

The targeting vector V1 also includes a resistance gene for screening positive clones, namel y the coding sequence of neomycin phosphotransferase (Neo) , with two site-specific recombinati on systems (Frt) arranged in the same direction and installed on both sides of the resistance gene, forming a Neo cassette. The connection between the 5′ end of the Neo cassette and mouse seque nce is designed as 5'-caacaaatgaaacccaccaactattccccaaaca aaacaa

TCTCGAGGTCGACG GTATCGATAAGCTTGATATCGAATTCCGAAG-3' (SEQ ID NO: 8) , where the last "a" in the s equence "aaacaa" is the last nucleotide of the mouse sequence, and the first "A" in the sequence"

is the first nucleotide of the Neo cassette. The connection between the 3′ end of the Ne o cassette and the mouse gene is designed as 5'-CTCTAGAAAGTATAGGAACTTCATCAGTC AGGTACATAATGGTG GATCC

agacattggacacctactttgcagtcgcctttctt-3' (SEQ ID NO: 9) , w here the last "C" in the sequence " GATCC" is the last nucleotide of the Neo cassette, and the first "a" in the sequence

is the first nucleotide of the mouse sequence. In addition, a negative selection marker (the encoding gene for diphtheria toxin A subunit (DTA) ) was also constructed downstream of the 3′ homology arm of the targeting vector. The mRNA sequence of the humaniz ed mouse BCMA is shown in SEQ ID NO: 10, and the expressed protein sequence is shown in S EQ ID NO: 11.

The construction of the targeting vector can be carried out by conventional methods, such as enzyme cleavage and ligation. The constructed targeting vector was preliminarily verified by enzyme digestion, and then sent to a sequencing company for sequencing verification. The correct target vector verified by sequencing was electroporated into embryonic stem cells of C57BL/6 mice. The obtained cells were screened using the positive clone selection marker. PCR and Southern Blot technology were used to detect and confirm the integration of the exogenous sequences, and to select the correct positive cloned cells.

After verification, the correct positive cloned cells (black mice) were introduced into the isolated blastocysts (white mice) according to techniques known in the art. The obtained chimeric blastocysts were transferred into cell culture medium for short-term culture and then transplanted to the fallopian tubes of recipient female mice (white mice) to produce F0 generation chimeric mice (black and white) . The F0 generation chimeric mice were backcrossed with the wild-type mice to obtain the F1 generation mice, and then the F1 generation heterozygous mice were bred to each other to obtain the F2 generation homozygous mice. The positive clones can also be mated with the Flp tool mice to remove the positive clone selection marker gene, and then cross-mated to obtain BCMA gene humanized homozygous mice. The genotype of the offspring mouse somatic cells can be verified by PCR (primers are shown in the table below) , and the PCR data of an exemplary F1 generation mouse (the Neo marker gene has been removed) is shown in FIG. 4. As shown by the PCR data, F1-01, F1-04 and F1-05 mice were positive heterozygous mice. Thus, using the present method, the BCMA gene humanized mice without random insertion can be obtained and can be stably passaged.

Table 3 F1 generation genotype PCR detection primer sequence and size of recombinant fragment

In addition, the CRISPR/Cas system can also be used for gene editing, following the targeting strategy shown in Figure 5. As shown, the targeting vector V2 contains the homology arm sequences upstream and downstream of the mouse BCMA gene, as well as the human BCMA sequence. The upstream homology arm sequence (5′ homology arm, SEQ ID NO: 14) is the same as the nucleotide sequence from nucleotide 11129848 to 11131816 of the NCBI accession number NC_000082.7, and the downstream homology arm sequence (3′ homology arm, SEQ ID NO: 15) is identical to the nucleotide sequence from nucleotide 11133050 to 11133997 of NCBI accession number NC_000082.7. The nucleotide sequence of human BCMA sequence is shown in SEQ ID NO: 5.

Given that human BCMA and murine BCMA have multiple isoforms or transcripts, the methods described herein can be applied to other isoforms or transcripts.

The construction of the targeting vector can be carried out by conventional methods, such as enzyme cleavage and ligation, de novo synthesis and the like. The constructed targeting vector was preliminarily verified by enzyme digestion, and then sent to a sequencing company for sequencing verification. Sequencing-validated targeting vectors were used for subsequent experiments.

The target sequence determines the binding specificity of the sgRNA and the efficiency of the induced Cas9 cleavage. Therefore, target sequence design is the key for constructing sgRNA expression vectors. The target sequence of each sgRNA on the BCMA gene is as follows:

sgRNA1 target site (SEQ ID NO: 16) : 5′-GGAAACACTGTTGCGCCATGAGG-3′

sgRNA2 target site (SEQ ID NO: 17) : 5′-GCTGAGGACTCGCACTTACTTGG-3′

sgRNA3 target site (SEQ ID NO: 18) : 5′-TTGGAACATCGCAAGTGACACGG-3′

sgRNA4 target site (SEQ ID NO: 19) : 5′-CCTCAGCTGTCGCTTCTTGTGGG-3′

sgRNA5 target site (SEQ ID NO: 20) : 5′-CACTTACTTGGATCACAGTAAGG-3′

sgRNA6 target site (SEQ ID NO: 21) : 5′-TGAATGTGCGTTAGGGGACCTGG-3′

sgRNA7 target site (SEQ ID NO: 22) : 5′-TGTCGGGAAGCCGTCATGCCTGG-3′

sgRNA8 target site (SEQ ID NO: 23) : 5′-CGCTCATGAATGTGCGTTAGGGG-3′

sgRNA9 target site (SEQ ID NO: 24) : 5′-ATTCATGAGCGTCTTACTGGGGG-3′

sgRNA10 target site (SEQ ID NO: 25) : 5′-ACGCTCATGAATGTGCGTTAGGG-3′

sgRNA11 target site (SEQ ID NO: 26) : 5′-CACATTCATGAGCGTCTTACTGG-3′

The UCA kit was used to detect the activity of the sgRNAs designed and synthesized based on the above target sequences. The detection results are shown in Table 4 and Figure 6. It can be seen that the sgRNAs had different activities, sgRNA3 and sgRNA9 were selected for subsequent experiments. Oligonucleotides (nuclease cleavage site) were added to the 5'end and a complementary strand to obtain a forward oligonucleotide and a reverse oligonucleotide (see Table 5 for the sequences) . After annealing, the annealed products were ligated into the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) to obtain expression vectors pT7-BCMA-3 and pT7-BCMA-9.

Table 4 sgRNA activity detection results (UCA)

Table 5 sgRNA3 and sgRNA9 sequence list

The pT7-sgRNA vector was synthesized, which included a DNA fragment containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 35) , and was ligated to the backbone vector (Takara, Catalog number: 3299) after restriction enzyme digestion (EcoRI and BamHI) . The resulting plasmid was confirmed by sequencing.

The pre-mixed Cas9 mRNA, the targeting vector, and in vitro transcription products of the pT7-BCMA-3, pT7-BCMA-9 plasmids (using Ambion in vitro transcription kit to carry out the transcription according to the method provided in the product instruction) were injected into the cytoplasm or nucleus of mouse fertilized eggs with a microinjection instrument. The embryo microinjection was carried out according to the method described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2006. The injected fertilized eggs were then transferred to a culture medium to culture for a short time and then was transplanted into the oviduct of the recipient mouse to produce the genetically modified mice (F0 generation) . The mouse population was further expanded by cross-breeding and self-breeding to establish stable homozygous mouse lines with genetically-modified BCMA gene locus.

The genotype of somatic cells of F0 generation mice can be identified by conventional methods, e.g., by PCR analysis. The identification results of some F0 generation mice are shown in FIG. 7. Considering the 5′ end primer detection result and the 3′ end primer detection result, the mouse numbered F0-01 was identified as positive. The PCT primer sequences are shown in the table below.

Table 6 F0 generation genotype PCR detection primer sequence and size of recombinant fragments

Primer L-GT-F is located on the left side of the 5′ homology arm, R-GT-R is located on the right side of the 3′ homology arm, and both Mut-R and R-GT-F are located on the human BCMA sequence.

The F0 BCMA gene humanized mice identified as positive were mated with wild-type mice to obtain F1 generation mice. F1 generation mice can be genotyped using the same PCR method (primer sequences are shown in Table 6) . Exemplary test results are shown in FIG. 8. As shown, 13 mice numbered F1-27, F1-30, F1-31, F1-32, F1-34, F1-35, F1-36, F1-41, F1-42, F1-43, F1-44, F1-45 and F1-46 were positive mice.

Southern blot was performed on mice identified as positive by PCR in the F1 generation to confirm the presence of random insertions. Specifically, mouse tail genomic DNA was extracted, digested with StuI or BglII restriction enzyme, transferred to a membrane, and then hybridized with probes. The 5′ probe and the 3′ probe are located on the 5′ homology arm and outside the 3′ homology arm, respectively. The lengths of the specific probes and fragment sizes are shown in Table 7. The results of Southern blot detection are shown in FIG. 9. The results from the 3′ probe detection and 5′ probe detection showed that, except for F1-35, F1-36, F1-45, and F1-46 (abnormal bands are not shown) , none of the 8 mice of F1-27, F1-30, F1-31, F1-034, F1-41, F1-42, F1-43, and F1-44 contained random inserations. Thus, these 8 mice were positive heterozygous mice with no random insertions. Thus, genetically engineered mice with BCMA gene humanization without random insertion can be obtained and can be stably passaged.

Table 7 Specific probes and lengths of target fragments

Probes are as follows:

5′Probe-F (SEQ ID NO: 38) : 5′-TCCTCCTGTCTTTCCTCTGCTGTCA-3′,

5′Probe-R (SEQ ID NO: 39) : 5′-GCAGATTCTCTGTGGGAGTTCCCTG-3′;

3′Probe-F (SEQ ID NO: 40) : 5′-GGAAACAGTGGTTACGGTCAAACGC-3′,

3′Probe-R (SEQ ID NO: 41) : 5′-CACCGTGTGACAAGATGACTGGGTA-3′;

The heterozygous F1 generation mice identified as positive were mated with each other to obtain the F2 generation BCMA gene humanized homozygous mice.

The expression of humanized BCMA mRNA in positive mice can be confirmed by conventional detection methods, such as RT-PCR. Specifically, a 13-week-old wild-type C57BL/6 female mouse and a BCMA gene humanized homozygous female mouse prepared in this examples were selected, respectively, and spleen tissues were collected after euthanasia (cervical dislocation) . Primers (sequences are shown in the table below) were used to detect the mRNA expression in spleen cells of C57BL/6 mice and BCMA gene humanized homozygous mice. The results are shown in FIG. 10. As can be seen from the figure, only murine BCMA mRNA was detected in wild-type C57BL/6 mice (Fig. 10A) ; and only humanized BCMA mRNA was detected in BCMA humznied homogenous mice (Fig. 10B) .

Table 8 RT-PCR primer sequences and recombinant fragment sizes

Further, insertion/deletion mutations are randomly generated through the repair of chromosomal homologous recombination based on the double-strand break of genomic DNA caused by Cas9 cleavage. This may lead to gene knockout mice with loss of BCMA protein function. A pair of primers were designed to detect BCMA gene knockout mice. The detection results are shown in FIG. 11. The KO-12, KO-13, KO-14, KO-15 and KO-16 mice were further verified by sequencing for BCMA knockout mice. The primers were located on the left side of the 5′-end target site and on the right side of the 3′-end target site, respectively. The primer sequences and the sizes of the recombinant fragments are shown the table below.

Table 9: PCR primer sequences and recombinant fragment sizes for genotyping of BCMA knockout mice

Example 2: Preparation of double humanized or multiple double humanized mice

The humanized BCMA mouse prepared by the methods described herein can also be used to prepare a double-or multi-gene humanized mouse model. For example, in Example 1, the embryonic stem cells used for blastocyst microinjection can be selected from mice containing PD-1, PD-L1, IL4R, IL6R, IL17, CD3, CD28, CD38, or other genetic modifications. Alternatively, the embryonic stem cells of BCMA gene humanized or knockout mice can be selected for gene editing, to obtain a double-gene or multi-gene humanized mouse model comprising humanized BCMA and other genetic modifications. In addition, it is also possible to breed the homozygous or heterozygous BCMA transgenic mice obtained by the methods described herein with other genetically modified homozygous or heterozygous mice, and the offspring can be screened. According to Mendel's law, it is possible to generate double-gene or multi-gene modified heterozygous mice comprising humanized BCMA gene and other genetic modifications. Then the heterozygous mice can be bred with each other to obtain homozygous double-gene or multi-gene humanized mice. These double-gene or multi-gene modified mice can be used to verify the in vivo efficacy of human BCMA and other gene regulators.

Example 3: Drug efficacy verification

The BCMA gene humanized mice or the multi-gene modified mice prepared by this method can be used to evaluate the efficacy of an antibody targeting human BCMA. For example, the BCMA gene humanized homozygous mice can be used to construct a tumor animal model. Here, the mice are divided into control or treatment groups. The treatment group is injected with antibody drugs targeting human BCMA, and the control group is injected with an equal volume of normal saline. Then, the body weight, tumor volume and tumor-related indicators of each group of mice are monitored, to evaluate the safety and in vivo efficacy of antibody drugs.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric BCMA.
The animal of claim 1, wherein the sequence encoding the human or chimeric BCMA is operably linked to an endogenous regulatory element at the endogenous BCMA gene locus in the at least one chromosome.
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric BCMA comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human BCMA (NP_001183.2 (SEQ ID NO: 2) ) .
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric BCMA comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 11.
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric BCMA comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to amino acids 1-47 or 1-54 of SEQ ID NO: 2.
The animal of any one of claims 1-5, wherein the animal is a mammal, e.g., a monkey, a rodent, or a mouse.
The animal of any one of claims 1-6, wherein the animal is a mouse.
The animal of any one of claims 1-7, wherein the animal does not express endogenous BCMA.
The animal of any one of claims 1-8, wherein the animal has one or more cells expressing human or chimeric BCMA.
The animal of any one of claims 1-9, wherein the animal has one or more cells expressing human or chimeric BCMA, and a human BCMA ligand (e.g., APRIL or BAFF) can bind to the expressed human or chimeric BCMA.
The animal of any one of claims 1-9, wherein the animal has one or more cells expressing human or chimeric BCMA, and an endogenous BCMA ligand (e.g., APRIL or BAFF) can bind to the expressed human or chimeric BCMA.
A genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous BCMA with a sequence encoding a corresponding region of human BCMA at an endogenous BCMA gene locus.
The animal of claim 12, wherein the sequence encoding the corresponding region of human BCMA is operably linked to an endogenous regulatory element at the endogenous BCMA locus, and one or more cells of the animal expresses a chimeric BCMA.
The animal of claim 12 or 13, wherein the animal does not express endogenous BCMA.
The animal of any one of claims 12-14, wherein the replaced sequence encodes all or a portion of the extracellular region of endogenous BCMA.
The animal of any one of claims 12-15, wherein the animal has one or more cells expressing a chimeric BCMA having an extracellular region, a transmembrane region, and a cytoplasmic region, wherein the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%identical to the extracellular region of human BCMA.
The animal of claim 16, wherein the extracellular region of the chimeric BCMA has a sequence that has at least 10, 20, 30, 40, or 47 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human BCMA.
The animal of any one of claims 12-17, wherein the sequence encoding a region of endogenous BCMA comprises exon 1 and/or exon 2, or a part thereof, of the endogenous BCMA gene.
The animal of claim 18, wherein the animal is a mouse, and the sequence encoding a region of endogenous BCMA starts within exon 1 and ends within exon 2 of the endogenous mouse BCMA gene.
The animal of any one of claims 12-19, wherein the animal is heterozygous with respect to the replacement at the endogenous BCMA gene locus.
The animal of any one of claims 12-19, wherein the animal is homozygous with respect to the replacement at the endogenous BCMA gene locus.
A method for making a genetically-modified, non-human animal, comprising:

replacing in at least one cell of the animal, at an endogenous BCMA gene locus, a sequence encoding a region of an endogenous BCMA with a sequence encoding a corresponding region of human BCMA.
The method of claim 22, wherein the sequence encoding the corresponding region of human BCMA comprises exon 1 and/or exon 2, or a part thereof, of a human BCMA gene.
The method of claim 22 or 23, wherein the sequence encoding the corresponding region of human BCMA starts within exon 1 and ends within exon 2 of a human BCMA gene.
The method of any one of claims 22-24, wherein the sequence encoding the corresponding region of human BCMA encodes amino acids 1-47 or 1-54 of SEQ ID NO: 2.
The method of any one of claims 22-25, wherein the region of an endogenous BCMA is located within the extracellular region.
The method of any one of claims 22-26, wherein the sequence encoding a region of endogenous BCMA comprises exon 1 and/or exon 2, or a part thereof, of the endogenous BCMA gene.
The method of claim 27, wherein the animal is a mouse, and the sequence encoding a region of an endogenous BCMA starts within exon 1 and ends within exon 2 of the endogenous mouse BCMA gene.
A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric BCMA polypeptide, wherein the chimeric BCMA polypeptide comprises at least 25 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human BCMA, wherein the animal expresses the chimeric BCMA.
The animal of claim 29, wherein the chimeric BCMA polypeptide has at least 40 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human BCMA extracellular region.
The animal of claim 29 or 30, wherein the chimeric BCMA polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 1-47 or 1-54 of SEQ ID NO: 2.
The animal of any one of claims 29-31, wherein the nucleotide sequence is operably linked to an endogenous BCMA regulatory element of the animal.
The animal of any one of claims 29-32, wherein the chimeric BCMA polypeptide comprises an endogenous BCMA transmembrane region and/or an endogenous BCMA cytoplasmic region.
The animal of any one of claims 29-33, wherein the nucleotide sequence is integrated to an endogenous BCMA gene locus of the animal.
The animal of any one of claims 29-34, wherein the chimeric BCMA has at least one mouse BCMA activity and/or at least one human BCMA activity.
A method of making a genetically-modified non-human animal cell that expresses a chimeric BCMA, the method comprising:

replacing at an endogenous BCMA gene locus, a nucleotide sequence encoding a region of endogenous BCMA with a nucleotide sequence encoding a corresponding region of human BCMA, thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the chimeric BCMA, wherein the non-human animal cell expresses the chimeric BCMA.
The method of claim 36, wherein the animal is a mammal, e.g., a monkey, a rodent, or a mouse.
The method of claim 36 or 37, wherein the chimeric BCMA comprises:

a humanized BCMA extracellular region; and

a transmembrane and/or a cytoplasmic region of endogenous BCMA.
The method of any one of claims 36-38, wherein the nucleotide sequence encoding the chimeric BCMA is operably linked to an endogenous BCMA regulatory region, e.g., promoter.
The animal of any one of claims 1-21 and 29-35, wherein the animal further comprises a sequence encoding an additional human or chimeric protein.
The animal of claim 40, wherein the additional human or chimeric protein is tumor necrosis factor ligand superfamily member 13 (APRIL) , programmed cell death protein 1 (PD-1) , IL4, Colony Stimulating Factor 1 (CSF1) , IL34, C-C Motif Chemokine Receptor 2 (CCR2) , CD40, C-X-C Motif Chemokine Receptor 4 (CXCR4) , Vascular Endothelial Growth Factor (VEGF) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , Lymphocyte Activating 3 (LAG-3) , B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , CD27, CD28, CD47, CD 137, CD 154, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , Signal regulatory protein α (SIRPα) or TNF Receptor Superfamily Member 4 (OX40) .
The animal of claim 40, wherein the additional human or chimeric protein is PD-1, and the animal expresses the human or chimeric PD-1.
The method of any one of claims 22-28 and 36-39, wherein the animal further comprises a sequence encoding an additional human or chimeric protein.
The method of claim 43, wherein the additional human or chimeric protein is APRIL, PD-1, IL4, CSF1, IL34, CCR2, CD40, CXCR4, VEGF, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPα or OX40.
The method of claim 43, wherein the additional human or chimeric protein is PD-1, and the animal expresses the human or chimeric PD-1.
A method of determining effectiveness of an anti-BCMA antibody for the treatment of cancer, comprising:

administering the anti-BCMA antibody to the animal of any one of claims 1-21, 29-35, or 40-42, wherein the animal has a cancer; and

determining the inhibitory effects of the anti-BCMA antibody to the cancer.
The method of claim 46, wherein the cancer comprises one or more cells that express BCMA.
The method of claim 46 or 47, wherein the cancer comprises one or more cancer cells that are injected into the animal.
The method of any one of claims 46-48, wherein determining the inhibitory effects of the anti-BCMA antibody to the cancer involves detecting tumor volume or signals (e.g., fluorescence) of tumor cells in the animal.
The method of any one of claims 46-49, wherein the cancer is hematologic malignancies (e.g., multiple myeloma, chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL) , and Hodgkin lymphoma) , tonsil cancer, lymph node cancer, duodenum cancer, colon cancer, stomach cancer, rectum cancer, spleen cancer, salivary gland cancer, small instestine cancer, appendix cancer, thymus cancer, breast cancer, urinary bladder cancer, or gallbladder cancer.
A method of determining effectiveness of an anti-BCMA antibody and an additional therapeutic agent for the treatment of cancer, comprising

administering the anti-BCMA antibody and the additional therapeutic agent to the animal of any one of claims 1-21, 29-35, or 40-42, wherein the animal has a cancer; and

determining the inhibitory effects on the cancer.
The method of claim 51, wherein the animal further comprises a sequence encoding a human or chimeric programmed cell death protein 1 (PD-1) .
The method of claim 51 or 52, wherein the animal further comprises a sequence encoding a human or chimeric programmed death-ligand 1 (PD-L1) .
The method of any one of claims 51-53, wherein the additional therapeutic agent is an anti-PD-1 antibody or an anti-PD-L1 antibody.
The method of any one of claims 51-54, wherein the cancer comprises one or more cancer cells that express BCMA, PD-L1, or PD-L2.
The method of any one of claims 51-55, wherein the cancer is caused by injection of one or more cancer cells into the animal.
The method of any one of claims 51-56, wherein determining the inhibitory effects of the treatment involves detecting tumor volume or signals (e.g., fluorescence) of the cancer cells in the animal.
The method of any one of claims 51-57, wherein the animal has cancer is hematologic malignancies (e.g., multiple myeloma, chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL) , and Hodgkin lymphoma) , tonsil cancer, lymph node cancer, duodenum cancer, colon cancer, stomach cancer, rectum cancer, spleen cancer, salivary gland cancer, small instestine cancer, appendix cancer, thymus cancer, breast cancer, urinary bladder cancer, or gallbladder cancer.
A protein comprising an amino acid sequence, wherein the amino acid sequence is one of the following:

(a) an amino acid sequence set forth in SEQ ID NO: 2 or 11;

(b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 2 or 11;

(c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 2 or 11;

(d) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 2 or 11 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and

(e) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 2 or 11.
A nucleic acid comprising a nucleotide sequence, wherein the nucleotide sequence is one of the following:

(a) a sequence that encodes the protein of claim 59;

(b) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 14 or 15;

(c) a sequence that is at least 90%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 14 or 15; and

(d) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 14 or 15.
A cell comprising the protein of claim 59 and/or the nucleic acid of claim 60.
An animal comprising the protein of claim 59 and/or the nucleic acid of claim 60.
A method of determining toxicity of an anti-BCMA antibody, the method comprising administering the anti-BCMA antibody to the animal of any one of claims 1-21, 29-35, or 40-42; and

determining weight change of the animal.
The method of claim 63, the method further comprising performing a blood test (e.g., determining red blood cell count) .