WO2023098729A1

WO2023098729A1 - Genetically modified non-human animal with human or chimeric genes

Info

Publication number: WO2023098729A1
Application number: PCT/CN2022/135477
Authority: WO
Inventors: Ruili LV; Chong Li; Zhiyuan Shen
Original assignee: Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
Priority date: 2021-11-30
Filing date: 2022-11-30
Publication date: 2023-06-08
Also published as: CN115873902A

Abstract

Provided are genetically modified non-human animals that express a human or chimeric (e.g., humanized) CD200 and/or CD200R, and methods of use thereof.

Description

GENETICALLY MODIFIED NON-HUMAN ANIMAL WITH HUMAN OR CHIMERIC GENES

CLAIM OF PRIORITY

This application claims the benefit of Chinese Patent Application App. No. 202111449904.9, filed on November 30, 2021. The entire contents of the foregoing application are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) CD200 and/or CD200R, and methods of use thereof.

BACKGROUND

The traditional drug research and development typically use in vitro screening approaches. However, these screening approaches cannot provide the body environment (such as tumor microenvironment, stromal cells, extracellular matrix components and immune cell interaction, etc. ) , resulting in a higher rate of failure in drug development. In addition, in view 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 or chimeric CD200 and/or CD200 receptor proteins. The animal model can express human or chimeric CD200 (e.g., humanized CD200) protein and/or human or chimeric CD200R (e.g., humanized CD200R) protein in its body. It can be used in the studies on the function of CD200 and CD200R genes, and can be used in the screening and evaluation of CD200/CD200R signaling pathway modulators (e.g., anti-human CD200 antibodies or anti-human CD200R 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, and cancer therapy for human CD200/CD200R 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 CD200/CD200R 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 cell surface transmembrane glycoprotein CD200 receptor 1 (CD200R) . In some embodiments, the sequence encoding the human or chimeric CD200R is operably linked to an endogenous regulatory element at the endogenous CD200R gene locus in the at least one chromosome. In some embodiments, the sequence encoding a human or chimeric CD200R comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human CD200R (NP_740750.1 (SEQ ID NO: 8) ) . In some embodiments, the sequence encoding a human or chimeric CD200R 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: 15. In some embodiments, the sequence encoding a human or chimeric CD200R comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to amino acids 29-243 of SEQ ID NO: 8. In some embodiments, the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat. In some embodiments, the animal is a mouse. In some embodiments, the animal does not express endogenous CD200R or expresses a decreased level of endogenous CD200R as compared to CD200R expression level in a wild-type animal. In some embodiments, the animal has one or more cells expressing human or chimeric CD200R. In some embodiments, the animal has one or more cells expressing human or chimeric CD200R, and the expressed human or chimeric CD200R can interact with a human OX-2 membrane glycoprotein (CD200) , activating downstream signaling pathways. In some embodiments, the animal has one or more cells expressing human or chimeric CD200R, and the expressed human or chimeric CD200R can interact with an endogenous CD200, activating downstream signaling pathways.

In one aspect, the disclosure is related to a genetically-modified, non-human animal, in some embodiments, the genome of the animal comprises a replacement of a sequence encoding a region of endogenous CD200R with a sequence encoding a corresponding region of human CD200R at an endogenous CD200R gene locus. In some embodiments, the sequence encoding the corresponding region of human CD200R is operably linked to an endogenous regulatory element at the endogenous CD200R locus, and one or more cells of the animal expresses a human or chimeric CD200R. In some embodiments, the animal does not express endogenous CD200R or expresses a decreased level of endogenous CD200R as compared to CD200R expression level in a wild-type animal. In some embodiments, the replaced sequence encodes the extracellular region of CD200R. In some embodiments, the animal has one or more cells expressing a chimeric CD200R having a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region, in some embodiments, 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 CD200R (NP_740750.1 (SEQ ID NO: 8) ) . In some embodiments, the extracellular region of the chimeric CD200R has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 211, 212, 213, 214, or 215 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human CD200R (e.g., amino acids 29-243 of SEQ ID NO: 8) . In some embodiments, the sequence encoding a region of endogenous CD200R comprises exon 2, exon 3, exon 4, and/or exon 5, or a part thereof, of the endogenous CD200R gene. In some embodiments, the animal is a mouse. In some embodiments, the animal is heterozygous or homozygous with respect to the replacement at the endogenous CD200R gene locus.

In one aspect, the disclosure is related to a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a humanized CD200R polypeptide, in some embodiments, the humanized CD200R polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human CD200R, in some embodiments, the animal expresses the humanized CD200R polypeptide. In some embodiments, the humanized CD200R polypeptide has at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 211, 212, 213, 214, or 215 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of human CD200R extracellular region (e.g., amino acids 29-243 of SEQ ID NO: 8) . In some embodiments, the humanized CD200R polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 29-243 of SEQ ID NO: 8. In some embodiments, the humanized CD200R polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 1-25 and 239-326 of SEQ ID NO: 7. In some embodiments, the nucleotide sequence is operably linked to an endogenous CD200R regulatory element of the animal. In some embodiments, the chimeric CD200R polypeptide comprises an endogenous CD200R signal peptide, an endogenous CD200R transmembrane region, and/or an endogenous CD200R cytoplasmic region. In some embodiments, the nucleotide sequence is integrated to an endogenous CD200R gene locus of the animal. In some embodiments, the humanized CD200R polypeptide has at least one mouse CD200R activity and/or at least one human CD200R activity.

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 CD200R gene locus, a sequence encoding a region of endogenous CD200R with a sequence encoding a corresponding region of human CD200R. In some embodiments, the sequence encoding the corresponding region of human CD200R comprises a portion of exon 2, exon 3, exon 4, and a portion of exon 5, of a human CD200R gene. In some embodiments, the sequence encoding the corresponding region of human CD200R encodes amino acids 29-243 of SEQ ID NO: 8. In some embodiments, the region comprises the extracellular region of CD200R. In some embodiments, the animal is a mouse, and the sequence encoding a region of endogenous CD200R comprises a portion of exon 2, exon 3, exon 4, and a portion of exon 5 of the endogenous CD200R gene.

In one aspect, the disclosure is related to a method of making a genetically-modified animal cell that expresses a chimeric CD200R, the method comprising: replacing at an endogenous CD200R gene locus, a nucleotide sequence encoding a region of endogenous CD200R with a nucleotide sequence encoding a corresponding region of human CD200R, thereby generating a genetically-modified animal cell that includes a nucleotide sequence that encodes the chimeric CD200R, in some embodiments, the animal cell expresses the chimeric CD200R. In some embodiments, the animal is a mouse. In some embodiments, the chimeric CD200R comprises a human or humanized CD200R extracellular region; and a signal peptide, a transmembrane and/or a cytoplasmic region of mouse CD200R. In some embodiments, the nucleotide sequence encoding the chimeric CD200R is operably linked to an endogenous CD200R 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 CD200, programmed cell death protein 1 (PD-1) , programmed death-ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , and/or CD73.

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 OX-2 membrane glycoprotein (CD200) . In some embodiments, the sequence encoding the human or chimeric CD200 is operably linked to an endogenous regulatory element at the endogenous CD200 gene locus in the at least one chromosome. In some embodiments, the sequence encoding a human or chimeric CD200 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human CD200 (NP_001004196.2; SEQ ID NO: 2) . In some embodiments, the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat. In some embodiments, the animal is a mouse. In some embodiments, the animal does not express endogenous CD200 or expresses a decreased level of endogenous CD200 as compared to CD200 expression level in a wild-type animal. In some embodiments, the animal has one or more cells expressing human or chimeric CD200. In some embodiments, the animal has one or more cells expressing human or chimeric CD200, and the expressed human or chimeric CD200 can interact with a human CD200R, activating downstream signaling pathways. In some embodiments, the animal has one or more cells expressing human or chimeric CD200, and the expressed human or chimeric CD200 can interact with an endogenous CD200R, activating downstream signaling pathways.

In one aspect, the disclosure is related to a genetically-modified, non-human animal, in some embodiments, the genome of the animal comprises a replacement of a sequence encoding a region of endogenous CD200 with a sequence encoding a corresponding region of human CD200 at an endogenous CD200 gene locus. In some embodiments, the sequence encoding the corresponding region of human CD200 is operably linked to an endogenous regulatory element at the endogenous CD200 locus, and one or more cells of the animal express a human or chimeric CD200. In some embodiments, the animal does not express endogenous CD200 or expresses a decreased level of endogenous CD200 as compared to CD200 expression level in a wild-type animal. In some embodiments, the replaced sequence encodes the full-length protein of CD200. In some embodiments, the animal is a mouse, and the replaced endogenous CD200 region comprises a portion of exon 1, exon 2, exon 3, exon 4, and/or a portion of exon 5 of the endogenous mouse CD200 gene. In some embodiments, the animal is heterozygous or homozygous with respect to the replacement at the endogenous CD200 gene locus.

In one aspect, the disclosure is related to a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a humanized CD200 polypeptide, in some embodiments, the humanized CD200 polypeptide comprises at least 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 291, 292, 293, or 294 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human CD200, in some embodiments, the animal expresses the humanized CD200 polypeptide. In some embodiments, the nucleotide sequence is operably linked to an endogenous CD200 regulatory element of the animal. In some embodiments, the nucleotide sequence is integrated to an endogenous CD200 gene locus of the animal. In some embodiments, the humanized CD200 polypeptide has at least one mouse CD200 activity and/or at least one human CD200 activity.

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 CD200 gene locus, a sequence encoding a region of an endogenous CD200 with a sequence encoding a corresponding region of human CD200. In some embodiments, the sequence encoding the corresponding region of human CD200 comprises a portion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or a portion of exon 7 of a human CD200 gene. In some embodiments, the sequence encoding the corresponding region of human CD200 encodes a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 2. In some embodiments, the animal is a mouse, and the locus is a portion of exon 1, exon 2, exon 3, exons 4, and/or a portion of exon 5 of the mouse CD200 gene.

In one aspect, the disclosure is related to a method of making a genetically-modified non-human animal cell that expresses a human or chimeric CD200, the method comprising: replacing, at an endogenous mouse CD200 gene locus, a nucleotide sequence encoding a region of endogenous CD200 with a nucleotide sequence encoding a corresponding region of human CD200, thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the human or chimeric CD200, in some embodiments, the animal cell expresses the human or chimeric CD200. In some embodiments, the animal is a mouse. In some embodiments, the nucleotide sequence encoding the human or chimeric CD200 is operably linked to an endogenous CD200 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 cell surface transmembrane glycoprotein CD200 receptor 1 (CD200R) , programmed cell death protein 1 (PD-1) , programmed death-ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , and/or CD73.

In one aspect, the disclosure is related to a method of determining effectiveness of a therapeutic agent for the treatment of cancer, comprising: a) administering the therapeutic agent to the animal as described herein, in some embodiments, the animal has a tumor; and b) determining inhibitory effects of the therapeutic agent to the tumor. In some embodiments, the therapeutic agent is an anti-CD200 antibody or an anti-CD200R antibody. In some embodiments, the tumor comprises one or more cancer cells that are injected into the animal. In some embodiments, determining inhibitory effects of the anti-CD200R antibody to the tumor involves measuring the tumor volume in the animal. In some embodiments, the cancer is melanoma, ovarian cancer, myeloid leukemia, B cell malignancy, endocrine malignancy (e.g., small cell lung carcinoma) , bladder cancer, or breast cancer.

In one aspect, the disclosure is related to a method of determining effectiveness of an anti-CD200 antibody or an anti-CD200R antibody, and an additional therapeutic agent for the treatment of cancer, comprising a) administering the anti-CD200 antibody or the anti-CD200R antibody, and the additional therapeutic agent to the animal as described herein, in some embodiments, the animal has a tumor; and b) determining inhibitory effects on the tumor. In some embodiments, the animal further comprises a sequence encoding a human or chimeric PD-1, a human or chimeric PD-L1, and/or a human or chimeric CTLA4. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the tumor comprises one or more tumor cells that express PD-L1 and/or CD200. In some embodiments, the tumor comprises one or more cancer cells that are injected into the animal. In some embodiments, determining inhibitory effects of the treatment involves measuring the tumor volume in the animal. In some embodiments, the animal has melanoma, ovarian cancer, myeloid leukemia, B cell malignancy, endocrine malignancy (e.g., small cell lung carcinoma) , bladder cancer, or breast cancer.

In one aspect, the disclosure is related to a method of determining toxicity of a therapeutic agent comprising: a) administering the therapeutic agent to the animal as described herein; and b) determining effects of the therapeutic agent to the animal. In some embodiments, the therapeutic agent is an anti-CD200 antibody or an anti-CD200R antibody. In some embodiments, determining effects of the therapeutic agent to the animal involves measuring the body weight, red blood cell count, hematocrit, and/or hemoglobin of the animal.

In one aspect, the disclosure is related to a protein comprising an amino acid sequence, in some embodiments, the amino acid sequence is one of the following: (a) an amino acid sequence set forth in SEQ ID NO: 1, 2, 7, 8, or 15; (b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 1, 2, 7, 8, or 15; (c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1, 2, 7, 8, or 15; (d) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 1, 2, 7, 8, or 15 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: 1, 2, 7, 8, or 15.

In one aspect, the disclosure is related to a nucleic acid comprising a nucleotide sequence, in some embodiments, the nucleotide sequence is one of the following: (a) a sequence that encodes the protein as described herein; (b) SEQ ID NO: 3, 4, 5, 6, 9, 10, 13, 14, 16, 17, 18, 19, 35, 36, 37, or 38; (c) a sequence that is at least 90%identical to SEQ ID NO: 3, 4, 5, 6, 9, 10, 13, 14, 16, 17, 18, 19, 35, 36, 37, or 38; 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, 9, 10, 13, 14, 16, 17, 18, 19, 35, 36, 37, or 38.

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

The disclosure further relates to a CD200 and/or CD200R genomic DNA sequence of a humanized mouse, a DNA sequence obtained by a reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence; a construct expressing the amino acid sequence thereof; a cell comprising the construct thereof; a tissue comprising the cell thereof.

The disclosure further relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the method as described herein in the development of a product related to an immunization processes of human cells, the manufacture of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.

The disclosure also relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the method 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 further relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the methods as described herein, in the screening, verifying, evaluating or studying the CD200 and/or CD200R gene function, human CD200 and/or CD200R antibodies, the drugs or efficacies for human CD200 and/or CD200R targeting sites, and the drugs for immune-related diseases and antitumor drugs.

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 mouse and human CD200 gene loci.

FIG. 2 is a schematic diagram showing humanized CD200 gene locus.

FIG. 3 is a schematic diagram showing a CD200 gene targeting strategy.

FIG. 4 shows Southern Blot results of cells after recombination using the LR Probe, Neo Probe, A1 Probe and A Probe, respectively. WT is a wild-type control.

FIG. 5 is a schematic diagram showing the FRT recombination process in CD200 gene humanized mice.

FIGS. 6A-6D show mouse tail PCR identification results of F1 generation mice by primer pairs WT-F/WT-R, WT-F/Mut-R, Frt-F/Frt-R, and Flp-F2/Flp-R2, respectively. M is a marker. WT is a wild-type control. H ₂O is a water control. PC is a positive control.

FIG. 7 is a schematic diagram showing mouse and human CD200R gene loci.

FIG. 8 is a schematic diagram showing humanized CD200R gene locus.

FIG. 9 is a schematic diagram showing a CD200R gene targeting strategy.

FIG. 10 is a schematic diagram showing the FRT recombination process in CD200R gene humanized mice.

FIG. 11 is a schematic diagram showing a CD200R gene targeting strategy.

FIGS. 12A-12B show mouse tail PCR identification results of F1 generation mice by primers R-GT-F and R-GT-R. M is a marker. WT is a wild-type control. H ₂O is a water control.

FIG. 13 shows Southern Blot results of cells after recombination using the A Probe (5’) and 3’ Probe, respectively. WT is a wild-type control.

FIG. 14 shows the alignment between human CD200 amino acid sequence (NP_001004196.2; SEQ ID NO: 2) and mouse CD200 amino acid sequence (NP_034948.3; SEQ ID NO: 1) .

FIG. 15 shows the alignment between human CD200 amino acid sequence (NP_001004196.2; SEQ ID NO: 2) and rat CD200 amino acid sequence (NP_113706.2; SEQ ID NO: 60) .

FIG. 16 shows the alignment between human CD200R amino acid sequence (NP_740750.1; SEQ ID NO: 8) and mouse CD200R amino acid sequence (NP_067300.1; SEQ ID NO: 7) .

FIG. 17 shows the alignment between human CD200R amino acid sequence (NP_740750.1; SEQ ID NO: 8) and rat CD200R amino acid sequence (NP_076443.1; SEQ ID NO: 61) .

DETAILED DESCRIPTION

This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) CD200R, and methods of use thereof.

Tumor-associated inflammation and immune responses are major contributors in regulating tumor growth and progression and establishing a tumor microenvironment (TME) . Tumor-associated myeloid cells (TAMCs) are a group of cells that play key roles in inducing tumor-associated inflammation/angiogenesis, activating tumor invasion/metastasis and regulating tumor-specific T cell responses. Therefore, to better understand cancer pathogenesis and pave the way for developing effective cancer immune therapy, identification and characterization of key pathways that regulate TAMCs in the TME is of critical importance. In this regard, accumulating evidence suggests that CD200-CD200 receptor (CD200R) interaction may be important in regulating the TME. Therefore, CD200 and CD200R are regarded as a potential biomarker and therapeutic target for cancer.

Experimental animal models are an indispensable research tool for studying the effects of antibodies targeting anti-CD200R antibodies and anti-CD200 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.

CD200R

Inhibitory immune receptors control the magnitude and duration of an immune response and thereby prevent collateral damage. CD200 Receptor 1 (also known as cell surface transmembrane glycoprotein CD200 receptor 1, CD200R or OX2R) is an immune inhibitory receptor that is expressed on myeloid cells, T cells and B cells. The major ligand for CD200R is CD200. CD200 is expressed on immune cells and also non-immune cells such as vasculature and neurons. Additionally, in mice two other ligands for CD200R, iSec1 and iSec2, are reported to be expressed in secretory cells of the gut. CD200R is similar in structure to CD200, is located in close proximity to CD200 on the chromosome (in mouse and human) and probably evolved by genetic duplication of CD200. Unlike CD200, more than one isoform of CD200R exists, although the best characterized is CD200R1, which is expressed on cells of the monocyte/myeloid lineage and some T cell subsets. The expression of CD200R is significantly up-regulated when human monocytes are induced to differentiate into dendritic cells in vitro in the presence of GM-CSF and IL-4. In humans, CD200R1 is presumed to represent the only expressed functional receptor for CD200.

CD200R signaling is shown to suppress anti-tumor immunity, and CD200R expression and function is altered in autoimmunity. Besides autoimmunity and cancer, CD200R is implicated in both viral and bacterial immunity. CD200R, the cognate ligand for CD200, is also an immunoglobulin superfamily (IgSF) protein. The expression pattern of mouse and human CD200R is similar, with strong expression in macrophages, neutrophils and mast cells. Unlike most of the IgSF receptors, CD200R lacks ITIM domains. However, its cytoplasmic tail contains three tyrosine residues, and the third tyrosine residue is located within an NPXY motif, which is phosphorylated upon ligation of the CD200R. This leads to the recruitment and phosphorylation of Dok-2 and 1, which then bind to RasGAP and SHIP. In macrophages and mast cells, this cascade has been shown to inhibit the phosphorylation of ERK, P38 and JNK, and the activation of myeloid cells. CD200R signaling in macrophage appears to limit autoimmune inflammation in animal models of multiple sclerosis, arthritis, and lung injury caused by viral infection, as CD200-deficient mice exhibit hyper active macrophages with significant increases in disease severity. Notably, CD200R-deficient mice were more susceptible to arthritis, presumably due to enhanced macrophage functions. These findings suggest that CD200-CD200R pathway is mainly involved in regulating the functions of myeloid lineages of cells.

A detailed description of CD200R and its function can be found, e.g., in Liu, J. Q., et al. "CD200-CD200R pathway in the regulation of tumor immune microenvironment and immunotherapy. " Tumor Microenvironment (2020) : 155-165; and Timmerman, L.M., et al. "Identification of a novel conserved signaling motif in CD200 receptor required for its inhibitory function. " PloS One 16.3 (2021) : e0244770; each of which is incorporated by reference in its entirety.

In human genomes, CD200R gene (Gene ID: 131450) locus has seven exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7 (FIG. 7) . The CD200R protein also has, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. The nucleotide sequence for human CD200R mRNA is NM_170780.3, and the amino acid sequence for human CD200R is NP_740750.1 (SEQ ID NO: 8) . The location for each exon and each region in human CD200R nucleotide sequence and amino acid sequence is listed below:

Table 1

The human CD200R gene (Gene ID: 131450) is located in Chromosome 3 of the human genome, which is located from 112921205 to 112975103 (GRCh38. p13 (GCF_000001405.39) ) . The 5’-UTR is from 112974858 to 112975090, exon 1 is from 112975090 to 112974791, intron 1 is from 112974790 to 112931172, exon 2 is from 112931171 to 112931106, intron 2 is from 112931105 to 112929508, exon 3 is from 112929507 to 112929190, intron 3 is from 112929189 to 112929065, exon 4 is from 112929064 to 112928816, intron 4 is from 112928815 to 112925194, exon 5 is from 112925193 to 112925085, intron 5 is from 112925084 to 112924536, exon 6 is from 112924535 to 112924490, intron 6 is from 112924489 to 112923800, exon 7 is from 112923799 to 112921209, and the 3’-UTR is from 112921209 to 112923676, based on transcript NM_170780.3. All relevant information for human CD200R locus can be found in the NCBI website with Gene ID: 131450, which is incorporated by reference herein in its entirety.

In mice, CD200R gene locus has seven exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7 (FIG. 7) . The mouse CD200R protein also has, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. The nucleotide sequence for mouse CD200R mRNA is NM_021325.3, the amino acid sequence for mouse CD200R is NP_067300.1 (SEQ ID NO: 7) . The location for each exon and each region in the mouse CD200R nucleotide sequence and amino acid sequence is listed below:

Table 2

The mouse CD200R gene (Gene ID: 57781) is located in Chromosome 16 of the mouse genome, which is located from 44586099 to 44615340 (GRCm39 (GCF_000001635.27) ) . The 5’-UTR is from 44586099 to 44586371, exon 1 is from 44586099 to 44586435, intron 1 is from 44586436 to 44609108, exon 2 is from 44609109 to 44609171, intron 2 is from 44609172 to 44609909, exon 3 is from 44609910 to 44610224, intron 3 is from 44610225 to 44610335, exon 4 is from 44610336 to 44610587, intron 4 is from 44610588 to 44613089, exon 5 is from 44613090 to 44613189, intron 5 is from 44613190 to 44613712, exon 6 is from 44613713 to 44613758, intron 6 is from 44613759 to 44614562, exon 7 is from 44614563 to 44615341, and the 3’-UTR is from 44614704 to 44615341, based on transcript NM_021325.3. All relevant information for mouse CD200R locus can be found in the NCBI website with Gene ID: 57781, which is incorporated by reference herein in its entirety.

FIG. 16 shows the alignment between human CD200R amino acid sequence (NP_740750.1; SEQ ID NO: 8) and mouse CD200R amino acid sequence (NP_067300.1; SEQ ID NO: 7) . Thus, the corresponding amino acid residue or region between human and mouse CD200R can be found in FIG. 16.

CD200R genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for CD200R (or CD200R1) in Rattus norvegicus (rat) is 64357, the gene ID for CD200R in Macaca mulatta (Rhesus monkey) is 708734 , the gene ID for CD200R in Equus caballus (horse) is 100071456, and the gene ID for CD200R in Sus scrofa (pig) is 100155169. 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. 17 shows the alignment between human CD200R amino acid sequence (NP_740750.1; SEQ ID NO: 8) and rat CD200R amino acid sequence (NP_076443.1; SEQ ID NO: 61) . Thus, the corresponding amino acid residue or region between human and rodent CD200R can be found in FIG. 17.

The present disclosure provides human or chimeric (e.g., humanized) CD200R nucleotide sequence and/or amino acid sequences. In some embodiments, the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, signal peptide, 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, exon 4, exon 5, exon 6, exon 7, signal peptide, 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, 450, 500, 550, 600, 610, 620, 630, 635, 636, 637, 638, 639, 640, 645, 650, 700, 800, 900, 1000, 1500, 1800, 2000, 2500, 3000, 3500, 3600 nucleotides, 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, 190, 200, 201, 202, 203, 204, 205, 210, 211, 212, 213, 214, 215, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 325, or 326 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, exon 4, exon 5, exon 6, exon 7, signal peptide, extracellular region, transmembrane region, or cytoplasmic region. In some embodiments, a region, a portion, or the entire sequence of mouse exon 1, exon 2, exon 3, exon 5, exon 6, and/or exon 7 (e.g., a portion of exon 2, exon 3, exon 4, and a portion of exon 5) are replaced by a region, a portion, or the entire sequence of the human exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 (e.g., a portion of exon 2, exon 3, exon 4, and a portion of exon 5) .

In some embodiments, a “region” or “portion” of the signal peptide, extracellular region, transmembrane region, cytoplasmic region, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 is deleted.

In some embodiments, the present disclosure is related to a genetically-modified, non-human animal whose genome comprises a chimeric (e.g., humanized ) CD200R nucleotide sequence. In some embodiments, the chimeric (e.g., humanized ) CD200R nucleotide sequence encodes a CD200R protein comprising a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the signal peptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 1-25 of SEQ ID NO: 7. In some embodiments, the signal peptide comprises all or part of endogenous CD200R signal peptide. In some embodiments, the extracellular region comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 29-243 of SEQ ID NO: 8. In some embodiments, the extracellular region comprises all or part of human CD200R extracellular region. In some embodiments, the transmembrane region comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 239-259 of SEQ ID NO: 7. In some embodiments, the transmembrane region comprises all or part of endogenous CD200R transmembrane region. In some embodiments, the cytoplasmic region comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 260-326 of SEQ ID NO: 7. In some embodiments, the cytoplasmic region comprises all or part of endogenous CD200R cytoplasmic region. In some embodiments, the genome of the animal comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 9, 10, 13, 14, or 35, 36, 37, 38.

In some embodiments, the genetically-modified non-human animal described herein comprises a sequence encoding a human or humanized CD200R protein. In some embodiments, the CD200R protein comprises, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the humanized CD200R protein comprises a human or humanized signal peptide. In some embodiments, the humanized CD200R protein comprises an endogenous signal peptide. In some embodiments, the humanized CD200R protein comprises a human or humanized extracellular region. In some embodiments, the humanized CD200R protein comprises an endogenous extracellular region. In some embodiments, the humanized CD200R protein comprises a human or humanized transmembrane region. In some embodiments, the humanized CD200R protein comprises an endogenous transmembrane region. In some embodiments, the humanized CD200R protein comprises a human or humanized cytoplasmic region. In some embodiments, the humanized CD200R protein comprises an endogenous cytoplasmic region. In some embodiments, the humanized CD200R protein comprises an endogenous signal peptide, a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the humanized CD200R protein comprises an endogenous sequence that corresponds to amino acids 1-25 and 239-326 of SEQ ID NO: 7.

In some embodiments, the genetically-modified non-human animal described herein comprises a human or humanized CD200R gene. In some embodiments, the humanized CD200R gene comprises 7 exons. In some embodiments, the humanized CD200R gene comprises endogenous exon 1, humanized exon 2, human exon 3, human exon 4, humanized exon 5, endogenous exon 6, and/or endogenous exon 7. In some embodiments, the humanized CD200R gene comprises 6 introns. In some embodiments, the humanized CD200R gene comprises endogenous intron 1, human intron 2, human intron 3, human intron 4 (optionally disrupted by Neo cassette or one or two Frt recombination sites) , endogenous intron 5, and/or endogenous intron 6. In some embodiments, the humanized CD200R gene comprises human or humanized 5’ UTR. In some embodiments, the humanized CD200R gene comprises human or humanized 3’ UTR. In some embodiments, the humanized CD200R gene comprises endogenous 5’ UTR. In some embodiments, the humanized CD200R gene comprises endogenous 3’ UTR.

Thus, in some embodiments, the present disclosure also provides a chimeric (e.g., humanized) CD200R 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 CD200R mRNA sequence (e.g., NM_021325.3) , mouse CD200R amino acid sequence (e.g., SEQ ID NO: 7) , or a portion thereof (e.g., 5’ UTR, exon 1, a portion of exon 2, a portion of exon 5, exon 6, exon 7, and 3’ UTR) ; 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 CD200R mRNA sequence (e.g., NM_170780.3) , human CD200R amino acid sequence (e.g., SEQ ID NO: 8) , or a portion thereof (e.g., a portion of exon 2, exons 3-4, and a portion of exon 5) .

In some embodiments, the sequence encoding amino acids 26-238 of mouse CD200R (SEQ ID NO: 7) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human CD200R (e.g., amino acids 29-243 of human CD200R (SEQ ID NO: 8) ) .

In some embodiments, the sequence encoding amino acids 1-238 of mouse CD200R (SEQ ID NO: 7) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human CD200R (e.g., amino acids 1-243 of human CD200R (SEQ ID NO: 8) ) .

In some embodiments, the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse CD200R 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 part of or the entire mouse CD200R nucleotide sequence (e.g., a portion of exon 2, exons 3-4, and a portion of exon 5 of NM_021325.3) .

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 part of or the entire mouse CD200R nucleotide sequence (e.g., 5’ UTR, exon 1, a portion of exon 2, a portion of exon 5, exons 6-7, and 3’ UTR of NM_021325.3) .

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 different from part of or the entire human CD200R nucleotide sequence (e.g., 5’ UTR, exon 1, a portion of exon 2, a portion of exon 5, exons 6-7, and 3’ UTR of NM_170780.3) .

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 part of or the entire human CD200R nucleotide sequence (e.g., a portion (at least 20 bp) of exon 2, exons 3-4, and a portion (at least 10 bp) of exon 5 of NM_170780.3) .

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, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 211, 212, 213, 214, 215, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 325, or 326 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from part of or the entire mouse CD200R amino acid sequence (e.g., amino acids 26-238 of NP_067300.1 (SEQ ID NO: 7) ) .

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, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 211, 212, 213, 214, 215, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 325, or 326 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire mouse CD200R amino acid sequence (e.g., amino acids 1-25 and 239-326 of NP_067300.1 (SEQ ID NO: 7) ) .

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, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 211, 212, 213, 214, 215, 216, 217, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 321, 322, 323, 324, or 325 amino acid residues, e.g., contiguous or non- contiguous amino acid residues) that is different from part of or the entire human CD200R amino acid sequence (e.g., amino acids 1-28 and 244-325 of NP_740750.1 (SEQ ID NO: 8) ) .

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, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 211, 212, 213, 214, 215, 216, 217, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 321, 322, 323, 324, or 325 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire human CD200R amino acid sequence (e.g., amino acids 29-243 of NP_740750.1 (SEQ ID NO: 8) ) .

The present disclosure also provides a humanized CD200R 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: 7, 8, or 15;

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: 7, 8, or 15;

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: 7, 8, or 15 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: 7, 8, or 15;

e) an amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 7, 8, or 15 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: 7, 8, or 15.

a) all or part of amino acids 1-243 or 29-243 of SEQ ID NO: 8;

b) an amino acid sequence have a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%to amino acids 1-243 or 29-243 of SEQ ID NO: 8;

c) an amino acid sequence that is different from amino acids 1-243 or 29-243 of SEQ ID NO: 8 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 amino acids 1-243 or 29-243 of SEQ ID NO: 8.

a) all or part of amino acids 1-25 and/or 239-326 of SEQ ID NO: 7;

b) an amino acid sequence have a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%to amino acids 1-25 and/or 239-326 of SEQ ID NO: 7;

c) an amino acid sequence that is different from amino acids 1-25 and/or 239-326 of SEQ ID NO: 7 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 amino acids 1-25 and/or 239-326 of SEQ ID NO: 7.

The present disclosure also relates to a CD200R 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: 9, 10, 13, 14, 35, 36, 37, 38, or a nucleic acid sequence encoding a homologous CD200R amino acid sequence of a humanized mouse CD200R;

b) a nucleic acid sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 9, 10, 13, 14, 35, 36, 37, or 38 under a low stringency condition or a strict stringency condition;

c) 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: 9, 10, 13, 14, 35, 36, 37, or 38;

d) 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: 7, 8, 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%, 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: 7, 8, or 15;

f) 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: 7, 8, or 15 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and/or

g) 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: 7, 8, or 15.

The present disclosure further relates to a CD200R genomic DNA sequence of a humanized mouse. The DNA sequence is obtained by 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: 13 or 14.

CD200

OX-2 membrane glycoprotein (CD200) , also known as MRC OX-2, is a highly conserved, type I transmembrane glycoprotein related structurally to the B7 family of costimulatory receptors. It contains two extracellular immunoglobulin domains, a single transmembrane region, and a short cytoplasmic tail lacking signaling motifs. The molecule is expressed by resting dendritic cells, thymocytes, endothelial cells, neurons and osteoblast precursors (OBp) , as well as by activated B and T cells. Recent studies have revealed that CD200 is also over-expressed in a variety of human cancer cells including human melanoma, ovarian cancer, myeloid leukemia, some B cell malignancies, and a majority of endocrine malignancies such as small cell lung carcinoma. Expression of CD200 is regulated at the transcriptional level by C/EBP-β which also regulates IFN-γ, IL-6, IL-1 and TNF-α-induced responses. IFN-γ and TNF-α have been shown to induce CD200 expression in an NF-kappaB, STAT1 and IRF-1 dependent manner.

A detailed description of CD200 and its function can be found, e.g., in Liu, J.Q., et al. "CD200-CD200R pathway in the regulation of tumor immune microenvironment and immunotherapy. " Tumor Microenvironment (2020) : 155-165; and Kotwica-Mojzych, K., et al. "CD200: CD200R interactions and their importance in immunoregulation. " International Journal of Molecular Sciences 22.4 (2021) : 1602; each of which is incorporated by reference in its entirety.

In human genomes, CD200 gene (Gene ID: 4345) locus has seven exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7 (FIG. 1) . The CD200 protein also has, from N- terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. The nucleotide sequence for human CD200 mRNA is NM_001004196.4, and the amino acid sequence for human CD200 is NP_001004196.2 (SEQ ID NO: 2) . The location for each exon and each region in human CD200 nucleotide sequence and amino acid sequence is listed below:

Table 3

The human CD200 gene (Gene ID: 4345) is located in Chromosome 3 of the human genome, which is located from 112332573 to 112362812 (NC_000003.12 (112332573 to112362812) ) . The 5’-UTR is from 112333156 to 112333212, exon 1 is from 112333156 to 112333224, intron 1 is from 112333225 to 112335942, exon 2 is from 112335943 to 112336017, intron 2 is from 112336018 to 112340901, exon 3 is from 112340902 to 112340983, intron 3 is from 112340984 to 112344961, exon 4 is from 112344962 to 112345288, intron 4 is from 112345289 to 112347557, exon 5 is from 112347558 to 112347830, intron 5 is from 112347831 to 112349711, exon 6 is from 112349712 to 112349819, intron 6 is from 112349820 to 112361542, exon 7 is from 112361543 to 112362132, and the 3’-UTR is from 112361551 to 112362132, based on transcript NM_001004196.4. All relevant information for human CD200 locus can be found in the NCBI website with Gene ID: 4345, which is incorporated by reference herein in its entirety.

In mice, CD200 gene locus has six exons, exon 1, exon 2, exon 3, exon 4, exon 5, and exon 6 (FIG. 1) . The mouse CD200 protein also has, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. The nucleotide sequence for mouse CD200 mRNA is NM_010818.3, the amino acid sequence for mouse CD200 is NP_034948.3 (SEQ ID NO: 1) . The location for each exon and each region in the mouse CD200 nucleotide sequence and amino acid sequence is listed below:

Table 4

The mouse CD200 gene (Gene ID: 17470) is located in Chromosome 16 of the mouse genome, which is located from 45202474 to 45229567 (GRCm39 (GCF_000001635.27) ) . The 5’-UTR is from 45229231 to 45229416, exon 1 is from 45229416 to 45229219, intron 1 is from 45229218 to 45220667, exon 2 is from 45220666 to 45220585, intron 2 is from 45220584 to 45217659, exon 3 is from 45217658 to 45217332, intron 3 is from 45217331 to 45215229, exon 4 is from 45215228 to 45214956, intron 4 is from 45214955 to 45212752, exon 5 is from 45212751 to 45212608, intron 5 is from 45212607 to 45203821, exon 6 is from 45203820 to 45202498, and the 3’-UTR is from 45202498 to 45203820, and 45, 212, 608, based on transcript NM_010818.3. All relevant information for mouse CD200 locus can be found in the NCBI website with Gene ID: 17470, which is incorporated by reference herein in its entirety.

FIG. 14 shows the alignment between human CD200 amino acid sequence (NP_001004196.2; SEQ ID NO: 2) and mouse CD200 amino acid sequence (NP_034948.3; SEQ ID NO: 1) . Thus, the corresponding amino acid residue or region between human and mouse CD200 can be found in FIG. 14.

CD200 genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for CD200 in Rattus norvegicus (rat) is 24560, the gene ID for CD200 in Macaca mulatta (Rhesus monkey) is 708110, the gene ID for CD200 in Bos taurus (cattle) is 534910, and the gene ID for CD200 in Sus scrofa (pig) is 100626182. 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. 15 shows the alignment between human CD200 amino acid sequence (NP_001004196.2; SEQ ID NO: 2) and rat CD200 amino acid sequence (NP_113706.2; SEQ ID NO: 60) . Thus, the corresponding amino acid residue or region between human and rodent CD200 can be found in FIG. 15.

The present disclosure provides human or chimeric (e.g., humanized) CD200 nucleotide sequence and/or amino acid sequences. In some embodiments, the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, signal peptide, 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, exon 4, exon 5, exon 6, signal peptide, 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, 450, 500, 600, 700, 800, 830, 850, 880, 900, 1000, 1500, 1800, 2000, 2200, or 2300 nucleotides, 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, 190, 200, 210, 220, 230, 240, 250, 260, 270, 275, 276, 277, 278, 280, 285, 290, 291, 292, 293, or 294 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, exon 4, exon 5, exon 6, exon 7, signal peptide, extracellular region, transmembrane region, or cytoplasmic region. In some embodiments, a region, a portion, or the entire sequence of mouse exon 1, exon 2, exon 3, exon 5, and/or exon 6 (e.g., a portion of exon 1, exons 2-4, and a portion of exon 5) are replaced by a region, a portion, or the entire sequence of the human exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 (e.g., a portion of exon 1, exons 2-6, and a portion of exon 7) .

In some embodiments, a “region” or “portion” of the signal peptide, extracellular region, transmembrane region, cytoplasmic region, exon 1, exon 2, exon 3, exon 4, exon 5, and/or exon 6 is deleted.

In some embodiments, the present disclosure is related to a genetically-modified, non-human animal whose genome comprises a chimeric (e.g., humanized) CD200 nucleotide sequence. In some embodiments, the chimeric (e.g., humanized) CD200 nucleotide sequence encodes a CD200 protein comprising an extracellular region (including a signal peptide) , a transmembrane region, and a cytoplasmic region. In some embodiments, the extracellular region (including the signal peptide) comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 1-257 of SEQ ID NO: 2. In some embodiments, the signal peptide comprises all or part of human CD200 signal peptide. In some embodiments, the extracellular region comprises all or part of human CD200 extracellular region. In some embodiments, the transmembrane region comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 258-284 of SEQ ID NO: 2. In some embodiments, the transmembrane region comprises all or part of human CD200 transmembrane region. In some embodiments, the cytoplasmic region comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 285-294 of SEQ ID NO: 2. In some embodiments, the cytoplasmic region comprises all or part of human CD200 cytoplasmic region. In some embodiments, the genome of the animal comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 3, 4, 5, 6, 16, 17, 18, or 19.

In some embodiments, the genetically-modified non-human animal described herein comprises a sequence encoding a human or humanized CD200 protein. In some embodiments, the CD200 protein comprises, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the humanized CD200 protein comprises a human or humanized signal peptide. In some embodiments, the humanized CD200 protein comprises an endogenous signal peptide. In some embodiments, the humanized CD200 protein comprises a human or humanized extracellular region. In some embodiments, the humanized CD200 protein comprises an endogenous extracellular region. In some embodiments, the humanized CD200 protein comprises a human or humanized transmembrane region. In some embodiments, the humanized CD200 protein comprises an endogenous transmembrane region. In some embodiments, the humanized CD200 protein comprises a human or humanized cytoplasmic region. In some embodiments, the humanized CD200 protein comprises an endogenous cytoplasmic region. In some embodiments, the human or humanized CD200 protein comprises SEQ ID NO: 2.

In some embodiments, the genetically-modified non-human animal described herein comprises a human or humanized CD200 gene. In some embodiments, the humanized CD200 gene comprises 7 exons. In some embodiments, the humanized CD200 gene comprises humanized exon 1, human exon 2, human exon 3, human exon 4, human exon 5, human exon 6, humanized exon 7, and/or endogenous exon 8 (e.g., exon 6 of mouse CD200 gene) . In some embodiments, the humanized CD200 gene comprises 7 introns. In some embodiments, the humanized CD200 gene comprises human intron 1, human intron 2, human intron 3, human intron 4, human intron 5, human intron 6 (optionally disrupted by Neo cassette or one or two Frt recombination sites) , and/or endogenous intron 7 (e.g., intron 5 of mouse CD200 gene) . In some embodiments, the humanized CD200 gene comprises human or humanized 5’ UTR. In some embodiments, the humanized CD200 gene comprises human or humanized 3’ UTR. In some embodiments, the humanized CD200 gene comprises endogenous 5’ UTR. In some embodiments, the humanized CD200 gene comprises endogenous 3’ UTR.

Thus, in some embodiments, the present disclosure also provides a chimeric (e.g., humanized) CD200 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 CD200 mRNA sequence (e.g., NM_010818.3) , mouse CD200 amino acid sequence (e.g., SEQ ID NO: 1) , or a portion thereof (e.g., 5’ UTR, a portion of exon 1, a portion of exon 5, exon 6, and 3’ UTR) ; 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 CD200 mRNA sequence (e.g., NM_001004196.4) , human CD200 amino acid sequence (e.g., SEQ ID NO: 2) , or a portion thereof (e.g., a portion of exon 1, exons 2-6, and a portion of exon 7) .

In some embodiments, the sequence encoding amino acids 1-278 of mouse CD200 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human CD200 (e.g., amino acids 1-294 of human CD200 (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 CD200 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 part of or the entire mouse CD200 nucleotide sequence (e.g., a portion of exon 1, exons 2-4, and a portion of exon 5 of NM_010818.3) .

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 part of or the entire mouse CD200 nucleotide sequence (e.g., 5’ UTR, a portion of exon 1, a portion of exon 5, exon 6, and 3’ UTR of NM_010818.3) .

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 different from part of or the entire human CD200 nucleotide sequence (e.g., 5’ UTR, a portion of exon 1, a portion of exon 7, and 3’ UTR of NM_001004196.4) .

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 part of or the entire human CD200 nucleotide sequence (e.g., a portion (at least 5 bp) of exon 1, exons 2-6, and a portion (at least 1 bp) of exon 7 of NM_001004196.4) .

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, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 275, 276, 277, or 278 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from part of or the entire mouse CD200 amino acid sequence (e.g., NP_034948.3 (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, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 275, 276, 277, or 278 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire mouse CD200 amino acid sequence (e.g., NP_034948.3 (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, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 291, 292, 293, or 294 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from part of or the entire human CD200 amino acid sequence (e.g., NP_001004196.2 (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, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 291, 292, 293, or 294 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire human CD200 amino acid sequence (e.g., NP_001004196.2 (SEQ ID NO: 2) ) .

The present disclosure also provides a humanized CD200 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: 1 or 2;

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: 1 or 2;

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: 1 or 2 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: 1 or 2;

e) an amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 1 or 2 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: 1 or 2.

The present disclosure also relates to a CD200 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, 16, 17, 18, 19, or a nucleic acid sequence encoding a homologous CD200 amino acid sequence of a humanized mouse CD200;

b) a nucleic acid sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 3, 4, 5, 6, 16, 17, 18, or 19, under a low stringency condition or a strict stringency condition;

c) 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, 16, 17, 18, or 19;

d) 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: 1 or 2;

e) 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: 1 or 2;

f) 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: 1 or 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and/or

g) 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: 1 or 2.

The present disclosure further relates to a CD200 genomic DNA sequence of a humanized mouse. The DNA sequence is obtained by 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 or 6.

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: 1, 2, 7, 8, or 15, and has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 1, 2, 7, 8, or 15 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: 1, 2, 7, 8, or 15 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: 5, 6, 13, or 14, and encodes a polypeptide that has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 5, 6, 13, or 14 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: 3, 4, 5, 6, 9, 10, 13, 14, 16, 17, 18, 19, 35, 36, 37, or 38 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, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, or 5000 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, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 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 example, 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) CD200 and/or CD200R from an endogenous non-human CD200 and/or CD200R loci.

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 CD200 and/or CD200R loci 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 wild-type 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 CD200R gene or a humanized CD200R nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human CD200R gene, at least one or more portions of the gene or the nucleic acid is from a non-human CD200R gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes an CD200R protein. The encoded CD200R protein is functional or has at least one activity of the human CD200R protein or the non-human CD200R protein, e.g., interacting with human or non-human CD200. In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized CD200 gene or a humanized CD200 nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human CD200 gene, at least one or more portions of the gene or the nucleic acid is from a non-human CD200 gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes an CD200 protein. The encoded CD200 protein is functional or has at least one activity of the human CD200 protein or the non-human CD200 protein, e.g., interacting with human or non-human CD200R.

In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized CD200R protein or a humanized CD200R 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 CD200R protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human CD200R protein. The humanized CD200R protein or the humanized CD200R polypeptide is functional or has at least one activity of the human CD200R protein or the non-human CD200R protein. In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized CD200 protein or a humanized CD200 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 CD200 protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human CD200 protein. The humanized CD200 protein or the humanized CD200 polypeptide is functional or has at least one activity of the human CD200 protein or the non-human CD200 protein.

In some embodiments, any of the cytoplasmic region described herein is human or humanized. In some embodiments, any of the transmembrane region described herein is human or humanized. In some embodiments, any of the extracellular region described herein is human or humanized. In some embodiments, any of the signal peptide described herein is human or humanized.

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 non-human animal is a rodent. In some embodiments, the non-human animal is a mouse having a 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 (C57BL/10Cr and C57BL/Ola) , C58, CBA/Br, CBA/Ca, CBA/J, CBA/st, or CBA/H background.

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 CD200 and/or CD200R 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 mice, SCID mice, NOD/SCID mice, IL2Rγ knockout 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 Rag1 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 CD200 and/or CD200R loci, 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 mice, SCID mice, NOD/SCID mice, IL-2Rγ knockout mice, NOD/SCID/γc ^null mice, nude mice, Rag1 and/or Rag2 knockout mice, NOD-Prkdc ^scid IL-2rγ ^null mice, NOD-Rag 1 ^-/--IL2rg ^-/- (NRG) mice, Rag 2 ^-/--IL2rg ^-/- (RG) 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 CD200 and/or CD200R coding sequences with human mature CD200 and/or CD200R coding sequences.

Genetically modified non-human animals can comprise a modification at an endogenous non-human CD200 and/or CD200R loci. In some embodiments, the modification can comprise a human nucleic acid sequence encoding at least a portion of a mature CD200 and/or CD200R proteins (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the mature CD200 and/or CD200R protein sequences) . 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 CD200 and/or CD200R loci in the germline of the animal.

Genetically modified animals can express a human CD200R and/or a chimeric (e.g., humanized) CD200R from endogenous mouse loci, wherein the endogenous mouse CD200R gene has been replaced with a human CD200R gene and/or a nucleotide sequence that encodes a region of human CD200R 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 CD200R extracellular region sequence. In various embodiments, an endogenous non-human CD200R locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a mature CD200R protein.

In some embodiments, the genetically modified mice can express the human CD200R and/or chimeric CD200R (e.g., humanized CD200R) from an endogenous locus that is under control of mouse promoters and/or mouse regulatory elements. The replacement (s) at the endogenous mouse locus provides non-human animals that express human CD200R or chimeric CD200R (e.g., humanized CD200R) 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 CD200R or the chimeric CD200R (e.g., humanized CD200R) expressed in animal can maintain one or more functions of the wild-type mouse or human CD200R in the animal. For example, the expressed CD200R can bind to human or non-human CD200. Furthermore, in some embodiments, the animal does not express endogenous CD200R. In some embodiments, the animal expresses a decreased level of endogenous CD200R as compared to a wild-type animal. As used herein, the term “endogenous CD200R” refers to CD200R protein that is expressed from an endogenous CD200R 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 amino acids 29-243 of human CD200R (NP_740750.1; SEQ ID NO: 8) . 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: 15.

The genome of the genetically modified animal can comprise a replacement at an endogenous CD200R gene locus of a sequence encoding a region of endogenous CD200R with a sequence encoding a corresponding region of human CD200R. In some embodiments, the sequence that is replaced is any sequence within the endogenous CD200R gene locus, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, 5’-UTR, 3’-UTR, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, or any combination thereof. In some embodiments, the sequence that is replaced is within the regulatory region of the endogenous CD200R gene. In some embodiments, the sequence that is replaced is a portion of exon 2, exons 3-4, and a portion of exon 5, of an endogenous mouse CD200R gene locus.

The genetically modified animal can have one or more cells expressing a human or chimeric CD200R (e.g., humanized CD200R) having, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the signal peptide comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the signal peptide of endogenous CD200R (e.g., amino acids 1-25 of SEQ ID NO: 7) . In some embodiments, the signal peptide of the humanized CD200R has a sequence that has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids (e.g., contiguously or non-contiguously) that are identical to the signal peptide of endogenous (e.g., mouse) CD200R. In some embodiments, the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the extracellular region of human CD200R (e.g., amino acids 29-243 of SEQ ID NO: 8) . In some embodiments, the extracellular region of the humanized CD200R has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 211, 212, 213, 214, or 215 amino acids (e.g., contiguously or non-contiguously) that are identical to the extracellular region of human CD200R. In some embodiments, the extracellular region described herein includes the signal peptide. In some embodiments, the extracellular region described herein does not include the signal peptide. Because human CD200R and non-human CD200R (e.g., mouse CD200R) sequences, in many cases, are different, antibodies that bind to human CD200R will not necessarily have the same binding affinity with non-human CD200R or have the same effects to non-human CD200R. Therefore, the genetically modified animal having a human or a humanized extracellular region can be used to better evaluate the effects of anti-human CD200R antibodies in an animal model.

In some embodiments, the transmembrane comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the transmembrane region of endogenous CD200R (e.g., amino acids 239-259 of SEQ ID NO: 7) . In some embodiments, the transmembrane region of the humanized CD200R has a sequence that has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids (contiguously or non-contiguously) that are identical to the transmembrane region of endogenous CD200R (e.g., mouse CD200R) . In some embodiments, the cytoplasmic comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the cytoplasmic of endogenous CD200R (e.g., amino acids 260-326 of SEQ ID NO: 7) . In some embodiments, the cytoplasmic region of the humanized CD200R has a sequence that has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 65, 66, or 67 amino acids (contiguously or non-contiguously) that are identical to the cytoplasmic region of endogenous CD200R (e.g., mouse CD200R) . In some embodiments, the entire transmembrane region and the entire cytoplasmic region of the humanized CD200R described herein are derived from endogenous sequence.

In some embodiments, the genome of the genetically modified animal comprises a sequence encoding an amino acid sequence that corresponds to a portion or the entire sequence of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of human CD200R; a portion or the entire sequence of the extracellular region of human CD200R; or a portion or the entire sequence of amino acids 29-243 of SEQ ID NO: 8.

In some embodiments, the genome of the genetically modified animal comprises a portion of exon 2, exons 3-4, and a portion of exon 5 of human CD200R gene. In some embodiments, the portion of exon 2 includes at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 46, 47, 48, 49, 50, 55, 60, 65, or 66 nucleotides. In some embodiments, the portion of exon 2 includes 49 nucleotides. In some embodiments, the portion of exon 2 includes a nucleotide of at least 20 bp. In some embodiments, the portion of exon 5 includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 105, 106, 107, 108, or 109 nucleotides. In some embodiments, the portion of exon 5 includes 29 nucleotides. In some embodiments, the portion of exon 5 includes a nucleotide of at least 10 bp.

In some embodiments, the non-human animal can have, at an endogenous CD200R gene locus, a nucleotide sequence encoding a chimeric human/non-human CD200R polypeptide, wherein a human portion of the chimeric human/non-human CD200R polypeptide comprises the entire human CD200R extracellular region, and wherein the animal expresses a functional CD200R on a surface of a cell of the animal. The human portion of the chimeric human/non-human CD200R polypeptide can comprise an amino acid sequence encoded by a portion of exon 2, exons 3-4, and/or a portion of exon 5 of human CD200R. In some embodiments, the human portion of the chimeric human/non-human CD200R polypeptide can comprise a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 29-243 of SEQ ID NO: 8. In some embodiments, the transmembrane region includes a sequence corresponding to the entire or part of amino acids 239-259 of SEQ ID NO: 7. In some embodiments, the cytoplasmic region includes a sequence corresponding to the entire or part of amino acids 260-326 of SEQ ID NO: 7. In some embodiments, the chimeric human/non-human CD200R polypeptide comprises a signal peptide, which includes a sequence corresponding to the entire or part of amino acids 1-25 of SEQ ID NO: 7.

In some embodiments, the non-human portion of the chimeric human/non-human CD200R polypeptide comprises the entire signal peptide, the entire transmembrane region, and/or the entire cytoplasmic region of an endogenous non-human CD200R polypeptide.

In some embodiments, the non-human animal described herein can generate a soluble CD200R. In some embodiments, the soluble CD200R is cleaved from any of the CD200R protein (e.g., humanized CD200R) described herein.

Furthermore, the genetically modified animal can be heterozygous with respect to the replacement at the endogenous CD200R locus, or homozygous with respect to the replacement at the endogenous CD200R locus.

In some embodiments, the humanized CD200R locus lacks a human CD200R 5’-UTR. In some embodiment, the humanized CD200R locus comprises an endogenous (e.g., mouse) 5’-UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3’-UTR. In appropriate cases, it may be reasonable to presume that the mouse and human CD200R genes appear to be similarly regulated based on the similarity of their 5’-flanking sequence. As shown in the present disclosure, humanized CD200R mice that comprise a replacement at an endogenous mouse CD200R locus, which retain mouse regulatory elements but comprise a humanization of CD200R encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized CD200R are grossly normal.

Genetically modified animals can express a human CD200 and/or a chimeric (e.g., humanized) CD200 from endogenous mouse loci, wherein the endogenous mouse CD200 gene has been replaced with a human CD200 gene and/or a nucleotide sequence that encodes a region of human CD200 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 CD200 sequence. In various embodiments, an endogenous non-human CD200 locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a mature CD200 protein.

In some embodiments, the genetically modified mice can express the human CD200 and/or chimeric CD200 (e.g., humanized CD200) from an endogenous locus that is under control of mouse promoters and/or mouse regulatory elements. The replacement (s) at the endogenous mouse locus provides non-human animals that express human CD200 or chimeric CD200 (e.g., humanized CD200) 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 CD200 or the chimeric CD200 (e.g., humanized CD200) expressed in animal can maintain one or more functions of the wild-type mouse or human CD200 in the animal. For example, the expressed CD200 can bind to human or non-human CD200R. Furthermore, in some embodiments, the animal does not express endogenous CD200. In some embodiments, the animal expresses a decreased level of endogenous CD200 as compared to a wild-type animal. As used herein, the term “endogenous CD200” refers to CD200 protein that is expressed from an endogenous CD200 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 CD200 (NP_001004196.2; SEQ ID NO: 2) .

The genome of the genetically modified animal can comprise a replacement at an endogenous CD200 gene locus of a sequence encoding a region of endogenous CD200 with a sequence encoding a corresponding region of human CD200. In some embodiments, the sequence that is replaced is any sequence within the endogenous CD200 gene locus, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, 5’-UTR, 3’-UTR, intron 1, intron 2, intron 3, intron 4, intron 5, or any combination thereof. In some embodiments, the sequence that is replaced is within the regulatory region of the endogenous CD200 gene. In some embodiments, the sequence that is replaced is a portion of exon 1, exons 2-4, and a portion of exon 5, of an endogenous mouse CD200 gene locus.

The genetically modified animal can have one or more cells expressing a human or chimeric CD200 (e.g., humanized CD200) having, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the signal peptide comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the signal peptide of human CD200. In some embodiments, the extracellular region (including the signal peptide) comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the extracellular region (including the signal peptide) of human CD200 (e.g., amino acids 1-257 of SEQ ID NO: 2) . In some embodiments, the extracellular region (including the signal peptide) of the humanized CD200 has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 255, 256, or 257 amino acids (e.g., contiguously or non-contiguously) that are identical to the extracellular region (including the signal peptide) of human CD200. In some embodiments, the extracellular region described herein includes the signal peptide. In some embodiments, the extracellular region described herein does not include the signal peptide. Because human CD200 and non-human CD200 (e.g., mouse CD200) sequences, in many cases, are different, antibodies that bind to human CD200 will not necessarily have the same binding affinity with non-human CD200 or have the same effects to non-human CD200. Therefore, the genetically modified animal having a human or a humanized extracellular region can be used to better evaluate the effects of anti-human CD200 antibodies in an animal model.

In some embodiments, the transmembrane comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the transmembrane region of human CD200 (e.g., amino acids 258-284 of SEQ ID NO: 2) . In some embodiments, the transmembrane region of the humanized CD200 has a sequence that has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids (contiguously or non-contiguously) that are identical to the transmembrane region of human CD200. In some embodiments, the cytoplasmic comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the cytoplasmic of human CD200 (e.g., amino acids 285-294 of SEQ ID NO: 2) . In some embodiments, the cytoplasmic region of the humanized CD200 has a sequence that has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (contiguously or non-contiguously) that are identical to the cytoplasmic region of human CD200.

In some embodiments, the genome of the genetically modified animal comprises a sequence encoding an amino acid sequence that corresponds to a portion or the entire sequence of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of human CD200; a portion or the entire sequence of human CD200; or a portion or the entire sequence of SEQ ID NO: 2.

In some embodiments, the genome of the genetically modified animal comprises a portion of exon 1, exons 2-6, and a portion of exon 7 of human CD200 gene. In some embodiments, the portion of exon 1 includes at least 5, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 46, 47, 48, 49, 50, 55, 60, 65, 66, 67, 68, or 69 nucleotides. In some embodiments, the portion of exon 1 includes 12 nucleotides. In some embodiments, the portion of exon 1 includes a nucleotide of at least 5 bp. In some embodiments, the portion of exon 7 includes 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, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1250, 1260, or 1270 nucleotides. In some embodiments, the portion of exon 7 includes 5 nucleotides. In some embodiments, the portion of exon 7 includes a nucleotide of at least 1 bp.

In some embodiments, the non-human animal described herein can generate a soluble CD200. In some embodiments, the soluble CD200 is cleaved from any of the CD200 protein (e.g., humanized CD200) described herein.

Furthermore, the genetically modified animal can be heterozygous with respect to the replacement at the endogenous CD200 locus, or homozygous with respect to the replacement at the endogenous CD200 locus.

In some embodiments, the humanized CD200 locus lacks a human CD200 5’-UTR. In some embodiment, the humanized CD200 locus comprises an endogenous (e.g., mouse) 5’-UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3’-UTR. In appropriate cases, it may be reasonable to presume that the mouse and human CD200 genes appear to be similarly regulated based on the similarity of their 5’-flanking sequence. As shown in the present disclosure, humanized CD200 mice that comprise a replacement at an endogenous mouse CD200 locus, which retain mouse regulatory elements but comprise a humanization of CD200 encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized CD200 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 CD200R gene and/or a humanized CD200 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 CD200 and/or CD200R 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 FIGS. 2, 3, 5, 8, 9, 10, and 11) . In some embodiments, a non-human mammal expressing human or humanized CD200 and/or CD200R is provided. In some embodiments, the tissue-specific expression of human or humanized CD200 and/or CD200R protein is provided.

In some embodiments, the expression of human or humanized CD200 and/or CD200R in a genetically modified animal is controllable, as by the addition of a specific inducer or repressor substance. In some embodiments, the specific inducer is selected from Tet-Off System/Tet-On System, or Tamoxifen System.

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 be 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 cells 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 CD200 and/or CD200R proteins 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 CD200 and/or CD200R proteins.

In another aspect, the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous CD200R gene, wherein the disruption of the endogenous CD200R gene comprises deletion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7, or part thereof of the endogenous CD200R gene.

In some embodiments, the disruption of the endogenous CD200R gene comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7 of the endogenous CD200R gene.

In some embodiments, the disruption of the endogenous CD200R gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, and intron 6 of the endogenous CD200R gene.

In some embodiments, the deletion can comprise deleting 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, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 5000, 10000, 15000, 20000, 25000, 26000, 27000, 28000, 29000, or more nucleotides.

In some embodiments, the disruption of the endogenous CD200R gene comprises the deletion of 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, 10, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nucleotides of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 (e.g., deletion of at least 20 nucleotides from exon 2, exons 3-4, and at least 5 nucleotides from exon 5) .

In another aspect, the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous CD200 gene, wherein the disruption of the endogenous CD200 gene comprises deletion of exon 1, exon 2, exon 3, exon 4, exon 5, and/or exon 6, or part thereof of the endogenous CD200 gene.

In some embodiments, the disruption of the endogenous CD200 gene comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, and exon 6 of the endogenous CD200 gene.

In some embodiments, the disruption of the endogenous CD200 gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, and intron 5 of the endogenous CD200 gene.

In some embodiments, the deletion can comprise deleting 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, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 5000, 10000, 15000, 20000, 25000, 26000, 27000, or more nucleotides.

In some embodiments, the disruption of the endogenous CD200 gene comprises the deletion of 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, 10, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nucleotides of exon 1, exon 2, exon 3, exon 4, exon 5, and/or exon 6 (e.g., deletion of at least 5 nucleotides from exon 1, exons 2-4, and at least 50 nucleotides from exon 5) .

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 CD200R 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 CD200R 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 from the position 44605076 to the position 44609119 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 from the position 44613110 to the position 44616609 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 from the position 44607693 to the position 44609119 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 from the position 44613110 to the position 44614556 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 more than about 4 kb, about 4.5 kb, about 5 kb, about 5.5 kb, about 6 kb, about 6.5 kb, about 7 kb, about 7.5 kb, or about 8 kb.

In some embodiments, the region to be altered is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of CD200R gene (e.g., a portion of exon 2, exons 3-4, and a portion of exon 5 of mouse CD200R gene) .

The targeting vector can further include one or more selectable markers, e.g., positive or negative selectable markers. In some embodiments, the positive selectable marker is a Neo gene or Neo cassette. In some embodiments, the negative selectable marker is a DTA gene.

In some embodiments, the sequence of the 5’ arm is shown in SEQ ID NO: 9; and the sequence of the 3’ arm is shown in SEQ ID NO: 10. In some embodiments, the sequence of the 5’ arm is shown in SEQ ID NO: 11; and the sequence of the 3’ arm is shown in SEQ ID NO: 12.

In some embodiments, the sequence is derived from human (e.g., 112925165-112931154 of NCBI accession number NC_000003.12; or positions 331-975 of NM_170780.3) . For example, the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human CD200R gene, preferably exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of the human CD200R gene. In some embodiments, the nucleotide sequence of the humanized CD200R encodes the entire or the part of human CD200R protein with the NCBI accession number NP_740750.1 (SEQ ID NO: 8) .

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 CD200 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 CD200 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 region to be altered (5’ arm) is selected from the nucleotides from the position 45229231 to the position 45232783 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 from the position 45208864 to the position 45212611 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 more than about 5 kb, about 10 kb, about 15 kb, about 20 kb, about 25 kb, about 26 kb, about 27 kb, about 28 kb, or about 29 kb.

In some embodiments, the region to be altered is exon 1, exon 2, exon 3, exon 4, exon 5, and/or exon 6 of CD200 gene (e.g., a portion of exon 1, exons 2-4, and a portion of exon 5 of mouse CD200 gene) .

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 is derived from human (e.g., 112333213-112361547 of NCBI accession number NC_000003.12; or positions 58-939 of NM_001004196.4) . For example, the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human CD200 gene, preferably exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of the human CD200 gene. In some embodiments, the nucleotide sequence of the humanized CD200 encodes the entire or the part of human CD200 protein with the NCBI accession number NP_001004196.2 (SEQ ID NO: 2) .

The disclosure also provides vectors for constructing a humanized animal model or a knock-out model. In some embodiments, the vectors comprise sgRNA sequence, wherein the sgRNA sequence target CD200R gene, and the sgRNA is unique on the target sequence of the gene to be altered, and meets the sequence arrangement rule of 5’-NNN (20) -NGG3’ or 5’-CCN-N (20) -3’ ; and in some embodiments, the targeting site of the sgRNA in the mouse CD200R gene is located on the exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, upstream of exon 1, or downstream of exon 7 (e.g., exon 2 and exon 5) of the mouse CD200R gene.

In some embodiments, the targeting sequences are shown as SEQ ID NOs: 58, 59, 43, 44, 45, 46, 47, and 48. Thus, the disclosure provides sgRNA sequences for constructing a genetic modified animal model. In some embodiments, the oligonucleotide sgRNA sequences are set forth in SEQ ID NOs: 58 and 59.

In some embodiments, the disclosure relates to a plasmid construct (e.g., pT7-sgRNA) including the sgRNA sequence, and/or a cell including the construct.

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. In some embodiments, the cell is an embryonic stem 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 CD200R gene locus, a sequence encoding a region of an endogenous CD200R with a sequence encoding a corresponding region of human or chimeric CD200R. In some embodiments, the disclosure provides replacing in at least one cell of the animal, at an endogenous CD200 gene locus, a sequence encoding a region of an endogenous CD200 with a sequence encoding a corresponding region of human or chimeric CD200. 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. 9 and FIG. 11 show humanization strategies for a mouse CD200R locus. FIG. 3 shows a humanization strategy for a mouse CD200 locus. The targeting strategies involve a vector comprising a 5’ homologous arm, a human CD200 or CD200R gene fragment, and a 3’ homologous arm. The process can involve replacing endogenous CD200 or CD200R 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 CD200 or CD200R sequence with human CD200 or CD200R sequence.

Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of replacing at an endogenous CD200R locus (or site) , a nucleic acid encoding a region of endogenous CD200R with a sequence encoding a corresponding region of human CD200R. The sequence can include a region (e.g., a part or the entire region) of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of a human CD200R gene. In some embodiments, the sequence includes a portion of exon 2, exons 3-4, and a portion of exon 5 of a human CD200R gene (e.g., nucleic acids 331-975 of NM_170780.3) . In some embodiments, the region includes the extracellular region of human CD200R (e.g., amino acids 29-243 of SEQ ID NO: 8) . In some embodiments, the endogenous CD200R locus is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of mouse CD200R. In some embodiments, the sequence includes exon 1, a portion of exon 2, a portion of exon 5, and exons 6-7 of mouse CD200R gene (e.g., nucleic acids 1-348 and 988-1891 of NM_021325.3) .

In some embodiments, the methods of modifying a CD200R locus of a mouse to express a chimeric human/mouse CD200R peptide can include the steps of replacing at the endogenous mouse CD200R locus a nucleotide sequence encoding a mouse CD200R with a nucleotide sequence encoding a human CD200R, thereby generating a sequence encoding a chimeric human/mouse CD200R.

In some embodiments, the nucleotide sequence encoding the chimeric human/mouse CD200R can include a first nucleotide sequence encoding the signal peptide of mouse CD200R; a second nucleotide sequence encoding the extracellular region of human CD200R; and a third nucleotide sequence encoding the transmembrane region and the cytoplasmic region of mouse CD200R. 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.

Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of replacing at an endogenous CD200 locus (or site) , a nucleic acid encoding a region of endogenous CD200 with a sequence encoding a corresponding region of human CD200. The sequence can include a region (e.g., a part or the entire region) of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of a human CD200 gene. In some embodiments, the sequence includes a portion of exon 1, exons 2-6, and a portion of exon 7 of a human CD200 gene (e.g., nucleic acids 58-939 of NM_001004196.4) . In some embodiments, the region includes the full-length protein of human CD200 (e.g., SEQ ID NO: 2) . In some embodiments, the endogenous CD200 locus is exon 1, exon 2, exon 3, exon 4, exon 5, and/or exon 6 of mouse CD200. In some embodiments, the sequence includes a portion of exon 1, a portion of exon 5, and exon 6 of mouse CD200 gene (e.g., nucleic acids 1-186 and 1021-2347 of NM_010818.3) .

In some embodiments, the methods of modifying a CD200 locus of a mouse to express a chimeric human/mouse CD200 peptide can include the steps of replacing at the endogenous mouse CD200 locus a nucleotide sequence encoding a mouse CD200 with a nucleotide sequence encoding a human CD200, thereby generating a sequence encoding a chimeric human/mouse CD200.

The present disclosure further provides a method for establishing CD200 and/or CD200R gene humanized animal models, 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 pseudopregnancy (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, methods of making the genetically modified animal comprises modifying the coding frame of the non-human animal’s CD200 or CD200R gene, e.g., by inserting a nucleotide sequence (e.g., DNA or cDNA sequence) encoding human or humanized CD200 or CD200R protein, e.g., immediately after the endogenous regulatory element of the non-human animal’s CD200 or CD200R gene. For example, one or more functional region sequences of the non-human animal’s CD200 or CD200R gene can be knocked out, or inserted with a sequence, such that the non-human animal cannot express or expresses a decreased level of endogenous CD200 or CD200R protein. In some embodiments, the coding frame of the modified non-human animal’s CD200R gene can be all or part of the nucleotide sequence from exon 1 to exon 7 of the non-human animal’s CD200R gene. In some embodiments, the coding frame of the modified non-human animal’s CD200 gene can be all or part of the nucleotide sequence from exon 1 to exon 6 of the non-human animal’s CD200 gene.

In some embodiments, methods of making the genetically modified animal comprises inserting a nucleotide sequence encoding human or humanized CD200 or CD200R protein and/or an auxiliary sequence after the endogenous regulatory element of the non-human animal’s CD200 or CD200R gene. In some embodiments, the auxiliary sequence can be a stop codon, such that the CD200 and/or CD200R gene humanized animal model can express human or humanized CD200 and/or CD200R proteins in vivo, but does not express non-human animal’s CD200 and/or CD200R proteins. In some embodiments, the auxiliary sequence includes WPRE (WHP Posttranscriptional Response Element) , loxP, and/or polyA.

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

(1) providing a plasmid comprising a human CD200R gene fragment, flanked by a 5’ homologous arm and a 3’ homologous arm, wherein the 5’ and 3’ homologous arms target an endogenous CD200R gene;

(2) providing one or more small guide RNAs (sgRNAs) that target the endogenous CD200R 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 CD200R 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 and a 3’-terminal targeting site.

In some embodiments, the sequence encoding the humanized CD200R protein is operably linked to an endogenous regulatory element at the endogenous CD200R gene locus.

In some embodiments, the genetically-modified animal does not express an endogenous CD200R protein.

(1) providing a plasmid comprising a human or chimeric CD200R gene fragment, flanked by a 5’ homologous arm and a 3’ homologous arm, wherein the 5’ and 3’ homologous arms target an endogenous CD200R gene;

(2) providing one or more small guide RNAs (sgRNAs) that target the endogenous CD200R gene; and

(3) modifying genome of a fertilized egg or an embryonic stem cell by inserting the human or chimeric CD200R gene fragment into the genome.

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 CD200 and/or CD200R proteins, 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 CD200 and/or CD200R, which are useful for testing agents that can decrease or block the interaction between CD200 and CD200R, between CD200R and anti-human CD200R antibodies, or between CD200 and anti-human CD200 antibodies, testing whether an agent can increase or decrease the immune response, and/or determining whether an agent is a CD200R agonist or antagonist, and whether an agent is a CD200 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 (e.g., breast cancer) or a blood cell tumor (e.g., a lymphocyte tumor, a B or T cell tumor) .

In some embodiments, the genetically modified animals can be used for determining effectiveness of a therapeutic agent (e.g., an anti-CD200R antibody or an anti-CD200 antibody) for the treatment of cancer. In some embodiments, the methods involve administering the therapeutic agent to the animal as described herein, wherein the animal has a cancer or tumor; and determining inhibitory effects of the therapeutic agent to the cancer or 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 addition, a delicate balance is required for these antibodies, as CD200 and CD200R are also expressed on many other cells. Thus, it is important that the human or humanized CD200 and CD200R function in a largely similar way as compared to the endogenous proteins, so that the results in the humanized animals can be used to predict the efficacy or toxicity of these therapeutic agents in the human. In some embodiments, the anti-CD200 antibody can directly target cancer cells or tumor-associated cells expressing CD200, e.g., by inducing complement mediated cytotoxicity (CMC) or antibody dependent cellular cytotoxicity (ADCC) to kill the cancer cells.

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-CD200R or the anti-CD200 antibody inhibits CD200/CD200R signaling pathways. In some embodiments, the anti-CD200R antibody or the anti-CD200 antibody does not inhibit CD200/CD200R signaling pathways. In some embodiments, the anti-CD200R or the anti-CD200 antibody activates CD200/CD200R signaling pathways. In some embodiments, the anti-CD200R antibody or the anti-CD200 antibody does not activate CD200/CD200R signaling pathways. In some embodiments, the therapeutic agent described herein can block the interaction between CD200 and CD200R.

In some embodiments, the genetically modified animals can be used for determining whether an anti-CD200R antibody is a CD200R agonist or antagonist. In some embodiments, the genetically modified animals can be used for determining whether an anti-CD200 antibody is a CD200 agonist or antagonist. In some embodiments, the methods as described herein are also designed to determine the effects of the agent (e.g., anti-CD200R antibodies or anti-CD200 antibodies) on CD200R or CD200, e.g., 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 cytotoxicity (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.

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 therapeutic agent (e.g., an anti-CD200R antibody or an anti-CD200 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 described herein is lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, glioma, 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, myeloproliferation abnormal syndromes, and sarcomas. 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 the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma , and chondrosarcoma. In a specific embodiment, the tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer, or liver cancer.

In some embodiments, the anti-CD200R antibody or the anti-CD200 antibody is designed for treating various autoimmune diseases, including rheumatoid arthritis, Crohn’s disease, systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel diseases (IBD) , ulcerative colitis, or scleroderma. In some embodiments, the anti-CD200R antibody or the anti-CD200 antibody is designed for treating various immune disorders, including allergy, asthma, and/or atopic dermatitis. Thus, the methods as described herein can be used to determine the effectiveness of an anti-CD200R antibody or the anti-CD200 antibody in inhibiting immune response. In some embodiments, the immune disorders described herein is allergy, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or neurological disorders, etc.

The present disclosure also provides methods of determining toxicity of an antibody (e.g., anti-CD200R antibody or an anti-CD200 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. In some embodiments, the antibody can decrease the red blood cells (RBC) , hematocrit, or hemoglobin by more than 20%, 30%, 40%, or 50%. In some embodiments, the animals can have a weight that is at least 5%, 10%, 20%, 30%, or 40%smaller than the weight of the control group (e.g., average weight of the animals that are not treated with the antibody) .

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 CD200 and/or CD200R gene functions, human CD200 and/or CD200R antibodies, drugs for human CD200 and/or CD200R targeting sites, the drugs or efficacies for human CD200 and/or CD200R 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 CD200 and/or CD200R gene humanized non-human animals prepared by the methods described herein, the CD200 and/or CD200R gene humanized non-human animals 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 CD200 and/or CD200R proteins, 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 CD200/CD200R-associated diseases described herein. In some embodiments, the TCA-T, CAR- T, and/or other immunotherapies provides an evaluation method for treating the CD200/CD200R-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 CD200 and/or CD200R genes and a sequence encoding an additional human or chimeric protein.

In some embodiments, the additional human or chimeric protein can be programmed cell death protein 1 (PD-1) , programmed death-ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , CD73, T cell immunoreceptor with Ig and ITIM domains (TIGIT) , CD226, tumor necrosis factor alpha (TNF-α) , B And T Lymphocyte Associated (BTLA) , CD27, CD28, CD47, CD137, CD154, Glucocorticoid-Induced TNFR-Related Protein (GITR) , and/or Signal regulatory protein α (SIRPα) .

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 CD200R gene or chimeric CD200R gene as described herein to obtain a genetically modified non-human animal;

(b) mating 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 PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, CD73, TIGIT, CD226, TNF-α, BTLA, CD27, CD28, CD47, CD137, CD154, GITR, and/or SIRPα. Some of these genetically modified non-human animal are described, e.g., e.g., in PCT/CN2018/091846, PCT/CN2017/090320, PCT/CN2017/099577, PCT/CN2017/099575, PCT/CN2017/099576, PCT/CN2017/099574, PCT/CN2017/110435, PCT/CN2017/120388, PCT/CN2017/117984, PCT/CN2018/091845, PCT/CN2017/110494, and PCT/CN2019/119793; each of which is incorporated herein by reference in its entirety.

In some embodiments, the CD200 and/or CD200R humanization is directly performed on a genetically modified animal having a human or chimeric PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, CD73, TIGIT, CD226, TNF-α, BTLA, CD27, CD28, CD47, CD137, CD154, GITR, and/or SIRPα 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-CD200R antibody or an anti-CD200 antibody, and an additional therapeutic agent for the treatment of cancer. The methods include administering the anti-CD200R antibody or the anti-CD200 antibody and the additional therapeutic agent 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 PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, CD73, TIGIT, CD226, TNF-α, BTLA, CD27, CD28, CD47, CD137, CD154, GITR, and/or SIRPα. 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 cancers as described herein, e.g., a solid tumor, gynecologic cancer, breast cancer, colorectal cancer, gastric adenocarcinoma, lung adenocarcinoma, pancreatic cancer, or head and neck 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.

BglII, DraIII, EcoRV, BspHI, and StuI restriction enzymes were purchased from NEB (Catalog numbers: R0144S, R3510S, R3195S, R0517S, and R0187S, respectively) .

C57BL/6 mice and Flp transgenic mice were purchased from the China Food and Drugs Research Institute National Rodent Experimental Animal Center.

Brilliant Violet 510 ^TM anti-mouse CD45 Antibody was purchased from BioLegend (Catalog number: 103138) .

PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody was purchased from BioLegend (Catalog number: 108426) .

FITC anti-mouse F4/80 was purchased from BioLegend (Catalog number: 123108) .

V450 Rat Anti-mouse CD11b was purchased from BD Horizon (Catalog number: 560455) .

APC anti-mouse CD200R (OX2R) Antibody was purchased from BioLegend (Catalog number: 123915) .

PE anti-human CD200R Antibody was purchased from BioLegend (Catalog number: 329305) .

Zombie NIR ^TM Fixable Viability Kit was purchased from BioLegend (Catalog number: 423106) .

Purified anti-mouse CD16/32 was purchased from BioLegend (Catalog number: 101302) .

FITC anti-Mouse CD19 was purchased from BioLegend (Catalog number: 115506) .

CD200 Monoclonal Antibody (OX90) , PE, eBioscience ^TM was purchased from Invitrogen (Catalog number: 12-5200-82) .

CD200 Monoclonal Antibody (OX104) , APC, eBioscience ^TM was purchased from Invitrogen (Catalog number: 17-9200-41) .

PerCP anti-mouse CD45 Antibody was purchased from BioLegend (Catalog number: 103130) .

Alexa

700 anti-mouse CD3 Antibody was purchased from BioLegend (Catalog number: 100216) .

Brilliant Violet 785 ^TM anti-mouse/human CD11b Antibody was purchased from BioLegend (Catalog number: 101243) .

Brilliant Violet 650 ^TM anti-mouse Ly-6G Antibody was purchased from BioLegend (Catalog number: 127641) .

PE/Cyanine7 anti-mouse F4/80 Antibody was purchased from BioLegend (Catalog number: 123114) .

Rat IgG2a kappa Isotype Control (eBR2a) , PE, eBioscience ^TM was purchased from Invitrogen (Catalog number: 12-4321-80) .

APC Mouse IgG2a, κ Isotype Ctrl Antibody was purchased from BioLegend (Catalog number: 400220) .

PE Mouse IgG1, κ Isotype Ctrl Antibody was purchased from BioLegend (Catalog number: 400112) .

Mouse IgG1 kappa Isotype Control (P3.6.2.8.1) , APC, eBioscience ^TM was purchased from Invitrogen (Catalog number: 17-4714-82) .

EXAMPLE 1: Mice with humanized CD200 gene

In this example, a non-human animal (e.g., a mouse) was modified to include a nucleotide sequence encoding human CD200 protein, and the obtained genetically-modified non-human animal can express a human or humanized CD200 protein in vivo. The mouse CD200 gene (NCBI Gene ID: 17470, Primary source: MGI: 1196990, UniProt ID: Q80VX2) is located at 45202474 to 45229567 of chromosome 16 (NC_000082.7) , and the human CD200 gene (NCBI Gene ID: 4345, Primary source: HGNC: 7203, UniProt ID: P41217-3) is located at 112332573 to 112362812 of chromosome 3 (NC_000003.12) . The mouse CD200 transcript is NM_010818.3, and the corresponding protein sequence NP_034948.3 is set forth in SEQ ID NO: 1. The human CD200 transcript is NM_001004196.4, and the corresponding protein sequence NP_001004196.2 is set forth in SEQ ID NO: 2. Mouse and human CD200 gene loci are shown in FIG. 1.

All or part of nucleotide sequences encoding human CD200 protein can be introduced into the mouse endogenous CD200 locus, so that the mouse expresses human or humanized CD200 protein. Specifically, using gene-editing techniques, under control of mouse CD200 gene regulatory elements, a sequence (about 16.6 kb) starting from within exon 1 and ending within exon 5 of mouse CD200 gene was replaced with a corresponding sequence (about 28.3 kb) starting from within exon 1 and ending within exon 7 of human CD200 gene, to obtain a humanized CD200 gene locus as shown in FIG. 2, thereby humanizing mouse CD200 gene.

As shown in the schematic diagram of the targeting strategy in FIG. 3, the targeting vector contains homologous arm sequences upstream and downstream of the mouse CD200 gene, and an “A Fragment” containing DNA sequences of human CD200 gene. Specifically, sequence of the upstream homologous arm (5’ homologous arm, SEQ ID NO: 3) is identical to nucleotide sequence of 45229231-45232783 of NCBI accession number NC_000082.7, and sequence of the downstream homologous arm (3’ homologous arm, SEQ ID NO: 4) is identical to nucleotide sequence of 45208864-45212611 of NCBI accession number NC_000082.7. The genomic DNA sequence from human CD200 gene (SEQ ID NO: 5) is identical to nucleotide sequence of 112333213-112361547 of NCBI accession number NC_000003.12. The connection between the 5’ end of the A fragment and the mouse sequence was designed as:

wherein the last “G” in sequence

is the last nucleotide of the mouse sequence, the first “A” in sequence “ ATGG” is the first nucleotide of the human sequence. The connection between the 3’ end of the A fragment and the mouse sequence was designed as:

wherein the last “C” in sequence

is the last nucleotide of the human sequence, and the “T” in sequence “ TAAG” is the first nucleotide of the mouse sequence.

The targeting vector also includes an antibiotic resistance gene for positive clone screening (neomycin phosphotransferase gene, or Neo) , and two Frt recombination sites flanking the antibiotic resistance gene, that formed a Neo cassette (within the A Fragment) . The connection between the 5’ end of the Neo cassette and the human sequence was designed as:

wherein the last “A” in sequence

is the last nucleotide of the human sequence, and the first “A” in sequence “ ATCA” is the first nucleotide of the Neo cassette. The connection between the 3’ end of the Neo cassette and the human sequence was designed as:

wherein the last “C” in sequence

is the last nucleotide of the Neo cassette, and the “G” in sequence “ GATA” is the first nucleotide of the human sequence. In addition, a coding gene with a negative selectable marker (a gene encoding diphtheria toxin A subunit (DTA) ) was also constructed downstream of the 3' homologous arm of the targeting vector. The mRNA sequence of the engineered mouse CD200 after humanization and its encoded protein sequence are shown in SEQ ID NO: 6 and SEQ ID NO: 2, respectively.

Given that human CD200 has multiple isoforms or transcripts, the methods described herein can be applied to other isoforms or transcripts. The targeting vector was constructed, e.g., by restriction enzyme digestion and ligation. The constructed targeting vector sequences were preliminarily confirmed by restriction enzyme digestion, and then verified by sequencing. Embryonic stem cells of C57BL/6 mice were transfected with the correct targeting vector by electroporation. The positive selectable marker genes were used to screen the cells, and the integration of exogenous genes was confirmed by PCR and Southern Blot.

The following primers were used in PCR detection: WT-F: 5’-GTCTCTTCCTCCACACTAGAGGAGC-3’ (SEQ ID NO: 28) , Mut-R: 5’-GAGGATGCGCCAGCTTCGTCAGC-3’ (SEQ ID NO: 30) .

The clones identified as positive by PCR were then verified by Southern Blot to verify whether the genetic modification introduced any random insertions. Specifically, genomic DNA from the mouse tail was extracted, which was digested with BglII, DraIII, EcoRV, or BspHI restriction enzyme. The digested genomic DNA was then transferred to a membrane and hybridized with respective probes. The restriction enzymes, probes, and the size of target fragment sizes are shown in the table below.

Table 5. Enzymes and probes used in Southern Blot

Restriction enzyme	Probe	Wild-type fragment size	Recombinant fragment size
BglII	LR Probe	6.1 kb	7.0 kb
DraIII	Neo Probe	--	11.7 kb
EcoRV	A1 Probe	--	16.5 kb
BspHI	A Probe	--	19.0 kb

The Southern Blot detection results are shown in FIG. 4. The results indicate that mice numbered ES-02, ES-03, ES-04, ES-06, ES-09, and ES-10 were verified as positive clones without random insertions.

The following primers were used for probe synthesis in Southern Blot assays:

LR Probe-F (SEQ ID NO: 20) : 5’-TAAGGATCCCGTGGTTCCCATGC-3’,

LR Probe-R (SEQ ID NO: 21) : 5’-CTTATAGATGGCATTTCATAAGTATGC-3’;

Neo Probe-F (SEQ ID NO: 22) : 5’-GGATCGGCCATTGAACAAGAT-3’,

Neo Probe-R (SEQ ID NO: 23) : 5’-CAGAAGAACTCGTCAAGAAGGC-3’;

A1 Probe-F (SEQ ID NO: 24) : 5’-CTAAACCCCGGTGCCCTGACCTC-3’,

A1 Probe-R (SEQ ID NO: 25) : 5’-CATTCAGAGGAATTAATGCAATTGGCC-3’;

A Probe-F (SEQ ID NO: 26) : 5’-CTCGCTTTCTCCGGGAGAGCTCC-3’,

A Probe-R (SEQ ID NO: 27) : 5’-CCGCCTGTTTATGTGTAAAACGAGG-3’.

The positive clones that had been screened (black mice) were introduced into isolated blastocysts (white mice) , and the resulted chimeric blastocysts were transferred to a culture medium for short-term culture and then transplanted to the fallopian tubes of the recipient mother (white mice) to produce the F0 chimeric mice (black and white) . The F2 generation homozygous mice were obtained by backcrossing the F0 generation chimeric mice with wild-type mice to obtain the F1 generation mice, and then breeding the F1 generation heterozygous mice with each other. The positive mice were also bred with the Flp transgenic mice to remove the positive selectable marker genes (schematic diagram shown in FIG. 5) , and then the humanized homozygous mice with a humanized CD200 gene were obtained by breeding the heterozygous mice with each other.

The genotype of the somatic cells of the F1 generation mice can be identified by PCR analysis. The PCR primers are shown in the table below.

Table 6. PCR primer sequences and target fragment sizes

The identification results of some F1 generation mice are shown in FIGS. 6A-6D. The results showed that mice numbered F1-01, F1-02, F1-03, and F1-04 were verified as positive heterozygous mice, demonstrating that the methods described herein can be used to construct CD200 gene humanized mice that can be stably passaged without random insertions.

Expression of human CD200 protein in positive mice can be confirmed, e.g., by flow cytometry. Specifically, one 8-week-old female C57BL/6 wild-type mouse and one 8-week-old female CD200 gene humanized heterozygous mouse were selected, and spleen tissues were collected after euthanasia by cervical dislocation. B cells in the spleen tissues were stained with: Brilliant Violet 510 ^TM anti-mouse CD45 (mCD45; an anti-mouse CD45 antibody) , FITC anti-Mouse CD19 (mCD19; an anti-mouse CD19 antibody) , CD200 Monoclonal Antibody (OX90) , PE, eBioscience ^TM (mCD200; an anti-mouse CD200 antibody) , CD200 Monoclonal Antibody (OX104) , APC, eBioscience ^TM (hCD200; an anti-human CD200 antibody) , Zombie NIR ^TM Fixable Viability Kit, and/or Purified anti-mouse CD16/32, and then subjected to flow cytometry analysis to detect expression of CD200 protein.

The results showed that the spleen B cells of C57BL/6 mouse (characterized by mCD45+mCD19+) had 0.32%hCD200 positive cells (characterized by mCD45+ mCD19 + hCD200+) , and 42.6%mCD200 positive cells (characterized by mCD45+ mCD19 +mCD200+) . By contrast, there were31.6%hCD200-positive cells and 50.2%mCD200-positive cells in the spleen B cells of CD200 gene humanized heterozygous mouse. The results indicate that only mouse CD200 protein, but not human CD200 protein, can be detected in the wild-type C57BL/6 mouse; whereas human CD200 protein can only be detected in the CD200 gene humanized heterozygous mouse.

EXAMPLE 2: Mice with humanized CD200R gene

In this example, a non-human animal (e.g., a mouse) was modified to include a nucleotide sequence encoding human CD200R protein, and the obtained genetically-modified non-human animal can express a human or humanized CD200R protein in vivo. The mouse CD200R gene (NCBI Gene ID: 57781, Primary source: MGI: 1889024, UniProt ID: Q9ES57) is located at 44586099 to 44615340 of chromosome 16 (NC_000082.7) , and the human CD200R gene (NCBI Gene ID: 131450, Primary source: HGNC: 24235, UniProt ID: Q8TD46) is located at 112921205 to 112975103 of chromosome 3 (NC_000003.12) . The mouse CD200R transcript is NM_021325.3, and the corresponding protein sequence NP_067300.1 is set forth in SEQ ID NO: 7. The human CD200R transcript is NM_170780.3, and the corresponding protein sequence NP_740750.1 is set forth in SEQ ID NO: 8. Mouse and human CD200R gene loci are shown in FIG. 7.

All or part of nucleotide sequences encoding human CD200R protein can be introduced into the mouse endogenous CD200R locus, so that the mouse expresses human or humanized CD200R protein. Specifically, using gene-editing techniques, under control of mouse CD200R gene regulatory elements, a sequence (about 4.0 kb) starting from within exon 2 and ending within exon 5 of mouse CD200R gene was replaced with a corresponding sequence (about 6.0 kb) starting from within exon 2 and ending within exon 5 of human CD200R gene, to obtain a humanized CD200R gene locus as shown in FIG. 8, thereby humanizing mouse CD200R gene.

As shown in the schematic diagram of the targeting strategy in FIG. 9, the targeting vector contains homologous arm sequences upstream and downstream of the mouse CD200R gene, and an “A1 Fragment” containing DNA sequences of human CD200R gene. Specifically, sequence of the upstream homologous arm (5’ homologous arm, SEQ ID NO: 9) is identical to nucleotide sequence of 44605076-44609119 of NCBI accession number NC_000082.7, and sequence of the downstream homologous arm (3’ homologous arm, SEQ ID NO: 10) is identical to nucleotide sequence of 44613110-44616609 of NCBI accession number NC_000082.7. The genomic DNA sequence from human CD200R gene (SEQ ID NO: 13) is identical to nucleotide sequence of 112925165-112931154 of NCBI accession number NC_000003.12. The connection between the 5’ end of the A1 fragment and the mouse sequence was designed as:

wherein the last “T” in sequence

is the last nucleotide of the mouse sequence, the “A” in sequence “ ATGG” is the first nucleotide of the human sequence. The connection between the 3’ end of the A1 fragment and the mouse sequence was designed as:

wherein the last “A” in sequence

is the last nucleotide of the human sequence, and the first “T” in sequence “ TATA” is the first nucleotide of the mouse sequence.

wherein the last “C” in sequence

is the last nucleotide of the human sequence, and the first “G” in sequence “ GTCG” is the first nucleotide of the Neo cassette. The connection between the 3’ end of the Neo cassette and the human sequence was designed as:

wherein the last “C” in sequence

is the last nucleotide of the Neo cassette, and the “C” in sequence “ CAAT” is the first nucleotide of the human sequence. In addition, a coding gene with a negative selectable marker (a gene encoding diphtheria toxin A subunit (DTA) ) was also constructed downstream of the 3' homologous arm of the targeting vector. The mRNA sequence of the engineered mouse CD200R after humanization and its encoded protein sequence are shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

Given that human CD200R has multiple isoforms or transcripts, the methods described herein can be applied to other isoforms or transcripts. The targeting vector was constructed, e.g., by restriction enzyme digestion and ligation. The constructed targeting vector sequences were preliminarily confirmed by restriction enzyme digestion, and then verified by sequencing. Embryonic stem cells of C57BL/6 mice were transfected with the correct targeting vector by electroporation. The positive selectable marker genes were used to screen the cells, and the integration of exogenous genes was confirmed by PCR and Southern Blot. The clones identified as positive by PCR (primers shown in the table below) were then verified by Southern Blot. Clones verified to have no random insertion were subjected for subsequent experiments.

Table 7. PCR primer sequences and target fragment sizes

The positive clones that had been screened (black mice) were introduced into isolated blastocysts (white mice) , and the resulted chimeric blastocysts were transferred to a culture medium for short-term culture and then transplanted to the fallopian tubes of the recipient mother (white mice) to produce the F0 chimeric mice (black and white) . The F2 generation homozygous mice were obtained by backcrossing the F0 generation chimeric mice with wild-type mice to obtain the F1 generation mice, and then breeding the F1 generation heterozygous mice with each other. The positive mice were also bred with the Flp transgenic mice to remove the positive selectable marker genes (schematic diagram shown in FIG. 10) , and then the humanized homozygous mice with a humanized CD200R gene were obtained by breeding the heterozygous mice with each other.

In addition, the CRISPR/Cas system can also be used for gene editing, and the targeting strategy shown in FIG. 11 was designed. The targeting vector contains homologous arm sequences upstream and downstream of the mouse CD200R gene, and a human CD200R gene fragment. Specifically, sequence of the upstream homologous arm (5’ homologous arm, SEQ ID NO: 11) is 99.93%identical to nucleotide sequence of 44607693-44609119 of NCBI accession number NC_000082.7, wherein the position 44608624 of NCBI accession number NC_000082.7 is mutated from "C" to "G" ; and sequence of the downstream homologous arm (3’ homologous arm, SEQ ID NO: 12) is identical to nucleotide sequence of 44613110-44614556 of NCBI accession number NC_000082.7. The human CD200R gene fragment (SEQ ID NO: 13) is identical to identical to nucleotide sequence of 112925165-112931154 of NCBI accession number NC_000003.12. The connection between the 5’ end of the human CD200R DNA fragment and the mouse sequence was designed as SEQ ID NO: 35, and the connection between the 3’ end of the human CD200R DNA fragment and the mouse sequence was designed as SEQ ID NO: 36. The mRNA sequence of the engineered mouse CD200R after humanization and its encoded protein sequence are shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

The targeting vector was constructed, e.g., by restriction enzyme digestion, ligation, or direct synthesis. The constructed targeting vector sequences were preliminarily confirmed by restriction enzyme digestion, and then verified by sequencing. Targeting vectors with verified sequences were used for subsequent experiments.

Specific sgRNA sequences were designed and synthesized that recognize the targeting site. The targeting site sequences of the sgRNAs on the CD200R gene locus are as follows:

sgRNA1 targeting site: 5’-GAGTTAGAGGAGTGATACCCAGG -3’ (SEQ ID NO: 41) ;

sgRNA2 targeting site: 5’-GACCGGTACCACCTCTCCTCCGG -3’ (SEQ ID NO: 42) .

UCA kit was used to detect the activity of the sgRNAs. After confirming that the sgRNAs can induce efficient Cas9 cleavage, restriction enzyme cleavage sites were added to its 5' end and a complementary strand to obtain a forward oligonucleotide and a reverse oligonucleotide, as shown in the table below. After annealing, the products were ligated to the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) , to obtain expression vector pT7-CD200R-1 and pT7-CD200R-2.

Table 8. sgRNA1 and sgRNA2 sequence list

The pT7-sgRNA vector was synthesized, which included a DNA fragment containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 49) , 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-CD200R-1 and pT7-CD200R-2 plasmids (using Ambion ^TM 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 fertilized eggs of C57BL/6 mice 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. The genotype of the somatic cells of the F0 generation mice can be identified by PCR analysis. The PCR primers are shown in the table below.

Table 9. PCR primer sequences and target fragment sizes

Mice identified as positive by PCR were then subjected to Southern Blot detection. Those mice identified as positive by Southern Blot were further sequenced to confirm there was no random insertion.

Many methods can be used to verify the success of generating CD200R gene humanized mice. For example, the genotype of the somatic cells of the F1 generation mice can be identified by PCR analysis. The PCR primers are shown in the table below.

Table 10. PCR primer sequences and target fragment sizes

The identification results of some F1 generation mice are shown in FIGS. 12A-12B. The results indicate that mice numbered F1-01, F1-02, F1-03, F1-04, F1-05, F1-06, and F1-07 were verified as positive heterozygous mice.

The F1 generation mice identified as positive by PCR were further verified by Southern Blot to confirm whether there was random insertion. Specifically, genomic DNA from the mouse tail was extracted, which was digested with StuI or BspHI restriction enzyme. The digested genomic DNA was then transferred to a membrane and hybridized with respective probes. The restriction enzymes, probes, and the size of target fragment sizes are shown in the table below.

Table 11. Enzymes and probes used in Southern Blot

Restriction enzyme	Probe	Wild-type fragment size	Recombinant fragment size
StuI	A Probe (5’)	--	6.8 kb
BspHI	3’ Probe	9.4 kb	13.4 kb

The Southern Blot detection results are shown in FIG. 13. The results showed that mice numbered F1-01 and F1-02 were verified as positive clones without random insertions, demonstrating that the methods described herein can be used to construct CD200R gene humanized mice that can be stably passaged without random insertions. Finally, the F1 generation heterozygous mice were bred with each other to obtain the F2 generation homozygous mice.

The following primers were used for probe synthesis in Southern Blot assays:

A Probe (5’) -F: 5’-GTTCTTGCTTGTCTCTGAACCTTTG-3’ (SEQ ID NO: 54) ,

A Probe (5’) -R: 5’-CTGAGAGGTGATTAGTCCACCAAAG-3’ (SEQ ID NO: 55) ;

3’Probe-F: 5’-CAAGGATGAAATGCAGCCTTATGC-3’ (SEQ ID NO: 56) ,

3’Probe-R: 5’-CAGGTAGCAGCAGAGATTCCAAAC-3’ (SEQ ID NO: 57) .

Expression of humanized CD200R protein in positive mice can be confirmed, e.g., by flow cytometry. Specifically, one 6-week-old C57BL/6 wild-type mouse and one 6-week-old CD200R gene humanized heterozygous mouse were selected, and peritoneal exudate macrophages were isolated. The cells were stained with: Brilliant Violet 510 ^TM anti-mouse CD45 (mCD45; an anti-mouse CD45 antibody) , PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody (mGr-1; an anti-mouse Ly-6G/Ly-6C (Gr-1) antibody) , FITC anti-mouse F4/80 (mF4/80; an anti-mouse F4/80 antibody) , V450 Rat Anti-mouse CD11b (mCD11b; an anti-mouse CD11b antibody) , APC anti-mouse CD200R (OX2R) Antibody (mCD200R; an anti-mouse CD200R antibody) , PE anti-human CD200R Antibody (hCD200R; an anti-human CD200R antibody) , Zombie NIR ^TM Fixable Viability Kit, and/or Purified anti-mouse CD16/32, and then subject to flow cytometry analysis to detect expression of CD200R protein.

The results showed that the peritoneal exudate macrophages of C57BL/6 mouse (characterized by mCD45+mGr-1-mCD11b+mF4/80+) had 0.67%hCD200R positive cells (characterized by mCD45+mGr-1-mCD11b+mF4/80+hCD200R+) , and 58.6%mCD200R positive cells (characterized by mCD45+mGr-1-mCD11b+mF4/80+mCD200R+) . By contrast, there were 54.9%hCD200R-positive cells and 39.3%mCD200R-positive cells in the peritoneal exudate macrophages of CD200R gene humanized heterozygous mouse. The results indicate that only mouse CD200R protein, but not humanized CD200R protein, can be detected in the wild-type C57BL/6 mouse; whereas humanized CD200R protein can only be detected in the CD200R gene humanized heterozygous mouse.

EXAMPLE 3: Generation of double-or multi-gene humanized mice

The CD200 and/or CD200R gene humanized mice generated using the methods described herein can also be used to generate double-or multi-gene humanized mouse models. For example, in Example 1, the embryonic stem (ES) cells for blastocyst microinjection can be selected from mice comprising other genetic modifications such as modified (e.g., human or humanized) CD200R, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, and/or CD73 genes. Alternatively, embryonic stem cells from humanized CD200 mice described herein can be isolated, and gene recombination targeting technology can be used to obtain double-gene or multi-gene-modified mouse models of CD200 and other gene modifications. In addition, it is also possible to breed the homozygous or heterozygous CD200 gene humanized 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 modified (e.g., human or humanized) CD200 gene and other genetic modifications. Then the heterozygous mice can be bred with each other to obtain homozygous double-gene or multi-gene modified mice. The methods described above can also be applied to the CD200R gene humanized mice obtained using methods described in Example 2, to obtain homozygous mice having other modified genes.

For example, CD200/CD200R double-gene humanized mice can be obtained using the methods described above. Specifically, the CD200 gene humanized mice prepared in Example 1 was bred with the CD200R gene humanized mice in Example 2, to obtain the CD200/CD200R double-gene humanized mice. CD200 and CD200R protein expression in CD200/CD200R double-gene humanized homozygous mice was detected by flow cytometry. Specifically, one 8-week-old female wild-type C57BL/6 mouse (+/+) and one CD200/CD200R double-gene humanized homozygous mouse (H/H) were selected. Mouse spleen cells or ascitic fluid was harvested after cervical euthanasia. The cells were stained with: Purified anti-mouse CD16/32 Antibody, Zombie NIR ^TM Fixable Viability Kit, PerCP anti-mouse CD45 Antibody, Alexa

700 anti-mouse CD3 Antibody, FITC anti-Mouse CD19, Brilliant Violet 785 ^TM anti-mouse/human CD11b Antibody, Brilliant Violet 650 ^TM anti-mouse Ly-6G Antibody, PE/Cyanine7 anti-mouse F4/80 Antibody, CD200 Monoclonal Antibody (OX90) , PE, eBioscience ^TM, APC anti-mouse CD200R (OX2R) Antibody, Rat IgG2a kappa Isotype Control (eBR2a) , PE, eBioscience ^TM, APC Mouse IgG2a, κ Isotype Ctrl Antibody, PE anti-human CD200R Antibody, CD200 Monoclonal Antibody (OX104) , APC, eBioscience ^TM, PE Mouse IgG1, κ Isotype Ctrl Antibody, and/or Mouse IgG1 kappa Isotype Control (P3.6.2.8.1) , APC, eBioscience ^TM, and subject to flow cytometry analysis to detect expression of CD200 and CD200R proteins. The results are shown in the follow table.

Table 12. Detection of CD200 and CD200R proteins by flow cytometry

According to the above table, only mouse CD200 protein and CD200R protein were detected in the wild-type C57BL/6 mouse, whereas only human CD200 protein and humanized CD200R protein can be detected in the CD200/CD200R double-gene humanized homozygous mouse.

These double-gene or multi-gene modified mice can be used for in vivo validation of gene regulators targeting human CD200, CD200R, and other genes.

EXAMPLE 4: Verification of drug efficacy

The CD200 and/or CD200R gene humanized mice prepared by the methods described herein can be used to evaluate the efficacy of drugs targeting human CD200 and/or CD200R. For example, the CD200 and/or CD200R gene humanized homozygous mice can be subcutaneously inoculated with mouse colon cancer cells MC38. When the tumor volume reaches about 100 mm ³, the mice can be placed into a control group and one or more treatment groups according to the tumor volume. The treatment group mice can be administered with randomly selected drugs targeting human CD200 and/or CD200R (e.g., an anti-human CD200 antibody or an anti-human CD200R antibody) , and the control group mice can be injected with an equal volume of saline. The tumor volume and body weight of the mice can be measured regularly, and the in vivo safety and efficacy of the drugs can be effectively assessed by comparing the changes in the body weight of the mice and the tumor size.

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 cell surface transmembrane glycoprotein CD200 receptor 1 (CD200R) .
The animal of claim 1, wherein the sequence encoding the human or chimeric CD200R is operably linked to an endogenous regulatory element at the endogenous CD200R gene locus in the at least one chromosome.
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric CD200R comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human CD200R (NP_740750.1 (SEQ ID NO: 8) ) .
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric CD200R 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: 15.
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric CD200R comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to amino acids 29-243 of SEQ ID NO: 8.
The animal of any one of claims 1-5, wherein the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat.
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 CD200R or expresses a decreased level of endogenous CD200Ras compared to CD200R expression level in a wild-type animal.
The animal of any one of claims 1-8, wherein the animal has one or more cells expressing human or chimeric CD200R.
The animal of any one of claims 1-9, wherein the animal has one or more cells expressing human or chimeric CD200R, and the expressed human or chimeric CD200R can interact with a human OX-2 membrane glycoprotein (CD200) , activating downstream signaling pathways.
The animal of any one of claims 1-9, wherein the animal has one or more cells expressing human or chimeric CD200R, and the expressed human or chimeric CD200R can interact with an endogenous CD200, activating downstream signaling pathways.
A genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous CD200R with a sequence encoding a corresponding region of human CD200R at an endogenous CD200R gene locus.
The animal of claim 12, wherein the sequence encoding the corresponding region of human CD200R is operably linked to an endogenous regulatory element at the endogenous CD200R locus, and one or more cells of the animal expresses a human or chimeric CD200R.
The animal of claim 12 or 13, wherein the animal does not express endogenous CD200R or expresses a decreased level of endogenous CD200R as compared to CD200R expression level in a wild-type animal.
The animal of any one of claims 12-14, wherein the replaced sequence encodes the extracellular region of CD200R.
The animal of any one of claims 12-15, wherein the animal has one or more cells expressing a chimeric CD200R having a signal peptide, 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 CD200R (NP_740750.1 (SEQ ID NO: 8) ) .
The animal of claim 16, wherein the extracellular region of the chimeric CD200R has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 211, 212, 213, 214, or 215 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human CD200R (e.g., amino acids 29-243 of SEQ ID NO: 8) .
The animal of any one of claims 12-17, wherein the sequence encoding a region of endogenous CD200R comprises exon 2, exon 3, exon 4, and/or exon 5, or a part thereof, of the endogenous CD200R gene.
The animal of claim 18, wherein the animal is a mouse.
The animal of any one of claims 12-19, wherein the animal is heterozygous or homozygous with respect to the replacement at the endogenous CD200R gene locus.
A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a humanized CD200R polypeptide, wherein the humanized CD200R polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human CD200R, wherein the animal expresses the humanized CD200R polypeptide.
The animal of claim 21, wherein the humanized CD200R polypeptide has at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 211, 212, 213, 214, or 215 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of human CD200R extracellular region (e.g., amino acids 29-243 of SEQ ID NO: 8) .
The animal of claim 21 or 22, wherein the humanized CD200R polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 29-243 of SEQ ID NO: 8.
The animal of any one of claims 21-23, wherein the humanized CD200R polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 1-25 and 239-326 of SEQ ID NO: 7.
The animal of any one of claims 21-24, wherein the nucleotide sequence is operably linked to an endogenous CD200R regulatory element of the animal.
The animal of any one of claims 21-25, wherein the chimeric CD200R polypeptide comprises an endogenous CD200R signal peptide, an endogenous CD200R transmembrane region, and/or an endogenous CD200R cytoplasmic region.
The animal of any one of claims 21-26, wherein the nucleotide sequence is integrated to an endogenous CD200R gene locus of the animal.
The animal of any one of claims 21-27, wherein the humanized CD200R polypeptide has at least one mouse CD200R activity and/or at least one human CD200R activity.
A method for making a genetically-modified, non-human animal, comprising:

replacing in at least one cell of the animal, at an endogenous CD200R gene locus, a sequence encoding a region of endogenous CD200R with a sequence encoding a corresponding region of human CD200R.
The method of claim 29, wherein the sequence encoding the corresponding region of humanCD200R comprises a portion of exon 2, exon 3, exon 4, and a portion of exon 5, of a human CD200R gene.
The method of claim 29 or 30, wherein the sequence encoding the corresponding region of human CD200R encodes amino acids 29-243 of SEQ ID NO: 8.
The method of any one of claims 29-31, wherein the region comprises the extracellular region of CD200R.
The method of any one of claims 29-32, wherein the animal is a mouse, and the sequence encoding a region of endogenous CD200R comprises a portion of exon 2, exon 3, exon 4, and a portion of exon 5of the endogenous CD200R gene.
A method of making a genetically-modified animal cell that expresses a chimeric CD200R, the method comprising:

replacing at an endogenous CD200R gene locus, a nucleotide sequence encoding a region of endogenous CD200R with a nucleotide sequence encoding a corresponding region of human CD200R, thereby generating a genetically-modified animal cell that includes a nucleotide sequence that encodes the chimeric CD200R, wherein the animal cell expresses the chimeric CD200R.
The method of claim 34, wherein the animal is a mouse.
The method of claim 34 or 35, wherein the chimeric CD200R comprises a human or humanized CD200R extracellular region; and a signal peptide, a transmembrane and/or a cytoplasmic region of mouse CD200R.
The method of any one of 34-36, wherein the nucleotide sequence encoding the chimeric CD200R is operably linked to an endogenous CD200R regulatory region, e.g., promoter.
The animal of any one of claims 1-28, wherein the animal further comprises a sequence encoding an additional human or chimeric protein.
The animal of claim 38, wherein the additional human or chimeric protein is CD200, programmed cell death protein 1 (PD-1) , programmed death-ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , and/or CD73.
The method of any one of claims 29-37, wherein the animal or mouse further comprises a sequence encoding an additional human or chimeric protein.
The method of claim 40, wherein the additional human or chimeric protein is CD200, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, and/or CD73.
A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric OX-2 membrane glycoprotein (CD200) .
The animal of claim 42, wherein the sequence encoding the human or chimeric CD200 is operably linked to an endogenous regulatory element at the endogenous CD200 gene locus in the at least one chromosome.
The animal of claim 42 or 43, wherein the sequence encoding a human or chimeric CD200 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human CD200 (NP_001004196.2; SEQ ID NO: 2) .
The animal of any one of claims 42-44, wherein the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat.
The animal of any one of claims 42-45, wherein the animal is a mouse.
The animal of any one of claims 42-46, wherein the animal does not express endogenous CD200or expresses a decreased level of endogenous CD200as compared to CD200 expression level in a wild-type animal.
The animal of any one of claims 42-47, wherein the animal has one or more cells expressing human or chimeric CD200.
The animal of any one of claims 42-48, wherein the animal has one or more cells expressing human or chimeric CD200, and the expressed human or chimeric CD200 can interact with a human CD200R, activating downstream signaling pathways.
The animal of any one of claims 42-48, wherein the animal has one or more cells expressing human or chimeric CD200, and the expressed human or chimeric CD200 can interact with an endogenous CD200R, activating downstream signaling pathways.
A genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous CD200 with a sequence encoding a corresponding region of human CD200 at an endogenous CD200 gene locus.
The animal of claim 51, wherein the sequence encoding the corresponding region of human CD200 is operably linked to an endogenous regulatory element at the endogenous CD200 locus, and one or more cells of the animal express a human or chimeric CD200.
The animal of claim 51 or 52, wherein the animal does not express endogenous CD200or expresses a decreased level of endogenous CD200as compared to CD200 expression level in a wild-type animal.
The animal of any one of claims 51-53, wherein the replaced sequence encodes the full-length protein of CD200.
The animal of any one of claims 51-54, wherein the animal is a mouse, and the replaced endogenous CD200 region comprises a portion of exon 1, exon 2, exon 3, exon 4, and/or a portion of exon 5 of the endogenous mouse CD200 gene.
The animal of any one of claims 51-55, wherein the animal is heterozygous or homozygous with respect to the replacement at the endogenous CD200 gene locus.
A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a humanized CD200 polypeptide, wherein the humanized CD200 polypeptide comprises at least 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 291, 292, 293, or 294 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human CD200, wherein the animal expresses the humanized CD200 polypeptide.
The animal of claim 57, wherein the nucleotide sequence is operably linked to an endogenous CD200 regulatory element of the animal.
The animal of claim 57 or 58, wherein the nucleotide sequence is integrated to an endogenous CD200 gene locus of the animal.
The animal of any one of claims 57-59, wherein the humanized CD200 polypeptide has at least one mouse CD200 activity and/or at least one human CD200 activity.
A method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous CD200 gene locus, a sequence encoding a region of an endogenous CD200 with a sequence encoding a corresponding region of human CD200.
The method of claim 61, wherein the sequence encoding the corresponding region of human CD200 comprises a portion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or a portion of exon 7 of a human CD200 gene.
The method of claim 61 or 62, wherein the sequence encoding the corresponding region of human CD200 encodes a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 2.
The method of any one of claims 61-63, wherein the animal is a mouse, and the locus is a portion of exon 1, exon 2, exon 3, exons 4, and/or a portion of exon 5 of the mouse CD200 gene.
A method of making a genetically-modified non-human animal cell that expresses a human or chimeric CD200, the method comprising:

replacing, at an endogenous mouse CD200 gene locus, a nucleotide sequence encoding a region of endogenous CD200 with a nucleotide sequence encoding a corresponding region of human CD200, thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the human or chimeric CD200, wherein the animal cell expresses the human or chimeric CD200.
The method of claim 65, wherein the animal is a mouse.
The method of claim 65 or 66, wherein the nucleotide sequence encoding the human or chimeric CD200 is operably linked to an endogenous CD200 regulatory region, e.g., promoter.
The animal of any one of claims 42-60, wherein the animal further comprises a sequence encoding an additional human or chimeric protein.
The animal of claim 68, wherein the additional human or chimeric protein is cell surface transmembrane glycoprotein CD200 receptor 1 (CD200R) , programmed cell death protein 1 (PD-1) , programmed death-ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , and/or CD73.
The method of any one of claims 61-67, wherein the animal or mouse further comprises a sequence encoding an additional human or chimeric protein.
The method of claim 70, wherein the additional human or chimeric protein is CD200R, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, and/or CD73.
A method of determining effectiveness of a therapeutic agent for the treatment of cancer, comprising:

a) administering the therapeutic agent to the animal of any one of claims 1-28, 38, 39, 42-60, 68, and 69, wherein the animal has a tumor; and

b) determining inhibitory effects of the therapeutic agent to the tumor.
The method of claim 72, wherein the therapeutic agent is an anti-CD200 antibody or an anti-CD200R antibody.
The method of claim 72 or 73, wherein the tumor comprises one or more cancer cells that are injected into the animal.
The method of any one of claims 72-74, wherein determining inhibitory effects of the anti-CD200R antibody to the tumor involves measuring the tumor volume in the animal.
The method of any one of claims 72-75, wherein the cancer is melanoma, ovarian cancer, myeloid leukemia, B cell malignancy, endocrine malignancy (e.g., small cell lung carcinoma) , bladder cancer, or breast cancer.
A method of determining effectiveness of an anti-CD200 antibody or an anti-CD200R antibody, and an additional therapeutic agent for the treatment of cancer, comprising

a) administering the anti-CD200 antibody or the anti-CD200R antibody, and the additional therapeutic agent to the animal of any one of claims 1-28, 38, 39, 42-60, 68, and 69, wherein the animal has a tumor; and

b) determining inhibitory effects on the tumor.
The method of claim 77, wherein the animal further comprises a sequence encoding a human or chimeric PD-1, a human or chimeric PD-L1, and/or a human or chimeric CTLA4.
The method of claim 77 or 78, wherein the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody.
The method of any one of claims 77-79, wherein the tumor comprises one or more tumor cells that express PD-L1 and/or CD200.
The method of any one of claims 77-80, wherein the tumor comprises one or more cancer cells that are injected into the animal.
The method of any one of claims 77-81, wherein determining inhibitory effects of the treatment involves measuring the tumor volume in the animal.
The method of any one of claims 77-82, wherein the animal has melanoma, ovarian cancer, myeloid leukemia, B cell malignancy, endocrine malignancy (e.g., small cell lung carcinoma) , bladder cancer, or breast cancer.
A method of determining toxicity of a therapeutic agent comprising:

a) administering the therapeutic agent to the animal of any one of claims 1-28, 38, 39, 42-60, 68, and 69; and

b) determining effects of the therapeutic agent to the animal.
The method of claim 84, wherein the therapeutic agent is an anti-CD200 antibody or an anti-CD200R antibody.
The method of claim 84 or 85, wherein determining effects of the therapeutic agent to the animal involves measuring the body weight, red blood cell count, hematocrit, and/or hemoglobin of the animal.
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: 1, 2, 7, 8, or 15;

(b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 1, 2, 7, 8, or 15;

(c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1, 2, 7, 8, or 15;

(d) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 1, 2, 7, 8, or 15 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: 1, 2, 7, 8, or 15.
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 87;

(b) SEQ ID NO: 3, 4, 5, 6, 9, 10, 13, 14, 16, 17, 18, 19, 35, 36, 37, or 38;

(c) a sequence that is at least 90%identical to SEQ ID NO: 3, 4, 5, 6, 9, 10, 13, 14, 16, 17, 18, 19, 35, 36, 37, or 38; 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, 9, 10, 13, 14, 16, 17, 18, 19, 35, 36, 37, or 38.
A cell comprising the protein of claim 87 and/or the nucleic acid of claim 88.
An animal comprising the protein of claim 87 and/or the nucleic acid of claim 88.