WO2022253322A1

WO2022253322A1 - Genetically modified non-human animal with human or chimeric vsig4

Info

Publication number: WO2022253322A1
Application number: PCT/CN2022/096904
Authority: WO
Inventors: Ruili LV; Chengzhang SHANG; Yanhui NIE
Original assignee: Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
Priority date: 2021-06-02
Filing date: 2022-06-02
Publication date: 2022-12-08
Also published as: CN115043929A

Abstract

Provided are genetically modified non-human animals that express a human or chimeric (e.g., humanized) VSIG4, and methods of use thereof.

Description

GENETICALLY MODIFIED NON-HUMAN ANIMAL WITH HUMAN OR CHIMERIC VSIG4

CLAIM OF PRIORITY

This application claims the benefit of Chinese Patent Application App. No. 202110615989.7, filed on June 2, 2021 and Chinese Patent Application App. No. 202111470150.5, filed on December 3, 2021. The entire contents of the foregoing applications are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) VSIG4, and methods of use thereof.

BACKGROUND

The immune system has developed multiple mechanisms to prevent deleterious activation of immune cells. One such mechanism is the intricate balance between positive and negative costimulatory signals delivered to immune cells. Targeting the stimulatory or inhibitory pathways for the immune system is considered to be a potential approach for the treatment of various diseases, e.g., cancers and autoimmune diseases.

The traditional drug research and development for these stimulatory or inhibitory receptors 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 VSIG4 or chimeric VSIG4. The animal model can express human VSIG4 or chimeric VSIG4 (e.g., humanized VSIG4) protein in its body. It can be used in the studies on the function of VSIG4 gene, and can be used in the screening and evaluation of anti-human VSIG4 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 VSIG4 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 VSIG4 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 VSIG4 (V-set and immunoglobulin domain containing 4) . In some embodiments, the sequence encoding the human or chimeric VSIG4 is operably linked to an endogenous regulatory element at the endogenous VSIG4 gene locus in the at least one chromosome. In some embodiments, the sequence encoding a human or chimeric VSIG4 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human VSIG4 (NP_009199.1 (SEQ ID NO: 2) ) . In some embodiments, the sequence encoding a human or chimeric VSIG4 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 11. In some embodiments, the sequence encoding a human or chimeric VSIG4 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 20-281 of 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 VSIG4 or expresses a decreased level of endogenous VSIG4 as compared to VSIG4 expression level in a wild-type animal. In some embodiments, the animal has one or more cells expressing human or chimeric VSIG4. In some embodiments, the animal has one or more cells expressing human or chimeric VSIG4, and the expressed human or chimeric VSIG4 can interact with human complement C3b. In some embodiments, the animal has one or more cells expressing human or chimeric VSIG4, and the expressed human or chimeric VSIG4 can interact with endogenous complement C3b.

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 VSIG4 with a sequence encoding a corresponding region of human VSIG4 at an endogenous VSIG4 gene locus. In some embodiments, the sequence encoding the corresponding region of human VSIG4 is operably linked to an endogenous regulatory element at the endogenous VSIG4 locus, and one or more cells of the animal expresses a human or chimeric VSIG4. In some embodiments, the animal does not express endogenous VSIG4 or expresses a decreased level of endogenous VSIG4 as compared to VSIG4 expression level in a wild-type animal. In some embodiments, the replaced sequence encodes the extracellular region of VSIG4, optionally without the signal peptide. In some embodiments, the animal has one or more cells expressing a chimeric VSIG4 having 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 VSIG4 (NP_009199.1 (SEQ ID NO: 2) ) . In some embodiments, the extracellular region of the chimeric VSIG4 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, 260, 261, 262, 263 or 264 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human VSIG4. In some embodiments, the sequence encoding a region of endogenous VSIG4 comprises exon 2, exon 3, exon 4, and/or exon 5, or a part thereof, of the endogenous VSIG4 gene. In some embodiments, the animal is a mouse. In some embodiments, the animal is heterozygous with respect to the replacement at the endogenous VSIG4 gene locus. In some embodiments, the animal is homozygous with respect to the replacement at the endogenous VSIG4 gene locus.

In one aspect, the disclosure is related to a method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous VSIG4 gene locus, a sequence encoding a region of endogenous VSIG4 with a sequence encoding a corresponding region of human VSIG4. In some embodiments, the sequence encoding the corresponding region of human VSIG4 comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8, or a part thereof, of a human VSIG4 gene. In some embodiments, the sequence encoding the corresponding region of human VSIG4 comprises a portion of exon 2, exon 3, exon 4, exon 5, and a portion of exon 6, of a human VSIG4 gene. In some embodiments, the sequence encoding the corresponding region of human VSIG4 encodes amino acids 20-281 of SEQ ID NO: 2. In some embodiments, the region is located within the extracellular region of VSIG4. In some embodiments, the sequence encoding a region of endogenous VSIG4 comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7, or a part thereof, of the endogenous VSIG4 gene. In some embodiments, the animal is a mouse, and the sequence encoding a region of endogenous VSIG4 comprises a portion of exon 2, exon 3, exon 4, and a portion of exon 5 of the endogenous VSIG4 gene.

In one aspect, the disclosure is related to a genetically-modified, non-human animal, in some embodiments, the genome of the animal comprises an insertion of a sequence encoding a human or chimeric VSIG4 at an endogenous VSIG4 gene locus. In some embodiments, the sequence encoding the human or chimeric VSIG4 is operably linked to an endogenous regulatory element at the endogenous VSIG4 locus, and one or more cells of the animal expresses a human or chimeric VSIG4. In some embodiments, the animal does not express endogenous VSIG4 or expresses a decreased level of endogenous VSIG4 as compared to VSIG4 expression level in a wild-type animal. In some embodiments, the sequence encoding a human or chimeric VSIG4 is inserted within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 (e.g., exon 2) of endogenous VSIG4 gene.

In some embodiments, the inserted sequence comprises, optionally from 5’ end to 3’ end: a) optionally a sequence encoding a self-cleaving peptide (e.g., P2A) , b) the sequence encoding a human or chimeric VSIG4, c) a regulatory sequence of endogenous VSIG4 gene, and d) optionally an auxiliary sequence (e.g., WPRE, STOP, and/or polyA) . In some embodiments, the sequence encoding a human or chimeric VSIG4 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 30. In some embodiments, the self-cleaving peptide is P2A, T2A, E2A, or F2A. In some embodiments, the sequence encoding a self-cleavage peptide is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 29. In some embodiments, the regulatory sequence of endogenous VSIG4 gene comprises 3’ UTR of endogenous VSIG4 gene. In some embodiments, the regulatory sequence at 3’ end of endogenous VSIG4 gene is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 31. In some embodiments, the auxiliary sequence is a STOP sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 32. In some embodiments, the animal is a mouse. In some embodiments, the animal is heterozygous with respect to the insertion at the endogenous VSIG4 gene locus. In some embodiments, the animal is homozygous with respect to the insertion at the endogenous VSIG4 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 human VSIG4 polypeptide, in some embodiments, the animal expresses the human VSIG4 polypeptide. In some embodiments, the human VSIG4 polypeptide is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding a human VSIG4 polypeptide is operably linked to an endogenous VSIG4 regulatory element of the animal.

In one aspect, the disclosure is related to a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric VSIG4 polypeptide, in some embodiments, the chimeric VSIG4 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human VSIG4, in some embodiments, the animal expresses the chimeric VSIG4 polypeptide. In some embodiments, the chimeric VSIG4 polypeptide has at least 50, at least 80, at least 100, at least 150, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 261, at least 262, at least 263 or at least 264 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human VSIG4 extracellular region. In some embodiments, the chimeric VSIG4 polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 20-281 of SEQ ID NO: 2. In some embodiments, the nucleotide sequence is operably linked to an endogenous VSIG4 regulatory element of the animal. In some embodiments, the chimeric VSIG4 polypeptide comprises an endogenous VSIG4 cytoplasmic region and/or an endogenous VSIG4 transmembrane region, optionally an endogenous signal peptide. In some embodiments, the nucleotide sequence is integrated to an endogenous VSIG4 gene locus of the animal. In some embodiments, the chimeric VSIG4 polypeptide has at least one mouse VSIG4 activity and/or at least one human VSIG4 activity.

In one aspect, the disclosure is related to a method of making a genetically-modified animal cell that expresses a chimeric VSIG4, the method comprising: replacing at an endogenous VSIG4 gene locus, a nucleotide sequence encoding a region of endogenous VSIG4 with a nucleotide sequence encoding a corresponding region of human VSIG4, thereby generating a genetically-modified animal cell that includes a nucleotide sequence that encodes the chimeric VSIG4, in some embodiments, the animal cell expresses the chimeric VSIG4. In some embodiments, the animal is a mouse. In some embodiments, the chimeric VSIG4 comprises a human or humanized VSIG4 extracellular region; and a transmembrane and/or a cytoplasmic region of mouse VSIG4. In some embodiments, the nucleotide sequence encoding the chimeric VSIG4 is operably linked to an endogenous VSIG4 regulatory region, e.g., promoter.

In one aspect, the disclosure is related to a method of making a genetically-modified animal cell that expresses a human or chimeric VSIG4, the method comprising: inserting at an endogenous VSIG4 gene locus (e.g., exon 2 of endogenous VSIG4 gene) , a nucleotide sequence comprising, optionally from 5’ end to 3’ end: a) optionally a sequence encoding a self-cleaving peptide (e.g., P2A) ; b) a sequence encoding a human or chimeric VSIG4; c) a regulatory sequence at 3’ end of endogenous VSIG4 gene; and d) optionally an auxiliary sequence (e.g., WPRE, STOP, and/or polyA) , thereby generating a genetically-modified animal cell that includes a nucleotide sequence that encodes the human or chimeric VSIG4, in some embodiments, the animal cell expresses the human or chimeric VSIG4. In some embodiments, the animal is a mouse.

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 programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , IL4, IL4 receptor (IL4R) , IL6, IL6 receptor (IL6R) , IL17, IL17 receptor (IL17R) , C-C Motif Chemokine Receptor 5 (CCR5) , or C-C Motif Chemokine Receptor 8 (CCR8) .

In one aspect, the disclosure is related to a method of determining effectiveness of an anti-VSIG4 antibody for the treatment of cancer, comprising: a) administering the anti-VSIG4 antibody to the animal as described herein, in some embodiments, the animal has a tumor; and b) determining inhibitory effects of the anti-VSIG4 antibody to the tumor. In some embodiments, the tumor comprises one or more cells that express VSIG4. 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-VSIG4 antibody to the tumor involves measuring the tumor volume in the animal. In some embodiments, the cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, or prostate cancer.

In one aspect, the disclosure is related to a method of determining effectiveness of an anti-VSIG4 antibody and an additional therapeutic agent for the treatment of cancer, comprising a) administering the anti-VSIG4 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 programmed cell death protein 1 (PD-1) . In some embodiments, the animal further comprises a sequence encoding a human or chimeric programmed death-ligand 1 (PD-L1) . In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the tumor comprises one or more tumor cells that express PD-L1. In some embodiments, the tumor is caused by injection of one or more cancer cells 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 bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and/or prostate cancer.

In one aspect, the disclosure is related to a method of determining effectiveness of an anti-VSIG4 antibody for treating an autoimmune disorder, comprising: a) administering the anti-VSIG4 antibody to the animal as described herein, in some embodiments, the animal has the autoimmune disorder; and b) determining effects of the anti-VSIG4 antibody for treating the auto-immune disease. In some embodiments, the autoimmune disorder is rheumatoid arthritis, Crohn’s disease, systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel diseases (IBD) , ulcerative colitis, and/or scleroderma.

In one aspect, the disclosure is related to a method of determining effectiveness of an anti-VSIG4 antibody for treating an immune disorder, comprising: a) administering the anti- VSIG4 antibody to the animal as described herein, in some embodiments, the animal has the immune disorder; and b) determining effects of the anti-VSIG4 antibody for treating the immune disorder. In some embodiments, the immune disorder is allergy, asthma, and/or atopic dermatitis.

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, or 11;

(b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 1, 2, or 11;

(c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1, 2, or 11;

(d) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 1, 2, or 11 byno 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, or 11.

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, 7, 8, 9, 10, 12, 13, 27, 28, 29, 30, 31, 32, 33, 34, 56, 57, 58, or 59;

(c) a sequence that is at least 90%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 27, 28, 29, 30, 31, 32, 33, 34, 56, 57, 58, or 59; and

(d) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 27, 28, 29, 30, 31, 32, 33, 34, 56, 57, 58, or 59.

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.

In another aspect, the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous VSIG4 gene, wherein the disruption of the endogenous VSIG4 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 VSIG4 gene.

In some embodiments, the disruption of the endogenous VSIG4 gene comprises deletion of one or more exons or part of exons selected from the group consisting of exon 2, exon 3, exon 4, exon 5 of the endogenous VSIG4 gene.

In some embodiments, the disruption of the endogenous VSIG4 gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 2, intron 3, and intron 4 of the endogenous VSIG4 gene.

In some embodiments, wherein 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, 10, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 498, or more nucleotides.

In some embodiments, the disruption of the endogenous VSIG4 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 300 nucleotides from exon 2, exon 3, exon 4, and at least 5 nucleotides from exon 5) .

The disclosure further relates to a VSIG4 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 VSIG4 gene function, human VSIG4 antibodies, the drugs or efficacies for human VSIG4 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 VSIG4 36 gene loci.

FIG. 2 is a schematic diagram showing humanized VSIG4 gene locus using method one.

FIG. 3 is a schematic diagram showing a VSIG4 gene targeting strategy using targeting vector V1.

FIG. 4 is a schematic diagram showing a VSIG4 gene targeting strategy using targeting vector V2.

FIG. 5 is a schematic diagram showing humanized VSIG4 gene locus using method two.

FIG. 6 is a schematic diagram showing a VSIG4 gene targeting strategy using targeting vector V3.

FIG. 7 is a schematic diagram showing a VSIG4 gene targeting strategy using targeting vector V4.

FIG. 8 shows relative Cas9/sgRNA activity using sgRNA1-sgRNA6. Con is a negative control. PC is a positive control.

FIGS. 9A-9B show mouse tail PCR identification results of F0 generation mice by primer pairs L-GT-F/L-GT-R and R-GT-F/R-GT-R, respectively. M is a marker. WT is a wild-type control. H ₂O is a water control.

FIGS. 10A-10B show mouse tail PCR identification results of F1 generation mice by primer pairs L-GT-F/L-GT-R and R-GT-F/R-GT-R, respectively. M is a marker. WT is a wild-type control. H ₂O is a water control.

FIG. 11 shows Southern Blot results of cells after recombination using the 5’ Probe and 3’ Probe. WT is a wild-type control.

FIGS. 12A-12C show RT-PCR detection results of mouse VSIG4 mRNA, humanized VSIG4 mRNA, and GAPDH mRNA, respectively, in liver tissues of wild-type C57BL/6 mice (+/+) and VSIG4 gene humanized heterozygous mice (H/+) . H ₂O is a water control. GAPDH is an internal reference.

FIG. 13A-13B show percentages of leukocyte subtypes and T cell subtypes, respectively, in the spleen of C57BL/6 wild-type mice (+/+) and VSIG4 gene humanized homozygous mice (H/H) .

FIG. 14A-14B show percentages of leukocyte subtypes and T cell subtypes, respectively, in the lymph nodes of C57BL/6 wild-type mice (+/+) and VSIG4 gene humanized homozygous mice (H/H) .

FIG. 15A-15B show percentages of leukocyte subtypes and T cell subtypes, respectively, in the peripheral blood of C57BL/6 wild-type mice (+/+) and VSIG4 gene humanized homozygous mice (H/H) .

FIG. 16A shows the body weight of VSIG4 gene humanized homozygous mice that were xenografted with mouse colon cancer cells (MC38) , and then treated with anti-human VSIG4 antibody Ab1 (G2) . G1 group mice were injected with hIgG1 as a control.

FIG. 16B shows the tumor volume of VSIG4 gene humanized homozygous mice that were xenografted with mouse colon cancer cells (MC38) , and then treated with anti-human VSIG4 antibody Ab1 (G2) . G1 group mice were injected with hIgG1 as a control.

FIG. 17 shows the alignment using EMBOSS Needle between human VSIG4 amino acid sequence (NP_009199.1; SEQ ID NO: 2) and mouse VSIG4 amino acid sequence (NP_808457.1; SEQ ID NO: 1) .

FIG. 18 shows the alignment using EMBOSS Needle between human VSIG4 amino acid sequence (NP_009199.1; SEQ ID NO: 2) and rat VSIG4 amino acid sequence (NP_001020175.1; SEQ ID NO: 60) .

DETAILED DESCRIPTION

This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) VSIG4, and methods of use thereof.

The V-set Ig domain-containing 4 (VSIG4) is an immune checkpoint molecule which belongs to B7-related family member. VSIG4 is physiologically expressed on tissue-resident macrophages, including alveolar macrophages in the lung and Kupffer cells in the liver. It shares a set of conserved amino acids with the B7 family members, and contains one complete IgV-type domain and a truncated IgC-type domain. VSIG4 has been known to block the alternative complement pathway by binding to the convertase subunit C3b. Moreover, it inhibits CD4+ and CD8+ T cell proliferation by ligating a receptor to the T cells. Initially, VSIG4 expression has been studied regarding the pathogenesis of inflammatory diseases such as rheumatoid arthritis, atherosclerosis, and chronic HBV-hepatitis. Additional studies reported that VSIG4 expression is involved in lung cancer development and associated with poor prognosis of high grade glioma. Therefore, VSIG4 is regarded as a potential biomarker and therapeutic target for cancer and immune disorders.

Experimental animal models are an indispensable research tool for studying the effects of these antibodies (e.g., anti-VSIG4 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.

VSIG4

T cell responses are regulated by a complex network of activating and inhibitory signals. Recognition ofpeptides presented by MHC molecules is usually not sufficient for full T cell activation, but additional signals from costimulatory molecules are required. The most prominent costimulatory molecule expressed on T cells is CD28, interacting with the B7 family members CD80 and CD86. Engagement of CD28 facilitates T cell activation by enhancing TCR-mediated signaling and reducing the number of TCRs that need to be engaged for activation. Members of the CD28/B7 families have been identified, such as ICOS, PD-1, CTLA-4, BTLA, B7-H3, and B7-H4.

The V-set and Ig domain-containing 4 (VSIG4) , also known as CRIg or Z39Ig, was identified as another B7 family-related protein. In contrast to other B7 family members, which contain 2 IgG domains, VSIG4 contains 1 complete IgV-type domain (or Ig-like 1 domain) and a truncated IgC-type domain (or Ig-like 2 domain) . In vitro experiments showed that VSIG4 is at least as potent at inhibiting T cell responses as PD-L1. Furthermore, VSIG4 inhibited proliferation of mouse as well as human T cells. In vivo, administration of VSIG4-Ig fusion molecules was able to inhibit the induction of CTL responses as well as the development of Th cell-dependent IgG responses. Hence, VSIG4 is a potent negative regulator of T cell responses.

VSIG4 is a membrane protein belonging to complement receptor of the immunoglobulin superfamily (CRIg) . By binding complement component C3b, VSIG4 mediates clearance of C3b-opsonized pathogens, such as Listeria monocytogenes and Staphylococcus aureus. The expression of VSIG4 is restricted to tissue macrophages, including peritoneal macrophages and liver-residential Kupffer cells. Moreover, VSIG4 marks a subset of macrophages that associates with diabetes resistance. VSIG4 can functionally inhibit IL-2 production and T-cell proliferation by binding an unidentified T-cell ligand or receptor. Interestingly, experiments showed that a VSIG4-Fc fusion protein can protect against development of experimental arthritis, experimental autoimmune uveoretinitis, and immune-mediated liver injuries, suggesting that VSIG4 can deliver anti-inflammatory signals.

A detailed description of VSIG4 and its function can be found, e.g., in Roh, J., et al. "The immune checkpoint molecule V-set Ig domain-containing 4 is an independent prognostic factor for multiple myeloma. " Oncotarget 8.35 (2017) : 58122; Vogt, L., et al. "VSIG4, a B7 family-related protein, is a negative regulator of T cell activation. " The Journal of clinical investigation 116.10 (2006) : 2817-2826; Helmy, K.Y., et al. "CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens. " Cell 124.5 (2006) : 915-927; van Lookeren Campagne, M., et al. "Pathogen clearance and immune adherence “revisited” : immuno-regulatory roles for CRIg. " Seminars in immunology. Vol. 37. Academic Press, 2018; each of which is incorporated by reference in its entirety.

In human genomes, VSIG4 gene (Gene ID: 11326) locus has eight exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 (FIG. 1) . The VSIG4 protein also has a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. The nucleotide sequence for human VSIG4 mRNA is NM_007268.3, and the amino acid sequence for human VSIG4 is NP_009199.1 (SEQ ID NO: 2) . The location for each exon and each region in human VSIG4 nucleotide sequence and amino acid sequence is listed below:

Table 1

The human VSIG4 gene (Gene ID: 11326) is located in Chromosome X of the human genome, which is located from 66021738 to 66040092 of NC_000023.11 (GRCh38. p13 (GCF_000001405.39) ) . The 5’ UTR is from 66, 040, 080 to 66, 039, 999, Exon 1 is from 66, 040, 080 to 66, 039, 944, the first Intron is from 66, 039, 943 to 66, 033, 831, Exon 2 is from 66, 033, 830 to 66, 033, 474, the second Intron is from 66, 033, 473 to 66, 032, 750, Exon 3 is from 66, 032, 749 to 66, 032, 468, the third Intron is from 66, 032, 467 to 66, 028, 113, Exon 4 is from 66, 028, 112 to 66, 028, 050, the forth Intron is from 66, 028, 049 to 66, 027, 527, Exon 5 is from 66, 027, 526 to 66, 027, 449, the fifth Intron is from 66, 027, 448 to 66, 025, 130, Exon 6 is from 66, 025, 129 to 66, 025, 025, the sixth Intron is from 66, 025, 024 to 66, 022, 863, Exon 7 is from 66, 022, 862 to 66, 022, 841, the seventh Intron is from 66, 022, 840 to 66, 022, 501, Exon 8 is from 66, 022, 500 to 66, 021, 738, the 3’UTR is from 66, 022, 262 to 66, 021, 738, based on transcript NM_007268.3. All relevant information for human VSIG4 locus can be found in the NCBI website with Gene ID: 11326, which is incorporated by reference herein in its entirety.

In mice, VSIG4 gene locus has seven exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7 (FIG. 1) . The mouse VSIG4 protein also has a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. The nucleotide sequence for mouse VSIG4 mRNA is NM_1 77789.5, the amino acid sequence for mouse VSIG4 is NP_808457.1 (SEQ ID NO: 1) . The location for each exon and each region in the mouse VSIG4 nucleotide sequence and amino acid sequence is listed below:

Table 2

The mouse VSIG4 gene (Gene ID: 278180) is located in Chromosome X of the mouse genome, which is located from 95290807 to 95337044 ofNC_000086.8 (GRCm39 (GCF_000001635.27) ) . The 5’ UTR is from 95, 337, 044 to 95, 336, 975, Exon 1 is from 95, 337, 044 to 95, 336, 920, the first Intron is from 95, 336, 919 to 95, 334, 453, Exon 2 is from 95, 334, 452 to 95, 334, 096, the second Intron is from 95, 334, 095 to 95, 313, 219, Exon 3 is from 95, 313, 218 to 95, 313, 162, the third Intron is from 95, 313, 161 to 95, 312, 739, Exon 4 is from 95, 312, 738 to 95, 312, 661, the forth Intron is from 95, 312, 660 to 95, 293, 586, Exon 5 is from 95, 293, 585 to 95, 293, 481, the fifth Intron is from 95, 293, 480 to 95, 291, 842, Exon 6 is from 95, 291, 841 to 95, 291, 820, the sixth Intron is from 95, 291, 819 to 95, 291, 497, Exon 7 is from 95, 291, 496 to 95, 290, 809, and the 3’UTR is from 95, 291, 326 to 95, 290, 809, based on transcript NM_177789.5. All relevant information for mouse VSIG4 locus can be found in the NCBI website with Gene ID: 278180, which is incorporated by reference herein in its entirety.

FIG. 17 shows the alignment between human VSIG4 amino acid sequence (NP_009199.1; SEQ ID NO: 2) and mouse VSIG4 amino acid sequence (NP_808457.1; SEQ ID NO: 1) . Thus, the corresponding amino acid residue or region between human and mouse VSIG4 can be found in FIG. 17.

VSIG4 genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for VSIG4 in Rattus norvegicus (rat) is 312102, the gene ID for VSIG4 in Macaca mulatta (Rhesus monkey) is 709685, the gene ID for VSIG4 in Canis lupus familiaris (dog) is 491925, and the gene ID for VSIG4 in Sus scrofa (pig) is 100156239. 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. 18 shows the alignment between human VSIG4 amino acid sequence (NP_009199.1; SEQ ID NO: 2) and rat VSIG4 amino acid sequence (NP_001020175.1; SEQ ID NO: 60) . Thus, the corresponding amino acid residue or region between human and rodent VSIG4 can be found in FIG. 18.

The present disclosure provides human or chimeric (e.g., humanized) VSIG4 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 regions, and/or cytoplasmic regions 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, or 498 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, or 166 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 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) 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, exon 7, and/or exon 8 (e.g., a portion of exon 2, exons 3-5, and a portion of exon 6) .

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 ) VSIG4 nucleotide sequence. In some embodiments, the chimeric (e.g., humanized ) VSIG4 nucleotide sequence encodes a VSIG4 protein comprising an extracellular region. In some embodiments, the extracellular region described herein is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 20-281 of SEQ ID NO: 2. In some embodiments, the extracellular region comprises the entire or part of human VSIG4 extracellular 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, 7, 8, 9, 10, 12, 13, 27, 28, 29, 30, 31, 32, 33, 34, 56, 57, 58, or 59.

In some embodiments, the genetically-modified non-human animal described herein comprises a sequence encoding a human or humanized VSIG4 protein. In some embodiments, the VSIG4 protein comprises a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the humanized VSIG4 protein comprises a human or humanized VSIG4 signal peptide. For example, the human or humanized VSIG4 signal peptide comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 1-19 of SEQ ID NO: 2. In some embodiments, the humanized VSIG4 protein comprises an endogenous VSIG4 signal peptide. For example, the endogenous VSIG4 signal peptide comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 1-19 of SEQ ID NO: 1. In some embodiments, the humanized VSIG4 protein comprises a human or humanized VSIG4 extracellular region. For example, the human or humanized VSIG4 extracellular region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 20-281 or 20-283 of SEQ ID NO: 2. In some embodiments, the humanized VSIG4 protein comprises an endogenous VSIG4 extracellular region. For example, the endogenous VSIG4 extracellular region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 20-187 of SEQ ID NO: 1. In some embodiments, the humanized VSIG4 protein comprises a human or humanized VSIG4 transmembrane region. For example, the human or humanized VSIG4 transmembrane region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 284-304 of SEQ ID NO: 2. In some embodiments, the humanized VSIG4 protein comprises an endogenous VSIG4 transmembrane region. For example, the endogenous VSIG4 transmembrane region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 188-210 of SEQ ID NO: 1. In some embodiments, the humanized VSIG4 protein comprises a human or humanized VSIG4 cytoplasmic region. For example, the human or humanized VSIG4 cytoplasmic region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 305-399 of SEQ ID NO: 2. In some embodiments, the humanized VSIG4 protein comprises an endogenous VSIG4 cytoplasmic region. For example, the endogenous VSIG4 cytoplasmic region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 211-280 of SEQ ID NO: 1. In some embodiments, the human or humanized VSIG4 protein comprises a human or humanized Ig-like 1 domain, which corresponds to amino acids 21-131 of SEQ ID NO: 2. In some embodiments, the human or humanized VSIG4 protein comprises a human or humanized Ig-like 2 domain, which corresponds to amino acids 143-226 of SEQ ID NO: 2.

In some embodiments, the genetically-modified non-human animal described herein comprises a human or humanized VSIG4 gene. In some embodiments, the humanized VSIG4 gene comprises 8 exons. In some embodiments, the humanized VSIG4 gene comprises endogenous exon 1 (e.g., mouse VSIG4 exon 1) , human or humanized exon 2 (e.g., chimeric exon including part of mouse VSIG4 exon 2 and part of human VSIG4 exon 2) , human or humanized exon 3 (e.g., human VSIG4 exon 3) , human or humanized exon 4 (e.g., human VSIG4 exon 4) , human or humanized exon 5 (e.g., human VSIG4 exon 5) , human or humanized exon 6 (e.g., chimeric exon including part of human VSIG4 exon 6 and part of mouse VSIG4 exon 5) , endogenous exon 7 (e.g., mouse VSIG4 exon 6) , and/or endogenous exon 8 (e.g., mouse VSIG4 exon 7) . In some embodiments, the humanized VSIG4 gene comprises endogenous intron 1, human or humanized intron 2, human or humanized intron 3, human or humanized intron 4, human or humanized intron 5, endogenous intron 6, and/or endogenous intron 7. In some embodiments, the humanized VSIG4 gene comprises human or humanized 5’ UTR. In some embodiments, the humanized VSIG4 gene comprises human or humanized 3’ UTR. In some embodiments, the humanized VSIG4 gene comprises endogenous 5’ UTR. In some embodiments, the humanized VSIG4 gene comprises endogenous 3’ UTR.

Thus, in some embodiments, the present disclosure also provides a chimeric (e.g., humanized) VSIG4 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 VSIG4 mRNA sequence (e.g., NM_177789.5) , mouse VSIG4 amino acid sequence (e.g., SEQ ID NO: 1) , or a portion thereof (e.g., exon 1, a portion of exon 2, a portion of exon 5, exon 6, and exon 7) ; 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 VSIG4 mRNA sequence (e.g., NM_007268.3) , human VSIG4 amino acid sequence (e.g., SEQ ID NO: 2) , or a portion thereof (e.g., a portion of exon 2, exons 3-5, and a portion of exon 6) .

In some embodiments, the sequence encoding amino acids 20-185 of mouse VSIG4 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human VSIG4 (e.g., amino acids 20-281 of human VSIG4 (SEQ ID NO: 2) ) .

In some embodiments, the sequence encoding amino acids 1-185 of mouse VSIG4 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human VSIG4 (e.g., amino acids 1-281 of human VSIG4 (SEQ ID NO: 2) ) .

In some embodiments, the sequence encoding amino acids 20-187 of mouse VSIG4 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human VSIG4 (e.g., amino acids 20-283 of human VSIG4 (SEQ ID NO: 2) ) .

In some embodiments, the sequence encoding amino acids 1-280 of mouse VSIG4 (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human VSIG4 (e.g., amino acids 1-399 of human VSIG4 (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 VSIG4 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 VSIG4 nucleotide sequence (e.g., a portion of exon 2, exons 3-4, and a portion of exon 5 NM_177789.5) .

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 VSIG4 nucleotide sequence (e.g., exon 1, a portion ofexon 2, a portion of exon 5, exon 6, and exon 7 of NM_177789.5) .

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 VSIG4 nucleotide sequence (e.g., exon 1, a portion of exon 2, a portion of exon 6, exon 7, and exon 8 of NM_007268.3) .

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 VSIG4 nucleotide sequence (e.g., a portion ofexon 2 (e.g., at least 100, 130, 150, 170, 200, 220, 250, 270, 300, 320, 350, 351, 352, 353, 354, 355, 356, or 357 bp) , exons 3-5, and a portion of exon 6 (e.g., at least 5, 6, 7, 8, 9, 10, 20, 50, 60, 70, 80, 90, 100, or 105bp) of NM_007268.3; alternatively, a portion of exon 1 (e.g., at least 10, 20, 30, 40, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, 100, 110, 120, 130, or 137 bp) , exons 2-7, and a portion ofexon 8 (e.g., at least 100, 150, 200, 210, 220, 230, 235, 236, 237, 238, 239, 240, 250, 300, 400, 500, 600, 700, 750, 760, or 763 bp) of NM_007268.3) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from part of or the entire mouse VSIG4 amino acid sequence (e.g., amino acids 20-185 ofNP_808457.1 (SEQ ID NO: 1) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire mouse VSIG4 amino acid sequence (e.g., amino acids 1-19 and 186-280 ofNP_808457.1 (SEQ ID NO: 1) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from part of or the entire human VSIG4 amino acid sequence (e.g., amino acids 1-19 and 282-399 ofNP_009199.1 (SEQ ID NO: 2) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire human VSIG4 amino acid sequence (e.g., amino acids 20-281 ofNP_009199.1 (SEQ ID NO: 2) ) .

In some embodiments, a region, a portion, or the entire sequence of human exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 is inserted to the animal genome. In some embodiments, the sequence is inserted within exon 2 of endogenous VSIG4 gene.

The present disclosure also provides a humanized VSIG4 mouse amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

a) an amino acid sequence shown in SEQ ID NO: 1, 2, or 11;

b) an amino acid sequence having a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 1, 2, or 11;

c) an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is able to hybridize to a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 1, 2, or 11 under a low stringency condition or a strict stringency condition;

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

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

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

The present disclosure also provides a humanized VSIG4 amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

a) all or part of amino acids 20-281 or 1-399 of SEQ ID NO: 2;

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 20-281 or 1-399 of SEQ ID NO: 2;

c) an amino acid sequence that is different from amino acids 20-281 or 1-399 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and

d) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to amino acids 20-281 or 1-399 of SEQ ID NO: 2.

The present disclosure also relates to a VSIG4 nucleic acid (e.g., DNA or RNA) sequence, wherein the nucleic acid sequence can be selected from the group consisting of:

a) a nucleic acid sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 27, 28, 29, 30, 31, 32, 33, 34, 56, 57, 58, or 59, or a nucleic acid sequence encoding a homologous VSIG4 amino acid sequence of a humanized mouse VSIG4;

b) a nucleic acid sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 27, 28, 29, 30, 31, 32, 33, 34, 56, 57, 58, or 59 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, 7, 8, 9, 10, 12, 13, 27, 28, 29, 30, 31, 32, 33, 34, 56, 57, 58, or 59;

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, 2, or 11;

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, 2, or 11;

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, 2, or 11 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and/or

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, 2, or 11.

The present disclosure further relates to a VSIG4 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, 10, 30, or 33.

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, or 11, and has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 1, 2, or 11 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing homology is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 1, 2, or 11 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

The disclosure also provides a nucleotide sequence that has a homology of at least 90%, or at least 90%identical to the sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10, and encodes a polypeptide that has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 27, 28, 29, 30, 31, 32, 33, 34, 56, 57, 58, or 59 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, 7, 8, 9, 10, 12, 13, 27, 28, 29, 30, 31, 32, 33, 34, 56, 57, 58, or 59 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

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

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

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

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For 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) VSIG4 from an endogenous non-human VSIG4 locus.

Genetically modified animals

As used herein, the term “genetically-modified non-human animal” refers to a non-human animal having exogenous DNA in at least one chromosome of the animal’s genome. In some embodiments, at least one or more cells, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%of cells of the genetically-modified non-human animal have the exogenous DNA in its genome. The cell having exogenous DNA can be various kinds of cells, e.g., an endogenous cell, a somatic cell, an immune cell, a T cell, a B cell, an antigen presenting cell, a macrophage, a dendritic cell, a germ cell, a blastocyst, or an endogenous tumor cell. In some embodiments, genetically-modified non-human animals are provided that comprise a modified endogenous VSIG4 locus that comprises an exogenous sequence (e.g., a human sequence) , e.g., a replacement of one or more non-human sequences with one or more human sequences. The animals are generally able to pass the modification to progeny, i.e., through germline transmission.

As used herein, the term “chimeric gene” or “chimeric nucleic acid” refers to a gene or a nucleic acid, wherein two or more portions of the gene or the nucleic acid are from different species, or at least one of the sequences of the gene or the nucleic acid does not correspond to the 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 VSIG4 gene or a humanized VSIG4 nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human VSIG4 gene, at least one or more portions of the gene or the nucleic acid is from a non-human VSIG4 gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes an VSIG4 protein. The encoded VSIG4 protein is functional or has at least one activity of the human VSIG4 protein or the non-human VSIG4 protein, e.g., inhibiting T cell responses; inhibiting T cell proliferation; mediating clearance of C3b-opsonized pathogens; inhibiting IL-2 production; delivering anti-inflammatory signals.

In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized VSIG4 protein or a humanized VSIG4 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 VSIG4 protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human VSIG4 protein. The humanized VSIG4 protein or the humanized VSIG4 polypeptide is functional or has at least one activity of the human VSIG4 protein or the non-human VSIG4 protein.

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

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

In some embodiments, the animal is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In some embodiments, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm) , 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac) , 129S7, 129S8, 129T1, 129T2. These mice are described, e.g., in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999) ; Auerbach et al., Establishment and Chimera Analysis of 129/SvEv-and C57BL/6-Derived Mouse Embryonic Stem Cell Lines (2000) , both of which are incorporated herein by reference in the entirety. In some embodiments, the genetically modified mouse is a mix of the 129 strain and the C57BL/6 strain. In some embodiments, the mouse is a mix of the 129 strains, or a mix of the BL/6 strains. In some embodiments, the mouse is a BALB strain, e.g., BALB/c strain. In some embodiments, the mouse is a mix of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50%BALB/c-50%12954/Sv; or 50%C57BL/6-50%129) . In some embodiments, the 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 VSIG4 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 ofhematopoietic 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, IL2Rγknockout mice, NOD/SCID/γcnull mice (Ito, M. et al., NOD/SCID/γcnull 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 VSIG4 locus, and further comprises a modification that compromises, inactivates, or destroys the immune system (or one or more cell types of the immune system) of the non-human animal in whole or in part. In some embodiments, modification is, e.g., selected from the group consisting of a modification that results in NOD 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 VSIG4 coding sequence with human mature VSIG4 coding sequence.

Genetically modified non-human animals that comprise a modification of an endogenous non-human VSIG4 locus. In some embodiments, the modification can comprise a human nucleic acid sequence encoding at least a portion of a mature VSIG4 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the mature VSIG4 protein sequence) . Although genetically modified cells are also provided that can comprise the modifications described herein (e.g., ES cells, somatic cells) , in many embodiments, the genetically modified non-human animals comprise the modification of the endogenous VSIG4 locus in the germline of the animal.

Genetically modified animals can express a human VSIG4 and/or a chimeric (e.g., humanized) VSIG4 from endogenous mouse loci, wherein the endogenous mouse VSIG4 gene has been replaced with a human VSIG4 gene and/or a nucleotide sequence that encodes a region of human VSIG4 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 VSIG4 sequence. In various embodiments, an endogenous non-human VSIG4 locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a mature VSIG4 protein.

In some embodiments, the genetically modified mice express the human VSIG4 and/or chimeric VSIG4 (e.g., humanized VSIG4) from endogenous loci that are under control of mouse promoters and/or mouse regulatory elements. The replacement (s) at the endogenous mouse loci provide non-human animals that express human VSIG4 or chimeric VSIG4 (e.g., humanized VSIG4) 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 VSIG4 or the chimeric VSIG4 (e.g., humanized VSIG4) expressed in animal can maintain one or more functions of the wild-type mouse or human VSIG4 in the animal. For example, human or non-human VSIG4 ligands (e.g., complement C3b) can bind to the expressed VSIG4. Furthermore, in some embodiments, the animal does not express endogenous VSIG4. In some embodiments, the animal expresses a decreased level of endogenous VSIG4 as compared to a wild-type animal. As used herein, the term “endogenous VSIG4” refers to VSIG4 protein that is expressed from an endogenous VSIG4 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 VSIG4 (NP_009199.1) (SEQ ID NO: 2) . In some embodiments, the genome comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 11.

The genome of the genetically modified animal can comprise a replacement at an endogenous VSIG4 gene locus of a sequence encoding a region of endogenous VSIG4 with a sequence encoding a corresponding region of human VSIG4. In some embodiments, the sequence that is replaced is any sequence within the endogenous VSIG4 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, etc. In some embodiments, the sequence that is replaced is within the regulatory region of the endogenous VSIG4 gene. In some embodiments, the sequence that is replaced is exon 2, exon 3, exon 4, exon 5, or a portion thereof, of an endogenous mouse VSIG4 gene locus.

The genome of the genetically modified animal can comprise an insertion at an endogenous VSIG4 gene locus a sequence encoding all or part of human VSIG4. In some embodiments, the sequence that is inserted comprises one or more sequences selected from, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, 5’-UTR, 3’UTR, the first intron, the second intron, and the third intron, the fourth intron, the fifth intron, the sixth intron, and/or the seventh intron, etc. In some embodiments, the sequence that is inserted is within the regulatory region of the endogenous VSIG4 gene. In some embodiments, the sequence that is inserted is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8, or part thereof, of a human VSIG4 gene. In some embodiments, the sequence that is inserted comprises a cDNA sequence encoding full-length human VSIG4 protein.

In some embodiments, the sequence is inserted within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of endogenous VSIG4 gene (e.g., mouse VSIG4 gene) . In some embodiments, the sequence is inserted within exon 2 of mouse VSIG4 gene, e.g., between two nucleic acids corresponding to position 208 and position 209 of NM_177789.5. In some embodiments, the animal can transiently express a fusion protein comprising, from N-terminus to C-terminus, amino acids 1-46 of SEQ ID NO: 1, P2A, and amino acids 1-100 of SEQ ID NO: 2. In some embodiments, the sequence encoding P2A is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 29. Upon self-cleavage of P2A, the fusion protein can generate two polypeptides: one polypeptide comprises amino acid 1-46 of SEQ ID NO: 1, and the other polypeptide comprises amino acids 1-399 of SEQ ID NO: 2.

In some embodiments, the inserted sequence includes, from 5’ end to 3’ end, the following sequences: optionally a sequence encoding a self-cleaving peptide, a sequence encoding all or part of human VSIG4 protein, a regulatory sequence of endogenous VSIG4 gene, and/or optionally a STOP sequence. In some embodiments, the self-cleaving peptide is selected from the group consisting of P2A, T2A, E2A, and F2A. In some embodiments, the self-cleaving peptide is P2A, and the corresponding encoding sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 29. In some embodiments, the sequence encoding all or part of human VSIG4 protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 30, and/or its encoded protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 2. In some embodiments, the sequence encoding all or part of human VSIG4 protein is a cDNA sequence corresponding to nucleic acids 83-1282 of NM_007268.3. In some embodiments, the regulator sequence of endogenous VSIG4 gene comprises, from 5’ end to 3’ end, 3’ UTR of endogenous VSIG4 gene (e.g., 3’ UTR of NM_177789.5) , and a nucleic acid sequence at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, or 139 bp downstream of the 3’ UTR. In some embodiments, the regulatory sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 31. In some embodiments, the STOP sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 32.

The genetically modified animal can have one or more cells expressing a human or chimeric VSIG4 (e.g., humanized VSIG4) having 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%, 99%identical to the extracellular region of human VSIG4. In some embodiments, the extracellular region of the humanized VSIG4 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, 260, 261, 262, 263, or 264 amino acids (e.g., contiguously or non-contiguously) that are identical to human VSIG4. Because human VSIG4 and non-human VSIG4 (e.g., mouse VSIG4) sequences, in many cases, are different, antibodies that bind to human VSIG4 will not necessarily have the same binding affinity with non-human VSIG4 or have the same effects to non-human VSIG4. Therefore, the genetically modified animal having a human or a humanized extracellular region can be used to better evaluate the effects of anti-human VSIG4 antibodies in an animal model. In some embodiments, the genome of the genetically modified animal comprises a sequence encoding an amino acid sequence that corresponds to a portion or the entire sequence of exon 2, exon 3, exon 4, exon 5, and/or exon 6 of human VSIG4, a portion or the entire sequence of extracellular region of human VSIG4, or a portion or the entire sequence of amino acids 20-281 of SEQ ID NO: 2.

In some embodiments, the genome of the genetically modified animal comprises a portion of exon 2, exons 3-5, and a portion of exon 6 of human VSIG4 gene. In some embodiments, the portion of exon 2 includes at least 10, 15, 20, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, or 355 nucleotides. In some embodiments, the portion of exon 6 includes at least 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides.

In some embodiments, the non-human animal can have, at an endogenous VSIG4 gene locus, a nucleotide sequence encoding a chimeric human/non-human VSIG4 polypeptide, wherein a human portion of the chimeric human/non-human VSIG4 polypeptide comprises a portion of human VSIG4 extracellular domain, and wherein the animal expresses a functional VSIG4 on a surface of a cell of the animal. The human portion of the chimeric human/non-human VSIG4 polypeptide can comprise an amino acid sequence encoded by a portion of exon 2, exons 3-5, and/or a portion of exon 6 of human VSIG4. In some embodiments, the human portion of the chimeric human/non-human VSIG4 polypeptide can comprise a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 20-281 of SEQ ID NO: 2.

In some embodiments, the non-human portion of the chimeric human/non-human VSIG4 polypeptide comprises transmembrane and/or cytoplasmic regions of an endogenous non-human VSIG4 polypeptide. In some embodiments, the non-human portion of the chimeric human/non-human VSIG4 polypeptide can comprise a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 1-19 and/or 186-280 of SEQ ID NO: 1. In some embodiments, the transmembrane region includes a sequence corresponding to the entire or part of amino acids 188-210 of SEQ ID NO: 1. In some embodiments, the cytoplasmic region includes a sequence corresponding to the entire or part of amino acids 211-280 of SEQ ID NO: 1.

Furthermore, the genetically modified animal can be heterozygous with respect to the replacement at the endogenous VSIG4 locus, or homozygous with respect to the replacement at the endogenous VSIG4 locus.

In some embodiments, the humanized VSIG4 locus lacks a human VSIG4 5’-UTR. In some embodiment, the humanized VSIG4 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 VSIG4 genes appear to be similarly regulated based on the similarity of their 5’-flanking sequence. As shown in the present disclosure, humanized VSIG4 mice that comprise a replacement or insertion at an endogenous mouse VSIG4 locus, which retain mouse regulatory elements but comprise a humanization of VSIG4 encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized VSIG4 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 VSIG4 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 VSIG4 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. 2-7) . In some embodiments, a non-human mammal expressing human or humanized VSIG4 is provided. In some embodiments, the tissue-specific expression of human or humanized VSIG4 protein is provided.

In some embodiments, the expression of human or humanized VSIG4 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 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 VSIG4 protein can be detected by a variety of methods.

There are many analytical methods that can be used to detect exogenous DNA, including methods at the level of nucleic acid (including the mRNA quantification approaches using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, and in situ hybridization) and methods at the protein level (including histochemistry, immunoblot analysis and in vitro binding studies) . In addition, the expression level of the gene of interest can be quantified by ELISA techniques well known to those skilled in the art. Many standard analysis methods can be used to complete quantitative measurements. For example, transcription levels can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis (RNAdot) analysis. Immunohistochemical staining, flow cytometry, Western blot analysis can also be used to assess the presence of human or humanized VSIG4 protein.

Vectors

The present disclosure relates to a targeting vector, comprising: a) a DNA fragment homologous to the 5' end of a region to be altered (5' arm) , which is selected from the VSIG4 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 VSIG4 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_000086.8; 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_000086.8.

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 95334451 to the position 95338756 of the NCBI accession number NC_000086.8; 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 95286193 to the position 95290531 of the NCBI accession number NC_000086.8.

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 95334451 to the position 95335151 of the NCBI accession number NC_000086.8; 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 95291492 to the position 95293577 of the NCBI accession number NC_000086.8.

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 95334370 to the position 95338100 of the NCBI accession number NC_000086.8; 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 95329107 to the position 95332689 of the NCBI accession number NC_000086.8.

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 95334370 to the position 95335701 of the NCBI accession number NC_000086.8; 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 95332829 to the position 95334369 of the NCBI accession number NC_000086.8.

In some embodiments, the length of the selected genomic nucleotide sequence in the targeting vector can be more than about 3 kb, about 4 kb, about 5 kb, about 6 kb, about 7 kb, about 8 kb, about 8.7 kb, about 9 kb, about 10 kb. In some embodiments, the length of the inserted sequence in the targeting vector can be more than about 1 kb, about 2 kb, about 3 kb, about 4 kb, or about 5 kb.

In some embodiments, the region to be altered is exon 2, exon 3, exon 4, and/or exon 5 of VSIG4 gene (e.g., a portion of exon 2, exon 3, exon 4, and a portion of exon 5 of mouse VSIG4 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: 3; and the sequence of the 3' arm is shown in SEQ ID NO: 4. In some embodiments, the sequence of the 5' arm is shown in SEQ ID NO: 12; and the sequence of the 3' arm is shown in SEQ ID NO: 13. In some embodiments, the sequence of the 5' arm is shown in SEQ ID NO: 56; and the sequence of the 3' arm is shown in SEQ ID NO: 57. In some embodiments, the sequence of the 5' arm is shown in SEQ ID NO: 27; and the sequence of the 3' arm is shown in SEQ ID NO: 28.

In some embodiments, the sequence is derived from human (e.g., 66025122-66033828 of NC_000023.11) . For example, the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human VSIG4, preferably exon 2, exon 3, exon 4, exon 5, and/or exon 6 of the human VSIG4. In some embodiments, the nucleotide sequence of the humanized VSIG4 encodes the entire or the part of human VSIG4 protein with the NCBI accession number NP_009199.1 (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 VSIG4 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 VSIG4 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 of the mouse VSIG4 gene. In some embodiments, the sgRNAs target exon 2.

In some embodiments, the targeting sequences are shown as SEQ ID NOS: 14-25. 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: 14-25. In some embodiments, the oligonucleotide sgRNA sequences targeting 5' end of the endogenous VSIG4 gene are set forth in SEQ ID NOs: 14-19. In some embodiments, the oligonucleotide sgRNA sequences targeting 3' end of the endogenous VSIG4 gene are set forth in SEQ ID NOs: 20-25. In some embodiments, the oligonucleotide sgRNA sequence targeting 5' end of the endogenous VSIG4 gene is set forth in SEQ ID NO: 18.

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 VSIG4 gene locus, a sequence encoding a region of an endogenous VSIG4 with a sequence encoding a corresponding region of human or chimeric VSIG4. In some embodiments, the replacement occurs in a germ cell, a somatic cell, a blastocyst, or a fibroblast, etc. The nucleus of a somatic cell or the fibroblast can be inserted into an enucleated oocyte.

FIG. 3 and FIG. 4 show humanization strategies for a mouse VSIG4 locus. In FIG. 3 and FIG. 4, the targeting strategy involves a vector comprising the 5' end homologous arm, human VSIG4 gene fragment, 3' homologous arm. The process can involve replacing endogenous VSIG4 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 VSIG4 sequence with human VSIG4 sequence.

FIG. 6 and FIG. 7 show humanization strategies for a mouse VSIG4 locus. In FIG. 6 and FIG. 7, the targeting strategy involves a vector comprising the 5' end homologous arm, human VSIG4 gene fragment, 3' homologous arm. The process can involve inserting within endogenous VSIG4 gene locus (e.g., exon 2 of mouse VSIG3 gene) , a sequence encoding all or part of human VSIG4 by homologous recombination. In some embodiments, the cleavage at the insertion site (e.g., by zinc finger nucleases, TALEN or CRISPR) can result in DNA double strands break, and the homologous recombination is used to insert the sequence encoding all or part of human VSIG4.

Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of replacing at an endogenous VSIG4 locus (or site) , a nucleic acid encoding a sequence encoding a region of endogenous VSIG4 with a sequence encoding a corresponding region of human VSIG4. 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, exon 7, and/or exon 8 of a human VSIG4 gene. In some embodiments, the sequence includes a portion of exon 2, exons 3-5, and a portion of exon 6 of a human VSIG4 gene (e.g., nucleic acids 140-925 of NM_007268.3) . In some embodiments, the region is located within the extracellular region of VSIG4 (e.g., amino acids 20-281 of SEQ ID NO: 2) . In some embodiments, the endogenous VSIG4 locus is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of mouse VSIG4 gene. In some embodiments, the sequence includes a portion of exon 2, exon 3, exon 4, and a portion of exon 5 of mouse VSIG4 gene (e.g., nucleic acids 128-625 of NM_177789.5) .

In some embodiments, the methods of modifying a VSIG4 locus of a mouse to express a chimeric human/mouse VSIG4 peptide can include the steps of replacing at the endogenous mouse VSIG4 locus a nucleotide sequence encoding a mouse VSIG4 with a nucleotide sequence encoding a human VSIG4, thereby generating a sequence encoding a chimeric human/mouse VSIG4.

In some embodiments, the nucleotide sequence encoding the chimeric human/mouse VSIG4 can include a first nucleotide sequence encoding a signal peptide of mouse VSIG4 (e.g., a sequence corresponding to amino acids 1-19 of SEQ ID NO: 1) ; a second nucleotide sequence encoding a portion of an extracellular region of human VSIG4 (e.g., a sequence corresponding to amino acids 20-281 of SEQ ID NO: 2) ; and a third nucleotide sequence encoding a portion of the extracellular region, a transmembrane region, and a cytoplasmic region of mouse VSIG4 (e.g., a sequence corresponding to amino acids 186-280 of SEQ ID NO: 1) .

In some embodiments, the methods for making a genetically modified, humanized animal, can include the step of inserting at an endogenous VSIG4 locus (or site) , a nucleic acid sequence encoding all or part of human VSIG4. For example, the nucleic acid sequence can include exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 of human VSIG4 gene. In some embodiments, the nucleic acid sequence comprises a cDNA sequence encoding full-length human VSIG4. In some embodiments, the insertion site is located within exon 2 of endogenous VSIG4 gene (e.g., mouse VSIG4 gene) . In some embodiments, the sequence is inserted immediately after the start codon.

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

The present disclosure further provides a method for establishing a VSIG4 gene humanized animal model, involving the following steps:

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

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

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

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

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

In some embodiments, the non-human mammal in step (c) is a female with 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 VSIG4 gene, e.g., by inserting a nucleotide sequence (e.g., cDNA sequence) encoding human or humanized VSIG4 protein after the endogenous regulatory element of the non-human animal's VSIG4 gene. For example, one or more functional region sequences of the non-human animal's VSIG4 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 VSIG4 protein. In some embodiments, the coding frame of the modified non-human animal's VSIG4 gene can be all or part of the nucleotide sequence from exon 1 to exon 7 (e.g., exon 2) of the non-human animal's VSIG4 gene.

In some embodiments, methods of making the genetically modified animal comprises inserting a nucleotide sequence encoding human or humanized VSIG4 protein and/or an auxiliary sequence after the endogenous regulatory element of the non-human animal's VSIG4 gene. In some embodiments, the auxiliary sequence can be a stop codon, such that the VSIG4 gene humanized animal model can express human or humanized VSIG4 protein in vivo, but does not express non-human animal's VSIG4 protein. In some embodiments, the auxiliary sequence includes WPRE (WHP Posttranscriptional Response Element) , STOP, and/or polyA (e.g., SV40 polyA, or BGH polyA) . In some embodiments, the auxiliary sequence is a sequence that can terminate transcription and/or translation of the inserted nucleotide sequence.

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

(1) providing a plasmid comprising a human VSIG4 gene fragment, flanked by a 5' homology arm and a 3' homology arm, wherein the 5' and 3' homology arms target an endogenous VSIG4 gene;

(2) providing two small guide RNAs (sgRNAs) that target the endogenous VSIG4 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 VSIG4 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 VSIG4 protein is operably linked to an endogenous regulatory element at the endogenous VSIG4 gene locus.

In some embodiments, the genetically-modified animal does not express an endogenous VSIG4 protein.

(1) providing a plasmid comprising a human or chimeric VSIG4 gene fragment, flanked by a 5' homology arm and a 3' homology arm, wherein the 5' and 3' homology arms target an endogenous VSIG4 gene;

(2) providing one small guide RNAs (sgRNAs) that target the endogenous VSIG4 gene;

(3) modifying genome of a fertilized egg or an embryonic stem cell by inserting the human or chimeric VSIG4 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 VSIG4 protein, e.g., in a physiologically appropriate manner, provide a variety of uses that include, but are not limited to, developing therapeutics for human diseases and disorders, and assessing the toxicity and/or the efficacy of these human therapeutics in the animal models.

In various aspects, genetically modified animals are provided that express human or humanized VSIG4, which are useful for testing agents that can decrease or block the interaction between VSIG4 and VSIG4 ligands (e.g., complement C3b) or the interaction between VSIG4 and anti-human VSIG4 antibodies, testing whether an agent can increase or decrease the immune response, and/or determining whether an agent is an VSIG4 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 (aknock-in or knockout) . In various embodiments, the genetically modified non-human animals further comprise an impaired immune system, e.g., a non-human animal genetically modified to sustain or maintain a human xenograft, e.g., a human solid tumor or a blood cell tumor (e.g., a lymphocyte tumor, a B or T cell tumor) . In some embodiments, the anti-VSIG4 antibody blocks or inhibits the VSIG4-related signaling pathway.

In some embodiments, the anti-VSIG4 antibody described herein can block the interaction between VSIG4 and its ligand. In some embodiments, the VSIG4 ligand is complement C3b.

In some embodiments, the genetically modified animals can be used for determining effectiveness of an anti-VSIG4 antibody for the treatment of cancer. The methods involve administering the anti-VSIG4 antibody (e.g., anti-human VSIG4 antibody) to the animal as described herein, wherein the animal has a tumor; and determining inhibitory effects of the anti-VSIG4 antibody to the tumor. The inhibitory effects that can be determined include, e.g., a decrease of tumor size or tumor volume, a decrease of tumor growth, a reduction of the increase rate of tumor volume in a subject (e.g., as compared to the rate of increase in tumor volume in the same subject prior to treatment or in another subject without such treatment) , a decrease in the risk of developing a metastasis or the risk of developing one or more additional metastasis, an increase of survival rate, and an increase of life expectancy, etc. The tumor volume in a subject can be determined by various methods, e.g., as determined by direct measurement, MRI or CT. In addition, a delicate balance is required for these antibodies, as VSIG4 is also expressed on many other cells. Thus, it is important that the humanized VSIG4 functions in a largely similar way as compared to the endogenous VSIG4, 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-VSIG4 antibody can directly target cancer cells expressing VSIG4, 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-VSIG4 antibody prevents complement C3b from binding to VSIG4. In some embodiments, the anti-VSIG4 antibody does not prevent complement C3b from binding to VSIG4.

In some embodiments, the genetically modified animals can be used for determining whether an anti-VSIG4 antibody is a VSIG4 agonist or antagonist. In some embodiments, the methods as described herein are also designed to determine the effects of the agent (e.g., anti-VSIG4 antibodies) on VSIG4, e.g., whether the agent can stimulate immune cells or inhibit immune cells (e.g., T cells, B cells, or NK cells) , whether the agent can increase or decrease the production of cytokines, whether the agent can activate or deactivate immune cells (e.g., T cells, B cells, or NK cells) , 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, or autoimmune diseases.

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

In some embodiments, the anti-VSIG4 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 VSIG4 antibody is designed for treating myeloid leukemia (AML) , breast cancer, colorectal cancer, gastric cancer, cervical cancer, glioblastoma, and/or pancreatic ductal adenocarcinoma (PDAC) . In some embodiments, the cancer is selected from a bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

In some embodiments, the anti-VSIG4 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-VSIG4 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-VSIG4 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-VSIG4 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 VSIG4 gene function, human VSIG4 antibodies, drugs for human VSIG4 targeting sites, the drugs or efficacies for human VSIG4 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 VSIG4 gene humanized non-human animal prepared by the methods described herein, the VSIG4 gene humanized non-human animal described herein, the double-or multi-humanized non-human animal generated by the methods described herein (or progeny thereof) , a non-human animal expressing the human or humanized VSIG4 protein, or the tumor-bearing or inflammatory animal models described herein. In some embodiments, the TCR-T, CAR-T, and/or other immunotherapies can treat the VSIG4-associated diseases described herein. In some embodiments, the TCA-T, CAR-T, and/or other immunotherapies provides an evaluation method for treating the VSIG4-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 VSIG4 gene 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 cell death ligand 1 (PD-L1) , IL4, IL4 receptor (IL4R) , IL6, IL6 receptor (IL6R) , IL17, IL17 receptor (IL17R) , C-C Motif Chemokine Receptor 5 (CCR5) , C-C Motif Chemokine Receptor 8 (CCR8) , tumor necrosis factor alpha (TNFα) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , Lymphocyte Activating 3 (LAG-3) , B And T Lymphocyte Associated (BTLA) , CD27, CD28, CD47, CD137, CD154, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , Signal regulatory protein α (SIRPα) , or TNF Receptor Superfamily Member 4 (TNFRSF4 or OX40) .

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

(a) using the methods of introducing human VSIG4 gene or chimeric VSIG4 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, IL4, IL4R, IL6, IL6R, IL 17, IL7R, CCR5, CCR8, TNFα, CTLA-4, LAG-3, BTLA, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPα, or OX40. Some of these genetically modified non-human animal are described, e.g., in PCT/CN2017/090320, PCT/CN2017/099577, PCT/CN2017/099575, PCT/CN2017/099576, PCT/CN2017/099574, PCT/CN2017/106024, PCT/CN2017/110494, PCT/CN2017/110435, PCT/CN2017/120388, PCT/CN2018/081628, PCT/CN2018/081629; each of which is incorporated herein by reference in its entirety.

In some embodiments, the VSIG4 humanization is directly performed on a genetically modified animal having a human or chimeric PD-1, PD-L1, IL4, IL4R, IL6, IL6R, IL17, IL7R, CCR5, CCR8, TNFα, CTLA-4, LAG-3, BTLA, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPα, or OX40 gene.

As these proteins may involve different mechanisms, a combination therapy that targets two or more of these proteins thereof may be a more effective treatment. In fact, many related clinical trials are in progress and have shown a good effect. The genetically modified animal model with two or more human or humanized genes can be used for determining effectiveness of a combination therapy that targets two or more of these proteins, e.g., an anti-VSIG4 antibody and an additional therapeutic agent for the treatment of cancer. The methods include administering the anti-VSIG4 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, IL4, IL4R, IL6, IL6R, IL17, IL7R, CCR5, CCR8, TNFα, CTLA-4, LAG-3, BTLA, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPα, or OX40. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab) , an anti-PD-1 antibody (e.g., nivolumab) , or an anti-PD-L1 antibody.

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

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

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

EXAMPLES

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

Materials and Methods

The following materials were used in the following examples.

BbsI, EcoRI, BamHI, AseI, and BspHI restriction enzymes were purchased from NEB (Catalog numbers: R0539L, R0101M, R0136M, R0526M, and R0517L, respectively) .

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

Ambion ^TM in vitro transcription kit (MEGAshortscript ^TM T7 Transcription Kit) was purchased from Thermo Fisher Scientific. The catalog number is AM1354.

Cas9mRNA was purchased from SIGMA (Catalog number: CAS9MRNA-1EA) .

UCA kit was obtained from Biocytogen Pharmaceuticals (Beijing) Co., Ltd. The catalog number is BCG-DX-001.

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

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

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

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

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

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

PE-VSIG4 Monoclonal Antibody (NLA14) was purchased from eBioscience (Catalog number: 12-5752-82) .

APC-VSIG4 Monoclonal Antibody (JAV4) was purchased from eBioscience (Catalog number: 17-5757-41) .

Brilliant Violet 421 ^TM anti-mouse CD4 Antibody was purchased from BioLegend (Catalog number: 100438) .

PE anti-mouse CD8a Antibody was purchased from BioLegend (Catalog number: 100708) .

PE/Cy ^TM 7 Mouse anti-mouse NK1.1 Antibody (BD Pharmingen ^TM) was purchased from BD Biosciences (Catalog number: 552878) .

APC anti-mouse/rat Foxp3 Antibody was purchased from eBioscience (Catalog number: 17-5773-82) .

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

PerCP/Cy5.5 anti-mouse TCRβ chain Antibody (BD Pharmingen ^TM) was purchased from BD Biosciences (Catalog number: 553174) .

Brilliant Violet 605 ^TManti-mouse CD11c Antibody was purchased from BioLegend (Catalog number: 117334) .

PE anti-mouse/human CD11b Antibody was purchased from BioLegend (Catalog number: 101208) .

APC/Cy7 anti-mouse CD45 antibody was purchased from BioLegend (Catalog number: 103116) .

PerCP/Cyanine5.5 anti-mouse CD3ε antibody was purchased from BioLegend (Catalog number: 100328) .

FITC anti-mouse CD4 antibody was purchased from BioLegend (Catalog number: 100406) .

Brilliant Violet 711 ^TM anti-mouse CD8a antibody was purchased from BioLegend (Catalog number: 100748) .

PerCP anti-mouse/human CD11b antibody was purchased from BioLegend (Catalog number: 101230) .

Anti-Mo/Rt FoxP3PE/Cy ^TM 7 antibody was purchased from eBioscience (Catalog number: 25-5773-82) .

Pacific Blue ^TM anti-mouse I-A/I-E antibody was purchased from BioLegend (Catalog number: 107620) .

PE/Cyanine7 anti-mouse CD206 (MMR) antibody was purchased from BioLegend (Catalog number: 141720) .

Brilliant Violet 711 ^TM anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody was purchased from BioLegend (Catalog number: 108443) .

EXAMPLE 1: Generation of mice with humanized VSIG4 gene (method one)

The genome of a non-human animal (e.g., a mouse) can be modified to include a nucleic acid sequence encoding all or a part of a human VSIG4 protein, such that the genetically modified non-human animal can express a human or humanized VSIG4 protein. The mouse VSIG4 gene (NCBI Gene ID: 278180, Primary source: MGI: 2679720, UniProt ID: F6TUL9) is located at 95290807 to 95337044 of chromosome X (NC_000086.8) , and the human VSIG4 gene (NCBI Gene ID: 11326, Primary source: HGNC: 17032, UniProt ID: Q9Y279) is located at 66021738 to 66040092 of chromosome X (NC_000023.11) . The mouse VSIG4 transcript is NM_177789.5, and the corresponding protein sequence NP_808457.1 is set forth in SEQ ID NO: 1. The human VSIG4 transcript is NM_007268.3, and the corresponding protein sequence NP_009199.1 is set forth in SEQ ID NO: 2. Mouse and human VSIG4 gene loci are shown in FIG. 1.

All or part of nucleotide sequences encoding human VSIG4 protein can be introduced into the mouse endogenous VSIG4 locus, so that the mouse expresses human or humanized VSIG4 protein. Specifically, under the control of the mouse VSIG4 gene regulatory element, a nucleotide sequence encoding human VSIG4 protein was used to replace the corresponding mouse sequence using gene-editing techniques, to obtain a humanized VSIG4 gene locus as shown in FIG. 2, thereby humanizing mouse VSIG4 gene.

As shown in the schematic diagram of the targeting strategy in FIG. 3, the targeting vector V1 contains homologous arm sequences upstream and downstream of the mouse VSIG4 gene, and an “A1 Fragment” containing DNA sequences of human VSIG4 gene. Specifically, sequence of the upstream homologous arm (5′ homologous arm, SEQ ID NO: 3) is identical to nucleotide sequence of 95334451-95338756 of NCBI accession number NC_000086.8, and sequence of the downstream homologous arm (3′ homologous arm, SEQ ID NO: 4) is identical to nucleotide sequence of 95286193-95290531 of NCBI accession number NC_000086.8. The A1 Fragment contains a human genomic DNA sequence from VSIG4 genes (SEQ ID NO: 5) , which is identical to nucleotide sequence of 66025122-66033828 of NCBI accession number NC_000023.11.

The connection between the 5’ end of the human VSIG4 sequence in the A1 Fragment and the mouse sequence was designed as: 5’-GTCTGTGTTTGCCTTTTTGACTTTGTTCCCTGTTT TAGGC

ATCCTGGAAGTGC CAGAGAGTGTAACAGGACCTT-3’ (SEQ ID NO: 6) , wherein the “C” in sequence “ TAGGC” is the last nucleotide of the mouse sequence, and the first “C” in sequence

is the first sequence of the human sequence. The connection between the 3’ end of the human VSIG4 sequence and mouse sequence was designed as: 5’-CAAATACCACTTGATCTTGTTTTACCCTCTAGGAA AGAGC

ATCTTTGCCATAATCTTCATCATCTCCCTTTGCT -3’ (SEQ ID NO: 7) , wherein the “C” in sequence “ AGAGC” is the last nucleotide of the human sequence, and the first “C” in sequence

is the first nucleotide of the mouse sequence.

The targeting vector V1 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. The connection between the 5’ end of the Neo cassette and the human sequence was designed as: 5’-TTATTAGATATTTTCTTTATATACATTTCAAATGC TATCC

CGAATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAA -3’ (SEQ ID NO: 8) , wherein the last “C” in sequence “ TATCC” is the last nucleotide of the human sequence, and the “G” in sequence

is the first nucleotide of the Neo cassette. The connection between the 3’ end of the Neo cassette and the mouse sequence was designed as: 5’-GAAAGTATAGGAACTTCATCAGTCAGGTACATAATGGTGGATCCA GTACT

GTTCCCTATACCCTCCCTCCGCCCTGCTCCCCTAC -3’ (SEQ ID NO: 9) , wherein the last “T” in sequence “ GTACT” is the last nucleotide of the Neo cassette, and the “T” in sequence

is the first nucleotide of the mouse 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 V1. The mRNA sequence of the engineered mouse VSIG4 after humanization and its encoded protein sequence are shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively.

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 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, and then the humanized homozygous mice with a humanized VSIG4 gene were obtained by breeding the heterozygous mice with each other.

The CRISPR/Cas system can also be introduced for gene editing, and the targeting strategy shown in FIG. 4 was designed. The targeting vector V2 contains homologous arm sequences upstream and downstream of the mouse VSIG4 gene, and an “A2 Fragment” containing DNA sequences of human VSIG4 gene. Specifically, sequence of the upstream homologous arm (5′ homologous arm, SEQ ID NO: 12) is identical to nucleotide sequence of 95334451-95335151 of NCBI accession number NC_000086.8, and sequence of the downstream homologous arm (3′ homologous arm, SEQ ID NO: 13) is identical to nucleotide sequence of 95291492-95293577 of NCBI accession number NC_000086.8. The A2 Fragment contains a human genomic DNA sequence from VSIG4 genes (SEQ ID NO: 5) .

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. Targeting vectors with verified sequences were used for subsequent experiments.

The target sequences are important for the targeting specificity of sgRNAs and the efficiency of Cas9-induced cleavage. Specific sgRNA sequences were designed and synthesized that recognize the 5’ end targeting site and 3’ end targeting site. The targeting site sequence of each sgRNA on the VSIG4 gene locus is as follows:

sgRNA1 targeting site (SEQ ID NO: 14) : 5’-AGGGTGGGGTGGCCTAAAACAGG-3’

sgRNA2 targeting site (SEQ ID NO: 15) : 5’-CAGGTCCCTGTCACACTCTCAGG-3’

sgRNA3 targeting site (SEQ ID NO: 16) : 5’-GCAAGTTTTGGTGAAATGGCTGG-3’

sgRNA4 targeting site (SEQ ID NO: 17) : 5’-CAGGAATCACTATACATGTGAGG-3’

sgRNA5 targeting site (SEQ ID NO: 18) : 5’-ATCTTCCTACGTGACTCCACTGG-3’

sgRNA6 targeting site (SEQ ID NO: 19) : 5’-TGCATCTATGATCCCCTGAGAGG-3’

sgRNA7 targeting site (SEQ ID NO: 20) : 5’-AAATAAGACCAAGTGCTATTTGG-3’

sgRNA8 targeting site (SEQ ID NO: 21) : 5’-GACTTTAATACCAACATACCAGG-3’

sgRNA9 targeting site (SEQ ID NO: 22) : 5’-GGTCTTGCTACTATCATACCAGG-3’

sgRNA10 targeting site (SEQ ID NO: 23) : 5’-AGCTGTTAGTTATCCTATAATGG-3’

sgRNA11 targeting site (SEQ ID NO: 24) : 5’-TGACCAAGGATTATAGTAACAGG-3’

sgRNA12 targeting site (SEQ ID NO: 25) : 5’-ACATAGGCTTCTCTCAATATTGG-3’

UCA kit was used to detect the activity of each sgRNA, and two sgRNAs with high activity and strong specificity were selected. Restriction enzyme cleavage sites were added to their 5′ ends and a complementary strand to obtain a forward oligonucleotide and a reverse oligonucleotide. After annealing, the products were ligated to the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) , respectively, to obtain expression vectors pT7-VSIG4.

The pT7-sgRNA vector was synthesized, which included a DNA fragment containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 26) , 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-VSIG4 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 F0 generation positive VSIG4 gene humanized mice were bred with wild-type mice to obtain F1 generation mice, and then the F1 generation mice were bred with each other to obtain F2 generation VSIG4 gene humanized homozygous mice.

EXAMPLE 2: Generation of mice with humanized VSIG4 gene (method two)

In addition, a nucleotide sequence encoding human VSIG4 protein can also be inserted into exon 2 of the mouse VSIG4 gene by gene editing technology. Specifically, a sequence encoding a linker peptide (e.g., P2A) can be used to connect the mouse sequence and human sequence, to obtain a humanized VSIG4 gene locus shown in FIG. 5, thereby humanizing mouse VSIG4 gene.

As shown in the schematic diagram of the targeting strategy in FIG. 6, the targeting vector V3 contains homologous arm sequences upstream and downstream of the mouse VSIG4 gene, and an “A3 Fragment” containing DNA sequences of human VSIG4 gene. Specifically, sequence of the upstream homologous arm (5′ homologous arm, SEQ ID NO: 56) is identical to nucleotide sequence of 95334370-95338100 of NCBI accession number NC_000086.8, and sequence of the downstream homologous arm (3′ homologous arm, SEQ ID NO: 57) is identical to nucleotide sequence of 95329107-95332689 of NCBI accession number NC_000086.8. The A3 Fragment contains, from 5’ end to 3’ end, a P2A-encoding sequence (SEQ ID NO: 29) , a human VSIG4 nucleotide sequence (SEQ ID NO: 30) , a mouse VSIG4 nucleotide sequence (SEQ ID NO: 31) , and a STOP sequence (SEQ ID NO: 32) . The human VSIG4 nucleotide sequence is identical to nucleotide sequences of 83-1282 of NCBI accession number NM_007268.3, and the mouse VSIG4 nucleotide sequence is identical to nucleotide sequences of 95290668-95291327 of NCBI accession number NC_000086.8. The connection between the STOP sequence and the mouse sequence was designed as: 5’-GTAAGTAAGCTTGGGCTGCAGGTCGAGGGACCTA GTCGAC

CTACAGGCAAGTTTTGGTGAAATGGCTGGTAAGAC-3’ (SEQ ID NO: 58) , wherein the last “C” in sequence “ GTCGAC” is the last nucleotide of the STOP sequence, and the first “A” in sequence

is the first nucleotide of the mouse sequence.

The targeting vector V3 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. The connection between the 5’ end of the Neo cassette and the mouse sequence was designed as: 5’-CTGACCTGTGTCTTTTTATAGCAGTATTGTGCTG GTTTTG

CCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGGT -3’ (SEQ ID NO: 59) , wherein the last “G” in sequence “ GTTTTG” is the last nucleotide of the mouse sequence, and the “G” in sequence

is the first nucleotide of the Neo cassette. The connection between the 3’ end of the Neo cassette and the mouse sequence was designed as: 5’-TTCTCTAGAAAGTATAGGAACTTCATCAGTCAGGTACATAATGGTG GATCC

TATATTTCTGTGAAATGTTCCAAAATCTGATATTG -3’ (SEQ ID NO: 34) , wherein the last “C” in sequence “ GATCC” is the last nucleotide of the Neo cassette, and the “C” in sequence

is the first nucleotide of the mouse 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 V3. The mRNA sequence of the engineered mouse VSIG4 after humanization and its encoded protein sequence are shown in SEQ ID NO: 33 and SEQ ID NO: 2, respectively.

The CRISPR/Cas system can also be introduced for gene editing, and the targeting strategy shown in FIG. 7 was designed. The targeting vector V4 contains homologous arm sequences upstream and downstream of the mouse VSIG4 gene, and an “A4 Fragment” containing DNA sequences of human VSIG4 gene. Specifically, sequence of the upstream homologous arm (5′ homologous arm, SEQ ID NO: 27) is identical to nucleotide sequence of 95334370-95335701 of NCBI accession number NC_000086.8, and sequence of the downstream homologous arm (3′ homologous arm, SEQ ID NO: 28) is identical to nucleotide sequence of 95332829-95334369 of NCBI accession number NC_000086.8. The A4 Fragment contains, from 5’ end to 3’ end, a sequence encoding P2A (SEQ ID NO: 29) , a human VSIG4 nucleotide sequence (SEQ ID NO: 30) , a mouse VSIG4 nucleotide sequence (SEQ ID NO: 31) , and a STOP sequence (SEQ ID NO: 32) . The human VSIG4 nucleotide sequence is identical to nucleotide sequences of 83-1282 of NCBI accession number NM_007268.3, and the mouse VSIG4 nucleotide sequence is identical to nucleotide sequences of 95290668-95291327 of NCBI accession number NC_000086.8. The mRNA sequence of the engineered mouse VSIG4 after humanization and its encoded protein sequence are shown in SEQ ID NO: 33 and SEQ ID NO: 2, respectively.

Given that human VSIG4 has multiple isoforms or transcripts, the methods described herein can be applied to other isoforms or transcripts.

Specific sgRNA sequences were designed and synthesized that recognize the targeting site. The targeting site sequence of each sgRNA on the VSIG4 gene locus is as follows:

sgRNA1 targeting site (SEQ ID NO: 14) : 5’-AGGGTGGGGTGGCCTAAAACAGG-3’

sgRNA2 targeting site (SEQ ID NO: 15) : 5’-CAGGTCCCTGTCACACTCTCAGG-3’

sgRNA3 targeting site (SEQ ID NO: 16) : 5’-GCAAGTTTTGGTGAAATGGCTGG-3’

sgRNA4 targeting site (SEQ ID NO: 17) : 5’-CAGGAATCACTATACATGTGAGG-3’

sgRNA5 targeting site (SEQ ID NO: 18) : 5’-ATCTTCCTACGTGACTCCACTGG-3’

sgRNA6 targeting site (SEQ ID NO: 19) : 5’-TGCATCTATGATCCCCTGAGAGG-3’

UCA kit was used to detect the activity of each sgRNA, and the results are shown in the table below and FIG. 8. sgRNA6 was selected for subsequence experiments. Restriction enzyme cleavage sites were added to its 5′ ends and a complementary strand to obtain a forward oligonucleotide and a reverse oligonucleotide. After annealing, the products were ligated to the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) , respectively, to obtain expression vectors pT7-VSIG4-6.

Table 3. sgRNA activity test results

Con.	1.00±0.07
PC	180.84±2.69
sgRNA1	43.20±0.33
sgRNA2	32.95±0.45
sgRNA3	31.16±1.52
sgRNA4	104.96±2.53
sgRNA5	63.59±1.53
sgRNA6	66.51±3.13

Table 4.

The pre-mixed Cas9 mRNA, the targeting vector, and in vitro transcription products of the pT7-VSIG4-18 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 identification results of some F0 generation mice are shown in FIGS. 9A-9B. Combined with the PCR results and the further verification results by sequencing, five mice numbered F0-01, F0-02, F0-03, F0-04, and F0-05 were identified as positive mice. The PCR primers are shown in the table below.

Table 5. PCR primer sequences for F0 generation genotyping and recombinant fragment sizes

The primer L-GT-F is located on the left side of the 5′ homologous arm, R-GT-R is located on the right side of the 3′ homologous arm, and both L-GT-R and R-GT-F are located on the human VSIG4 sequence.

The positive VSIG4 gene humanized mice of F0 generation were bred with wild-type mice to obtain F1 generation mice, which can be genotyped using the same method by PCR. The PCR primer sequences are shown in the table below. As shown in FIGS. 10A-10B, 8 mice numbered F1-0010, F1-0012, F1-0013, F1-0018, F1-0019, F1-0022, F1-0023, and F1-0082 were identified as positive mice.

Table 6. PCR primer sequences for F1 generation genotyping and recombinant fragment sizes

Southern Blot was performed on F1 generation mice identified as positive by PCR to confirm ifrandom insertions were introduced. Specifically, mouse tail genomic DNA was extracted, and digested with BspHI or AseI restriction enzyme, transferred to a membrane, and then hybridized with probes. The 5′ probe and the 3′ probe are located on the upstream of the 5′ homologous arm and on the A3 fragment, respectively. The specific probes and the target fragment sizes are shown in the table below.

Table 7. Specific probes and target fragment sizes

The detection results of Southern Blot are shown in FIG. 11. In view of the hybridization results by 3’ probe and 5’ probe, seven F1 generation mice numbered F1-0010, F1-0012, F1-0013, F1-0019, F1-0022, F1-0023, and F1-0082 were confirmed to be positive heterozygotes and no random insertions were detected. This indicates that the method described above can be used to generate genetically-modified VSIG4 gene humanized mice that can be stably passaged without random insertions.

The following primers were used to synthesize probes in Southern Blot assays:

5’ Probe-F (SEQ ID NO: 46) : 5’-CTTCTCACGATCAACCCAGAACAG-3’,

5’ Probe-R (SEQ ID NO: 47) : 5’-GCATAAACATAGCCCATCATTGCTG-3’;

3’ Probe-F (SEQ ID NO: 48) : 5’-AACTGATGAATGGGAGCAGTGGTGG-3’,

3’ Probe-R (SEQ ID NO: 49) : 5’-GCAGACACTCTATGCCTGTGTGGAG-3’.

The heterozygous mice identified as positive in the F1 generation were bred with each other to obtain the F2 generation VSIG4 gene humanized homozygous mice.

EXAMPLE 3: Detection of human or humanized VSIG4 protein expression in vivo in VSIG4 gene humanized mice

Expression of human or humanized VSIG4 in positive mice can be confirmed, e.g., by RT-PCR or flow cytometry. Specifically, a 6-week-old wild-type C57BL/6 female mouse and a 6-week-old VSIG4 gene humanized heterozygous female mouse prepared in Example 2 were selected. Liver tissues were collected after euthanasia by cervical dislocation. The primer sequences shown in the table below were used for RT-PCR detection, and the detection results are shown in FIGS. 12A-12C. In the wild-type C57BL/6 mouse, only mouse VSIG4 mRNA was detected (FIG. 12A) , and no humanized VSIG4 mRNA was detected (FIG. 12B) . In the VSIG4 gene humanized heterozygous mouse, both mouse VSIG4 mRNA (FIG. 12A) and humanized VSIG4 mRNA (FIG. 12B) were detected.

Table 8. RT-PCR primer sequences and target fragment sizes

Expression of humanized VSIG4 protein in positive mice was also detected by flow cytometry. Specifically, one 11-week-old wild-type C57BL/6 male mouse and one 11-week-old VSIG4 gene humanized homozygous male mouse prepared in Example 2 were selected. Macrophages in peritoneal lavage fluid were extracted to prepare single-cell suspension. The cells were stained with Purified anti-mouse CD16/32 Antibody (an anti-mouse FcγR antibody) , 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; a neutrophil marker antibody) , V450 Rat Anti-mouse CD11b (mCD11b; a monocyte marker antibody) , FITC anti-mouse F4/80 (mF4/80; a macrophage marker antibody) , PE-VSIG4 Monoclonal Antibody (mVSIG4; an anti-mouse VSIG4 antibody) and/or APC-VSIG4 Monoclonal Antibody (hVSIG4; an anti-human VSIG4 antibody) , followed by flow cytometry detection.

The results showed that 19.2%of macrophages (characterized as mCD45+ mGr-1-mCD11b+ mF4/80+) in the peritoneal lavage fluid of the wild-type C57BL/6 mouse expressed mouse VSIG4 protein (characterized as mCD45+ mGr-1-mCD11b+ mF4/80+ mVSIG4+) , and 0.064%expressed humanized VSIG4 protein (characterized as mCD45+ mGr-1-mCD11b+mF4/80+ hVSIG4+) . Within the macrophages in the peritoneal lavage fluid of VSIG4 gene humanized homozygous mouse, 15.7%expressed humanized VSIG4 protein and 0.047%expressed mouse VSIG4 protein. The results indicate that VSIG4 gene humanized mice successfully expressed humanized VSIG4 protein.

The immunophenotyping of leukocyte subtypes and T cell subtypes in mice was further detected by flow cytometry. Specifically, three 7-week-old female wild-type C57BL/6 mice and three 7-week-old VSIG4 gene humanized homozygous mice prepared in Example 2 were selected. The spleen, lymph nodes and peripheral blood were collected after euthanasia by cervical dislocation. The cells were stained with Brilliant Violet 510 ^TM anti-mouse CD45 Antibody (mCD45; an anti-mouse CD45 antibody) , PerCP anti-mouse Ly-6G/Ly-6C Antibody (mGr-1; an anti-mouse Gr-1 antibody) , Brilliant Violet 421 ^TM anti-mouse CD4 Antibody (mCD4; an anti-mouse CD4 antibody) , FITC anti-mouse F4/80 Antibody (mF4/80; an anti-mouse F4/80 antibody) , PE anti-mouse CD8a Antibody (mCD8; an anti-mouse CD8 antibody) , PE/Cy ^TM 7 Mouse anti-mouse NK1.1 Antibody (mNK1.1; an anti-mouse NK1.1 antibody) , APC anti-mouse/rat Foxp3 Antibody (mFoxp3; an anti-mouse Foxp3 antibody) , FITC anti-Mouse CD19 Antibody (mCD19; an anti-mouse CD19 antibody) , PerCP/Cy5.5 anti-mouse TCR β chain Antibody (mTCRβ; an anti-mouse TCR β antibody) , Brilliant Violet 605 ^TM anti-mouse CD11c Antibody (mCD11c; an anti-mouse CD11c antibody) , and PE anti-mouse/human CD11b Antibody (mCD11b; and/or an anti-mouse/human CD11b antibody) , followed by flow cytometry detection.

T cells were characterized by mCD45+ mTCRβ+, B cells were characterized by mCD45+mCD19+, NK cells were characterized by mCD45+ mTCRβ-mNK1.1+, dendritic cells were characterized by mCD45+ mTCRβ-mCD11c+, granulocytes were characterized by mCD45+mGr-1+, monocytes and macrophages were characterized by mCD45+ mGr-1-mCD11b+mF4/80+, helper T cells (CD4+T cells) were characterized by mCD45+ mCD4+ mFoxp3+, killer T cells (CD8+ T cells) were characterized by mCD45+ mCD8+.

The detection results of spleen, lymph nodes and peripheral blood are shown in FIGS. 13A-13B, FIGS. 14A-14B, and FIGS. 15A-15B, respectively. The results showed that the leukocyte subtypes of VSIG4 gene humanized homozygous mice (H/H) , including T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes, and microphages, were basically the same as those of C57BL/6 wild-type mice. In addition, the percentages of T cell subtypes, including CD4+ T cells, CD8+ T cells, and Treg cells, were basically the same as those of C57BL/6 wild-type mice. The results indicate that the humanization of VSIG4 gene did not affect the differentiation, development and distribution of leukocytes and T cells in mice.

EXAMPLE 4. In vivo efficacy verification

Ten 7-week-old female VSIG4 gene humanized homozygous mice obtained in Example 2 were selected and subcutaneously inoculated with mouse colon cancer cells MC38 (5 × 10 ⁵ cells per mouse) . When the tumor volume grew to about 100-150 mm ³, the mice were randomly placed into a control group (G1) and a treatment group (G2) . Each group had 65 mice. The control group mice were injected with hIgG1 (10 mg/kg) , whereas the treatment group mice were injected with an anti-human VSIG4 antibody (Ab1, prepared by immunizing mice using methods described in Janeway′s Immunobiology (9th Edition) ) . The mice were administered by intraperitoneal injection (i.p. ) on the grouping day. The frequency of administration was twice a week (BIW) , and there were 6 times of administrations in total. The tumor volume was measured twice a week and the body weight of the mice was weighed as well. Euthanasia was performed when the tumor volume of the mouse reached 3000 mm ³.

The mouse body weight and tumor volume are shown in FIG. 16A and FIG. 16B, respectively. The results showed that at the end of the experiment, the control group mice and the treatment group mice were in good health, and the body weight increased during the experimental period (FIG. 16A) . With respect to the tumor volume (FIG. 16B) , tumors in both control and treatment groups continued to grow during the experimental period. Compared with the control group mice, the tumor volume growth in the treatment group mice slowed down, indicating that the anti-human VSIG4 antibody can inhibit tumor growth.

Further, based on the above in vivo efficacy verification results, the tumor cells of the control group mice (G1) and the treatment group mice (G2) were collected respectively at the end of the experiment. The tumor-infiltrating lymph node cells (TILs) were isolated for flow cytometry detection to analyze the immune cell types. The cells were stained with APC/Cy7 anti-mouse CD45 antibody (mCD45; an anti-mouse CD45 antibody) , PerCP/Cyanine5.5 anti-mouse CD3ε antibody (mCD3; an anti-mouse CD3 antibody) , FITC anti-mouse CD4 antibody (mCD4; an anti-mouse CD4 antibody) , Brilliant Violet 711 ^TM anti-mouse CD8a antibody (mCD8; an anti-mouse CD8 antibody) , FITC anti-mouse F4/80 antibody (mF4/80; an anti-mouse F4/80 antibody) , PerCP anti-mouse/human CD11b antibody (mCD11b; an anti-mouse/human CD11b antibody) , Anti-Mo/Rt FoxP3PE/Cy ^TM 7 antibody (mFoxP3; an anti-mouse FoxP3 antibody) , Pacific Blue ^TM anti-mouse I-A/I-E antibody (mI-A/I-E; an anti-mouse I-A/I-E antibody) , PE/Cyanine7 anti-mouseCD206 (MMR) antibody (mCD206; an anti-mouse CD206 antibody) , and/or Brilliant Violet 711 ^TM anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody (mGr-1; an anti-mouse Gr-1 antibody) , followed by flow cytometry detection. The results are shown in the table below.

Table 9. Detection results of TILs in mice of control group and treatment group

Group	CTL	Th	Tregs	MDSC	M1	M2	M1/M2
G1	11.92%	6.38%	3.33%	9.13%	40.46%	40.44%	1.45
G2	13.43%	7.96%	3.96%	7.66%	53.18%	29.82%	2.33

Specifically, T cells were characterized by mCD45+ mCD3+, helper T cells (Th cells) were characterized by mCD45+ mCD3+ mCD4+, regulatory T cells (Tregs cells) were characterized by mCD45+ mCD3+ mCD4+ mCD8-mFoxP3+, cytotoxic T cells lymphocytes (CTL) were characterized by mCD45+ mCD3+ mCD8+, M1 macrophages (M1) were characterized by mCD45+ mCD11b+ mF4/80+ mI-A/I-E+ mCD206-, M2 macrophages (M2) were characterized by mCD45+ mCD11b+ mF4/80+ mI-A/I-E-mCD206+, and myeloid-derived suppressor cells (MDSCs) were characterized by mCD45+ mCD11b+ mF4/80-mGr-1+.

As shown in the table above, compared with the control group mice, the percentages of CTL cells, Th cells, Tregs cells, M1 macrophages and M1/M2 ratio increased, while the percentages of MDSCs and M2 macrophages decreased in the treatment group mice. The results indicate that the anti-human VSIG4 antibody inhibited tumor growth.

The above results indicate that the VSIG4 gene humanized mice can be used as an animal model for the validation and screening of anti-human VSIG4 antibodies in vivo.

EXAMPLE 5. Generation of double-or multi-gene humanized mice

The VSIG4 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) PD-1, PD-L1, IL4, IL4R, IL6, IL6R, IL17, IL7R, CCR5, and/or CCR8 genes. Alternatively, embryonic stem cells from humanized VSIG4 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 VSIG4 and other gene modifications. In addition, it is also possible to breed the homozygous or heterozygous VSIG4 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) VSIG4 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. These double-gene or multi-gene modified mice can be used for in vivo validation of gene regulators targeting human VSIG4 and other genes.

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 VSIG4 (V-set and immunoglobulin domain containing 4) .
The animal of claim 1, wherein the sequence encoding the human or chimeric VSIG4 is operably linked to an endogenous regulatory element at the endogenous VSIG4 gene locus in the at least one chromosome.
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric VSIG4 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human VSIG4 (NP_009199.1 (SEQ ID NO: 2) ) .
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric VSIG4 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 11.
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric VSIG4 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 20-281 of SEQ ID NO: 2.
The animal of any one of claims 1-5, wherein the animal is a mammal, e.g., a monkey, a rodent, 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 VSIG4 or expresses a decreased level of endogenous VSIG4 as compared to VSIG4 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 VSIG4.
The animal of any one of claims 1-9, wherein the animal has one or more cells expressing human or chimeric VSIG4, and the expressed human or chimeric VSIG4 can interact with human complement C3b.
The animal of any one of claims 1-9, wherein the animal has one or more cells expressing human or chimeric VSIG4, and the expressed human or chimeric VSIG4 can interact with endogenous complement C3b.
A genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous VSIG4 with a sequence encoding a corresponding region of human VSIG4 at an endogenous VSIG4 gene locus.
The animal of claim 12, wherein the sequence encoding the corresponding region of human VSIG4 is operably linked to an endogenous regulatory element at the endogenous VSIG4 locus, and one or more cells of the animal expresses a human or chimeric VSIG4.
The animal of claim 12 or 13, wherein the animal does not express endogenous VSIG4 or expresses a decreased level of endogenous VSIG4 as compared to VSIG4 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 VSIG4, optionally without the signal peptide.
The animal of any one of claims 12-15, wherein the animal has one or more cells expressing a chimeric VSIG4 having an extracellular region, a transmembrane region, and a cytoplasmic region, wherein the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%identical to the extracellular region of human VSIG4 (NP_009199.1 (SEQ ID NO: 2) ) .
The animal of claim 16, wherein the extracellular region of the chimeric VSIG4 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, 260, 261, 262, 263 or 264 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human VSIG4.
The animal of any one of claims 12-17, wherein the sequence encoding a region of endogenous VSIG4 comprises exon 2, exon 3, exon 4, and/or exon 5, or a part thereof, of the endogenous VSIG4 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 with respect to the replacement at the endogenous VSIG4 gene locus.
The animal of any one of claims 12-19, wherein the animal is homozygous with respect to the replacement at the endogenous VSIG4 gene locus.
A method for making a genetically-modified, non-human animal, comprising:

replacing in at least one cell of the animal, at an endogenous VSIG4 gene locus, a sequence encoding a region of endogenous VSIG4 with a sequence encoding a corresponding region of human VSIG4.
The method of claim 22, wherein the sequence encoding the corresponding region of human VSIG4 comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8, or a part thereof, of a human VSIG4 gene.
The method of claim 22 or 23, wherein the sequence encoding the corresponding region of human VSIG4 comprises a portion of exon 2, exon 3, exon 4, exon 5, and a portion of exon 6, of a human VSIG4 gene.
The method of any one of claims 22-24, wherein the sequence encoding the corresponding region of human VSIG4 encodes amino acids 20-281 of SEQ ID NO: 2.
The method of claim 24, wherein the region is located within the extracellular region of VSIG4.
The method of any one of claims 22-26, wherein the sequence encoding a region of endogenous VSIG4 comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7, or a part thereof, of the endogenous VSIG4 gene.
The method of any one of claims 22-27, wherein the animal is a mouse, and the sequence encoding a region of endogenous VSIG4 comprises a portion of exon 2, exon 3, exon 4, and a portion of exon 5 of the endogenous VSIG4 gene.
A genetically-modified, non-human animal, wherein the genome of the animal comprises an insertion of a sequence encoding a human or chimeric VSIG4 at an endogenous VSIG4 gene locus.
The animal of claim 29, wherein the sequence encoding the human or chimeric VSIG4 is operably linked to an endogenous regulatory element at the endogenous VSIG4 locus, and one or more cells of the animal expresses a human or chimeric VSIG4.
The animal of claim 29 or 30, wherein the animal does not express endogenous VSIG4 or expresses a decreased level of endogenous VSIG4 as compared to VSIG4 expression level in a wild-type animal.
The animal of any one of claims 29-31, wherein the sequence encoding a human or chimeric VSIG4 is inserted within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 (e.g., exon 2) of endogenous VSIG4 gene.
The animal of any one of claims 29-32, wherein the inserted sequence comprises, optionally from 5’ end to 3’ end:

a) optionally a sequence encoding a self-cleaving peptide (e.g., P2A) ,

b) the sequence encoding a human or chimeric VSIG4,

c) a regulatory sequence of endogenous VSIG4 gene, and

d) optionally an auxiliary sequence (e.g., WPRE, STOP, and/or polyA) .
The animal of any one of claims 29-33, wherein the sequence encoding a human or chimeric VSIG4 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 30.
The animal of claim 33 or 34, wherein the self-cleaving peptide is P2A, T2A, E2A, or F2A.
The animal of any one of claims 33-35, wherein the sequence encoding a self-cleavage peptide is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 29.
The animal of any one of claims 33-36, wherein the regulatory sequence of endogenous VSIG4 gene comprises 3’ UTR of endogenous VSIG4 gene.
The animal of any one of claims 33-37, wherein the regulatory sequence at 3’ end of endogenous VSIG4 gene is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 31.
The animal of any one of claims 33-38, wherein the auxiliary sequence is a STOP sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 32.
The animal of any one of claims 29-39, wherein the animal is a mouse.
The animal of any one of claims 29-40, wherein the animal is heterozygous with respect to the insertion at the endogenous VSIG4 gene locus.
The animal of any one of claims 29-40, wherein the animal is homozygous with respect to the insertion at the endogenous VSIG4 gene locus.
A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a human VSIG4 polypeptide, wherein the animal expresses the human VSIG4 polypeptide.
The animal of claim 43, wherein the human VSIG4 polypeptide is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 2.
The animal of claim 43 or 44, wherein the nucleotide sequence encoding a human VSIG4 polypeptide is operably linked to an endogenous VSIG4 regulatory element of the animal.
A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric VSIG4 polypeptide, wherein the chimeric VSIG4 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human VSIG4, wherein the animal expresses the chimeric VSIG4 polypeptide.
The animal of claim 46, wherein the chimeric VSIG4 polypeptide has at least 50, at least 80, at least 100, at least 150, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 261, at least 262, at least 263 or at least 264 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human VSIG4 extracellular region.
The animal of claim 46 or 47, wherein the chimeric VSIG4 polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 20-281 of SEQ ID NO: 2.
The animal of any one of claims 46-48, wherein the nucleotide sequence is operably linked to an endogenous VSIG4 regulatory element of the animal.
The animal of any one of claims 46-49, wherein the chimeric VSIG4 polypeptide comprises an endogenous VSIG4 cytoplasmic region and/or an endogenous VSIG4 transmembrane region, optionally an endogenous signal peptide.
The animal of any one of claims 46-50, wherein the nucleotide sequence is integrated to an endogenous VSIG4 gene locus of the animal.
The animal of any one of claims 46-51, wherein the chimeric VSIG4 polypeptide has at least one mouse VSIG4 activity and/or at least one human VSIG4 activity.
A method of making a genetically-modified animal cell that expresses a chimeric VSIG4, the method comprising:

replacing at an endogenous VSIG4 gene locus, a nucleotide sequence encoding a region of endogenous VSIG4 with a nucleotide sequence encoding a corresponding region of human VSIG4, thereby generating a genetically-modified animal cell that includes a nucleotide sequence that encodes the chimeric VSIG4, wherein the animal cell expresses the chimeric VSIG4.
The method of claim 53, wherein the animal is a mouse.
The method of claim 53 or 54, wherein the chimeric VSIG4 comprises a human or humanized VSIG4 extracellular region; and a transmembrane and/or a cytoplasmic region of mouse VSIG4.
The method of any one of 53-55, wherein the nucleotide sequence encoding the chimeric VSIG4 is operably linked to an endogenous VSIG4 regulatory region, e.g., promoter.
A method of making a genetically-modified animal cell that expresses a human or chimeric VSIG4, the method comprising:

inserting at an endogenous VSIG4 gene locus (e.g., exon 2 of endogenous VSIG4 gene) , a nucleotide sequence comprising, optionally from 5’ end to 3’ end:

a) optionally a sequence encoding a self-cleaving peptide (e.g., P2A) ;

b) a sequence encoding a human or chimeric VSIG4;

c) a regulatory sequence at 3’ end of endogenous VSIG4 gene; and

d) optionally an auxiliary sequence (e.g., WPRE, STOP, and/or polyA) ,

thereby generating a genetically-modified animal cell that includes a nucleotide sequence that encodes the human or chimeric VSIG4, wherein the animal cell expresses the human or chimeric VSIG4.
The method of claim 57, wherein the animal is a mouse.
The animal of any one of claims 1-21, and 29-52, wherein the animal further comprises a sequence encoding an additional human or chimeric protein.
The animal of claim 59, wherein the additional human or chimeric protein is programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , IL4, IL4 receptor (IL4R) , IL6, IL6 receptor (IL6R) , IL17, IL17 receptor (IL17R) , C-C Motif Chemokine Receptor 5 (CCR5) , or C-C Motif Chemokine Receptor 8 (CCR8) .
The method of any one of claims 22-28 and 53-58, wherein the animal or mouse further comprises a sequence encoding an additional human or chimeric protein.
The method of claim 42, wherein the additional human or chimeric protein is PD-1, PD-L1, IL4, IL4R, IL6, IL6R, IL17, IL7R, CCR5, or CCR8.
A method of determining effectiveness of an anti-VSIG4 antibody for the treatment of cancer, comprising:

a) administering the anti-VSIG4 antibody to the animal of any one of claims 1-21, 29-52, 59, and 60, wherein the animal has a tumor; and

b) determining inhibitory effects of the anti-VSIG4 antibody to the tumor.
The method of claim 63, wherein the tumor comprises one or more cells that express VSIG4.
The method of claim 63 or 64, wherein the tumor comprises one or more cancer cells that are injected into the animal.
The method of any one of claims 63-65, wherein determining inhibitory effects of the anti-VSIG4 antibody to the tumor involves measuring the tumor volume in the animal.
The method of any one of claims 63-66, wherein the cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, or prostate cancer.
A method of determining effectiveness of an anti-VSIG4 antibody and an additional therapeutic agent for the treatment of cancer, comprising

a) administering the anti-VSIG4 antibody and the additional therapeutic agent to the animal of any one of claims 1-21, 29-52, 59, and 60, wherein the animal has a tumor; and

b) determining inhibitory effects on the tumor.
The method of claim 68, wherein the animal further comprises a sequence encoding a human or chimeric programmed cell death protein 1 (PD-1) .
The method of claim 68 or 69, wherein the animal further comprises a sequence encoding a human or chimeric programmed death-ligand 1 (PD-L1) .
The method of any one of claims 68-70, wherein the additional therapeutic agent is an anti-PD-1 antibody or an anti-PD-L1 antibody.
The method of any one of claims 68-71, wherein the tumor comprises one or more tumor cells that express PD-L1.
The method of any one of claims 68-72, wherein the tumor is caused by injection of one or more cancer cells into the animal.
The method of any one of claims 68-73, wherein determining inhibitory effects of the treatment involves measuring the tumor volume in the animal.
The method of any one of claims 68-74, wherein the animal has bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and/or prostate cancer.
A method of determining effectiveness of an anti-VSIG4 antibody for treating an autoimmune disorder, comprising:

a) administering the anti-VSIG4 antibody to the animal of any one of claims 1-21, 29-52, 59, and 60, wherein the animal has the autoimmune disorder; and

b) determining effects of the anti-VSIG4 antibody for treating the auto-immune disease.
The method of claim 76, wherein the autoimmune disorder is rheumatoid arthritis, Crohn’s disease, systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel diseases (IBD) , ulcerative colitis, and/or scleroderma.
A method of determining effectiveness of an anti-VSIG4 antibody for treating an immune disorder, comprising:

a) administering the anti-VSIG4 antibody to the animal of any one of claims 1-21, 29-52, 59, and 60, wherein the animal has the immune disorder; and

b) determining effects of the anti-VSIG4 antibody for treating the immune disorder.
The method of claim 78, wherein the immune disorder is allergy, asthma, and/or atopic dermatitis.
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, or 11;

(b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 1, 2, or 11;

(c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1, 2, or 11;

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

(e) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 1, 2, or 11.
A nucleic acid comprising a nucleotide sequence, wherein the nucleotide sequence is one of the following:

(a) a sequence that encodes the protein of claim 80;

(b) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 27, 28, 29, 30, 31, 32, 33, 34, 56, 57, 58, or 59;

(c) a sequence that is at least 90%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 27, 28, 29, 30, 31, 32, 33, 34, 56, 57, 58, or 59; and

(d) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 27, 28, 29, 30, 31, 32, 33, 34, 56, 57, 58, or 59.