WO2021209050A1

WO2021209050A1 - Genetically modified non-human animal with human or chimeric tnfsf9 and/or 4-1bb

Info

Publication number: WO2021209050A1
Application number: PCT/CN2021/087867
Authority: WO
Inventors: Yuelei SHEN; Jiawei Yao; Yanan GUO; yang BAI; Lei Zhao
Original assignee: Biocytogen Jiangsu Co., Ltd.; Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
Priority date: 2020-04-17
Filing date: 2021-04-16
Publication date: 2021-10-21
Also published as: CN113234139A

Abstract

Disclosed are genetically modified non-human animals that express a human or chimeric (e.g., humanized) TNFSF9 and/or 4-1BB, and methods of use thereof.

Description

GENETICALLY MODIFIED NON-HUMAN ANIMAL WITH HUMAN OR CHIMERIC TNFSF9 AND/OR 4-1BB

CLAIM OF PRIORITY

This application claims the benefit of Chinese Patent Application App. No. CN 202010305793.3, filed on April 17, 2020. The entire contents of the foregoing application are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) TNFSF9 and/or 4-1BB, 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 pathways 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

Because the amino acid sequences of human 4-1BB and TNFSF9 are significantly different from the corresponding proteins in rodents (e.g., the sequence identity between human and mouse 4-1BB protein sequences is only about 58%, and the sequence identity between human and mouse TNFSF9 protein sequences is only about 39%) , antibodies targeting human 4-1BB or TNFSF9 protein usually do not recognize mouse 4-1BB or TNFSF9 protein. Therefore, wild-type mice cannot be used to screen and evaluate the efficacy of drugs targeting the 4-1BB/TNFSF9 signaling pathway. In order to make pre-clinical trials more effective and minimize the failure rate of research and development, there is an urgent need in the field to develop a humanized non-human animal model of 4-1BB and/or TNFSF9.

This disclosure is related to an animal model with human TNFSF9 or chimeric TNFSF9. The animal model can express human TNFSF9 or chimeric TNFSF9 (e.g., humanized TNFSF9) protein in its body. It can be used in the studies on the function of TNFSF9 gene, and can be used in the screening and evaluation of anti-human TNFSF9 antibodies. This disclosure is also related to an animal model with human 4-1BB or chimeric 4-1BB. The animal model can express human 4-1BB or chimeric 4-1BB (e.g., humanized 4-1BB) protein in its body. It can be used in the studies on the function of 4-1BB gene, and can be used in the screening and evaluation of anti-human 4-1BB antibodies. In some embodiments, the disclosure is related to 4-1BB/TNFSF9 double gene humanized mice.

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 TNFSF9 and/or 4-1BB 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 TNFSF9 and/or 4-1BB protein and a platform for screening drugs, e.g., antibodies, against cancers.

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 TNF Superfamily Member 9 (TNFSF9) . In some embodiments, the disclosure is related to a genetically-modified, non-human animal having one or more cells (e.g., somatic cells or germline cells) whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric TNFSF9.

In some embodiments, the sequence encoding the human or chimeric TNFSF9 is operably linked to an endogenous regulatory element at the endogenous TNFSF9 gene locus in the at least one chromosome.

In some embodiments, the sequence encoding a human or chimeric TNFSF9 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human TNFSF9 (NP_003802.1 (SEQ ID NO: 4) ) .

In some embodiments, the sequence encoding a human or chimeric TNFSF9 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: 12.

In some embodiments, the sequence encoding a human or chimeric TNFSF9 comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to amino acids 26-254 of SEQ ID NO: 4.

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

In some embodiments, the animal does not express endogenous TNFSF9.

In some embodiments, the animal has one or more cells expressing human or chimeric TNFSF9.

In some embodiments, the animal has one or more cells expressing human or chimeric TNFSF9, and a human TNF Receptor Superfamily Member 9 (4-1BB) can bind to the expressed human or chimeric TNFSF9. In some embodiments, the animal has one or more cells expressing human or chimeric TNFSF9, and an endogenous 4-1BB can bind to the expressed human or chimeric TNFSF9.

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 TNFSF9 with a sequence encoding a corresponding region of human TNFSF9 at an endogenous TNFSF9 gene locus.

In some embodiments, the sequence encoding the corresponding region of human TNFSF9 is operably linked to an endogenous regulatory element at the endogenous TNFSF9 locus, and one or more cells of the animal expresses a chimeric TNFSF9.

In some embodiments, the animal does not express endogenous TNFSF9.

In some embodiments, the replaced sequence encodes all or a portion of the transmembrane region and/or extracellular region of endogenous TNFSF9.

In some embodiments, the animal has one or more cells expressing a chimeric TNFSF9 having a cytoplasmic region, a transmembrane region, and an extracellular 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 TNFSF9. In some embodiments, the extracellular region of the chimeric TNFSF9 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, or 205 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human TNFSF9. In some embodiments, the transmembrane region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%identical to the transmembrane region of human TNFSF9. In some embodiments, the transmembrane region of the chimeric TNFSF9 has a sequence that has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or 21 contiguous amino acids that are identical to a contiguous sequence present in the transmembrane region of human TNFSF9.

In some embodiments, the sequence encoding a region of endogenous TNFSF9 comprises exon 1, exon 2, and/or exon 3, or a part thereof, of the endogenous TNFSF9 gene. In some embodiments, the animal is a mouse, and the sequence encoding a region of endogenous TNFSF9 starts within exon 1 and ends within exon 3 of the endogenous mouse TNFSF9 gene.

In some embodiments, the animal is heterozygous with respect to the replacement at the endogenous TNFSF9 gene locus. In some embodiments, the animal is homozygous with respect to the replacement at the endogenous TNFSF9 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 TNFSF9 gene locus, a sequence encoding a region of an endogenous TNFSF9 with a sequence encoding a corresponding region of human TNFSF9.

In some embodiments, the sequence encoding the corresponding region of human TNFSF9 comprises exon 1, exon 2, and/or exon 3, or a part thereof, of a human TNFSF9 gene.

In some embodiments, the sequence encoding the corresponding region of human TNFSF9 starts within exon 1 and ends within exon 3 of a human TNFSF9 gene.

In some embodiments, the sequence encoding the corresponding region of human TNFSF9 encodes amino acids 26-254 of SEQ ID NO: 4.

In some embodiments, the region of an endogenous TNFSF9 is located within the transmembrane region and/or extracellular region of endogenous TNFSF9.

In some embodiments, the sequence encoding a region of an endogenous TNFSF9 is exon 1, exon 2, and/or exon 3, or a part thereof, of the endogenous TNFSF9 gene. In some embodiments, the animal is a mouse, and the sequence encoding a region of an endogenous TNFSF9 starts within exon 1 and ends within exon 3 of the endogenous mouse TNFSF9 gene.

In one aspect, the disclosure is related to a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric TNFSF9 polypeptide, in some embodiments, the chimeric TNFSF9 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human TNFSF9, in some embodiments, the animal expresses the chimeric TNFSF9 polypeptide.

In some embodiments, the chimeric TNFSF9 polypeptide has at least 50, at least 100, or at least 200 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human TNFSF9 transmembrane region and/or extracellular region.

In some embodiments, the chimeric TNFSF9 polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 26-254 of SEQ ID NO: 4.

In some embodiments, the nucleotide sequence is operably linked to an endogenous TNFSF9 regulatory element of the animal.

In some embodiments, the chimeric TNFSF9 polypeptide comprises an endogenous TNFSF9 cytoplasmic region.

In some embodiments, the nucleotide sequence is integrated to an endogenous TNFSF9 gene locus of the animal.

In some embodiments, the chimeric TNFSF9 polypeptide has at least one mouse TNFSF9 activity and/or at least one human TNFSF9 activity.

In one aspect, the disclosure is related to a method of making a genetically-modified non-human animal cell that expresses a chimeric TNFSF9, the method comprising: replacing at an endogenous TNFSF9 gene locus, a nucleotide sequence encoding a region of endogenous TNFSF9 with a nucleotide sequence encoding a corresponding region of human TNFSF9, thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the chimeric TNFSF9, in some embodiments, the animal cell expresses the chimeric TNFSF9.

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

In some embodiments, the chimeric TNFSF9 comprises a cytoplasmic region of endogenous TNFSF9; and a transmembrane region and/or an extracellular region of human TNFSF9.

In some embodiments, the nucleotide sequence encoding the chimeric TNFSF9 is operably linked to an endogenous TNFSF9 regulatory region, e.g., promoter.

In some embodiments, the animal further comprises a sequence encoding an additional human or chimeric protein. In some embodiments, the additional human or chimeric protein is TNF Receptor Superfamily Member 9 (4-1BB) , programmed cell death protein 1 (PD-1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , Lymphocyte Activating 3 (LAG-3) , B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , CD27, CD28, CD47, CD154, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , Signal regulatory protein α (SIRPα) or TNF Receptor Superfamily Member 4 (OX40) .

In some embodiments, the additional human or chimeric protein is TNF Receptor Superfamily Member 9 (4-1BB) , and the animal expresses the human or chimeric 4-1BB.

In one aspect, the disclosure is related to a method of determining effectiveness of a therapeutic agent for treating cancer, comprising: a) administering the therapeutic agent to the animal as described herein, in some embodiments, the animal has a tumor; and b) determining the inhibitory effects of the therapeutic agent to the tumor. In some embodiments, the therapeutic agent is an anti-TNFSF9 antibody or an anti-4-1BB antibody. In some embodiments, the tumor comprises one or more cells that express TNFSF9 and/or 4-1BB. In some embodiments, the tumor comprises one or more cancer cells that are injected into the animal. In some embodiments, determining the inhibitory effects of the therapeutic agent to the tumor involves measuring the tumor volume in the animal. In some embodiments, the cancer is solid tumor, refractory solid tumor, B-cell lymphoma, non-Hodgkin’s lymphoma, metastatic solid tumor, breast cancer, colorectal cancer, melanoma, non-small cell lung cancer (NSCLC) , small cell lung cancer (SCLC) , bladder cancer, renal cancer, ovarian cancer, prostate cancer, melanoma, or multiple myeloma.

In one aspect, the disclosure is related to a method of determining effectiveness of a therapeutic agent for treating an autoimmune disorder, comprising: a) administering the therapeutic agent to the animal as described herein, in some embodiments, the animal has the autoimmune disorder; and b) determining effects of the therapeutic agent for treating the auto-immune disease. In some embodiments, the therapeutic agent is an anti-TNFSF9 antibody or an anti-4-1BB antibody. In some embodiments, the autoimmune disorder is multiple sclerosis, diabetes, encephalomyelitis, rheumatoid arthritis, lupus, allergic conjunctivitis, or inflammatory bowel disease.

In one aspect, method of determining effectiveness of a therapeutic agent for treating an immune disorder, comprising: a) administering the therapeutic agent to the animal as described herein, in some embodiments, the animal has the immune disorder; and b) determining effects of the therapeutic agent for treating the immune disease. In some embodiments, the therapeutic agent is an anti-TNFSF9 antibody or an anti-4-1BB antibody. In some embodiments, the immune disorder is allergy, asthma, and/or atopic dermatitis.

In one aspect, the disclosure is related to a method of determining toxicity of a therapeutic agent, the method comprising a) administering the therapeutic agent to the animal as described herein; and b) determining weight change of the animal.

In one aspect, the disclosure is related to a method of determining toxicity of a therapeutic agent, the method comprising a) administering the therapeutic agent to the animal as described herein; and b) determining one or more biochemical parameters of the animal. In some embodiments, the one or more biochemical parameters comprise the serum levels of alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) of the animal.

In one aspect, the disclosure is related to a method of determining toxicity of a therapeutic agent, the method comprising a) administering the therapeutic agent to the animal as described herein; and b) determining organ damage of the animal. In some embodiments, the organ is selected from the group consisting of liver, kidney, brain, heart, spleen, lung, and skin. In some embodiments, the organ is liver, and liver damage is evaluated via the percentage of lesion site area over total area of liver. In some embodiments, the organ is isolated from the animal before the determining step. In some embodiments, the organ is stained, e.g., by haematoxylin and eosin (H&E) .

In some embodiments, the therapeutic agent is an anti-TNFSF9 antibody or an anti-4-1BB antibody.

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: 2, 4, 12, 52, 54, and/or 56; (b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 2, 4, 12, 52, 54, and/or 56; (c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 2, 4, 12, 52, 54, and/or 56; (d) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 2, 4, 12, 52, 54, and/or 56 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and (e) an amino acid sequence that comprises a substitution, a deletion and /or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 2, 4, 12, 52, 54, and/or 56.

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: 1, 3, 5-11, 13, 50, 51, 53, 55, and/or 57; (c) a sequence that is at least 90 %identical to SEQ ID NO: 1, 3, 5-11, 13, 50, 51, 53, 55, and/or 57; and (d) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1, 3, 5-11, 13, 50, 51, 53, 55, and/or 57.

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

The disclosure further relates to a TNFSF9 and/or 4-1BB 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 TNFSF9 and/or 4-1BB gene function, human TNFSF9 antibodies, human 4-1BB antibodies, the drugs or efficacies for human TNFSF9 and/or 4-1BB targeting sites, and the drugs for immune-related diseases and antitumor drugs.

In another aspect, the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous TNFSF9 gene, wherein the disruption of the endogenous TNFSF9 gene comprises deletion of exon 1, exon 2, and/or exon 3, or part thereof of the endogenous TNFSF9 gene.

In some embodiments, the disruption of the endogenous TNFSF9 gene comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3 of the endogenous TNFSF9 gene.

In some embodiments, the disruption of the endogenous TNFSF9 gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1 and intron 2 of the endogenous TNFSF9 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, 500, 550, 600, 650, or more nucleotides.

In some embodiments, the disruption of the endogenous TNFSF9 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, and/or exon 3 (e.g., deletion of a nucleotide sequence starting from the first nucleotide of the transmembrane region-encoding sequence to the last nucleotide of the extracellular region-encoding sequence) .

In one aspect, the disclosure relates to genetically-modified, non-human animals whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric TNF Receptor Superfamily Member 9 (4-1BB) . In some embodiments, the sequence encoding the human or chimeric 4-1BB is operably linked to an endogenous regulatory element at the endogenous 4-1BB gene locus in the at least one chromosome. In some embodiments, the sequence encoding a human or chimeric 4-1BB comprises a sequence encoding an amino acid sequence that is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human 4-1BB (NP_001552.2 (SEQ ID NO: 54) ) . In some embodiments, the sequence encoding a human or chimeric 4-1BB comprises a sequence encoding an amino acid sequence that is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 56. In some embodiments, the sequence encoding a human or chimeric 4-1BB comprises a sequence encoding an amino acid sequence that corresponds to amino acids 1-184 of SEQ ID NO: 54.

In some embodiments, the animal is a mammal, e.g., a monkey, a rodent or a mouse. In some embodiments, the animal is a C57BL/6 mouse. In some embodiments, the animal does not express endogenous 4-1BB. In some embodiments, the animal has one or more cells expressing human or chimeric 4-1BB. In some embodiments, the expressed human or chimeric 4-1BB can bind to or interact with human protein TNFSF9 (also known as 4-1BB ligand, or 4-1BBL) . In some embodiments, the expressed human or chimeric 4-1BB can bind to or interact with endogenous TNFSF9.

In one aspect, the disclosure relates to genetically-modified, non-human animals, wherein the genome of the animals comprises a replacement, at an endogenous 4-1BB gene locus, of a sequence encoding a region of endogenous 4-1BB with a sequence encoding a corresponding region of human 4-1BB. In some embodiments, the sequence encoding the corresponding region of human 4-1BB is operably linked to an endogenous regulatory element at the endogenous 4-1BB locus, and one or more cells of the animal expresses a chimeric 4-1BB. In some embodiments, the animal does not express endogenous 4-1BB. In some embodiments, the locus of endogenous 4-1BB is the extracellular region of 4-1BB. In some embodiments, the animal has one or more cells expressing a chimeric 4-1BB 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 4-1BB. In some embodiments, the extracellular region of the chimeric 4-1BB has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human 4-1BB. In some embodiments, the chimeric 4-1BB has an endogenous 4-1BB transmembrane region and/or an endogenous 4-1BB cytoplasmic region. In some embodiments, the sequence encoding a region of endogenous 4-1BB comprises exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8, or a part thereof, of the endogenous 4-1BB gene. In some embodiments, the animal is a mouse, and the sequence encoding a region of endogenous 4-1BB starts within exon 2 and ends within exon 7 of the endogenous mouse 4-1BB gene. In some embodiments, the animal is heterozygous with respect to the replacement at the endogenous 4-1BB gene locus. In some embodiments, the animal is homozygous with respect to the replacement at the endogenous 4-1BB gene locus.

In one aspect, the disclosure relates to methods for making a genetically-modified, non-human animal. The methods involve replacing in at least one cell of the animal, at an endogenous 4-1BB gene locus, a sequence encoding a region of an endogenous 4-1BB with a sequence encoding a corresponding region of human 4-1BB. In some embodiments, the sequence encoding the corresponding region of human 4-1BB comprises exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and/or exon 9, or a part thereof, of a human 4-1BB gene. In some embodiments, the sequence encoding the corresponding region of human 4-1BB starts within exon 3 and ends within exon 8 of a human 4-1BB gene. In some embodiments, the sequence encoding the corresponding region of human 4-1BB encodes amino acids 1-184 of SEQ ID NO: 54. In some embodiments, the region is located within the extracellular region of 4-1BB. In some embodiments, the sequence encoding a region of an endogenous 4-1BB is exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8, or a part thereof, of the endogenous 4-1BB gene. In some embodiments, the animal is a mouse, and the sequence encoding a region of an endogenous 4-1BB starts within exon 2 and ends within exon 7 of the endogenous mouse 4-1BB gene.

In one aspect, the disclosure relates to non-human animals comprising at least one cell comprising a nucleotide sequence encoding a chimeric 4-1BB polypeptide, wherein the chimeric 4-1BB polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human 4-1BB, wherein the animal expresses the chimeric 4-1BB polypeptide. In some embodiments, the chimeric 4-1BB polypeptide has at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human 4-1BB extracellular region. In some embodiments, the chimeric 4-1BB polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 1-184 of SEQ ID NO: 54. In some embodiments, the nucleotide sequence is operably linked to an endogenous 4-1BB regulatory element of the animal. In some embodiments, the chimeric 4-1BB polypeptide comprises an endogenous 4-1BB transmembrane region and/or an endogenous 4-1BB cytoplasmic region. In some embodiments, the nucleotide sequence is integrated to an endogenous 4-1BB gene locus of the animal. In some embodiments, the chimeric 4-1BB has at least one mouse 4-1BB activity (e.g., interacting with mouse TNFSF9, and promoting immune responses in mice) and/or at least one human 4-1BB activity (e.g., interacting with human TNFSF9, and promoting immune responses in human) .

In one aspect, the disclosure relates to method of making a genetically-modified non-human animal cell that expresses a chimeric 4-1BB, the method comprising: replacing at an endogenous 4-1BB gene locus, a nucleotide sequence encoding a region of endogenous 4-1BB with a nucleotide sequence encoding a corresponding region of human 4-1BB, thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the chimeric 4-1BB, wherein the animal cell expresses the chimeric 4-1BB. In some embodiments, the animal is a mammal, e.g., a monkey, a rodent, or a mouse. In some embodiments, the chimeric 4-1BB comprises an extracellular region of human 4-1BB comprising a human signal peptide sequence; and a transmembrane and/or a cytoplasmic region of endogenous 4-1BB. In some embodiments, the chimeric 4-1BB comprises a signal peptide sequence (e.g., a mouse signal peptide sequence or a human signal peptide sequence) , an extracellular region of mouse 4-1BB, an extracellular region of human 4-1BB, a transmembrane and/or a cytoplasmic region of a mouse 4-1BB. In some embodiments, the nucleotide sequence encoding the chimeric 4-1BB is operably linked to an endogenous 4-1BB regulatory region, e.g., promoter.

In some embodiments, the animals further comprise a sequence encoding an additional human or chimeric protein. In some embodiments, the additional human or chimeric protein is TNF Superfamily Member 9 (TNFSF9) , programmed cell death protein 1 (PD-1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , Lymphocyte Activating 3 (LAG-3) , B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , CD27, CD28, CD47, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) , or TNF Receptor Superfamily Member 4 (TNFRSF4 or OX40) . In some embodiments, the additional human or chimeric protein is TNF Superfamily Member 9 (TNFSF9) , and the animal expresses the human or chimeric TNFSF9.

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. 1A is a schematic diagram showing mouse TNFSF9 gene locus.

FIG. 1B is a schematic diagram showing human TNFSF9 gene locus.

FIG. 2 is a schematic diagram showing humanized TNFSF9 gene locus.

FIG. 3 is a schematic diagram showing a TNFSF9 gene targeting strategy.

FIG. 4 shows Southern Blot results of cells after recombination. CL-01, CL-02, CL-03, and CL-04 are clone numbers. WT is a wild-type control.

FIG. 5 is a schematic diagram showing the FRT recombination process.

FIG. 6 shows PCR identification results of F1 generation mice by primers WT-F and Mut-R. F1-01, F1-02, F1-03, F1-04, F1-05, F1-06, F1-07, F1-08, F1-09, and F1-10 are mouse numbers. M is a marker. PC is a positive control. WT is a wild-type control. H ₂O is a water control.

FIG. 7 is a schematic diagram showing a TNFSF9 gene targeting strategy by the CRISPR method.

FIG. 8A shows activity testing results for 5’ end targeting sites of sgRNA1, and sgRNA3-sgRNA7. PC is positive control. Con is negative control.

FIG. 8B shows activity testing results for 3’ end targeting sites of sgRNA9-sgRNA16. PC is positive control. Con is negative control.

FIG. 9A shows PCR identification results of F0 generation mice by primers L-GT-F and L-GT-R. WT is a wild-type control. H ₂O is a water control. F0-01, F0-02, F0-03, and F0-04 are mouse numbers. WT is a wild-type control. H ₂O is a water control.

FIG. 9B shows PCR identification results of F0 generation mice by primers R-GT-F and R-GT-R. WT is a wild-type control. H ₂O is a water control. F0-01, F0-02, F0-03, and F0-04 are mouse numbers. WT is a wild-type control. H ₂O is a water control.

FIG. 10A shows PCR identification results of F1 generation mice by primers L-GT-F and L-GT-R. F1-01, F1-02, F1-03, F1-04, F1-05, F1-06, and F1-07 are mouse numbers. WT is a wild-type control. H ₂O is a water control.

FIG. 10B shows PCR identification results of F1 generation mice by primers R-GT-F and R-GT-R. F1-01, F1-02, F1-03, F1-04, F1-05, F1-06, and F1-07 are mouse numbers. WT is a wild-type control. H ₂O is a water control.

FIG. 11 shows Southern Blot analysis result of F1 generation mice by P1 or P2 probe. F1-01, F1-02, and F1-03 are mouse numbers. WT is a wild-type control.

FIG. 12 shows PCR identification results of TNFSF9 gene knockout mice. Mouse tail genomic DNA was used for PCR identification. KO-1, KO-2, and KO-3 are mouse numbers. PC is a positive control. WT is a wild-type control. H ₂O is a water control.

FIG. 13A is a schematic diagram showing mouse 4-1BB gene locus.

FIG. 13B is a schematic diagram showing human 4-1BB gene locus.

FIG. 14 is a schematic diagram showing humanized 4-1BB gene locus.

FIG. 15 shows the average body weight of humanized 4-1BB homozygous mice that were xenografted with mouse colon cancer cells (MC38) , and then treated with anti-human 4-1BB antibody Ab2 at 3 mg/kg (G2) . Saline solution was administered as a control (G1) .

FIG. 16 shows the body weight change of humanized 4-1BB homozygous mice that were xenografted with mouse colon cancer cells (MC38) , and then treated with anti-human 4-1BB antibody Ab2 at 3 mg/kg (G2) . Saline solution was administered as a control (G1) .

FIG. 17 shows the tumor volume of humanized 4-1BB homozygous mice that were xenografted with mouse colon cancer cells (MC38) , and then treated with anti-human 4-1BB antibody Ab2 at 3 mg/kg (G2) . Saline solution was administered as a control (G1) .

FIG. 18 shows the average body weight of TNFSF9/4-1BB double-gene humanized homozygous mice that were xenografted with mouse colon cancer cells (MC38) , and then treated with anti-human 4-1BB antibody Urelumab at 0.1 mg/kg (G2) , 0.3 mg/kg (G3) , or 1.0 mg/kg (G4) . Human IgG4 (hIgG4) was administered as a control antibody (G1) .

FIG. 19 shows the body weight change of TNFSF9/4-1BB double-gene humanized homozygous mice that were xenografted with mouse colon cancer cells (MC38) , and then treated with anti-human 4-1BB antibody Urelumab at 0.1 mg/kg (G2) , 0.3 mg/kg (G3) , or 1.0 mg/kg (G4) . Human IgG4 (hIgG4) was administered as a control antibody (G1) .

FIG. 20 shows the tumor volume of TNFSF9/4-1BB double-gene humanized homozygous mice that were xenografted with mouse colon cancer cells (MC38) , and then treated with anti-human 4-1BB antibody Urelumab at 0.1 mg/kg (G2) , 0.3 mg/kg (G3) , or 1.0 mg/kg (G4) . Human IgG4 (hIgG4) was administered as a control antibody (G1) .

FIG. 21A shows the serum alanine aminotransferase (ALT) detection results in 4-1BB gene humanized homozygous mice (G1-G3) or TNFSF9/4-1BB double-gene humanized homozygous mice (G4-G6) that were administered with anti-human 4-1BB antibody Urelumab. The serum was collected on the 21st day after the first administration. Human IgG4 (hIgG4) was administered as a control antibody (G1 and G4) .

FIG. 21B shows the serum aspartate aminotransferase (AST) detection results in 4-1BB gene humanized homozygous mice (G1-G3) or TNFSF9/4-1BB double-gene humanized homozygous mice (G4-G6) that were administered with anti-human 4-1BB antibody Urelumab. The serum was collected on the 21st day after the first administration. Human IgG4 (hIgG4) was administered as a control antibody (G1 and G4) .

FIG. 22A shows the H&E staining results from the liver of 4-1BB gene humanized homozygous mice (G1-G3) that were administered with anti-human 4-1BB antibody Urelumab. The mouse liver was stained on the 21st day after the first administration. Human IgG4 (hIgG4) was administered as a control antibody (G1) .

FIG. 22B shows the H&E staining results from the liver of TNFSF9/4-1BB double-gene humanized homozygous mice (G4-G6) that were administered with anti-human 4-1BB antibody Urelumab. The mouse liver was stained on the 21st day after the first administration. Human IgG4 (hIgG4) was administered as a control antibody (G4) .

FIG. 23 shows the alignment between mouse TNFSF9 amino acid sequence (NP_033430.1; SEQ ID NO: 2) and human TNFSF9 amino acid sequence (NP_003802.1; SEQ ID NO: 4) .

FIG. 24 shows the alignment between rat TNFSF9 amino acid sequence (NP_852049.1; SEQ ID NO: 58) and human TNFSF9 amino acid sequence (NP_003802.1; SEQ ID NO: 4) .

FIG. 25 shows the alignment between mouse 4-1BB amino acid sequence (NP_035742.1; SEQ ID NO: 52) and human 4-1BB amino acid sequence (NP_001552.2; SEQ ID NO: 54) .

FIG. 26 shows the alignment between rat 4-1BB amino acid sequence (NP_001020944.1; SEQ ID NO: 59) and human 4-1BB amino acid sequence (NP_001552.2; SEQ ID NO: 54) .

DETAILED DESCRIPTION

This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) TNFSF9 and/or 4-1BB, and methods of use thereof.

The tumor necrosis factor superfamily of ligands (TNFSF) and receptors (TNFRSF) provide key communication signals between various cell types during development especially in the skin, bone and lymphoid organs, and maintain organ homeostasis and initiate tissue responses. The TNF related ligands are defined by structural homology in their ectodomain, assembling into trimers that form a highly efficient receptor clustering and signal initiating mechanism. TNF receptors share a conserved ectodomain defined by a cysteine-rich signature. High affinity binding of their specific TNFSF ligands induces clustering of receptors expressed in the cognate target cell that in turn initiates signal transduction pathways culminating in cellular responses. The cytosolic signaling domain subdivides TNFRSF into those utilizing the death domain or receptors engaging the TRAF family of ubiquitin E3 ligases, or lack a cytosolic domain and function as decoy receptor. Depending upon the specific cellular circumstance the outcome of TNFR signaling may be cellular life, death or differentiation.

The tumor necrosis factor (TNF) superfamily is comprised of 27 ligands that all share the hallmark extracellular TNF homology domain (THD) . This THD triggers formation of non-covalent homotrimers. TNF ligands are typically expressed as type II transmembrane proteins, but in most ligands the extracellular domain can be subject to proteolytic processing into a soluble ligand. TNF ligands exert their biological function by binding to and activation of members of the TNF receptor (TNFR) superfamily. These TNFRs are typically expressed as trimeric type I transmembrane proteins and contain one to six cysteine-rich domains (CRDs) in their extracellular domain.

The TNF ligand superfamily has diverse functions in the immune system, one of which is the induction of apoptotic cell death in target cells. This function is performed by a family subgroup coined the Death Inducing Ligands, comprising the archetypal member TNF, FasL, and TRAIL. These Death Inducing Ligands bind to and activate cognate members of a TNFR subgroup termed the Death Receptors (DRs) . DRs are characterized by the hallmark intracellular Death Domain (DD) that transmits the apoptotic signal.

Another important function of the TNF superfamily is the provision of co-stimulatory signals at distinct stages of an immune response. Such co-stimulatory signaling is initiated upon TNFL/TNFR interaction and subsequent recruitment of members of the adaptor protein family of TNF receptor associated factor (TRAFs) . The TRAF family consists of 6 members and is characterized by a highly conserved C-terminal domain that is responsible for trimer formation and interaction with the TNF receptors. The N-terminal domain is less conserved and is responsible for downstream proinflammatory and prosurvival signal transduction. Typical signaling pathways activated by TRAFs are NFκB, PI3K, and PKB. Various co-stimulatory TNFL/TNFR pairs, including CD40L/CD40, CD70/CD27, 4-1BBL/4-1BB, and OX40L/OX40, have gained prominence as possible targets for cancer immunotherapy, in particular with the aim of induction or (re) activation of antitumor T-cell immunity.

The transmission of 4-1BB/TNFSF9 signal is bidirectional, i.e., after 4-1BB binds to its ligand TNFSF9, it transmits a co-stimulatory signal (positive signal) through the receptor 4-1BB, which leads to a series of biological effects in the cell; at the same time, 4-1BB can also transmit reverse signals through its ligand TNFSF9, and play a negative regulatory role in the activation of T cells. This feature enables the 4-1BB/TNFSF9 signaling pathway to promote the proliferation of antigen-sensitized T cells, especially CD8+ T cells, and control the development of diseases such as tumors and viral infections. In addition, the 4-1BB/TNFSF9 signaling pathway can also inhibit the damage of autoreactive CD4+ T cells to the body, thereby preventing the occurrence of autoimmune diseases.

A detailed description of the TNF superfamily and functions thereof can be found, e.g., in Ward-Kavanagh, Lindsay K., et al. "The TNF receptor superfamily in co-stimulating and co-inhibitory responses. " Immunity 44.5 (2016) : 1005-1019; Bremer, Edwin. "Targeting of the tumor necrosis factor receptor superfamily for cancer immunotherapy. " International Scholarly Research Notices 2013 (2013) ; Locksley, Richard M., et al., "The TNF and TNF receptor superfamilies: integrating mammalian biology. " Cell 104.4 (2001) : 487-501; each of which is incorporated herein by reference in its entirety. Thus, antibodies targeting the tumor necrosis factor superfamily members can be potentially used for treating cancers.

Experimental animal models are an indispensable research tool for studying the effects of these antibodies (e.g., TNFSF9 or 4-1BB 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.

Particularly, the present disclosure demonstrates that a replacement with human TNFSF9 sequence at an endogenous TNFSF9 locus under control of endogenous regulatory elements provides a physiologically appropriate expression pattern and level that results in a useful humanized animal. As shown in the present disclosure, while the human TNFSF9 sequence is quite different from the animal TNFSF9 sequence (see e.g., FIGS. 23-24) , the human TNFSF9 gene sequences are properly spliced in the animal, and the expressed human TNFSF9 is functional and can properly interact with the endogenous TNFSF9 receptor. The present disclosure also demonstrates that a replacement with human 4-1BB sequence at an endogenous 4-1BB locus under control of endogenous regulatory elements provides a physiologically appropriate expression pattern and level that results in a useful humanized animal. As shown in the present disclosure, while the human 4-1BB sequence is quite different from the animal 4-1BB sequence (see e.g., FIGS. 25-26) , the human 4-1BB gene sequences are properly spliced in the animal, and the expressed human 4-1BB is functional and can properly interact with the endogenous 4-1BB receptor. Both genetically modified animals that are heterozygous or homozygous for humanized TNFSF9 and/or 4-1BB are grossly normal and can be used to evaluate the efficacy of anti-human ILIB or anti-human 4-1BB antibodies in an cancer model.

Unless otherwise specified, the practice of the methods described herein can take advantage of the techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology.

TNFSF9

TNFSF9 (also known as 4-1BB ligand, 4-1BBL, CD137L, tumor necrosis factor ligand superfamily member 9) is a type II transmembrane protein of the TNF superfamily primarily on antigen-presenting cells, such as IFN-γ activated macrophages, CD40 ligand activated B cells, monocytes, T cells, dendritic cells (DC) , and B cells. TNFSF9 on the cell membrane can transmit a reverse signal, thereby inhibiting the proliferation of activated T cells and inducing their apoptosis. The reverse signal can also induce monocyte activation, promote the secretion of IL-6, IL-8 and TNF-Ade, and prolong cell survival. In addition, the reverse signal can stimulate the maturation of DC derived from CD34+ hematopoietic stem cells. Northern blot analysis revealed multiple TNFSF9 transcripts in brain, placenta, lung, skeletal muscle, and kidney, as well as in activated T cells, transformed B cells, and monocyte lines.

The presence of a ligand for 4-1BB was first confirmed in the EL4 cell line through its binding with a fusion 4-1BB/Fc protein, the 4-1BBL gene was then cloned through the screening of an EC1 cDNA expression library. Murine 4-1BBL consists of 309 amino acid polypeptide. Hydrophobicity analysis predicted that amino acids 83-103 were a signal hydrophobic domain, while the absence of a signal sequence suggested that 4-1BBL was a type II membrane glycoprotein with an extracellular carboxyterminal domain. The 4-1BBL gene maps to murine chromosome 17.

Human 4-1BBL was first isolated in 1994. A fusion protein consisting of the extracellular portion of human 4-1BB coupled to the Fc region of human immunoglobulin (Ig) G1 was used to identify and clone the gene for human 4-1BBL from an activated CD4+ T-cell clone using a direct expression cloning strategy. Sequence analysis revealed that human 4-1BBL consisted of 254 amino acids and shared a 39%identity with murine 4-1BBL. The cysteine residues were not conserved between mice and humans, which raised the possibility that these two genes may in fact represent two distinct ligands for 4-1BB.

The membranous form of 4-1BBL exists as a trimer, and upon engagement with its receptor on T cells, it delivers a robust costimulatory signal. 4-1BBL was found to be expressed following stimulation on professional APCs including DCs and macrophages as well as activated B cells in both human and mice. Human 4-1BBL message was detected as early as 30 minutes following stimulation through immobilized CD3 monoclonal antibody (mAb) and peaks at 1 hour. 4-1BBL was also present at high levels in the sera of some patients with hematological diseases35 as well as on some carcinoma cell lines.

A detailed description of TNFSF9 and its function can be found, e.g., in Cheuk, Adam TC, et al., "Role of 4-1BB: 4-1BB ligand in cancer immunotherapy. " Cancer Gene Therapy 11.3 (2004) : 215-226; and Li, Yan, et al., "Limited cross-linking of 4-1BB by 4-1BB ligand and the agonist monoclonal antibody Utomilumab. " Cell Reports 25.4 (2018) : 909-920; each of which is incorporated by reference in its entirety.

In human genomes, TNFSF9 gene locus has 3 exons, exon 1, exon 2, and exon 3 (FIG. 1B) . The nucleotide sequence for human TNFSF9 mRNA is NM_003811.4 (SEQ ID NO: 3) , and the amino acid sequence for human TNFSF9 is NP_003802.1 (SEQ ID NO: 4) . The location for each exon and each region in human TNFSF9 nucleotide sequence and amino acid sequence is listed below:

Table 1

The human TNFSF9 gene (Gene ID: 8744) is located in Chromosome 19 of the human genome, which is located from 6531026 to 6535924, of NC_000019.10 (GRCh38. p13 (GCF_000001405.39) ) . The 5’-UTR is from 6,531,026 to 6,531,035, exon 1 is from 6,531,026 to 6,531,303, the first intron is from 6,531,304 to 6,532,785, exon 2 is from 6,532,786 to 6,532,816, the second intron is from 6,532,817 to 6,534,599, exon 3 is from 6,534,600 to 6,535,924, and the 3’-UTR is from 6,535,067 to 6,535,924, based on transcript NM_003811.4. All relevant information for human TNFSF9 locus can be found in the NCBI website with Gene ID: 8744, which is incorporated by reference herein in its entirety.

According to the UniProt Database (UniProt ID: P41273) , the cytoplasmic region of human TNFSF9 corresponds to amino acids 1-28 of SEQ ID NO: 4, the transmembrane region of human TNFSF9 corresponds to amino acids 29-49 of SEQ ID NO: 4, and the extracellular region of human TNFSF9 corresponds to amino acids 50-254 of SEQ ID NO: 4.

In mice, TNFSF9 gene locus has 3 exons, exon 1, exon 2, and exon 3 (FIG. 1A) . The nucleotide sequence for mouse TNFSF9 mRNA is NM_009404.3 (SEQ ID NO: 1) , the amino acid sequence for mouse TNFSF9 is NP_033430.1 (SEQ ID NO: 2) . The location for each exon and each region in the mouse TNFSF9 nucleotide sequence and amino acid sequence is listed below:

Table 2

The mouse TNFSF9 gene (Gene ID: 21950) is located in Chromosome 17 of the mouse genome, which is located from 57105287 to 57107758 of NC_000083.6 (GRCm38. p6 (GCF_000001635.26) ) . The 5’-UTR is from 57,105,325 to 57,105,431, exon 1 is from 57,105,325 to 57,105,860, the first intron is from 57,105,861 to 57,106,249, exon 2 is from 57,106,250 to 57,106,286, the second intron is from 57,106,287 to 57,107,040 , exon 3 is from57,107,041 to 57,107,757, and the 3’-UTR is from 57,107,505 to 57,107,757, based on transcript NM_009404.3. All relevant information for mouse Tnfsf9 locus can be found in the NCBI website with Gene ID: 21950, which is incorporated by reference herein in its entirety.

According to the UniProt Database (UniProt ID: P41274) , the cytoplasmic region of mouse TNFSF9 corresponds to amino acids 1-82 of SEQ ID NO: 2, the transmembrane region of mouse TNFSF9 corresponds to amino acids 83-103 of SEQ ID NO: 2, and the extracellular region of mouse TNFSF9 corresponds to amino acids 104-309 of SEQ ID NO: 2.

FIG. 23 shows the alignment between mouse TNFSF9 amino acid sequence (NP_033430.1; SEQ ID NO: 2) and human TNFSF9 amino acid sequence (NP_003802.1; SEQ ID NO: 4) . Thus, the corresponding amino acid residue or region between mouse and human TNFSF9 can be found in FIG. 23.

TNFSF9 genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for TNFSF9 in Rattus norvegicus (rat) is 353218, the gene ID for TNFSF9 in Macaca mulatta (Rhesus monkey) is 700588, the gene ID for TNFSF9 in Sus scrofa (pig) is 100736831, the gene ID for TNFSF9 in Canis lupus familiaris (dog) is 476729, and the gene ID for TNFSF9 in Felis catus (domestic cat) is 101087207. 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. 24 shows the alignment between rodent TNFSF9 amino acid sequence (NP_852049.1; SEQ ID NO: 58) and human TNFSF9 amino acid sequence (NP_003802.1; SEQ ID NO: 4) . Thus, the corresponding amino acid residue or region between rodent and human TNFSF9 can be found in FIG. 24.

The present disclosure provides human or chimeric (e.g., humanized) TNFSF9 nucleotide sequence and/or amino acid sequences. In some embodiments, the entire sequence of mouse exon 1, exon 2, exon 3, cytoplasmic region, transmembrane region, and/or extracellular region are replaced by the corresponding human sequence. In some embodiments, a “region” or “portion” of mouse exon 1, exon 2, exon 3, cytoplasmic region, transmembrane region, and/or extracellular region are replaced by the corresponding human sequence. In some embodiments, a “region” or “portion” of mouse exon 1, exon 2, exon 3, cytoplasmic region, transmembrane region, and/or extracellular region are replaced by a sequence encoding a “region” or “portion” of the corresponding human sequence. The term “region” or “portion” can refer to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 500, or 600 nucleotides (contiguous or non-contiguous) , or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues (contiguous or non-contiguous) . 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, cytoplasmic region, transmembrane region, or extracellular region. In some embodiments, a region, a portion, or the entire sequence of mouse exon 1, exon 2, and/or exon 3 (e.g., a portion of exon 1, exon 2, and a portion of exon 3) are replaced by a sequence comprising the human exon 1, exon 2, and/or exon 3 (e.g., a portion of exon 1, exon 2, and a portion of exon 3) sequence.

In some embodiments, the present disclosure also provides a chimeric (e.g., humanized) TNFSF9 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 TNFSF9 mRNA sequence (e.g., SEQ ID NO: 1) , mouse TNFSF9 amino acid sequence (e.g., SEQ ID NO: 2) , or a portion thereof (e.g., a portion of exon 1, and a portion of exon 3) ; 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 TNFSF9 mRNA sequence (e.g., SEQ ID NO: 3) , human TNFSF9 amino acid sequence (e.g., SEQ ID NO: 4) , or a portion thereof (e.g., a portion of exon 1, exon 2, and a portion of exon 3) .

In some embodiments, the sequence encoding amino acids 83-309 of mouse TNFSF9 (SEQ ID NO: 2) is replaced or inactivated. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human TNFSF9 (e.g., amino acids 26-254, or amino acids 29-254 of human TNFSF9 (SEQ ID NO: 4) ) .

In some embodiments, the sequence encoding amino acids 104-309 of mouse TNFSF9 (SEQ ID NO: 2) is replaced or inactivated. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human TNFSF9 (e.g., amino acids 50-254 of human TNFSF9 (SEQ ID NO: 4) ) .

In some embodiments, the sequence encoding the entirety or a portion of the transmembrane region and/or the extracellular region of mouse TNFSF9 (SEQ ID NO: 2) is replaced or inactivated. In some embodiments, the sequence is replaced by a sequence encoding the entirety or a portion of the corresponding regions of human TNFSF9 (SEQ ID NO: 4) . In some embodiments, the corresponding regions of human TNFSF9 comprise a portion of the cytoplasmic region (e.g., at least 1, at least 2, or at least 3 amino acids in connection to the transmembrane region) , the entirety of the transmembrane region, and the entirety of the extracellular region of human TNFSF9. In some embodiments, the corresponding regions of human TNFSF9 comprise a portion of the cytoplasmic region, comprising a sequence that is at least 90%or 100%identical to amino acids 26-28 of SEQ ID NO: 24.

In some embodiments, the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse TNFSF9 promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from a portion of or the entire mouse TNFSF9 nucleotide sequence (e.g., a portion of exon 1, exon 2, and a portion of exon 3 of NM_009404.3 (SEQ ID NO: 1) ) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire mouse TNFSF9 nucleotide sequence (e.g., a portion of exon 1, and a portion of exon 3 of NM_009404.3 (SEQ ID NO: 1) ) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from a portion of or the entire human TNFSF9 nucleotide sequence (e.g., a portion of exon 1, and a portion of exon 3 of NM_003811.4 (SEQ ID NO: 3) ) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire human TNFSF9 nucleotide sequence (e.g., a portion of exon 1, exon 2, and a portion of exon 3 of NM_003811.4 (SEQ ID NO: 3) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from a portion of or the entire mouse TNFSF9 amino acid sequence (e.g., an amino acid sequence encoded by a portion of exon 1, exon 2, and a portion of exon 3 of NM_009404.3 (SEQ ID NO: 1) ; or NP_033430.1 (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 a portion of or the entire mouse TNFSF9 amino acid sequence (e.g., an amino acid sequence encoded by a portion of exon 1 of NM_009404.3 (SEQ ID NO: 1) ; or NP_033430.1 (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 different from a portion of or the entire human TNFSF9 amino acid sequence (e.g., an amino acid sequence encoded by a portion of exon 1 of NM_003811.4 (SEQ ID NO: 3) ; or NP_003802.1 (SEQ ID NO: 4) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as a portion of or the entire human TNFSF9 amino acid sequence (e.g., an amino acid sequence encoded by a portion of exon 1, exon 2, and a portion of exon 3 of NM_003811.4 (SEQ ID NO: 3) ; or NP_003802.1 (SEQ ID NO: 4) ) .

4-1BB

4-1BB (also known as CD137, or tumor necrosis factor receptor superfamily member 9 (TNFRSF9) ) is a glycosylated type I membrane protein composed of four cysteine-rich pseudo repeats (CRDs) forming the extracellular domain, a short helical transmembrane domain, and a cytoplasmic signaling domain. The extracellular domains of TNFRs range from one to four CRDs and typically form elongated structures.

4-1BB is an inducible costimulatory receptor expressed on activated T cells (e.g., CD4+or CD8+ T cells) and natural killer (NK) cells. 4-1BB is also expressed on the surface of monocytes, activated macrophages and dendritic cells. In addition, the expression of 4-1BB can also be detected on the surface of some non-immune cells, such as malignant tumor vascular endothelial cells. In the presence of TCR signal, 4-1BB costimulatory signal can cooperate with CD28 costimulatory signal to maintain the activation state of T cells and inhibit activation-induced cell death (AICD) . The CD28 costimulatory signal mainly works in the early stage of T cell activation, promoting the proliferation of T cells and maintaining their short-term survival, while the 4-1BB costimulatory signal mainly works in the later stage of T cell activation.

4-1BB ligation on T cells triggers a signaling cascade that results in upregulation of antiapoptotic molecules, cytokine secretion, and enhanced effector function. In dysfunctional T cells that have a decreased cytotoxic capacity, 4-1BB ligation demonstrates a potent ability to restore effector functions.

On NK cells, 4-1BB signaling can increase antibody-dependent cell-mediated cytotoxicity. Agonistic monoclonal antibodies targeting 4-1BB have been developed to harness 4-1BB signaling for cancer immunotherapy. On T cells, 4-1BB is transiently expressed after T-cell receptor engagement and, when 4-1BB is engaged by the natural or artificial ligand, provides CD28-independent costimulation resulting in enhanced proliferation and Th1 cytokine production. The major biological ligand, 4-1BBL, is expressed on activated professional antigen presenting cells (APCs) , including dendritic cells (DCs) and macrophages as well as B cells. Ligation of 4-1BB recruits TNFR-associated factor (TRAF) 1 and TRAF2 and induces signaling through the master transcription factor NF-κB and MAPKs. TRAF1 seems to be essential for ERK 3 and NF-κB activation downstream of 4-1BB. Interestingly, upon ligation with agonist mAbs, 4-1BB rapidly internalizes to an endosomal compartment, from which it keeps signaling through this pathway. 4-1BB signaling ultimately contributes to the secretion of interleukin 2 (IL-2) and interferon γ (IFN-γ) and upregulation of the antiapoptotic Bcl-2 family members Bcl-xL and Bfl-1, which provide strong protection against activation-induced T-cell death.

A detailed description of 4-1BB and its function can be found, e.g., in Chester, Cariad, et al., "Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. " Blood 131.1 (2018) : 49-57; Chin, S. Michael, et al. "Structure of the 4-1BB/4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab. " Nature Communications 9.1 (2018) : 1-13; Bartkowiak, Todd, et al., "4-1BB agonists: multi-potent potentiators of tumor immunity. " Frontiers in oncology 5 (2015) : 117; Wen et al., "4-1BB ligand-mediated costimulation of human T cells induces CD4 and CD8 T cell expansion, cytokine production, and the development of cytolytic effector function, " The Journal of Immunology 168.10 (2002) : 4897-4906; Broll et al., "CD137 expression in tumor vessel walls: high correlation with malignant tumors, " American Journal of Clinical Pathology 115.4 (2001) : 543-549; and Palazón et al., "Agonist anti-CD137 mAb act on tumor endothelial cells to enhance recruitment of activated T lymphocytes, " Cancer Research 71.3 (2011) : 801-811; each of which is incorporated by reference in its entirety.

In human genomes, 4-1BB gene locus has 9 exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and exon 9 (FIG. 13B) . The nucleotide sequence for human 4-1BB mRNA is NM_001561.5 (SEQ ID NO: 53) , and the amino acid sequence for human 4-1BB is NP_001552.2 (SEQ ID NO: 54) . The location for each exon and each region in human 4-1BB nucleotide sequence and amino acid sequence is listed below:

Table 3

The human 4-1BB gene (Gene ID: 3604) is located in Chromosome 1 of the human genome, which is located from 7915871 to 7941607 of NC_000001.11 (GRCh38. p13 (GCF_000001405.39) ) . The 5’-UTR is from 7940784 to 7940839and from 7939995 to 7940078, exon 1 is from 7,940,839 to7,940,784, the first intron is from 7,940,783 to 7,940,079, exon 2 is from 7,940,078 to 7,939,895, the second intron is from 7,939,894 to 7,938,829, exon 3 is from 7,938,828 to 7,938,721, the third intron is from 7,938,720 to 7,938,331, exon 4 is from 7,938,330 to 7,938,193, the forth intron is from 7,938,192 to 7,937,757, exon 5 is from7,937,756 to 7,937,690, the fifth intron is from 7,937,689 to 7,935,144, exon 6 is from 7,935,143 to 7,935,013, the sixth intron is from 7,935,012 to 7,933,297, exon 7 is from 7,933,296 to 7,933,162, the seventh intron is from 7,933,161 to 7,920,924, exon 8 is from 7,920,923 to 7,915,871, and the 3’-UTR is from 7915871 to 7920833, based on transcript NM_001561.5. All relevant information for human 4-1BB locus can be found in the NCBI website with Gene ID: 3604, which is incorporated by reference herein in its entirety.

According to the UniProt Database (UniProt ID: Q07011) , the extracellular region (excluding signal peptide region) of human 4-1BB corresponds to amino acids 24-186 of SEQ ID NO: 54, the transmembrane region of human 4-1BB corresponds to amino acids 187-213 of SEQ ID NO: 54, and the cytoplasmic region of human 4-1BB corresponds to amino acids 214-255 of SEQ ID NO: 54.

In mice, 4-1BB gene locus has 8 exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 (FIG. 13A) . The nucleotide sequence for mouse 4-1BB mRNA is NM_011612.2 (SEQ ID NO: 51) , the amino acid sequence for mouse 4-1BB is NP_035742.1 (SEQ ID NO: 52) . The location for each exon and each region in the mouse 4-1BB nucleotide sequence and amino acid sequence is listed below:

Table 4

The mouse 4-1BB gene (Gene ID: 21942) is located in Chromosome 4 of the mouse genome, which is located from 150920155 to 150946104, of NC_000070.6 (GRCm38. p4 (GCF_000001635.24) ) . The 5’-UTR is from 150,920,190 to 150,920,260 and 150,929,821 to 150,929,845, exon 1 is from 150,920,190 to 150,920,260, the first intron is from 150,920,261 to 150,929,820, exon 2 is from 150,929,821 to 150,929,945, the second intron is from 150,929,946 to 150,930,728, exon 3 is from 150,930,729 to 150,930,833, the third intron is from 150,930,834 to 150,932,307, exon 4 is from 150,932,308 to 150,932,445, the fourth intron is from 150,932,446 to 150,933,032, exon 5 is from 150,933,033 to 150,933,102, the fifth intron is from 150,933,103 to 150,934,286, exon 6 is from 150,934,287 to 150,934,411, the sixth intron is from 150,934,412 to 150,935,421, exon 7 is from the 150,935,422 to 150,935,556, the seventh intron is from 150,935,557 to 150,944,772, exon 8 is from the 150,944, 773 to 150,946,102, and 3’-UTR is from 150,944,871 to 150,946,102, based on transcript NM_011612.2. All relevant information for mouse 4-1BB locus can be found in the NCBI website with Gene ID: 21942, which is incorporated by reference herein in its entirety.

According to the UniProt Database (UniProt ID: P20334) , the extracellular region (excluding signal peptide region) of mouse 4-1BB corresponds to amino acids 24-187 of SEQ ID NO: 52, the transmembrane region of mouse 4-1BB corresponds to amino acids 188-208 of SEQ ID NO: 52, and the cytoplasmic region of mouse 4-1BB corresponds to amino acids 209-256 of SEQ ID NO: 52.

FIG. 25 shows the alignment between mouse 4-1BB amino acid sequence (NP_035742.1; SEQ ID NO: 52) and human 4-1BB amino acid sequence (NP_001552.2; SEQ ID NO: 54) . Thus, the corresponding amino acid residue or region between mouse and human 4-1BB can be found in FIG. 25.

4-1BB genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for 4-1BB in Rattus norvegicus (rat) is 500590, the gene ID for 4-1BB in Equus caballus (horse) is 100058657, the gene ID for 4-1BB in Sus scrofa (pig) is 100519368, the gene ID for 4-1BB in Macaca fascicularis (crab-eating macaque) is 102127961, and the gene ID for 4-1BB in Cavia porcellus (domestic guinea pig) is 100730923. 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. 26 shows the alignment between rodent 4-1BB amino acid sequence (NP_001020944.1; SEQ ID NO: 59) and human 4-1BB amino acid sequence (NP_001552.2; SEQ ID NO: 54) . Thus, the corresponding amino acid residue or region between rodent and human 4-1BB can be found in FIG. 26.

The present disclosure provides human or chimeric (e.g., humanized) 4-1BB 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, exon 8, 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, exon 8, 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, exon 8, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region are replaced by a sequence encoding a “region” or “portion” of the corresponding human sequence. The term “region” or “portion” can refer to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 500, or 600 nucleotides (contiguous or non-contiguous) , or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues (contiguous or non-contiguous) . 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, exon 8, exon 9, 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, exon 7, and/or exon 8 (e.g., a portion of exon 2, exons 3-6, and a portion of exon 7) are replaced by a sequence comprising the human exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and/or exon 9 (e.g., a portion of exon 3, exons 4-7, and a portion of exon 8) sequence.

In some embodiments, the present disclosure also provides a chimeric (e.g., humanized) 4-1BB 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 4-1BB mRNA sequence (e.g., SEQ ID NO: 51) , mouse 4-1BB amino acid sequence (e.g., SEQ ID NO: 52) , or a portion thereof (e.g., exon 1, a portion of exon 2, a portion of exon 7, and exon 8) ; 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 4-1BB mRNA sequence (e.g., SEQ ID NO: 53) , human 4-1BB amino acid sequence (e.g., SEQ ID NO: 54) , or a portion thereof (e.g., a portion of exon 3, exons 4-7, and a portion of exon 8) .

In some embodiments, the sequence encoding amino acids 1-183 of mouse 4-1BB (SEQ ID NO: 52) is replaced or inactivated. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human 4-1BB (e.g., amino acids 1-184 of human 4-1BB (SEQ ID NO: 54) ) .

In some embodiments, the sequence encoding amino acids 24-183 of mouse 4-1BB (SEQ ID NO: 52) is replaced or inactivated. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human 4-1BB (e.g., amino acids 24-184 of human 4-1BB (SEQ ID NO: 54) ) .

In some embodiments, the sequence encoding amino acids 1-187 or amino acids 24-187 of mouse 4-1BB (SEQ ID NO: 52) is replaced or inactivated. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human 4-1BB (e.g., amino acids 1-186 or amino acids 24-186 of human 4-1BB (SEQ ID NO: 54) ) .

In some embodiments, the sequence encoding the entirety or a portion of the extracellular region (with or without the signal peptide) of mouse 4-1BB (SEQ ID NO: 52) is replaced or inactivated. In some embodiments, the sequence is replaced by a sequence encoding the entirety or a portion of the corresponding region of human 4-1BB (SEQ ID NO: 54) . In some embodiments, the corresponding region of human 4-1BB comprises the entirety or a portion of the extracellular region (with or without the signal peptide) of human 4-1BB. In some embodiments, the corresponding region of human 4-1BB comprise a portion of the cytoplasmic region comprising a sequence that is at least 90%or 100%identical to amino acids 182-184 of SEQ ID NO: 54.

In some embodiments, the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse 4-1BB promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from a portion of or the entire mouse 4-1BB nucleotide sequence (e.g., a portion of exon 2, exons 3-6, and a portion of exon 7 of NM_011612.2 (SEQ ID NO: 51) ) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire mouse 4-1BB nucleotide sequence (e.g., exon 1, a portion of exon 2, a portion of exon 7, and exon 8 of NM_011612.2 (SEQ ID NO: 51) ) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from a portion of or the entire human 4-1BB nucleotide sequence (e.g., exons 1-2, a portion of exon 3, a portion of exon 8, and exon 9 of NM_001561.5 (SEQ ID NO: 53) ) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire human 4-1BB nucleotide sequence (e.g., a portion of exon 3, exons 4-7, and a portion of exon 8 of NM_001561.5 (SEQ ID NO: 53) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from a portion of or the entire mouse 4-1BB amino acid sequence (e.g., an amino acid sequence encoded by a portion of exon 2, exons 3-6, and a portion of exon 7 of NM_011612.2 (SEQ ID NO: 51) ; or NP_035742.1 (SEQ ID NO: 52) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as a portion of or the entire mouse 4-1BB amino acid sequence (e.g., an amino acid sequence encoded by a portion of exon 7, and exon 8 of NM_011612.2 (SEQ ID NO: 51) ; or NP_035742.1 (SEQ ID NO: 52) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from a portion of or the entire human 4-1BB amino acid sequence (e.g., an amino acid sequence encoded by a portion of exon 8, and exon 9 of NM_001561.5 (SEQ ID NO: 53) ; or NP_001552.2 (SEQ ID NO: 54) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as a portion of or the entire human 4-1BB amino acid sequence (e.g., amino acid sequence encoded by a portion of exon 3, exons 4-7, and a portion of exon 8 of NM_001561.5 (SEQ ID NO: 53) ; or NP_001552.2 (SEQ ID NO: 54) ) .

The present disclosure also provides a human or humanized TNFSF9 amino acid sequence, or a human or humanized 4-1BB amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

a) an amino acid sequence shown in SEQ ID NO: 2, 4, 12, 52, 54, or 56;

b) an amino acid sequence having a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 2, 4, 12, 52, 54, or 56;

c) an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is able to hybridize to a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 2, 4, 12, 52, 54, or 56, under a low stringency condition or a strict stringency condition;

d)an amino acid sequence having a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 2, 4, 12, 52, 54, or 56;

e) an amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 2, 4, 12, 52, 54, or 56, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and

f) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 2, 4, 12, 52, 54, or 56.

The present disclosure also relates to a TNFSF9 nucleic acid (e.g., DNA or RNA) sequence, or a 4-1BB 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: 1, 3, 5-11, 13, 50, 51, 53, 55, or 57; a nucleic acid sequence encoding a homologous TNFSF9 amino acid sequence of a humanized mouse TNFSF9; or a nucleic acid sequence encoding a homologous 4-1BB amino acid sequence of a humanized mouse 4-1BB;

b) a nucleic acid sequence that is shown in SEQ ID NO: 1, 3, 5-11, 13, 50, 51, 53, 55, or 57;

c) a nucleic acid sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5-11, 13, 50, 51, 53, 55, or 57 under a low stringency condition or a strict stringency condition;

d) a nucleic acid sequence that has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5-11, 13, 50, 51, 53, 55, or 57;

e) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 2, 4, 12, 52, 54, or 56;

f) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%with, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 2, 4, 12, 52, 54, or 56;

g) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence is different from the amino acid sequence shown in SEQ ID NO: 2, 4, 12, 52, 54, or 56 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and

h) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence comprises a substitution, a deletion and /or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 2, 4, 12, 52, 54, or 56.

The present disclosure also relates to a TNFSF9 protein sequence, wherein the amino acid sequence of the TNFSF9 protein can be selected from the group consisting of:

a) all or part of the amino acid sequence shown in amino acids 26-254 of SEQ ID NO: 4;

b) an amino acid sequence having a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in amino acids 26-254 of SEQ ID NO: 4;

c) an amino acid sequence that is different from the amino acid sequence shown in amino acids 26-254 of SEQ ID NO: 4, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and

d) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to the amino acid sequence shown in amino acids 26-254 of SEQ ID NO: 4.

The present disclosure also relates to a 4-1BB protein sequence, wherein the amino acid sequence of the TNFSF9 protein can be selected from the group consisting of:

a) all or part of the amino acid sequence shown in amino acids 1-184 of SEQ ID NO: 54;

b) an amino acid sequence having a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in amino acids 1-184 of SEQ ID NO: 54;

c) an amino acid sequence that is different from the amino acid sequence shown in amino acids 1-184 of SEQ ID NO: 54, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and

d) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to the amino acid sequence shown in amino acids 1-184 of SEQ ID NO: 54.

The present disclosure also relates to a humanized TNFSF9 gene sequence, wherein the transcribed mRNA sequence of the humanized TNFSF9 gene can be selected from the group consisting of:

a) all or part of the nucleotide sequence shown in SEQ ID NO: 11;

b) a nucleotide sequence that at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or at least 99%identical to the nucleotide sequence shown in SEQ ID NO: 11;

c) a nucleotide sequence that is different from the nucleotide sequence shown in SEQ ID NO: 11 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; and

d) a nucleotide sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to the nucleotide sequence shown at SEQ ID NO: 11.

The present disclosure also relates to a humanized 4-1BB gene sequence, wherein the transcribed mRNA sequence of the humanized TNFSF9 gene can be selected from the group consisting of:

a) all or part of the nucleotide sequence shown in SEQ ID NO: 55 or 57;

b) a nucleotide sequence that at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or at least 99%identical to the nucleotide sequence shown in SEQ ID NO: 55 or 57;

c) a nucleotide sequence that is different from the nucleotide sequence shown in SEQ ID NO: 55 or 57 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; and

d) a nucleotide sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to the nucleotide sequence shown at SEQ ID NO: 55 or 57.

The present disclosure further relates to an TNFSF9 genomic DNA sequence of a humanized mouse TNFSF9, or an 4-1BB genomic DNA sequence of a humanized mouse 4-1BB. The DNA sequence is obtained by a reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence homologous to the sequence shown in SEQ ID NO: 7.

The disclosure also provides an amino acid sequence that has a homology of at least 90%with, or at least 90%identical to the sequence shown in SEQ ID NO: 2, 4, 12, 52, 54, or 56, and has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 2, 4, 12, 52, 54, or 56, is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing homology is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 2, 4, 12, 52, 54, or 56, 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: 1, 3, or 11, and encodes a polypeptide that has TNFSF9 protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 1, 3, 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 50%, 55%, 60%, 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: 51, 53, 55, or 57, and encodes a polypeptide that has 4-1BB protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 51, 53, 55, or 57 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: 1, 3, 5-11, 13, 50, 51, 53, 55, or 57 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

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

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

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

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

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

Cells, tissues, and animals (e.g., mouse) are also provided that comprise the nucleotide sequences as described herein, as well as cells, tissues, and animals (e.g., mouse) that express human or chimeric (e.g., humanized) TNFSF9 and/or 4-1BB from an endogenous non-human TNFSF9 locus and/or an endogenous non-human 4-1BB 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 TNFSF9 and/or 4-1BB 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 TNFSF9 gene or a humanized TNFSF9 nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human TNFSF9 gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes a human or humanized TNFSF9 protein. The encoded TNFSF9 protein is functional or has at least one activity of the human TNFSF9 protein and/or the non-human TNFSF9 protein, e.g., interacting with human or non-human 4-1BB; inhibiting proliferation of activated T cells; inducing apoptosis; inducing monocyte activation; promoting secretion of IL-6, IL-8, and/or TNF-Ade; stimulating maturation of DC derived from CD34+ hematopoietic stem cells; and/or upregulating the immune response.

In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized 4-1BB gene or a humanized 4-1BB nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human 4-1BB gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes a human or humanized 4-1BB protein. The encoded 4-1BB protein is functional or has at least one activity of the human 4-1BB protein and/or the non-human 4-1BB protein, e.g., interacting with human or non-human TNFSF9; regulating T cell immunity; promoting T cell activation and/or proliferation; recruiting TNFR-associated factor (TRAF) 1 and TRAF2; maintaining the activation state of T cells; inhibiting activation-induced cell death (AICD) ; inducing interleukin 2 (IL-2) and/or interferon γ(IFN-γ) secretion; augmenting T cell cytotoxicity; and/or upregulating the immune response.

In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized TNFSF9 protein or a humanized TNFSF9 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 TNFSF9 protein. The human TNFSF9 protein or the humanized TNFSF9 protein is functional or has at least one activity of the human TNFSF9 protein or the non-human TNFSF9 protein.

In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized 4-1BB protein or a humanized 4-1BB 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 4-1BB protein. The human 4-1BB protein or the humanized 4-1BB protein is functional or has at least one activity of the human 4-1BB protein or the non-human 4-1BB protein.

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

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

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

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

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

The animal can have one or more other genetic modifications, and/or other modifications, that are suitable for the particular purpose for which the humanized TNFSF9 and/or 4-1BB animal is made. For example, suitable mice for maintaining a xenograft (e.g., a human cancer or tumor) , can have one or more modifications that compromise, inactivate, or destroy the immune system of the non-human animal in whole or in part. Compromise, inactivation, or destruction of the immune system of the non-human animal can include, for example, destruction of hematopoietic cells and/or immune cells by chemical means (e.g., administering a toxin) , physical means (e.g., irradiating the animal) , and/or genetic modification (e.g., knocking out one or more genes) . Non-limiting examples of such mice include, e.g., NOD-Prkdcscid IL-2rγ ^null NOD mice, NOD-Rag 1-/--IL2rg-/- (NRG) mice, Rag 2-/--IL2rg-/- (RG) mice, SCID mice, NOD/SCID mice, IL2Rγ knockout mice, NOD/SCID/γc ^null 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 TNFSF9 and/or 4-1BB locus, and further comprises a modification that compromises, inactivates, or destroys the immune system (or one or more cell types of the immune system) of the non-human animal in whole or in part. In some embodiments, modification is, e.g., selected from the group consisting of a modification that results in NOD-Prkdcscid IL-2rγ ^null NOD mice, NOD-Rag 1-/--IL2rg-/- (NRG) mice, Rag 2-/--IL2rg-/- (RG) mice, NOD mice, SCID mice, NOD/SCID mice, IL-2Rγ knockout mice, NOD/SCID/γc null mice, nude mice, Rag1 and/or Rag2 knockout mice, and a combination thereof. These genetically modified animals are described, e.g., in US20150106961, which is incorporated herein by reference in its entirety.

In some embodiments, the genetically modified non-human animal comprises a modification of an endogenous non-human TNFSF9 locus. In some embodiments, the modification can comprise a human nucleic acid sequence encoding at least a portion of a mature TNFSF9 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to the mature TNFSF9 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 TNFSF9 locus in the germline of the animal. In some embodiments, the genetically modified non-human animal comprises a modification of an endogenous non-human 4-1BB locus. In some embodiments, the modification can comprise a human nucleic acid sequence encoding at least a portion of a mature 4-1BB protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to the mature 4-1BB 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 4-1BB locus in the germline of the animal.

In some embodiments, the genetically modified mice express a human TNFSF9 and/or a chimeric (e.g., humanized) TNFSF9 from endogenous mouse loci, wherein the endogenous mouse TNFSF9 gene has been replaced with a human TNFSF9 gene and/or a nucleotide sequence that encodes a region of human TNFSF9 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 TNFSF9 sequence. In various embodiments, an endogenous non-human TNFSF9 locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a mature TNFSF9 protein. In some embodiments, the genetically modified mice express a human 4-1BB and/or a chimeric (e.g., humanized) 4-1BB from endogenous mouse loci, wherein the endogenous mouse 4-1BB gene has been replaced with a human 4-1BB gene and/or a nucleotide sequence that encodes a region of human 4-1BB 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 4-1BB sequence. In various embodiments, an endogenous non-human 4-1BB locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a mature 4-1BB protein.

In some embodiments, the genetically modified mice express the human TNFSF9 and/or chimeric TNFSF9 (e.g., humanized TNFSF9) 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 TNFSF9 or chimeric TNFSF9 (e.g., humanized TNFSF9) 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 TNFSF9 or the chimeric TNFSF9 (e.g., humanized TNFSF9) expressed in animal can maintain one or more functions of the wild-type mouse or human TNFSF9 in the animal. For example, human or non-human 4-1BB can bind to the expressed TNFSF9, and stimulate immune response. Furthermore, in some embodiments, the animal does not express endogenous TNFSF9. As used herein, the term “endogenous TNFSF9” refers to TNFSF9 protein that is expressed from an endogenous TNFSF9 nucleotide sequence of the non-human animal (e.g., mouse) before any genetic modification.

In some embodiments, the genetically modified mice express the human 4-1BB and/or chimeric 4-1BB (e.g., humanized 4-1BB) 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 4-1BB or chimeric 4-1BB (e.g., humanized 4-1BB) 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 4-1BB or the chimeric 4-1BB (e.g., humanized 4-1BB) expressed in animal can maintain one or more functions of the wild-type mouse or human 4-1BB in the animal. For example, human or non-human TNFSF9 can bind to the expressed 4-1BB, and stimulate immune response. Furthermore, in some embodiments, the animal does not express endogenous 4-1BB. As used herein, the term “endogenous 4-1BB” refers to 4-1BB protein that is expressed from an endogenous 4-1BB 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 TNFSF9 (e.g., NP_003802.1 (SEQ ID NO: 4) ) . 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: 12.

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 4-1BB (e.g., NP_001552.2 (SEQ ID NO: 54) ) . 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: 56.

The genome of the genetically modified animal can comprise a replacement at an endogenous TNFSF9 gene locus of a sequence encoding a region of endogenous TNFSF9 with a sequence encoding a corresponding region of human TNFSF9. In some embodiments, the sequence that is replaced is any sequence within the endogenous TNFSF9 gene locus, e.g., exon 1, exon 2, exon 3, 5’-UTR, 3’-UTR, the first intron, and the second intron, etc. In some embodiments, the sequence that is replaced is within the regulatory region of the endogenous TNFSF9 gene. In some embodiments, the sequence that is replaced is exon 1, exon 2, exon 3, or a part thereof, of an endogenous mouse TNFSF9 gene locus. In some embodiments, the sequence that is replaced is starts within exon 1 and ends within exon 3 of an endogenous mouse TNFSF9 gene locus. In some embodiments, the sequence that is replaced is from exon 1 to exon 3 of an endogenous mouse TNFSF9 gene locus.

The genome of the genetically modified animal can comprise a replacement at an endogenous 4-1BB gene locus of a sequence encoding a region of endogenous 4-1BB with a sequence encoding a corresponding region of human 4-1BB. In some embodiments, the sequence that is replaced is any sequence within the endogenous 4-1BB gene locus, 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, the seventh intron, etc. In some embodiments, the sequence that is replaced is within the regulatory region of the endogenous 4-1BB gene. In some embodiments, the sequence that is replaced is exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or a part thereof, of an endogenous mouse 4-1BB gene locus. In some embodiments, the sequence that is replaced starts within exon 2 and ends within exon 7 of an endogenous mouse 4-1BB gene locus. In some embodiments, the sequence that is replaced is from exon 2 to exon 7 of an endogenous mouse 4-1BB gene locus.

The genetically modified animal can have one or more cells expressing a human or chimeric TNFSF9 (e.g., humanized TNFSF9) having a cytoplasmic region, a transmembrane region, and/or an extracellular 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 TNFSF9. In some embodiments, the extracellular region of the humanized TNFSF9 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, or 200 amino acids (e.g., contiguously or non-contiguously) that are identical to human TNFSF9. In some embodiments, the transmembrane region of the humanized TNFSF9 comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the transmembrane region of human TNFSF9. In some embodiments, the transmembrane region of the humanized TNFSF9 has a sequence that has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids (e.g., contiguously or non-contiguously) that are identical to human TNFSF9. In some embodiments, the cytoplasmic region of the humanized TNFSF9 comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to amino acids 1-82 of SEQ ID NO: 2.

Because human TNFSF9 and non-human TNFSF9 (e.g., mouse TNFSF9) sequences, in many cases, are different, antibodies that bind to human TNFSF9 will not necessarily have the same binding affinity with non-human TNFSF9 or have the same effects to non-human TNFSF9. Therefore, the genetically modified animal having a human or a humanized extracellular region can be used to better evaluate the effects of anti-human TNFSF9 antibodies in an animal model. In some embodiments, the genome of the genetically modified animal comprises a sequence encoding an amino acid sequence that corresponds to part or the entire sequence of exon 1, exon 2, and/or exon 3 of human TNFSF9; part or the entire sequence of extracellular region of human TNFSF9; or part or the entire sequence of amino acids 26-254, or 50-254 of SEQ ID NO: 4.

In some embodiments, the non-human animal can have, at an endogenous TNFSF9 gene locus, a nucleotide sequence encoding a chimeric human/non-human TNFSF9 polypeptide, wherein a human portion of the chimeric human/non-human TNFSF9 polypeptide comprises the entirety or a portion of human TNFSF9 extracellular domain, and wherein the animal expresses a functional TNFSF9 on a surface of a cell (e.g., APC cell) of the animal. The human portion of the chimeric human/non-human TNFSF9 polypeptide can comprise the entirety or a portion of exon 1, exon 2, and/or exon 3 of human TNFSF9. In some embodiments, the human portion of the chimeric human/non-human TNFSF9 polypeptide can comprise a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 26-254, or 50-254 of SEQ ID NO: 4.

In some embodiments, the non-human portion of the chimeric human/non-human TNFSF9 polypeptide comprises a cytoplasmic region of an endogenous non-human TNFSF9 polypeptide. There may be several advantages that are associated with the cytoplasmic region of an endogenous non-human TNFSF9 polypeptide. For example, once 4-1BB or an anti-TNFSF9 antibody binds to TNFSF9, they can properly transmit extracellular signals into the cells and initiate the downstream pathway. In some embodiments, a few cytoplasmic amino acids that are close to the transmembrane region of TNFSF9 are also derived from human sequence. These amino acids can also be important for transmembrane signal transmission.

The genetically modified animal can have one or more cells expressing a human or chimeric 4-1BB (e.g., humanized 4-1BB) having an extracellular region, a transmembrane region, and/or 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 4-1BB. In some embodiments, the extracellular region of the humanized 4-1BB has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 amino acids (e.g., contiguously or non-contiguously) that are identical to human 4-1BB. In some embodiments, the extracellular region of the humanized 4-1BB comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to amino acids 1-184 of SEQ ID NO: 54. In some embodiments, the humanized TNFSF9 comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to amino acids 184-256 of SEQ ID NO: 52.

Because human 4-1BB and non-human 4-1BB (e.g., mouse 4-1BB) sequences, in many cases, are different, antibodies that bind to human 4-1BB will not necessarily have the same binding affinity with non-human 4-1BB or have the same effects to non-human 4-1BB. Therefore, the genetically modified animal having a human or a humanized extracellular region can be used to better evaluate the effects of anti-human 4-1BB antibodies in an animal model. In some embodiments, the genome of the genetically modified animal comprises a sequence encoding an amino acid sequence that corresponds to part or the entire sequence of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and/or exon 9 of human 4-1BB; part or the entire sequence of extracellular region of human 4-1BB (with or without signal peptide) ; or part or the entire sequence of amino acids 1-184, 24-184, 1-186, or 24-186 of SEQ ID NO: 54.

In some embodiments, the non-human animal can have, at an endogenous 4-1BB gene locus, a nucleotide sequence encoding a chimeric human/non-human 4-1BB polypeptide, wherein a human portion of the chimeric human/non-human 4-1BB polypeptide comprises the entirety or a portion of human 4-1BB extracellular domain, and wherein the animal expresses a functional 4-1BB on a surface of a cell (e.g., activated T cell) of the animal. The human portion of the chimeric human/non-human 4-1BB polypeptide can comprise the entirety or a portion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and/or exon 9 of human 4-1BB. In some embodiments, the human portion of the chimeric human/non-human 4-1BB polypeptide can comprise a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 1-184, 24-184, 1-186, or 24-186 of SEQ ID NO: 54.

In some embodiments, the non-human portion of the chimeric human/non-human 4-1BB polypeptide comprises transmembrane and/or cytoplasmic regions of an endogenous non-human 4-1BB polypeptide. There may be several advantages that are associated with the transmembrane and/or cytoplasmic region of an endogenous non-human 4-1BB polypeptide. For example, once 4-1BB or an anti-4-1BB antibody binds to 4-1BB, they can properly transmit extracellular signals into the cells and initiate the downstream pathway. In some embodiments, a few extracellular amino acids that are close to the transmembrane region of 4-1BB are also derived from human sequence. These amino acids can also be important for transmembrane signal transmission.

In some embodiments, the genetically modified animal does not express endogenous TNFSF9. In some embodiments, the genetically modified animal expresses a decreased level of endogenous TNFSF9 as compared to a wild-type animal. In some embodiments, the genetically modified animal does not express endogenous 4-1BB. In some embodiments, the genetically modified animal expresses a decreased level of endogenous 4-1BB as compared to a wild-type animal.

Furthermore, the genetically modified animal can be heterozygous with respect to the replacement at the endogenous TNFSF9 locus, or homozygous with respect to the replacement at the endogenous TNFSF9 locus. Furthermore, the genetically modified animal can be heterozygous with respect to the replacement at the endogenous 4-1BB locus, or homozygous with respect to the replacement at the endogenous 4-1BB locus.

In some embodiments, the humanized TNFSF9 locus lacks a human TNFSF9 5’-UTR. In some embodiment, the humanized TNFSF9 locus comprises a rodent (e.g., mouse) 5’-UTR. In some embodiments, the humanization comprises a human 3’-UTR. In some embodiments, the humanization comprises a mouse 3’-UTR. In appropriate cases, it may be reasonable to presume that the mouse and human TNFSF9 genes appear to be similarly regulated based on the similarity of their 5’-flanking sequence. As shown in the present disclosure, humanized TNFSF9 mice that comprise a replacement at an endogenous mouse TNFSF9 locus, which retain mouse regulatory elements but comprise a humanization of TNFSF9 encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized TNFSF9 are grossly normal.

In some embodiments, the humanized 4-1BB locus lacks a human 4-1BB 5’-UTR. In some embodiment, the humanized 4-1BB locus comprises a rodent (e.g., mouse) 5’-UTR. In some embodiments, the humanization comprises a human 3’-UTR. In some embodiments, the humanization comprises a mouse 3’-UTR. In appropriate cases, it may be reasonable to presume that the mouse and human 4-1BB genes appear to be similarly regulated based on the similarity of their 5’-flanking sequence. As shown in the present disclosure, humanized 4-1BB mice that comprise a replacement at an endogenous mouse 4-1BB locus, which retain mouse regulatory elements but comprise a humanization of 4-1BB encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized 4-1BB 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 TNFSF9 gene. In some embodiments, the non-human mammal expresses a protein encoded by a humanized 4-1BB 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 TNFSF9 in the genome of the 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 4-1BB in the genome of the mammal.

In some embodiments, the non-human mammal comprises the genetic construct as described herein (e.g., gene construct as shown in FIG. 2 or FIG. 14) . In some embodiments, a non-human mammal expressing human or humanized TNFSF9 is provided. In some embodiments, a non-human mammal expressing human or humanized 4-1BB is provided. In some embodiments, the tissue-specific expression of human or humanized TNFSF9 protein is provided. In some embodiments, the tissue-specific expression of human or humanized 4-1BB protein is provided.

In some embodiments, the expression of human or humanized TNFSF9 in a genetically modified animal is controllable, as by the addition of a specific inducer or repressor substance. In some embodiments, the expression of human or humanized 4-1BB in a genetically modified animal is controllable, as by the addition of a specific inducer or repressor substance. In some embodiments, the specific inducer is selected from Tet-Off System/Tet-On System, or Tamoxifen System.

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

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

The present disclosure also provides a cell line or primary cell culture derived from the non-human mammal or a progeny thereof. A model based on cell culture can be prepared, for example, by the following methods. Cell cultures can be obtained by way of isolation from a non-human mammal, alternatively cell can be obtained from the cell culture established using the same constructs and the standard cell transfection techniques. The integration of genetic constructs containing DNA sequences encoding human TNFSF9 and/or 4-1BB 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 TNFSF9 and/or 4-1BB protein.

Vectors

In one aspect, 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 TNFSF9 gene genomic DNAs in the length of 100 to 12,000 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 TNFSF9 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_000083.6; 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_000083.6.

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 57095327 to the position 57105677 of the NCBI accession number NC_000083.6; 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 57108062 to the position 57112881 of the NCBI accession number NC_000083.6.

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 57104184 to the position 57105677 of the NCBI accession number NC_000083.6; 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 57107505 to the position 57108979 of the NCBI accession number NC_000083.6.

In some embodiments, the length of the selected genomic nucleotide sequence in the targeting vector can be more than about 1 kb, about 1.5 kb, about 2 kb, about 2.5 kb, 3 kb, about 3.5 kb, about 4 kb, about 4.5 kb, about 5 kb, about 5.5 kb, or about 6 kb.

In some embodiments, the region to be altered is exon 1, exon 2, and/or exon 3 of TNFSF9 gene (e.g., a portion of exon 1, exon 2, and a portion of exon 3 of mouse TNFSF9 gene) .

The targeting vector can further include a selected gene marker.

In some embodiments, the sequence of the 5’ arm is shown in SEQ ID NO: 5; and the sequence of the 3’ arm is shown in SEQ ID NO: 6. In some embodiments, the sequence of the 5’ arm is shown in SEQ ID NO: 50; and the sequence of the 3’ arm is shown in SEQ ID NO: 13.

In some embodiments, the desired/donor DNA sequence is derived from human (e.g., 6531112-6535066 of NC_000019.10) . For example, the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human TNFSF9, preferably comprising exon 1, exon 2, and/or exon 3, or a part thereof, of the human TNFSF9. In some embodiments, the nucleotide sequence of the humanized TNFSF9 gene encodes the entire or the part of human TNFSF9 protein with the NCBI accession number NP_003802.1 (SEQ ID NO: 4) . In some embodiments, the desired/donor DNA sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 7.

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., non-homologous 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 TNFSF9 gene locus, a sequence encoding a region of an endogenous TNFSF9 with a sequence encoding a corresponding region of human or chimeric TNFSF9. In some embodiments, the disclosure provides replacing in at least one cell of the animal, at an endogenous 4-1BB gene locus, a sequence encoding a region of an endogenous 4-1BB with a sequence encoding a corresponding region of human or chimeric 4-1BB. 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. 7 show a humanization strategy for a mouse TNFSF9 locus. Both of the targeting strategies involve a vector comprising the 5’ end homologous arm, human TNFSF9 gene fragment, 3’ homologous arm. The process can involve replacing endogenous TNFSF9 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 TNFSF9 sequence with human TNFSF9 sequence.

Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of replacing at an endogenous TNFSF9 locus (or site) , a nucleic acid encoding a sequence encoding a region of endogenous TNFSF9 with a sequence encoding a corresponding region of human TNFSF9. The sequence can include a region (e.g., a part or the entire region) of exon 1, exon 2, and/or exon 3 of an endogenous TNFSF9 gene. In some embodiments, the sequence includes a region of exon 1, exon 2, and a region of exon 3 of a human TNFSF9 gene (e.g., a sequence encoding amino acids 26-254 of SEQ ID NO: 4) . In some embodiments, the endogenous TNFSF9 locus is exon 1, exon 2, and/or exon 3 of mouse TNFSF9 gene (e.g., a sequence encoding amino acids 83-309 of SEQ ID NO: 2) .

In some embodiments, the methods of modifying a TNFSF9 locus of a mouse to express a chimeric human/mouse TNFSF9 peptide can include the steps of replacing at the endogenous mouse TNFSF9 locus a nucleotide sequence encoding a mouse TNFSF9 with a nucleotide sequence encoding a human TNFSF9, thereby generating a sequence encoding a chimeric human/mouse TNFSF9.

In some embodiments, provided herein is a genetically-modified non-human animal whose genome comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 8, 9, or 10.

FIG. 14 shows a humanized mouse 4-1BB gene locus. The targeting strategy involves a vector comprising the 5’ end homologous arm, human 4-1BB gene fragment, 3’ homologous arm. The process can involve replacing endogenous 4-1BB 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 4-1BB sequence with human 4-1BB sequence.

Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of replacing at an endogenous 4-1BB locus (or site) , a nucleic acid encoding a sequence encoding a region of endogenous 4-1BB with a sequence encoding a corresponding region of human 4-1BB. 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 an endogenous 4-1BB gene. In some embodiments, the sequence includes a region of exon 3, exon 4, exon 5, exon 6, exon 7, and a region of exon 8 of a human 4-1BB gene (e.g., a sequence encoding amino acids 1-184 of SEQ ID NO: 54) . In some embodiments, the endogenous 4-1BB locus is exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of mouse 4-1BB gene (e.g., a sequence encoding amino acids 1-183 of SEQ ID NO: 52) .

In some embodiments, the methods of modifying a 4-1BB locus of a mouse to express a chimeric human/mouse 4-1BB peptide can include the steps of replacing at the endogenous mouse 4-1BB locus a nucleotide sequence encoding a mouse 4-1BB with a nucleotide sequence encoding a human 4-1BB, thereby generating a sequence encoding a chimeric human/mouse 4-1BB.

In some embodiments, the nucleotide sequences as described herein do not overlap with each other (e.g., the 5’ homologous arm, the “A fragment” , and/or the 3’ homologous arm 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 TNFSF9 and/or 4-1BB gene humanized animal model, involving the following steps:

(a) providing the cell (e.g. an embryonic stem cell) based on the methods described herein;

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

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

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

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

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

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

Embryonic stem cells can come from any non-human animal, e.g., any non-human animal as described herein. In some embodiments, the embryonic stem cells are derived from rodents. The genetic construct can be introduced into an embryonic stem cell by microinjection of DNA. For example, by way of culturing an embryonic stem cell after microinjection, a cultured embryonic stem cell 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.

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 TNFSF9 gene, and the sgRNA is unique on the target sequence of the gene to be altered. In some embodiments, the sgRNA meets the sequence arrangement rule of 5’-NNN (20) -NGG3’ or 5’-CCN-N (20) -3’ . In some embodiments, the targeting site of the sgRNA in the mouse TNFSF9 gene is located on the exon 1, exon 2, exon 3, intron 1, intron 2, upstream of exon 1, or downstream of exon 3 of the mouse TNFSF9 gene. In some embodiments, the 5’ targeting site is located on exon 1 of the mouse TNFSF9. In some embodiments, the 3’ targeting site is located on exon 3 of the mouse TNFSF9 gene.

In some embodiments, the 5’ targeting site sequences of the sgRNA are shown as SEQ ID NOs: 14-21, and the sgRNA recognizes the 5’ targeting site. In some embodiments, the 3’ targeting sequences for the sgRNA are shown as SEQ ID NOs: 22-29 and the sgRNA recognizes the 3’ targeting site. In some embodiments, the 5’ targeting sequence is SEQ ID NO: 14 and the 3’ targeting sequence is SEQ ID NO: 23. 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: 30-37.

In some embodiments, the disclosure provides DNA sequences encoding the sgRNAs. 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.

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.

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 TNFSF9 and/or 4-1BB 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 TNFSF9, which are useful for testing agents that can decrease or block the interaction between TNFSF9 and TNFSF9 receptors (e.g., 4-1BB) or the interaction between TNFSF9 and anti-human TNFSF9 antibodies, testing whether an agent can increase or decrease the immune response, and/or determining whether an agent is an TNFSF9 agonist or antagonist. In various aspects, genetically modified animals are provided that express human or humanized 4-1BB, which are useful for testing agents that can decrease or block the interaction between 4-1BB and 4-1BB ligands (e.g., TNFSF9) or the interaction between 4-1BB and anti-human 4-1BB antibodies, testing whether an agent can increase or decrease the immune response, and/or determining whether an agent is an 4-1BB agonist or antagonist. The genetically modified animals can be, e.g., an animal model of a human disease, e.g., the disease is induced genetically (a knock-in or knockout) . In various embodiments, the genetically modified non-human animals further comprise an impaired immune system, e.g., a non-human animal genetically modified to sustain or maintain a human xenograft, e.g., a human solid tumor or a blood cell tumor (e.g., a lymphocyte tumor, e.g., a B or T cell tumor) .

In one aspect, the disclosure also provides methods of determining effectiveness of an TNFSF9 antagonist (e.g., an anti-TNFSF9 antibody) for reducing inflammation. The methods involve administering the TNFSF9 antagonist to the animal described herein, wherein the animal has an inflammation; and determining effects of the TNFSF9 antagonist for reducing the inflammation. In one aspect, the disclosure also provides methods of determining effectiveness of an 4-1BB antagonist (e.g., an anti-4-1BB antibody) for reducing inflammation. The methods involve administering the 4-1BB antagonist to the animal described herein, wherein the animal has an inflammation; and determining effects of the 4-1BB antagonist for reducing the inflammation.

In one aspect, the disclosure also provides methods of determining effectiveness of an TNFSF9 antagonist (e.g., an anti-TNFSF9 antibody) for treating an immune disorder (e.g., an autoimmune disorder or allergic disorder) . The methods involve administering the TNFSF9 antagonist to the animal described herein, wherein the animal has an immune disorder; and determining effects of the TNFSF9 antagonist for treating the immune disorder. In one aspect, the disclosure also provides methods of determining effectiveness of an 4-1BB antagonist (e.g., an anti-4-1BB antibody) for treating an immune disorder (e.g., an autoimmune disorder or allergic disorder) . The methods involve administering the 4-1BB antagonist to the animal described herein, wherein the animal has an immune disorder; and determining effects of the 4-1BB antagonist for treating the immune disorder.

In some embodiments, the genetically modified animals can be used for determining effectiveness of an anti-TNFSF9 antibody for treating cancer. The methods involve administering the anti-TNFSF9 antibody (e.g., anti-human TNFSF9 antibody) to the animal as described herein, wherein the animal has a tumor; and determining the inhibitory effects of the anti-TNFSF9 antibody to the tumor. In some embodiments, the genetically modified animals can be used for determining effectiveness of an anti-4-1BB antibody for treating cancer. The methods involve administering the anti-4-1BB antibody (e.g., anti-human 4-1BB antibody) to the animal as described herein, wherein the animal has a tumor; and determining the inhibitory effects of the anti-4-1BB antibody to the tumor. The inhibitory effects that can be determined include, e.g., a decrease of tumor size or tumor volume, a decrease of tumor growth, a reduction of the increase rate of tumor volume in a subject (e.g., as compared to the rate of increase in tumor volume in the same subject prior to treatment or in another subject without such treatment) , a decrease in the risk of developing a metastasis or the risk of developing one or more additional metastasis, an increase of survival rate, and an increase of life expectancy, etc. The tumor volume in a subject can be determined by various methods, e.g., as determined by direct measurement, MRI or CT.

In some embodiments, the tumor comprises one or more cancer cells (e.g., human or mouse cancer cells) that are injected into the animal. In some embodiments, the anti-TNFSF9 antibody prevents 4-1BB from binding to TNFSF9. In some embodiments, the anti-TNFSF9 antibody does not prevent 4-1BB from binding to TNFSF9. In some embodiments, the anti-4-1BB antibody prevents TNFSF9 from binding to 4-1BB. In some embodiments, the anti-4-1BB antibody does not prevent TNFSF9 from binding to 4-1BB.

In some embodiments, the genetically modified animals can be used for determining whether an anti-TNFSF9 antibody is a TNFSF9 agonist or antagonist. In some embodiments, the genetically modified animals can be used for determining whether an anti-4-1BB antibody is a 4-1BB agonist or antagonist. 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., an immune disorder, an allergy, 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-TNFSF9 antibody or the anti-4-1BB 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 anti-TNFSF9 antibody or the anti-4-1BB antibody is designed for treating breast cancer, non-small-cell lung cancer (NSCLC) , colorectal cancer, gastric cancer, hepatocellular carcinoma (HCC) , hepatobiliary cancer, pancreatic cancer, lung cancer, prostate cancer, kidney cancer, ovarian cancer, uterine cancer, endometrial cancer, cervical cancer, head and neck cancer, brain cancer, glioma, gingivitis and salivary cancer, skin cancer, squamous cell carcinoma, blood cancer, lymphoma, urothelial cancer, oral cancer, or bone cancer. In some embodiments, the anti-TNFSF9 or anti-4-1BB antibody is designed for treating solid tumor. In some embodiments, the anti-TNFSF9 or anti-4-1BB antibody is designed for treating metastatic solid tumors. In some embodiments, the anti-TNFSF9 or anti-4-1BB antibody is designed for reducing tumor growth, metastasis, and/or angiogenesis. In some embodiments, the anti-TNFSF9 or anti-4-1BB antibody is designed for treating hematopoietic malignancies.

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

In some embodiments, the antibody is designed for treating various autoimmune diseases, immune disorder, immune-related diseases, or allergy (e.g., asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, primary thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, self-immune liver disease, diabetes, pain, or neurological disorders) . The autoimmune diseases also include psoriasis, allergic rhinitis, sinusitis, asthma, rheumatoid arthritis, atopic dermatitis, chronic obstructive pulmonary disease (COPD) , chronic bronchitis, emphysema, eczema, osteoarthritis, rheumatoid arthritis, systemic lupus erythematosus, polymyalgia rheumatica, autoimmune hemolytic anemia, systemic vasculitis, pernicious anemia, inflammatory bowel disease, ulcerative colitis, Crohn's disease, or multiple sclerosis. Thus, the methods as described herein can be used to determine the effectiveness of an antibody in inhibiting immune response.

In some embodiments, the antibody is designed for reducing inflammation (e.g., inflammatory bowel disease, chronic inflammation, asthmatic inflammation, periodontitis, or wound healing) . Thus, the methods as described herein can be used to determine the effectiveness of an antibody for reducing inflammation. In some embodiments, the inflammation described herein is an acute inflammation or chronic inflammation. In some embodiments, the inflammation described herein includes, but not limited to, degenerative inflammation, exudative inflammation (e.g., serous inflammation, fibrinitis, suppurative inflammation, hemorrhagic inflammation, necrotitis, or catarrhal inflammation) , proliferative inflammation, or specific inflammation (e.g., tuberculosis, Syphilis, leprosy, or lymphogranuloma) .

The present disclosure also provides methods of determining toxicity of an antibody (e.g., an anti-TNFSF9 or anti-4-1BB 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 is administered at a dose level of about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg.

In some embodiments, one or more biochemical parameters of the animal are determined. In some embodiments, the biochemical parameters include hepatic biochemical parameters or serum biochemical parameters. Details can be found, e.g., in Ramaiah, Shashi K. "A toxicologist guide to the diagnostic interpretation of hepatic biochemical parameters. " Food and Chemical Toxicology 45.9 (2007) : 1551-1557, which is incorporated herein by reference in its entirety.

In some embodiments, the biochemical parameters are the serum levels of alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) . In some embodiments, the serum ALT level of the animal is less than or about 80 U/L, less than or about 70 U/L, less than or about 60 U/L, less than or about 50 U/L, less than or about 40 U/L, less than or about 30 U/L. In some embodiments, the serum AST level of the animal is less than or about 130 U/L, less than or about 120 U/L, less than or about 110 U/L, less than or about 100 U/L, less than or about 90 U/L, less than or about 80 U/L, less than or about 70 U/L, less than or about 60 U/L, less than or about 50 U/L. In some embodiments, the serum ALT or AST level of the animal treated by the antibody described herein (e.g., an anti-TNFSF9 or anti-4-1BB antibody) is less than 150%, less than 140%, less than 130%, less than 120%, or less than 110%as compared to that of an animal treated by a control antibody (e.g., an antibody with the same immunoglobulin isotype) .

In some embodiments, the organ damage of the animal is determined, e.g., damage of liver, kidney, brain, heart, spleen, lung, or skin. Some biomarkers of organ failure can be found, e.g., in Dao, Long, et al., "The Organ Trail: A Review of Biomarkers of Organ Failure. " Frontiers in Oncology 10 (2020) , which is incorporated herein by reference in its entirety.

In some embodiments, liver damage is evaluated for liver lesions, e.g., hepatic perivascular cell infiltration and/or chronic inflammation. In some embodiments, the liver lesions are quantitatively determined by measuring the percentage of the lesion site area over total area of liver. In some embodiments, the percentage of liver lesions of the animal treated by the antibody described herein (e.g., an anti-TNFSF9 or anti-4-1BB antibody) is less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%of the total area of liver. In some embodiments, the liver lesions of the animal treated by the antibody described herein (e.g., an anti-TNFSF9 or anti-4-1BB antibody) are evaluated as moderate, mild, slight, or NVL according to Table 16.

In some embodiments, the antibody described herein (e.g., an anti-TNFSF9 or anti-4-1BB antibody) is not considered as toxic when administered at a dose level less than 20 mg/kg, 15 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, or 1 mg/kg.

In some embodiments, the animal described herein is a TNFSF9/4-1BB double-gene humanized mouse. In some embodiments, the animal described herein is a 4-1BB gene humanized mouse. In some embodiments, the animal described herein is a TNFSF9 gene humanized mouse. In some embodiments, the TNFSF9/4-1BB double-gene humanized mouse is more sensitive to hepatotoxicity (e.g., induced by administration of any of the antibodies described herein) than the 4-1BB or TNFSF9 single-gene humanized mouse.

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 TNFSF9 gene function, human TNFSF9 antibodies, drugs for human TNFSF9 targeting sites, the drugs or efficacies for human TNFSF9 targeting sites, the drugs for immune-related diseases and antitumor drugs. 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 4-1BB gene function, human 4-1BB antibodies, drugs for human 4-1BB targeting sites, the drugs or efficacies for human 4-1BB 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 TNFSF9 and/or 4-1BB gene humanized non-human animal prepared by the methods described herein, the TNFSF9 and/or 4-1BB 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 TNFSF9 and/or 4-1BB 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 TNFSF9-associated or 4-1BB-associated diseases described herein. In some embodiments, the TCR-T, CAR-T, and/or other immunotherapies provides an evaluation method for treating the TNFSF9-associated or 4-1BB-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 TNFSF9 and/or 4-1BB gene and a sequence encoding an additional human or chimeric protein.

In some embodiments, the additional human or chimeric protein can be TNF Receptor Superfamily Member 9 (4-1BB) , TNF Superfamily Member 9 (TNFSF9) , programmed cell death protein 1 (PD-1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , Lymphocyte Activating 3 (LAG-3) , IL15 receptor, B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , CD3, CD27, CD28, CD47, CD154, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , Signal regulatory protein α (SIRPα) or TNF Receptor Superfamily Member 4 (OX40) .

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 TNFSF9 gene (or human 4-1BB gene) or chimeric TNFSF9 gene (or chimeric 4-1BB 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 4-1BB, TNFSF9, PD-1, CTLA-4, LAG-3, IL15 receptor, BTLA, PD-L1, CD3, CD27, CD28, CD47, 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 TNFSF9 and/or 4-1BB humanization is directly performed on a genetically modified animal having a human or chimeric PD-1, CTLA-4, LAG-3, IL15 receptor, BTLA, PD-L1, CD3, CD27, CD28, CD47, 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-TNFSF9 or anti-4-1BB antibody and an additional therapeutic agent for the treatment of cancer or an immune disorder. The methods include administering the anti-TNFSF9 or anti-4-1BB 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, CTLA-4, LAG-3, IL15 receptor, BTLA, PD-L1, CD3, CD27, CD28, CD47, 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, and/or an anti-CTLA-4 antibody. In some embodiments, the tumor comprises one or more tumor cells that express TNFSF9, 4-1BB, PD-1, and/or PD-L1.

In some embodiments, the combination treatment is designed for treating various cancer as described herein, e.g., breast cancer, non-small-cell lung cancer (NSCLC) , colorectal cancer, gastric cancer, hepatocellular carcinoma (HCC) , hepatobiliary cancer, pancreatic cancer, lung cancer, prostate cancer, kidney cancer, ovarian cancer, uterine cancer, endometrial cancer, cervical cancer, head and neck cancer, brain cancer, glioma, gingivitis and salivary cancer, skin cancer, squamous cell carcinoma, blood cancer, lymphoma, urothelial cancer, oral cancer, or bone cancer.

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

EXAMPLES

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

Materials and Methods

The following materials were used in the following examples.

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

UCA kit was obtained from Beijing Biocytogen Co., Ltd. (Catalog number: BCG-DX-001) .

Ambion in vitro transcription kit MEGAshortscript ^TM Kit was purchased from Thermo Fisher Scientific (Catalog number: AM1354) .

Cas9 mRNA was purchased from SIGMA (Catalog number: CAS9MRNA-1EA) .

StuI, EcoRI, BamHI, and NcoI restriction enzymes were purchased from NEB (Catalog numbers: R0187M, R0101M, R0136M, and R0193M, respectively) .

EXAMPLE 1: Mice with humanized TNFSF9 gene

In this example, a non-human animal (e.g., a mouse) was modified to include a nucleotide sequence encoding human TNFSF9 protein, and the obtained genetically-modified non-human animal can express a human or humanized TNFSF9 protein in vivo. The mouse TNFSF9 gene (NCBI Gene ID: 21950, Primary source: MGI: 1101058, UniProt ID: P41274) is located at 57105287 to 57107758 of chromosome 17 (NC_000083.6) , and the human TNFSF9 gene (NCBI Gene ID: 8744, Primary source: HGNC: 11939, UniProt ID: P41273) is located at 6531026 to 6535924 of chromosome 19 (NC_000019.10) . The mouse TNFSF9 transcript sequence NM_009404.3 is set forth in SEQ ID NO: 1, and the corresponding protein sequence NP_033430.1 is set forth in SEQ ID NO: 2. The human TNFSF9 transcript sequence NM_003811.4 is set forth in SEQ ID NO: 3, and the corresponding protein sequence NP_003802.1 is set forth in SEQ ID NO: 4. Mouse and human TNFSF9 gene loci are shown in FIG. 1A and FIG. 1B, respectively.

A nucleotide sequence encoding human TNFSF9 protein can be introduced into the endogenous mouse TNFSF9 locus, such that the mouse can express a human or humanized TNFSF9 protein. Specifically, mouse cells can be modified by various gene-editing techniques, for example, replacement of specific mouse TNFSF9 gene sequences with human TNFSF9 gene sequences at the endogenous mouse TNFSF9 locus. For example, a 1827 bp sequence of the mouse TNFSF9 gene starting within exon 1 and ending within exon 3 was replaced with the corresponding human DNA sequence to obtain a humanized TNFSF9 gene locus as shown in FIG. 2, thereby humanizing mouse TNFSF9 gene.

As shown in the schematic diagram of the targeting strategy in FIG. 3, the targeting vector has homologous arm sequences upstream and downstream of mouse TNFSF9 gene locus, and an “A fragment” comprising a nucleotide sequence encoding a portion of human TNFSF9 protein. Specifically, the upstream homologous arm sequence (5’ homologous arm, SEQ ID NO: 5)is identical to nucleotide sequence of 57095327-57105677 of NCBI accession number NC_000083.6, and the downstream homologous arm sequence (3’ homologous arm, SEQ ID NO: 6) is identical to nucleotide sequence of 57108062-57112881 of NCBI accession number NC_000083.6. The “A fragment” contains a 3955 bp genomic DNA sequence (SEQ ID NO: 7) starting from within exon 1 and ending within exon 3 of the human TNFSF9 gene, which is identical to nucleotide sequence of the 6531112-6535066 of the NCBI accession number NC_000019.10. The connection between the downstream of the human TNFSF9 gene sequence and the mouse TNFSF9 gene (within the A fragment) was designed as: 5’-CGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGG AATAA

ATCCTTCTTGTG ACTCCTAGTTGCTAAGTCCTCAA -3’ (SEQ ID NO: 8) , wherein the last “A” of sequence “ AATAA” is the last nucleotide of human sequence, and the “G” of sequence

is the first nucleotide of mouse sequence.

The targeting vector also included 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’-ATTCCCCAGGGGAGTGCCTATACCAGATCTTAAAA TAATT

CGAATTCCGAAGTT CCTATTCTCTAGAAAGTATAG-3’ (SEQ ID NO: 9) , wherein the last “T” of sequence “ TAATT” is the last nucleotide of mouse sequence, and the “G” of sequence

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

AAGTGGCTCTGTA CCACTATCCCTTTTTTGAGACA-3’ (SEQ ID NO: 10) , wherein the last “C” of sequence “ GATCC” is the last nucleotide of the Neo cassette, and the first “G” of sequence

is the first nucleotide of mouse sequence. In addition, a coding gene with a negative selectable marker (a gene encoding diphtheria toxin A subunit (DTA) ) was also inserted downstream of the 3' homologous arm of the targeting vector. The modified humanized mouse TNFSF9 mRNA sequence is shown as SEQ ID NO: 11, and the expressed protein has an amino acid sequence shown in SEQ ID NO: 12.

The targeting vector was constructed, e.g., by restriction enzyme digestion and ligation. The constructed targeting vector sequence was preliminarily verified by restriction enzyme digestion, followed by verification by sequencing. The correct targeting vector was electroporated and transfected into embryonic stem cells of C57BL/6 mice. The positive selectable marker gene was used to screen the cells, and the integration of exogenous genes was confirmed by PCR and Southern Blot, thereby screening the correct positive clone cells.

Specifically, PCR primers CL-F and CL-R were used for PCR identification of the screened positive clones. The positive clones identified by PCR were further confirmed by Southern Blot (digested with StuI restriction enzyme and then hybridized with a probe) to screen out correct positive clone cells. The length of the probes and the size of target fragments are shown in the table below, and the results are shown in FIG. 4. Four clones with numbers CL-01, CL-02, CL-03, and CL-04 were confirmed as positive clones. The positive clones were further verified by sequencing, and no random insertions were detected.

Table 5. Probe and target fragment size

Restriction enzyme	Probe	Wild-type fragment size	Recombinant sequence fragment size
StuI	P1	-	13.8 kb

The following primers were used in PCR:

CL-F (SEQ ID NO: 39) : 5’-AGCTCAGTAGGTTCCATGCCCAGTA-3’

CL-R (SEQ ID NO: 40) : 5’-GAAGGTGCTGGGAGGAGTGTCTTG-3’

The following primers were used to synthesize the P1 probe in Southern Blot assays:

P1-F (SEQ ID NO: 43) : 5’-CCGACCCTCGGTAGCTGGTCTC-3’

P1-R (SEQ ID NO: 44) : 5’-CTCCCGTGCAAGACGGAGAAGGAG-3’

The positive clones that had been screened (black mice) were introduced into isolated blastocysts (white mice) , and the resulted chimeric blastocysts were transferred to a culture medium for short-term culture and then transplanted to the fallopian tubes of the recipient mother (white mice) to produce the F0 chimeric mice (black and white) . The F2 generation homozygous mice were obtained by backcrossing the F0 generation chimeric mice with wild-type mice to obtain the F1 generation mice, and then breeding the F1 generation heterozygous mice with each other. The positive mice were also bred with the Flp mice to remove the positive selectable marker gene (the process diagram is shown in FIG. 5) , and then the humanized TNFSF9 homozygous mice were obtained by breeding the heterozygous mice with each other. The genotype of the progeny mice can be identified by PCR using primers shown in the table below. The identification results of exemplary F1 generation mice (Neo cassette-removed) are shown in FIG. 6, and a total of 10 mice, with numbers from F1-01 to F1-10, were identified as positive heterozygous clones.

Table 6. F1 generation genotype identification primer sequences and target fragment length

In addition, CRISPR/Cas gene editing technology was also used to obtain the TNFSF9 gene humanized mice. For example, to generate the TNFSF9 gene humanized mice with the humanized mouse TNFSF9 gene locus shown in FIG. 2, a targeting strategy was designed as shown in FIG. 7. The targeting vector has an upstream homologous arm sequence (5’ homologous arm; SEQ ID NO: 50) , a downstream homologous arm sequence (3’ homologous arm; SEQ ID NO: 13) , and a fragment of human TNFSF9 gene sequence in between. Specifically, the 5’ homologous arm is identical to nucleotide sequence of 57104184-57105677 of NCBI accession number NC_000083.6, and the 3’ homologous arm is identical to nucleotide sequence of 57107505-57108979 of NCBI accession number NC_000083.6. The fragment of human TNFSF9 gene sequence is identical to SEQ ID NO: 7. The targeting vector was constructed, e.g., by restriction enzyme digestion and ligation, or synthesized directly. The constructed targeting vector sequence was preliminarily verified by restriction enzyme digestion, then verified by sequencing. The correct targeting vector verified by sequencing was used for subsequent experiments.

Specific sgRNA sequences were designed and synthesized that recognize the 5’ end targeting sites (sgRNA1-sgRNA8) and 3’ end targeting sites (sgRNA9-sgRNA16) . The 5'end targeting sites are located on exon 1 of the mouse TNFSF9 gene; and the 3'end targeting site are located on exon 3 of the mouse TNFSF9 gene. The targeting site sequence of each sgRNA on the TNFSF9 gene locus is as follows:

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

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

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

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

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

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

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

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

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

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

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

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

sgRNA13 targeting site (SEQ ID NO: 26) : 5’-GGCTGGCCACCGCCTCAGTGTGG-3’

sgRNA14 targeting site (SEQ ID NO: 27) : 5’-CTCCATGGAGAACAAGTTAGTGG-3’

sgRNA15 targeting site (SEQ ID NO: 28) : 5’-GGTCTGAGGGCTTATCTGCATGG-3’

sgRNA16 targeting site (SEQ ID NO: 29) : 5’-CCCAGGATGCATACAGAGACTGG-3’

Table 7. UCA detection results

The UCA kit was used to detect the activities of each sgRNA. As shown in FIGS. 8A-8B and the above table, the results showed that the sgRNAs had different activities. In particular, sgRNA9, sgRNA14, and sgRNA16 exhibited relatively low activities, which may be caused by sequence variations of their targeting sites. However, the relative activities of sgRNA9, sgRNA14, and sgRNA16 were still significantly higher than that of the negative control (Con) . It is therefore concluded that sgRNA9, sgRNA14 and sgRNA16 can suffice the requirement for gene editing experiment. sgRNA1 and sgRNA10 were randomly selected for subsequent experiments. Oligonucleotides were added to the 5’ end and a complementary strand to obtain a forward oligonucleotide and a reverse oligonucleotide (See the table below for the sequences) . After annealing, the products were ligated to the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) , respectively, to obtain expression vectors PT7-sgRNA1 and pT7-sgRNA10.

Table 8.

The pT7-sgRNA vector was synthesized, which included a DNA fragment containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 38) , 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-sgRNA1, pT7-sgRNA10 plasmids (using Ambion in vitro transcription kit to carry out the transcription according to the method provided in the product instruction) were injected into the cytoplasm or nucleus of mouse fertilized eggs with a microinjection instrument. The embryo microinjection was carried out according to the method described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2006. The injected fertilized eggs were then transferred to a culture medium to culture for a short time and then was transplanted into the oviduct of the recipient mouse to produce the genetically modified mice (F0 generation) . The mouse population was further expanded by cross-breeding and self-breeding to establish stable homozygous mouse lines with genetically-modified TNFSF9 gene locus.

The genotype of somatic cells of F0 generation mice can be identified, e.g., by PCR analysis. The identification results of some F0 generation mice are shown in FIGS. 9A-9B. In view of the 5'end primer detection result and the 3'end primer detection result using primers shown in the table below, the four mice with numbers from F0-01 to F0-04 in FIGS. 9A-9B were identified as positive mice. The four positive clone mice were further confirmed by sequencing. The following primers were used in the PCR:

Table 9. F0 generation PCR detection primer sequence and target fragment length

The positive F0 generation TNFSF9 gene humanized mice generated using the targeting strategy shown in FIG. 3 were bred with wild-type mice to generate F1 generation mice. The same method (e.g., PCR) was used for genotype identification of the F1 generation mice. As shown in FIGS. 10A-10B, the 7 mice numbered from F1-01 to F1-07 were all identified as positive mice.

The F1 generation mice were further analyzed by Southern Blot (See the table below for the length of specific probes and target fragments) , to confirm whether random insertions were introduced. Specifically, mouse tail genomic DNA was extracted, digested with NcoI or StuI restriction enzyme, transferred to a membrane, and then hybridized with probes. Probes P1 and P2 are located on the human sequence and downstream of the 3’ homologous arm, respectively.

Table 10. The length of the specific probes and target fragments

Restriction Enzyme	Probe	Wild-type fragment size	Recombinant sequence fragment size
StuI	P1	-	13.8 kb
NcoI	P2	9.3 kb	11.3 kb

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

P1-F (SEQ ID NO: 43) : 5’-CCGACCCTCGGTAGCTGGTCTC-3’

P1-R (SEQ ID NO: 44) : 5’-CTCCCGTGCAAGACGGAGAAGGAG-3’

P2-F (SEQ ID NO: 45) : 5’-TGAGCTGTTGGGAGACCTTGACTTA-3’

P2-R (SEQ ID NO: 46) : 5’-GGAGTTGACTCAGTGGTCAGCACTTA-3’

The detection result of Southern Blot is shown in FIG. 11. In view of the hybridization results by P1 and P2 probes, no random insertions were detected in the three F1 generation mice numbered F1-01, F1-02, and F1-03. The results indicate that this method can be used to generate genetically-modified TNFSF9 gene humanized mice that can be passaged stably without random insertions.

The expression of humanized TNFSF9 protein in TNFSF9 gene humanized mice can be confirmed, e.g., by flow cytometry.

In addition, due to the double-strand break of genomic DNA caused by Cas9 cleavage, insertion/deletion mutations can be randomly generated through chromosome homologous recombination repair, which may result in knockout mice with loss of TNFSF9 protein function. A pair of primers shown in the table below were designed to detect gene-knockout mice. After PCR amplification, wild-type knockout mice should produce a PCR band with a length of about 420 bp. As shown in FIG. 12, three mice numbered KO-1, KO-2, and KO-3 were identified as TNFSF9 gene knockout mice. The primers are located on the upstream of the 5’ end targeting site and downstream of the 3’ end targeting site, with sequences shown as follows:

Table 11. KO mouse PCR detection primer sequences and target fragment length

EXAMPLE 2: Mice with humanized 4-1BB gene

In this example, a non-human animal (e.g., a mouse) was modified to include a nucleotide sequence encoding human 4-1BB protein, and the obtained genetically-modified non-human animal can express a human or humanized 4-1BB protein in vivo. The mouse 4-1BB gene (NCBI Gene ID: 21942, Primary source: MGI: 1101059, UniProt ID: P20334) is located at 150920155 to 150946104 of chromosome 4 (NC_000070.6) , and the human 4-1BB gene (NCBI Gene ID: 3604, Primary source: HGNC: 11924, UniProt ID: Q07011) is located at 7915871 to 7941607 of chromosome 1 (NC_000001.11) . The mouse 4-1BB transcript sequence NM_011612.2 is set forth in SEQ ID NO: 51, and the corresponding protein sequence NP_035742.1 is set forth in SEQ ID NO: 52. The human 4-1BB transcript sequence NM_001561.5 is set forth in SEQ ID NO: 53, and the corresponding protein sequence NP_001552.2 is set forth in SEQ ID NO: 54. Mouse and human 4-1BB gene loci are shown in FIG. 13A and FIG. 13B, respectively.

A nucleotide sequence encoding human 4-1BB protein can be introduced into the endogenous mouse 4-1BB locus, such that the mouse can express a human or humanized 4-1BB protein. Specifically, mouse cells can be modified by various gene-editing techniques, for example, replacement of specific mouse 4-1BB gene sequences with human 4-1BB gene sequences at the endogenous mouse 4-1BB locus. For example, a 6706 bp sequence of the mouse 4-1BB gene starting within exon 2 and ending within exon 7 was replaced with a sequence of the human 4-1BB gene starting within exon 3 and ending within exon 8, to obtain the F1 generation heterozygous mice with humanized 4-1BB gene. The F1 generation heterozygous mice were bred with each other and humanized homozygous mice with humanized 4-1BB gene were obtained after multiple generations of screening. The schematic diagram of the genetically-modified humanized mouse 4-1BB gene locus is shown in FIG. 14. The humanized mouse 4-1BB mRNA sequence is shown as SEQ ID NO: 55, and the expressed protein has an amino acid sequence shown in SEQ ID NO: 56. In particular, the humanized mouse 4-1BB mRNA sequence comprises a sequence shown in SEQ ID NO: 57, which is identical to nucleic acids 262-813 of human 4-1BB mRNA NM_001561.5 (SEQ ID NO: 53) . The 4-1BB gene humanized mice were generated using similar methods as described in Example 1. Details can be found, e.g., in U.S. Application Publication No. 20190343095A1, which is incorporated herein by reference in its entirety.

In one experiment, the 4-1BB gene humanized homozygous mice (4-8 week old) were subcutaneously injected with mouse colon cancer cell MC38 (5 × 10 ⁵ cells in 100 μl P Phosphate-buffered saline (PBS) ) . After the tumor grew to a volume of about 100 mm ³, the mice were placed into a control group and a treatment group (6 mice in each group) according to the tumor volume. An anti-human 4-1BB antibody Ab2 (obtained by immunizing mice; See Janeway’s Immunobiology. Garland Science, 2016 (9th edition) ) was randomly selected to inject mice (at a dose level of 3 mg/kg) in the treatment group. The control group mice were injected with an equal volume of saline solution. The frequency of administration was once every three days (6 times of administrations in total) . The tumor volume was measured twice a week and body weight of the mice was recorded as well. Euthanasia was performed when tumor volume of a mouse reached 3000 mm ³.

Overall, the mice in each group were grossly healthy. At the end of the experimental period (25 days post grouping) , body weight of all the treatment and control group mice increased. The body weight and body weight change of the mice during the entire experimental period are shown in FIG. 15 and FIG. 16, respectively. According to the results of tumor volume measurements (FIG. 17) , the tumors of the control group mice continued growing during the experimental period, while the tumor volume growth in the treatment group mice was significantly reduced as compared to that of the control group mice.

The table below lists the results for this experiment, including the tumor volumes on Day 0 (at the time of grouping) , Day 18 (18 days post grouping) , and Day 25 (25 days post grouping, or the last day of the experiment) ; the survival status of the mice; the number of tumor-free mice; the Tumor Growth Inhibition value (TGI _TV%) ; and the statistical difference (P value) of mouse body weight and tumor volume between the treatment group and control group.

Table 12.

As shown in the table above and FIG. 15, at the end of the experiment (Day 25) , the body weight of each group increased, indicating that the animals tolerated the anti-human 4-1BB antibody Ab2 well. According to the measurement results of tumor volume, the tumor volume of the control group mice continued growing during the experiment. However, among the 6 mice in the treatment group, 3 mice (50 %) were tumor-free at the end of the experiment. At the end of the experiment, the average tumor volume of the control group mice (G1) was 3501 ± 458 mm ³, and the average tumor volume of the treatment group mice (G2) was 84 ± 76 mm ³. The results showed that the tumor volume of the mice in the treatment group was significantly smaller than that of the control group. In addition, the TGI _TV%was 102.7%for the treatment group mice, indicating that the anti-human 4-1BB antibody Ab2 has a significant inhibitory effect on tumor growth (TGI _TV%> 60%) .

The above research results indicate that humanized 4-1BB mice can be used as a living animal model for in vivo pharmacodynamics research; screening, evaluation and treatment of human 4-1BB signaling pathway modulators; evaluation of the in vivo effectiveness and therapeutic effect of 4-1BB-targeting drugs.

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

The humanized TNFSF9 mouse prepared by the methods described herein can also be used to prepare a double-or multi-gene humanized mouse model. For example, in Example 1, the embryonic stem cells used for blastocyst microinjection can be selected from mice containing 4-1BB, PD-1, PD-L1, OX40, or other genetic modifications. Alternatively, the embryonic stem cells of TNFSF9 gene humanized mice can be selected for gene editing, to obtain a double-gene or multi-gene humanized mouse model comprising humanized TNFSF9 and/or 4-1BB and other genetic modifications. In addition, it is also possible to breed the homozygous or heterozygous TNFSF9 transgenic mice obtained by the methods described herein with other genetically modified homozygous or heterozygous mice, and the offspring can be screened. According to Mendel’s law, it is possible to generate double-gene or multi-gene modified heterozygous mice comprising humanized TNFSF9 and/or 4-1BB gene and other genetic modifications. Then the heterozygous mice can be bred with each other to obtain homozygous double-gene or multi-gene humanized mice. These double-gene or multi-gene modified mice can be used to verify the in vivo efficacy of human TNFSF9, 4-1BB, and other gene regulators. Specifically, TNFSF9 and 4-1BB homozygous mice were used for breeding, and after multiple generations of screening, TNFSF9/4-1BB double-gene humanized mice can be obtained.

The mice disclosed above can be used to prepare multiple human disease models for testing the in vivo efficacy of human-specific antibodies. For example, TNFSF9 and/or 4-1BB gene humanized mice can be used to evaluate the pharmacodynamics, pharmacokinetics, and in vivo therapeutic effect of various disease models of human-specific TNFSF9 and/or 4-1BB signaling pathway drugs.

EXAMPLE 4: Evaluation of drug efficacy in a tumor model using TNFSF9/4-1BB double-gene humanized mice

The mice with humanized TNFSF9 and/or 4-1BB genes prepared herein were used to construct tumor models, which can be used to test the efficacy of drugs targeting human TNFSF9 and/or 4-1BB. Specifically, 8-week-old female TNFSF9/4-1BB double-gene humanized homozygous mice prepared in Example 3 were selected. The mice were subcutaneously injected with mouse colon cancer cell MC38 (5×10 ⁵ cells) . After the tumor grew to a volume of about 100 mm ³, the mice were placed into a control group and three treatment groups (5 mice in each group) . The treatment group mice were administered by intraperitoneal injection (i. p. ) with difference doses of Urelumab (See PCT Application Publication No. WO2005035584A1) . The control group mice were injected with a control antibody with the same immunoglobulin isotype, i.e., human IgG4. The frequency of administration was twice a week (6 times of administrations in total) . The tumor volume was measured twice a week and body weight of the mice was recorded as well. Euthanasia was performed when tumor volume of a mouse reached 3000 mm ³. Specific grouping and administration details are shown in the table below. The mouse body weight, body weight change, and tumor volume measurement results during the experimental period are shown in FIGS. 18-20, respectively.

Table 13.

The table below lists the results for this experiment, including the tumor volumes at Day 0 (at the time of grouping) , Day 14 (14 days post grouping) , and Day 21 (21 days post grouping) ; the survival status of the mice; ratio of tumor-free mice; the Tumor Growth Inhibition value (TGI _TV%) ; and the statistical difference (P value) of mouse body weight and tumor volume between the treatment groups and control group.

Table 14.

As shown in FIGS. 18-19 and the above table, the mice in each group were grossly healthy during the experimental period. On Day 21, body weight of both the control group mice (G1) and the treatment group mice (G2, G3 and G4) showed an increasing trend (FIG. 18) , and there was no significant difference (P > 0.05) , indicating that the anti-human 4-1BB antibody Urelumab was well tolerated in mice without obvious toxic effects. As shown in FIG. 20 and the above table, the tumor volume of the treatment group mice was smaller than that of the control group mice in each stage of the experimental period. On Day 21, the tumor volume of the mice in the G2, G3, and G4 groups were 1906 ± 219 mm ³, 1437 ± 184 mm ³, and 796 ± 181 mm ³, respectively, each of which was reduced as compared to the tumor volume of 1983 ± 329 mm ³ of the control group mice. In particular, tumor volume of the G4 group mice was significantly different from that of the control group mice (P < 0.05) . The results showed that different doses of Urelumab exhibited different tumor suppressive effects in humanized TNFSF9/4-1BB mice, in a dose-dependent manner.

EXAMPLE 5: Evaluation of the drug toxicity in a mouse model using 4-1BB humanized mice and TNFSF9/4-1BB double-gene humanized mice

The mice with humanized TNFSF9 and/or 4-1BB genes prepared herein were used to construct mouse models, which can be used to test the toxicity of drugs targeting human TNFSF9 and/or 4-1BB. Specifically, 8-week-old female 4-1BB gene humanized homozygous mice prepared in Example 2 (or “4-1BB mice” ) , and 8-week-old female TNFSF9/4-1BB double-gene humanized homozygous mice prepared in Example 3 (or “TNFSF9/4-1BB mice” ) were selected. The mice were randomly placed into two control groups and four treatment groups (5 mice in each group) . The treatment group mice were administered by intraperitoneal injection (i.p. ) with difference doses of Urelumab (1 mg/kg or 20 mg/kg) . The control group mice were injected at a dose level of 20 mg/kg with a control antibody with the same immunoglobulin isotype, i.e., human IgG4. The frequency of administration was three times a week (4 times of administrations in total) . Specific grouping and administration details are shown in the table below. Blood was collected on the 21st day after the first administration, and the serum was collected by centrifugation to detect alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations. Mouse liver tissues were also collected, fixed, and stained by haematoxylin and eosin (H&E) for pathological analysis.

Table 15.

During the experimental period, the mice in each group were grossly healthy, and their body weight increased without significant difference (P > 0.05) . ALT and AST test results are shown in FIG. 21A and FIG. 21B, respectively. In 4-1BB mice, 20 mg/kg Urelumab treatment (G3) significantly increased the ALT level as compared to that of the control group mice (G1) . However, there was no significant change of the AST level after 20 mg/kg Urelumab treatment. In TNFSF9/4-1BB mice, 20 mg/kg Urelumab treatment (G6) significantly increased the AST level as compared to that of the control group mice (G4) . However, there was no significant change of the ALT level after 20 mg/kg Urelumab treatment. In both 4-1BB mice and TNFSF9/4-1BB mice, there was no significant change of ALT and AST levels in the treatment groups administered with 1 mg/kg Urelumab (G2 and G5) . The above results indicate that when Urelumab is administered at a dose level of 20 mg/kg and the aforementioned frequency, TNFSF9/4-1BB double-gene humanized mice are more sensitive to hepatotoxicity than 4-1BB gene humanized mice.

Table 16. Pathology Evaluation Form

Evaluation Criteria: Hepatic perivascular cell infiltration or chronic inflammation.

sight (+) : Lesion site accounts for about 5%of the total area

mild (++) : Lesion site accounts for about 5～20%of the total area

moderate (+++) : Lesion site accounts for about 20～40%of the total area

severe (++++) : Lesion site accounts for about 40%of the total area

Normal: NVL (no visible lesion)

The microscopic images after H&E staining and pathological scores are shown in FIGS. 22A-22B, and the above table, respectively. With respect to the 4-1BB mice, there were no obvious abnormal changes in the liver of the G2 group mice; all 5 mice in the G3 group showed abnormal changes, e.g., cell infiltration or chronic inflammation around hepatic blood vessels, and the lesions were mild. With respect to the TNFSF9/4-1BB mice, 3 out of 5 mice in the G5 group showed pathological changes (slight in 2 mice, and mild in 1 mouse) ; all 5 mice in the G6 group showed a moderate level of pathological changes in the liver. In addition, the severity and incidence of liver pathology in the G6 group mice were significantly higher than those of the G5 group mice.

Overall, the above results showed that, at the same dosing frequency as described above, the mice administered with 20 mg/kg Urelumab were more likely to have hepatic perivascular cell infiltration or chronic inflammation, as compared to the mice administered with 1 mg/kg Urelumab. In addition, TNFSF9/4-1BB double-gene humanized mice were more sensitive to hepatotoxicity than 4-1BB gene humanized mice.

The above experiments demonstrated that the humanized TNFSF9/4-1BB mice prepared by the methods described herein can be used for screening of anti-human TNFSF9 and/or 4-1BB antibodies and in vivo drug efficacy testing, and can be used as a living substitute model for in vivo research, as well as screening, evaluation and treatment for human TNFSF9 /4-1BB signaling pathway modulators.

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 TNF Superfamily Member 9 (TNFSF9) .
The animal of claim 1, wherein the sequence encoding the human or chimeric TNFSF9 is operably linked to an endogenous regulatory element at the endogenous TNFSF9 gene locus in the at least one chromosome.
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric TNFSF9 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human TNFSF9 (NP_003802.1 (SEQ ID NO: 4) ) .
The animal of any one of claims 1-3, wherein the sequence encoding a human or chimeric TNFSF9 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: 12.
The animal of claim 1-4, wherein the sequence encoding a human or chimeric TNFSF9 comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to amino acids 26-254 of SEQ ID NO: 4.
The animal of any one of claims 1-5, wherein the animal is a mammal, e.g., a monkey, a rodent, or a mouse.
The animal of claim 6, wherein the mammal is a mouse.
The animal of any one of claims 1-7, wherein the animal does not express endogenous TNFSF9.
The animal of any one of claims 1-8, wherein the animal has one or more cells expressing human or chimeric TNFSF9.
The animal of any one of claims 1-9, wherein the animal has one or more cells expressing human or chimeric TNFSF9, and a human TNF Receptor Superfamily Member 9 (4-1BB) can bind to the expressed human or chimeric TNFSF9.
The animal of any one of claims 1-10, wherein the animal has one or more cells expressing human or chimeric TNFSF9, and an endogenous 4-1BB can bind to the expressed human or chimeric TNFSF9.
A genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous TNFSF9 with a sequence encoding a corresponding region of human TNFSF9 at an endogenous TNFSF9 gene locus.
The animal of claim 12, wherein the sequence encoding the corresponding region of human TNFSF9 is operably linked to an endogenous regulatory element at the endogenous TNFSF9 locus, and one or more cells of the animal expresses a chimeric TNFSF9.
The animal of claim 12 or 13, wherein the animal does not express endogenous TNFSF9.
The animal of any one of claims 12-14, wherein the replaced sequence encodes all or a portion of the transmembrane region and/or extracellular region of endogenous TNFSF9.
The animal of any one of claims 12-15, wherein the animal has one or more cells expressing a chimeric TNFSF9 having a cytoplasmic region, a transmembrane region, and an extracellular 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 TNFSF9.
The animal of claim 16, wherein the extracellular region of the chimeric TNFSF9 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, or 200 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human TNFSF9.
The animal of claim 16 or 17, wherein the transmembrane region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%identical to the transmembrane region of human TNFSF9.
The animal of any one of claims 16-18, wherein the transmembrane region of the chimeric TNFSF9 has a sequence that has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids that are identical to a contiguous sequence present in the transmembrane region of human TNFSF9.
The animal of any one of claims 12-19, wherein the sequence encoding a region of endogenous TNFSF9 comprises exon 1, exon 2, and/or exon 3, or a part thereof, of the endogenous TNFSF9 gene.
The animal of any one of claims 12-20, wherein the animal is a mouse, and the sequence encoding a region of endogenous TNFSF9 starts within exon 1 and ends within exon 3 of the endogenous mouse TNFSF9 gene.
The animal of any one of claims 12-21, wherein the animal is heterozygous with respect to the replacement at the endogenous TNFSF9 gene locus.
The animal of any one of claims 12-22, wherein the animal is homozygous with respect to the replacement at the endogenous TNFSF9 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 TNFSF9 gene locus, a sequence encoding a region of an endogenous TNFSF9 with a sequence encoding a corresponding region of human TNFSF9.
The method of claim 24, wherein the sequence encoding the corresponding region of human TNFSF9 comprises exon 1, exon 2, and/or exon 3, or a part thereof, of a human TNFSF9 gene.
The method of claim 24 or 25, wherein the sequence encoding the corresponding region of human TNFSF9 starts within exon 1 and ends within exon 3 of a human TNFSF9 gene.
The method of any one of claims 24-26, wherein the sequence encoding the corresponding region of human TNFSF9 encodes amino acids 26-254 of SEQ ID NO: 4.
The method of any one of claims 24-27, wherein the region of an endogenous TNFSF9 is located within the transmembrane region and/or extracellular region of endogenous TNFSF9.
The method of any one of claims 24-28, wherein the sequence encoding a region of an endogenous TNFSF9 is exon 1, exon 2, and/or exon 3, or a part thereof, of the endogenous TNFSF9 gene.
The method of any one of claims 24-29, wherein the animal is a mouse, and the sequence encoding a region of an endogenous TNFSF9 starts within exon 1 and ends within exon 3 of the endogenous mouse TNFSF9 gene.
A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric TNFSF9 polypeptide, wherein the chimeric TNFSF9 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human TNFSF9, wherein the animal expresses the chimeric TNFSF9 polypeptide.
The animal of claim 31, wherein the chimeric TNFSF9 polypeptide has at least 50, at least 100, or at least 200 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human TNFSF9 transmembrane region and/or extracellular region.
The animal of claim 31 or 32, wherein the chimeric TNFSF9 polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 26-254 of SEQ ID NO: 4.
The animal of any one of claims 31-33, wherein the nucleotide sequence is operably linked to an endogenous TNFSF9 regulatory element of the animal.
The animal of any one of claims 31-34, wherein the chimeric TNFSF9 polypeptide comprises an endogenous TNFSF9 cytoplasmic region.
The animal of any one of claims 31-35, wherein the nucleotide sequence is integrated to an endogenous TNFSF9 gene locus of the animal.
The animal of any one of claims 31-36, wherein the chimeric TNFSF9 polypeptide has at least one mouse TNFSF9 activity and/or at least one human TNFSF9 activity.
A method of making a genetically-modified non-human animal cell that expresses a chimeric TNFSF9, the method comprising:

replacing at an endogenous TNFSF9 gene locus, a nucleotide sequence encoding a region of endogenous TNFSF9 with a nucleotide sequence encoding a corresponding region of human TNFSF9, thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the chimeric TNFSF9, wherein the animal cell expresses the chimeric TNFSF9.
The method of claim 39, wherein the animal is a mammal, e.g., a monkey, a rodent, or a mouse.
The method of claim 38 or 39, wherein the chimeric TNFSF9 comprises a cytoplasmic region of endogenous TNFSF9; and a transmembrane region and/or an extracellular region of human TNFSF9.
The method of any one of claims 38-40, wherein the nucleotide sequence encoding the chimeric TNFSF9 is operably linked to an endogenous TNFSF9 regulatory region, e.g., promoter.
The animal of any one of claims 1-23 and 31-37, wherein the animal further comprises a sequence encoding an additional human or chimeric protein.
The animal of claim 42, wherein the additional human or chimeric protein is TNF Receptor Superfamily Member 9 (4-1BB) , programmed cell death protein 1 (PD-1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , Lymphocyte Activating 3 (LAG-3) , B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , CD27, CD28, CD47, CD154, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , Signal regulatory protein α (SIRPα) or TNF Receptor Superfamily Member 4 (OX40) .
The animal of claim 42 or 43, wherein the additional human or chimeric protein is TNF Receptor Superfamily Member 9 (4-1BB) , and the animal expresses the human or chimeric 4-1BB.
The method of any one of claims 24-30 and 38-41, wherein the animal or mouse further comprises a sequence encoding an additional human or chimeric protein.
The method of claim 45, wherein the additional human or chimeric protein is 4-1BB, PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD47, CD154, TIGIT, TIM-3, GITR, SIRPαor OX40.
The method of claim 45 or 46, wherein the additional human or chimeric protein is TNF Receptor Superfamily Member 9 (4-1BB) , and the animal expresses the human or chimeric 4-1BB.
A method of determining effectiveness of a therapeutic agent for treating cancer, comprising:

a) administering the therapeutic agent to the animal of any one of claims 1-23, 31-37, and 42-44, wherein the animal has a tumor; and

b) determining the inhibitory effects of the therapeutic agent to the tumor.
The method of claim 48, wherein the therapeutic agent is an anti-TNFSF9 antibody or an anti-4-1BB antibody.
The method of claim 48 or 49, wherein the tumor comprises one or more cells that express TNFSF9 and/or 4-1BB.
The method of any one of claims 48-50, wherein the tumor comprises one or more cancer cells that are injected into the animal.
The method of any one of claims 48-51, wherein determining the inhibitory effects of the therapeutic agent to the tumor involves measuring the tumor volume in the animal.
The method of any one of claims 48-52, wherein the cancer is solid tumor, refractory solid tumor, B-cell lymphoma, non-Hodgkin’s lymphoma, metastatic solid tumor, breast cancer, colorectal cancer, melanoma, non-small cell lung cancer (NSCLC) , small cell lung cancer (SCLC) , bladder cancer, renal cancer, ovarian cancer, prostate cancer, melanoma, or multiple myeloma.
A method of determining effectiveness of a therapeutic agent for treating an autoimmune disorder, comprising:

a) administering the therapeutic agent to the animal of any one of claims 1-23, 31-37, and 42-44, wherein the animal has the autoimmune disorder; and

b) determining effects of the therapeutic agent for treating the auto-immune disease.
The method of claim 54, wherein the therapeutic agent is an anti-TNFSF9 antibody or an anti-4-1BB antibody.
The method of claim 54 or 55, wherein the autoimmune disorder is multiple sclerosis, diabetes, encephalomyelitis, rheumatoid arthritis, lupus, allergic conjunctivitis, or inflammatory bowel disease.
A method of determining effectiveness of a therapeutic agent for treating an immune disorder, comprising:

a) administering the therapeutic agent to the animal of any one of claims 1-23, 31-37, and 42-44, wherein the animal has the immune disorder; and

b) determining effects of the therapeutic agent for treating the immune disease.
The method of claim 57, wherein the therapeutic agent is an anti-TNFSF9 antibody or an anti-4-1BB antibody.
The method of claim 57 or 58, wherein the immune disorder is allergy, asthma, and/or atopic dermatitis.
A method of determining toxicity of a therapeutic agent, the method comprising

a) administering the therapeutic agent to the animal of any one of claims 1-23, 31-37, and 42-44; and

b) determining weight change of the animal.
A method of determining toxicity of a therapeutic agent, the method comprising

a) administering the therapeutic agent to the animal of any one of claims 1-23, 31-37, and 42-44; and

b) determining one or more biochemical parameters of the animal.
The method of claim 61, wherein the one or more biochemical parameters comprise the serum levels of alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) of the animal.
A method of determining toxicity of a therapeutic agent, the method comprising

a) administering the therapeutic agent to the animal of any one of claims 1-23, 31-37, and 42-44; and

b) determining organ damage of the animal.
The method of claim 63, wherein the organ is selected from the group consisting of liver, kidney, brain, heart, spleen, lung, and skin.
The method of claim 64, wherein the organ is liver, and liver damage is evaluated by measuring the percentage of lesion site area over total area of liver.
The method of any one of claims 63-65, wherein the organ is isolated from the animal before the determining step.
The method of any one of claims 63-66, wherein the organ is stained, e.g., by haematoxylin and eosin (H&E) .
The method of any one of claims 60-67, wherein the therapeutic agent is an anti-TNFSF9 antibody or an anti-4-1BB antibody.
A protein comprising an amino acid sequence, wherein the amino acid sequence is one of the following:

(a) an amino acid sequence set forth in SEQ ID NO: 2, 4, 12, 52, 54, and/or 56;

(b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 2, 4, 12, 52, 54, and/or 56;

(c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 2, 4, 12, 52, 54, and/or 56;

(d) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 2, 4, 12, 52, 54, and/or 56 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and

(e) an amino acid sequence that comprises a substitution, a deletion and /or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 2, 4, 12, 52, 54, and/or 56.
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 69;

(b) SEQ ID NO: 1, 3, 5-11, 13, 50, 51, 53, 55, and/or 57;

(c) a sequence that is at least 90 %identical to SEQ ID NO: 1, 3, 5-11, 13, 50, 51, 53, 55, and/or 57; and

(d) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1, 3, 5-11, 13, 50, 51, 53, 55, and/or 57.
A cell comprising the protein of claim 69 and/or the nucleic acid of claim 70.
An animal comprising the protein of claim 69 and/or the nucleic acid of claim 70.