WO2023041035A1

WO2023041035A1 - Genetically modified non-human animal with human or chimeric genes

Info

Publication number: WO2023041035A1
Application number: PCT/CN2022/119313
Authority: WO
Inventors: Linlin Wang; Chang Liu; Ruili LV
Original assignee: Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
Priority date: 2021-09-18
Filing date: 2022-09-16
Publication date: 2023-03-23
Also published as: CN116063450A

Abstract

Provided are genetically modified non-human animals that express a human or chimeric (e.g., humanized) IL12A, IL12B, IL12RB1, and/or IL12RB2, and methods of use thereof.

Description

GENETICALLY MODIFIED NON-HUMAN ANIMAL WITH HUMAN OR CHIMERIC GENES

CLAIM OF PRIORITY

This application claims the benefit of Chinese Patent Application App. No. 202111101912.4, filed on September 18, 2021 and Chinese Patent Application App. No. 202210191673.4, filed on February 28, 2022. The entire contents of the foregoing applications are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) IL12 and/or IL12 receptor proteins, and methods of use thereof.

BACKGROUND

The traditional drug research and development typically use in vitro screening approaches. However, these screening approaches cannot provide the body environment (such as tumor microenvironment, stromal cells, extracellular matrix components and immune cell interaction, etc. ) , resulting in a higher rate of failure in drug development. In addition, in view of the differences between humans and animals, the test results obtained from the use of conventional experimental animals for in vivo pharmacological test may not reflect the real disease state and the interaction at the targeting sites, resulting in that the results in many clinical trials are significantly different from the animal experimental results.

Therefore, the development of humanized animal models that are suitable for human antibody screening and evaluation will significantly improve the efficiency of new drug development and reduce the cost for drug research and development.

SUMMARY

This disclosure is related to an animal model with human or chimeric IL12 and/or IL12 receptor proteins. The animal model can express human or chimeric IL12A (e.g., humanized IL12A) protein, human or chimeric IL12B (e.g., humanized IL12B) protein, human or chimeric IL12RB1 (e.g., humanized IL12RB1) protein, and/or human or chimeric IL12RB2 (e.g., humanized IL12RB2) protein in its body. It can be used in the studies on the function of IL12 and IL12 receptor genes, and can be used in the screening and evaluation of IL12/IL12R signaling pathway modulators (e.g., anti-human IL12 antibodies, anti-human IL12R antibodies, or human IL12 protein or variants thereof) . 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 IL12/IL12R 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 IL12/IL12R protein and a platform for screening cancer drugs.

In one aspect, the disclosure is related to a genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric interleukin-12 subunit alpha (IL12A) . In some embodiments, the sequence encoding the human or chimeric IL12A is operably linked to an endogenous regulatory element (e.g., endogenous 5'UTR and/or 3'UTR) at the endogenous IL12A gene locus in the at least one chromosome. In some embodiments, the sequence encoding a human or chimeric IL12A comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human IL12A (NP_000873.2; SEQ ID NO: 2) . In some embodiments, the human or chimeric IL12A forms a functional IL12 heterodimer with an endogenous or human IL12B. In some embodiments, the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat. In some embodiments, the animal is a mouse. In some embodiments, the animal does not express endogenous IL12A or expresses a decreased level of endogenous IL12A. In some embodiments, the animal has one or more cells expressing human or chimeric IL12A. In some embodiments, the animal has one or more cells expressing human or chimeric IL12A, and endogenous IL12 receptor can bind to the IL12 heterodimer comprising the expressed human or chimeric IL12A. In some embodiments, the animal has one or more cells expressing human or chimeric IL12A, and human IL12 receptor can bind to the IL12 heterodimer comprising the expressed human or chimeric IL12A.

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 IL12A with a sequence encoding a corresponding region of human IL12A at an endogenous IL12A gene locus. In some embodiments, the sequence encoding the corresponding region of human IL12A is operably linked to an endogenous regulatory element at the endogenous IL12A locus, and one or more cells of the animal express a human or chimeric IL12A. In some embodiments, the animal does not express endogenous IL12A or expresses a decreased level of endogenous IL12A. In some embodiments, the replaced sequence encodes the full-length protein of IL12A. In some embodiments, the animal is a mouse, and the replaced endogenous IL12A region is a portion ofexon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or a portion ofexon 7 of the endogenous mouse IL12A gene. In some embodiments, the animal is heterozygous or homozygous with respect to the replacement at the endogenous IL12A gene locus.

In one aspect, the disclosure is related to a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric IL12A polypeptide, in some embodiments, the chimeric IL12A polypeptide comprises at least 50, 100, 150, 200, 210, 220, 230, 240, or 250 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL12A, in some embodiments, the animal expresses the chimeric IL12A polypeptide. In some embodiments, the nucleotide sequence is operably linked to an endogenous IL12A regulatory element of the animal. In some embodiments, the nucleotide sequence is integrated to an endogenous IL12A gene locus of the animal. In some embodiments, the chimeric IL12A polypeptide has at least one mouse IL12A activity and/or at least one human IL12A activity.

In one aspect, the disclosure is related to a method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous IL12A gene locus, a sequence encoding a region of an endogenous IL12A with a sequence encoding a corresponding region of human IL12A. In some embodiments, the sequence encoding the corresponding region of human IL12A comprises a portion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or a portion of exon 7 of a human IL12A gene. In some embodiments, the sequence encoding the corresponding region of human IL12A comprises at least 50, 100, 200, or 300 nucleotides ofexon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of a human IL12A gene. In some embodiments, the sequence encoding the corresponding region of human IL12A encodes a sequence that is at least 90%identical to SEQ ID NO: 2. In some embodiments, the animal is a mouse, and the locus is a portion ofexon 1, exon 2, exon 3, exons 4, exon 5, exon 6, and/or a portion ofexon 7 of the mouse IL12A gene.

In one aspect, the disclosure is related to a method of making a genetically-modified non-human animal cell that expresses a human or chimeric IL12A, the method comprising: replacing, at an endogenous mouse IL12A gene locus, a nucleotide sequence encoding a region of endogenous IL12A with a nucleotide sequence encoding a corresponding region of human IL12A, thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the human or chimeric IL12A, in some embodiments, the animal cell expresses the human or chimeric IL12A. In some embodiments, the animal is a mouse. In some embodiments, the nucleotide sequence encoding the human or chimeric IL12A is operably linked to an endogenous IL12A regulatory region, e.g., promoter.

In some embodiments, the animal further comprises a sequence encoding an additional human or chimeric protein, e.g., Interleukin-12 subunit beta (IL12B) , Interleukin-12 receptor subunit beta-1 (IL12RB1) , Interleukin 12 receptor subunit beta 2 (IL12RB2) , Interleukin-23 (IL23) , programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , and/or CD73. In some embodiments, the additional human or chimeric protein is IL12B.

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 interleukin-12 subunit beta (IL12B) . In some embodiments, the sequence encoding the human or chimeric IL12B is operably linked to an endogenous regulatory element at the endogenous IL12B gene locus in the at least one chromosome. In some embodiments, the sequence encoding the human or chimeric IL12B is operably linked to an endogenous 5'UTR and a human 3'UTR. In some embodiments, the sequence encoding a human or chimeric IL12B comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human IL12B (NP_002178.2; SEQ ID NO: 10) . In some embodiments, the human or chimeric IL12B forms a functional IL12 heterodimer with an endogenous or human IL12A. In some embodiments, the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat. In some embodiments, the animal is a mouse. In some embodiments, the animal does not express endogenous IL12B or expresses a decreased level of endogenous IL12B. In some embodiments, the animal has one or more cells expressing human or chimeric IL12B. In some embodiments, the animal has one or more cells expressing human or chimeric IL12B, and endogenous IL12 receptor can bind to the IL12 heterodimer comprising the expressed human or chimeric IL12B. In some embodiments, the animal has one or more cells expressing human or chimeric IL12B, and human IL12 receptor can bind to the IL12 heterodimer comprising the expressed human or chimeric IL12B.

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 IL12B with a sequence encoding a corresponding region of human IL12B at an endogenous IL12B gene locus. In some embodiments, the sequence encoding the corresponding region of human IL12B is operably linked to an endogenous 5'UTR and a human 3'UTR at the endogenous IL12B locus, and one or more cells of the animal express a human or chimeric IL12B. In some embodiments, the animal does not express endogenous IL12B or expresses a decreased level of endogenous IL12B. In some embodiments, the replaced sequence encodes the full-length protein of IL12B. In some embodiments, the animal is a mouse, and the replaced endogenous IL12B region is exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8, or a portion thereof, of the endogenous mouse IL12B gene. In some embodiments, the animal is heterozygous or homozygous with respect to the replacement at the endogenous IL12B gene locus.

In one aspect, the disclosure is related to a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric IL12B polypeptide, in some embodiments, the chimeric IL12B polypeptide comprises at least 50, 100, 150, 200, 210, 220, 230, 240, or 250 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL12B, in some embodiments, the animal expresses the chimeric IL12B polypeptide. In some embodiments, the nucleotide sequence is operably linked to an endogenous IL12B regulatory element of the animal. In some embodiments, the nucleotide sequence is integrated to an endogenous IL12B gene locus of the animal. In some embodiments, the chimeric IL12B polypeptide has at least one mouse IL12B activity and/or at least one human IL12B activity.

In one aspect, the disclosure is related to a method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous IL12B gene locus, a sequence encoding a region of an endogenous IL12B with a sequence encoding a corresponding region of human IL12B. In some embodiments, the sequence encoding the corresponding region of human IL12B comprises, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8, or a portion thereof, of a human IL12B gene. In some embodiments, the sequence encoding the corresponding region of human IL12B comprises at least 50, 100, 200, or 300 nucleotides ofexon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 of a human IL12B gene. In some embodiments, the sequence encoding the corresponding region of human IL12B encodes a sequence that is at least 90%identical to SEQ ID NO: 10. In some embodiments, the animal is a mouse, and the locus is exon 2, exon 3, exons 4, exon 5, exon 6, exon 7, and/or exon 8 of the mouse IL12B gene.

In one aspect, the disclosure is related to a method of making a genetically-modified non-human animal cell that expresses a human or chimeric IL12B, the method comprising: replacing, at an endogenous mouse IL12B gene locus, a nucleotide sequence encoding a region of endogenous IL12B with a nucleotide sequence encoding a corresponding region of human IL12B, thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the human or chimeric IL12B, in some embodiments, the animal cell expresses the human or chimeric IL12B. In some embodiments, the animal is a mouse. In some embodiments, the nucleotide sequence encoding the human or chimeric IL12B is operably linked to an endogenous IL12B regulatory region, e.g., promoter.

In some embodiments, the animal further comprises a sequence encoding an additional human or chimeric protein, e.g., Interleukin-12 subunit alpha (IL12A) , Interleukin-12 receptor subunit beta-1 (IL12RB1) , Interleukin 12 receptor subunit beta 2 (IL12RB2) , Interleukin-23 (IL23) , programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , and/or CD73. In some embodiments, the additional human or chimeric protein is IL12A.

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 interleukin-12 receptor subunit beta-1 (IL12RB1) . In some embodiments, the sequence encoding the human or chimeric IL12RB1 is operably linked to an endogenous regulatory element at the endogenous IL12RB1 gene locus in the at least one chromosome. In some embodiments, the sequence encoding a human or chimeric IL12RB1 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to amino acids 24-570 of human IL12RB1 (NP_005526.1; SEQ ID NO: 18) . In some embodiments, the sequence encoding a human or chimeric IL12RB1 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: 27. In some embodiments, the human or chimeric IL12RB1 forms a functional IL12 receptor with an endogenous or human IL12RB2. In some embodiments, the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat. In some embodiments, the animal is a mouse. In some embodiments, the animal does not express endogenous IL12RB1 or expresses a decreased level of endogenous IL12RB1. In some embodiments, the animal has one or more cells expressing human or chimeric IL12RB1. In some embodiments, the animal has one or more cells expressing human or chimeric IL12RB1, and endogenous IL12 can bind to the IL12 receptor comprising the expressed human or chimeric IL12RB1. In some embodiments, the animal has one or more cells expressing human or chimeric IL12RB1, and human IL12 can bind to the IL12 receptor comprising the expressed human or chimeric IL12RB1.

In one aspect, the disclosure is related to a genetically-modified, non-human animal, in some embodiments, the genome of the animal comprises an insertion of a sequence encoding a human or chimeric IL12RB1 at an endogenous IL12RB1 gene locus. In some embodiments, the sequence encoding a human or chimeric IL12RB1 does not include a sequence encoding the signal peptide of IL12RB1. In some embodiments, the sequence encoding a human or chimeric IL12RB1 is operably linked to an endogenous regulatory element at the endogenous IL12RB1 locus, and one or more cells of the animal express the human or chimeric IL12RB1. In some embodiments, the animal does not express endogenous IL12RB1 or expresses a decreased level of endogenous IL12RB1. In some embodiments, the sequence encoding a human or chimeric IL12RB1 is inserted within exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, and/or exon 16 of endogenous IL12RB1 gene. In some embodiments, the sequence encoding a human or chimeric IL12RB1 is inserted within exon 1 and/or intron 1 of endogenous IL12RB1 gene. In some embodiments, the sequence encoding a human or chimeric IL12RB1 is inserted immediately after the last nucleotide encoding the signal peptide of endogenous IL12RB1 (e.g., a nucleotide corresponding to position 160 of NM_008353.2) . In some embodiments, a sequence encoding the N-

terminal

1, 2, 3, or 4 amino acids of endogenous IL12RB1 extracellular region is deleted from exon 1 of endogenous IL12RB1 gene. In some embodiments, a sequence corresponding to positions 161-170 of NM_008353.2 and the downstream 425 nucleotides within intron 1 (e.g., position 71261263 to position 71261687 of NC_000074.7) are deleted. In some embodiments, the inserted sequence comprises, optionally from 5'end to 3'end: a) a sequence encoding the extracellular region and transmembrane region of a human IL12RB1; b) a sequence encoding the cytoplasmic region of an endogenous IL12RB1; and c) optionally one or more auxiliary sequences (e.g., WPRE, STOP, and/or polyA) . In some embodiments, the sequence encoding the extracellular region and transmembrane region of a human IL12RB1 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 21, and the sequence encoding the cytoplasmic region of an endogenous IL12RB1 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 48. In some embodiments, the one or more auxiliary sequences comprise, optionally from 5'end to 3'end: a WPRE sequence and a STOP sequence. In some embodiments, the WPRE sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 24 and the STOP sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 25. In some embodiments, the animal is heterozygous or homozygous with respect to the insertion at the endogenous IL12RB1 gene locus.

In one aspect, the disclosure is related to a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric IL12RB1 polypeptide, in some embodiments, the chimeric IL12RB1 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL12RB1, in some embodiments, the animal expresses the chimeric IL12RB1 polypeptide. In some embodiments, the chimeric IL12RB1 polypeptide has at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 510, 520, 530, 540, 545, 546, or 547 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of human IL12RB1 extracellular and transmembrane regions. In some embodiments, the chimeric IL12RB1 polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 24-570 of SEQ ID NO: 18. In some embodiments, the nucleotide sequence is operably linked to an endogenous IL12RB1 regulatory element of the animal. In some embodiments, the chimeric IL12RB1 polypeptide comprises an endogenous IL12RB1 cytoplasmic region, and optionally an endogenous signal peptide. In some embodiments, the nucleotide sequence is integrated to an endogenous IL12RB1 gene locus of the animal. In some embodiments, the chimeric IL12RB1 polypeptide has at least one mouse IL12RB1 activity and/or at least one human IL12RB1 activity.

In one aspect, the disclosure is related to a method of making a genetically-modified non-human animal cell that expresses a chimeric IL12RB1, the method comprising: inserting at an endogenous IL12RB1 gene locus (e.g., exon 1 of endogenous IL12RB1 gene) , a nucleotide sequence comprising, optionally from 5'end to 3'end: a) a sequence encoding the extracellular region and transmembrane region of a human IL12RB1; b) a sequence encoding the cytoplasmic region of an endogenous IL12RB1; and c) optionally one or more auxiliary sequences (e.g., WPRE, STOP, and/or polyA) , thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the chimeric IL12RB1, in some embodiments, the animal cell expresses the chimeric IL12RB1. In some embodiments, the animal is a mouse. In some embodiments, the nucleotide sequence encoding the chimeric IL12RB1 polypeptide is operably linked to an endogenous IL12RB1 regulatory region, e.g., promoter.

In some embodiments, the animal further comprises a sequence encoding an additional human or chimeric protein, e.g., Interleukin 12 receptor subunit beta 2 (IL12RB2) , Interleukin-12 subunit alpha (IL12A) , Interleukin-12 subunit beta (IL12B) , Interleukin-23 (IL23) , programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , and/or CD73. In some embodiments, the additional human or chimeric protein is IL12RB2.

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 interleukin-12 receptor subunit beta-2 (IL12RB2) . In some embodiments, the sequence encoding the human or chimeric IL12RB2 is operably linked to an endogenous regulatory element at the endogenous IL12RB2 gene locus in the at least one chromosome. In some embodiments, the sequence encoding a human or chimeric IL12RB2 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to amino acids 28-622 of human IL12RB2 (NP_001361188.1; SEQ ID NO: 29) . In some embodiments, the sequence encoding a human or chimeric IL12RB2 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: 36. In some embodiments, the human or chimeric IL12RB2 forms a functional IL12 receptor with an endogenous or human IL12RB1. In some embodiments, the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat. In some embodiments, the animal is a mouse. In some embodiments, the animal does not express endogenous IL12RB2 or expresses a decreased level of endogenous IL12RB2. In some embodiments, the animal has one or more cells expressing human or chimeric IL12RB2. In some embodiments, the animal has one or more cells expressing human or chimeric IL12RB2, and endogenous IL12 can bind to the IL12 receptor comprising the expressed human or chimeric IL12RB2. In some embodiments, the animal has one or more cells expressing human or chimeric IL12RB2, and human IL12 can bind to the IL12 receptor comprising the expressed human or chimeric IL12RB2.

In one aspect, the disclosure is related to a genetically-modified, non-human animal, in some embodiments, the genome of the animal comprises an insertion of a sequence encoding a human or chimeric IL12RB2 at an endogenous IL12RB2 gene locus. In some embodiments, the sequence encoding a human or chimeric IL12RB2 does not include a sequence encoding the signal peptide of IL12RB2. In some embodiments, the sequence encoding a human or chimeric IL12RB2 is operably linked to an endogenous regulatory element at the endogenous IL12RB2 locus, and one or more cells of the animal express the human or chimeric IL12RB2. In some embodiments, the animal does not express endogenous IL12RB2 or expresses a decreased level of endogenous IL12RB2. In some embodiments, the sequence encoding a human or chimeric IL12RB2 is inserted within exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, and/or exon 16 of endogenous IL12RB2 gene. In some embodiments, the sequence encoding a human or chimeric IL12RB2 is inserted within exon 2 and/or intron 2 of endogenous IL12RB2 gene. In some embodiments, the sequence encoding a human or chimeric IL12RB2 is inserted immediately after the last nucleotide encoding the signal peptide of endogenous IL12RB2 (e.g., a nucleotide corresponding to position 257 of NM_008354.4) . In some embodiments, a sequence encoding the N-

terminal

1, 2, or 3 amino acids of endogenous IL12RB2 extracellular domain is deleted from exon 2 of endogenous IL12RB2 gene. In some embodiments, a sequence corresponding to positions 258-264 of NM_008354.4 and the downstream 409 nucleotides within intron 2 (e.g., position 67338450 to position 67338858 of NC_000072.7) are deleted. In some embodiments, the inserted sequence comprises, optionally from 5'end to 3'end: a) a sequence encoding the extracellular region of a human IL12RB2; b) a sequence encoding the transmembrane region and cytoplasmic region of an endogenous IL12RB2; and c) optionally one or more auxiliary sequences (e.g., WPRE, STOP, and/or polyA) . In some embodiments, the sequence encoding the extracellular region of a human IL12RB2 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 34, and the sequence encoding the cytoplasmic region and transmembrane region of an endogenous IL12RB2 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 35. In some embodiments, the one or more auxiliary sequences comprise, optionally from 5'end to 3'end: a WPRE sequence and a STOP sequence. In some embodiments, the WPRE sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 32 and the STOP sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 33. In some embodiments, the animal is heterozygous or homozygous with respect to the insertion at the endogenous IL12RB2 gene locus.

In one aspect, the disclosure is related to a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric IL12RB2 polypeptide, in some embodiments, the chimeric IL12RB2 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL12RB2, in some embodiments, the animal expresses the chimeric IL12RB2 polypeptide. In some embodiments, the chimeric IL12RB2 polypeptide has at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 591, 592, 593, 594, 595, 596, 597, 598, or 599 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL12RB2 extracellular region. In some embodiments, the chimeric IL12RB2 polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 28-622 of SEQ ID NO: 29. In some embodiments, the nucleotide sequence is operably linked to an endogenous IL12RB2 regulatory element of the animal. In some embodiments, the chimeric IL12RB2 polypeptide comprises endogenous IL12RB2 transmembrane and cytoplasmic regions, and optionally an endogenous signal peptide. In some embodiments, the nucleotide sequence is integrated to an endogenous IL12RB2 gene locus of the animal. In some embodiments, the chimeric IL12RB2 polypeptide has at least one mouse IL12RB2 activity and/or at least one human IL12RB2 activity.

In one aspect, the disclosure is related to a method of making a genetically-modified non-human animal cell that expresses a chimeric IL12RB2, the method comprising: inserting at an endogenous II 12RB2 gene locus (e.g., exon 2 of endogenous IL12RB2 gene) , a nucleotide sequence comprising, optionally from 5'end to 3'end: a) a sequence encoding the extracellular region of a human IL12RB2; b) a sequence encoding the transmembrane region and cytoplasmic region of an endogenous IL12RB2; and c) optionally one or more auxiliary sequences (e.g., WPRE, STOP, and/or polyA) , thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the chimeric IL12RB2, in some embodiments, the animal cell expresses the chimeric IL12RB2. In some embodiments, the animal is a mouse. In some embodiments, the nucleotide sequence encoding the chimeric IL12RB2 polypeptide is operably linked to an endogenous IL12RB2 regulatory region, e.g., promoter.

In some embodiments, the animal further comprises a sequence encoding an additional human or chimeric protein, e.g., Interleukin 12 receptor subunit beta 1 (IL12RB1) , Interleukin-12 subunit alpha (IL12A) , Interleukin-12 subunit beta (IL12B) , Interleukin-23 (IL23) , programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , and/or CD73. In some embodiments, the additional human or chimeric protein is IL12RB1.

In one aspect, the disclosure is related to a method of determining effectiveness of a therapeutic agent for treating cancer, comprising: administering the therapeutic agent to the animal as described herein, in some embodiments, the animal has a tumor; and determining inhibitory effects of the therapeutic agent to the tumor. In some embodiments, the therapeutic agent is an IL12 signaling pathway modulator (e.g., an antibody or antigen-binding fragment that binds to IL12A, IL12B, IL12RB1, and/or IL12RB2) . In some embodiments, the therapeutic agent is a human IL12 protein or variant thereof, or an antibody targeting an immune checkpoint molecule (e.g., PD-1) . In some embodiments, the tumor comprises one or more cancer cells that are injected into the animal. In some embodiments, determining inhibitory effects of the therapeutic agent to the tumor involves measuring the tumor volume in the animal. In some embodiments, the cancer is bladder cancer, melanoma, leukemia, hepatocellular carcinoma, breast cancer, sarcoma, head and neck cancer, colon cancer, lung cancer, liver cancer, or brain cancer.

In one aspect, the disclosure is related to a method of determining effectiveness of an IL12 signaling pathway modulator and an additional therapeutic agent for the treatment of cancer, comprising administering the IL12 signaling pathway modulator and the additional therapeutic agent to the animal as described herein, in some embodiments, the animal has a tumor; and determining inhibitory effects on the tumor. In some embodiments, the animal further comprises a sequence encoding a human or chimeric programmed cell death protein 1 (PD-1) and/or a human or chimeric programmed death-ligand 1 (PD-L1) . In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the tumor comprises one or more tumor cells that express PD-L1. In some embodiments, the tumor is caused by injection of one or more cancer cells into the animal. In some embodiments, determining inhibitory effects of the treatment involves measuring the tumor volume in the animal. In some embodiments, the animal has bladder cancer, melanoma, leukemia, hepatocellular carcinoma, breast cancer, sarcoma, head and neck cancer, colon cancer, lung cancer, liver cancer, or brain cancer.

In one aspect, the disclosure is related to a method of determining effectiveness of an IL12 signaling pathway modulator for treating an autoimmune disorder, comprising: administering the IL12 signaling pathway modulator to the animal as described herein; and determining effects of the IL12 signaling pathway modulator. In some embodiments, the autoimmune disorder is asthma, systemic lupus erythematosus (SLE) , rheumatoid arthritis (RA) , Sjogren′s syndrome (SS) , multiple sclerosis (MS) , Crohn′s disease (CD) , inflammatory bowel disease (IBD) , or psoriasis.

In one aspect, the disclosure is related to a method of determining effectiveness of an IL12 signaling pathway modulator for reducing inflammation, comprising: administering the IL12 signaling pathway modulator to the animal as described herein; and determining effects of the IL12 signaling pathway modulator.

In one aspect, the disclosure is related to a method of determining toxicity of an IL12 signaling pathway modulator, the method comprising administering the IL12 signaling pathway modulator to the animal as described herein; and determining effects of the IL12 signaling pathway modulator to the animal. In some embodiments, determining effects of the therapeutic agent to the animal involves measuring the body weight, red blood cell count, hematocrit, and/or hemoglobin of the animal.

In one aspect, the disclosure is related to a protein comprising an amino acid sequence, In some embodiments, the amino acid sequence is one of the following:

(a) an amino acid sequence set forth in SEQ ID NO: 1, 2, 9, 10, 17, 18, 27, 28, 29, or 36;

(b) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1, 2, 9, 10, 17, 18, 27, 28, 29, or 36;

(c) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 1, 2, 9, 10, 17, 18, 27, 28, 29, or 36 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and

(d) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 1, 2, 9, 10, 17, 18, 27, 28, 29, or 36.

In one aspect, the disclosure is related to a nucleic acid comprising a nucleotide sequence, in some embodiments, the nucleotide sequence is one of the following:

(a) a sequence that encodes the protein as described herein;

(b) SEQ ID NO: 3, 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 26, 30, 31, 34, 35, 37, 38, 48, or 65; and

(c) a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 26, 30, 31, 34, 35, 37, 38, 48, or 65.

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

The disclosure further relates to 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 IL12A, IL12B, IL12RB1, and/or IL12RB2 gene functions, human IL12A, IL12B, IL12RB1, and/or IL12RB2 antibodies, drugs or efficacies for human IL12A, IL12B, IL12RB1, and/or IL12RB2 targeting sites, the drugs for immune-related diseases and antitumor drugs.

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

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

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing mouse and human IL12A gene loci.

FIG. 2 is a schematic diagram showing humanized IL12A gene locus.

FIG. 3 is a schematic diagram showing a IL12A gene targeting strategy.

FIG. 4 shows Southern Blot results of cells after recombination using the IL12A-5'Probe, IL12A-3'Probe, and IL12A-3'Neo Probe. WT is a wild-type control.

FIG. 5 is a schematic diagram showing the FRT recombination process in IL 12A gene humanized mice.

FIGS. 6A-6D show mouse tail PCR identification results of F1 generation mice by primer pairs IL 12A-WT-F/IL 12A-WT-R, IL 12A-Mut-F/IL 12A-WT-R, IL 12A-Frt-F/IL 12A-Frt-R, and IL 12A-Flp-F/IL 12A-Flp-R, respectively. 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 mouse and human IL 12B gene loci.

FIG. 8 is a schematic diagram showing humanized IL 12B gene locus.

FIG. 9 is a schematic diagram showing a IL 12B gene targeting strategy.

FIG. 10 shows Southern Blot results of cells after recombination using the IL 12B-5' Probe, IL 12B-3' Probe, and Neo Probe. WT is a wild-type control.

FIG. 11 is a schematic diagram showing the FRT recombination process in IL 12B gene humanized mice.

FIGS. 12A-12D show mouse tail PCR identification results of F1 generation mice by primer pairs IL 12B-WT-F/IL 12B-WT-R, IL 12B-WT-F/IL 12B-Mut-R, IL 12B-Frt-F/IL 12B-Frt-R, and IL 12B-Flp-F/IL 12B-Flp-R, respectively. M is a marker. PC is a positive control. WT is a wild-type control. H ₂O is a water control.

FIG. 13 is a schematic diagram showing mouse and human IL 12RB1 gene loci.

FIG. 14 is a schematic diagram showing humanized IL 12RB1 gene locus.

FIG. 15 is a schematic diagram showing a IL 12RB1 gene targeting strategy.

FIG. 16 shows Southern Blot results of cells after recombination using the IL 12RB1-5' Probe, IL 12RB1-3' Probe, and Neo Probe. WT is a wild-type control.

FIG. 17 is a schematic diagram showing the FRT recombination process in IL 12RB1 gene humanized mice.

FIGS. 18A-18B show mouse tail PCR identification results of F1 generation mice by primer pairs IL 12RB1-WT-F/IL 12RB1-Mut-R and IL 12RB1-Frt-F/IL 12RB1-Frt-R, respectively. M is a marker. PC1 and PC2 are a positive control. WT is a wild-type control. H ₂O is a water control.

FIG. 19 is a schematic diagram showing mouse and human IL 12RB2 gene loci.

FIG. 20 is a schematic diagram showing humanized IL 12RB2 gene locus.

FIG. 21 is a schematic diagram showing a IL 12RB2 gene targeting strategy.

FIG. 22 shows Southern Blot results of cells after recombination using the IL 12RB2-5' Probe, IL 12RB2-3' Probe, and Neo Probe. WT is a wild-type control.

FIG. 23 is a schematic diagram showing the FRT recombination process in IL 12RB2 gene humanized mice.

FIGS. 24A-24D show mouse tail PCR identification results of F1 generation mice by primer pairs IL 12RB2-WT-F/IL 12RB2-WT-R, IL 12RB2-Frt-F/IL 12RB2-Frt-R, IL 12RB2-WT-F/IL 12RB2-Mut-R, and Flp-F/Flp-R, respectively. M is a marker. WT is a wild-type control. H ₂O is a water control.

FIG. 25A shows the expression level of mouse IL-12 protein in wild-type C57BL/6 mice (+/+) and IL 12A/IL 12B double-gene humanized mice (H/H) .

FIG. 25B shows the expression level of human IL-12 protein in wild-type C57BL/6 mice (+/+) and IL 12A/IL 12B double-gene humanized mice (H/H) .

FIG. 26A shows RT-PCR results of mouse IL 12A (mIL 12A) mRNA, human IL 12A (hIL 12A) mRNA, and GAPDH mRNA in wild-type C57BL/6 mice (+/+) and IL 12A/IL 12B double-gene humanized mice (H/H) . H ₂O is a water control.

FIG. 26B shows RT-PCR results of mouse IL 12B (mIL 12B) mRNA, human IL 12B (hIL 12B) mRNA, and GAPDH mRNA in wild-type C57BL/6 mice (+/+) and IL 12A/IL 12B double-gene humanized mice (H/H) . H ₂O is a water control.

FIG. 27A shows RT-PCR results of mouse IL 12RB1 (mIL 12RB1) mRNA, human IL 12RB1 (hIL 12RB1) mRNA, and GAPDH mRNA in wild-type C57BL/6 mice (+/+) and IL 12A/IL 12B double-gene humanized mice (H/H) . H ₂O is a water control. mHPRT is an internal control to confirm no contamination from mouse genomic DNA.

FIG. 27B shows RT-PCR results of mouse IL 12RB2 (mIL 12RB2) mRNA, human IL 12RB2 (hIL 12RB2) mRNA, and GAPDH mRNA in wild-type C57BL/6 mice (+/+) and IL 12A/IL 12B double-gene humanized mice (H/H) . H ₂O is a water control. mHPRT is an internal control to confirm no contamination from mouse genomic DNA.

FIG. 28A shows RT-PCR results of mouse IL 12A (mIL 12A) mRNA, human IL 12A (hIL 12A) mRNA, and GAPDH mRNA in wild-type C57BL/6 mice (+/+) and IL 12A/IL 12B/IL 12RB 1/IL 12RB2 four-gene humanized homozygous mice (H/H) . H ₂O is a water control.

FIG. 28B shows RT-PCR results of mouse IL 12B (mIL 12B) mRNA, human IL 12B (hIL 12B) mRNA, and GAPDH mRNA in wild-type C57BL/6 mice (+/+) and IL 12A/IL 12B/IL 12RB 1/IL 12RB2 four-gene humanized homozygous mice (H/H) . H ₂O is a water control.

FIG. 28C shows RT-PCR results of mouse IL 12RB1 (mIL 12RB1) mRNA, human IL 12RB1 (hIL 12RB1) mRNA, and GAPDH mRNA in wild-type C57BL/6 mice (+/+) and IL 12A/IL 12B/IL 12RB 1/IL 12RB2 four-gene humanized homozygous mice (H/H) . H ₂O is a water control.

FIG. 28D shows RT-PCR results of mouse IL 12RB2 (mIL 12RB2) mRNA, human IL 12RB2 (hIL 12RB2) mRNA, and GAPDH mRNA in wild-type C57BL/6 mice (+/+) and IL 12A/IL 12B/IL 12RB 1/IL 12RB2 four-gene humanized homozygous mice (H/H) . H ₂O is a water control.

FIG. 29A shows the expression level of mouse IL 12 (p70) protein in wild-type C57BL/6 mice (+/+) and IL 12A/IL 12B/IL 12RB 1/IL 12RB2 four-gene humanized homozygous mice (H/H) .

FIG. 29B shows the expression level of human IL 12 (p70) protein in wild-type C57BL/6 mice (+/+) and IL 12A/IL 12B/IL 12RB1/IL 12RB2 four-gene humanized homozygous mice (H/H) .

FIG. 30 shows the secretion level of mouse IFN-γ (mIFN-γ) by the spleen CD4+ T cells of wild-type C57BL/6 mice and IL 12A/IL 12B/IL 12RB1/IL 12RB2 four-gene humanized homozygous mice after treatment of anti-mCD3ε, anti-mCD28, and different concentrations of mIL 12 or hIL 12.

FIG. 31A shows the percentages of leukocyte subtypes in the spleen of C57BL/6 wild-type mice (+/+) and IL 12A/IL 12B/IL 12RB1/IL 12RB2 four-gene humanized homozygous mice (H/H) .

FIG. 31B shows the percentages of T cell subtypes in the spleen of C57BL/6 wild-type mice (+/+) and IL 12A/IL 12B/IL 12RB1/IL 12RB2 four-gene humanized homozygous mice (H/H) .

FIG. 32A shows the percentages of leukocyte subtypes in the lymph nodes of C57BL/6 wild-type mice (+/+) and IL 12A/IL 12B/IL 12RB1/IL 12RB2 four-gene humanized homozygous mice (H/H) .

FIG. 32B shows the percentages of T cell subtypes in the lymph nodes of C57BL/6 wild-type mice (+/+) and IL 12A/IL 12B/IL 12RB1/IL 12RB2 four-gene humanized homozygous mice (H/H) .

FIG. 33A shows the percentages of leukocyte subtypes in the peripheral blood of C57BL/6 wild-type mice (+/+) and IL 12A/IL 12B/IL 12RB1/IL 12RB2 four-gene humanized homozygous mice (H/H) .

FIG. 33B shows the percentages of T cell subtypes in the peripheral blood of C57BL/6 wild-type mice (+/+) and IL 12A/IL 12B/IL 12RB1/IL 12RB2 four-gene humanized homozygous mice (H/H) .

FIG. 34 shows the tumor volume of IL 12A/IL 12B/IL 12RB1/IL 12RB2 four-gene humanized homozygous mice that were inoculated with MC38 cells, and then treated with PBS (G1) , an anti-mouse PD-1 antibody (G2) , a human IL 12 protein variant (G3) , and a human IL 12 protein (G4) , respectively.

FIG. 35 shows the body weight of IL 12A/IL 12B/IL 12RB1/IL 12RB2 four-gene humanized homozygous mice that were inoculated with MC38 cells, and then treated with PBS (G1) , an anti-mouse PD-1 antibody (G2) , a human IL 12 protein variant (G3) , and a human IL 12 protein (G4) , respectively.

FIG. 36 shows the alignment between human IL 12A amino acid sequence (NP_000873.2; SEQ ID NO: 2) and mouse IL 12A amino acid sequence (NP_032377.1; SEQ ID NO: 1) .

FIG. 37 shows the alignment between human IL 12A amino acid sequence (NP_000873.2; SEQ ID NO: 2) and rat IL 12A amino acid sequence (NP_445842.1; SEQ ID NO: 111) .

FIG. 38 shows the alignment between human IL 12B amino acid sequence (NP_002178.2; SEQ ID NO: 10) and mouse IL 12B amino acid sequence (NP_001290173.1; SEQ ID NO: 9) .

FIG. 39 shows the alignment between human IL 12B amino acid sequence (NP_002178.2; SEQ ID NO: 10) and rat IL 12B amino acid sequence (NP_072133.1; SEQ ID NO: 112.

FIG. 40 shows the alignment between human IL 12RB1 amino acid sequence (NP_005526.1; SEQ ID NO: 18) and mouse IL 12RB1 amino acid sequence (NP_032379.2; SEQ ID NO: 17) .

FIG. 41 shows the alignment between human IL 12RB1 amino acid sequence (NP_005526.1; SEQ ID NO: 18) and rat IL 12RB1 amino acid sequence (NP_001164075.1; SEQ ID NO: 113) .

FIG. 42 shows the alignment between human IL 12RB2 amino acid sequence (NP_001361188.1; SEQ ID NO: 29) and mouse IL 12RB2 amino acid sequence (NP_032380.1; SEQ ID NO: 28) .

FIG. 43 shows the alignment between human IL 12RB2 amino acid sequence (NP_001361188.1; SEQ ID NO: 29) and rat IL 12RB2 amino acid sequence (NP_001178679.1; SEQ ID NO: 70) .

DETAILED DESCRIPTION

This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) IL12A, and methods of use thereof.

Cytokines are among the chief players in controlling immune responses, and cytokine-based approaches for cancer therapy have been pursued in a number of ways. In that respect, the immunomodulatory cytokine IL-12, a key member of the IL-12 family of cytokines, emerged as a potent inducer of antitumor immunity. IL-12 was originally identified in 1989 as a natural killer (NK) cell-stimulatory factor with multiple biologic effects on peripheral blood lymphocytes. It is mainly produced by antigen-presenting cells (APCs) such as dendritic cells (DCs) , monocytes, macrophages and B cells upon Toll-like receptor engagement. Thus, IL-12 is secreted as an early pro-inflammatory cytokine in response to infections. Additional amplifying signals such as interferon-γ (IFN-γ) , IL-15, or cluster of differentiation (CD) 40L-CD40 cell-cell interactions are necessary for the optimal production of biologically active IL-12. Conversely, IL-12 is negatively regulated through cytokines such as IL-10 and transforming growth factor-β1 (TGF-β1) . IL-12 is a heterodimer with a molecular weight of 70 kDa consisting of a heavy (p40) and a light (p35) chain subunit, which are covalently linked by disulfide bonds. While p40 is produced in abundance by phagocytic cells, p35 is ubiquitously and constitutively expressed only at low levels and is thought to require p40 co-expression for secretion of the biologically active cytokine.

The sensing of IL-12 is mediated through the heterodimeric IL-12 receptor (IL-12R) composed of IL-12RB1 and IL-12RB2. Co-expression of both receptor subunits is required for the generation of high-affinity binding sites for IL-12. The IL-12R complex is found on NK cells, NK T and activated T cells but has also been detected on cell types of myeloid origin and tonsillar B cells. Naive T cells express IL-12RB1 but not IL-12RB2, which is critical for the signal transduction downstream of the receptor complex. Upon activation of T cells via the T-cell receptor, both IL-12 receptor chains are induced, which is additionally enhanced by IL-12 itself, IFN-γ, tumor necrosis factor-α (TNF-α) and anti-CD28 costimulation. Successful triggering of the receptor activates the Janus kinase-STAT (signal transducer and activator of transcription) signaling pathway, predominantly leading to STAT4 phosphorylation, which mediates subsequent cellular responses.

IL-12 has a key role in the regulation of inflammation by linking innate and adaptive immune responses. IL-12 release by microbe-sensing APCs results in subsequent activation and proliferation of NK and T cells and promotes their effector functions by inducing the transcription of cytokines and cytolytic factors such as perforin and granzyme B. Moreover, IL-12 polarizes T cells into a type 1 helper T (Th1) effector cell phenotype. Th1 polarization is further pronounced by IL-12 inhibiting the developmental program of type 2 helper T cells and interference with the differentiation of regulatory T cells (Tregs) and Th17 cells induced by TGF-β. Additionally, IL-12 programs effector T cells for optimal generation of effector memory T cells and T follicular helper cells. Direct effects of IL-12 on APCs have also been reported. Even though the activation of IL-12R in these cells did not involve the canonical STAT pathway, it increased their ability to present poorly immunogenic tumor peptides. Therefore, IL12 and IL12 receptor are regarded as potential therapeutic target for cancer.

Experimental animal models are an indispensable research tool for studying the effects of these antibodies (e.g., anti-IL12A 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 tech

nologies, 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.

IL12

IL-12 is a heterodimeric molecule composed of an alpha chain (IL12A, or the p35 subunit) and a beta chain (IL12B, or the p40 subunit) covalently linked by a disulfide bridge to form the biologically active 70 kDa dimer. Biologically, IL-12 is an inflammatory cytokine that is produced in response to infection by a variety of cells of the immune system, including phagocytic cells, B cells and activated dendritic cells. IL-12 plays an essential role in mediating the interaction of the innate and adaptive arms of the immune system, acting on T-cells and natural killer (NK) cells to enhance the proliferation and activity of cytotoxic lymphocytes and the production of other inflammatory cytokines, especially interferon-gamma (IFN-gamma) .

A detailed description of IL12 and its function can be found, e.g., in Tugues, S., et al. "New insights into IL-12-mediated tumor suppression. " Cell Death &Differentiation 22.2 (2015) : 237-246; Nguyen, K.G., et al. "Localized interleukin-12 for cancer immunotherapy. " Frontiers in immunology 11 (2020) : 575597; Colombo, M.P., et al. "Interleukin-12 in anti-tumor immunity and immunotherapy. " Cytokine &growth factor reviews 13.2 (2002) : 155-168; Habiba, U.E., et al. "The multifaceted role of IL-12 in cancer. " Advances in Cancer Biology-Metastasis (2022) : 100053; and Hamza, T., et al. "Interleukin 12 a key immunoregulatory cytokine in infection applications. " International Journal of Molecular Sciences 11.3 (2010) : 789-806; each of which is incorporated by reference in its entirety.

As discussed above, IL-12 is a heterodimeric molecule composed of IL12A and IL12B. The present disclosure provides genetically modified non-human animals comprising human or chimeric (e.g., humanized) IL12A and/or IL12B.

IL12A

In human genomes, IL12A gene (Gene ID: 3592) locus has seven exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7 (FIG. 1) . The IL12A protein also has a signal peptide. The nucleotide sequence for human IL12A mRNA is NM_000882.4, and the amino acid sequence for human IL12A is NP_000873.2 (SEQ ID NO: 2) . The location for each exon and each region in human IL12A nucleotide sequence and amino acid sequence is listed below:

Table 1

The human IL12A gene (Gene ID: 3592) is located in Chromosome 3 of the human genome, which is located from 159988835 to 159996019 of NC_000003.12. The 5' UTR is from 159988835 to 159989056, Exon 1 is from 159, 988, 835 to 159, 989, 174, the first intron is from 159, 989, 175 to 159, 990, 166, Exon 2 is from 159, 990, 167 to 159, 990, 312, the second intron is from 159, 990, 313 to 159, 993, 011, Exon 3 is from 159, 993, 012 to 159, 993, 125, the third intron is from 159, 993, 012 to 159, 993, 125, Exon 4 is from 159, 993, 451 to 159, 993, 492, the fourth intron is from 159, 993, 493 to 159, 993, 567, Exon 5 is from 159, 993, 568 to 159, 993, 609, the fifth intron is from 159, 993, 610 to 159, 993, 700, Exon 6 is from 159, 993, 844 to 159, 993, 701, the sixth intron is from 159, 995, 403 to 159, 993, 845, Exon 7 is from 159, 996, 019 to 159, 996, 019, and the 3'UTR is from 159995560 to 159996019, based on transcript NM_000882.4. All relevant information for mouse IL12A locus can be found in the NCBI website with Gene ID: 3592.

In mice, IL12A gene locus has seven exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7 (FIG. 1) . The mouse IL12A protein also has a signal peptide. The nucleotide sequence for mouse IL12A mRNA is NM_008351.3, the amino acid sequence for mouse IL12A is NP_032377.1 (SEQ ID NO: 1) . The location for each exon and each region in the mouse IL12A nucleotide sequence and amino acid sequence is listed below:

Table 2

The mouse IL12A gene (Gene ID: 16159) is located in Chromosome 3 of the mouse genome, which is located from 68597977 to 68605883 of NC_000069.7. The 5' UTR is from 68598757 to 68598883, Exon 1 is from 68, 598, 757 to 68, 598, 899, the first intron is from 68, 598, 900 to 68, 599, 355, Exon 2 is from 68, 599, 356 to 68, 599, 489, the second intron is from 68, 599, 490 to 68, 601, 493, Exon 3 is from 68, 601, 494 to 68, 601, 607, the third intron is from 68, 601, 608 to 68, 602, 522, Exon 4 is from 68, 602, 523 to 68, 602, 564, the fourth intron is from 68, 602, 565 to 68, 602, 637, Exon 5 is from 68, 602, 638 to 68, 602, 679, the fifth intron is from 68, 602, 680 to 68, 602, 769, Exon 6 is from 68, 602, 770 to 68, 602, 913, the sixth intron is from 68, 602, 914 to 68, 605, 227, Exon 7 is from 68, 605, 228 to 68, 605, 876, and the 3'UTR is from 68605384 to 68605876, based on transcript NM_008351.3. All relevant information for mouse IL12A locus can be found in the NCBI website with Gene ID: 16159, which is incorporated by reference herein in its entirety.

FIG. 36 shows the alignment between human IL12A amino acid sequence (NP_000873.2; SEQ ID NO: 2) and mouse IL12A amino acid sequence (NP_032377.1; SEQ ID NO: 1) . Thus, the corresponding amino acid residue or region between human and mouse IL12A can be found in FIG. 36.

IL12A genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for IL12A in Rattus norvegicus (rat) is 84405, the gene ID for IL12A in Macaca mulatta (Rhesus monkey) is 703205, the gene ID for IL12A in Canis lupus familiaris (dog) is 403977, and the gene ID for IL12A in Sus scrofa (pig) is 397053. 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. 37 shows the alignment between human IL12A amino acid sequence (NP_000873.2; SEQ ID NO: 2) and rat IL12A amino acid sequence (NP_445842.1; SEQ ID NO: 111) . Thus, the corresponding amino acid residue or region between human and rodent IL12A can be found in FIG. 37.

The present disclosure provides human or chimeric (e.g., humanized) IL12A 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, and/or exon 7 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, and/or exon 7 are replaced by the corresponding human sequence. The term “region” or “portion” can refer to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, or 1400 nucleotides, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acid residues. In some embodiments, the “region” or “portion” can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%identical to exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7. In some embodiments, a region, a portion, or the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 (e.g., a portion of exon 1, exons 2-6, and a portion of exon 7) are replaced by a region, a portion, or the entire sequence of the human exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 (e.g., a portion of exon 1, exons 2-6, and a portion of exon 7) .

In some embodiments, a “region” or “portion” of endogenous exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 is deleted.

Thus, in some embodiments, the present disclosure also provides a chimeric (e.g., humanized) IL12A 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 IL12A mRNA sequence (e.g., NM_008351.3) , mouse IL12A amino acid sequence (e.g., SEQ ID NO: 1) , or a portion thereof (e.g., 5' UTR, a portion of exon 1, a portion of exon 7, and 3' UTR) ; and in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%of the sequence are identical to or derived from human IL12A mRNA sequence (e.g., NM_000882.4) , human IL12A amino acid sequence (e.g., SEQ ID NO: 2) , or a portion thereof (e.g., a portion of exon 1, exons 2-6, and a portion of exon 7) .

In some embodiments, the sequence encoding amino acids 1-215 of mouse IL12A (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human IL12A (e.g., amino acids 1-253 of human IL12A (SEQ ID NO: 2) ) .

In some embodiments, the sequence encoding amino acids 23-215 of mouse IL12A (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human IL12A (e.g., amino acids 57-253 of human IL12A (SEQ ID NO: 2) ) .

In some embodiments, the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse IL12A promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from part of or the entire mouse IL12A nucleotide sequence (e.g., a portion of exon 1, exons 2-6, and a portion of exon 7 NM_008351.3) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as part of or the entire mouse IL12A nucleotide sequence (e.g., 5' UTR, a portion of exon 2, a portion of exon 7, and 3' UTR of NM_008351.3) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from part of or the entire human IL12A nucleotide sequence (e.g., 5' UTR, a portion of exon 1, a portion of exon 7, and 3' UTR of NM_000882.4) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as part of or the entire human IL12A nucleotide sequence (e.g., a portion of exon 1, exons 2-6, and a portion of exon 7 of NM_000882.4) .

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 different from part of or the entire mouse IL12A amino acid sequence (e.g., SEQ ID NO: 1) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire mouse IL12A amino acid sequence (e.g., SEQ ID NO: 1) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from part of or the entire human IL12A amino acid sequence (e.g., SEQ ID NO: 2) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire human IL12A amino acid sequence (e.g., SEQ ID NO: 2) .

The present disclosure also provides a humanized IL12A mouse amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

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

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

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

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

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

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

The present disclosure also relates to a IL12A nucleic acid (e.g., DNA or RNA) sequence, wherein the nucleic acid sequence can be selected from the group consisting of:

a) a nucleic acid sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, or 8, or a nucleic acid sequence encoding a homologous IL12A amino acid sequence of a humanized mouse IL12A;

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

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

d) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 1 or 2;

e) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%with, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 1 or 2;

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

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

The present disclosure further relates to a IL12A genomic DNA sequence of a humanized mouse. The DNA sequence is obtained by reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence homologous to the sequence shown in SEQ ID NO: 5 or 8.

IL12B

In human genomes, IL12B gene (Gene ID: 3593) locus has eight exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 (FIG. 7) . The IL12B protein also has a signal peptide. The nucleotide sequence for human IL12B mRNA is NM_002187.3, and the amino acid sequence for human IL12B is NP_002178.2 (SEQ ID NO: 10) . The location for each exon and each region in human IL12B nucleotide sequence and amino acid sequence is listed below:

Table 3

The human IL12B gene (Gene ID: 3593) is located in Chromosome 5 of the human genome, which is located from 159314780 to 159330487 of NC_000005.10. The 5' UTR is from 159, 330, 487 to 159, 330, 432, Exon 1 is from 159, 330, 487 to 159, 330, 432, the first intron is from 159, 330, 431 to 159, 326, 783, Exon 2 is from 159, 326, 782 to 159, 326, 695, the second intron is from 159, 326, 694 to 159, 323, 330, Exon 3 is from 159, 323, 329 to 159, 323, 054, the third intron is from 159, 323, 053 to 159, 322, 512, Exon 4 is from 159, 322, 511 to 159, 322, 394, the fourth intron is from 159, 322, 393 to 159, 320, 521, Exon 5 is from 159, 320, 520 to 159, 320, 306, the fifth intron is from 159, 320, 305 to 159, 318, 894, Exon 6 is from 159, 318, 893 to 159, 318, 736, the sixth intron is from 159, 316, 817 to 159, 318, 735, Exon 7 is from 159, 316, 816 to 159, 316, 685, the seventh intron is from 159, 316, 684 to 159, 316, 101, Exon 8 is from 159, 316, 100 to 159, 314, 780, and the 3'UTR is from 159, 316, 100 to 159, 314, 780, based on transcript NM_002187.3. All relevant information for mouse IL12B locus can be found in the NCBI website with Gene ID: 3593.

In mice, IL12B gene locus has eight exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 (FIG. 7) . The mouse IL12B protein also has a signal peptide. The nucleotide sequence for mouse IL12B mRNA is NM_001303244.1, the amino acid sequence for mouse IL12B is NP_001290173.1 (SEQ ID NO: 9) . The location for each exon and each region in the mouse IL12B nucleotide sequence and amino acid sequence is listed below:

Table 4

The mouse IL12B gene (Gene ID: 16160) is located in Chromosome 11 of the mouse genome, which is located from 44290890 to 44305504 of NC_000077.7. The 5' UTR is from 44, 290, 890 to 44, 290, 945, Exon 1 is from 44, 290, 890 to 44, 290, 945, the first intron is from 44, 290, 946 to 44, 294, 863, Exon 2 is from 44, 294, 864 to 44, 294, 951, the second intron is from 44, 294, 952 to 44, 298, 634, Exon 3 is from 44, 298, 635 to 44, 298, 901, the third intron is from 44, 298, 902 to 44, 299, 287, Exon 4 is from 44, 299, 288 to 44, 299, 405, the fourth intron is from 44, 299, 406 to 44, 300, 958, Exon 5 is from 44, 300, 959 to 44, 301, 176, the fifth intron is from 44, 301, 177 to 44, 301, 777, Exon 6 is from 44, 301, 778 to 44, 301, 956, the sixth intron is from 44, 301, 957 to 44, 303, 344, Exon 7 is from 44, 303, 345 to 44, 303, 469, the seventh intron is from 44, 303, 470 to 44, 304, 050, Exon 8 is from 44, 304, 051 to 44, 304, 860, and the 3'UTR is from 44304064 to 44304860, base d on transcript NM_001303244.1. All relevant information for mouse IL12B locus can be found in the NCBI website with Gene ID: 16160, which is incorporated by reference herein in its entirety.

FIG. 38 shows the alignment between human IL12B amino acid sequence (NP_002178.2; SEQ ID NO: 10) and mouse IL12B amino acid sequence (NP_001290173.1; SEQ ID NO: 9) . Thus, the corresponding amino acid residue or region between human and mouse IL12B can be found in FIG. 38.

IL12B genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for IL12B in Rattus norvegicus (rat) is 64546, the gene ID for IL12B in Macaca mulatta (Rhesus monkey) is 694747, the gene ID for IL12B in Canis lupusfamiliaris (dog) is 403976, and the gene ID for IL12B in Sus scrofa (pig) is 397076. 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. 39 shows the alignment between human IL12B amino acid sequence (NP_002178.2; SEQ ID NO: 10) and rat IL12B amino acid sequence (NP_072133.1; SEQ ID NO: 112) . Thus, the corresponding amino acid residue or region between human and rodent IL12B can be found in FIG. 39.

The present disclosure provides human or chimeric (e.g., humanized) IL12B 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, and/or exon 8 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, and/or exon 8 are replaced by the corresponding human sequence. The term “region” or “portion” can refer to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, or 2400 nucleotides, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, or 330 amino acid residues. In some embodiments, the “region” or “portion” can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%identical to exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or exon 8. 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., exons 2-8) are replaced by a region, a portion, or the entire sequence of the human exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 (e.g., exons 2-8) .

In some embodiments, a “region” or “portion” of endogenous exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 is deleted.

Thus, in some embodiments, the present disclosure also provides a chimeric (e.g., humanized) IL12B 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 IL12B mRNA sequence (e.g., NM_001303244.1) , mouse IL12B amino acid sequence (e.g., SEQ ID NO: 9) , or a portion thereof (e.g., 5' UTR and exon 1) ; 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 IL12B mRNA sequence (e.g., NM_002187.3) , human IL12B amino acid sequence (e.g., SEQ ID NO: 10) , or a portion thereof (e.g., exons 2-8 and 3' UTR) .

In some embodiments, the sequence encoding amino acids 1-335 of mouse IL12B (SEQ ID NO: 9) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human IL12B (e.g., amino acids 1-328 of human IL12B (SEQ ID NO: 10) ) .

In some embodiments, the sequence encoding amino acids 23-335 of mouse IL12B (SEQ ID NO: 9) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human IL12B (e.g., amino acids 23-328 of human IL12B (SEQ ID NO: 10) ) .

In some embodiments, the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse IL12B promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from part of or the entire mouse IL12B nucleotide sequence (e.g., exons 2-8 and 3' UTR of NM_001303244.1) .

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 part of or the entire mouse IL12B nucleotide sequence (e.g., 5' UTR and exon 1 of NM_001303244.1) .

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 part of or the entire human IL12B nucleotide sequence (e.g., 5' UTR and exon 1 of NM_002187.3) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as part of or the entire human IL12B nucleotide sequence (e.g., exons 2-8 and 3' UTR of NM_002187.3) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from part of or the entire mouse IL12B amino acid sequence (e.g., SEQ ID NO: 9) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire mouse IL12B amino acid sequence (e.g., SEQ ID NO: 9) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from part of or the entire human IL12B amino acid sequence (e.g., SEQ ID NO: 10) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire human IL12B amino acid sequence (e.g., SEQ ID NO: 10) .

The present disclosure also provides a humanized IL12B mouse amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

a) an amino acid sequence shown in SEQ ID NO: 9 or 10;

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: 9 or 10;

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: 9 or 10 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: 9 or 10;

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

f) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 9 or 10.

The present disclosure also relates to a IL12B 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: 11, 12, 13, 14, 15, or 16, or a nucleic acid sequence encoding a homologous IL12B amino acid sequence of a humanized mouse IL12B;

b) a nucleic acid sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 11, 12, 13, 14, 15, or 16 under a low stringency condition or a strict stringency condition;

c) a nucleic acid sequence that has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the nucleotide sequence as shown in SEQ ID NO: 11, 12, 13, 14, 15, or 16;

d) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 9 or 10;

e) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%with, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 9 or 10;

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

g) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence comprises a substitution, a deletion and/or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 9 or 10.

The present disclosure further relates to a IL12B genomic DNA sequence of a humanized mouse. The DNA sequence is obtained by reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence homologous to the sequence shown in SEQ ID NO: 13 or 16.

IL12R

IL-12 signals through a receptor complex of IL-12RB1 and IL-12RB2 expressed on natural killer (NK) cells and T cells. Dimerization of the IL-12 receptor induces activation of the receptor-associated JAK molecules JAK2 and Tyk2, which trans-phosphorylate one another as well as tyrosine residues in the intracellular domain of IL-12RB2, which serve as docking sites for the SH2-containing STAT4. Receptor-associated STAT4 proteins are then phosphorylated before translocating to the nucleus, where they promote the expression of interferon gamma (IFNγ) and the polarization of CD4+ T cells toward a T helper 1 (Th1) phenotype.

A detailed description of IL12 receptor and its function can be found, e.g., in Presky, D. H., et al. "A functional interleukin 12 receptor complex is composed of two β-type cytokine receptor subunits. " Proceedings of the National Academy of Sciences 93.24 (1996) : 14002-14007; Robinson, R.T. "IL12Rβ1: the cytokine receptor that we used to know. " Cytokine 71.2 (2015) : 348-359; and Glassman, C.R., et al. "Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells. " Cell 184.4 (2021) : 983-999; each of which is incorporated by reference in its entirety.

As discussed above, IL-12 receptor (IL12R) is a heterodimeric molecule composed of IL12RB1 (or IL12Rβ1) and IL12RB2 (or IL12Rβ2) . The present disclosure provides genetically modified non-human animals comprising human or chimeric (e.g., humanized) IL12RB1 and/or IL12RB2.

IL12RB1

In human genomes, IL12RB1 gene (Gene ID: 3594) locus has seventeen exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and exon 17 (FIG. 13) . The IL12RB1 protein also has a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. The nucleotide sequence for human IL12RB1 mRNA is NM_005535.3, and the amino acid sequence for human IL12RB1 is NP_005526.1 (SEQ ID NO: 18) . The location for each exon and each region in human IL12RB1 nucleotide sequence and amino acid sequence is listed below:

Table 5

The human IL12RB1 gene (Gene ID: 3594) is located in Chromosome 19 of the human genome, which is located from 18058994 to 18099027 of NC_000019.10. The 5' UTR is from 18086824 to 18086934, Exon 1 is from 18086934 to 18086760, the first intron is from 18086759 to 18083492, Exon 2 is from 18083491 to 18083432, the second intron is from 18083431 to 18082265, Exon 3 is from 18082264 to 18082150, the third intron is from 18082149 to 18081002, Exon 4 is from 18081001 to 18080832, the fourth intron is from 18080831 to 18077656, Exon 5 is from 18077655 to 18077516, the fifth intron is from 18077515 to 18076328, Exon 6 is from 18076327 to 18076297, the sixth intron is from 18076296 to 18075869, Exon 7 is from 18075868 to 18075749, the seventh intron is from 18075748 to 18073600, Exon 8 is from 18073599 to 18073517, the eighth intron is from 18073516 to 18072350, Exon 9 is from 18072349 to 18072112, the ninth intron is from 18072111 to 18069714, Exon 10 is from 18069713 to 18069546, the tenth intron is from 18069545 to 18068527, Exon 11 is from 18068526 to 18068389, the eleventh intron is from 18068388 to 18066698, Exon 12 is from 18066697 to 18066542, the twelfth intron is from 18066541 to 18064011, Exon 13 is from 18064010 to 18063876, the thirteenth intron is from 18063875 to 18062278, Exon 14 is from 18062277 to 18062181, the fourteenth intron is from 18062180 to 18061198, Exon 15 is from 18061197 to 18061122, the fifteenth intron is from 18061121 to 18060086, Exon 16 is from 18060085 to 18059894, the sixteenth intron is from 18059893 to 18059614, Exon 17 is from 18059613 to 18058995, and the 3'UTR is from 18058995 to 18059607, based on transcript NM_005535.3. All relevant information for mouse IL12rb 1 locus can be found in the NCBI website with Gene ID: 3594.

In mice, IL12RB1 gene locus has sixteen exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, and exon 16 (FIG. 13) . The mouse IL12RB1 protein also has a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. The nucleotide sequence for mouse IL12RB1 mRNA is NM_008353.2, the amino acid sequence for mouse IL12RB1 is NP_032379.2 (SEQ ID NO: 17) . The location for each exon and each region in the mouse IL12RB1 nucleotide sequence and amino acid sequence is listed below:

Table 6

The mouse TFR1 gene (Gene ID: 16161) is located in Chromosome 8 of the mouse genome, which is located from 71261005 to 71276186 of NC_000074.7. The 5' UTR is from 71261093 to 71261195, Exon 1 is from 71261093 to 71261262, the first intron is from 71261263 to 71262474, Exon 2 is from 71262475 to 71262534, the second intron is from 71262535 to 71263198, Exon 3 is from 71263199 to 71263313, the third intron is from 71263314 to 71263682, Exon 4 is from 71263683 to 71263876, the fourth intron is from 71263877 to 71265121, Exon 5 is from 71265122 to 71265258, the fifth intron is from 71265259 to 71265948, Exon 6 is from 71265949 to 71266015, the sixth intron is from 71266016 to 71266296, Exon 7 is from 71266297 to 71266422, the seventh intron is from 71266423 to 71266709, Exon 8 is from 71266710 to 71266792, the eighth intron is from 71266793 to 71267080, Exon 9 is from 71267081 to 71267318, the ninth intron is from 71267319 to 71268345, Exon 10 is from 71268346 to 71268513, the tenth intron is from 71268514 to 71269076, Exon 11 is from 71269077 to 71269211, the eleventh intron is from 71269212 to 71269527, Exon 12 is from 71269528 to 71269683, the twelfth intron is from 71269684 to 71269855, Exon 13 is from 71269856 to 71269990, the thirteenth intron is from 71269991 to 71271968, Exon 14 is from 71271969 to 71272065, the fourteenth intron is from 71272066 to 71272325, Exon 15 is from 71272326 to 71272407, the fifteenth intron is from 71272408 to 71273158, Exon 16 is from 71273159 to 71274068, and the 3'UTR is from 71273516 to 71274068, based on transcript NM_008353.2. All relevant information for mouse IL12RB1 locus can be found in the NCBI website with Gene ID: 16161, which is incorporated by reference herein in its entirety.

FIG. 40 shows the alignment between human IL12RB1 amino acid sequence (NP_005526.1; SEQ ID NO: 18) and mouse IL12RB1 amino acid sequence (NP_032379.2; SEQ ID NO: 17) . Thus, the corresponding amino acid residue or region between human and mouse IL12RB1 can be found in FIG. 40.

IL12RB1 genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for IL12RB1 in Rattus norvegicus (rat) is 171333, the gene ID for IL12RB1 in Macaca mulatta (Rhesus monkey) is 574199, the gene ID for IL12RB1 in Canis lupusfamiliaris (dog) is 484828, and the gene ID for IL12RB1 in Sus scrofa (pig) is 100271900. 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. 41 shows the alignment between human IL12RB1 amino acid sequence (NP_005526.1; SEQ ID NO: 18) and rat IL12RB1 amino acid sequence (NP_001164075.1; SEQ ID NO: 113) . Thus, the corresponding amino acid residue or region between human and rodent IL12RB1 can be found in FIG. 41.

The present disclosure provides human or chimeric (e.g., humanized) IL12RB1 nucleotide sequence and/or amino acid sequences. In some embodiments, the entire sequence of mouse exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region are replaced by human sequences. In some embodiments, a “region” or “portion” of mouse exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, signal peptide, extracellular region, transmembrane regions, and/or cytoplasmic regions are replaced by human sequences. 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, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, or 2800 nucleotides, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 amino acid residues. In some embodiments, the “region” or “portion” can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%identical to exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, signal peptide, extracellular region, transmembrane region, or cytoplasmic region. In some embodiments, a region, a portion, or the entire sequence of mouse exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16 (e.g., a portion of exon 1 and a portion of intron 1) is replaced by a sequence including a region, a portion, or the entire sequence of the human exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and/or exon 17 (e.g., a portion of exon 2, exons 3-13, and a portion of exon 14) .

In some embodiments, a “region” or “portion” of the signal peptide, extracellular region, transmembrane region, cytoplasmic region, exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, and/or exon 16 of endogenous IL12RB1 protein or endogenous IL12RB1 gene is deleted.

Thus, in some embodiments, the present disclosure also provides a chimeric (e.g., humanized) IL12RB1 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 IL12RB1 mRNA sequence (e.g., NM_008353.2) , mouse IL12RB1 amino acid sequence (e.g., SEQ ID NO: 17) , or a portion thereof (e.g., a portion of exon 1, a portion of exon 14, exon 15, and a portion of exon 16) ; 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 IL12RB1 mRNA sequence (e.g., NM_005535.3) , human IL12RB1 amino acid sequence (e.g., SEQ ID NO: 18) , or a portion thereof (e.g., a portion of exon 2, exons 3-13, and a portion of exon 14) .

In some embodiments, the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse IL12RB1 promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from part of or the entire mouse IL12RB1 nucleotide sequence (e.g., all or a portion of exons 1-14 of NM_008353.2) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as part of or the entire mouse IL12RB1 nucleotide sequence (e.g., a portion of exon 1, a portion of exon 14, exon 15, and a portion of exon 16 of NM_008353.2) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from part of or the entire human IL12RB1 nucleotide sequence (e.g., all or a portion of exons 1-2 and 14-17 of NM_005535.3) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as part of or the entire human IL12RB1 nucleotide sequence (e.g., a portion of exon 2, exons 3-13, and a portion of exon 14 of NM_005535.3) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from part of or the entire mouse IL12RB1 amino acid sequence (e.g., amino acids 20-591 of NP_032379.2 (SEQ ID NO: 17) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire mouse IL12RB1 amino acid sequence (e.g., amino acids 1-19 and 592-738 of NP_032379.2 (SEQ ID NO: 17) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from part of or the entire human IL12RB1 amino acid sequence (e.g., amino acids 1-23 and 571-662 of NP_005526.1 (SEQ ID NO: 18) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire human IL12RB1 amino acid sequence (e.g., amino acids 24-570 of NP_005526.1 (SEQ ID NO: 18) ) .

The present disclosure also provides a humanized IL12RB1 mouse amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

a) an amino acid sequence shown in SEQ ID NO: 17, 18, or 27;

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: 17, 18, or 27;

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: 17, 18, or 27 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: 17, 18, or 27;

e) an amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 17, 18, or 27 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

f) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 17, 18, or 27.

The present disclosure also provides a humanized IL12RB1 amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

a) all or part of amino acids 24-570 of SEQ ID NO: 18;

b) an amino acid sequence have a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%to amino acids 24-570 of SEQ ID NO: 18;

c) an amino acid sequence that is different from amino acids 24-570 of SEQ ID NO: 18 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and

d) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to amino acids 24-570 of SEQ ID NO: 18.

The present disclosure also relates to a IL12RB1 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: 19, 20, 21, 22, 23, 26, or 48, or a nucleic acid sequence encoding a homologous IL12RB1 amino acid sequence of a humanized mouse IL12RB1;

b) a nucleic acid sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 19, 20, 21, 22, 23, 26, or 48 under a low stringency condition or a strict stringency condition;

c) a nucleic acid sequence that has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the nucleotide sequence as shown in SEQ ID NO: 19, 20, 21, 22, 23, 26, or 48;

d) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 17, 18, or 27;

e) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%with, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 17, 18, or 27;

f) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence is different from the amino acid sequence shown in SEQ ID NO: 17, 18, or 27 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and/or

g) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence comprises a substitution, a deletion and/or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 17, 18, or 27.

The present disclosure further relates to a IL12RB1 genomic DNA sequence of a humanized mouse. The DNA sequence is obtained by reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence homologous to the sequence shown in SEQ ID NO: 21, 26, or 48.

IL12RB2

In human genomes, IL12RB2 gene (Gene ID: 3595) locus has seventeen exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and exon 17 (FIG. 19) . The IL12RB2 protein also has a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. The nucleotide sequence for human IL12RB2 mRNA is NM_001374259.2, and the amino acid sequence for human IL12RB2 is NP_001361188.1 (SEQ ID NO: 29) . The location for each exon and each region in human IL12RB2 nucleotide sequence and amino acid sequence is listed below:

Table 7

The human IL12RB2 gene (Gene ID: 3595) is located in Chromosome 1 of the human genome, which is located from 67307351 to 67398724 of NC_000001.11. The 5' UTR is from 67,307,873 to 67,307,967, from 67,313,913 to 67,314,000, and from 67320333 to 67320368, Exon 1 is from 67307873 to 67307967, the first intron is from 67307968 to 67313912, Exon 2 is from 67313913 to 67314000, the second intron is from 67314001 to 67320332, Exon 3 is from 67320333 to 67320444, the third intron is from 67320445 to 67321601, Exon 4 is from 67321602 to 67321889, the fourth intron is from 67321890 to 67326734, Exon 5 is from 67326735 to 67326849, the fifth intron is from 67326850 to 67328199, Exon 6 is from 67328200 to 67328384, the sixth intron is from 67328385 to 67329586, Exon 7 is from 67329587 to 67329729, the seventh intron is from 67329730 to 67330659, Exon 8 is from 67330660 to 67330810, the eighth intron is from 67330811 to 67338623, Exon 9 is from 67338624 to 67338703, the ninth intron is from 67338704 to 67350869, Exon 10 is from 67350870 to 67351089, the tenth intron is from 67351090 to 67367824, Exon 11 is from 67367825 to 67368025, the eleventh intron is from 67368026 to 67372435, Exon 12 is from 67372436 to 67372534, the twelfth intron is from 67372535 to 67372624, Exon 13 is from 67372625 to 67372783, the thirteen intron is from 67372784 to 67379985, Exon 14 is from 67379986 to 67380123, the fourteenth intron is from 67380124 to 67386578, Exon 15 is from 67386579 to 67386669, the fifteenth intron is from 67386670 to 67390028, Exon 16 is from 67390029 to 67390128, the sixteenth intron is from 67390129 to 67395546, Exon 17 is from 67395547 to 67398724, and the 3' UTR is from 67396090 to 67398724, based on transcript NM_001374259.2. All relevant information for mouse IL12RB2 locus can be found in the NCBI website with Gene ID: 3595.

In mice, IL12RB2 gene locus has sixteen exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, and exon 16 (FIG. 19) . The mouse IL12RB2 protein also has a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. The nucleotide sequence for mouse IL12RB2 mRNA is NM_008354.4, the amino acid sequence for mouse IL12RB2 is NP_032380.1 (SEQ ID NO: 28) . The location for each exon and each region in the mouse IL12RB2 nucleotide sequence and amino acid sequence is listed below:

Table 8

The mouse IL12RB2 gene (Gene ID: 16162) is located in Chromosome 6 of the human genome, which is located from 67263914 to 67353277 of NC_000003.12. The 5' UTR is from 67,353,172 to 67,353,019, and from 67338935 to 67338968, Exon 1 is from 67353172 to 67353019, the first intron is from 67353018 to 67338969, Exon 2 is from 67338968 to 67338859, the second intron is from 67338858 to 67337804, Exon 3 is from 67337803 to 67337477, the third intron is from 67337476 to 67334775, Exon 4 is from 67334774 to 67334660, the fourth intron is from 67334659 to 67333760, Exon 5 is from 67333759 to 67333566, the fifth intron is from 67333565 to 67331852, Exon 6 is from 67331851 to 67331709, the sixth intron is from 67331708 to 67328374, Exon 7 is from 67328373 to 67328223, the seventh intron is from 67328222 to 67313751, Exon 8 is from 67313750 to 67313671, the eighth intron is from 67313670 to 67293392, Exon 9 is from 67293391 to 67293175, the ninth intron is from 67293174 to 67286143, Exon 10 is from 67286142 to 67285942, the tenth intron is from 67285941 to 67280782, Exon 11 is from 67280781 to 67280683, the eleventh intron is from 67280682 to 67280603, Exon 12 is from 67280602 to 67280444, the twelfth intron is from 67280443 to 67276030, Exon 13 is from 67276029 to 67275892, the thirteenth intron is from 67275891 to 67275224, Exon 14 is from 67275223 to 67275133, the fourteenth intron is from 67275132 to 67272296, Exon 15 is from 67272295 to 67272196, the fifteenth intron is from 67272195 to 67269721, Exon 16 is from 67269720 to 67269002, and the 3' UTR is from 67269002 to 67269186, based on transcript NM_008354.4. All relevant information for mouse IL12rb2 locus can be found in the NCBI website with Gene ID.: 16162.

FIG. 42 shows the alignment between human IL12RB2 amino acid sequence (NP_001361188.1; SEQ ID NO: 29) and mouse IL12RB2 amino acid sequence (NP_032380.1; SEQ ID NO: 28) . Thus, the corresponding amino acid residue or region between human and mouse IL12RB2 can be found in FIG. 42.

IL12RB2 genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for IL12RB2 in Rattus norvegicus (rat) is 171334, the gene ID for IL12RB2 in Macaca mulatta (Rhesus monkey) is 700677, the gene ID for IL12RB2 in Canis lupus familiaris (dog) is 489550, and the gene ID for IL12RB2 in Sus scrofa (pig) is 397178. 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. 43 shows the alignment between human IL12RB2 amino acid sequence (NP_001361188.1; SEQ ID NO: 29) and rat IL12RB2 amino acid sequence (NP_001178679.1; SEQ ID NO: 70) . Thus, the corresponding amino acid residue or region between human and rodent IL12RB2 can be found in FIG. 43.

The present disclosure provides human or chimeric (e.g., humanized) IL12RB2 nucleotide sequence and/or amino acid sequences. In some embodiments, the entire sequence of mouse exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region are replaced by human sequences. In some embodiments, a “region” or “portion” of mouse exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, signal peptide, extracellular region, transmembrane regions, and/or cytoplasmic regions are replaced by human sequences. 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, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 4000, or 5000 nucleotides, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 850 amino acid residues. In some embodiments, the “region” or “portion” can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%identical to exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, signal peptide, extracellular region, transmembrane region, or cytoplasmic region. In some embodiments, a region, a portion, or the entire sequence of mouse exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16 (e.g., a portion of exon 2 and a portion of intron 2) is replaced by a sequence including a region, a portion, or the entire sequence of the human exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and/or exon 17 (e.g., a portion of exon 4, exons 5-14, and a portion of exon 15) .

In some embodiments, a “region” or “portion” of the signal peptide, extracellular region, transmembrane region, cytoplasmic region, exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, and/or exon 16 of endogenous IL12RB2 protein or endogenous IL12RB2 gene is deleted.

Thus, in some embodiments, the present disclosure also provides a chimeric (e.g., humanized) IL12RB2 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 IL12RB2 mRNA sequence (e.g., NM_008354.4) , mouse IL12RB2 amino acid sequence (e.g., SEQ ID NO: 28) , or a portion thereof (e.g., exon 1, a portion of exon 2, a portion ofexon 14, exon 15, and a portion exon 16) ; 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 IL12RB2 mRNA sequence (e.g., NM_001374259.2) , human IL12RB2 amino acid sequence (e.g., SEQ ID NO: 29) , or a portion thereof (e.g., a portion of exon 4, human exons 5-14, and a portion of exon 15) .

In some embodiments, the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse IL12RB2 promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from part of or the entire mouse IL12RB2 nucleotide sequence (e.g., all or a portion of exons 2-14 of NM_008354.4) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as part of or the entire mouse IL12RB2 nucleotide sequence (e.g., exon 1, a portion ofexon 2, a portion of exon 14, exon 15, and a portion exon 16 of NM_008354.4) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from part of or the entire human IL12RB2 nucleotide sequence (e.g., all or a portion ofexons 1-4 and 15-17 of NM_001374259.2) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as part of or the entire human IL12RB2 nucleotide sequence (e.g., a portion of exon 4, human exons 5-14, and a portion of exon 15 of NM_001374259.2) .

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 part of or the entire mouse IL12RB2 amino acid sequence (e.g., amino acids 24-637 of NP_032380.1 (SEQ ID NO: 28) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire mouse IL12RB2 amino acid sequence (e.g., amino acids 1-23 and 638-874 of NP_032380.1 (SEQ ID NO: 28) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from part of or the entire human IL12RB2 amino acid sequence (e.g., amino acids 1-23 and 623-862 of NP_001361188.1 (SEQ ID NO: 29) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire human IL12RB2 amino acid sequence (e.g., amino acids 24-622 or 28-622 of NP_001361188.1 (SEQ ID NO: 29) ) .

The present disclosure also provides a humanized IL12RB2 mouse amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

a) an amino acid sequence shown in SEQ ID NO: 28, 29, or 36;

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: 28, 29, or 36;

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: 28, 29, or 36 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: 28, 29, or 36;

e) an amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 28, 29, or 36 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

f) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 28, 29, or 36.

The present disclosure also provides a humanized IL12RB2 amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

a) all or part of amino acids 24-622 or 28-622 of SEQ ID NO: 29;

b) an amino acid sequence have a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%to amino acids 24-622 or 28-622 of SEQ ID NO: 29;

c) an amino acid sequence that is different from amino acids 24-622 or 28-622 of SEQ ID NO: 29 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and

d) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to amino acids 24-622 or 28-622 of SEQ ID NO: 29.

The present disclosure also relates to a IL12RB2 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: 30, 31, 34, 35, 37, 38, or 65, or a nucleic acid sequence encoding a homologous IL12RB2 amino acid sequence of a humanized mouse IL12RB2;

b) a nucleic acid sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 30, 31, 34, 35, 37, 38, or 65 under a low stringency condition or a strict stringency condition;

c) a nucleic acid sequence that has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the nucleotide sequence as shown in SEQ ID NO: 30, 31, 34, 35, 37, 38, or 65;

d) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 28, 29, or 36;

e) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%with, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 28, 29, or 36;

f) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence is different from the amino acid sequence shown in SEQ ID NO: 28, 29, or 36 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and/or

g) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence comprises a substitution, a deletion and/or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 28, 29, or 36.

The present disclosure further relates to a IL12RB2 genomic DNA sequence of a humanized mouse. The DNA sequence is obtained by reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence homologous to the sequence shown in SEQ ID NO: 34, 35, or 65.

The disclosure also provides an amino acid sequence that has a homology of at least 90%with, or at least 90%identical to the sequence shown in SEQ ID NO: 1, 2, 9, 10, 17, 18, 27, 28, 29, or 36, and has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 1, 2, 9, 10, 17, 18, 27, 28, 29, or 36 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing homology is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 1, 2, 9, 10, 17, 18, 27, 28, 29, or 36 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

The disclosure also provides a nucleotide sequence that has a homology of at least 90%, or at least 90%identical to the sequence shown in SEQ ID NO: 5, 8, 13, 16, 21, 26, 34, 35, 48, or 65, and encodes a polypeptide that has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 5, 8, 13, 16, 21, 26, 34, 35, 48, or 65 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing homology is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 26, 30, 31, 34, 35, 37, 38, 48, or 65 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, or200 amino acid residues.

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

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

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

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

Cells, tissues, and animals (e.g., mouse) are also provided that comprise the nucleotide sequences as described herein, as well as cells, tissues, and animals (e.g., mouse) that express human or chimeric (e.g., humanized) IL12A from an endogenous non-human IL12A locus, human or chimeric (e.g., humanized) IL12B from an endogenous non-human IL12B locus, human or chimeric (e.g., humanized) IL12RB1 from an endogenous non-human IL12RB1 locus, and/or human or chimeric (e.g., humanized) IL12RB2 from an endogenous non-human IL12RB2 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 NK 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 IL12A, IL12B, IL12RB1, and/or IL12RB2 loci that comprise 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, or an insertion of one or more human and/or non-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 IL12A gene or a humanized IL12A nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human IL12A gene, at least one or more portions of the gene or the nucleic acid is from a non-human IL12A gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes an IL12A protein. The encoded IL12A protein is functional or has at least one activity of the human IL12A protein or the non-human IL12A protein, e.g., interacting with IL12B to form a functional IL12 heterodimer.

In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized IL12B gene or a humanized IL12B nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human IL12B gene, at least one or more portions of the gene or the nucleic acid is from a non-human IL12B gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes an IL12B protein. The encoded IL12B protein is functional or has at least one activity of the human IL12B protein or the non-human IL12B protein, e.g., interacting with IL12A to form a functional IL12 heterodimer.

In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized IL12RB1 gene or a humanized IL12RB1 nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human IL12RB1 gene, at least one or more portions of the gene or the nucleic acid is from a non-human IL12RB1 gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes an IL12RB1 protein. The encoded IL12RB1 protein is functional or has at least one activity of the human IL12RB1 protein or the non-human IL12RB1 protein, e.g., interacting with IL12RB2 to form a functional IL12 receptor.

In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized IL12RB2 gene or a humanized IL12RB2 nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human IL12RB2 gene, at least one or more portions of the gene or the nucleic acid is from a non-human IL12RB2 gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes an IL12RB2 protein. The encoded IL12RB2 protein is functional or has at least one activity of the human IL12RB2 protein or the non-human IL12RB2 protein, e.g., interacting with IL12RB1 to form a functional IL12 receptor.

In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized IL12A protein or a humanized IL12A 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 IL12A protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human IL12A protein. The humanized IL12A protein or the humanized IL12A polypeptide is functional or has at least one activity of the human IL12A protein or the non-human IL12A protein.

In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized IL12B protein or a humanized IL12B 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 IL12B protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human IL12B protein. The humanized IL12B protein or the humanized IL12B polypeptide is functional or has at least one activity of the human IL12B protein or the non-human IL12B protein.

In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized IL12RB1 protein or a humanized IL12RB1 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 IL12RB1 protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human IL12RB1 protein. The humanized IL12RB1 protein or the humanized IL12RB1 polypeptide is functional or has at least one activity of the human IL12RB1 protein or the non-human IL12RB1 protein.

In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized IL12RB2 protein or a humanized IL12RB2 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 IL12RB2 protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human IL12RB2 protein. The humanized IL12RB2 protein or the humanized IL12RB2 polypeptide is functional or has at least one activity of the human IL12RB2 protein or the non-human IL12RB2 protein.

In some embodiments, the extracellular region is human or humanized. In some embodiments, the transmembrane region is human or humanized. In some embodiments, the cytoplasmic region is human or humanized.

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

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

In some embodiments, the animal is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In some embodiments, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm) , 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac) , 129S7, 129S8, 129T1, 129T2. These mice are described, e.g., in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999) ; Auerbach et al., Establishment and Chimera Analysis of 129/SvEv-and C57BL/6-Derived Mouse Embryonic Stem Cell Lines (2000) , both of which are incorporated herein by reference in the entirety. In some embodiments, the genetically modified mouse is a mix of the 129 strain and the C57BL/6 strain. In some embodiments, the mouse is a mix of the 129 strains, or a mix of the BL/6 strains. In some embodiments, the mouse is a BALB strain, e.g., BALB/c strain. In some embodiments, the mouse is a mix of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50%BALB/c-50%12954/Sv; or 50%C57BL/6-50%129) . In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse having a BALB/c, A, A/He, A/J, A/WySN, AKR, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL (C57BL/10Cr and C57BL/Ola) , C58, CBA/Br, CBA/Ca, CBA/J, CBA/st, or CBA/H background.

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

The animal can have one or more other genetic modifications, and/or other modifications, that are suitable for the particular purpose for which the humanized IL12A, IL12B, IL12RB1, and/or IL12RB2 animal is made. For example, suitable mice for maintaining a xenograft (e.g., a human cancer or tumor) , can have one or more modifications that compromise, inactivate, or destroy the immune system of the non-human animal in whole or in part. Compromise, inactivation, or destruction of the immune system of the non-human animal can include, for example, destruction of hematopoietic cells and/or immune cells by chemical means (e.g., administering a toxin) , physical means (e.g., irradiating the animal) , and/or genetic modification (e.g., knocking out one or more genes) . Non-limiting examples of such mice include, e.g., NOD mice, SCID mice, NOD/SCID mice, IL2Rγ knockout mice, NOD/SCID/γc ^null mice (Ito, M. et al., NOD/SCID/γc ^null mouse: an excellent recipient mouse model for engraftment of human cells, Blood 100 (9) : 3175-3182, 2002) , nude mice, and Rag1 and/or Rag2 knockout mice. These mice can optionally be irradiated, or otherwise treated to destroy one or more immune cell type. Thus, in various embodiments, a genetically modified mouse is provided that can include a humanization of at least a portion of an endogenous non-human IL12A, IL12B, IL12RB1, and/or IL12RB2 loci, and further comprises a modification that compromises, inactivates, or destroys the immune system (or one or more cell types of the immune system) of the non-human animal in whole or in part. In some embodiments, modification is, e.g., selected from the group consisting of a modification that results in NOD mice, SCID mice, NOD/SCID mice, IL-2Rγ knockout mice, NOD/SCID/γc ^null mice, nude mice, Rag1 and/or Rag2 knockout mice, NOD- Prkdcscid IL-2rγ ^null mice, NOD-Rag 1 ^-/--IL2rg ^-/- (NRG) mice, Rag 2 ^-/--IL2rg ^-/- (RG) mice, and a combination thereof. These genetically modified animals are described, e.g., in US20150106961, which is incorporated herein by reference in its entirety. In some embodiments, the mouse can include a replacement of all or part of mature IL12A coding sequence with human mature IL12A coding sequence. In some embodiments, the mouse can include a replacement of all or part of mature IL12B coding sequence with human mature IL12B coding sequence. In some embodiments, the mouse can include an insertion of a chimeric (e.g., human/non-human) IL12RB1 coding sequence at an endogenous IL12RB1 locus. In some embodiments, the mouse can include an insertion of a chimeric (e.g., human/non-human) IL12RB2 coding sequence at an endogenous IL12RB2 locus.

Genetically modified non-human animals can comprise a modification at endogenous non-human IL12A, IL12B, IL12RB1, and/or IL12RB2 loci. In some embodiments, the modification can comprise a human nucleic acid sequence encoding at least a portion of a mature IL12A protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the mature IL12A, IL12B, IL12RB1, or IL12RB2 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 IL12A, IL12B, IL12RB1, and/or IL12RB2 loci in the germline of the animal.

In some embodiments, the present disclosure is related to a genetically-modified, non-human animal whose genome comprises a human or chimeric (e.g., humanized ) IL12A nucleotide sequence. In some embodiments, the human or chimeric (e.g., humanized) IL12A nucleotide sequence encodes a IL12A protein that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 2. In some embodiments, the genome of the animal comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 3, 4, 5, 6, 7, or 8.

In some embodiments, the genetically-modified non-human animal described herein comprises a sequence encoding a human or humanized IL12A protein. In some embodiments, the humanized IL12A protein comprises a human or humanized signal peptide. In some embodiments, the humanized IL12A protein comprises an endogenous signal peptide.

In some embodiments, the genetically-modified non-human animal described herein comprises a human or humanized IL12A gene. In some embodiments, the humanized IL12A gene comprises 7 exons. In some embodiments, the humanized IL12A gene comprises humanized exon 1, human exon 2, human exon 3, human exon 4, human exon 5, human exon 6, and/or humanized exon 7. In some embodiments, the humanized IL12A gene comprises 6 introns. In some embodiments, the humanized IL12A gene comprises human intron 1, human intron 2, human intron 3, human intron 4, human intron 5, and/or human intron 6. In some embodiments, the humanized IL12A gene comprises human or humanized 5' UTR. In some embodiments, the humanized IL12A gene comprises human or humanized 3' UTR. In some embodiments, the humanized IL12A gene comprises endogenous 5' UTR. In some embodiments, the humanized IL12A gene comprises endogenous 3' UTR.

In some embodiments, the genetically modified animals can express a human IL12A and/or a chimeric (e.g., humanized) IL12A from endogenous mouse loci, wherein the endogenous mouse IL12A gene has been replaced with a human IL12A gene and/or a nucleotide sequence that encodes a region of human IL12A 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 IL12A sequence. In various embodiments, an endogenous non-human IL12A locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a mature IL12A protein.

In some embodiments, the genetically modified mice can express the human IL12A and/or chimeric IL12A (e.g., humanized IL12A) 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 IL12A or chimeric IL12A (e.g., humanized IL12A) 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 IL12A or the chimeric IL12A (e.g., humanized IL12A) expressed in animal can maintain one or more functions of the wild-type mouse or human IL12A in the animal. For example, the expressed IL12A can bind to human or non-human IL12B. Furthermore, in some embodiments, the animal does not express endogenous IL12A. In some embodiments, the animal expresses a decreased level of endogenous IL12A as compared to a wild-type animal. As used herein, the term “endogenous IL12A” refers to IL12A protein that is expressed from an endogenous IL12A 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 IL12A (NP_000873.2; SEQ ID NO: 2) . In some embodiments, the genome comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 5 or 8.

The genome of the genetically modified animal can comprise a replacement at an endogenous IL12A gene locus of a sequence encoding a region of endogenous IL12A with a sequence encoding a corresponding region of human IL12A. In some embodiments, the sequence that is replaced is any sequence within the endogenous IL12A gene locus, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, 5’-UTR, 3’-UTR, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, or any combination thereof. In some embodiments, the sequence that is replaced is within the regulatory region of the endogenous IL12A gene. In some embodiments, the sequence that is replaced is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7, or a portion thereof, of an endogenous mouse IL12A gene locus.

The genetically modified animal can have one or more cells expressing a human or chimeric IL12A (e.g., humanized IL12A) . In some embodiments, the human or chimeric IL12A has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 251, 252, or 253 amino acids (e.g., contiguously or non-contiguously) that are identical to human IL12A (e.g., SEQ ID NO: 2) .

In some embodiments, the genome of the genetically modified animal comprises a sequence that corresponds to a portion or the entire sequence of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of human IL12A gene; a portion or the entire sequence of human IL12A gene; or a portion or the entire sequence of SEQ ID NO: 8.

In some embodiments, the genome of the genetically modified animal comprises a portion of exon 1, exons 2-6, and a portion of exon 7 of human IL12A gene. In some embodiments, the portion of exon 1 includes at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 115, 116, 117, 118, 119, 120, 150, 200, 250, 300, 320, or 340 nucleotides. In some embodiments, the portion of exon 2 includes 118 nucleotides. In some embodiments, the portion of exon 2 includes a nucleotide of at least 50 bp. In some embodiments, the portion of exon 7 includes at least 10, 20, 30, 40, 50, 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 100, 110, 120, 130, 140, 150, 155, 156, 157, 158, 159, 160, 190, 200, 300, 400, 500, 600, or 616 nucleotides. In some embodiments, the portion of exon 7 includes 156 nucleotides. In some embodiments, the portion of exon 7 includes a nucleotide of at least 50 bp. In some embodiments, the replaced sequence encodes the coding sequence of human IL12A (e.g., positions 223-984 of NM_000882.4) .

Furthermore, the genetically modified animal can be heterozygous with respect to the replacement at the endogenous IL12A locus, or homozygous with respect to the replacement at the endogenous IL12A locus.

In some embodiments, the humanized IL12A locus lacks a human IL12A 5’-UTR. In some embodiment, the humanized IL12A locus comprises an endogenous (e.g., mouse) 5'-UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3'-UTR. In appropriate cases, it may be reasonable to presume that the mouse and human IL12A genes appear to be similarly regulated based on the similarity of their 5'-flanking sequence. As shown in the present disclosure, humanized IL12A mice that comprise a replacement at an endogenous mouse IL12A locus, which retain mouse regulatory elements but comprise a humanization of IL12A encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized IL12A are grossly normal.

In another aspect, the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal's endogenous IL12A gene, wherein the disruption of the endogenous IL12A gene comprises deletion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7, or part thereof of the endogenous IL12A gene.

In some embodiments, the disruption of the endogenous IL12A gene comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7 of the endogenous IL12A gene.

In some embodiments, the disruption of the endogenous IL12A gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, and intron 6 of the endogenous IL12A 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, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 7500, 7900, 8000, or more nucleotides.

In some embodiments, the disruption of the endogenous IL12A gene comprises the deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 10, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nucleotides of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 (e.g., deletion of at least 50 nucleotides from exon 2, exons 2-6, and at least 50 nucleotides from exon 7) .

The disclosure further relates to a IL12A 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.

In some embodiments, the present disclosure is related to a genetically-modified, non-human animal whose genome comprises a human or chimeric (e.g., humanized ) IL12B nucleotide sequence. In some embodiments, the human or chimeric (e.g., humanized ) IL12B nucleotide sequence encodes a IL12B protein that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 10. In some embodiments, the genome of the animal comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 11, 12, 13, 14, 15, or 16.

In some embodiments, the genetically-modified non-human animal described herein comprises a sequence encoding a human or humanized IL12B protein. In some embodiments, the humanized IL12B protein comprises a human or humanized signal peptide. In some embodiments, the humanized IL12B protein comprises an endogenous signal peptide.

In some embodiments, the genetically-modified non-human animal described herein comprises a human or humanized IL12B gene. In some embodiments, the humanized IL12B gene comprises 8 exons. In some embodiments, the humanized IL12B gene comprises endogenous exon 1, human exon 2, human exon 3, human exon 4, human exon 5, human exon 6, human exon 7, and/or human exon 8. In some embodiments, the humanized IL12B gene comprises 7 introns. In some embodiments, the humanized IL12B gene comprises endogenous intron 1, human intron 2, human intron 3, human intron 4, human intron 5, human intron 6, and/or human intron 7. In some embodiments, the humanized IL12B gene comprises human or humanized 5' UTR. In some embodiments, the humanized IL12B gene comprises human or humanized 3' UTR. In some embodiments, the humanized IL12B gene comprises endogenous 5' UTR. In some embodiments, the humanized IL12B gene comprises endogenous 3' UTR. In some embodiments, the humanized IL12B gene comprises endogenous 5' UTR and human 3' UTR.

In one aspect, the genetically modified animals can express a human IL12B and/or a chimeric (e.g., humanized) IL12B from endogenous mouse loci, wherein the endogenous mouse IL12B gene has been replaced with a human IL12B gene and/or a nucleotide sequence that encodes a region of human IL12B 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 IL12B sequence. In various embodiments, an endogenous non-human IL12B locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a mature IL12B protein.

In some embodiments, the genetically modified mice can express the human IL12B and/or chimeric IL12B (e.g., humanized IL12B) 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 IL12B or chimeric IL12B (e.g., humanized IL12B) 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 IL12B or the chimeric IL12B (e.g., humanized IL12B) expressed in animal can maintain one or more functions of the wild-type mouse or human IL12B in the animal. For example, the expressed IL12B can bind to human or non-human IL12A. Furthermore, in some embodiments, the animal does not express endogenous IL12B. In some embodiments, the animal expresses a decreased level of endogenous IL12B as compared to a wild-type animal. As used herein, the term “endogenous IL12B” refers to IL12B protein that is expressed from an endogenous IL12B 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 IL12B (NP_002178.2; SEQ ID NO: 10) . In some embodiments, the genome comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 13 or 16.

The genome of the genetically modified animal can comprise a replacement at an endogenous IL12B gene locus of a sequence encoding a region of endogenous IL12B with a sequence encoding a corresponding region of human IL12B. In some embodiments, the sequence that is replaced is any sequence within the endogenous IL12B gene locus, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, 5’-UTR, 3’-UTR, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, or any combination thereof. In some embodiments, the sequence that is replaced is within the regulatory region of the endogenous IL12B gene. In some embodiments, the sequence that is replaced is exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8, or a portion thereof, of an endogenous mouse IL12B gene locus.

The genetically modified animal can have one or more cells expressing a human or chimeric IL12B (e.g., humanized IL12B) . In some embodiments, the human or chimeric IL12B has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 325, 326, 327, or 328 amino acids (e.g., contiguously or non-contiguously) that are identical to human IL12B (e.g., SEQ ID NO: 10) .

In some embodiments, the genome of the genetically modified animal comprises a sequence that corresponds to a portion or the entire sequence of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 of human IL12B gene; a portion or the entire sequence of human IL12B; or a portion or the entire sequence of SEQ ID NO: 16.

In some embodiments, the genome of the genetically modified animal comprises exons 2-8 of human IL12B gene. In some embodiments, the replaced sequence includes 3’ UTR of endogenous IL12B gene.

Furthermore, the genetically modified animal can be heterozygous with respect to the replacement at the endogenous IL12B locus, or homozygous with respect to the replacement at the endogenous IL12B locus.

In some embodiments, the humanized IL12B locus lacks a human IL12B 5'-UTR. In some embodiment, the humanized IL12B locus comprises an endogenous (e.g., mouse) 5'-UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3'-UTR. In appropriate cases, it may be reasonable to presume that the mouse and human IL12B genes appear to be similarly regulated based on the similarity of their 5'-flanking sequence. As shown in the present disclosure, humanized IL12B mice that comprise a replacement at an endogenous mouse IL12B locus, which retain mouse regulatory elements but comprise a humanization of IL12B encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized IL12B are grossly normal.

In another aspect, the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal's endogenous IL12B gene, wherein the disruption of the endogenous IL12B gene comprises deletion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8, or part thereof of the endogenous IL12B gene.

In some embodiments, the disruption of the endogenous IL12B gene comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 of the endogenous IL12B gene.

In some embodiments, the disruption of the endogenous IL12B gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7 of the endogenous IL12B 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, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, or more nucleotides.

In some embodiments, the disruption of the endogenous IL12B 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 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8.

The disclosure further relates to a IL12B 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.

In some embodiments, the present disclosure is related to a genetically-modified, non-human animal whose genome comprises a chimeric (e.g., humanized ) IL12RB1 nucleotide sequence. In some embodiments, the chimeric (e.g., humanized ) IL12RB1 nucleotide sequence encodes a IL12RB1 protein comprising an endogenous IL12RB1 signal peptide, a human or humanized IL12RB1 extracellular region, a human or humanized IL12RB1 transmembrane regions, and an endogenous IL12RB1 cytoplasmic region. In some embodiments, the encoded protein comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 17, 18, or 27. In some embodiments, the genome of the animal comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, 26, or 48.

In some embodiments, the genetically-modified non-human animal described herein comprises a sequence encoding a human or humanized IL12RB1 protein. In some embodiments, the IL12RB1 protein comprises a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the humanized IL12RB1 protein comprises a human or humanized IL12RB1 signal peptide. For example, the human or humanized IL12RB1 signal peptide comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 1-23 of SEQ ID NO: 18. In some embodiments, the humanized IL12RB1 protein comprises an endogenous IL12RB1 signal peptide. For example, the endogenous IL12RB1 signal peptide comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 1-19 of SEQ ID NO: 17. In some embodiments, the humanized IL12RB1 protein comprises a human or humanized IL12RB1 extracellular region. For example, the human or humanized IL12RB1 extracellular region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 24-545 of SEQ ID NO: 18.In some embodiments, the humanized IL12RB1 protein comprises an endogenous IL12RB1 extracellular region. For example, the endogenous IL12RB1 extracellular region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 20-565 of SEQ ID NO: 17. In some embodiments, the humanized IL12RB1 protein comprises a human or humanized IL12RB1 transmembrane region. For example, the human or humanized IL12RB1 transmembrane region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 546-570 of SEQ ID NO: 18. In some embodiments, the humanized IL12RB1 protein comprises an endogenous IL12RB1 transmembrane region. For example, the endogenous IL12RB1 transmembrane region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 566-591 of SEQ ID NO: 17. In some embodiments, the humanized IL12RB1 protein comprises a human or humanized IL12RB1 cytoplasmic region. For example, the human or humanized IL12RB1 cytoplasmic region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 571-662 of SEQ ID NO: 18. In some embodiments, the humanized IL12RB1 protein comprises an endogenous IL12RB1 cytoplasmic region. For example, the endogenous IL12RB1 cytoplasmic region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 592-738 of SEQ ID NO: 17.

In some embodiments, the genetically-modified non-human animal described herein comprises a human or humanized IL12RB1 gene. In some embodiments, the humanized IL12RB1 gene comprises from 5' end to 3' end: a portion (e.g., nucleotides 1-160) of endogenous exon 1, a portion (e.g., nucleotides 181-235) of human exon 2, human exons 3-13, a portion (e.g., nucleotides 1730-1821) of human exon 14, a portion (e.g., nucleotides 1877-1881) of endogenous exon 14, endogenous exon 15, and a portion (e.g., nucleotides 1964-2320) of endogenous exon 16. In some embodiments, the humanized IL12RB1 gene further includes a WPRE sequence and/or a STOP sequence. In some embodiments, the humanized IL12RB1 gene comprises human or humanized 5' UTR. In some embodiments, the humanized IL12RB1 gene comprises human or humanized 3' UTR. In some embodiments, the humanized IL12RB1 gene comprises endogenous 5' UTR. In some embodiments, the humanized ILI2RB1 gene comprises endogenous 3' UTR.

In some embodiments, the genetically-modified non-human animal described herein comprises an insertion in its genome, at an endogenous IL12RB1 gene locus, of a sequence encoding a human or humanized IL12RB1 protein. In some embodiments, the inserted sequence comprises one or more sequences selected from: all or a portion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and/or exon 17 of human IL12RB1 gene; and/or all or a portion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, and/or exon 16 of endogenous IL12RB1 gene (e.g., mouse IL12RB1 gene) . In some embodiments, the inserted sequence is a cDNA sequence. In some embodiments, the inserted sequence includes a portion of human exon 2, human exons 3-13, a portion of human exon 14, a portion of endogenous exon 14, endogenous exon 15, a portion of endogenous exon 16, a WPRE sequence, and/or a STOP sequence. In some embodiments, the inserted sequence does not encode a IL12RB1 signal peptide. In some embodiments, the inserted sequence encodes a human IL12RB1 extracellular region, a human IL12RB1 transmembrane region, and an endogenous IL12RB1 cytoplasmic region.

In some embodiments, the insertion described herein is between any two nucleotides within exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, and/or exon 16 of endogenous IL12RB1 gene (e.g., mouse IL12RB1 gene) . In some embodiments, the insertion is between any two nucleotides within exon 1 and/or intron 1 of endogenous IL12RB1 gene. For example, the insertion is between any two of the nucleotides selected from the group consisting of positions 160-170 of exon 1 and the downstream 426 nucleotides within intron 1 of endogenous IL12RB1 gene. In some embodiments, the insertion is immediately after the last nucleotide encoding the signal peptide of endogenous IL12RB1, e.g., between position 160 and position 161 of NM_008353.2.

In some embodiments, the genetically-modified non-human animal described herein comprises deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from a sequence corresponding to positions 161-170 of NM_008353.2. In some embodiments, the deleted sequence encodes the N-

terminal

1, 2, 3, or 4 amino acids of endogenous IL12RB1 extracellular region. In some embodiments, the deletion also includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 410, 420, 421, 422, 423, 424, or 425 nucleotides of first 425 nucleotides in intron 1 of endogenous IL12RB1 gene (e.g., mouse IL12RB1 gene) .

In some embodiments, the genetically modified animals can express a chimeric (e.g., humanized) IL12RB1 from endogenous mouse loci, wherein a sequence encoding the extracellular region and transmembrane region of human IL12RB1, and the cytoplasmic region of endogenous IL12RB1 is inserted within exon 1 and/or intron 1 of endogenous IL12RB1 gene. In some embodiments, the chimeric IL12RB1 includes an endogenous IL12RB1 signal peptide. In some embodiments, the extracellular region and transmembrane region of human IL12RB1 comprises an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to amino acids 24-570 of SEQ ID NO: 18. In various embodiments, an endogenous non-human IL12RB1 locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least the extracellular region and/or transmembrane region of human IL12RB1 protein.

In some embodiments, the genetically modified mice can express the chimeric IL12RB1 (e.g., humanized IL12RB1) from endogenous loci that are under control of mouse promoters and/or mouse regulatory elements. The insertion at the endogenous mouse loci provides non- human animals that express chimeric IL12RB1 (e.g., humanized IL12RB1) 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 chimeric IL12RB1 (e.g., humanized IL12RB1) expressed in animal can maintain one or more functions of the wild-type mouse or human IL12RB1 in the animal. For example, the expressed IL12RB1 can bind to human or non-human IL12RB2. Furthermore, in some embodiments, the animal does not express endogenous IL12RB1. In some embodiments, the animal expresses a decreased level of endogenous IL12RB1 as compared to a wild-type animal. As used herein, the term “endogenous IL12RB1” refers to IL12RB1 protein that is expressed from an endogenous IL12RB1 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 IL12RB1 (NP_005526.1; SEQ ID NO: 18) . 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: 27.

The genome of the genetically modified animal can comprise an insertion at an endogenous IL12RB1 gene locus. In some embodiments, the sequence is inserted between two nucleotides within any sequence of the endogenous IL12RB1 gene locus, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, 5’-UTR, 3’-UTR, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, and intron 15. In some embodiments, the sequence is inserted within the regulatory region of the endogenous IL12RB1 gene. In some embodiments, the sequence is inserted within exon 1 and intron 1 of an endogenous mouse IL12RB1 gene locus.

The genetically modified animal can have one or more cells expressing a human or chimeric IL12RB1 (e.g., humanized IL12RB1) having, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the extracellular region of human IL12RB1. In some embodiments, the extracellular region of the humanized IL12RB1 has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 521, or 522 amino acids (e.g., contiguously or non-contiguously) that are identical to the extracellular region of human IL12RB1. Because human IL12RB1 and non-human IL12RB1 (e.g., mouse IL12RB1) sequences, in many cases, are different, antibodies that bind to human IL12RB1 will not necessarily have the same binding affinity with non-human IL12RB1 or have the same effects to non-human IL12RB1. Therefore, the genetically modified animal having a human or a humanized extracellular region can be used to better evaluate the effects of anti-human IL12RB1 antibodies in an animal model.

In some embodiments, the transmembrane region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the transmembrane region of human IL12RB1. In some embodiments, the transmembrane region of the humanized IL12RB1 has a sequence that has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids (contiguously or non-contiguously) that are identical to the transmembrane region of human IL12RB1. While not intending to be bound by any theory, it is believed that keeping sequences encoding a human IL12RB1 transmembrane region in the chimeric IL12RB1 gene (e.g., humanized IL12RB1 gene) can avoid splicing sites, thereby improving the success rate of constructing the genetically modified IL12RB1 gene locus.

In some embodiments, the cytoplasmic comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the cytoplasmic of endogenous IL12RB1. In some embodiments, the cytoplasmic region of the humanized IL12RB1 has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 145, 146, or 147 amino acids (contiguously or non-contiguously) that are identical to the cytoplasmic region of endogenous IL12RB1. In some embodiments, the entire extracellular region and transmembrane region of the humanized IL12RB1 described herein are derived from human sequence. In some embodiments, the entire signal peptide and cytoplasmic region of the humanized IL12RB1 described herein are derived from endogenous sequence (e.g., mouse sequence) .

In some embodiments, the genome of the genetically modified animal comprises a sequence that corresponds to a portion or the entire sequence of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14 of human IL12RB1 gene; a sequence encoding the extracellular region and the transmembrane region of human IL12RB1; or a portion or the entire sequence of SEQ ID NO: 26.

In some embodiments, the genome of the genetically modified animal comprises a portion of exon 2, exons 3-13, and a portion of exon 14 of human IL12RB1 gene. In some embodiments, the portion of exon 2 includes at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, or 60 nucleotides. In some embodiments, the portion of exon 2 includes 55 nucleotides. In some embodiments, the portion of exon 2 includes a nucleotide sequence of at least 20 bp. In some embodiments, the portion of exon 14 includes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, or 97 nucleotides. In some embodiments, the portion of exon 14 includes 92 nucleotides. In some embodiments, the portion of exon 14 includes a nucleotide sequence of at least 20 bp. In some embodiments, the human sequence encodes the entire extracellular region and transmembrane region of human IL12RB1.

In some embodiments, the non-human animal can have, at an endogenous IL12RB1 gene locus, a nucleotide sequence encoding a chimeric human/non-human IL12RB1 polypeptide, wherein a human portion of the chimeric human/non-human IL12RB1 polypeptide comprises the entire human IL12RB1 extracellular domain and the entire human IL12RB1 transmembrane region, and wherein the animal expresses a functional IL12RB1 on a surface of a cell (e.g., T cell or NK cell) of the animal. The human portion of the chimeric human/non-human IL12RB1 polypeptide can comprise an amino acid sequence encoded by a portion of exon 2, exons 3-13, and/or a portion of exon 14 of human IL12RB1 gene. In some embodiments, the human portion of the chimeric human/non-human IL12RB1 polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 24-570 of SEQ ID NO: 18. In some embodiments, the cytoplasmic region includes a sequence corresponding to the entire or part of amino acids 592-738 of SEQ ID NO: 17. In some embodiments, the chimeric human/non-human IL12RB1 polypeptide comprises a signal peptide, which includes a sequence corresponding to the entire or part of amino acids 1-19 of SEQ ID NO: 17.

In some embodiments, the non-human portion of the chimeric human/non-human IL12RB1 polypeptide comprises the entire signal peptide and the entire cytoplasmic region of an endogenous non-human IL12RB1 polypeptide.

Furthermore, the genetically modified animal can be heterozygous with respect to the insertion at the endogenous IL12RB1 locus, or homozygous with respect to the insertion at the endogenous IL12RB1 locus.

In some embodiments, the humanized IL12RB1 locus lacks a human IL12RB1 5’-UTR. In some embodiment, the humanized IL12RB1 locus comprises an endogenous (e.g., mouse) 5'-UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3'-UTR. In appropriate cases, it may be reasonable to presume that the mouse and human IL12RB1 genes appear to be similarly regulated based on the similarity of their 5'-flanking sequence. As shown in the present disclosure, humanized IL12RB1 mice that comprise an insertion at an endogenous mouse IL12RB1 locus, which retain mouse regulatory elements but comprise a humanization of IL12RB1 encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized IL12RB1 are grossly normal.

In another aspect, the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal's endogenous IL12RB1 gene, wherein the disruption of the endogenous IL12RB1 gene comprises deletion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, and/or exon 16, or part thereof of the endogenous IL12RB1 gene.

In some embodiments, the disruption of the endogenous IL12RB1 gene comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, and exon 16 the endogenous IL12RB1 gene.

In some embodiments, the disruption of the endogenous IL12RB1 gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14 and intron 15 of the endogenous IL12RB1 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, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, or more nucleotides.

In some embodiments, the disruption of the endogenous IL12RB1 gene comprises the deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 10, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nucleotides of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, and exon 16.

In some embodiments, the disruption of the endogenous IL12RB1 gene is caused by insertion of a sequence including one or more auxiliary sequences (e.g., WPRE, Stop, and/or polyA) . The insertion can cause frameshift, mutation, or truncation of the endogenous IL12RB1 coding sequence, such that the level of transcription and/or translation of endogenous IL12RB1 gene is decreased (e.g., by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) .

The disclosure further relates to a IL12RB1 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.

In some embodiments, the present disclosure is related to a genetically-modified, non-human animal whose genome comprises a cNmeric (e.g., humanized) IL12RB2 nucleotide sequence. In some embodiments, the chimeric (e.g., humanized) IL12RB2 nucleotide sequence encodes a IL12RB2 protein comprising an endogenous IL12RB2 signal peptide, a human or humanized IL12RB2 extracellular region, an endogenous transmembrane regions, and an endogenous IL12RB2 cytoplasmic region. In some embodiments, the encoded protein comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 28, 29, or 36. In some embodiments, the genome of the animal comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 30, 31, 32, 33, 34, 35, 37, 38, or 65.

In some embodiments, the genetically-modified non-human animal described herein comprises a sequence encoding a human or humanized IL12RB2 protein. In some embodiments, the IL12RB2 protein comprises a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the humanized IL12RB2 protein comprises a human or humanized IL12RB2 signal peptide. For example, the human or humanized IL12RB2 signal peptide comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 1-23 of SEQ ID NO: 29. In some embodiments, the humanized IL12RB2 protein comprises an endogenous IL12RB2 signal peptide. For example, the endogenous IL12RB2 signal peptide comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 1-23 of SEQ ID NO: 28. In some embodiments, the humanized IL12RB2 protein comprises a human or humanized IL12RB2 extracellular region. For example, the human or humanized IL12RB2 extracellular region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 24-622 or 28-622 of SEQ ID NO: 29. In some embodiments, the humanized IL12RB2 protein comprises an endogenous IL12RB2 extracellular region. For example, the endogenous IL12RB2 extracellular region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 24-637 of SEQ ID NO: 28. In some embodiments, the humanized IL12RB2 protein comprises a human or humanized IL12RB2 transmembrane region. For example, the human or humanized IL12RB2 transmembrane region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 623-643 of SEQ ID NO: 29. In some embodiments, the humanized IL12RB2 protein comprises an endogenous IL12RB2 transmembrane region. For example, the endogenous IL12RB2 transmembrane region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 638-658 of SEQ ID NO: 28. In some embodiments, the humanized IL12RB2 protein comprises a human or humanized IL12RB2 cytoplasmic region. For example, the human or humanized IL12RB2 cytoplasmic region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 644-682 of SEQ ID NO: 29. In some embodiments, the humanized IL12RB2 protein comprises an endogenous IL12RB2 cytoplasmic region. For example, the endogenous IL12RB2 cytoplasmic region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100%identical to amino acids 659-874 of SEQ ID NO: 28.

In some embodiments, the genetically-modified non-human animal described herein comprises a human or humanized IL12RB2 gene. In some embodiments, the humanized IL12RB2 gene comprises from 5’ end to 3’ end: endogenous exon 1, a portion (e.g., nucleotides 155-257) of endogenous exon 2, a portion (e.g., nucleotides 301 -583) of human exon 4, human exons 5-14, a portion (e.g., nucleotides 2075-2085) of human exon 15, a portion (e.g., nucleotides 2100-2179) of endogenous exon 14, endogenous exon 15, and a portion (e.g., nucleotides 2280-2813) of endogenous exon 16. In some embodiments, the humanized IL12RB2 gene comprises endogenous intron 1. In some embodiments, the humanized IL12RB2 gene further includes a WPRE sequence and/or a STOP sequence. In some embodiments, the humanized IL12RB2 gene comprises human or humanized 5’ UTR. In some embodiments, the humanized IL12RB2 gene comprises human or humanized 3’ UTR. In some embodiments, the humanized IL12RB2 gene comprises endogenous 5’ UTR. In some embodiments, the humanized IL12RB2 gene comprises endogenous 3’ UTR.

In some embodiments, the genetically-modified non-human animal described herein comprises an insertion in its genome, at an endogenous IL12RB2 gene locus, of a sequence encoding a human or humanized IL12RB2 protein. In some embodiments, the inserted sequence comprises one or more sequences selected from: all or a portion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and/or exon 17 of human IL12RB2 gene; and/or all or a portion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, and/or exon 16 of endogenous IL12RB2 gene (e.g., mouse IL12RB2 gene) . In some embodiments, the inserted sequence is a cDNA sequence. In some embodiments, the inserted sequence includes a portion (e.g., nucleotides 301-583) of human exon 4, human exons 5-14, a portion (e.g., nucleotides 2075-2085) of human exon 15, a portion (e.g., nucleotides 2100-2179) of endogenous exon 14, endogenous exon 15, and a portion (e.g., nucleotides 2280-2813) of endogenous exon 16, a WPRE sequence, and/or a STOP sequence. In some embodiments, the inserted sequence does not encode a IL12RB2 signal peptide. In some embodiments, the inserted sequence encodes a human IL12RB2 extracellular region, an endogenous IL12RB2 transmembrane region, and an endogenous IL12RB2 cytoplasmic region.

In some embodiments, the insertion described herein is between any two nucleotides within exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, and/or exon 16 of endogenous IL12RB2 gene (e.g., mouse IL12RB2 gene) . In some embodiments, the insertion is between any two nucleotides within exon 2 and/or intron 2 of endogenous IL12RB2 gene. For example, the insertion is between any two of the nucleotides selected from the group consisting of positions 258-264 of exon 2 and the downstream 410 nucleotides within intron 2 of endogenous IL12RB2 gene. In some embodiments, the insertion is immediately after the last nucleotide encoding the signal peptide of endogenous IL12RB2, e.g., between position 257 and position 258 of NM_008354.4.

In some embodiments, the genetically-modified non-human animal described herein comprises deletion of at least 1, 2, 3, 4, 5, 6, or 7 nucleotides from a sequence corresponding to positions 258-264 of NM_008354.4. In some embodiments, the deleted sequence encodes the N-

terminal

1, 2, or 3 amino acids of endogenous IL12RB2 extracellular region. In some embodiments, the deletion also includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 405, 406, 407, 408, 409, or 410 nucleotides of first 410 nucleotides in intron 2 of endogenous IL12RB2 gene (e.g., mouse IL12RB2 gene) .

In some embodiments, the genetically modified animals can express a chimeric (e.g., humanized) IL12RB2 from endogenous mouse loci, wherein a sequence encoding the extracellular region of human IL12RB2, and the transmembrane and cytoplasmic regions of endogenous IL12RB2 is inserted within exon 2 and/or intron 2 of endogenous IL12RB2 gene. In some embodiments, the chimeric IL12RB2 includes an endogenous IL12RB2 signal peptide. In some embodiments, the extracellular region of human IL12RB2 comprises an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to amino acids 24-622 or 28-622 of SEQ ID NO: 29. In various embodiments, an endogenous non-human IL12RB2 locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least the extracellular region of human IL12RB2 protein.

In some embodiments, the genetically modified mice can express the chimeric IL12RB2 (e.g., humanized IL12RB2) from endogenous loci that are under control of mouse promoters and/or mouse regulatory elements. The insertion at the endogenous mouse loci provides non-human animals that express chimeric IL12RB2 (e.g., humanized IL12RB2) 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 chimeric IL12RB2 (e.g., humanized IL12RB2) expressed in animal can maintain one or more functions of the wild-type mouse or human IL12RB2 in the animal. For example, the expressed IL12RB2 can bind to human or non-human IL12RB1. Furthermore, in some embodiments, the animal does not express endogenous IL12RB2. In some embodiments, the animal expresses a decreased level of endogenous IL12RB2 as compared to a wild-type animal. As used herein, the term “endogenous IL12RB2” refers to IL12RB2 protein that is expressed from an endogenous IL12RB2 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 IL12RB2 (NP_001361188.1; SEQ ID NO: 29) . 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: 36.

The genome of the genetically modified animal can comprise an insertion at an endogenous IL12RB2 gene locus. In some embodiments, the sequence is inserted between two nucleotides within any sequence of the endogenous IL12RB2 gene locus, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, 5’-UTR, 3’-UTR, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, and intron 15. In some embodiments, the sequence is inserted within the regulatory region of the endogenous IL12RB2 gene. In some embodiments, the sequence is inserted within exon 2 and intron 2 of an endogenous mouse IL12RB2 gene locus.

The genetically modified animal can have one or more cells expressing a human or chimeric IL12RB2 (e.g., humanized IL12RB2) having, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the extracellular region of human IL12RB2. In some embodiments, the extracellular region of the humanized IL12RB2 has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 595, 596, 597, 598, or 599 amino acids (e.g., contiguously or non-contiguously) that are identical to the extracellular region of human IL12RB2. Because human IL12RB2 and non-human IL12RB2 (e.g., mouse IL12RB2) sequences, in many cases, are different, antibodies that bind to human IL12RB2 will not necessarily have the same binding affinity with non-human IL12RB2 or have the same effects to non-human IL12RB2. Therefore, the genetically modified animal having a human or a humanized extracellular region can be used to better evaluate the effects of anti-human IL12RB2 antibodies in an animal model.

In some embodiments, the transmembrane comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the transmembrane region of endogenous IL12RB2. In some embodiments, the transmembrane region of the humanized IL12RB2 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, or21 amino acids (contiguously or non-contiguously) that are identical to the transmembrane region of endogenous IL12RB2. In some embodiments, the cytoplasmic comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the cytoplasmic of endogenous IL12RB2. In some embodiments, the cytoplasmic region of the humanized IL12RB2 has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 215, or 216 amino acids (contiguously or non-contiguously) that are identical to the cytoplasmic region of endogenous IL12RB2. In some embodiments, the entire extracellular region of the humanized IL12RB2 described herein are derived from human sequence. In some embodiments, the entire signal peptide, transmembrane and cytoplasmic regions of the humanized IL12RB2 described herein are derived from endogenous sequence (e.g., mouse sequence) .

In some embodiments, the genome of the genetically modified animal comprises a sequence that corresponds to a portion or the entire sequence of exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, and/or exon 15 of human IL12RB2 gene; a sequence encoding the extracellular region of human IL12RB2; or a portion or the entire sequence of SEQ ID NO: 65.

In some embodiments, the genome of the genetically modified animal comprises a portion of exon 4, exons 5-14, and a portion of exon 15 of human IL12RB2 gene. In some embodiments, the portion of exon 4 includes at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 281, 282, 283, 284, 285, or 288 nucleotides. In some embodiments, the portion of exon 4 includes 283 nucleotides. In some embodiments, the portion of exon 4 includes a nucleotide sequence of at least 100 bp. In some embodiments, the portion of exon 4 starts from any one of the nucleotides encoding the N-terminal 1-5 (e.g., 1, 2, 3, 4, or 5) amino acids of IL12RB2 extracellular region and ends at the last nucleotide of exon 4. In some embodiments, the portion of exon 15 includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 90, or 91 nucleotides. In some embodiments, the portion of exon 15 includes 11 nucleotides. In some embodiments, the portion of exon 15 includes a nucleotide sequence of at least 5 bp. In some embodiments, the human sequence encodes the entire extracellular region of human IL12RB2, optionally without the N-

terminal

1, 2, 3, or 4 amino acids of the extracellular region. While not intending to be bound by any theory, it is believed that removing sequences encoding the N-

terminal

1, 2, 3, or 4 amino acids of the IL12RB2 extracellular region can avoid splicing sites, thereby improving the success rate of constructing the genetically modified IL12RB2 gene locus.

In some embodiments, the non-human animal can have, at an endogenous IL12RB2 gene locus, a nucleotide sequence encoding a chimeric human/non-human IL12RB2 polypeptide, wherein a human portion of the chimeric human/non-human IL12RB2 polypeptide comprises the entire human IL12RB2 extracellular domain, and wherein the animal expresses a functional IL12RB2 on a surface of a cell (e.g., T cell or NK cell) of the animal. The human portion of the chimeric human/non-human IL12RB2 polypeptide can comprise an amino acid sequence encoded by a portion of exon 4, exons 5-14, and/or a portion of exon 15 of human IL12RB2 gene. In some embodiments, the human portion of the chimeric human/non-human IL12RB2 polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 24-622 or 28-622 of SEQ ID NO: 29. In some embodiments, the transmembrane region includes a sequence corresponding to the entire or part of amino acids 638-658 of SEQ ID NO: 28. In some embodiments, the cytoplasmic region includes a sequence corresponding to the entire or part of amino acids 659-874 of SEQ ID NO: 28. In some embodiments, the chimeric human/non-human IL12RB2 polypeptide comprises a signal peptide, which includes a sequence corresponding to the entire or part of amino acids 1-23 of SEQ ID NO: 28.

In some embodiments, the non-human portion of the chimeric human/non-human IL12RB2 polypeptide comprises the entire signal peptide, the entire transmembrane region, and the entire cytoplasmic region of an endogenous non-human IL12RB2 polypeptide.

Furthermore, the genetically modified animal can be heterozygous with respect to the insertion at the endogenous IL12RB2 locus, or homozygous with respect to the insertion at the endogenous IL12RB2 locus.

In some embodiments, the humanized IL12RB2 locus lacks a human IL12RB2 5’-UTR. In some embodiment, the humanized IL12RB2 locus comprises an endogenous (e.g., mouse) 5’-UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3’-UTR. In appropriate cases, it may be reasonable to presume that the mouse and human IL12RB2 genes appear to be similarly regulated based on the similarity of their 5’-flanking sequence. As shown in the present disclosure, humanized IL12RB2 mice that comprise an insertion at an endogenous mouse IL12RB2 locus, which retain mouse regulatory elements but comprise a humanization of IL12RB2 encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized IL12RB2 are grossly normal.

In another aspect, the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous IL12RB2 gene, wherein the disruption of the endogenous IL12RB2 gene comprises deletion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, and/or exon 16, or part thereof of the endogenous IL12RB2 gene.

In some embodiments, the disruption of the endogenous IL12RB2 gene comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, and exon 16 the endogenous IL12RB2 gene.

In some embodiments, the disruption of the endogenous IL12RB2 gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14 and intron 15 of the endogenous IL12RB2 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, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 3000, 40000, 50000, 60000, 70000, 80000, 90000, or more nucleotides.

In some embodiments, the disruption of the endogenous IL12RB2 gene comprises the deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 10, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nucleotides of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, and exon 16.

In some embodiments, the disruption of the endogenous IL12RB2 gene is caused by insertion of a sequence including one or more auxiliary sequences (e.g., WPRE, Stop, and/or polyA) . The insertion can cause frameshift, mutation, or truncation of the endogenous IL12RB2 coding sequence, such that the level of transcription and/or translation of endogenous IL12RB2 gene is decreased (e.g., by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) .

The disclosure further relates to a IL12RB2 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 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 humanized IL12A, IL12B, IL12RB1, and/or IL12RB2 genes.

In addition, the present disclosure also relates to a tumor beating 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 beating 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 beating 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 c in the genome of the animal.

In some embodiments, the non-human mammal comprises a humanized IL12A gene having the genetic construct as described herein (e.g., gene construct as shown in FIGS. 2, 3, and 5) . In some embodiments, the non-human mammal comprises a humanized IL12B gene having the genetic construct as described herein (e.g., gene construct as shown in FIGS. 8, 9, and 11) . In some embodiments, the non-human mammal comprises a humanized IL12RB1 gene having the genetic construct as described herein (e.g., gene construct as shown in FIGS. 14, 15, and 17) . In some embodiments, the non-human mammal comprises a humanized IL12RB2 gene having the genetic construct as described herein (e.g., gene construct as shown in FIGS. 20, 21, and 23) . In some embodiments, a non-human mammal expressing human or humanized IL12A, IL12B, IL12RB1, and/or IL12RB2 is provided. In some embodiments, the tissue-specific expression of human or humanized IL12A, IL12B, IL12RB1, and/or IL12RB2 proteins is provided.

In some embodiments, the expression of human or humanized IL12A, IL12B, IL12RB1, and/or IL12RB2 in a genetically modified animal is controllable, as by the addition of a specific inducer or repressor substance. In some embodiments, the specific inducer is selected from Tet-Off System/Tet-On System, or Tamoxifen System.

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

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

The present disclosure also provides a cell line or primary cell culture derived from the non-human mammal or a progeny thereof. A model based on cell culture can be prepared, for example, by the following methods. Cell cultures can be obtained by way of isolation from a non-human mammal, alternatively cells can be obtained from the cell culture established using the same constructs and the standard cell transfection techniques. The integration of genetic constructs containing DNA sequences encoding human IL12A, IL12B, IL12RB1, and/or IL12RB2 proteins can be detected by a variety of methods.

There are many analytical methods that can be used to detect exogenous DNA, including methods at the level of nucleic acid (including the mRNA quantification approaches using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, and in situ hybridization) and methods at the protein level (including histochemistry, immunoblot analysis and in vitro binding studies) . In addition, the expression level of the gene of interest can be quantified by ELISA techniques well known to those skilled in the art. Many standard analysis methods can be used to complete quantitative measurements. For example, transcription levels can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis (RNAdot) analysis. Immunohistochemical staining, flow cytometry, Western blot analysis can also be used to assess the presence of human or humanized IL12A, IL12B, IL12RB1, and/or IL12RB2 proteins.

In some embodiments, provided herein is a genetically-modified non-human animal having two or more human or humanized genes selected from IL12A, IL12B, IL12RB1, and IL12RB2.

Vectors

The present disclosure relates to a targeting vector, comprising: a) a DNA fragment homologous to the 5’ end of a region to be altered (5’ arm) , which is selected from the IL12A gene genomic DNAs in the length of 100 to 10,000 nucleotides; b) a desired/donor DNA sequence encoding a donor region; and c) a second DNA fragment homologous to the 3’ end of the region to be altered (3’ arm) , which is selected from the IL12A 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_000069.7; c) the DNA fragment homologous to the 3’ end of the region to be altered (3’ arm) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000069.7.

In some embodiments, a) the DNA fragment homologous to the 5’ end of a region to be altered (5’ arm) is selected from the nucleotides from the position 68594689 to the position 68598883 of the NCBI accession number NC_000069.7; c) the DNA fragment homologous to the 3’ end of the region to be altered (3’ arm) is selected from the nucleotides from the position 68606338 to the position 68609962 of the NCBI accession number NC_000069.7.

In some embodiments, the length of the selected genomic nucleotide sequence in the targeting vector can be more than about 2 kb, 2.5 kb, 3 kb, 3.5 kb, 3 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7kb, 7.5 kb, 8 kb, 8.5 kb, or 9 kb.

In some embodiments, the region to be altered is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7 of IL12A gene (e.g., a portion of exon 1, exons 2-6, and a portion of exon 7 of mouse IL12A gene) .

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

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

In some embodiments, the inserted sequence is derived from human (e.g., 159989057-159995559 of NC_000003.12) . For example, the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human IL12A gene, preferably exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of the human IL12A. In some embodiments, the nucleotide sequence of the humanized IL12A encodes the entire or the part of human IL12A protein with the NCBI accession number NP_000873.2 (SEQ ID NO: 2) .

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 IL12B gene genomic DNAs in the length of 100 to 10,000 nucleotides; b) a desired/donor DNA sequence encoding a donor region; and c) a second DNA fragment homologous to the 3’ end of the region to be altered (3’ arm) , which is selected from the IL12B 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_000077.7; c) the DNA fragment homologous to the 3’ end of the region to be altered (3’ arm) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000077.7.

In some embodiments, a) the DNA fragment homologous to the 5’ end of a region to be altered (5’ arm) is selected from the nucleotides from the position 44291022 to the position 44294863 of the NCBI accession number NC_000077.7; c) the DNA fragment homologous to the 3’ end of the region to be altered (3’ arm) is selected from the nucleotides from the position 44305162 to the position 44309763 of the NCBI accession number NC_000077.7.

In some embodiments, the length of the selected genomic nucleotide sequence in the targeting vector can be more than about 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, or 15 kb.

In some embodiments, the region to be altered is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 of IL12B gene (e.g., exons 2-8 of mouse IL12B gene) .

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

In some embodiments, the inserted sequence is derived from human (e.g., 159314313-159326782 of NC_000005.10) . For example, the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human IL12B gene, preferably exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 of the human IL12B. In some embodiments, the nucleotide sequence of the humanized IL12B encodes the entire or the part of human IL12B protein with the NCBI accession number NP_002178.2 (SEQ ID NO: 10) .

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 IL12RB1 gene genomic DNAs in the length of 100 to 10,000 nucleotides; b) a desired/donor DNA sequence encoding a donor region; and c) a second DNA fragment homologous to the 3’ end of the region to be altered (3’ arm) , which is selected from the IL12RB1 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’ ann) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000074.7; c) the DNA fragment homologous to the 3’ end of the region to be altered (3’ ann) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000074.7.

In some embodiments, a) the DNA fragment homologous to the 5’ end of a region to be altered (5’ arm) is selected from the nucleotides from the position 71257214 to the position 71261252 of the NCBI accession number NC_000074.7; c) the DNA fragment homologous to the 3’ end of the region to be altered (3’ ann) is selected from the nucleotides from the position 71261688 to the position 71266063 of the NCBI accession number NC_000074.7.

In some embodiments, the length of the selected genomic nucleotide sequence in the targeting vector can be more than about 1.5 kb, 2 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kb, 3 kb, 3.1 kb, 3.2 kb, or 3.3 kb.

In some embodiments, the region to be altered is exon 1 and/or intron 1 of endogenous IL12RB1 gene (e.g., positions 161-170 of NM_008353.2 and the downstream 425 nucleotides within intron 1 of mouse IL12RB1 gene) .

In some embodiments, the sequence of the 5’ arm is shown in SEQ ID NO: 19; and the sequence of the 3’ arm is shown in SEQ ID NO: 20.

In some embodiments, the inserted sequence is derived from human (e.g., nucleic acids 181-1821 of NM_005535.3) and mouse (e.g., nucleic acids 1877-2320 of NM_008353.2) . For example, the target region in the targeting vector comprises a portion (e.g., nucleotides 1-160) of endogenous IL12RB1 exon 1, a portion (e.g., nucleotides 181-235) of human IL12RB1 exon 2, human IL12RB1 exons 3-13, a portion (e.g., nucleotides 1730-1821) of human IL12RB1 exon 14, a portion (e.g., nucleotides 1877-1881) of endogenous IL12RB1 exon 14, endogenous IL12RB1 exon 15, and a portion (e.g., nucleotides 1964-2320) of endogenous IL12RB1 exon 16. In some embodiments, the nucleotide sequence of the humanized IL12RB1 encodes a IL12RB1 protein with amino acid sequence set forth in SEQ ID NO: 27.

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 IL12RB2 gene genomic DNAs in the length of 100 to 10,000 nucleotides; b) a desired/donor DNA sequence encoding a donor region; and c) a second DNA fragment homologous to the 3’ end of the region to be altered (3’ arm) , which is selected from the IL12RB2 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_000072.7; c) the DNA fragment homologous to the 3’ end of the region to be altered (3’ arm) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000072.7.

In some embodiments, a) the DNA fragment homologous to the 5’ end of a region to be altered (5’ arm) is selected from the nucleotides from the position 67338866 to the position 67343067 of the NCBI accession number NC_000072.7; c) the DNA fragment homologous to the 3’ end of the region to be altered (3’ arm) is selected from the nucleotides from the position 67334283 to the position 67338449 of the NCBI accession number NC_000072.7.

In some embodiments, the length of the selected genomic nucleotide sequence in the targeting vector can be more than about 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kb, 3 kb, 3.1 kb, 3.2 kb, 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb. 3.8 kb, 3.9 kb, or 4.0 kb.

In some embodiments, the region to be altered is exon 2 and/or intron 2 of endogenous IL12RB2 gene (e.g., positions 258-264 of NM_008354.4 and the downstream 409 nucleotides within intron 2 of mouse IL12RB2 gene) .

In some embodiments, the sequence of the 5’ arm is shown in SEQ ID NO: 30; and the sequence of the 3’ arm is shown in SEQ ID NO: 31.

In some embodiments, the inserted sequence is derived from human (e.g., nucleic acids 301-2085 of NM_001374259.2) and mouse (e.g., nucleic acids 2100-2813 of NM_008354.4) . For example, the target region in the targeting vector comprises endogenous IL12RB2 exon 1, a portion (e.g., nucleotides 155-257) of endogenous IL12RB2exon 2, a portion (e.g., nucleotides 301-583) of human IL12RB2 exon 4, human IL12RB2 exons 5-14, a portion (e.g., nucleotides 2075-2085) of human IL12RB2 exon 15, a portion (e.g., nucleotides 2100-2179) of endogenous IL12RB2 exon 14, endogenous IL12RB2 exon 15, and a portion (e.g., nucleotides 2280-2813) of endogenous IL12RB2 exon 16. In some embodiments, the nucleotide sequence of the humanized IL12RB2 encodes a IL12RB2 protein with amino acid sequence set forth in SEQ ID NO: 36.

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

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

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

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

Methods of making genetically modified animals

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

Thus, in some embodiments, the disclosure provides replacing in at least one cell of the animal, at an endogenous IL12A gene locus, a sequence encoding a region of an endogenous IL12A with a sequence encoding a corresponding region of human or chimeric IL12A. In some embodiments, the replacement occurs in a germ cell, a somatic cell, a blastocyst, or a fibroblast, etc. The nucleus of a somatic cell or the fibroblast can be inserted into an enucleated oocyte.

FIG. 3 shows a humanization strategy for a mouse IL12A locus. In FIG. 3, the targeting strategy involves a vector comprising the 5’ end homologous arm, human IL12A gene fragment, 3’ homologous arm. The process can involve replacing endogenous IL12A 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 IL12A sequence with human IL12A sequence.

Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of replacing at an endogenous IL12A locus (or site) , a nucleic acid encoding a region of endogenous IL12A with a sequence encoding a corresponding region of human IL12A. The sequence can include a region (e.g., a part or the entire region) of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of a human IL12A gene. In some embodiments, the sequence includes a portion of exon 1, exons 2-6, and a portion of exon 7 of a human IL12A gene (e.g., nucleic acids 223-984 of NM_000882.4) . In some embodiments, the region includes the entire coding sequence (CDS) of human IL12A (e.g., SEQ ID NO: 2) . In some embodiments, the endogenous IL12A locus is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of mouse IL12A. In some embodiments, the sequence includes a portion of exon 1, exons 2-6, and a portion of exon 7 of mouse IL12A gene (e.g., nucleic acids 128-775 of NM_008351.3) .

In some embodiments, the methods of modifying a IL12A locus of a mouse to express a chimeric human/mouse IL12A peptide can include the steps of replacing at the endogenous mouse IL12A locus a nucleotide sequence encoding a mouse IL12A with a nucleotide sequence encoding a human IL12A, thereby generating a sequence encoding a chimeric human/mouse IL12A.

In some embodiments, the nucleotide sequence encoding the chimeric human/mouse IL12A can include a first nucleotide sequence including the 5' UTR of mouse IL12A gene; a second nucleotide sequence including the entire coding sequence of human IL12A gene; and/or a third nucleotide sequence including the 3' UTR of mouse IL12A gene.

In some embodiments, the disclosure provides replacing in at least one cell of the animal, at an endogenous IL12B gene locus, a sequence encoding a region of an endogenous IL12B with a sequence encoding a corresponding region of human or chimeric IL12B. In some embodiments, the replacement occurs in a germ cell, a somatic cell, a blastocyst, or a fibroblast, etc. The nucleus of a somatic cell or the fibroblast can be inserted into an enucleated oocyte.

FIG. 9 shows a humanization strategy for a mouse IL12B locus. In FIG. 9, the targeting strategy involves a vector comprising the 5' end homologous arm, human IL12B gene fragment, 3' homologous arm. The process can involve replacing endogenous IL12B 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 IL12B sequence with human IL12B sequence.

Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of replacing at an endogenous IL12B locus (or site) , a nucleic acid encoding a region of endogenous IL12B with a sequence encoding a corresponding region of human IL12B. The sequence can include a region (e.g., a part or the entire region) of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 of a human IL12B gene. In some embodiments, the sequence includes exons 2-8 of a human IL12B gene (e.g., nucleic acids 57-2364 of NM_002187.3) . In some embodiments, the region includes the entire coding sequence (CDS) of human IL12B (e.g., SEQ ID NO: 10) . In some embodiments, the endogenous IL12B locus is exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 of mouse IL12B. In some embodiments, the sequence includes exons 2-8 of mouse IL12B gene (e.g., nucleic acids 57-2505 of NM_001303244.1) .

In some embodiments, the methods of modifying a IL12B locus of a mouse to express a chimeric human/mouse IL12B peptide can include the steps of replacing at the endogenous mouse IL12B locus a nucleotide sequence encoding a mouse IL12B with a nucleotide sequence encoding a human IL12B, thereby generating a sequence encoding a chimeric human/mouse IL12B.

In some embodiments, the nucleotide sequence encoding the chimeric human/mouse IL12B can include a first nucleotide sequence including the 5' UTR of mouse IL12B gene; and a second nucleotide sequence including the entire coding sequence and 3' UTR of human IL12B gene.

In some embodiments, the disclosure provides inserting in at least one cell of the animal, at an endogenous IL12RB1 gene locus (e.g., exon 1 and/or intron 1 of endogenous IL12RB1 gene) , a sequence encoding the extracellular and transmembrane regions of human IL12RB1, and the cytoplasmic region of endogenous IL12RB1. In some embodiments, the insertion 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. 15 shows a humanization strategy for a mouse IL12RB1 locus. In FIG. 15, the targeting strategy involves a vector comprising the 5' end homologous arm, a chimeric IL12RB1 sequence, 3' homologous arm. The process can involve inserting the chimeric IL12RB1 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 insert the chimeric IL12RB1 sequence within the endogenous IL12RB1 gene locus.

Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of inserting at an endogenous IL12RB1 locus (or site) , a nucleic acid encoding the extracellular and transmembrane regions of human IL12RB1, and the cytoplasmic region of endogenous IL12RB1. The sequence can include a portion (e.g., nucleotides 1-160) of endogenous IL12RB1 exon 1, a portion (e.g., nucleotides 181-235) of human IL12RB1 exon 2, human IL12RB1 exons 3-13, a portion (e.g., nucleotides 1730-1821) of human IL12RB1 exon 14, a portion (e.g., nucleotides 1877-1881) of endogenous IL12RB1 exon 14, endogenous IL12RB1 exon 15, and a portion (e.g., nucleotides 1964-2320) of endogenous IL12RB1 exon 16. In some embodiments, the sequence includes nucleic acids 1-160 and 1877-2320 of NM_008353.2 and nucleic acids 181-1821 of NM_005535.3.

In some embodiments, the methods of modifying a IL12RB1 locus of a mouse to express a chimeric human/mouse IL12RB1 peptide can include the steps of inserting at the endogenous mouse IL12RB1 locus a nucleotide sequence encoding a chimeric (e.g., humanized) IL12RB1 protein, thereby generating a sequence encoding a chimeric human/mouse IL12RB1.

In some embodiments, the nucleotide sequence encoding the chimeric human/mouse IL12RB1 can include a first nucleotide sequence encoding the signal peptide of mouse IL12RB1; a second nucleotide sequence encoding the extracellular and transmembrane regions of human IL12RB1; and a third nucleotide sequence encoding the cytoplasmic region of mouse IL12RB1.

In some embodiments, the disclosure provides inserting in at least one cell of the animal, at an endogenous IL12RB2 gene locus (e.g., exon 2 and/or intron 2 of endogenous IL12RB2 gene) , a sequence encoding all or part of the extracellular region of human IL12RB2, and the transmembrane and cytoplasmic regions of endogenous IL12RB2. In some embodiments, the insertion 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. 21 shows a humanization strategy for a mouse IL12RB2 locus. In FIG. 21, the targeting strategy involves a vector comprising the 5' end homologous arm, a chimeric IL12RB2 sequence, 3' homologous arm. The process can involve inserting the chimeric IL12RB2 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 insert the chimeric IL12RB2 sequence within the endogenous IL12RB2 gene locus.

Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of inserting at an endogenous IL12RB2 locus (or site) , a nucleic acid encoding the extracellular region of human IL12RB2, and the transmembrane and cytoplasmic regions of endogenous IL12RB2. The sequence can include endogenous IL12RB2 exon 1, a portion (e.g., nucleotides 155-257) of endogenous IL12RB2exon 2, a portion (e.g., nucleotides 301-583) of human IL12RB2 exon 4, human IL12RB2 exons 5-14, a portion (e.g., nucleotides 2075-2085) of human IL12RB2 exon 15, a portion (e.g., nucleotides 2100-2179) of endogenous IL12RB2 exon 14, endogenous IL12RB2 exon 15, and a portion (e.g., nucleotides 2280-2813) of endogenous IL12RB2 exon 16. In some embodiments, the sequence includes nucleic acids 1-257 and 2100-2813 of NM_008354.4 and nucleic acids 301-2085 of NM_001374259.2.

In some embodiments, the methods of modifying a IL12RB2 locus of a mouse to express a chimeric human/mouse IL12RB2 peptide can include the steps of inserting at the endogenous mouse IL12RB2 locus a nucleotide sequence encoding a chimeric (e.g., humanized) IL12RB2 protein, thereby generating a sequence encoding a chimeric human/mouse IL12RB2.

In some embodiments, the nucleotide sequence encoding the chimeric human/mouse IL12RB2 can include a first nucleotide sequence encoding the signal peptide of mouse IL12RB2; a second nucleotide sequence encoding all or part of the extracellular region of human IL12RB2; and a third nucleotide sequence encoding the transmembrane and cytoplasmic regions of mouse IL12RB2.

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

The present disclosure further provides a method for establishing IL12A, IL12B, IL12RB1, and/or IL12RB2 gene humanized animal models, involving the following steps:

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

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

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

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

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

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

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

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

In some embodiments, methods of making the genetically modified animal comprises modifying the coding frame of the non-human animal's IL12A, IL12B, IL12RB1, and/or IL12RB2 genes, e.g., by inserting a nucleotide sequence (e.g., DNA or cDNA sequence) encoding human or humanized IL12A, IL12B, IL12RB1, and/or IL12RB2 proteins immediately after the endogenous regulatory element of the non-human animal's IL12A, IL12B, IL12RB1, and/or IL12RB2 genes. For example, one or more functional region sequences of the non-human animal's IL12A, IL12B, IL12RB1, and/or IL12RB2 genes can be knocked out, or inserted with a sequence, such that the non-human animal cannot express or expresses a decreased level of endogenous IL12A, IL12B, IL12RB1, and/or IL12RB2 proteins. In some embodiments, the coding frame of the modified non-human animal's IL12A gene can be all or part of the nucleotide sequence from exon 1 to exon 7 of the non-human animal's IL12A gene. In some embodiments, the coding frame of the modified non-human animal's IL12B gene can be all or part of the nucleotide sequence from exon 2 to exon 8 of the non-human animal's IL12B gene. In some embodiments, the coding frame of the modified non-human animal's IL12RB1 gene can be all or part of the nucleotide sequence from exon 1 to exon 17 of the non-human animal's IL12RB1 gene. In some embodiments, the coding frame of the modified non-human animal's IL12RB2 gene can be all or part of the nucleotide sequence from exon 3 to exon 17 of the non-human animal's IL12RB2 gene.

In some embodiments, methods of making the genetically modified animal comprises inserting a nucleotide sequence encoding human or humanized IL12A, IL12B, IL12RB1, and/or IL12RB2 proteins and/or an auxiliary sequence after the endogenous regulatory element of the non-human animal's IL12A, IL12B, IL12RB1, and/or IL12RB2 genes. In some embodiments, the auxiliary sequence can be a stop codon, such that the IL12A, IL12B, IL12RB1, and/or IL12RB2 gene humanized animal models can express human or humanized IL12A, IL12B, IL12RB1, and/or IL12RB2 proteins in vivo, but does not express non-human animal's IL12A, IL12B, IL12RB1, and/or IL12RB2 proteins. In some embodiments, the auxiliary sequence includes WPRE (WHP Posttranscriptional Response Element) and/or polyA.

Methods of using genetically modified animals

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

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

Genetically modified animals that express human or humanized IL12A, IL12B, IL12RB1, and/or IL12RB2 proteins, e.g., in a physiologically appropriate manner, provide a variety of uses that include, but are not limited to, developing therapeutics for human diseases and disorders, and assessing the toxicity and/or the efficacy of these human therapeutics in the animal models.

In various aspects, genetically modified animals are provided that express human or humanized IL12A, IL12B, IL12RB1, and/or IL12RB2, which are useful for testing agents that can decrease or block the interaction between the interaction between IL12 (or variant thereof) and IL12 receptor, the interaction between IL12 and anti-human IL12 antibodies, and the interaction between IL12 receptor and anti-IL12 receptor antibodies, testing whether an agent can increase or decrease the immune response, and/or determining whether an agent is an IL12/IL12R agonist or antagonist. The genetically modified animals can be, e.g., an animal model of a human disease, e.g., the disease is induced genetically (a knock-in or knockout) . In various embodiments, the genetically modified non-human animals further comprise an impaired immune system, e.g., a non-human animal genetically modified to sustain or maintain a human xenograft, e.g., a human solid tumor (e.g., breast cancer) or a blood cell tumor (e.g., a lymphocyte tumor, a B or T cell tumor) . In some embodiments, the anti-IL12 antibody or anti-IL12 receptor antibody blocks or inhibits the IL12/IL12R-related signaling pathway.

In some embodiments, the anti-IL12 antibody described herein can block the interaction between IL12A and IL12B, thereby inhibiting formation of a functional IL12 heterodimer. In some embodiments, the anti-IL12R antibody described herein can block the interaction between IL12RB1 and IL12RB2, thereby inhibiting formation of a functional IL12 receptor.

In some embodiments, the genetically modified animals can be used for determining effectiveness of a therapeutic agent (e.g., human IL12 protein or variant thereof) for the treatment of cancer. In some embodiments, the methods involve administering the therapeutic agent (e.g., human IL12 protein or variant thereof) to the animal as described herein, wherein the animal has a cancer or tumor; and determining inhibitory effects of the therapeutic agent to the cancer or tumor. The inhibitory effects that can be determined include, e.g., a decrease of tumor size or tumor volume, a decrease of tumor growth, a reduction of the increase rate of tumor volume in a subject (e.g., as compared to the rate of increase in tumor volume in the same subject prior to treatment or in another subject without such treatment) , a decrease in the risk of developing a metastasis or the risk of developing one or more additional metastasis, an increase of survival rate, and an increase of life expectancy, etc. The tumor volume in a subject can be determined by various methods, e.g., as determined by direct measurement, MRI or CT. In addition, a delicate balance is required for these antibodies, as IL12 and IL12 receptor are also expressed on many other cells. Thus, it is important that the humanized IL12 and/or IL12 receptor functions in a largely similar way as compared to the endogenous IL12 and/or IL12 receptor, so that the results in the humanized animals can be used to predict the efficacy or toxicity of these therapeutic agents in the human.

In some embodiments, the tumor comprises one or more cancer cells (e.g., human or mouse cancer cells) that are injected into the animal. In some embodiments, the therapeutic agent inhibits IL12/IL12R signaling pathways. In some embodiments, the therapeutic agent does not inhibit IL12/IL12R signaling pathways.

In some embodiments, the genetically modified animals can be used for determining whether an anti-IL12 or anti-IL12R antibody is an agonist or antagonist. In some embodiments, the methods as described herein are also designed to determine the functional effects of the therapeutic agent (e.g., human IL12 protein or variant thereof; or antibodies targeting immune checkpoint molecule (e.g., PD-1) ) , e.g., whether the agent can induce production of IFN-γ, whether the agent can activate T cells and NK cells, whether the agent can induce differentiation of

T cells into Th1 cells, whether the agent has anti-angiogenic activity, whether the agent can upregulate the immune response or downregulate immune response, and/or whether the agent can induce complement mediated cytotoxicity (CMC) or antibody dependent cellular cytotoxicity (ADCC) . In some embodiments, the genetically modified animals can be used for determining the effective dosage of a therapeutic agent for treating a disease in the subject, e.g., cancer.

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

In some embodiments, the therapeutic agent (e.g., human IL12 protein or variant thereof) is designed for treating various cancers. As used herein, the term “cancer” refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term “tumor” as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the agents described herein are designed for treating or diagnosing a carcinoma in a subject. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

In some embodiments, the cancer described herein is lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myeloproliferation abnormal syndromes, and sarcomas. In some embodiments, the leukemia is selected from acute lymphocytic (lymphoblasfic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia. In some embodiments, the lymphoma is selected from Hodgkin′s lymphoma and non-Hodgkin′s lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom macroglobulinemia. In some embodiments, the sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In a specific embodiment, the tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer, or liver cancer.

In some embodiments, the cancer described herein is bladder cancer, melanoma, leukemia, hepatocellular carcinoma, breast cancer, sarcoma, head and neck cancer, colon cancer, lung cancer, liver cancer, or brain cancer.

In some embodiments, the genetically modified animals can be used for determining effectiveness of a therapeutic agent (e.g., human IL12 protein or variant thereof) for the treatment of various autoimmune diseases, including rheumatoid arthritis, Crohn's disease, systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel diseases (IBD) , ulcerative colitis, or scleroderma. In some embodiments, the genetically modifled animals can be used for determining effectiveness of a therapeutic agent (e.g., human IL12 protein or variant thereof) for the treatment of various immune disorders, including allergy, asthma, and/or atopic dermatitis. Thus, the methods as described herein can be used to determine the effectiveness of an anti-IL12 or anti-IL12R antibody in inhibiting immune response. In some embodiments, the immune disorders described herein is allergy, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or neurological disorders.

The present disclosure also provides methods of determining toxicity of a therapeutic agent (e.g., human IL12 protein or variant thereof) . The methods involve administering the antibody to the animal as described herein. The animal is then evaluated for its weight change, red blood cell count, hematocrit, and/or hemoglobin. In some embodiments, the antibody can decrease the red blood cells (RBC) , hematocrit, or hemoglobin by more than 20%, 30%, 40%, or 50%. In some embodiments, the animals can have a weight that is at least 5%, 10%, 20%, 30%, or 40%smaller than the weight of the control group (e.g., average weight of the animals that are not treated with the antibody) .

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

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

The disclosure also relates to the use of the animal model generated through the methods as described herein in the screening, verifying, evaluating or studying the IL12A, IL12B, IL12RB1, and/or IL12RB2 gene functions, human IL12A, IL12B, IL12RB1, and/or IL12RB2 antibodies, drugs or efficacies for human IL12A, IL12B, IL12RB1, and/or IL12RB2 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 IL12A, IL12B, IL12RB1, and/or IL12RB2 gene humanized non-human animal prepared by the methods described herein, the IL12A, IL12B, IL12RB1, and/or IL12RB2 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 IL12A, IL12B, IL12RB1, and/or IL12RB2 proteins, or the tumor-bearing or inflammatory animal models described herein. In some embodiments, the TCR-T, CAR-T, and/or other immunotherapies can treat the IL12/IL12R-associated diseases described herein. In some embodiments, the TCA-T, CAR-T, and/or other immunotherapies provides an evaluation method for treating the IL12/IL12R-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 IL12A, IL12B, IL12RB1, and/or IL12RB2 genes and a sequence encoding an additional human or chimeric protein.

In some embodiments, the additional human or chimeric protein can be Interleukin-23 (IL23) , programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , CD73, tumor necrosis factor alpha (TNFα) , B And T Lymphocyte Associated (BTLA) , CD27, CD28, CD47, CD137, CD154, CD226, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , and/or Signal regulatory protein α (SIRPα) .

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

(a) using the methods of introducing human IL12A, IL12B, IL12RB1, and/or IL12RB2 genes or chimeric IL12A, IL12B, IL12RB1, and/or IL12RB2 genes 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 IL23, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, CD73, TNFα, BTLA, CD27, CD28, CD47, CD137, CD154, CD226, TIGIT, GITR, and/or SIRPα. 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 IL12A, IL12B, IL12RB1, and/or IL12RB2 humanizations are directly performed on a genetically modified animal having a human or chimeric IL23, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, CD73, TNFα, BTLA, CD27, CD28, CD47, CD137, CD154, CD226, TIGIT, GITR, and/or SIRPα gene.

As these proteins may involve different mechanisms, a combination therapy that targets two or more of these proteins thereof may be a more effective treatment. In fact, many related clinical trials are in progress and have shown a good effect. The genetically modified animal model with two or more human or humanized genes can be used for determining effectiveness of a combination therapy that targets two or more of these proteins, e.g., human IL12 protein or variant thereof, and an additional therapeutic agent for the treatment of cancer. The methods include administering the human IL12 protein or variant thereof 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 IL23, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, CD73, TNFα, BTLA, CD27, CD28, CD47, CD137, CD154, CD226, TIGIT, GITR, and/or SIRPα. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab) , an anti-PD-1 antibody (e.g., nivolumab) , or an anti-PD-L1 antibody.

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

In some embodiments, the combination treatment is designed for treating various cancers as described herein, e.g., bladder cancer, melanoma, leukemia, hepatocellular carcinoma, breast cancer, sarcoma, head and neck cancer, colon cancer, lung cancer, liver cancer, or brain cancer.

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

EXAMPLES

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

Materials and Methods

The following materials were used in the following examples.

ELISA MAX ^TM Deluxe Set Mouse IFN-γ was purchased from BioLegend (Catalog number: 430804) .

Mouse IL-12, research grade was purchased from Mi ltenyi Biotec (Catalog number: 130-096-708) .

Brilliant Violet 510 ^TM anti-mouse CD45 was purchased from Miltenyi Biotec (Catalog number: 130-096-798) .

Mouse IL-12 (p70) ELISA KIT was purchased from BioLegend (Catalog number: 433607) .

Human IL-12 (p70) ELISA KIT was purchased from BioLegend (Catalog number: 431707) .

InVivoMAb anti-mouse CD3 was purchased from Bio X Cell (Catalog number: BP0001-1) .

InVivoMAb anti-mouse CD28 was purchased from Bio X Cell (Catalog number: BE0015-1) .

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

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

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

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

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

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

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

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

PerCP/Cy5.5 anti-mouse TCRβ chain was purchased from BioLegend (Catalog number: 109228) .

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

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

EXAMPLE 1: Mice with humanized IL12A gene

In this example, a non-human animal (e.g., a mouse) was modified to include a nucleotide sequence encoding human IL12A protein, and the obtained genetically-modified non-human animal can express a human or humanized IL12A protein in vivo. The mouse IL12A gene (NCBI Gene ID: 16159, Primary source: MGI: 96539, UniProt ID: P43431) is located at 68597977 to 68605883 of chromosome 3 (NC_000069.7) , and the human IL12A gene (NCBI Gene ID: 3592, Primary source: HGNC: 5969, UniProt ID: O60595) is located at 159988835 to 159996019 of chromosome 3 (NC_000003.12) . The mouse IL12A transcript is NM_008351.3, and the corresponding protein sequence NP_032377.1 is set forth in SEQ ID NO: 1. The human IL12A transcript is NM_000882.4, and the corresponding protein sequence NP_000873.2 is set forth in SEQ ID NO: 2. Mouse and human IL12A gene loci are shown in FIG. 1.

All or part of nucleotide sequences encoding human IL12A protein can be introduced into the mouse endogenous IL12A locus, so that the mouse expresses human or humanized IL12A protein. Specifically, using gene-editing techniques, a nucleotide sequence (e.g., DNA or cDNA sequence) of the human IL12A gene can be used to replace the corresponding mouse sequence at the mouse endogenous IL12A locus. For example, a sequence starting from within exon 1 and ending within exon 7 of mouse IL12A gene was replaced with a corresponding sequence starting from within exon 1 and ending within exon 7 of human IL12A gene, to obtain a humanized IL12A gene locus as shown in FIG. 2, thereby humanizing mouse IL12A gene.

As shown in the schematic diagram of the targeting strategy in FIG. 3, the targeting vector contains homologous arm sequences upstream and downstream of the mouse IL12A gene, and an “A1 Fragment” containing DNA sequences of human IL12A gene. Specifically, sequence of the upstream homologous arm (5’ homologous arm, SEQ ID NO: 3) is identical to nucleotide sequence of 68594689-68598883 of NCBI accession number NC_000069.7, and sequence of the downstream homologous arm (3’ homologous arm, SEQ ID NO: 4) is identical to nucleotide sequence of 68606338-68609962 of NCBI accession number NC_000069.7. The human genomic DNA sequence from IL12A genes (SEQ ID NO: 5) is identical to nucleotide sequence of 159989057-159995559 of NCBI accession number NC_000003.12.

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

(SEQ ID NO: 6) , wherein the last “C” in sequence “ CCCTC” is the last nucleotide of the mouse sequence, and the first “C” in sequence

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

(SEQ ID NO: 7) , wherein the “C” in sequence “ ATATC” is the last nucleotide of the Neo cassette, and the first “C” in sequence

is the first nucleotide of the mouse sequence. In addition, a coding gene with a negative selectable marker (a gene encoding diphtheria toxin A subunit (DTA) ) was also constructed downstream of the 3′ homologous arm of the targeting vector. The mRNA sequence of the engineered mouse IL12A after humanization and its encoded protein sequence are shown in SEQ ID NO: 8 and SEQ ID NO: 2, respectively.

The targeting vector was constructed, e.g., by restriction enzyme digestion and ligation. The constructed targeting vector sequences were preliminarily confirmed by restriction enzyme digestion, and then verified by sequencing. Embryonic stem cells of C57BL/6 mice were transfected with the correct targeting vector by electroporation. The positive selectable marker genes were used to screen the cells, and the integration of exogenous genes was confirmed by PCR and Southern Blot. PCR primers are shown in the table below.

Table 9. PCR primer sequences and target fragment sizes

Specifically, after mouse embryonic stem cells were transfected with targeting vectors, the clones identified as positive by PCR were then verified by Southern Blot (cell DNA was digested with BamHI, EcoRV, or BglII; and hybridized with three probes) to screen out correct positive clone cells. The restriction enzymes, probes, and the size of target fragment sizes are shown in the table below. The Southern Blot detection results are shown in FIG. 4. The results indicate that among the thirteen positive clones identified by PCR, ES-03, ES-05, ES-06, ES-07, ES-08, ES-09, ES-10, ES-11, and ES-12 were verified as positive heterozygous clones without random insertions.

Table 10. Enzymes and probes used in Southern Blot

Restriction enzyme	Probe	Wild-type fragment size	Recombinant fragment size
BamHI	IL12A-5’ probe	18.8 kb	13.5 kb
EcoRV	IL12A-3’ probe	17.7 kb	11.5 kb
BglII	Neo probe	--	8.3 kb

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

IL12A-5’ Probe:

IL12A-5’ Probe-F: 5’-GGCAAGTGTCATGATGTAGCC-3’ (SEQ ID NO: 39) ,

IL12A-5’ Probe-R: 5’-CTCTGCTCTAATACTCAGTGTGAAAC-3’ (SEQ ID NO: 40) ;

IL12A-3’ Probe:

IL12A-3’ Probe-F: 5’-ACCACTGAATTTGCATAATCC-3’ (SEQ ID NO: 41) ,

IL12A-3’ Probe-R: 5’-CAGTGAGGCCAGGCAGTCCT-3’ (SEQ ID NO: 42) ;

IL12A-3’ Neo Probe:

Neo Probe-F: 5’-GGATCGGCCATTGAACAAGA-3’ (SEQ ID NO: 43) ,

Neo Probe-R: 5’-CAGAAGAACTCGTCAAGAAGGC-3’ (SEQ ID NO: 44) .

The positive clones that had been screened (black mice) were introduced into isolated blastocysts (white mice) , and the resulted chimeric blastocysts were transferred to a culture medium for short-term culture and then transplanted to the fallopian tubes of the recipient mother (white mice) to produce the F0 chimeric mice (black and white) . The F2 generation homozygous mice were obtained by backcrossing the F0 generation chimeric mice with wild-type mice to obtain the F1 generation mice, and then breeding the F1 generation heterozygous mice with each other. The positive mice were also bred with the Flp transgenic mice to remove the positive selectable marker genes (schematic diagram shown in FIG. 5) , and then the humanized homozygous mice with a humanized IL12A gene were obtained by breeding the heterozygous mice with each other. The identification results of exemplary F1 generation mice (Neo cassette removed) are shown in FIGS. 6A-6D, wherein four mice numbered F1-01, F1-02, F1-03, and F1-04 were identified as positive heterozygous mice. The genotype of the IL12A gene humanized mice were verified by PCR using primers shown in the table below. The above results showed that the methods described herein can be used to generate IL12A gene humanized mice that can be stably passaged without random insertions.

Table 11. PCR primer sequences and target fragment sizes

EXAMPLE 2: Mice with humanized IL12B gene

In this example, a non-human animal (e.g., a mouse) was modified to include a nucleotide sequence encoding human IL12B protein, and the obtained genetically-modified non-human animal can express a human or humanized IL12B protein in vivo. The mouse IL12B gene (NCBI Gene ID: 16160, Primary source: MGI: 96540 UniProt ID: P43432) is located at 44290890 to 44305504 of chromosome 11 (NC_000077.7) , and the human IL12B gene (NCBI Gene ID: 3593, Primary source: HGNC: 5970, UniProt ID: P02786) is located at 159314780 to 159330487 of chromosome 5 (NC_000005.10) . The mouse IL12B transcript is NM_001303244.1, and the corresponding protein sequence NP_001290173.1 is set forth in SEQ ID NO: 9. The human IL12B transcript is NM_002187.3, and the corresponding protein sequence NP_002178.2 is set forth in SEQ ID NO: 10. Mouse and human IL12B gene loci are shown in FIG. 7.

All or part of nucleotide sequences encoding human IL12B protein can be introduced into the mouse endogenous IL12B locus, so that the mouse expresses human or humanized IL12B protein. Specifically, using gene-editing techniques, a nucleotide sequence (e.g., DNA or cDNA sequence) of the human IL12B gene can be used to replace the corresponding mouse sequence at the mouse endogenous IL12B locus. For example, a sequence of 12470 bp spanning from exon 2 to exon 8 of mouse IL12B gene was replaced with a corresponding sequence spanning from exon 2 to exon 8 of human IL12B gene, to obtain a humanized IL12B gene locus as shown in FIG. 8, thereby humanizing mouse IL12B gene.

As shown in the schematic diagram of the targeting strategy in FIG. 9, the targeting vector contains homologous arm sequences upstream and downstream of the mouse IL12B gene, and an “A2 Fragment” containing DNA sequences of human IL12B gene. Specifically, sequence of the upstream homologous arm (5’ homologous arm, SEQ ID NO: 11) is identical to nucleotide sequence of 44291022-44294863 of NCBI accession number NC_000077.7, and sequence of the downstream homologous arm (3’ homologous arm, SEQ ID NO: 12) is identical to nucleotide sequence of 44305162-44309763 of NCBI accession number NC_000077.7. The human genomic DNA sequence from IL12B genes (SEQ ID NO: 13) is identical to nucleotide sequence of 159314313-159326782 of NCBI accession number NC_000005.10.

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

(SEQ ID NO: 14) , wherein the last “C” in sequence “ CACTC” is the last nucleotide of the human sequence, and the first “A” in sequence

(SEQ ID NO: 15) , wherein the “C” in sequence “ ATATC” is the last nucleotide of the Neo cassette, and the “C” in sequence

is the first nucleotide of the mouse sequence. In addition, a coding gene with a negative selectable marker (agene encoding diphtheria toxin A subunit (DTA) ) was also constructed downstream of the 3′ homologous arm of the targeting vector. The mRNA sequence of the engineered mouse IL12B after humanization and its encoded protein sequence are shown in SEQ ID NO: 16 and SEQ ID NO: 10, respectively.

The targeting vector was constructed, e.g., by restriction enzyme digestion and ligation. The constructed targeting vector sequences were preliminarily confirmed by restriction enzyme digestion, and then verified by sequencing. Embryonic stem cells of C57BL/6 mice were transfected with the correct targeting vector by electroporation. The positive selectable marker genes were used to screen the cells, and the integration of exogenous genes was confirmed by PCR and Southern Blot. Specifically, after mouse embryonic stem cells were transfected with targeting vectors, the clones identified as positive by PCR were then verified by Southern Blot (cell DNA was digested with KpnI, EcoRV, or SspI; and hybridized with three probes) to screen out correct positive clone cells. The restriction enzymes, probes, and the size of target fragment sizes are shown in the table below. The Southern Blot detection results are shown in FIG. 10. The results indicate that among the nine positive clones identified by PCR, ES-02, ES-05, ES-06, and ES-08 were verified as positive heterozygous clones without random insertions.

Table 12. Enzymes and probes used in Southern Blot

The following PCR primers were used:

IL12B-F1: 5’-CATCAGACCAGGCAGCTCGCAGC-3’ (SEQ ID NO: 53) ,

IL12B-R1: 5’-CCCAAGAGTCCTGGCTTAGAAGTG-3’ (SEQ ID NO: 54) ;

IL12B-F2: 5’-AACTGTTCGCCAGGCTCAAG-3’ (SEQ ID NO: 55) ,

IL12B-R2: 5’-GGGGCTGCCCATATTGGTCTTGC-3’ (SEQ ID NO: 56) .

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

IL12B-5’ Probe:

IL12B-5’ Probe-F: 5’-TATGTCTAGCTCAGTTCATGCTG-3’ (SEQ ID NO: 57) ,

IL12B-5’ Probe-R: 5’-TACAGAGGGAATATAGACGTCGA-3’ (SEQ ID NO: 58) ;

IL12B-3’ Probe:

IL12B-3’ Probe-F: 5’-CCCAACAACTTCCCACAAAGG-3’ (SEQ ID NO: 59) ,

IL12B-3’ Probe-R: 5’-CAGCTATTGCCAGCGATCCGG-3’ (SEQ ID NO: 60) ;

Neo Probe:

Neo Probe-F: 5’-GGATCGGCCATTGAACAAGA-3’ (SEQ ID NO: 43) ,

Neo Probe-R: 5’-CAGAAGAACTCGTCAAGAAGGC-3’ (SEQ ID NO: 44) .

The positive clones that had been screened (black mice) were introduced into isolated blastocysts (white mice) , and the resulted chimeric blastocysts were transferred to a culture medium for short-term culture and then transplanted to the fallopian tubes of the recipient mother (white mice) to produce the F0 chimeric mice (black and white) . The F2 generation homozygous mice were obtained by backcrossing the F0 generation chimeric mice with wild-type mice to obtain the F1 generation mice, and then breeding the F1 generation heterozygous mice with each other. The positive mice were also bred with the Flp transgenic mice to remove the positive selectable marker genes (schematic diagram shown in FIG. 11) , and then the humanized homozygous mice with a humanized IL12B gene were obtained by breeding the heterozygous mice with each other. The identification results of exemplary F1 generation mice (Neo cassette removed) are shown in FIGS. 12A-12D, wherein one mouse numbered F1-01 was identified as a positive heterozygous mouse. The genotype of the IL12B gene humanized mice were verified by PCR using primers shown in the table below. The above results showed that the methods described herein can be used to generate IL12B gene humanized mice that can be stably passaged without random insertions.

Table 13. PCR primer sequences and target fragment sizes

EXAMPLE 3: Mice with humanized IL12RB1 gene

In this example, a non-human animal (e.g., a mouse) was modified to include a nucleotide sequence encoding human IL12RB1 protein, and the obtained genetically-modified non-human animal can express a human or humanized IL12RB1 protein in vivo. The mouse IL12RB1 gene (NCBI Gene ID: 16161, Primary source: MGI: 104579, UniProt ID: Q60837) is located at 71261005 to 71276186 of chromosome 8 (NC_000074.7) , and the human IL12RB1 gene (NCBI Gene ID: 3594, Primary source: HGNC: 5971, UniProt ID: P42701) is located at 18058994 to 18099027 of chromosome 19 (NC_000019.10) . The mouse IL12RB1 transcript is NM_008353.2, and the corresponding protein sequence NP_032379.2 is set forth in SEQ ID NO: 17. The human IL12RB1 transcript is NM_005535.3, and the corresponding protein sequence NP_005526.1 is set forth in SEQ ID NO: 18. Mouse and human IL12RB1 gene loci are shown in FIG. 13.

All or part of nucleotide sequences encoding human IL12RB1 protein can be introduced into the mouse endogenous IL12RB1 locus, so that the mouse expresses human or humanized IL12RB1 protein. Specifically, using gene-editing techniques, a nucleotide sequence including part of human IL12RB1 gene and part of mouse IL12RB1 gene can be used to replace part of exon 1 and intron 1 of mouse IL12RB1 gene, and transcriptional regulatory elements WPRE sequence and Stop sequence can be added, to obtain a humanized IL12RB1 gene locus as shown in FIG. 14, thereby humanizing mouse IL12RB1 gene.

As shown in the schematic diagram of the targeting strategy in FIG. 15, the targeting vector contains homologous arm sequences upstream and downstream of the mouse IL12RB1 gene, and an “A3 Fragment” containing a DNA sequence (SEQ ID NO: 21) of human IL12RB1 gene. Specifically, sequence of the upstream homologous arm (5’ homologous arm, SEQ ID NO: 19) is identical to nucleotide sequence of 71257214-71261252 of NCBI accession number NC_000074.7, and sequence of the downstream homologous arm (3’ homologous arm, SEQ ID NO: 20) is identical to nucleotide sequence of 71261688-71266063 of NCBI accession number NC_000074.7. The A3 Fragment includes from 5’ end to 3’ end: a human IL12RB1 nucleotide sequence (SEQ ID NO: 21) , a mouse IL12RB1 nucleotide sequence (SEQ ID NO: 48) , WPRE sequence (SEQ ID NO: 24) and Stop sequence (SEQ ID NO: 25) .

(SEQ ID NO: 22) , wherein the last “T” in sequence “ CATCT” is the last nucleotide of the mouse sequence, and the first “A” in sequence

(SEQ ID NO: 23) , wherein the last “T” in sequence “ TTAAT” is the last nucleotide of the Neo cassette, and the first “G” in sequence

is the first nucleotide of the mouse sequence. In addition, a coding gene with a negative selectable marker (agene encoding diphtheria toxin A subunit (DTA) ) was also constructed downstream of the 3′homologous arm of the targeting vector. The mRNA sequence of the engineered mouse IL12RB1 after humanization and its encoded protein sequence are shown in SEQ ID NO: 26 and SEQ ID NO: 27, respectively.

The targeting vector was constructed, e.g., by restriction enzyme digestion and ligation. The constructed targeting vector sequences were preliminarily confirmed by restriction enzyme digestion, and then verified by sequencing. Embryonic stem cells of C57BL/6 mice were transfected with the correct targeting vector by electroporation. The positive selectable marker genes were used to screen the cells, and the integration of exogenous genes was confirmed by PCR and Southern Blot. Specifically, after mouse embryonic stem cells were transfected with targeting vectors, the clones identified as positive by PCR were then verified by Southern Blot (cell DNA was digested with SpeI, AseI, or NdeI; and hybridized with three probes) to screen out correct positive clone cells. The restriction enzymes, probes, and the size of target fragment sizes are shown in the table below. The Southern Blot detection results are shown in FIG. 16. The results indicate that among the twelve positive clones identified by PCR, ES-01 to ES-11 were verified as positive heterozygous clones without random insertions.

Table 14. Enzymes and probes used in Southern Blot

The following PCR primers were used:

IL12RB1-F1: 5’-GCTCGAAGAAGCCACCACTATCACAC-3’ (SEQ ID NO: 71) ,

IL12RB1-R1: 5’-GGAGCTAAGGCAACACCGCAG-3’ (SEQ ID NO: 72) ;

IL12RB1-F2: 5’-CGCATTGTCTGAGTAGGTGTC-3’ (SEQ ID NO: 73) ,

IL12RB1-R2: 5’-CACCCTTGACTGTGACCTTGGC-3’ (SEQ ID NO: 74) .

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

IL12B-5’ Probe:

IL12RB1-5’ Probe-F: 5’-TCCAACAGTGACTATATTGTGAGTC-3’ (SEQ ID NO: 75) ,

IL12RB1-5’ Probe-R: 5’-TGAGCAACATCAGGCCAGGC-3’ (SEQ ID NO: 76) ;

IL12B-3’ Probe:

IL12RB1-3’ Probe-F: 5’-TAGAGAAAGCCAGGCAGCAGGAC-3’ (SEQ ID NO: 77) ,

IL12RB1-3’ Probe-R: 5’-TCAAGGGATAGATCGACTCACCGTT-3’ (SEQ ID NO: 78) ;

Neo Probe:

Neo Probe-F: 5’-GGATCGGCCATTGAACAAGA-3’ (SEQ ID NO: 43) ,

Neo Probe-R: 5’-CAGAAGAACTCGTCAAGAAGGC-3’ (SEQ ID NO: 44) .

The positive clones that had been screened (black mice) were introduced into isolated blastocysts (white mice) , and the resulted chimeric blastocysts were transferred to a culture medium for short-term culture and then transplanted to the fallopian tubes of the recipient mother (white mice) to produce the F0 chimeric mice (black and white) . The F2 generation homozygous mice were obtained by backcrossing the F0 generation chimeric mice with wild-type mice to obtain the F1 generation mice, and then breeding the F1 generation heterozygous mice with each other. The positive mice were also bred with the Flp transgenic mice to remove the positive selectable marker genes (schematic diagram shown in FIG. 17) , and then the humanized homozygous mice with a humanized IL12RB1 gene were obtained by breeding the heterozygous mice with each other. The identification results of exemplary F1 generation mice (Neo cassette removed) are shown in FIGS. 18A-18B, wherein four mice numbered F1-01, F1-02, F1-03, and F1-04 were identified as positive heterozygous mice. The genotype of the IL12RB1 gene humanized mice were verified by PCR using primers shown in the table below. The above results showed that the methods described herein can be used to generate IL12RB1 gene humanized mice that can be stably passaged without random insertions.

Table 15. PCR primer sequences and target fragment sizes

EXAMPLE 4: Mice with humanized IL12RB2 gene

In this example, a non-human animal (e.g., a mouse) was modified to include a nucleotide sequence encoding human IL12RB2 protein, and the obtained genetically-modified non-human animal can express a human or humanized IL12RB2 protein in vivo. The mouse IL12RB2 gene (NCBI Gene ID: 16162, Primary source: MGI: 1270861, UniProt ID: P97378) is located at 67263914 to 67353277 of chromosome 6 (NC_000072.7) , and the human IL12RB2 gene (NCBI Gene ID: 3595, Primary source: HGNC: 5972, UniProt ID: Q99665-1) is located at 67307351 to 67398724 of chromosome 1 (NC_000001.11) . The mouse IL12RB2 transcript is NM_008354.4, and the corresponding protein sequence NP_032380.1 is set forth in SEQ ID NO: 28. The human IL12RB2 transcript is NM_001374259.2, and the corresponding protein sequence NP_001361188.1 is set forth in SEQ ID NO: 29. Mouse and human IL12RB2 gene loci are shown in FIG. 19.

All or part of nucleotide sequences encoding human IL12RB2 protein can be introduced into the mouse endogenous IL12RB2 locus, so that the mouse expresses human or humanized IL12RB2 protein. Specifically, using gene-editing techniques, a nucleotide sequence including part of human IL12RB2 gene and part of mouse IL12RB2 gene can be used to replace part of exon 2 and intron 2 of mouse IL12RB2 gene, and transcriptional regulatory elements WPRE sequence and Stop sequence can be added, to obtain a humanized IL12RB2 gene locus as shown in FIG. 20, thereby humanizing mouse IL12RB2 gene.

As shown in the schematic diagram of the targeting strategy in FIG. 21, the targeting vector contains homologous arm sequences upstream and downstream of the mouse IL12RB2 gene, and an “A4 Fragment” containing DNA sequences of human IL12RB2 gene. Specifically, sequence of the upstream homologous arm (5’ homologous arm, SEQ ID NO: 30) is identical to nucleotide sequence of 67338866-67343067 of NCBI accession number NC_000072.7, and sequence of the downstream homologous arm (3’ homologous arm, SEQ ID NO: 31) is identical to nucleotide sequence of 67334283-67338449 of NCBI accession number NC_000072.7. The A4 Fragment includes from 5’ end to 3’ end: a human IL12RB2 nucleotide sequence (SEQ ID NO: 34) , a mouse IL12RB2 nucleotide sequence (SEQ ID NO: 35) , WPRE sequence (SEQ ID NO: 32) and Stop sequence (SEQ ID NO: 33) .

(SEQ ID NO: 37) , wherein the last “T” in sequence “ CTCAT” is the last nucleotide of the mouse sequence, and the first “A” in sequence

(SEQ ID NO: 38) , wherein the “T” in sequence “ GGCCT” is the last nucleotide of the Neo cassette, and the “G” in sequence

is the first nucleotide of the mouse sequence. In addition, a coding gene with a negative selectable marker (agene encoding diphtheria toxin A subunit (DTA) ) was also constructed downstream of the 3′homologous arm of the targeting vector. The mRNA sequence of the engineered mouse IL12RB2 after humanization and its encoded protein sequence are shown in SEQ ID NO: 65 and SEQ ID NO: 36, respectively.

The targeting vector was constructed, e.g., by restriction enzyme digestion and ligation. The constructed targeting vector sequences were preliminarily confirmed by restriction enzyme digestion, and then verified by sequencing. Embryonic stem cells of C57BL/6 mice were transfected with the correct targeting vector by electroporation. The positive selectable marker genes were used to screen the cells, and the integration of exogenous genes was confirmed by PCR and Southern Blot. Specifically, after mouse embryonic stem cells were transfected with targeting vectors, the clones identified as positive by PCR were then verified by Southern Blot (cell DNA was digested with BamHI, StuI, or EcoNI; and hybridized with three probes) to screen out correct positive clone cells. The restriction enzymes, probes, and the size of target fragment sizes are shown in the table below. The Southern Blot detection results are shown in FIG. 22. The results indicate that among the twelve positive clones identified by PCR, ES-01, ES-02, ES-03, ES-04, ES-05, ES-08, ES-09, ES-10, ES-11, and ES-12 were verified as positive heterozygous clones without random insertions.

Table 16. Enzymes and probes used in Southern Blot

The following PCR primers were used:

IL12RB2-F1: 5’-GCTCGACTAGAGCTTGCGGA -3’ (SEQ I D NO: 88) ,

IL12RB2-R1: 5’-GCTCACCTAGGTTCAGCTAGGCTG -3’ (SEQ ID NO: 89) ;

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

IL12RB2-5’ Probe:

IL12RB2-5’ Probe-F: 5’-CAGCTTTTGCCTGGTAAGAGACAGG-3’ (SEQ ID NO: 90) ,

IL12RB2-5’ Probe-R: 5’-CTTTCTAAAGGCATCTGATACTTGT-3’ (SEQ ID NO: 91) ;

IL12RB2-3’ Probe:

IL12RB2-3’ Probe-F: 5’-GACAGAAGCTAATGGGAAGTAAC-3’ (SEQ ID NO: 92) ,

IL12RB2-3’ Probe-R: 5’-GGTGTGAGTAGGAACTACAGC-3’ (SEQ ID NO: 93) ;

Neo Probe:

Neo Probe-F: 5’-GGATCGGCCATTGAACAAGA-3’ (SEQ ID NO: 43) ,

Neo Probe-R: 5’-CAGAAGAACTCGTCAAGAAGGC-3’ (SEQ ID NO: 44) .

The positive clones that had been screened (black mice) were introduced into isolated blastocysts (white mice) , and the resulted chimeric blastocysts were transferred to a culture medium for short-term culture and then transplanted to the fallopian tubes of the recipient mother (white mice) to produce the F0 chimeric mice (black and white) . The F2 generation homozygous mice were obtained by backcrossing the F0 generation chimeric mice with wild-type mice to obtain the F1 generation mice, and then breeding the F1 generation heterozygous mice with each other. The positive mice were also bred with the Flp transgenic mice to remove the positive selectable marker genes (schematic diagram shown in FIG. 23) , and then the humanized homozygous mice with a humanized IL12RB2 gene were obtained by breeding the heterozygous mice with each other. The identification results of exemplary F1 generation mice (Neo cassette removed) are shown in FIGS. 24A-24B, wherein two mice numbered F1-01 and F1-02 were identified as positive heterozygous mice. The genotype of the IL12RB2 gene humanized mice were verified by PCR using primers shown in the table below. The above results showed that the methods described herein can be used to generate IL12RB2 gene humanized mice that can be stably passaged without random insertions.

Table 17. PCR primer sequences and target fragment sizes

EXAMPLE 5. Generation of double-gene humanized mice

The IL12A, IL12B, IL12RB1, and IL12RB2 gene humanized mice generated using the methods described herein can also be used to generate double-or multi-gene humanized mouse models. For example, in Example 1, the embryonic stem (ES) cells for blastocyst microinjection can be selected from mice comprising other genetic modifications such as modified (e.g., human or humanized) IL12B, IL12RB1, IL12RB2, IL23, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3 and/or CD73 genes. Alternatively, embryonic stem cells from humanized IL12A, IL12B, IL12RB1, and/or IL12RB2 mice described herein can be isolated, and gene recombination targeting technology can be used to obtain double-gene or multi-gene-modified mouse models of IL12A, IL12B, IL12RB1, and/or IL12RB2 and other gene modifications. In addition, it is also possible to breed the homozygous or heterozygous IL12A, IL12B, IL12RB1, and/or IL12RB2 gene humanized mice obtained by the methods described herein with other genetically modified homozygous or heterozygous mice, and the offspring can be screened. According to Mendel’s law, it is possible to generate double-gene or multi-gene modified heterozygous mice comprising modified (e.g., human or humanized) IL12A, IL12B, IL12RB1, and/or IL12RB2 gene and other genetic modifications. Then the heterozygous mice can be bred with each other to obtain homozygous double-gene or multi-gene modified mice. These double-gene or multi-gene modified mice can be used for in vivo validation of gene regulators targeting human IL12A, IL12B, IL12RB1, and IL12RB2 and other genes.

For example, IL12A/IL12B double-gene humanized mice was prepared as follows. The IL12A gene humanized homozygous mice prepared in Example 1 and the IL12B gene humanized homozygous mice prepared in Example 2 were used for mating, and IL12A/IL12B double-gene humanized mice were obtained after progeny screening. The expression of human IL12 protein in positive mice can be confirmed by methods known in the art. e.g., ELISA. Specifically, three 12-week-old wild-type C57BL/6 mice and three 12-14 weeks old IL12A/IL12B double-gene humanized homozygous mice were selected. Each mouse was intraperitoneally injected with 7.5 μg of mCD3 and 4 μg of mCD28. Serum was collected after 3 hours. The mouse IL12 and human IL12 protein levels in diluted serum were detected using Mouse IL-12 (p70) ELISA KIT and Human IL-12 (p70) ELISA KIT, respectively. As shown in FIGS. 25A-25B, expression of human IL12 protein, but not mouse IL12 protein, was detected in IL12A/IL12B double-gene humanized homozygous mice. By contrast, expression of mouse IL12 protein, but not human IL12 protein, was detected in wild-type C57BL/6 mice.

The spleen tissues of wild-type C57BL/6 mice and IL12A/IL12B double-gene humanized homozygous mice were collected to detect the expression of IL12A, and the thymus tissues of the mice were collected to detect the expression of IL12B. Total RNA was reverse transcribed into cDNA using a reverse transcription kit, using primers mIL12A-RT-PCR-F1: 5’-CCGAAACCTGCTGAAGACCACAGAT-3’ (SEQ ID NO: 101) and mIL12A-RT-PCR-R1: 5’-TCTGTAAGGGTCTGCTTCTCCCACA-3’ (SEQ ID NO: 102) to amplify a mouse IL12A fragment (448 bp) ; and using primers mIL12B-RT-PCR-F1: 5’-GTTTGCCATCGTTTTGCTGGTGT-3’ (SEQ ID NO: 103) and mIL12B-RT-PCR-R1: 5’-AGGGGAACTGCTACTGCTCTTG-3’ (SEQ ID NO: 104) to amplify a mouse IL12B fragment (451 bp) . Primers hIL12A-RT-PCR-F1: 5’-ATGCTCCAGAAGGCCAGACAAACTC-3’ (SEQ ID NO: 105) and hIL12A-RT-PCR-R1: 5’-CAGGGCCTGCATCAGCTCATCAATA-3’ (SEQ ID NO: 106) were used to amplify a human IL12A fragment (360 bp) ; and primers hIL12B-RT-PCR-F1: 5’-CGAGGTTCTAAGCCATTCGCT-3’ (SEQ ID NO: 107) and hIL12B-RT-PCR-R1: 5’-CCACCTGCCGAGAATTCTTTAATG-3’ (SEQ ID NO: 108) were used to amplify a human IL12B fragment (476 bp) . In addition, primers GAPDH-F: 5’-TCACCATCTTCCAGGAGCGAGA -3’ (SEQ ID NO: 109) and GAPDH-R: 5’- GAAGGCCATGCCAGTGAGCTT -3' (SEQ ID NO: 110) were used to amplify a GAPDH fragment (479 bp) .

The detection results are shown in FIGS. 26A-26B. Only mouse IL12A mRNA was detected in the spleen of wild-type C57BL/6 mice (+/+) , and only mouse IL12B mRNA was detected in the thymus of the same mice. By contrast, only humanized IL12B mRNA was detected in the spleen cells of IL12A/IL12B double-gene humanized homozygous mice (H/H) , and only humanized IL12B mRNA was detected in the thymus of the same mice.

As another example, IL12RB1/IL12RB2 double-gene humanized mice was prepared as follows. The IL12RB1 gene humanized homozygous mice prepared in Example 3 and the IL12RB2 gene humanized homozygous mice prepared in Example 4 were used for mating, and IL12RB1/IL12RB2 double-gene humanized mice were obtained after progeny screening. The thymus tissues of wild-type C57BL/6 mice and IL12RB1/IL12RB2 double-gene humanized homozygous mice were collected. Total RNA was reverse transcribed into cDNA using a reverse transcription kit, using primers mIL12RB1-RT-PCR-F1: 5'-GGTGTCACAATCACACCGGC-3' (SEQ ID NO: 50) and mIL12RB1-RT-PCR-R1: 5'-CTTGGGGTTCTTGGAGGCG-3' (SEQ ID NO: 61) to amplify a mouse IL12RB1 fragment (871 bp) ; and using primers mIL12RB2-RT-PCR-F1: 5'-GGAAGATGAGGGGCAAGTGGTACTC-3' (SEQ ID NO: 62) and mIL12RB2-RT-PCR-R1: 5'-TGGTCCAGGAGGTGTGTCTTGTAGT-3' (SEQ ID NO: 69) to amplify a mouse IL12RB2 fragment (413 bp) . Primers hIL12RB1-RT-PCR-F1: 5'-CTGGACCAAGACGACCCCTC-3' (SEQ ID NO: 79) and hIL12RB1-RT-PCR-R1: 5'-TCTCGCGGGTACAACACCTC -3' (SEQ ID NO: 80) were used to amplify a human IL12RB1 fragment (989 bp) ; and primers hIL12RB2-RT-PCR-F1: 5'-CAGCCTCAGCTCTGTGAAATTCCCT -3' (SEQ ID NO: 86) and hIL12RB2-RT-PCR-R1: 5'-GAGGACTTCATGGATGATCAGGGGC -3' (SEQ ID NO: 87) were used to amplify a human IL12RB2 fragment (432 bp) .

The detection results are shown in FIGS. 27A-27B. Only mouse IL12RB1 and mouse IL12RB2 expression were detected in wild-type C57BL/6 mouse cells (+/+) . By contrast, only humanized IL12RB 1 and humanized IL12RB2 mRNA expression were detected in IL12RB1/IL12RB2 double-gene humanized homozygous mouse cells (H/H) .

EXAMPLE 6. Generation of multi-gene humanized mice

IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized mice was prepared as follows. The IL12A/IL12B double-gene humanized mice and IL12RB1/IL12RB2 double-gene humanized mice prepared as described herein were bred. After multiple generations of screenings, IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized mice were obtained. The thymus tissues of wild-type C57BL/6 mice and IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized homozygous mice were collected. Total RNA was reverse transcribed into cDNA using a reverse transcription kit, and amplification was performed using the same primers as previously described for RT-PCR of IL12A/IL12B double-gene homozygous mice and IL12RB1/IL12RB2 double-gene homozygous mice. The detection results are shown in FIGS. 28A-28D. Only mouse IL12A, mouse IL12B, mouse IL12RB1 and mouse IL12RB2 expression were detected in wild-type C57BL/6 mouse cells (+/+) . By contrast, only humanized IL12A, humanized IL12B, humanized IL12RB1 and humanized IL12RB2 mRNA expression were detected in IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized homozygous mouse cells (H/H) .

The expression of human IL12 proteins in positive mice was confirmed by ELISA. Specifically, three 10-week-old wild-type mice and three 10-week-old IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized homozygous mice were selected. Each mouse was intraperitoneally injected with 7.5 μg of mCD3 and 4 μg of mCD28. Serum was collected after 3 hours. The mouse IL12 and human IL12 protein levels in diluted serum were detected using Mouse IL-12 (p70) ELISA KIT and Human IL-12 (p70) ELISA KIT, respectively. As shown in FIGS. 29A-29B, expression of human IL12 protein, but not mouse IL12 protein, was detected in IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized homozygous mice (H/H; FIG. 29B) . By contrast, expression of mouse IL12 protein, but not human IL12 protein, was detected in wild-type C57BL/6 mice (+/+; FIG. 29A) .

Further, ELISA was used to detect the secretion of IFN-γ in wild-type mice and IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized homozygous mice. Specifically, three wild-type C57BL/6 mice and three IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized homozygous mice were selected. After euthanasia, mouse spleen tissue was collected and processed into single cell suspension. CD4+ T cells were sorted, which were treated with anti-mouse CD3ε antibody (anti-mCD3ε, concentration 0.6 μg/mL) , anti-mouse CD28 antibody (anti-mCD28, concentration 0.8 μg/mL) , and different concentrations of a mouse IL12 recombinant protein (mIL12 at a concentration of 0.01 μg/mL or 0.05 μg/mL) or a recombinant human IL12 protein (hIL12 at a concentration of 0.02 μg/mL or 0.2 μg/mL) , and cultured at 37℃ for 48 hours. The cell culture supernatant was collected for ELISA experiment to detect the secretion of mlFN-γ. The results are shown in FIG. 30. The results showed that under the stimulation of hIL12 and mIL12, increased mIFN-γ secretion was detected in both wild-type mice and IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized homozygous mice.

The above results indicate that the IL12A, IL12B, IL12RB1 and IL12RB2 proteins of the IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized mice described herein can be expressed normally and mediate downstream IFN-γ secretion.

Further, the in vivo immuno-phenotyping of IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized mice was detected by flow cytometry. Specifically, three 6-week-old female C57BL/6 wild-type mice (+/+) and IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized homozygous mice (H/H) prepared using the methods described herein were selected. After euthanasia by cervical dislocation, the spleen, lymph nodes and peripheral blood were collected. The cells were stained with Brilliant Violet 510 ^TM anti-mouse CD45 Antibody, PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody, Brilliant Violet 421 ^TM anti-mouse CD4 Antibody, FITC anti-mouse F4/80 Antibody, PE anti-mouse CD8a Antibody, PE/Cy ^TM 7 Mouse anti-mouse NK1.1 Antibody, APC anti-mouse/rat Foxp3 Antibody, FITC anti-Mouse CD19 Antibody, PerCP/Cy5.5 anti-mouse TCRβ chain, APC Hamster Anti-Mouse TCRβ Chain, Brilliant Violet 605 ^TM anti-mouse CD11c Antibody, or PE anti-mouse/human CD11b Antibody, and then subjected to flow cytometry analysis.

The detection results of leukocyte subtypes in the spleen and peripheral blood are shown in FIG. 3lA and FIG. 33A, respectively. The results showed that the percentages of T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells, granulocytes, dendritic cells (DC cells) , macrophages, monocytes, and other leukocyte subtypes in the spleen and peripheral blood of IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized mice were basically the same as those in C57BL/6 wild-type mice. The detection results of leukocyte subtypes in lymph nodes are shown in FIG. 32A. The results showed that the leukocyte subtypes, e.g., T cells, B cells, NK cells, CD4+ T cells, and CD8+ T cells in the lymph nodes of IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized mice were basically the same as those of C57BL/6 wild-type mice.

The detection results of T cell subtypes in the spleen, lymph nodes, and peripheral blood are shown in FIG. 3lB, FIG. 32B, and FIG. 33B, respectively. The results showed that the T cell subtypes, e.g., CD4+ T cells, CD8+ T cells, and Treg cells in the IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized mice were basically the same as those of C57BL/6 wild-type mice.

The results indicate that the humanization of IL12A, IL12B, IL12RB1, and IL12RB2 genes did not affect the differentiation, development and distribution of leukocytes and T cells in mice.

In addition, six 8-week-old female wild-type C57BL/6 mice and six IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized mice were selected, and peripheral blood was collected for blood routine and blood biochemical tests. Blood routine test indicators included: white blood cell count (WBC) , red blood cell count (RBC) , hematocrit (HCT) , hemoglobin (HGB) , mean corpuscular volume (MCV) , mean corpuscular hemoglobin (MCH) , mean corpuscular hemoglobin concentration (MCHC) , platelet count (PLT) , lymphocytes (LYMPH) , monocytes (MONO) , neutrophils (NEUT) , Red blood cell distribution width (RDW-SD) , red blood cell volume distribution width coefficient of variation (RDW-CV) , and mean platelet volume (MPV) . Blood biochemical test indicators included: alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , albumin (ALB) , blood glucose (GLU) , urea (UREA) , serum creatinine (CREA) , serum total cholesterol (TC) , and triglyceride (TG) . The blood routine test results (mean) and the blood biochemical test results are shown in the tables below.

Table 18. Blood routine test results

Table 19. Blood biochemical test results

As shown in the tables above, the results showed that humanization of IL12A, IL12B, IL12RB1, and IL12RB2 genes did not affect the composition and morphology of blood cells in mice, and the IL12A gene humanized mice had similar liver function as the wild type mice.

The mice described herein can also be used to induce multiple human disease models and test the in vivo efficacy of human-specific antibodies. For example, IL12A, IL12B, IL12RB1 and/or IL12RB2 gene humanized mice can be used to evaluate the efficacy and pharmacokinetics of human IL12-specific signaling pathway drugs and the in vivo therapeutic efficacy for various disease models known in the art.

Specifically, 7-9 week-old female IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized homozygous mice prepared herein were selected and subcutaneously inoculated with mouse colon cancer cells MC38 (5 × 10 ⁵ cells per mouse) . When the tumor volume grew to about 100 ± 50mm ³, the mice were randomly placed into a control group (G1; n = 4) and three treatment groups (G2, G3, and G4; n = 5) . The G1 group mice were injected with PBS; the G2 group mice were injected with an anti-mouse PD-1 antibody (mPD-1, prepared by immunizing mice using methods described in Janeway′s Immunobiology (9th Edition) ) ; the G3 group mice were injected with a human IL12 protein variant; and the G4 group mice were injected with a human IL12 protein (hIL12; purchased from Miltenyi Biotec (Catalog number: 130-096-798)) . The mice were administered by intraperitoneal injection (i.p. ) . Euthanasia was performed when the tumor volume of the mouse reached 3000 mm ³.

The main data and analysis results of each experiment are listed in the table below, including the tumor volume at the time of grouping (Day 0) , 14 days after grouping (Day 14) , 21 days after grouping (Day 21) , the survived mouse number, the Tumor Growth Inhibition value tumor (TGI _TV) on Day 21, and the statistical difference (P value) of tumor volume between the treated and control group mice.

Table 20. Tumor volume, survived mouse number and TGI _TV

FIG. 34 and FIG. 35 showed tumor volume and body weight of the mice, respectively. Overall, the animals in each group were healthy. On Day 21, the body weights of all the treatment group mice (G2-G4) and control group mice (G1) increased (FIG. 35) , and were not significantly different from each other (P ＞ 0.05) during the experimental period. The results indicate that the treatment group mice tolerated the mPD-1, the IL12 variant, and hIL12 well. According to the results shown in FIG. 34 and the table below, tumor volume of the treatment group mice was smaller than that of the control group mice at all times during the experimental period. For example, on Day 21, the tumor volumes of mice in the G2, G3 and G4 groups were 694 ± 177 mm ³, 1263 ± 174 mm ³ and 1548 ± 316 mm ³, respectively, which were all smaller than that of the control group of 1925 ± 127 mm ³. Different treatment groups showed different treatment results. For example, as compared to the control group (G1) , the tumor volume of the G3 group mice was significantly inhibited on Day 21 (P ＜ 0.05) . The results showed that the IL12A/IL12B/IL12RB1/IL12RB2 four-gene humanized homozygous mice prepared by the methods described herein can be used as an animal model for in vivo efficacy verification and screening of human IL12 protein or protein variants.

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 interleukin-12subunit alpha (IL12A) .
The animal of claim 1, wherein the sequence encoding the human or chimeric IL12A is operably linked to an endogenous regulatory element (e.g., endogenous 5’ UTR and/or 3’ UTR) at the endogenous IL12A gene locus in the at least one chromosome.
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric IL12A comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human IL12A (NP_000873.2; SEQ ID NO: 2) .
The animal of any one of claims 1-3, wherein the human or chimeric IL12A forms a functional IL12 heterodimer with an endogenous or human IL12B.
The animal of any one of claims 1-4, wherein the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat.
The animal of any one of claims 1-5, wherein the animal is a mouse.
The animal of any one of claims 1-6, wherein the animal does not express endogenous IL12A or expresses a decreased level of endogenous IL12A.
The animal of any one of claims 1-7, wherein the animal has one or more cells expressing human or chimeric IL12A.
The animal of any one of claims 1-8, wherein the animal has one or more cells expressing human or chimeric IL12A, and endogenous IL12 receptor can bind to the IL12heterodimer comprising the expressed human or chimeric IL12A.
The animal of any one of claims 1-8, wherein the animal has one or more cells expressing human or chimeric IL12A, and human IL12 receptor can bind to the IL12 heterodimer comprising the expressed human or chimeric IL12A.
A genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous IL12A with a sequence encoding a corresponding region of human IL12A at an endogenous IL12A gene locus.
The animal of claim 11, wherein the sequence encoding the corresponding region of human IL12A is operably linked to an endogenous regulatory element at the endogenous IL12A locus, and one or more cells of the animal express a human or chimeric IL12A.
The animal of claim 11 or 12, wherein the animal does not express endogenous IL12A or expresses a decreased level of endogenous IL12A.
The animal of any one of claims 11-13, wherein the replaced sequence encodes the full-length protein of IL12A.
The animal of any one of claims 11-14, wherein the animal is a mouse, and the replaced endogenous IL12A region is a portion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or a portion of exon 7 of the endogenous mouse IL12A gene.
The animal of any one of claims 11-15, wherein the animal is heterozygous or homozygous with respect to the replacement at the endogenous IL12A gene locus.
A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric IL12A polypeptide, wherein the chimeric IL12A polypeptide comprises at least 50, 100, 150, 200, 210, 220, 230, 240, or 250contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL12A, wherein the animal expresses the chimeric IL12A polypeptide.
The animal of claim 17, wherein the nucleotide sequence is operably linked to an endogenous IL12A regulatory element of the animal.
The animal of claim 17 or 18, wherein the nucleotide sequence is integrated to an endogenous IL12A gene locus of the animal.
The animal of any one of claims 17-19, wherein the chimeric IL12A polypeptide has at least one mouse IL12A activity and/or at least one human IL12A activity.
A method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous IL12A gene locus, a sequence encoding a region of an endogenous IL12A with a sequence encoding a corresponding region of human IL12A.
The method of claim 21, wherein the sequence encoding the corresponding region of human IL12A comprises a portion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or a portion of exon 7 of a human IL12A gene.
The method of claim 21 or 22, wherein the sequence encoding the corresponding region of human IL12A comprises at least 50, 100, 200, or 300 nucleotides of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of a human IL12A gene.
The method of any one of claims 21-23, wherein the sequence encoding the corresponding region of human IL12A encodes a sequence that is at least 90%identical to SEQ ID NO: 2.
The method of any one of claims 21-24, wherein the animal is a mouse, and the locus is a portion of exon 1, exon 2, exon 3, exons 4, exon 5, exon 6, and/or a portion of exon 7 of the mouse IL12A gene.
A method of making a genetically-modified non-human animal cell that expresses a human or chimeric IL12A, the method comprising:

replacing, at an endogenous mouse IL12A gene locus, a nucleotide sequence encoding a region of endogenous IL12A with a nucleotide sequence encoding a corresponding region of human IL12A, thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the human or chimeric IL12A, wherein the animal cell expresses the human or chimeric IL12A.
The method of claim 26, wherein the animal is a mouse.
The method of claim 26 or 27, wherein the nucleotide sequence encoding the human or chimeric IL12A is operably linked to an endogenous IL12A regulatory region, e.g., promoter.
The animal of any one of claims 1-20, wherein the animal further comprises a sequence encoding an additional human or chimeric protein, e.g., Interleukin-12 subunit beta (IL12B) , Interleukin-12 receptor subunit beta-1 (IL12RB1) , Interleukin 12 receptor subunit beta 2 (IL12RB2) , Interleukin-23 (IL23) , programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , and/or CD73.
The animal of claim 29, wherein the additional human or chimeric protein is IL12B.
The method of any one of claims 21-28, wherein the animal or mouse further comprises a sequence encoding an additional human or chimeric protein, e.g., IL12B, IL12RB1, IL12RB2, IL23, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, and/or CD73.
The method of claim 31, wherein the additional human or chimeric protein is IL12B.
A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric interleukin-12 subunit beta (IL12B) .
The animal of claim 33, wherein the sequence encoding the human or chimeric IL12B is operably linked to an endogenous regulatory element at the endogenous IL12B gene locus in the at least one chromosome.
The animal of claim 33 or 34, wherein the sequence encoding the human or chimeric IL12B is operably linked to an endogenous 5’ UTR and a human 3’ UTR.
The animal of any one of claims33-35, wherein the sequence encoding a human or chimeric IL12B comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human IL12B (NP_002178.2; SEQ ID NO: 10) .
The animal of any one of claims 33-36, wherein the human or chimeric IL12B forms a functional IL12 heterodimer with an endogenous or human IL12A.
The animal of any one of claims 33-37, wherein the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat.
The animal of any one of claims 33-38, wherein the animal is a mouse.
The animal of any one of claims 33-39, wherein the animal does not express endogenous IL12B or expresses a decreased level of endogenous IL12B.
The animal of any one of claims 33-40, wherein the animal has one or more cells expressing human or chimeric IL12B.
The animal of any one of claims 33-41, wherein the animal has one or more cells expressing human or chimeric IL12B, and endogenous IL12 receptor can bind to the IL12 heterodimer comprising the expressed human or chimeric IL12B.
The animal of any one of claims 33-41, wherein the animal has one or more cells expressing human or chimeric IL12B, and human IL12 receptor can bind to the IL12 heterodimer comprising the expressed human or chimeric IL12B.
A genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous IL12B with a sequence encoding a corresponding region of human IL12B at an endogenous IL12B gene locus.
The animal of claim 44, wherein the sequence encoding the corresponding region of human IL12B is operably linked to an endogenous 5’ UTR and a human 3’ UTR at the endogenous IL12B locus, and one or more cells of the animal express a human or chimeric IL12B.
The animal of claim 44 or 45, wherein the animal does not express endogenous IL12B or expresses a decreased level of endogenous IL12B.
The animal of any one of claims 44-46, wherein the replaced sequence encodes the full-length protein of IL12B.
The animal of any one of claims 44-47, wherein the animal is a mouse, and the replaced endogenous IL12B region isexon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8, or a portion thereof, of the endogenous mouse IL12B gene.
The animal of any one of claims 44-48, wherein the animal is heterozygous or homozygous with respect to the replacement at the endogenous IL12B gene locus.
A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric IL12B polypeptide, wherein the chimeric IL12B polypeptide comprises at least 50, 100, 150, 200, 210, 220, 230, 240, or 250 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL12B, wherein the animal expresses the chimeric IL12B polypeptide.
The animal of claim 50, wherein the nucleotide sequence is operably linked to an endogenous IL12B regulatory element of the animal.
The animal of claim 50 or 51, wherein the nucleotide sequence is integrated to an endogenous IL12B gene locus of the animal.
The animal of any one of claims 50-52, wherein the chimeric IL12B polypeptide has at least one mouse IL12B activity and/or at least one human IL12B activity.
A method for making a genetically-modified, non-human animal, comprising:

replacing in at least one cell of the animal, at an endogenous IL12B gene locus, a sequence encoding a region of an endogenous IL12B with a sequence encoding a corresponding region of human IL12B.
The method of claim 54, wherein the sequence encoding the corresponding region of human IL12B comprises, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8, or a portion thereof, of a human IL12B gene.
The method of claim 54 or 55, wherein the sequence encoding the corresponding region of human IL12B comprises at least 50, 100, 200, or 300 nucleotides of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 of a human IL12B gene.
The method of any one of claims 54-56, wherein the sequence encoding the corresponding region of human IL12B encodes a sequence that is at least 90%identical to SEQ ID NO: 10.
The method of any one of claims 54-57, wherein the animal is a mouse, and the locus is exon 2, exon 3, exons 4, exon 5, exon 6, exon 7, and/or exon 8 of the mouse IL12B gene.
A method of making a genetically-modified non-human animal cell that expresses a human or chimeric IL12B, the method comprising:

replacing, at an endogenous mouse IL12B gene locus, a nucleotide sequence encoding a region of endogenous IL12B with a nucleotide sequence encoding a corresponding region of human IL12B, thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the human or chimeric IL12B, wherein the animal cell expresses the human or chimeric IL12B.
The method of claim 59, wherein the animal is a mouse.
The method of claim59 or 60, wherein the nucleotide sequence encoding the human or chimeric IL12B is operably linked to an endogenous IL12B regulatory region, e.g., promoter.
The animal of any one of claims 33-53, wherein the animal further comprises a sequence encoding an additional human or chimeric protein, e.g., Interleukin-12 subunit alpha (IL12A) , Interleukin-12 receptor subunit beta-1 (IL12RB1) , Interleukin 12 receptor subunit beta 2 (IL12RB2) , Interleukin-23 (IL23) , programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , and/or CD73.
The animal of claim 62, wherein the additional human or chimeric protein is IL12A.
The method of any one of claims 54-61, wherein the animal or mouse further comprises a sequence encoding an additional human or chimeric protein, e.g., IL12A, IL12RB1, IL12RB2, IL23, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, and/or CD73.
The method of claim 64, wherein the additional human or chimeric protein is IL12A.
A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric interleukin-12 receptor subunit beta-1 (IL12RB1) .
The animal of claim 66, wherein the sequence encoding the human or chimeric IL12RB1 is operably linked to an endogenous regulatory element at the endogenous IL12RB1 gene locus in the at least one chromosome.
The animal of claim 66 or 67, wherein the sequence encoding a human or chimeric IL12RB1 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to amino acids 24-570 of human IL12RB1 (NP_005526.1; SEQ ID NO: 18) .
The animal of claim 66 or 67, wherein the sequence encoding a human or chimeric IL12RB1 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: 27.
The animal of any one of claims 66-69, wherein the human or chimeric IL12RB1 forms a functional IL12 receptor with an endogenous or human IL12RB2.
The animal of any one of claims 66-70, wherein the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat.
The animal of any one of claims 66-71, wherein the animal is a mouse.
The animal of any one of claims 66-72, wherein the animal does not express endogenous IL12RB1or expresses a decreased level of endogenous IL12RB1.
The animal of any one of claims 66-73, wherein the animal has one or more cells expressing human or chimeric IL12RB1.
The animal of any one of claims 66-74, wherein the animal has one or more cells expressing human or chimeric IL12RB1, and endogenous IL12 can bind to the IL12 receptor comprising the expressed human or chimeric IL12RB1.
The animal of any one of claims 66-74, wherein the animal has one or more cells expressing human or chimeric IL12RB1, and human IL12 can bind to the IL12 receptor comprising the expressed human or chimeric IL12RB1.
A genetically-modified, non-human animal, wherein the genome of the animal comprises an insertion of a sequence encoding a human or chimeric IL12RB1 at an endogenous IL12RB1 gene locus.
The animal of claim 77, wherein the sequence encoding a human or chimeric IL12RB1 does not include a sequence encoding the signal peptide of IL12RB1.
The animal of claim 77 or 78, wherein the sequence encoding a human or chimericIL12RB1 is operably linked to an endogenous regulatory element at the endogenous IL12RB1 locus, and one or more cells of the animal express the human or chimeric IL12RB1.
The animal of any one of claims77-79, wherein the animal does not express endogenous IL12RB1or expresses a decreased level of endogenous IL12RB1.
The animal of any one of claims 77-80, wherein the sequence encoding a human or chimeric IL12RB1 is inserted within exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, and/or exon 16 of endogenous IL12RB1 gene.
The animal of any one of claims 77-81, wherein the sequence encoding a human or chimeric IL12RB1 is inserted within exon 1 and/or intron 1 of endogenous IL12RB1 gene.
The animal of any one of claims 77-82, wherein the sequence encoding a human or chimeric IL12RB1 is inserted immediately after the last nucleotide encoding the signal peptide of endogenous IL12RB1 (e.g., a nucleotide corresponding to position 160 of NM_008353.2) .
The animal of claim 83, wherein a sequence encoding the N-terminal 1, 2, 3, or 4 amino acids of endogenous IL12RB1 extracellular region is deleted from exon 1 of endogenous IL12RB1 gene.
The animal of claim 83, wherein asequence corresponding to positions 161-170 of NM_008353.2 and the downstream 425 nucleotides within intron 1 (e.g., position 71261263 to position 71261687 of NC_000074.7) are deleted.
The animal of any one of claims 77-85, wherein the inserted sequence comprises, optionally from 5’ end to 3’ end:

a) a sequence encoding the extracellular region and transmembrane region of a human IL12RB1;

b) a sequence encoding the cytoplasmic region of an endogenous IL12RB1; and

c) optionally one or more auxiliary sequences (e.g., WPRE, STOP, and/or polyA) .
The animal of claim 86, wherein the sequence encoding the extracellular region and transmembrane region of a human IL12RB1 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 21, and the sequence encoding the cytoplasmic region of an endogenous IL12RB1 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 48.
The animal of claim 86 or 87, wherein the one or more auxiliary sequencescomprise, optionally from 5’ end to 3’ end: a WPRE sequence and a STOP sequence.
The animal of claim 88, wherein the WPRE sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 24 and the STOP sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 25.
The animal of any one of claims 77-89, wherein the animal is heterozygous or homozygous with respect to the insertion at the endogenous IL12RB1 gene locus.
A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric IL12RB1 polypeptide, wherein the chimeric IL12RB1 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL12RB1, wherein the animal expresses the chimeric IL12RB1 polypeptide.
The animal of claim 91, wherein the chimeric IL12RB1 polypeptide has at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 510, 520, 530, 540, 545, 546, or 547 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of human IL12RB1 extracellular and transmembrane regions.
The animal of claim 91 or 92, wherein the chimeric IL12RB1 polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 24-570 of SEQ ID NO: 18.
The animal of any one of claims 91-93, wherein the nucleotide sequence is operably linked to an endogenous IL12RB1 regulatory element of the animal.
The animal of any one of claims 91-94, wherein the chimeric IL12RB1 polypeptide comprises an endogenous IL12RB1 cytoplasmic region, and optionally an endogenous signal peptide.
The animal of any one of claims 91-95, wherein the nucleotide sequence is integrated to an endogenous IL12RB1 gene locus of the animal.
The animal of any one of claims 91-96, wherein the chimeric IL12RB1 polypeptide has at least one mouse IL12RB1 activity and/or at least one human IL12RB1 activity.
A method of making a genetically-modified non-human animal cell that expresses a chimeric IL12RB1, the method comprising:

inserting at an endogenous IL12RB1 gene locus (e.g., exon 1 of endogenous IL12RB1 gene) , a nucleotide sequence comprising, optionally from 5’ end to 3’ end:

a) a sequence encoding the extracellular region and transmembrane region of a human IL12RB1;

b) a sequence encoding the cytoplasmic region of an endogenous IL12RB1; and

c) optionally one or more auxiliary sequences (e.g., WPRE, STOP, and/or polyA) , thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the chimeric IL12RB1, wherein the animal cell expresses the chimeric IL12RB1.
The method of claim 98, wherein the animal is a mouse.
The method of claim 98 or 99, wherein the nucleotide sequence encoding the chimeric IL12RB1 polypeptide is operably linked to an endogenous IL12RB1 regulatory region, e.g., promoter.
The animal of any one of claims 66-97, wherein the animal further comprises a sequence encoding an additional human or chimeric protein, e.g., Interleukin 12 receptor subunit beta 2 (IL12RB2) , Interleukin-12 subunit alpha (IL12A) , Interleukin-12 subunit beta (IL12B) , Interleukin-23 (IL23) , programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , and/or CD73.
The animal of claim 101, wherein the additional human or chimeric protein is IL12RB2.
The method of any one of claims98-100, wherein the animal or mouse further comprises a sequence encoding an additional human or chimeric protein, e.g., IL12RB2, IL12A, IL12B, IL23, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, and/or CD73.
The method of claim 103, wherein the additional human or chimeric protein is IL12RB2.
A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric interleukin-12 receptor subunit beta-2 (IL12RB2) .
The animal of claim 105, wherein the sequence encoding the human or chimeric IL12RB2 is operably linked to an endogenous regulatory element at the endogenous IL12RB2 gene locus in the at least one chromosome.
The animal of claim 105 or 106, wherein the sequence encoding a human or chimeric IL12RB2 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to amino acids 28-622 of human IL12RB2 (NP_001361188.1; SEQ ID NO: 29) .
The animal of claim 105 or 106, wherein the sequence encoding a human or chimeric IL12RB2 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: 36.
The animal of any one of claims 105-108, wherein the human or chimeric IL12RB2 forms a functional IL12 receptor with an endogenous or human IL12RB1.
The animal of any one of claims 105-109, wherein the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat.
The animal of any one of claims 105-110, wherein the animal is a mouse.
The animal of any one of claims 105-111, wherein the animal does not express endogenous IL12RB2or expresses a decreased level of endogenous IL12RB2.
The animal of any one of claims 105-112, wherein the animal has one or more cells expressing human or chimeric IL12RB2.
The animal of any one of claims 105-113, wherein the animal has one or more cells expressing human or chimeric IL12RB2, and endogenous IL12 can bind to the IL12 receptor comprising the expressed human or chimeric IL12RB2.
The animal of any one of claims 105-114, wherein the animal has one or more cells expressing human or chimeric IL12RB2, and human IL12 can bind to the IL12 receptor comprising the expressed human or chimeric IL12RB2.
A genetically-modified, non-human animal, wherein the genome of the animal comprises an insertion of a sequence encoding a human or chimeric IL12RB2 at an endogenous IL12RB2 gene locus.
The animal of claim 116, wherein the sequence encoding a human or chimeric IL12RB2 does not include a sequence encoding the signal peptide of IL12RB2.
The animal of claim 116 or 117, wherein the sequence encoding a human or chimeric IL12RB2 is operably linked to an endogenous regulatory element at the endogenous IL12RB2 locus, and one or more cells of the animal express the human or chimeric IL12RB2.
The animal of any one of claims 116-118, wherein the animal does not express endogenous IL12RB2or expresses a decreased level of endogenous IL12RB2.
The animal of any one of claims 116-119, wherein the sequence encoding a human or chimeric IL12RB2 is inserted within exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, and/or exon 16 of endogenous IL12RB2 gene.
The animal of any one of claims 116-120, wherein the sequence encoding a human or chimeric IL12RB2 is inserted within exon 2 and/or intron 2 of endogenous IL12RB2 gene.
The animal of any one of claims 116-121, wherein the sequence encoding a human or chimeric IL12RB2 is inserted immediately after the last nucleotide encoding the signal peptide of endogenous IL12RB2 (e.g., a nucleotide corresponding to position 257 of NM_008354.4) .
The animal of claim 122, wherein a sequence encoding the N-terminal 1, 2, or 3 amino acids of endogenous IL12RB2 extracellular domain is deleted from exon 2 of endogenous IL12RB2 gene.
The animal of claim 122, wherein a sequence corresponding to positions 258-264 of NM_008354.4 and the downstream 409 nucleotides within intron 2 (e.g., position 67338450 to position 67338858 of NC_000072.7) are deleted.
The animal of any one of claims 116-124, wherein the inserted sequence comprises, optionally from 5’ end to 3’ end:

a) a sequence encoding the extracellular region of a human IL12RB2;

b) a sequence encoding the transmembrane region and cytoplasmic region of an endogenous IL12RB2; and

c) optionally one or more auxiliary sequences (e.g., WPRE, STOP, and/or polyA) .
The animal of claim 125, wherein the sequence encoding the extracellular region of a human IL12RB2 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 34, and the sequence encoding the cytoplasmic regionand transmembrane region of an endogenous IL12RB2 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 35.
The animal of claim 125 or 126, wherein the one or more auxiliary sequences comprise, optionally from 5’ end to 3’ end: a WPRE sequence and a STOP sequence.
The animal of claim 127, wherein the WPRE sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 32 and the STOP sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 33.
The animal of any one of claims 116-128, wherein the animal is heterozygous or homozygous with respect to the insertion at the endogenous IL12RB2 gene locus.
A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric IL12RB2 polypeptide, wherein the chimeric IL12RB2 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL12RB2, wherein the animal expresses the chimeric IL12RB2 polypeptide.
The animal of claim 130, wherein the chimeric IL12RB2 polypeptide has at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 591, 592, 593, 594, 595, 596, 597, 598, or 599contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL12RB2 extracellular region.
The animal of claim 130 or 131, wherein the chimeric IL12RB2 polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 28-622 of SEQ ID NO: 29.
The animal of any one of claims 130-132, wherein the nucleotide sequence is operably linked to an endogenous IL12RB2 regulatory element of the animal.
The animal of any one of claims 130-133, wherein the chimeric IL12RB2 polypeptide comprises endogenous IL12RB2transmembrane and cytoplasmic regions, and optionally an endogenous signal peptide.
The animal of any one of claims 130-134, wherein the nucleotide sequence is integrated to an endogenous IL12RB2 gene locus of the animal.
The animal of any one of claims 130-135, wherein the chimeric IL12RB2 polypeptide has at least one mouse IL12RB2 activity and/or at least one human IL12RB2 activity.
A method of making a genetically-modified non-human animal cell that expresses a chimeric IL12RB2, the method comprising:

inserting at an endogenous IL12RB2 gene locus (e.g., exon 2 of endogenous IL12RB2 gene) , a nucleotide sequence comprising, optionally from 5’ end to 3’ end:

a) a sequence encoding the extracellular region of a human IL12RB2;

b) a sequence encoding the transmembrane region and cytoplasmic region of an endogenous IL12RB2; and

c) optionally one or more auxiliary sequences (e.g., WPRE, STOP, and/or polyA) , thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the chimeric IL12RB2, wherein the animal cell expresses the chimeric IL12RB2.
The method of claim 137, wherein the animal is a mouse.
The method of claim 137 or 138, wherein the nucleotide sequence encoding the chimeric IL12RB2 polypeptide is operably linked to an endogenous IL12RB2 regulatory region, e.g., promoter.
The animal of any one of claims 105-136, wherein the animal further comprises a sequence encoding an additional human or chimeric protein, e.g., Interleukin 12 receptor subunit beta 1 (IL12RB1) , Interleukin-12 subunit alpha (IL12A) , Interleukin-12 subunit beta (IL12B) , Interleukin-23 (IL23) , programmed cell death protein 1 (PD-1) , programmed cell death ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , tumor necrosis factor receptor superfamily, member 4 (OX40) , lymphocyte-activation gene 3 (LAG3) , T-cell immunoglobulin and mucin-domain containing-3 (TIM3) , and/or CD73.
The animal of claim 110, wherein the additional human or chimeric protein is IL12RB1.
The method of any one of claims 137-139, wherein the animal or mouse further comprises a sequence encoding an additional human or chimeric protein, e.g., IL12RB1, IL12A, IL12B, IL23, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, and/or CD73.
The method of claim 142, wherein the additional human or chimeric protein is IL12RB1.
A method of determining effectiveness of a therapeutic agent for treating cancer, comprising:

administering the therapeutic agent to the animal of any one of claims 1-20, 29, 30, 33-53, 62, 63, 66-97, 101, 102, 105-136, 140, and 141, wherein the animal has a tumor; and

determining inhibitory effects of the therapeutic agent to the tumor.
The method of claim 144, wherein the therapeutic agent is an IL12 signaling pathway modulator (e.g., an antibody or antigen-binding fragment that binds to IL12A, IL12B, IL12RB1, and/or IL12RB2) .
The method of claim 144, wherein the therapeutic agent is a human IL12 protein or variant thereof, or an antibody targeting an immune checkpoint molecule (e.g., PD-1) .
The method of any one of claims 144-146, wherein the tumor comprises one or more cancer cells that are injected into the animal.
The method of any one of claims 144-147, wherein determining inhibitory effects of the therapeutic agent to the tumor involves measuring the tumor volume in the animal.
The method of any one of claims 144-148, wherein the cancer is bladder cancer, melanoma, leukemia, hepatocellular carcinoma, breast cancer, sarcoma, head and neck cancer, colon cancer, lung cancer, liver cancer, or brain cancer.
A method of determining effectiveness of an IL12 signaling pathway modulator and an additional therapeutic agent for the treatment of cancer, comprising

administering the IL12 signaling pathway modulator and the additional therapeutic agent to the animal of any one of claims 1-20, 29, 30, 33-53, 62, 63, 66-97, 101, 102, 105-136, 140, and 141, wherein the animal has a tumor; and

determining inhibitory effects on the tumor.
The method of claim 150, wherein the animal further comprises a sequence encoding a human or chimeric programmed cell death protein 1 (PD-1) and/or a human or chimeric programmed death-ligand 1 (PD-L1) .
The method of claim 150 or 151, wherein the additional therapeutic agent is an anti-PD-1 antibody or an anti-PD-L1 antibody.
The method of any one of claims 150-152, wherein the tumor comprises one or more tumor cells that express PD-L1.
The method of any one of claims 150-153, wherein the tumor is caused by injection of one or more cancer cells into the animal.
The method of any one of claims 150-154, wherein determining inhibitory effects of the treatment involves measuring the tumor volume in the animal.
The method of any one of claims 56-62, wherein the animal has bladder cancer,

melanoma, leukemia, hepatocellular carcinoma, breast cancer, sarcoma, head and neck cancer, colon cancer, lung cancer, liver cancer, or brain cancer.
A method of determining effectiveness of an IL12signaling pathway modulator for treating an autoimmune disorder, comprising:

administering the IL12 signaling pathway modulator to the animal of any one of claims 1-20, 29, 30, 33-53, 62, 63, 66-97, 101, 102, 105-136, 140, and 141; and

determining effects of the IL12 signaling pathway modulator.
The method of claim 157, wherein the autoimmune disorder is asthma, systemic lupus erythematosus (SLE) , rheumatoid arthritis (RA) , Sjogren's syndrome (SS) , multiple sclerosis (MS) , Crohn's disease (CD) , inflammatory bowel disease (IBD) , or psoriasis.
A method of determining effectiveness of an IL12 signaling pathway modulator for reducing inflammation, comprising:

administering the IL12 signaling pathway modulator to the animal of any one of claims 1-20, 29, 30, 33-53, 62, 63, 66-97, 101, 102, 105-136, 140, and 141; and

determining effects of the IL12 signaling pathway modulator.
A method of determining toxicity of an IL12 signaling pathway modulator, the method comprising

administering the IL12 signaling pathway modulator to the animal of any one of claims 1-20, 29, 30, 33-53, 62, 63, 66-97, 101, 102, 105-136, 140, and 141; and

determining effects of the IL12 signaling pathway modulator to the animal.
The method of claim 160, wherein determining effects of the therapeutic agent to the animal involves measuring the body weight, red blood cell count, hematocrit, and/or hemoglobin of the animal.
A protein comprising an amino acid sequence, wherein the amino acid sequence is one of the following:

(a) an amino acid sequence set forth in SEQ ID NO: 1, 2, 9, 10, 17, 18, 27, 28, 29, or 36;

(b) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1, 2, 9, 10, 17, 18, 27, 28, 29, or 36;

(c) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 1, 2, 9, 10, 17, 18, 27, 28, 29, or 36 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and

(d) an amino acid sequence that comprises a substitution, a deletion and /or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 1, 2, 9, 10, 17, 18, 27, 28, 29, or 36.
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 162;

(b) SEQ ID NO: 3, 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 26, 30, 31, 34, 35, 37, 38, 48, or 65; and

(c) a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 26, 30, 31, 34, 35, 37, 38, 48, or 65.
A cell comprising the protein of claim 162 and/or the nucleic acid of claim 163.
An animal comprising the protein of claim 162 and/or the nucleic acid of claim 163.