RU2788523C2 - ANIMALS WITH HUMANIZED IL-4 AND IL-4Rα - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular to a rodent for the expression of a humanized IL-4 protein, where the rodent genome contains the replacement of the genomic DNA of the rodent IL-4 gene in the endogenous IL-4 locus of the rodent with the nucleic acid sequence of the human IL-4 gene to form a humanized IL-4 gene, to an embryonic stem cell and an embryo of the above rodent, as well as to a method for obtaining it. Also disclosed is a targeting vector for humanizing the endogenous IL-4 gene in a rodent.

EFFECT: invention enables effective screening for an antagonist specific for human IL-4.

13 cl, 10 dwg, 8 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application No. 61/989,757, filed May 7, 2014, the entire contents of which are incorporated herein by reference.

INCLUDING THE SEQUENCE LISTING VIA LINK

[0002] The sequence listing in a 16 KB ASCII text file called 31260_SEQ.txt, created on May 7, 2015 and filed with the US Patent and Trademark Office via EFS-Web, is incorporated herein by reference.

FIELD OF TECHNOLOGY

[0003] Non-human animals are disclosed herein that contain nucleic acid sequences encoding IL-4 and/or IL-4Rα proteins containing sequences from a human. Also disclosed herein are transgenic non-human animals that contain an IL-4 and/or IL-4Rα gene that is wholly or partially human. Also disclosed are non-human animals that express human or humanized IL-4 and/or IL-4Rα proteins. Also disclosed are methods for the preparation and use of non-human animals containing human or humanized IL-4 and/or IL-4Rα nucleic acid sequences.

BACKGROUND OF THE INVENTION

[0004] IL-4 and IL-4Rα are therapeutic targets for the treatment of various human diseases, disorders and conditions that are associated with abnormal T-helper type 2 (Th2) cells. The pharmacokinetics (PK) and pharmacodynamics (PD) of therapeutic molecules that target human IL-4 or human IL-4Rα proteins are typically evaluated in non-human animals, eg rodents, eg mice or rats. However, the PD of such therapeutic molecules cannot be properly determined in specific non-human animals because these therapeutic molecules do not target endogenous IL-4 or IL-4Rα proteins.

[0005] Moreover, evaluating the therapeutic efficacy of antagonists specific for human IL-4 and IL-4Rα proteins using various non-human animals as disease models associated with abnormal Th2 cells is problematic in non-human animals, in which such species-specific antagonists do not interact with endogenous IL-4 or IL-4Rα proteins.

[0006] Accordingly, there is a need for non-human animals, for example, rodents, for example, animals of the murine subfamily, such as mice or rats, in which the IL-4 and/or IL-4Rα genes of a non-human animal are completely or partially humanized or replaced (eg, at endogenous non-human loci) with human IL-4 and/or IL-4Rα genes containing sequences encoding human or humanized IL-4 and/or IL-4Rα proteins, respectively.

[0007] There is also a need for non-human animals containing IL-4 and/or IL-4Rα genes (e.g., humanized or human) in which the IL-4 and/or IL-4R genes are under non-human regulatory control. elements (eg, endogenous regulatory elements).

[0008] There is also a need for non-human animals with humanized sequences that express human or humanized IL-4 protein in blood, plasma, or serum at a concentration similar to that of IL-4 protein present in blood, plasma, or serum. blood of a non-human animal of the appropriate age group that expresses functional IL-4 protein, but does not contain human or humanized IL-4 genes, and/or expresses human or humanized IL-4Rα protein on immune cells, for example, B- and T- cells, at a level similar to that of the IL-4Rα protein expressed on immune cells, such as B and T cells, of a non-human animal of the appropriate age group, expressing functional IL-4Rα protein, but not containing the human or humanized IL- 4Ra.

BRIEF DESCRIPTION OF THE INVENTION

[0009] Non-human animals are provided that contain nucleic acid sequences encoding IL-4 and/or IL-4Rα proteins that contain a sequence from a human.

[0010] Transgenic non-human animals containing IL-4 and/or IL-4Rα genes that are fully or partially human are provided.

[0011] Non-human animals are provided that express human or humanized IL-4 and/or IL-4Rα proteins.

[0012] Non-human animals are provided that have a replacement (total or partial) of the endogenous IL-4 and/or IL-4Rα genes of a non-human animal.

[0013] Non-human animals are provided with humanized IL-4 and/or IL-4Rα (in whole or in part) at loci other than human endogenous IL-4 and/or IL-4Rα.

[0014] Non-human animals are provided having a human or humanized IL-4 gene, wherein the non-human animals do not express endogenous IL-4 protein, while the non-human animals express the human or humanized IL-4 protein in blood, plasma blood or serum at a concentration similar to that of an IL-4 protein present in the blood, plasma, or serum of a non-human animal of the appropriate age group that expresses a functional endogenous IL-4 protein but does not contain a human or humanized IL-4 gene .

[0015] Non-human animals are provided having a human or humanized IL-4Rα gene, wherein non-human animals do not express endogenous IL-4Rα protein, but express human or humanized IL-4Rα protein on immune cells, e.g., B- and T cells, at a level similar to that of the IL-4Rα protein present on immune cells, e.g., B and T cells, in a non-human animal of the appropriate age group that expresses functional endogenous IL-4Rα protein, but does not contain human or humanized IL-4Rα gene.

[0016] In one aspect, non-human animals are provided that contain a human or humanized IL-4 and/or IL-4Rα nucleic acid sequence.

[0017] In one aspect, genetically modified non-human animals are provided that are characterized by a substitution at an endogenous locus of an IL-4 and/or IL-4Rα gene encoding endogenous IL-4 and/or IL-4Rα with a gene encoding human or humanized proteins IL-4 and/or IL-4Rα. Rodents, e.g., mice or rats, are provided that are characterized by the replacement of the endogenous IL-4 gene at the endogenous rodent IL-4 locus with the human IL-4 gene and/or are characterized by the replacement of the endogenous IL-4Rα gene at the endogenous rodent IL-4Rα locus by the human IL-4 gene. 4Ra. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0018] In one aspect, genetically modified rodents, e.g., mice or rats, are provided, characterized by humanization of an endogenous rodent IL-4 gene, wherein the humanization involves replacing, at the endogenous rodent IL-4 locus, a rodent nucleic acid containing at least one exon of the IL gene. -4 rodent, a nucleic acid sequence containing at least one exon of the human IL-4 gene, with the formation of a modified IL-4 gene, while the expression of the modified IL-4 gene is under the control of rodent regulatory elements in the endogenous rodent IL-4 locus.

[0019] In one embodiment, the rodent is a mouse or rat. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0020] In one embodiment, the modified IL-4 gene encodes for a human or humanized IL-4 protein and contains a stretch from exon 1 starting at the ATG start codon to exon 4 of the human IL-4 gene.

[0021] In one embodiment, the rodent is a mouse that is unable to express mouse IL-4 protein.

[0022] In one embodiment, the rodent is a mouse that expresses the mouse IL-4Rα protein encoded by the endogenous mouse IL-4Rα gene.

[0023] In one embodiment, the rodent is a mouse that expresses a human or humanized IL-4Rα protein.

[0024] In one embodiment, the humanized IL-4Rα protein contains the ectodomain of the human IL-4Rα protein.

[0025] In one embodiment, the humanized IL-4Rα protein comprises a transmembrane domain and a cytoplasmic domain of a mouse IL-4Rα protein.

[0026] In one embodiment, the rodent is a mouse that is characterized by a substitution at the endogenous mouse IL-4Rα locus of a mouse nucleic acid containing at least one exon of the mouse IL-4Rα gene with a nucleic acid sequence containing at least one exon of the human gene IL-4Rα, with the formation of a modified IL-4Rα gene, while the expression of the modified IL-4Rα gene is under the control of mouse regulatory elements in the endogenous mouse IL-4Rα locus.

[0027] In one embodiment, the rodent is a mouse, where a contiguous genomic fragment of a mouse IL-4 sequence comprising a stretch from exon 1 starting at the ATG start codon to exon 4 of mouse IL-4 is replaced by a contiguous genomic fragment of a human IL-4 sequence. 4, containing the stretch from exon 1 starting with the ATG start codon to exon 4 of human IL-4.

[0028] In one embodiment, expression of a modified IL-4Rα gene encoding a human or humanized IL-4Rα protein is under the control of mouse regulatory elements at the endogenous mouse IL-4Rα locus.

[0029] In one aspect, genetically modified rodents, e.g., mice or rats, are provided, characterized by humanization of an endogenous rodent IL-4Rα gene, wherein the humanization involves replacing, at the endogenous rodent IL-4Rα locus, a rodent nucleic acid comprising an exon of the rodent IL-4Rα gene, a nucleic acid sequence encoding at least one exon of the human IL-4Rα gene to form a modified (i.e., humanized) IL-4Rα gene, wherein the expression of the modified, humanized IL-4Rα gene is under the control of rodent regulatory elements at the endogenous locus Rodent IL-4Rα.

[0030] In one embodiment, the rodent is a mouse or rat. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0031] In one embodiment, the modified IL-4Rα gene encodes for a human or humanized IL-4Rα protein and contains a stretch from exon 1 starting at the ATG start codon to exon 5 of the human IL-4Rα gene.

[0032] In one embodiment, the rodent is a mouse that is unable to express mouse IL-4Rα protein.

[0033] In one embodiment, the rodent is a mouse that expresses the mouse IL-4 protein encoded by the endogenous mouse IL-4 gene.

[0034] In one embodiment, the rodent is a mouse that expresses human or humanized IL-4 protein.

[0035] In one embodiment, the rodent is a mouse that is characterized by a substitution at the endogenous murine IL-4 locus of a mouse nucleic acid containing an exon of the mouse IL-4 gene with a nucleic acid sequence encoding at least one exon of the human IL-4 gene, with the formation of a modified IL-4 gene, while the expression of the modified IL-4 gene is under the control of mouse regulatory elements in the endogenous mouse IL-4 locus.

[0036] In one embodiment, the rodent is a mouse, and wherein the contiguous genomic fragment of the mouse IL-4Rα sequence, comprising the region from exon 1 starting at the ATG start codon to exon 5 of IL-4Rα, is replaced by a contiguous genomic fragment of the human IL sequence -4Rα, containing a stretch from exon 1, starting with the ATG start codon, to exon 5 of human IL-4Rα.

[0037] In one aspect, genetically modified rodents, such as a mouse or rat, are provided that express a human or humanized IL-4 protein, wherein the rodent that expresses the human or humanized IL-4 protein has a normal immune system, i. the number of immune cells, e.g., B and T cells, in the blood, plasma, or serum of a rodent expressing human or humanized IL-4 protein is similar to the number of immune cells, e.g., B and T cells, in the blood, blood plasma or serum of a rodent that expresses a functional endogenous IL-4 protein. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0038] In one aspect, genetically modified rodents, such as a mouse or rat, are provided that express an IL-4 protein encoded by a human or humanized IL-4 gene, wherein the human or humanized IL-4 protein is expressed in the rodent's blood serum. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0039] In one embodiment, the serum of a rodent that expresses a human or humanized IL-4 protein has approximately the same level of IL-4 protein as a rodent that expresses a functional endogenous IL-4 protein, e.g., mouse or rat wild type. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0040] In one embodiment, the mouse expresses human or humanized IL-4 protein in serum at a concentration of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 %, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200% of the level of IL-4 protein present in the blood serum of mice of the corresponding age group, expressing a functional endogenous IL-4 protein, but not characterized by the replacement of the endogenous IL-4 gene at the endogenous murine IL-4 locus with the human IL-4 gene.

[0041] In one embodiment, the mouse expresses human or humanized IL-4 protein in serum at a concentration of about 10% to about 200%, about 20% to about 150%, or about 30% to about 100% of the protein level. Mouse IL-4 present in the serum of mice of the appropriate age group expressing functional endogenous IL-4 protein, but not characterized by the replacement of the endogenous IL-4 gene at the endogenous IL-4 locus of the mouse with the human IL-4 gene.

[0042] In one aspect, genetically modified rodents, such as a mouse or rat, are provided that express a human or humanized IL-4Rα protein, wherein the rodent expresses the human or humanized IL-4Rα protein on immune cells, such as B and T cells. , at a level similar to that of the IL-4Rα protein present on immune cells, eg, B and T cells, of a rodent of an appropriate age group expressing a functional endogenous IL-4Rα protein. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0043] In one aspect, genetically modified rodents, such as a mouse or rat, are provided that express an IL-4Rα protein encoded by the human IL-4Rα gene, wherein the rodent expresses a human or humanized IL-4Rα protein on immune cells, such as B- and T cells. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0044] In one embodiment, immune cells, e.g., B and T cells, of a rodent that express human or humanized IL-4Rα protein have approximately the same level of IL-4Rα protein as immune cells, e.g., B- and T cells, a rodent expressing functional endogenous IL-4Rα protein, eg mouse or wild-type rat. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0045] In one embodiment, the mouse expresses human or humanized IL-4Rα protein on immune cells, e.g., B and T cells, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% of IL-4Rα protein present on immune cells, e.g., B and T cells, of mice of the appropriate age group, expressing a functional endogenous IL-4Rα protein, but not characterized by the replacement of the endogenous IL-4Rα gene at the mouse endogenous IL-4Rα locus with the human IL-4Rα gene.

[0046] In one embodiment, the mouse expresses human or humanized IL-4Rα protein on immune cells, such as B and T cells, at about 10% to about 200%, about 20% to about 150%, or about 30% to about 100% of the amount of mouse IL-4Rα protein present on immune cells, e.g., B and T cells, age appropriate mice expressing functional endogenous IL-4Rα protein but not characterized by endogenous IL- 4Rα at the endogenous mouse IL-4Rα locus by the human IL-4Rα gene.

[0047] In one aspect, a genetically modified rodent is provided comprising a humanized IL-4Rα gene comprising a replacement of the sequence encoding the rodent IL-4Rα ectodomain with a sequence encoding the human IL-4Rα ectodomain, wherein the humanized IL-4Rα gene contains a transmembrane IL-4Rα sequence. a rodent 4Rα and a rodent IL-4Rα cytoplasmic sequence, wherein the humanized IL-4Rα gene is under the control of endogenous rodent IL-4Rα regulatory elements at the endogenous IL-4Rα locus, and the rodent further comprises a humanized IL-4 gene encoding human or humanized an IL-4 protein, wherein the humanized IL-4 gene is under the control of endogenous rodent IL-4 regulatory elements at the endogenous IL-4 locus.

[0048] In one embodiment, the rodent is a mouse or rat. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0049] In one embodiment, the mouse is unable to express mouse IL-4 protein and is unable to express mouse IL-4Rα protein.

[0050] In one embodiment, the rodent regulatory elements or sequences at the endogenous rodent IL-4 locus and/or rodent IL-4Rα locus are derived from a mouse or rat.

[0051] In one embodiment, the rodent regulatory elements or sequences are endogenous rodent regulatory elements or sequences at the rodent IL-4 locus and/or the rodent IL-4Rα locus.

[0052] In one aspect, a non-human animal, such as a rodent, such as a mouse or rat, is provided that expresses human or humanized IL-4 and/or IL-4Rα proteins, wherein the non-human animal expresses human or humanized IL proteins. -4 and/or IL-4Rαc other than the human endogenous IL-4Rα locus and/or other than the human endogenous IL-4Rα locus. In an embodiment, the non-human animal is a rodent. In an embodiment, the rodent is a mouse. In an embodiment, the rodent is a rat.

[0053] In one aspect, a genetically modified mouse is provided that expresses a human or humanized IL-4 protein from an endogenous mouse IL-4 locus, wherein the endogenous mouse IL-4 gene has been wholly or partially replaced by a human IL-4 gene.

[0054] In one embodiment, a continuous mouse genomic nucleic acid sequence of approximately 6.3 kb. from the endogenous murine IL-4 locus, including the region from exon 1, starting with the ATG start codon, to exon 4 (including the 3'-untranslated region) and part of the 3' region below exon 4, is deleted and replaced with a human IL nucleic acid sequence -4, approximately 8.8 kb long, containing a region from exon 1, starting at the ATG start codon, to exon 4 (including the 3'-untranslated region) and part of the 3' region below exon 4 of the human IL- gene four. In a specific embodiment, the human IL-4 nucleic acid sequence that replaces the mouse genomic nucleic acid comprises a region from exon 1 starting at the ATG start codon to exon 4 and a portion of the 3' region below exon 4 of the human IL-4 gene from BAC RP11 -17K19 containing the human gene. In a specific embodiment, the modified IL-4 gene contains the 5' regulatory elements from mouse IL-4 and a region from exon 1 starting at the ATG start codon to exon 4 of human IL-4, i.e. sequences encoding the IL-4 protein.

[0055] In one aspect, a genetically modified mouse is provided that contains a nucleotide sequence encoding a human or humanized IL-4 protein, wherein the nucleotide sequence encoding a human or humanized IL-4 protein completely or partially replaces an endogenous nucleotide sequence encoding an endogenous protein mouse IL-4.

[0056] In one aspect, a genetically modified mouse is provided that expresses a human or humanized IL-4Rα protein from an endogenous mouse IL-4Rα locus, wherein the endogenous mouse IL-4Rα gene has been wholly or partially replaced by a human IL-4Rα gene.

[0057] In one embodiment, a continuous mouse genomic nucleic acid sequence of approximately 7.1 kb. in the endogenous murine IL-4Rα locus, including the stretch from exon 1, starting with the ATG start codon, to exon 5 and part of intron 5, is deleted and replaced with a human IL-4Rα nucleic acid sequence of approximately 15.6 kb. , containing a section from exon 1, starting with the start codon ATG, to exon 5 and part of intron 5 of the human IL-4Rα gene. In a specific embodiment, the human IL-4α nucleic acid sequence that replaces the mouse genomic nucleic acid comprises a stretch from exon 1 starting with the ATG start codon to exon 5 and a portion of intron 5 of the human IL-4α gene from BAC RP11-16E24 containing human gene. In a specific embodiment, the human IL-4Rα nucleic acid sequence replacing the mouse genomic nucleic acid contains the entire human IL-4Rα ectodomain encoding sequence.

[0058] In one aspect, a method for producing a humanized IL-4 rodent comprising replacing a rodent IL-4 gene sequence encoding a rodent IL-4 protein with a human IL-4 nucleic acid sequence comprising one or more exons of a human IL-4 gene sequence is provided. 4 to form a modified humanized IL-4 gene encoding a human or humanized IL-4 protein, wherein the substitution is at the endogenous rodent IL-4 locus, and the humanized IL-4 gene sequence containing one or more exons of the human IL-4 gene sequence. 4 and encoding a human or humanized IL-4 protein is operably linked to rodent regulatory elements or sequences (eg, 5' and/or 3' regulatory elements) at the endogenous rodent IL-4 locus.

[0059] In one embodiment, the rodent is a mouse or rat. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0060] In one embodiment, the rodent regulatory elements or sequences are derived from a mouse. In one embodiment, the rodent regulatory elements or sequences are from a rat.

[0061] In one embodiment, the rodent regulatory elements or sequences are endogenous rodent regulatory elements or sequences at the rodent IL-4 locus. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0062] In one embodiment, the human IL-4 nucleic acid sequence replacing the rodent IL-4 gene sequence contains at least one exon of the human IL-4 gene sequence. In other embodiments, the human IL-4 nucleic acid sequence replacing the rodent IL-4 gene sequence contains at least 2 or at least 3 exons of the human IL-4 gene sequence. In one embodiment, the human IL-4 nucleic acid sequence replacing the rodent IL-4 gene sequence contains all 4 exons of the human IL-4 gene sequence. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0063] In one embodiment, a human IL-4 nucleic acid sequence replacing a rodent IL-4 gene sequence encodes a protein that is about 85%, 90%, 95%, 96%, 97%, 98%, or about 99% % is identical to human IL-4 (eg, human IL-4 protein encoded by the nucleic acid set forth in GenBank Accession No. NM_000589.3s).

[0064] In one embodiment, the substitution is at the endogenous rodent IL-4 locus and the humanized IL-4 gene sequence comprising one or more exons of the human IL-4 gene sequence and encoding the human or humanized IL-4 protein is operably linked to the endogenous rodent regulatory elements or sequences (eg, 5' and/or 3' regulatory elements) at the endogenous rodent IL-4 locus.

[0065] In one aspect, a method is provided for producing a humanized IL-4 mouse, comprising replacing a mouse IL-4 gene sequence encoding a mouse IL-4 protein with a human IL-4 gene sequence to form a modified, humanized IL-4 gene encoding a human or a humanized IL-4 protein.

[0066] In one embodiment, the substitution is at an endogenous mouse IL-4 locus, and the resulting humanized IL-4 gene encoding a human or humanized IL-4 protein is operably linked to mouse regulatory elements or sequences (e.g., 5'- and/or 3'-regulatory elements) in the endogenous mouse IL-4 locus.

[0067] In one embodiment, the substitution is at an endogenous mouse IL-4 locus, wherein the humanized IL-4 gene encoding human or humanized IL-4 protein is operably linked to endogenous mouse regulatory elements or sequences (e.g., 5' - and/or 3'-regulatory elements) in the endogenous mouse IL-4 locus.

[0068] In one aspect, a method for producing a humanized IL-4Rα rodent comprising replacing a rodent IL-4Rα gene sequence encoding a rodent IL-4Rα protein with a human IL-4Rα nucleic acid sequence comprising one or more exons of a human IL-4Rα gene sequence is provided. 4Rα, with the formation of a modified humanized IL-4Rα gene encoding a human or humanized IL-4Rα protein, while the replacement is carried out at the endogenous rodent IL-4Rα locus and the sequence of the humanized IL-4Rα gene containing one or more exons of the sequence of the human IL-4Rα gene and encoding a human or humanized IL-4Rα protein is operably linked to rodent regulatory elements or sequences (eg, 5' and/or 3' regulatory elements) at the endogenous rodent IL-4Rα locus.

[0069] In one embodiment, the rodent is a mouse or rat. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0070] In one embodiment, the rodent regulatory elements or sequences are derived from a mouse. In one embodiment, the rodent regulatory elements or sequences are from a rat.

[0071] In one embodiment, the rodent regulatory elements or sequences are endogenous rodent regulatory elements or sequences at the rodent IL-4Rα locus. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0072] In one embodiment, the human IL-4Rα nucleic acid sequence replacing the rodent IL-4Rα gene sequence contains at least one exon of the human IL-4Rα gene sequence. In other embodiments, the human IL-4Rα nucleic acid sequence replacing the rodent IL-4Rα gene sequence contains at least 2, 3, 4, 5, 6, 7, or 8 exons of the human IL-4Rα gene sequence. In one embodiment, the human IL-4Rα nucleic acid sequence replacing the rodent IL-4Rα gene sequence contains all 9 exons of the human IL-4Rα gene sequence. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0073] In one embodiment, the human IL-4Rα nucleic acid sequence replacing the rodent IL-4Rα gene sequence encodes a protein that is about 85%, 90%, 95%, 96%, 97%, 98%, or about 99% % is identical to human IL-4Rα (eg, human IL-4Rα protein encoded by the nucleic acid set forth in GenBank Accession No. NM_000418.3).

[0074] In one embodiment, the human IL-4Rα nucleic acid sequence replacing the rodent IL-4Rα gene sequence contains at least one exon of the human IL-4Rα gene sequence encoding the human IL-4Rα protein ectodomain. In other embodiments, the human IL-4Rα nucleic acid sequence replacing the rodent IL-4Rα gene sequence contains at least 2, 3, or 4 exons of the human IL-4Rα gene sequence encoding the human IL-4Rα protein ectodomain. In one embodiment, the human IL-4Rα nucleic acid sequence replacing the rodent IL-4Rα gene sequence contains all 5 exons of the human IL-4Rα gene sequence encoding the human IL-4Rα protein ectodomain. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0075] In one embodiment, the human or humanized IL-4Rα gene sequence replacing the rodent IL-4Rα gene sequence encodes an IL-4Rα protein ectodomain that is approximately 85%, 90%, 95%, 96%, 97%, 98 % or approximately 99% identical to the ectodomain of a human IL-4Rα protein (eg, human IL-4Rα protein encoded by the nucleic acid set forth in GenBank Accession No. NM_000418.3).

[0076] In one embodiment, the substitution is at the endogenous rodent IL-4Rα locus and the humanized IL-4Rα gene sequence comprising one or more exons of the human IL-4Rα gene sequence and encoding the human or humanized IL-4Rα protein is operably linked to the endogenous rodent regulatory elements or sequences (eg, 5' and/or 3' regulatory elements) at the endogenous rodent IL-4Rα locus.

[0077] In one aspect, a method is provided for producing a humanized IL-4Rα mouse, comprising replacing a mouse IL-4Rα gene sequence encoding a mouse IL-4Rα protein with a human IL-4Rα nucleic acid sequence to form a humanized IL-4Rα gene encoding a human or humanized IL-4Rα protein.

[0078] In one embodiment, the substitution is at the endogenous murine IL-4Rα locus and the humanized IL-4Rα gene encoding human or humanized IL-4Rα protein is operably linked to mouse regulatory elements or sequences (e.g., 5'- and/or 3'-regulatory elements) in the endogenous mouse IL-4Rα locus.

[0079] In one embodiment, the substitution is at the mouse endogenous IL-4Rα locus and the humanized IL-4Rα gene encoding human or humanized IL-4Rα protein is operably linked to endogenous mouse regulatory elements or sequences (e.g., 5'- and/ or 3'-regulatory elements) in the endogenous mouse IL-4Rα locus.

[0080] In various aspects, the genetically modified non-human animals described herein, for example, rodents, such as mice or rats, carry genetic modifications in their germline.

[0081] In one aspect, there is provided an embryo of a non-human animal, such as a rodent, such as a mouse or rat, containing the genetic modification described herein.

[0082] In one aspect, a host embryo of a non-human animal, such as a rodent, such as a mouse or rat, is provided that contains a donor cell containing the genetic modification described herein.

[0083] In one aspect, a pluripotent or totipotent cell of a non-human animal, such as a rodent, such as a mouse or rat, is provided, comprising the genetic modification described herein. In one embodiment, the cell is a rodent cell. In one embodiment, the cell is a mouse cell. In one embodiment, the cell is a rodent ES cell. In one embodiment, the cell is a mouse ES cell.

[0084] In one aspect, a non-human animal ovum, such as a rodent, such as a mouse or rat, is provided, wherein the non-human animal ovum comprises a non-human ectopic chromosome, wherein the non-human ectopic chromosome comprises the genetic modification described in this document. In one embodiment, the non-human animal is a rodent. In one embodiment, the rodent is a mouse. In one embodiment, the rodent is a rat.

[0085] In one aspect, the mouse embryo, egg, or cell that is genetically modified to contain a human IL-4 gene or a human IL-4Rα gene is from a mouse that is a C57BL mouse 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 another embodiment, the mouse is a mouse of line 129 selected from the group consisting of lines that are 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/Svlm), 129S2, 129S4, 129S5, 129S9/ SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see e.g. Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10:836, see also Auerbach et al (2000 ) Establishment and Chimera Analysis of 129/SvEv-and C57BL/6-Derived Mouse Embryonic Stem Cell Lines). In a specific embodiment, the genetically modified mouse is a hybrid of the above line 129 and the above line C57BL/6. In another specific embodiment, the mouse is a hybrid of the above 129 lines or a hybrid of the above BL/6 lines. In a particular embodiment, line 129 in the hybrid is line 129S6 (129/SvEvTac). In another embodiment, the mouse is a BALB mouse, eg BALB/c. In yet another embodiment, the mouse is a hybrid of the BALB strain and another of the aforementioned strains. In one embodiment, the mouse is a Swiss or Swiss Webster mouse.

[0086] In various aspects, non-human animals containing the human or humanized IL-4R and/or IL-4 nucleic acid sequence are selected from mammals and birds. In one embodiment, the non-human animals are mammals. In one embodiment, mammals are a subfamily of mice.

[0087] In one aspect, a method for screening for an antagonist specific for human IL-4 or IL-4Rα is provided. The method is useful for identifying therapeutic candidates and evaluating therapeutic efficacy. The method includes administering an agent to a genetically modified rodent in which two sequences of IL-4 and IL-4Rα are humanized as described herein, determining the effect of the agent on biological function mediated by the IL-4/IL-4Rα signaling pathway, and identifying the agent as an antagonist specific for human IL-4 or IL-4Rα if it antagonizes function mediated by the IL-4/IL-4Rα signaling pathway in a genetically modified rodent.

[0088] In one embodiment, the agent contains an immunoglobulin variable domain that binds IL-4 or IL-4Rα. In one embodiment, the agent specifically binds human IL-4 or IL-4Rα, but not rodent IL-4 or IL-4Rα. In one embodiment, the agent is an antibody. In a particular embodiment, the agent is an antibody that specifically binds human IL-4Rα but not rodent IL-4Rα.

[0089] In one embodiment, the screening method uses a mouse with two humanized sequences that expresses a human IL-4 protein and a humanized IL-4Rα protein, where the humanized IL-4Rα protein contains a human IL-4Rα protein ectodomain associated with a transmembrane and cytoplasmic domains of endogenous mouse IL-4Rα protein, and where the mouse does not express mouse IL-4 or mouse IL-4Rα.

[0090] In some embodiments, the screening method comprises the steps of inducing a disease associated with IL-4/IL-4Rα signaling in a two-sequence humanized rodent described herein, administering an agent to the rodent, determining whether the agent reduces the severity of the disease, and identifying an agent as a human IL-4 or IL-4Rα specific antagonist suitable for treating a disease if the agent reduces the severity of the disease.

[0091] In some embodiments, an IL-4/IL-4Rα signaling-associated disease is airway inflammation that can be induced in a rodent by intranasal administration of an allergen (e.g., house dust mite extract) at one or more doses within a certain period of time. The effect of an agent can be determined by measuring whether the degree of inflammation of the airways (as reflected, for example, by accumulation of mucus, cellular infiltrate in bronchoalveolar lavage fluid and/or total IgE levels in the bloodstream) is reduced as a result of administration of the agent.

[0092] In some embodiments, the disease associated with IL-4/IL-4Rα signaling is skin inflammation or atopic dermatitis, which can be induced in a rodent by creating a skin lesion and exposing the damaged skin to an allergen (e.g., a bacterial toxin or house dust mite extract) in one or more doses over a period of time. The effect of an agent can be determined by measuring whether skin inflammation is reduced by administration of the agent.

[0093] In a further aspect, a non-human animal with the three humanized IL-4, IL-4Rα, and IL-33 genes described herein is used to evaluate pharmacodynamics (PD) and therapeutic efficacy of a compound or combination of compounds.

[0094] Each of the aspects and embodiments described herein may be used in conjunction unless expressly excluded or evident from the context of the embodiment or aspect.

BRIEF DESCRIPTION OF GRAPHICS

[0095] FIG. 1 is an illustration, not to scale, of IL-4 and IL-13 receptor signaling and the mechanism of action of dupilumab neutralizing a fully human monoclonal antibody that specifically binds to the human IL-4 receptor α chain (IL-4Rα).

и IL-4Rα

(2А) Ген IL-4 мыши (вверху), охватывающий кодирующий участок от экзона 1, начинающегося со стартового кодона ATG, до экзона 4 (включая 3'-нетранслируемый участок) и часть 3'-участка ниже экзона 4, подвергали делеции и заменяли кодирующим участком от экзона 1, начинающегося со стартового кодона ATG, до экзона 4 (включая 3'-нетранслируемый участков) и частью 3'-участка ниже экзона 4 человеческого гена IL-4 (внизу) вместе с фланкированной loxP кассетой с селектируемым маркером устойчивости к гигромицину, как указано. (2В) Ген IL-4Rα мыши (вверху), охватывающий кодирующий участок от экзона 1, начинающегося со стартового кодона ATG, до экзона 5 и часть интрона 5 подвергали делеции и заменяли кодирующим участком от экзона 1, начинающегося со стартового кодона ATG, до экзона 5 и частью интрона 5 человеческого гена IL-4Rα (внизу) и фланкированной loxP кассетой с селектируемым маркером устойчивости к неомицину, как указано.[0096] FIG. 2A-2B illustrate, not to scale, strategies for humanizing IL-4 loci.

and IL-4Rα

(2A) Mouse IL-4 gene (top), spanning the coding region from exon 1 starting at the ATG start codon to exon 4 (including the 3'-untranslated region) and part of the 3' region below exon 4, was deleted and replaced coding region from exon 1, starting at the ATG start codon, to exon 4 (including the 3'-untranslated regions) and part of the 3' region downstream of exon 4 of the human IL-4 gene (bottom), along with a loxP flanked cassette with a selectable marker of resistance to hygromycin, as indicated. (2B) Mouse IL-4Rα gene (top) spanning the coding region from exon 1 starting at the ATG start codon to exon 5 and part of intron 5 was deleted and replaced with the coding region from exon 1 starting from the ATG start codon to exon 5 and part of intron 5 of the human IL-4Rα gene (bottom) and loxP flanked neomycin resistance selectable marker cassette as indicated.

[0097] FIG. 3 shows expression of humanized IL-4Rα protein on B and T cells from mice with two humanized IL-4/IL-4Rα sequences (Il4 ^hu/hu /Il4ra ^hu/hu ).

[0098] FIG. 4 shows IL-4 and IL-13 ligand specificity and receptor functional properties using primary cells derived from mice with a humanized IL4Rα (Il4ra ^hu/hu ) sequence.

[0099] FIG. 5 shows IL-4 dependent IgE production in vivo in wild-type mice, but not mice with a humanized IL4Rα sequence (Il4ra ^hu/hu ).

[00100] FIG. 6 shows that dose-dependent induction of ex vivo mIgE production in mouse B cells (left panel) of IL-4 is blocked by dupilumab in a dose-dependent manner (right panel).

[00101] FIG. 7 shows that dupilumab dose-dependently prevents the development of IL-25-induced lung pathologies in vivo in mice with a humanized IL4Rα (Il4ra ^hu/hu ) sequence.

[00102] FIG. Figure 8 shows a design of an experiment evaluating the therapeutic efficacy of dupilumab in a house dust mite extract (HDM) induced lung inflammation model using mice with two humanized sequences of IL-4 and IL-4Rα (IL-4 ^hu/hu /IL-4R ^{hu /hu} ). "REGN668" refers to a human anti-human IL-4Rα monoclonal antibody, also known as dupilumab. "REGN129" means a solution of mouse IL-13Rα-2-Fc, which is a fusion protein from the ectodomain of mouse IL-13R2α and Fc.

[00103] FIG. 9 shows a design of an experiment evaluating the therapeutic efficacy of dupilumab in an HDM-induced lung inflammation model using mice with two humanized sequences of IL-4 and IL-4Rα (IL-4 ^hu/hu /IL-4R ^hu/hu ) and antibodies as an isotype control.

[00104] FIG. 10A-10C illustrate strategies for humanizing the mouse IL-33 locus. In FIG. 10A illustrates that the murine IL-33 gene (top) spanning the coding region from exon 2 starting at the ATG start codon to the stop codon of exon 8 is deleted and replaced with the coding region from exon 2 starting at the ATG codon to exon 8 (including the 3'-untranslated region) of the human IL-33 gene (bottom). In FIG. 10B shows the humanized IL-33 allele in mouse ES cell clone MAID 7060, which contains a loxP flanked neomycin resistance selectable marker cassette. In FIG. 10C shows the humanized IL-33 allele in mouse ES cell clone MAID 7061, in which the neomycin resistance selectable marker cassette was deleted, leaving loxP sites and cloning sites (77 bp) downstream of human IL-33 sequence, and the mouse 3' UTR remained below the loxP site.

DETAILED DESCRIPTION OF THE INVENTION

IL-4 and IL-4Rα as therapeutic targets

[00105] Allergic diseases are a spectrum of diseases that are emerging at an increasing rate, especially in developed countries. Atopic dermatitis, asthma, and allergic rhinitis are the most common inflammatory conditions among patients with allergic diseases; however, these patients often experience multiple clinical symptoms. Allergy pathogenesis is associated with abnormal immune responses to exogenous antigens (see Mueller et al. (2002) Structure, binding, and antagonists in the IL-4/IL-13 receptor system, Biochim Biophys Acta 1592:237-250).

[00106] Excessive production of antigen-specific IgE is an important component in initiating allergic inflammation. Polarization of abnormal T-helper type 2 (Th2) cells leads to enhanced IgE-mediated responses.

[00107] Interleukin-4 (IL-4) and interleukin-13 (IL-13), first identified in activated T cells, are major Th2 cytokines that play a major role in initiating and maintaining immune and inflammatory responses in allergic diseases.

[00108] IL-4 and IL-13 signaling is mediated by two different receptor complexes with a common subunit, the IL-4 receptor alpha (IL-4Rα), which may contribute to the crossover of biological responses between these two cytokines. See figure 1.

[00109] Interleukin-4/13 receptor signaling and mechanism of action of dupilumab. IL-4Rα forms two different heterodimeric receptor complexes to mediate the biological functions of IL-4 and IL-13 in a tissue-specific and reaction-specific manner. The type I receptor, consisting of IL-4Rα and a common cytokine receptor gamma chain (γC), is unique to IL-4. The type II receptor derived from IL-4Rα and IL-13Rα1 is the main receptor for IL-13 but is also functional for IL-4. In addition, IL-13 will bind to a second high affinity receptor, IL-13Rα2, which is usually defined as a decoy receptor or as having a possible profibrotic effect in full length form.

[00110] Dupilumab is a human IL-4Rα antagonist monoclonal antibody that suppresses IL-4 and IL-13 induced biological activities. Dupilumab blocks IL-4 signaling by preventing its binding to receptor subunits, while the inhibitory effect on IL-13 signaling appears to be mediated by preventing interaction with the dimeric receptor.

[00111] Dupilumab, a fully human monoclonal antibody to the common IL-4Rα subunit, was developed by Regeneron Pharmaceuticals, Inc. using mice VelocImmune ^® . Dupilumab is in clinical trials for the treatment of moderate to severe asthma and for the treatment of moderate to severe atopic dermatitis.

[00112] Evaluation of the efficacy of dupilumab in mouse models is fraught with many difficulties: (a) dupilumab does not recognize the mouse IL-4 cognate receptor; and (b) there is a lack of functional interaction between the IL-4 protein and the human IL-4 receptor.

IL-4 gene and protein

[00113] The IL-4 gene encodes a secreted IL-4 protein that plays an important role in the activation of B cells as well as other cell types (see Figure 1).

[00114] Human IL-4. Gene ID at NCBI: 3565; original source: HGNC:6014; reference sequence transcript: NM_000589.3; ID in UniProt: Р05112; genomic assembly: GRCh37; localization: chr5:132009743-132018576+thread.

[00115] The human IL-4 gene is located on chromosome 5 at 5q31.1. The human IL-4 gene contains 4 exons and encodes a 153 amino acid precursor polypeptide comprising the 24 amino acids of the signal peptide and the 129 amino acids of the mature IL-4 protein.

[00116] Mouse IL-4. Gene ID at NCBI: 16189; source: MGI:96556; reference sequence transcript: NM_021283.2; ID in UniProt: Р07750; genomic assembly: GRCm38; localization: chr11:53612350-53618606 - thread.

[00117] The mouse IL-4 gene is located on chromosome 11 at 11, 31, 97 cM. The mouse IL-4 gene contains 4 exons and encodes a 140 amino acid precursor polypeptide comprising the 20 amino acids of the signal peptide and 120 amino acids of the mature IL-4 protein.

IL-4Rα gene and protein

[00118] The IL-4Rα gene encodes the IL-4Rα transmembrane receptor protein, which is expressed primarily on B and T cells and is the receptor for IL-4 and IL-13 proteins (see Figure 1).

[00119] Human IL-4Rα. Gene ID at NCBI: 3566; source: MGI:6015; reference sequence transcript: NM_000418.3; ID in UniProt: Р24394; genomic assembly: GRCh37; localization: chr16:27351525-27367111+thread.

[00120] The human IL-4Rα gene is located on chromosome 16 at 16p12.1-p11.2. The human IL-4Rα gene contains 9 coding exons and encodes a precursor polypeptide of 825 amino acids, including 25 amino acids of the signal peptide and 800 amino acids of the mature IL-4Rα protein, while the first 207 amino acid residues of the mature protein constitute the extracellular domain. The extracellular domain (ie, ectodomain) of the human IL-4Rα protein is encoded by coding exons 1-5 of the human IL-4Rα gene.

[00121] Mouse IL-4Rα. Gene ID at NCBI: 16190; source: MGI: 105367; reference sequence transcript: NM_001008700.3; ID in UniProt: Р16382; genomic assembly: GRCm38; localization: chr11:125565655-125572745+thread.

[00122] The mouse IL-4Rα gene is located on chromosome 7 at region 7, 68.94 cM. The mouse IL-4Rα gene contains 9 coding exons and encodes an 810 amino acid precursor polypeptide comprising the 25 amino acids of the signal peptide and 785 amino acids of the mature IL-4Rα protein, with the first 208 amino acid residues of the mature protein constituting the extracellular domain. The extracellular domain (ie, ectodomain) of the mouse IL-4Rα protein is encoded by coding exons 1-5 of the mouse IL-4Rα gene.

Species specificity of IL-4 and IL-4Rα proteins

(См. также, например, Andrews et al. (2001) Reconstitution of a functional human type II IL-4/IL-13 receptor in mouse В cells: demonstration of species specificity, J Immunol. 166:1716-1722).[00123] As shown herein, mouse IL-4, not human, is functional in wild-type mice, and conversely, human IL-4, not mouse, is functional in mice with humanized IL-4Rα sequences

(See also, for example, Andrews et al. (2001) Reconstitution of a functional human type II IL-4/IL-13 receptor in mouse B cells: demonstration of species specificity, J Immunol. 166:1716-1722).

Species specificity of human IL-4 and IL-4Rα inhibitors

[00124] Candidate therapeutic molecules targeting IL-4 or IL-4Rα proteins are typically evaluated for pharmacokinetics (PK) and pharmacodynamics (PD) in non-human animals, e.g., rodents, e.g., mice or rats. Such therapeutic molecules are also being tested for in vivo therapeutic efficacy in non-human animals, eg rodents, eg mice or rats, as human models of diseases, disorders and conditions associated with abnormal Th2 cells.

[00125] However, therapeutic molecules specific for human IL-4 or IL-4Rα proteins, such as inhibitors specific for human IL-4 or IL-4Rα, cannot be assessed with sufficient accuracy in relation to PD or in vivo therapeutic efficacy in rodents, mice in particular, since they lack targets for these therapeutic molecules. This problem is not eliminated by the use of transgenic non-human animals, eg rodents, eg mice or rats, expressing human IL-4 or IL-4Rα proteins due to the aforementioned species specificity of the IL-4 protein.

[00126] Accordingly, in various embodiments, in order to evaluate the PD and in vivo therapeutic efficacy of antagonists or inhibitors specific for human IL-4 or IL-4Rα proteins in non-human animals, e.g., rodents, e.g., mice or rats, it is desirable replace endogenous IL-4 and/or IL-4Rα proteins with human IL-4 and/or IL-4Rα proteins.

[00127] In addition, in various embodiments, in order to avoid potential problems of overexpression or underexpression of human IL-4 and/or IL-4Rα proteins, it is necessary to insert human IL-4 and/or IL-4Rα genes into the genome of non-human animals, e.g. rodents, e.g. mice or rats, at endogenous IL-4 and/or IL-4Rα gene loci and place the expression of human IL-4 and/or IL-4Rα proteins in non-human animals, e.g. rodents, e.g. mice or rats, under the control of at least partially endogenous regulatory elements IL-4 and/or IL-4Rα.

Genetically modified non-human animals

[00128] Provided herein are genetically modified non-human animals in which endogenous IL-4 genes and/or IL-4Rα genes have been completely or partially replaced at the endogenous IL-4 locus and/or IL-4Rα locus with human IL- 4 and/or human IL-4Rα nucleic acid to form a modified IL-4 gene and/or a modified IL-4Rα gene that encode a human or humanized IL-4 protein and/or a human or humanized IL-4Rα protein.

[00129] As used herein, the phrase "non-human animal" refers to any vertebrate organism that is not a human. In some embodiments, the non-human animal is a mammal. In specific embodiments, the non-human animal is a rodent, such as a rat or mouse.

[00130] In one aspect, genetically modified rodents, such as mice or rats, are provided in which the endogenous rodent IL-4 gene is wholly or partially replaced at the endogenous IL-4 locus with a nucleic acid encoding human IL-4.

[00131] The replacement includes the replacement of at least one exon, i. e. one or more exons, in the rodent IL-4 gene, with a human nucleic acid sequence comprising at least one exon of the human IL-4 gene. In some embodiments, a rodent contiguous genomic fragment that includes the region from exon 1 starting with the ATG start codon to exon 4 of the rodent IL-4 gene is replaced by a human contiguous fragment that includes the region from exon 1 starting with the ATG start codon to exon 4 human IL-4 genes. In a particular embodiment, the rodent is a mouse, wherein the contiguous mouse genomic fragment is approximately 6.3 kb long. in the endogenous murine IL-4 locus, including the region from exon 1, starting with the ATG start codon, to exon 4 (including the 3'-untranslated region) and part of the 3' region below exon 4, is deleted and replaced with a human IL nucleic acid sequence -4, approximately 8.8 kb long, containing a region from exon 1, starting at the ATG start codon, to exon 4 (including the 3'-untranslated region) and part of the 3' region below exon 4 of the human IL- gene four.

[00132] In some embodiments, the replacement results in a modified humanized IL-4 gene at the endogenous IL-4 gene locus, wherein expression of the modified IL-4 gene is under the control of endogenous regulatory elements at the endogenous IL-4 locus. As used herein, the expression "regulatory elements" refers to transcriptional regulatory sequences, including both 5' transcriptional regulatory sequences such as promoter, enhancer, and transcription repressive elements, and 3' transcriptional regulatory sequences, regulating transcription, such as a transcription termination sequence. In some embodiments, expression of the modified IL-4 gene is under the control of endogenous 5' end regulatory elements. In other embodiments, expression of the modified IL-4 gene is under the control of endogenous 3'-terminal regulatory elements. In certain embodiments, expression of the modified IL-4 gene is under the control of endogenous 5' and 3' regulatory elements.

[00133] A modified humanized IL-4 gene generated at the endogenous IL-4 locus encodes a human or humanized IL-4 protein. The expression "humanized" refers to nucleic acids or proteins that contain portions or sequences of a gene or protein found in a non-human animal (for example, a rodent such as a mouse or rat) and also contain portions or sequences that are different from those occurring in a non-human animal, but instead correspond (are identical) to portions or sequences of analogues of a human gene or protein. The modified humanized IL-4 gene can encode an IL-4 protein that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or 100% identical to human IL-4 protein it (eg, human IL-4 protein encoded by the nucleic acid set forth in GenBank Accession No. NM_000589.3).

[00134] A genetically modified rodent with complete or partial replacement of the endogenous rodent IL-4 gene at the endogenous IL-4 locus with human IL-4 nucleic acid may be homozygous or heterozygous for the substitution. In some embodiments, the genetically modified rodent is heterozygous for the substitution, i. only one of the two copies of the endogenous rodent IL-4 gene was replaced with human IL-4 nucleic acid. In other embodiments, the genetically modified rodent is homozygous for the substitution, i. both copies of the endogenous rodent IL-4 gene were replaced with human IL-4 nucleic acid.

[00135] In a genetically modified rodent, human or humanized IL-4 protein is expressed in serum. In some embodiments, the genetically modified rodent does not express endogenous rodent IL-4 protein. In one embodiment, the serum of a rodent expressing a human or humanized IL-4 protein has approximately the same level of IL-4 protein as a rodent expressing a functional endogenous IL-4 protein, e.g., a wild type rodent (e.g., a rodent, expressing a functional endogenous IL-4 protein, but not containing a complete or partial replacement of the endogenous IL-4 gene at the endogenous IL-4 locus with human IL-4 nucleic acid). By "about the same level" is meant a level that is within 25%, 20%, 15%, 10%, 5% or less to either side (i.e., more or less) of the level in the wild-type rodent. In other embodiments, the rodent expresses human or humanized IL-4 protein in serum at a concentration of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100 %, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200% of the level of IL-4 protein present in the blood serum of a rodent of the corresponding age group expressing functional endogenous IL-4 protein, but not containing a complete or partial replacement of the endogenous IL-4 gene at the endogenous IL-4 locus with human IL-4 nucleic acid.

[00136] In some embodiments, a genetically modified rodent characterized by complete or partial replacement of the endogenous rodent IL-4 gene with human IL-4 nucleic acid and expressing human or humanized IL-4 protein in serum has a normal immune system, i. the number of immune cells, e.g., B and T cells, in the blood, plasma, or serum of a rodent expressing human or humanized IL-4 protein is similar to the number of immune cells, e.g., B and T cells, in the blood, blood plasma or serum of a rodent expressing a functional endogenous IL-4 protein and not characterized by complete or partial replacement of the endogenous rodent IL-4 gene with human IL-4 nucleic acid.

[00137] In additional embodiments, a genetically modified rodent characterized by a complete or partial replacement of the endogenous rodent IL-4 gene with a human IL-4 nucleic acid and expressing a human or humanized IL-4 protein also has a complete or partial replacement of the endogenous rodent IL-4Rα gene at the endogenous IL-4 Rα locus with human IL-4Rα nucleic acid and therefore also expresses human or humanized IL-4Rα protein.

[00138] In another aspect, genetically modified rodents, such as mice or rats, are provided in which the endogenous rodent IL-4Rα gene has been completely or partially replaced at the endogenous IL-4Rα locus with a human IL-4Rα nucleic acid.

[00139] The replacement involves the replacement of at least one exon, i. e. one or more exons, in the rodent IL-4Rα gene, with a human nucleic acid sequence comprising at least one exon of the human IL-4Rα gene. In some embodiments, the replacement involves replacing at least one of the exons of the rodent IL-4Rα gene encoding the rodent ectodomain with at least one of the exons of the human IL-4Rα gene encoding the human ectodomain. In some embodiments, the replacement involves replacing with a human nucleic acid sequence containing at least 2, 3, or 4 of the 5 ectodomain-encoding exons of the human IL-4Rα gene. In other embodiments, a continuous rodent genomic fragment that includes the stretch from exon 1 starting with the ATG start codon to exon 5 of the rodent IL-4Rα gene is replaced with a genomic fragment that includes the stretch from exon 1 starting with the ATG start codon to exon 5 human IL-4Rα gene. In a particular embodiment, the rodent is a mouse, wherein the contiguous mouse genomic fragment is approximately 7.1 kb long. at the endogenous murine IL-4Rα locus, including the stretch from exon 1, starting with the ATG start codon, to exon 5 and part of intron 5, is deleted and replaced with a human IL-4Rα nucleic acid sequence of approximately 15.6 kb. , containing a section from exon 1, starting with the start codon ATG, to exon 5 and part of intron 5 of the human IL-4Rα gene.

[00140] In some embodiments, the replacement results in a modified humanized IL-4Rα gene at the endogenous IL-4Rα gene locus, wherein expression of the modified IL-4Rα gene is under the control of endogenous regulatory elements at the endogenous IL-4Rα locus.

[00141] A modified humanized IL-4Rα gene derived from the endogenous IL-4Rα locus encodes a human or humanized IL-4Rα protein. In some embodiments, the modified humanized IL-4Rα gene encodes an IL-4Rα protein that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to human IL-4Rα protein or is 100% identical to it (eg, the human IL-4 protein encoded by the nucleic acid disclosed in GenBank Accession No. NM_000418.3). In other embodiments, the modified IL-4Rα gene encodes a humanized IL-4Rα protein that contains an ectodomain that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the human protein ectodomain. IL-4Rα or is 100% identical to it (for example, the human IL-4 protein encoded by the nucleic acid set forth in GenBank Accession No. NM_000418.3). In specific embodiments, the transmembrane and cytoplasmic domains of the humanized IL-4Rα protein are identical to the transmembrane and cytoplasmic domains of the endogenous rodent IL-4Rα protein.

[00142] A genetically modified rodent characterized by complete or partial replacement of the endogenous rodent IL-4Rα gene at the endogenous IL-4Rα locus with a human IL-4Rα nucleic acid may be homozygous or heterozygous for the substitution. In some embodiments, the genetically modified rodent is heterozygous for the substitution, i. only one of the two copies of the endogenous rodent IL-4Rα gene was replaced with human IL-4Rα nucleic acid. In other embodiments, the genetically modified rodent is homozygous for the substitution, i. both copies of the endogenous rodent IL-4Rα gene were replaced with human IL-4Rα nucleic acid.

[00143] In the genetically modified rodent disclosed herein, the human or humanized IL-4Rα protein is expressed on immune cells such as B and T cells. In some embodiments, the genetically modified rodent does not express endogenous rodent IL-4Rα protein. In one embodiment, immune cells of a rodent expressing human or humanized IL-4Rα protein have approximately the same level of IL-4Rα protein on immune cells as a rodent expressing functional endogenous IL-4Rα protein on immune cells, such as a wild rodent. type expressing functional, endogenous IL-4Rα protein and not expressing human or humanized IL-4Rα protein. In other embodiments, the rodent expresses human or humanized IL-4Rα protein on immune cells in an amount of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200% of the amount of IL-4Rα protein present on the immune cells of a rodent of the appropriate age group expressing a functional endogenous IL-4Rα protein, but not characterized by complete or partial replacement of the endogenous IL-4Rα gene with human IL-4Rα nucleic acid.

[00144] In some embodiments, a genetically modified rodent characterized by complete or partial replacement of the endogenous rodent IL-4Rα gene with human IL-4Rα nucleic acid and expressing human or humanized IL-4Rα protein has a normal immune system, i. the number of immune cells, e.g., B and T cells, in the blood, plasma, or serum of a rodent expressing human or humanized IL-4Rα protein is similar to the number of immune cells, e.g., B and T cells, in the blood, blood plasma or serum of a wild-type rodent (eg, a rodent expressing a functional endogenous IL-4Rα protein and not characterized by complete or partial replacement of the endogenous rodent IL-4Rα gene with human IL-4Rα nucleic acid).

[00145] In some embodiments, a genetically modified rodent characterized by complete or partial replacement of the endogenous rodent IL-4Rα gene with human IL-4Rα nucleic acid and expressing human or humanized IL-4Rα protein is capable of mediating IL-4 dependent signaling and IL-13 signal transduction and has functional properties to it. For example, a humanized IL-4Rα protein with the ectodomain of the human IL-4Rα protein, expressed on genetically modified rodent immune cells, interacts with human IL-4 and mediates human IL-4 dependent signaling via type I receptor formation (see FIG. 1 ). This humanized IL-4Rα protein with the ectodomain of the human IL-4Rα protein also interacts with human and mouse IL-13 and mediates human IL-13 dependent signaling through type II receptor formation (see FIG. 1). The functional properties of a humanized IL-4Rα protein expressed in a genetically modified rodent can be assessed using various assays known in the art, including those specifically described in the examples provided herein below, such as an assay that measures IL-4 -induced switch to the synthesis of the IgE class using primary B cells obtained from a genetically modified rodent.

[00146] In additional embodiments, a genetically modified rodent characterized by complete or partial replacement of the endogenous rodent IL-4Rα gene with a human IL-4Rα nucleic acid and expressing a human or humanized IL-4Rα protein is also characterized by complete or partial replacement of the endogenous rodent IL-4 gene at the endogenous IL-4 locus with human IL-4 nucleic acid and therefore also expresses human or humanized IL-4.

[00147] In a further aspect, dual sequence humanized rodents, e.g., mice or rats, have been provided in which the endogenous rodent IL-4 gene has been completely or partially replaced at the endogenous IL-4 locus with human IL-4 nucleic acid, and in which the endogenous IL-4 gene Rodent -4Rα has also been completely or partially replaced at the endogenous IL-4 Rα locus with human IL-4Rα nucleic acid. Such rodents with two humanized sequences may be homozygous or heterozygous for each substitution resulting in humanization. In a specific embodiment, a rodent with two humanized sequences is homozygous for both humanized IL-4 and humanized IL-4Rα.

[00148] Genetic modification of an endogenous rodent IL-4 gene in a rodent with two humanized sequences includes such modifications or substitutions as described herein above for a genetically modified rodent characterized by complete or partial replacement of the endogenous rodent IL-4 gene with human IL-4 nucleic acid. four. Similarly, genetic modification of an endogenous rodent IL-4Rα gene in a rodent with two humanized sequences includes such modifications or substitutions as described herein above for a genetically modified rodent characterized by complete or partial replacement of an endogenous rodent IL-4Rα gene with human IL- 4Ra. Thus, the features disclosed herein regarding the humanization of the rodent IL-4 gene and regarding the humanization of the rodent gene, respectively, are specifically included herein in relation to a rodent with two humanized sequences.

[00149] In specific embodiments, a rodent with two humanized sequences, such as a mouse or rat, expressing human IL-4 protein and humanized IL-4Rα protein, wherein the humanized IL-4Rα protein comprises the ectodomain of the human IL-4Rα protein and contains a transmembrane and cytoplasmic domains of the endogenous rodent IL-4Rα protein. In specific embodiments, expression of human IL-4 protein and humanized IL-4Rα protein is under the control of endogenous rodent regulatory sequences at the endogenous rodent IL-4 locus and the rodent IL-4Rα locus, respectively.

[00150] In some embodiments, the dual humanized sequence rodent has a normal immune system (i.e., the number of immune cells is about the same as that of the wild-type rodent), has about the same level of serum IL-4 protein, and expresses approximately the same amount of IL-4Rα protein on immune cells as a wild-type rodent, whereby a wild-type rodent is a rodent that expresses functional endogenous IL-4 protein and IL-4Rα protein and does not express human or humanized IL-4 protein or IL-4Rα protein.

[00151] In specific embodiments, the dual humanized sequence rodent is characterized by a functional IL-4 signaling pathway. By "functional IL-4 signaling pathway" is meant that both human or humanized IL-4 protein and human or humanized IL-4Rα protein are expressed in a rodent with two humanized sequences and interact with each other in a rodent with two humanized sequences with in order to effectively mediate downstream signaling and carry out the biological activities of the normal IL-4 signaling pathway. The biological activities of the normal IL-4 signaling pathway are described herein above and illustrated in Figure 1, including those activities that are mediated by the type I receptor, such as initiation and maintenance of Th2 differentiation, B cell activation and growth, switching synthesis between IgE classes and IgG4, and activities that are mediated by type II receptor signaling such as goblet cell hyperplasia, subepithelial fibrosis, and tissue remodeling. For example, a functional IL-4 signaling pathway in a rodent with two humanized sequences exhibits an inflammatory phenotype characterized by, for example, elevated circulating IgE levels, airway inflammation, and/or eosinophilic cell infiltrate in response to house dust mite extract administration, wherein the phenotype also seen in wild-type rodents without two humanized sequences.

Methods for producing a genetically modified non-human animal

[00152] A genetically modified non-human animal, such as a rodent, can be obtained using methods known in the art. For example, a targeting vector can be prepared that contains a human nucleic acid (such as the human IL-4 or IL-4Rα gene, in whole or in part) flanked below and above by non-human animal homologous regions. The targeting construct may also comprise a selectable drug resistance marker cassette (e.g., a loxP flanked hygromycin resistance selectable marker cassette that can then be removed with a transient Cre expression vector) that is positioned relative to the 3' end of the human nucleic acid. The targeting vector can be introduced into the genome of a non-human animal cell, such as an embryonic stem (ES) cell (such as mouse ES cells), for example, by electroporation. Precisely targeted ES cell clones can then be introduced into an early stage embryo (eg mouse embryo at 8 cell stage). Non-human animals descended entirely from precisely targeted ES cells are identified based on, for example, allelic analysis. For non-human animals for which suitable genetically modified ES cells are difficult to obtain, other methods can be used to produce a non-human animal containing the genetic modifications described herein. Such methods include, for example, modifying the genome of cells other than ES cells (e.g., a fibroblast or an induced pluripotent cell) and using nuclear transfer to move the modified genome into a suitable cell, such as an oocyte, and incubating the modified cell (e.g., a modified oocyte) in a non-human animal under suitable conditions to form an embryo.

Methods of using a genetically modified non-human animal

[00153] In one aspect, IL-4 and/or IL-4Rα genetically modified non-human animals disclosed herein are used to evaluate the pharmacodynamics (PD) and therapeutic efficacy of human IL-4 and/or IL-specific antagonists. -4Rα, for example, neutralizing antibodies to IL-4 and/or to IL-4Rα (eg, dupilumab), in various disease models, further illustrated in the examples below.

[00154] In some embodiments, the present invention provides a method for screening for an antagonist specific for human IL-4 or IL-4Rα using mice with the two humanized IL-4 and IL-4Rα genes disclosed herein.

[00155] By "antagonists of IL-4 or IL-4Rα" is meant molecules (e.g., antibodies) that block, suppress or inhibit one or more biological functions mediated by IL-4 or IL-4Rα.. "Antagonists specific to human "IL-4 or IL-4Rα" refers to antagonists that are specific for human IL-4 or IL-4Rα and have little or no effect on rodent IL-4 or IL-4Rα.

[00156] In specific embodiments, the screening method uses a mouse with two humanized sequences that expresses a human IL-4 protein and a humanized IL-4Rα protein, where the humanized IL-4Rα protein contains an ectodomain of a human IL-4Rα protein associated with a transmembrane and cytoplasmic domains of endogenous mouse IL-4Rα protein, and where the mouse does not express mouse IL-4 or IL-4Rα.

[00157] In some embodiments, a method for screening for an antagonist specific for human IL-4 or IL-4Rα comprises administering the agent to a genetically modified rodent with two humanized IL-4 and IL-4Rα genes as described herein, determining the effect of the agent by relation to biological function mediated by the IL-4/IL-4Rα signaling pathway and identifying the agent as an antagonist specific to human IL-4 or IL-4Rα if it antagonizes the function mediated by the IL-4/IL-4Rα signaling pathway , in a genetically modified rodent.

[00158] In one embodiment, the agent contains an immunoglobulin variable domain that binds IL-4 or IL-4Rα. In one embodiment, the agent specifically binds human IL-4 or IL-4Rα, but not rodent IL-4 or IL-4Rα. In one embodiment, the agent is an antibody. In a particular embodiment, the agent is an antibody that specifically binds human IL-4Rα but not rodent IL-4Rα.

[00159] In one embodiment, the screening method uses a mouse with two humanized sequences that expresses a human IL-4 protein and a humanized IL-4Rα protein, where the humanized IL-4Rα protein contains an ectodomain of a human IL-4Rα protein associated with a transmembrane and cytoplasmic domains of endogenous mouse IL-4Rα protein, and where the mouse does not express mouse IL-4 or mouse IL-4Rα.

[00160] In some embodiments, the screening method comprises the steps of inducing a disease associated with IL-4/IL-4Rα signaling in a two humanized sequence described rodent, administering an agent to the rodent, determining whether the agent reduces the severity of the disease, and identifying an agent as a human IL-4 or IL-4Rα specific antagonist suitable for treating a disease if the agent reduces the severity of the disease.

[00161] By "disease associated with IL-4/IL-4Rα signaling" is meant a disease in which a biological function mediated by IL-4/IL-4Rα signaling is implicated. Examples of diseases associated with IL-4/IL-4Rα signaling include, for example, inflammatory diseases or disorders such as asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD) (which may arise at least in part from cigarette smoke) , inflammatory bowel disease, multiple sclerosis, arthritis, allergic rhinitis, eosinophilic esophagitis, and psoriasis. Asthma can be eosinophilic or non-eosinophilic asthma and steroid sensitive or steroid resistant asthma.

[00162] In some embodiments, an IL-4/IL-4Rα signaling-associated disease is airway inflammation that can be induced in a rodent by intranasal administration of an allergen (e.g., house dust mite extract) at one or more doses within a certain period of time. The effect of an agent can be determined by measuring whether the degree of inflammation of the airways is reduced (as expressed, for example, by accumulation of mucus, eosinophilic cell infiltrate in bronchoalveolar lavage fluid, total IgE levels in the bloodstream, and/or a change in the expression profile, measured by microarray expression analysis ) due to the introduction of funds. The allergen used to induce airway inflammation and the test agent may be administered simultaneously or at different times. In some embodiments, the allergen is administered to the rodent in one or more doses, and the test agent is administered to the rodent after the rodent has received at least one dose of the allergen.

[00163] In some embodiments, the disease associated with IL-4/IL-4Rα signaling is skin inflammation or atopic dermatitis, which can be induced in a rodent by creating a skin lesion and exposing the damaged skin to an allergen (e.g., a bacterial toxin or house dust mite extract) in one or more doses over a period of time. The effect of an agent can be determined by measuring whether skin inflammation is reduced as a result of administration of the agent.

[00164] In a further aspect, non-human animals with three humanized sequences, i.e. non-human animals whose IL-4, IL-4Rα and IL-33 genes have been humanized are used to evaluate the pharmacodynamics (PD) and therapeutic efficacy of candidate compounds such as, for example, antagonists specific for human IL-4 and/or IL -4Rα, and antagonists specific to human IL-33.

[00165] By "antagonists of IL-33" is meant molecules (eg, antibodies) that block, suppress or inhibit one or more biological functions or signaling mediated by IL-33. "Antagonists specific for human IL-33" refers to antagonists that are specific for IL-33 and have little or no effect on rodent IL-33. IL-33 is known to stimulate signal transduction through ST2 and RAcP of IL-1, which is attenuated in the presence of an antagonist such as anti-IL-33 antibodies. Inhibition of IL-33 signaling through ST2 and IL-1 RAcP can be determined by assaying IL-33 signaling in in vitro or in vivo assays, such as those described in US Published Application 2014/0271658 A1, the entire contents of which are incorporated herein by reference. For example, an assay such as that described in U.S. Published Application 2014/0271658 A1 can be used to evaluate the effect of an anti-IL-33 antibody on lung inflammation in allergen-susceptible animals that are homozygous for the expression of human IL-33. An anti-IL-33 antibody effective as an IL-33 antagonist should tend to decrease the number of inflammatory cells in the lung and also tend to decrease the amount of cytokines such as IL-4 and IL-5.

[00166] In specific embodiments, a non-human tri-humanized sequence animal is used according to this document to evaluate candidate compounds, wherein the tri-humanized sequence animal is a tri-humanized mouse that expresses human IL-4 protein, a humanized protein IL-4Rα, which contains the ectodomain of the human IL-4Rα protein associated with the transmembrane and cytoplasmic domains of the mouse IL-4Rα protein and the human IL-33 protein, while the mouse does not express mouse IL-4, mouse IL-4Rα, or IL-33 mice.

[00167] In some embodiments, a non-human animal with three humanized sequences is used to evaluate the pharmacodynamics (PD) and therapeutic efficacy of a candidate compound, such as, for example, an antagonist specific for human IL-4 and/or IL-4Rα, or an antagonist specific to human IL-33. For example, an antibody specific for human IL-4, an antibody specific for human IL-4Rα, and an antibody specific for human IL-33 can be tested separately in a three-sequence humanized animal (such as a rodent, e.g., mouse or rat ), after which their PD profiles and therapeutic efficacy can be evaluated and compared.

[00168] In other embodiments, a non-human animal with three humanized sequences is used to evaluate the efficacy of a combination of compounds, e.g., a combination of an antagonist antibody specific for human IL-4 and/or IL-4Rα with an antagonist antibody specific for human IL-33, compared with the effectiveness of these compounds when used alone to determine, for example, whether a combination of compounds exhibits a synergistic therapeutic effect. In specific embodiments, the combination of an antibody specific for human IL-4 and an antibody specific for human IL-33 is tested in a non-human animal with three humanized sequences. In other specific embodiments, the combination of an antibody specific for human IL-4Rα and an antibody specific for human IL-33 is tested in a non-human animal with three humanized sequences.

[00169] To evaluate a candidate compound or combination of compounds, a disease associated with IL-4/IL-4Rα signaling and IL-33 signaling can be induced in an animal with three humanized sequences. Examples of diseases associated with IL-4/IL-4Rα signaling and IL-33 signaling include, for example, inflammatory diseases or disorders such as asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD) (which can occur at least least partly from cigarette smoke), inflammatory bowel disease, multiple sclerosis, arthritis, allergic rhinitis, eosinophilic esophagitis, and psoriasis. Asthma can be eosinophilic or non-eosinophilic asthma and steroid sensitive or steroid resistant asthma. The effect of a compound or combination of compounds can be evaluated in a similar manner to an animal characterized by IL-4/IL-4Rα dual humanization as described herein above.

[00170] The present invention is further illustrated by the following non-limiting examples.

Example 1

Replacement of Endogenous Mouse IL-4 Gene with Human IL-4 Gene

[00171] The 8.8 kb human IL-4 genome containing the coding portion from exon 1, starting at the ATG start codon, to exon 4 (including the 3'-untranslated region) and a portion of the 3' region below exon 4 of the human IL-4 gene, the 6.3 kb murine IL-4 gene locus was replaced, spanning the coding portion from exon 1, starting with the ATG start codon, to exon 4 (including the 3'-untranslated region) and part of the 3' region below exon 4. See Figure 2A.

[00172] A targeting construct to replace the mouse IL-4 gene with a human one in a single targeting step was constructed using VelociGene® engineering technology (see Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech, 21(6):652-659). Mouse IL-4 and human IL-4 DNA was obtained from bacterial artificial chromosome (BAC) clones bMQ-41A12 and RP11-17K19, respectively. Briefly, an SbfI linearized targeting construct obtained by gap-repair cloning and containing mouse IL-4 upstream and downstream of homologous regions flanking an 8.8 kb human IL-4 sequence extending from the ATG codon to exon 1 to exon 4 (including the 3' untranslated region), and part of the 3' region below exon 4 (genomic coordinates: GRCh37: chr5:132 009 743-132 018 576 (+ strand)), and a flanked selectable marker cassette hygromycin resistance were introduced by electroporation into F1H4 mouse embryonic stem (ES) cells (F1 hybrid C57BL/6×129). Finely targeted ES cells (MAID 879) were then electroporated with a transient Cre expression vector to remove the selectable drug resistance marker cassette. Clones of targeted ES cells without a drug resistance cassette (MAID 1553) were introduced into an 8-cell stage SW mouse embryo using the VelociMouse® method (see US Pat. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses, Nature Biotech 25(1):91-99). VelociMice® (F0 mice completely derived from a donor ES cell) carrying the humanized IL-4 gene were identified by genotyping for mouse allele loss and human allele gain using modified allele analysis (see Valenzuela et al. (2003) ).

[00173] Accurately targeted ES cell clones were identified using native allele loss assay (LONA) (Valenzuela et al. 2003), in which copy number of the native unmodified IL-4 gene was determined by two TaqMan™ quantitative polymerase chain reactions (qPCR ) with specific primers for mouse IL-4 gene sequences that were targeted for deletion. The qPCR reactions contained the following primer-probe sets (written 5' to 3' end): forward primer upstream of the target region, CATGCACGGA GATGGATGTG (SEQ ID NO:1); reverse primer upstream of the target region, GACCCCTCAG GTCCACTTAC C (SEQ ID NO: 2); probe above target site, FAM-AACGTCCTCA CAGCAACGA-MGB (SEQ ID NO: 3); forward primer below the target site, GTGCCCAGGT GTGCTCATG (SEQ ID NO: 4); reverse primer below the target region, CGCCTGCCTC STCATTTTAT C (SEQ ID NO: 5); probe below target site, FAM-ATCTGCTTCA CCATCCACT-MGB (SEQ ID NO: 6); whereby FAM refers to the 5-carboxyfluorescein fluorescent probe and BHQ refers to a black hole fluorescence quencher (Biosearch Technologies). DNA isolated from clones of ES cells that they had taken up as part of a targeted vector and incorporated into their genomes was combined with a TaqMan™ Gene Expression Master Mix (Life Technologies) according to the manufacturer's instructions in a 384-well PCR plate ( Optical 384-well MicroAmp™ Reaction Plate, Life Technologies) and cycled in Prism 7900HT from Applied Biosystems, which collected fluorescence data during PCR cycles and determined the threshold cycle (Ct), a fractional PCR cycle at which accumulated fluorescence reached a predetermined threshold. QPCR with specific primers upstream and downstream of IL-4 and two qPCRs for untargeted reference genes were performed for each DNA sample. Differences in Ct values (ΔCt) between each IL-4-specific qPCR and each qPCR for the reference gene were calculated, and then the difference between each ΔCt and the median ΔCt for all analyzed samples was calculated to obtain ΔΔCt values for each sample. The number of copies of the IL-4 gene in each sample was calculated using the following formula: number of copies = 2×2 ^-ΔΔCt . A clone subjected to precise targeted action that has lost one of its native copies will be characterized by the number of copies of the IL-4 gene equal to one. To prove that the human IL-4 gene sequence had replaced the deleted mouse IL-4 gene sequence in the humanized allele, a TaqMan™ qPCR assay was performed that included the following primer-probe sets (written 5' to 3' end ): forward primer specific for the human gene, GCCTGGACCA AGACTCTGT (SEQ ID NO: 7); reverse primer specific for the human gene, ACCGTGGGAC GGCTTCTTAC (SEQ ID NO: 8); probe specific for the human gene, FAM-CACCGAGTTG ACCGTAACAG ACATC-BHQ (SEQ ID NO: 9). To prove that the hygromycin resistance selectable marker cassette inserted with the human IL-4 gene sequence in the humanized allele, a TaqMan™ qPCR assay was performed that included the following primer-probe sets (written 5' to 3' end ): forward primer specific for the hygromycin resistance gene, TGCGGCCGAT CTTAGCC (SEQ ID NO: 10); reverse primer specific for the hygromycin resistance gene, TTGACCGATT CCTTGCGG (SEQ ID NO: 11); probe specific for the hygromycin resistance gene, FAM-ACGAGCGGGT TCGGCCCATT C-BHQ (SEQ ID NO: 12).

[00174] The same LONA assay was used to analyze DNA isolated from mouse tail biopsies derived from targeted ES cells to determine their IL-4 genotypes and confirm that the humanized IL-4 allele was passed on in the germline. Two offspring heterozygous for the substitution were crossed to produce a mouse homozygous for the substitution of the endogenous mouse IL-4 gene with the human IL-4 gene. Offspring homozygous for the substitution were used for phenotyping.

где последовательности человеческого IL-4 выделены курсивом, а последовательности, кодирующие IL-4, взяты в скобки. Нижний участок соединения последовательности, содержащей ген человеческого IL-4, и фланкированной loxP кассеты с селектируемым маркером устойчивости к гигромицину, конструировали так, что бы он находился в пределе

где последовательности человеческого IL-4 выделены курсивом, а последовательности, кодирующие IL-4, взяты в скобки. Нижний участок соединения последовательности фланкированной loxP кассеты с селектируемым маркером устойчивости к гигромицину и локуса IL-4 мыши конструировали так, что бы он находился в пределе

где последовательности кассеты, обеспечивающей устойчивость к гигромицину, выделены курсивом.[00175] The upper junction of the mouse IL-4 locus and the sequence containing the human IL-4 gene was designed to be in the range

where human IL-4 sequences are in italics and IL-4 encoding sequences are in parentheses. The lower junction of the sequence containing the human IL-4 gene and the loxP flanked hygromycin resistance selectable marker cassette was designed to be within the

where human IL-4 sequences are in italics and IL-4 encoding sequences are in parentheses. The lower junction of the sequence of the loxP flanked hygromycin resistance selectable marker cassette and the mouse IL-4 locus was designed to be in the range

where the cassette sequences conferring resistance to hygromycin are in italics.

Example 2

Replacement of the mouse endogenous IL-4Rα ectodomain gene sequence with the human IL-4Rα ectodomain gene sequence

[00176] The 15.6 kb human IL-4Rα gene, containing the region from exon 1, starting with the ATG start codon, to exon 5 and part of intron 5 of the human IL-4Rα gene, was replaced with the IL-4Rα gene locus mouse length 7.1 kb, covering the coding part from exon 1, starting with the start codon ATG, to exon 5 and part of intron 5. Mouse exons 6-9 were saved; however, only exons 1-5 (ie, ectodomains) were humanized. See Figure 2B.

[00177] A targeting construct to replace the mouse IL-4Rα gene with a human one in a single targeting step was constructed using VelociGene® engineering technology (see Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech, 21(6):652-659). Mouse IL-4Rα and human IL-4Rα DNA were obtained from bacterial artificial chromosome (BAC) clones RP23-136G14 and RP11-166E24, respectively. Briefly, a NotI linearized targeting construct obtained by gap repair cloning and containing the mouse IL-4Rα gene upstream and downstream of homologous regions flanking a 15.6 kb human IL-4Rα sequence extending from the ATG codon to exon 1 to exon 5, and part of intron 5 (genomic coordinates: GRCh37: chr16:27351525-27367111 (+ strand)), and a loxP flanked cassette with selectable neomycin resistance marker, were electroporated into mouse F1H4 embryonic stem (ES) cells (hybrid F1 C57BL/6 × 129). Finely targeted ES cells (MAID 803) were then electroporated with a transient Cre expression vector to remove the selectable drug resistance marker cassette. Clones of targeted ES cells without a drug resistance cassette (MAID 1444) were introduced into an 8-cell stage SW mouse embryo using the VelociMouse® method (see US Pat. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses, Nature Biotech 25(1):91-99). VelociMice® (F0 mice completely derived from a donor ES cell) with a humanized IL-4Rα gene were identified by genotyping for mouse allele loss and human allele gain using modified allele analysis (see Valenzuela et al. (2003)).

[00178] Accurately targeted ES cell clones were identified using native allele loss assay (LONA) (Valenzuela et al. 2003), in which copy number of the native unmodified IL-4Rα gene was determined by two TaqMan™ quantitative polymerase chain reactions (qPCR ) with specific primers for mouse IL-4Rα gene sequences that have been targeted for deletion. The qPCR reactions contained the following primer-probe sets (written 5' to 3'): forward primer upstream of the target region, CCGCTGGCAT GTGTATTGTG (SEQ ID NO: 16); reverse primer upstream of the target region, TGAGTGTGGG ACCCTCAAGA G (SEQ ID NO: 17); probe above target site, FAM-TGACCCAAGC CCTACATGGC CACT-BHQ (SEQ ID NO: 18); forward primer below the target site, TGAGGAGAGC TCACGGGAAT C (SEQ ID NO: 19); reverse primer below the target region, ACCATCCCC TGCGTTTCTG (SEQ ID NO: 20); probe below target site, FAM-TTGACACGCC AGCTACACTG CTCCA-BHQ (SEQ ID NO: 21); whereby FAM refers to the 5-carboxyfluorescein fluorescent probe and BHQ refers to a black hole fluorescence quencher (Biosearch Technologies). DNA isolated from clones of ES cells that they had taken up as part of a targeted vector and incorporated into their genomes was combined with a TaqMan™ Gene Expression Master Mix (Life Technologies) according to the manufacturer's instructions in a 384-well PCR plate ( Optical 384-well MicroAmp™ Reaction Plate, Life Technologies) and cycled in Prism 7900HT from Applied Biosystems, which collected fluorescence data during PCR cycles and determined the threshold cycle (Ct), a fractional PCR cycle at which accumulated fluorescence reached a predetermined threshold. QPCR with specific primers upstream and downstream of IL-4Rα and two qPCRs for untargeted reference genes were performed for each DNA sample. Differences in Ct values (ΔCt) between each IL-4Rα-specific qPCR and each qPCR for the reference gene were calculated, and then the difference between each ΔCt and the median ΔCt for each sample analyzed was calculated to obtain ΔΔCt values for each sample. The copy number of the IL-4Rα gene in each sample was calculated using the following formula: copy number = 2×2 ^-ΔΔCt . A precisely targeted clone that has lost one of its native copies will have a copy number of the IL-4Rα gene equal to one. To prove that the human IL-4Rα gene sequence had replaced the deleted mouse IL-4Rα gene sequence in the humanized allele, a TaqMan™ qPCR assay was performed that included the following primer-probe sets (written 5' to 3' end ): forward primer specific for the human gene, ACTGCGTCT CCGACTACAT G (SEQ ID NO: 22); reverse primer specific for the human gene, GAGCTCGGTG CTGCAATTG (SEQ ID NO: 23); probe specific for the human gene, FAM-TGGGACCATT CATCTTCCAC TCGCA-BHQ (SEQ ID NO: 24). To prove that the neomycin resistance selectable marker cassette inserted with the human IL-4Rα gene sequence in the humanized allele, a TaqMan™ qPCR assay was performed that included the following primer-probe sets (written 5' to 3' end ): forward primer specific for the neomycin resistance gene, GGTGGAGAGG CTATTCGGC (SEQ ID NO: 25); a reverse primer specific for the neomycin resistance gene, GAACACCGGCG GCATCAG (SEQ ID NO: 26); probe specific for the neomycin resistance gene, FAM-TGGGCACAAC AGACAATCGG CTG-BHQ (SEQ ID NO: 27).

[00179] The same LONA assay was used to analyze DNA isolated from mouse tail biopsies derived from targeted ES cells to determine their IL-4Rα genotypes and to confirm that the humanized IL-4Rα allele was transmitted in the germline. Two offspring heterozygous for the substitution were crossed to produce a mouse homozygous for the replacement of the endogenous mouse IL-4Rα gene with the human IL-4Rα gene. Offspring homozygous for the substitution were used for phenotyping.

где последовательности человеческого IL-4Rα выделены курсивом, а последовательности, кодирующие IL-4Rα, подчеркнуты. Нижний участок соединения последовательности, содержащей человеческий ген IL-4Rα, и фланкированной loxP кассеты с селектируемым маркером устойчивости к неомицину, сконструировали так, что бы он находился в пределе

где последовательности человеческого IL-4Rα выделены курсивом, а последовательности кассеты указаны нижним регистром. Нижний участок соединения последовательности фланкированной loxP кассеты с селектируемым маркером устойчивости к неомицину и локуса IL-4Rα мыши сконструировали так, чтобы он находился в пределе

где последовательности, кодирующие IL-4Rα мыши, заключены в скобки, а последовательности кассеты, обеспечивающей устойчивость к неомицину, указаны нижним регистром.[00180] The upper junction of the mouse IL-4Rα locus and the sequence containing the human IL-4Rα gene was designed to be in the range

where human IL-4Rα sequences are in italics and IL-4Rα encoding sequences are underlined. The lower junction of the sequence containing the human IL-4Rα gene and the loxP flanked neomycin resistance selectable marker cassette was designed to be in the range

where human IL-4Rα sequences are in italics and cassette sequences are in lowercase. The bottom junction of the sequence of the loxP flanked neomycin resistance selectable marker cassette and the mouse IL-4Rα locus was designed to be in the range

where sequences encoding mouse IL-4Rα are in parentheses and neomycin resistance cassette sequences are in lowercase.

Example 3

Obtaining mice with two humanized IL-4/IL-4Rα

[00181] Mice with two humanized IL-4/IL-4Rα (Il4 ^hu/hu /Il4ra ^hu/hu ) were generated as follows. The MAID 803 ES cell clone containing the humanized IL-4Rα gene and a loxP flanked cassette conferring neomycin resistance was electroporated with a Cre-expressing vector to remove the loxP flanked cassette conferring neomycin resistance to produce a MAID 1444 ES cell clone containing humanized IL-4Rα gene without selectable drug resistance marker cassette (see example 2). The same targeting construct that was used to generate the MAID 879 ES cell clone containing the humanized IL-4 gene and a loxP-flanked hygromycin resistance cassette (see Example 1) was inserted by electroporation into the MAID 1444 ES cell clone with 879 Het/1444 Het (Il4 ^+/hu /Il4ra ^+/hu ) ES cells, which were subsequently electroporated with a Cre-expression vector to remove the loxP flanked cassette conferring hygromycin resistance, to obtain an ES cell clone (MAID 1553/ 1444) containing humanized IL-4 and IL-4Rα genes. The MAID 1553/1444 ES cell clone lacking the drug resistance cassette was introduced into an 8 cell stage SW mouse embryo to produce dual humanized IL-4/IL-4Rα mice.

Example 4

Evaluation of the efficacy of dupilumab, a fully human anti-IL-4Rα mAb, in mice with gene substitutions for human IL-4 and IL4Rα

Ways

[00182] Genetically engineered mice were generated using ^VelociGene® technology to replace the full-length mouse IL-4 locus with 8.8 kb human IL-4 genomic sequences. (see example 1 and figure 2A), and gene extracellular domain (ie ectodomain) IL-4Rα mouse (CD124) fragment length of 15.6 kb. corresponding genomic DNA of human IL-4Rα (see example 2 and figure 2B).

[00183] Homozygous mice with a humanized IL-4Rα gene were validated for human gene expression and function. To determine human IL-4Rα expression in humanized mice, splenocytes from wild-type and humanized mice were harvested and treated with labeled fluorescent anti-mouse CD3, mouse CD19, human CD124, and mouse CD124 antibodies for fluorescent activated cell sorting (FACS) analysis. (See, for example, Blaeser et al. (2003) Targeted inactivation of the IL-4 receptor a chain I4R motif promotes allergic airway inflammation, J Exp Med 198(8): 1189-1200).

[00184] Primary cells derived from mice with humanized IL-4Rα were used to demonstrate ligand specificities and functional properties of the receptors. Bone marrow-derived macrophages were cultured using bone marrow cells from the femur of wild-type and humanized IL-4Rα mice in DMEM containing 10% fetal bovine serum and 20% conditioned medium with L-cells for 7 days. .

[00185] Cells were then separately treated with 20 ng/ml mouse IL-4, mouse IL-13, human IL-4, human IL-13, or solvent diluted in culture medium for 20 hours. Quadruple samples from each condition were taken for gene expression analysis.

[00186] Total RNA from these samples was extracted and amplified to cRNA by introducing Cy3-TP. Cy3-labeled cRNAs from each sample were then hybridized on a custom-designed chip from Agilent containing 43,538 60 nucleotide long oligonucleotide sequences spanning mouse transcriptomes. Data was read from scanned images of the chip using Feature Extraction 9.5 software from Agilent.

[00187] Differentially expressed genes among experimental groups were identified using Student's t-test (p<0.05, fold change ≥ 1.5). The expression cluster of these genes was obtained using the Pearson correlation coefficient clustering algorithm from GeneSpring GX7.3.

[00188] The IL-4 neutralizing effect of dupilumab was assessed using an in vitro IgE synthesis switch assay on primary B cells isolated from humanized IL-4Rα mice.

вводили (гидродинамически) большой объем раствора «голой» плазмидной ДНК, управляющей доставкой гена для экспрессии IL-25 мыши in vivo. (См., например, Liu et al. (1999) Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA, Gene Therapy 6:1258-1266.) Через 8 дней отбирали периферическую кровь для измерения уровней IgE (mIgE) мыши в сыворотке крови с использованием коммерческого набора для проведения ELISA (R&D systems, Миннесота).[00189] Wild-type (WT) mice and mice with humanized IL-4Rα

injected (hydrodynamically) a large volume of naked plasmid DNA solution directing gene delivery for in vivo mouse IL-25 expression. (See, for example, Liu et al. (1999) Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA, Gene Therapy 6:1258-1266.) After 8 days, peripheral blood was collected to measure IgE (mIgE) levels of mice in serum using a commercial ELISA kit (R&D systems, Minnesota).

) и (Δ) соответственно. Уровни IgE мыши в надосадочных жидкостях культуры клеток измеряли с использованием коммерческого набора для ELISA. (См, например, Moon et al. (1989) Regulation of IgG1 and IgE synthesis by interleukin 4 in mouse В cells, Scand I Immunol 30:355-361.)[00190] Primary B cells purified from humanized sequence mouse splenocytes were activated with bacterial LPS and mixed with increasing amounts of recombinant human IL-4 in a 7 day culture to induce a switch in synthesis between immunoglobulin classes. For the antibody blocking experiment, purified B cells were incubated with increasing doses of dupilumab for 30 minutes before addition of 0.167 nM recombinant human IL-4, and cultured for 7 days. IgE production in the absence of IL-4 or with an isotype control mAb is indicated by (

) and (Δ) respectively. Mouse IgE levels in cell culture supernatants were measured using a commercial ELISA kit. (See, for example, Moon et al. (1989) Regulation of IgG1 and IgE synthesis by interleukin 4 in mouse B cells, Scand I Immunol 30:355-361.)

[00191] Interleukin-25 (IL-25) is a cytokine produced by Th2 cells whose main activities are mediated through the production of IL-4 and IL-13, resulting in the induction of tissue-specific pathologies such as increased mucus production in the lungs and goblet cell hyperplasia. (See Fort et al. (2001) IL-25 induces IL-4, IL-5, and IL-13 and Th2-associated pathologies in vivo, Immunity 15(6):985-995.)

[00192] The absence of IL-13 provides protection to animals from IL-25-induced pathologies in the target organ. Thus, the IL-25 model of lung inflammation was used to evaluate the pharmacodynamic (PD) properties of dupilumab in vivo in mice containing humanized IL-4 and/or IL-4Rα genes.

[00193] The PD properties of type II receptor responses to dupilumab exposure were characterized using the IL-25 inflammation induction method by measuring mucus accumulation in the lungs of humanized IL-4Rα mice.

вводили с помощью гидродинамической доставки вектор экспрессии IL-25 мыши, а затем с помощью инъекции дупилумаб или mAb изотипического контроля в указанных дозах. Дополнительные дозы антител вводили через день в общем количестве 4 дозы. В день 8 собирали образцы тканей легкого от эвтаназированных мышей и обработанные срезы легкого окрашивали перйодной кислотой-реактивом Шиффа перед их оценкой «при заслеплении» в отношении патологических изменений.[00194] On day 0, WT mice and humanized IL-4Rα

administered by hydrodynamic delivery of the mouse IL-25 expression vector and then by injection of dupilumab or isotype control mAb at indicated doses. Additional doses of antibodies were administered every other day for a total of 4 doses. On day 8, lung tissue samples were collected from euthanized mice and treated lung sections were stained with periodic acid-Schiff's reagent before they were assessed "by blinding" for pathological changes.

results

(см. фигуру 3); (b) изменение специфичностей к лиганду IL-4 у мышей с гуманизированным IL-4Rα

(см. фиг. 4); и (с) функциональные способности в отношении сигнального пути IL-13 у гуманизированных по IL-4Rα

мышей (см. фигуру 4).[00195] Humanized IL-4Rα mice were characterized as demonstrating: (a) expression of human IL-4Rα on primary cells from mice with two humanized IL-4/IL-4Rα

(see figure 3); (b) change in IL-4 ligand specificities in mice with humanized IL-4Rα

(see Fig. 4); and (c) IL-13 signaling functionality in IL-4Rα humanized

mice (see figure 4).

экспрессировали аналогичные количества белка IL-4Rα на поверхности В- (CD19⁺, CD3^-) и Т- (CD19^-, CD3⁺) клеток.[00196] As shown in FIG. 3, which depicts marked IL-4Rα (CD124) profiles on gated B and T cell populations and the distribution of the corresponding unstained cell population, depicted by the shaded area, in wild-type and humanized IL-4Rα mice.

expressed similar amounts of IL-4Rα protein on the surface of B- (CD19 ⁺ , CD3 ^- ) and T- (CD19 ^- , CD3 ⁺ ) cells.

отвечали на IL-4 мыши, но не на человеческий, и отвечали как на IL-13 мыши, так и на человеческий. Как показано на фигуре 4 (справа), гуманизированные по IL-4Rα

мыши отвечали на человеческий IL-4, но не на IL-4 мыши, и отвечали как на IL-13 мыши, так и на человеческий IL-13.[00197] As shown in Figure 4 (left), wild-type mice

responded to mouse IL-4 but not human, and responded to both mouse and human IL-13. As shown in figure 4 (right), humanized for IL-4Rα

mice responded to human IL-4 but not to mouse IL-4, and responded to both mouse IL-13 and human IL-13.

[00198] These data show that IL-4, but not IL-13, is species-specific in wild-type and humanized IL-4Rα mice.

[00199] As shown in Figure 5, the role of IL-4 as a major factor mediating the synthesis switch from the IgE class was reinforced by the absence of elevated circulating levels of IgE following delivery of the mouse IL-25 gene to humanized IL-4Rα mice.

[00200] The monoclonal antibody dupilumab was studied in vitro and in vivo.

[00201] As shown in Figure 6, dupilumab prevented human IL-4-induced IgE production in primary B cell cultures derived from humanized IL-4Rα mice.

[00202] As shown in Figure 7, dupilumab dose-dependently reduced IL-25-induced lung pathologies at a dose of 10 mg/kg and above (25 mg/kg dose reduced pathology associated with mucus accumulation).

findings

[00203] These results demonstrate the pharmacological activity of dupilumab, a fully human anti-human IL-4Rα monoclonal antibody, in genetically engineered mice as a model of cytokine-induced inflammation.

[00204] Generation of genetically engineered mice with human IL-4 and/or IL-4Rα gene substitutions provides a powerful tool to assess the function of gene orthologs and the performance of candidate antibodies in vivo with limited cross-species reactivity.

Example 5

House dust mite extract (HDM) induced lung inflammation model

[00205] Chronic airway inflammation in humanized IL-4 and IL-R4α mice was induced by intranasal administration of house dust mite (HDM) extract (Greer laboratories). Briefly, mice were first sensitized by intranasal instillation of HDM suspension (20 μl at a concentration of 2.5 μg/ml) for 10 days. After a two week reversal interval, mice were re-exposed to the allergen by intranasal HDM 3 times a week from weeks 5 to 12. Treatment with dupilumab (an anti-IL4Rα antibody) was started at week 7 twice a week by subcutaneous injection until completion of the experiment at week 12. Tissue samples were taken for subsequent analyses. The experimental plan is shown in figure 8.

Demonstration of the therapeutic efficacy of dupilumab in an HDM-induced airway inflammation model using dual humanized IL-4 and IL-4Rα mice

[00206] Respiratory disease was induced in mice with two humanized IL-4 and IL-4Rα (IL-4 ^hu/hu /IL-4R ^hu/hu ) using the protocol described above. Histological analysis of lung tissue showed that intranasal instillation of HDM resulted in increased mucus production in the airways. Dupilumab treatment reduced mucus accumulation in mice exposed to the HDM allergen. Analysis of cellular infiltrate in bronchoalveolar lavage fluid (BALF) indicates that the number of eosinophils increased with HDM instillation and decreased with dupilumab treatment. Total IgE in the circulation of humanized mice was increased by HDM treatment, indicating a competent IL-4 signaling pathway. The use of dupilumab provided the opportunity to reduce the level of IgE. In contrast, the reference molecule, IL13R2α-Fc, which antagonized only IL-13 without interfering with IL-4 signaling, had comparable activities in reducing mucus accumulation and preventing eosinophilic infiltration. However, a differential effect was found in terms of circulating IgE levels between dupilumab and the IL-13 antagonist, IL13R2α-Fc. Blocking the IL-13 pathway alone was not sufficient to reduce HDM-induced IgE levels; while dupilumab reduced the production of IgE, the major pathological mediator of allergy, by blocking both the IL-4 and IL-13 pathways.

[00207] In a separate series of experiments, respiratory disease was induced in mice with two humanized IL-4 and IL-4Rα (IL-4 ^hu/hu /IL-4R ^hu/hu ) using the same protocol described above, except that that used different controls. In these experiments, an isotope control antibody of the same IgG isotype as dupilumab was used. The experimental design is depicted in Figure 9. mRNA was purified from total RNA using the Dynabeads mRNA isolation kit (Life Tech), and a specific strand library for RNA sequencing was generated from the mRNA using the Scriptseq RNA Library Preparation Kit (Illumina). The library was sequenced with a HiSeq 2000 (Illumina) at 33 bp read length. and data on gene expression levels were extracted from raw reads using the Clcbio RNA sequencing management system (Qiagen). Differentially expressed genes among experimental groups were identified using Student's t-test (p<0.05, fold change ≥1.5). The expression cluster of these genes was obtained using the Pearson correlation coefficient clustering algorithm from GeneSpring GX7.3. HDM was found to induce a change in lung gene expression in IL-4 and IL-4Rα doubly humanized mice and this change was blocked by dupilumab. Serum samples were taken from euthanized mice at the end of the treatment period. Mouse serum IgE levels were measured using a commercial ELISA kit (R&D systems). Statistical analysis was performed using the conventional method of one-way analysis of variance (ANOVA).

Example 6

An antigen-induced skin inflammation model

[00208] Chronic skin inflammation in mice with two humanized Il-4 and IL-4Rα can be induced using the following procedure. The fur on the back of mice with the humanized sequence was sheared with an electric clipper and then stripped with adhesive tape to create minor damage and cracks in the skin barrier. A gauze dressing moistened with an allergen solution (such as egg albumin with bacterial toxin or house dust mite extract) was applied to the skin for one week, followed by a two-week relief period. The procedure was repeated three times for a total of 7 weeks with induction as a result of skin lesions like atopic dermatitis. Treated mice are characterized by elevated IgE levels, pruritis, thickening of the epidermal layer, typical symptoms of atopic dermatitis.

Example 7

Characterization of PK profiles of anti-human IL-4Rα antibodies in mice expressing humanized IL-4Rα

[00209] This example describes experiments conducted to evaluate the PK profiles of REGN 668 (human anti-human IL-4Rα monoclonal antibody, also known as "dupilumab") and control antibody REGN646 (monkey surrogate, non-binding anti-mfIL-4R control antibody).

[00210] The mice used in these experiments were MAID 1444 (homozygous for humanized IL-4Rα, or "IL-4Rα HumIn", in which the IL-4Rα ectodomain is of human origin, and the transmembrane and cytoplasmic regions of the mouse) and wild-type mice ("WT") of the corresponding line (75% C57BL/6/25%129Sv) aged 20-23 weeks. The study group consisted of a total of 40 mice, male and female, with a cohort size per drug per dose of 5 homozygous and 5 WT of the respective strain. Antibodies (in PBS buffer) were administered to mice by subcutaneous injection at 10 mg/kg. Blood samples were taken for analysis on the day of injection (time "0" or day 0), 6 hours after injection and on day 1, day 3, day 7, day 10, day 14, day 21 and day 30, respectively.

[00211] Blood levels of drugs (ie, REGN668 or REGN646) were determined by assaying total human antibodies using an ELISA immunoassay. Briefly, goat anti-human IgG polyclonal antibody (Jackson ImmunoResearch, #109-005-098) was coated on 96-well plates to capture test human antibodies in serum, and then antibodies bound to the plate substrate were detected using goat anti-human polyclonal antibody. IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch, #109-035-098) and TMB substrate (BD Pharmingen). Serum samples were 6-fold dose serial dilutions per sample ranging from 1:100-1:243,000, and reference standards of the respective antibodies were 12-fold dose serial dilutions. Serum drug antibody concentrations were calculated from a calibration curve constructed using Graphpad Prism software.

[00212] The half-life of REGN 668 was found to be shortened in HumIn IL-4Rα mice compared to wild-type mice producing only mouse IL-4Rα protein. This difference in PK profiles can be explained by target-mediated interaction and clearance between monoclonal antibodies and the human IL-4α receptor. Thus, mice expressing human or humanized IL-4Rα provide a suitable mimic for characterizing the PK properties of anti-human IL-4Rα antibodies (eg, dupilumab) in a preclinical mouse model.

Uses of mice with humanized IL-4 and/or IL-4Rα

[00213] Mice with humanized IL-4 and/or IL-4Rα are applicable to evaluate the pharmacodynamics (PD) of antagonists specific for human IL-4 and/or IL-4Rα, for example, neutralizing antibodies to IL-4 and/or to IL-4Rα, such as dupilumab.

[00214] Pharmacokinetic (PK) and PD analyzes in IL-4 and/or IL-4Rα humanized mice were performed according to standard procedures known in the art.

[00215] Humanized IL-4 and/or IL-4Rα mice are useful for testing in vivo therapeutic efficacy of human IL-4 and/or IL-4Rα specific antagonists, e.g., anti-IL-4 and/or IL neutralizing antibodies -4Rα, for example, dupilumab, in various disease models known in the art, such as those shown herein above.

Example 8

Replacement of Endogenous Mouse IL-33 Gene with Human IL-33 Gene

[00216] Mouse IL-33 gene (NCBI gene ID: 77125, original source: MGI: 1924375; reference sequence transcript: NM_001164724.1; UniProt ID: Q8BVZ5; genomic assembly: NCBI37/mm9; localization: chr19:29999604-30035205 + strand) contains 8 exons and encodes a protein of 266 amino acids (GenBank accession no. NP_001158196.1).

[00217] Human IL-33 gene (NCBI gene ID: 90865, original source: HGNC: 16028; reference sequence transcript: NM_033439.3; UniProt ID: O95760; genomic assembly: GRCh37/hg19; localization: chr9:6215149-6257983 + strand) also contains 8 exons and encodes a protein of 270 amino acids (GenBank accession no. NP_254274.1).

[00218] The 16333 bp human genomic segment containing the stretch from exon 2 starting at the ATG start codon to exon 8 (including the 3'-untranslated region) of the human IL-33 gene was replaced with a mouse IL-33 gene locus of length 9381 bp, covering the region from exon 2, starting with the ATG start codon, to the coding part of exon 8, including the stop codon. See figure 10A.

[00219] A targeting construct to replace the mouse IL-33 gene with a human IL-33 genomic segment in a single targeting step was constructed using VelociGene® engineering technology (see Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech, 21(6):652-659) similar to the procedure described in Example 1 above to replace the mouse IL-4 gene with the human IL-4 genomic segment, except that the IL-4 DNA 33 mice and humans were derived from bacterial artificial chromosome (BAC) clones bMQ-350I18 and CTD-3015M15, respectively, and that the targeting vector contained a loxP flanked cassette with a neomycin resistance selectable marker (Figure 10B).

[00220] Accurately targeted ES cell clones (MAID 7060) were identified by native allele loss assay (LONA) (Valenzuela et al. 2003) in which copy number of the native unmodified IL-33 gene was determined by two quantitative polymerase chain reactions TaqMan™ (qPCR) with specific primers for mouse IL-33 gene sequences targeted for deletion. The qPCR reaction mixtures contained the following primer-probe sets (written 5' to 3' end):

above the target site ("mTU"):

direct primer,

reverse primer,

probe (MGB),

below the target area ("mTD"):

direct primer,

reverse primer,

probe (MGB),

[00221] To prove that the human IL-33 gene sequence had replaced the mouse IL-33 gene sequence in the humanized allele, a TaqMan™ qPCR assay was performed that included the following primer-probe sets (written 5'- to 3'- end):

above the target area ("hTU")

direct primer,

reverse primer,

probe (MGB),

below the target area ("hTD"):

direct primer,

reverse primer,

probe (MGB),

где последовательность человеческого IL-33 выделена курсивом, а старт-кодон ATG человеческого гена подчеркнут. Нижний участок соединения последовательности, содержащей геномную последовательность человеческого IL-33 и фланкированной loxP кассеты с селектируемым маркером устойчивости к неомицину (обозначенный «II» на фиг. 10В) конструировали так, чтобы он находился в пределе

где последовательность человеческого IL-33 выделена курсивом и участок соединения указан символом «/», а участок lox Р подчеркнут. Нижний участок соединения последовательности фланкированной loxP кассеты с селектируемым маркером устойчивости к неомицину и локуса IL-33 мыши (обозначенный «III» на фиг. 10С) конструировали так, чтобы он находился в пределе

где участок соединения показан символом «/», а участок loxP подчеркнут.[00222] The upper junction of the mouse IL-33 locus and the sequence containing the human IL-33 gene (labeled "I" in Figure 10B) was designed to be within the

where the human IL-33 sequence is in italics and the human ATG start codon is underlined. The lower junction of the sequence containing the genomic sequence of human IL-33 and the loxP flanked neomycin resistance selectable marker cassette (labeled "II" in FIG. 10B) was designed to be within the

where the sequence of human IL-33 is in italics and the connection site is indicated by the symbol "/", and the lox P plot is underlined. The bottom junction of the sequence of the loxP flanked neomycin resistance selectable marker cassette and the mouse IL-33 locus (labeled "III" in FIG. 10C) was designed to be within the

where the junction site is shown as "/" and the loxP section is underlined.

где 3'-конец последовательности человеческого IL-33 выделен курсивом перед первым символом «/», а 3'-последовательность IL-33 мыши выделена курсивом после второго символа «/» и участок loxP подчеркнут.[00223] Precisely targeted ES cells (MAID 7060) were then electroporated with a transient Cre expression vector to remove the selectable drug resistance marker cassette to generate ES cell clones without the drug resistance cassette (MAID 7061) . The top junction in these MAID 7061 ES cells (labeled "I" in Fig. 18C) was the same as in MAID 7060 ES cells. The bottom junction (labeled "II" in Fig. 18C) was designed to he was on the edge

where the 3' end of the human IL-33 sequence is in italics before the first "/" and the 3' mouse IL-33 sequence is in italics after the second "/" and the loxP region is underlined.

[00224] Precisely targeted ES cells (MAID 7060 or MAID 7061) were introduced into an 8-cell stage SW mouse embryo using the VelociMouse® method (See US Pat. ) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses, Nature Biotech.25(1):91-99). VelociMice® (F0 mice entirely derived from a donor ES cell) carrying the humanized IL-33 gene were identified by genotyping for mouse allele loss and human allele gain using modified allele analysis (see Valenzuela et al. (2003) ). The same LONA assay was used to analyze DNA isolated from mouse tail biopsies derived from targeted ES cells to determine their IL-33 genotypes and confirm that the humanized IL-33 allele was germline transmitted. . Two offspring heterozygous for the substitution were crossed to produce a mouse homozygous for the substitution of the endogenous mouse IL-33 gene with the human IL-33 gene.

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SEQUENCE LIST

<110> Wang, Li-Sien

Xue, Yingzi

Murphy, Andrew J.

Stephens, Sean

<120> ANIMALS WITH HUMANIZED IL-4 and IL-4a

<130> 31260 (T0029)

<150> 61/989757

<151> 2014-05-07

<160> 46

<170> PatentIn version 3.5

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<400> 15

tgccaagttc taattccatc agacctcgac ctgcagccgg cgcgccataa cttcgtataa 60

ggtatcctat acgaagttat ctcgaggagga gttcccaccc ttctcaagag cataatgcgc 120

agatcattaa gggacagatg caggctgggg agacggttca gcagttagga gtacctgttg 180

ctcttccaga ggacccaggt tcaattcccg gcactcacat agcagcttaa aacaataact 240

caagttctgg gggagctgat gctctcctct ggcctcctgt ggaggtacac agaccacatg 300

cctgtaggca agacacccac acacataaaa acaaaataaa ataaggatag aaaggccagg 360

gggatgaatc cagaggtaga agaaaactta ttccctggaa ttgtcctctg actcccctcc 420

caaaacctct aacacgca 438

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 16

ccgctggcat gtgtattgtg 20

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 17

tgagtgtggg accctcaaga g 21

<210> 18

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> other_characteristic

<222> (1)..(1)

<223> Labeled FAM: 5-carboxyfluorescein fluorescent probe

<220>

<221> other_characteristic

<222> (24)..(24)

<223> Labeled with BHQ: with a fluorescence quencher corresponding to the type of quencher

"black hole"

<400> 18

tgacccaagc cctacatggc cact 24

<210> 19

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 19

tgaggagagc tcacgggaat c 21

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 20

acccatctcc tgcgtttctg 20

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> other_characteristic

<222> (1)..(1)

<223> Labeled FAM: 5-carboxyfluorescein fluorescent probe

<220>

<221> other_characteristic

<222> (25)..(25)

<223> Labeled with BHQ: with a fluorescence quencher corresponding to the type of quencher

"black hole"

<400> 21

ttgacacgcc agctacactg ctcca 25

<210> 22

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 22

acctgcgtct ccgactacat g 21

<210> 23

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 23

gagctcggtg ctgcaattg 19

<210> 24

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> other_characteristic

<222> (1)..(1)

<223> Labeled FAM: 5-carboxyfluorescein fluorescent probe

<220>

<221> other_characteristic

<222> (25)..(25)

<223> Labeled with BHQ: with a fluorescence quencher corresponding to the type of quencher

"black hole"

<400> 24

tgggaccatt catcttccac tcgca 25

<210> 25

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 25

ggtggagagg ctattcggc 19

<210> 26

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 26

gaacacggcg gcatcag 17

<210> 27

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> other_characteristic

<222> (1)..(1)

<223> Labeled FAM: 5-carboxyfluorescein fluorescent probe

<220>

<221> other_characteristic

<222> (23)..(23)

<223> Labeled with BHQ: with a fluorescence quencher corresponding to the type of quencher

"black hole"

<400> 27

tgggcacaac agacaatcgg ctg 23

<210> 28

<211> 243

<212> DNA

<213> Artificial sequence

<220>

<223> Portion of the mouse IL-4Ralf locus and sequence containing the gene

IL-4Ralpha

<400> 28

tgggggaggg aggccatgac acaaatgaaa tggaccccgc tgacccagga tcagcatctg 60

cccactcttc tttctgcagg caccttttgt gtccccaatg gggtggcttt gctctgggct 120

cctgttccct gtgagctgcc tggtcctgct gcaggtggca agctctggta agtcaccact 180

tctcaatcat tcatttgttg gctattaatg gcgtgccagg gtcctgcagt atgtcacctg 240

gcc 243

<210> 29

<211> 243

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid sequence containing the bottom portion

compound sequence of human IL-4Ralpha, and flanked

loxP cassettes with neomycin resistance selectable marker

<400> 29

gtcagatcgt ggagggtctc ggacgagggt cctgaccctg ggtctccagt cctgggaagt 60

ggagcccagg ctgtaccagg gctgacctca gctcatggct cccgggctcg ataactataa 120

cggtcctaag gtagcgactc gagataactt cgtataatgt atgctatacg aagttatatg 180

catggcctcc gcgccgggtt ttggcgcctc ccgcgggcgc ccccctcctc acggcgagcg 240

ctg 243

<210> 30

<211> 390

<212> DNA

<213> Artificial sequence

<220>

<223> Part of a sequence flanked by a loxP selectable cassette

neomycin resistance marker, and the mouse IL-4Ralpha locus

<400> 30

tattgttttg ccaagttcta attccatcag acctcgacct gcagccccta gataacttcg 60

tataatgtat gctatacgaa gttatcctag gttggagctc tctgtagcca ggtaaccaag 120

ggtcccaggg gaacccccag tgtggacgcg gactgcacat gacacaggggc ggcctcccca 180

ttcatgactg ttttctctcct tgcagacttc cagctgcccc tgatacagcg ccttccactg 240

ggggtcacca tctcctgcct ctgcatcccg ttgttttgcc tgttctgtta cttcagcatt 300

accaagtgag ttcctgcttt ggctggtgtc tctggctggc ccttcagcag tgctctcaga 360

ggtcacagtc attgtgctgg ctgagaaaag 390

<210> 31

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 31

ttggactagt aacaagaagg gtagca 26

<210> 32

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 32

cctttcccat caccctctaa ctt 23

<210> 33

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 33

agctctggtg gacaga 16

<210> 34

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 34

tctctgccaa gctgcttatc c 21

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 35

ggctgcatgg aagaggtgaa 20

<210> 36

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 36

ctctccacaa atcg 14

<210> 37

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 37

caggcaggaa tagctgagat aatct 25

<210> 38

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 38

tgtggagcaa aaagtggttg at 22

<210> 39

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 39

cctgtgaata gtgataaac 19

<210> 40

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 40

cagttccaaa cgataggctc aa 22

<210> 41

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 41

ataattctgt gaagcatctg gtcttc 26

<210> 42

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 42

ctagagctgc tagtaaaa 18

<210> 43

<211> 160

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 43

atagccaagg ttgcttctga tgacttcagg tccatatagt tggattaatg ttatatttca 60

atcccacaga aacctgaaaa atgaagccta aaatgaagta ttcaaccaac aaaatttcca 120

cagcaaagtg gaagaacaca gcaagcaaag ccttgtgttt 160

<210> 44

<211> 160

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 44

tttatattat tgaataaagt atattttcca aatgtatgtg agactataat gattttatca 60

tatgatgact caatattctg ctcgagataa cttcgtataa tgtatgctat acgaagttat 120

atgcatggcc tccgcgccgg gttttggcgc ctcccgcggg 160

<210> 45

<211> 160

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 45

agcccctaga taacttcgta taatgtatgc tatacgaagt tatgctagta actataacgg 60

tcctaaggta gcgagctagc cgcctgtgcg ttctgggttg aatgacttaa tgcttccaac 120

tgaagaaagg gtaacagaga gaaagaaagc cattcttggc 160

<210> 46

<211> 237

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 46

tttatattat tgaataaagt atattttcca aatgtatgtg agactataat gattttatca 60

tatgatgact caatattctg ctcgagataa cttcgtataa tgtatgctat acgaagttat 120

gctagtaact ataacggtcc taaggtagcg agctagccgc ctgtgcgttc tgggttgaat 180

gacttaatgc ttccaactga agaaagggta acagagagaa agaaagccat tcttggc 237

<---

Claims (31)

1. Rodent for humanized IL-4 protein expression, wherein the rodent genome comprises replacing the genomic DNA of the rodent IL-4 gene at the endogenous rodent IL-4 locus with the nucleic acid sequence of the human IL-4 gene to form a humanized IL-4 gene, where the humanized IL-4 gene encodes a human IL-4 protein and contains a stretch from exon 1, starting with the ATG start codon, to exon 4 of the human IL-4 gene, wherein expression of the humanized IL-4 gene is under the control of rodent regulatory elements at the endogenous rodent IL-4 locus, and where the rodent is a mouse or a rat. 2. The rodent of claim 1, wherein the rodent is unable to express the rodent IL-4 protein. 3. The rodent according to claim 1 or 2, where the rodent is a mouse, and where the replacement is the replacement of genomic DNA containing a region from exon 1, starting from the ATG start codon to exon 4 of the mouse IL-4 gene, with genomic DNA containing the stretch from exon 1, starting with the ATG start codon, to exon 4 of the human IL-4 gene. 4. The rodent according to any one of paragraphs. 1-3, where a humanized IL-4Rα protein is expressed in a rodent containing the ectodomain of the human IL-4Rα protein. 5. The rodent of claim 4, wherein the humanized IL-4Rα protein comprises a transmembrane domain and a cytoplasmic domain of endogenous rodent IL-4Rα protein. 6. Rodent embryonic stem (ES) cell for producing a humanized IL-4 rodent containing replacing the genomic DNA of the rodent IL-4 gene at the endogenous rodent IL-4 locus with the nucleic acid sequence of the human IL-4 gene to form a humanized IL-4 gene, where the humanized IL-4 gene encodes a human IL-4 protein and contains a stretch from exon 1, starting with the ATG start codon, to exon 4 of the human IL-4 gene, wherein expression of the humanized IL-4 gene is under the control of rodent regulatory elements at the endogenous rodent IL-4 locus, and where the rodent ES cell is a mouse ES cell or a rat ES cell. 7. A rodent ES cell according to claim 6, wherein the rodent ES cell is a mouse ES cell. 8. A rodent ES cell according to claim 7, wherein the replacement is a replacement of genomic DNA containing a region from exon 1, starting with the ATG start codon, to exon 4 of the mouse IL-4 gene, with genomic DNA containing a region from exon 1, from the ATG start codon to exon 4 of the human IL-4 gene. 9. A rodent embryo for producing a humanized IL-4 rodent, comprising the rodent ES cell of claim 6, wherein the rodent embryo is a mouse embryo or a rat embryo. 10. Method for obtaining a rodent with humanized IL-4, including: (a) replacing the genomic DNA of the rodent IL-4 gene at the endogenous rodent IL-4 locus in the rodent ES cell with the human IL-4 gene nucleic acid sequence to form a humanized IL-4 gene, thereby obtaining a genetically modified rodent ES cell, where the humanized IL-4 gene encodes a human IL-4 protein and contains a stretch from exon 1, starting with the ATG start codon, to exon 4 of the human IL-4 gene, wherein expression of the humanized IL-4 gene is under the control of rodent regulatory elements at the endogenous rodent IL-4 locus, and where the rodent ES cell is a mouse ES cell or a rat ES cell; and (b) obtaining a humanized IL-4 rodent using the genetically modified ES cell from step (a), wherein the humanized IL-4 rodent is a mouse or a rat. 11. The method of claim 10, wherein the rodent is a mouse, and wherein the replacement is a replacement of the genomic DNA containing the region from exon 1, starting at the ATG start codon, to exon 4 of the mouse IL-4 gene, with the genomic DNA containing the region from exon 1, starting at the ATG start codon, to exon 4 of the human IL-4 gene. 12. A targeting vector for humanizing an endogenous IL-4 gene in a rodent, wherein the rodent is a mouse or rat, comprising a nucleic acid sequence of the human IL-4 gene flanked by nucleic acid sequences that are homologous to nucleic acid sequences at the rodent IL-4 locus and mediate the insertion of the nucleic acid sequence of the human IL-4 gene into the rodent IL-4 locus, where the nucleic acid sequence of the human IL-4 gene contains a section from the ATG start codon of exon 1 to exon 4 of the human IL-4 gene; where insertion of the nucleic acid sequence of the human IL-4 gene into the rodent IL-4 locus results in the replacement of a genomic fragment of the rodent IL-4 gene to form a humanized IL-4 gene, where the genomic fragment of the rodent IL-4 gene in which the replacement is made contains a region from the ATG start codon of exon 1 to exon 4 of the rodent IL-4 gene, and wherein expression of the humanized IL-4 gene is under the control of rodent IL-4 regulatory elements at the endogenous rodent IL-4 locus. 13. The targeting vector of claim 12, wherein the rodent is a mouse.

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