Transgenic animals expressing chimeric C-KIT proteins

Abstract

The present disclosure describes methods for producing transgenic cells or transgenic mice that express a chimeric c-KIT protein that can be expressed on mouse cells and bind to mouse stem cell factor (SCF), which can be inhibited by anti-human c-KIT inhibitors. The transgenic cells and transgenic mice can be used to test candidate anti-human c-KIT inhibitors.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 371,489, filed August 15, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] (Reference to the Electronic Sequence Listing) The contents of the electronic sequence listing (350546.xml, size: 18,701 bytes, and creation date: August 15, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] The tyrosine-protein kinase KIT, also known as c-KIT, CD117 (Cluster of Differentiation 117), or SCFR (mast / stem cell growth factor receptor), is expressed on the surface of hematopoietic stem cells, mast cells, neural crest-derived melanocytes, and germ cells. c-KIT binds to stem cell factor (SCF), forming a dimer that activates its intrinsic tyrosine kinase activity, which in turn phosphorylates and activates signaling molecules that propagate signals in the cell.

[0004] Anti-c-KIT antibodies have been developed that can be useful for inhibiting c-KIT-mediated signaling and resulting in the ablation of hematopoietic stem cells in a subject. Such ablation helps ensure the efficacy of subsequent hematopoietic stem cell engraftment. The development of such antibodies can be easily performed using animal models expressing human c-KIT protein. However, the introduction of cDNA encoding human c-KIT protein into animals can be unsuccessful. For example, human c-KIT protein may not be expressed in the desired cells, may not be correctly localized, or may not undergo the correct post-translational modifications. Furthermore, when activated, human c-KIT protein may not induce the correct downstream signaling pathways in animal cells.

[0005] The human c-KIT protein contains an extracellular domain (ECD) with five Ig-like domains (D1-D5) involved in ligand binding and homodimerization, a transmembrane domain, and an intracellular domain (ICD). Proteolytic processing of c-KIT involves N-linked glycosylation and phosphorylation at both tyrosine and serine residues. When SCF dimers bind to c-KIT, binding induces c-KIT dimerization, conformational changes in both the ECD and ICD, ICD transphosphorylation, and the binding of various partners.

[0006] Designing human-animal chimeric c-KIT proteins also presents significant challenges. For example, human and mouse c-KIT proteins share 90% sequence similarity. However, protein glycosylation and conformational changes in both the ECD and ICD required for activation may be impaired in chimeric proteins. Furthermore, it was unknown how chimeric proteins would function to induce cell proliferation in cells with different levels of endogenous c-KIT expression and how they would respond to mouse SCF. Summary of the Invention

[0007] The present disclosure describes methods for producing transgenic cells or mice expressing a chimeric c-KIT protein that is capable of being expressed on mouse cells and binding to mouse stem cell factor (SCF). Such binding can be inhibited by anti-human c-KIT inhibitors, and thus serves as a platform for inhibitor testing.

[0008] According to one embodiment of the present disclosure, there is provided a transgenic mouse cell comprising a nucleic acid encoding a chimeric c-KIT protein, the chimeric c-KIT protein comprising a mouse c-KIT signal peptide, a human c-KIT extracellular domain, a mouse c-KIT transmembrane domain, and a mouse c-KIT intracellular domain.

[0009] In some embodiments, the human c-KIT extracellular domain comprises the GNNK sequence. In some embodiments, the human c-KIT extracellular domain comprises the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In some embodiments, the human c-KIT extracellular domain comprises the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

[0010] In some embodiments, the nucleic acid comprises an exogenous portion and an endogenous portion. In some embodiments, the exogenous portion of the nucleic acid comprises coding sequences for a human c-KIT extracellular domain, a mouse c-KIT transmembrane domain, and a mouse c-KIT intracellular domain. In some embodiments, the exogenous portion is inserted into the genome of the mouse at a locus within exon 2 of the mouse c-KIT gene. In some embodiments, the locus is 3' to the last codon of the endogenous mouse c-KIT signal peptide. In some embodiments, the exogenous portion further comprises a stop codon. In some embodiments, the insertion is homozygous. In some embodiments, the cell is in vivo in a transgenic mouse.

[0011] In another embodiment, there is further provided a method for testing the activity of an inhibitor against human c-KIT, comprising contacting a candidate inhibitor with a transgenic mouse cell of the present disclosure and measuring c-KIT signaling in the cell.

[0012] Also provided is a transgenic mouse comprising the transgenic mouse cell of the present disclosure. Also provided is a method for testing the activity of an inhibitor against human c-KIT, comprising administering a candidate inhibitor to a transgenic mouse and measuring c-KIT signaling in the mouse.

[0013] In some embodiments, the candidate inhibitor is an anti-c-KIT antibody. In some embodiments, c-KIT signaling is measured along with cell proliferation.

[0014] In another embodiment, a method for preparing a transgenic mouse cell is provided, comprising introducing into a target mouse cell a construct comprising (i) a coding sequence encoding a partial chimeric protein comprising a human c-KIT extracellular domain, a mouse c-KIT transmembrane domain, and a mouse c-KIT intracellular domain, and (ii) flanking sequences that enable recombination integration of the coding sequence into a locus within an exon of an endogenous c-KIT gene in the genome of the mouse. In some embodiments, the exon is exon 2.

[0015] In some embodiments, the transgenic cells express a chimeric protein comprising a mouse c-KIT signal peptide, a human c-KIT extracellular domain, a mouse c-KIT transmembrane domain, and a mouse c-KIT intracellular domain. [Brief explanation of the drawings]

[0016] [Figure 1] The structures of the expression vectors tested for functional validation are shown. [Figure 2] 1 shows stem cell factor (SCF)-mediated survival and proliferation using mouse or human SCF. [Figure 3] A schematic diagram of the c-Kit expression vector for splicing verification is presented. [Figure 4A] The protocol for in-frame insertion of humanized c-Kit cDNA with the mouse signal peptide (hEC-mTM-mIC) (A) and after removal of the neo sequence (B) is shown. [Figure 4B] The protocol for in-frame insertion of humanized c-Kit cDNA with the mouse signal peptide (hEC-mTM-mIC) (A) and after removal of the neo sequence (B) is shown. [Figure 5A] Observations in transgenic mice regarding T cell precursors in the thymus (A) are presented. [Figure 5B] We present observations in transgenic mice regarding B cell precursors (B) in the bone marrow. [Figure 5C] Observations in transgenic mice regarding c-Kit expression in bone marrow HSPCs (C) are presented. [Figure 6] This shows that briquilimab prevented IgE-induced PSA in hmCD117 mice. hmCD117 mice were treated with briquilimab at 5 mg / kg three times a week for 3 weeks or a bolus dose of 25 mg / kg for 2 weeks, then sensitized with anti-DNP IgE and challenged with DNP-HSA 24 hours later. Data represent the mean ± standard error of the mean (SEM). Statistical analysis was performed by comparing group means from 0 to 60 minutes. **p<0.01 by Welch's t-test. DETAILED DESCRIPTION OF THE INVENTION

[0017] definition It should be noted that the term "a" or "an" entity refers to one or more of that entity. For example, "an antibody" is understood to refer to one or more antibodies. Similarly, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0018] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" and the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with well-known protecting / blocking groups, proteolytic cleavage, or modification with unnatural amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but need not be translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

[0019] As used herein with respect to cells, nucleic acids (such as DNA or RNA), the term "isolated" refers to molecules separated from other DNA or RNA, respectively, that are present in the natural source of the macromolecule. As used herein, the term "isolated" also refers to nucleic acids or peptides that are substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is meant to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to cells or polypeptides that are isolated from other cellular proteins or tissues. Isolated polypeptides are meant to encompass both purified and recombinant polypeptides.

[0020] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide containing molecule comprising at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such include, but are not limited to, a heavy or light chain complementarity determining region (CDR) or ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0021] The term "antibody fragment" or "antigen-binding fragment," as used herein, refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, or scFv. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and bispecific antibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0022] "Specifically bind" or "has specificity to" generally mean that an antibody binds to an epitope via its antigen-binding domain and that the binding involves some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope if it binds to the epitope via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have a higher specificity for a given epitope than antibody "B," or antibody "A" can be said to bind epitope "C" with greater specificity than it has for the related epitope "D."

[0023] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties that result therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand (the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in a sequence listing) and the non-coding strand (used as a template for transcription of the gene or cDNA) can be referred to as encoding the protein or other product of that gene or cDNA.

[0024] As used herein, "endogenous" refers to any substance that is derived from or produced within an organism, cell, tissue, or system.

[0025] As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside an organism, cell, tissue, or system.

[0026] As used herein, the term "expand" refers to an increase in number, such as an increase in the number of T cells. In one embodiment, T cells expanded ex vivo are increased in number compared to the number originally present in the culture. In another embodiment, T cells expanded ex vivo are increased in number compared to other cell types in the culture. As used herein, the term "ex vivo" refers to cells removed from a living organism (e.g., a human) and grown outside of the organism (e.g., in a culture dish, test tube, or bioreactor).

[0027] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0028] An "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression. Other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) that incorporate the recombinant polynucleotide.

[0029] As used herein, "homologous" refers to subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a subunit position in both of the two molecules is occupied by the same monomer subunit, e.g., if a position in each of the two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a linear function of the number of matching or homologous positions; for example, if half of the positions in the two sequences (e.g., five positions in a polymer 10 subunits long) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

[0030] As used herein, "identity" refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, e.g., between two polypeptide molecules. If two amino acid sequences have the same residue at the same position, e.g., if that position in each of the two polypeptide molecules is occupied by arginine, then they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; for example, if half of the positions in the two sequences (e.g., five positions in a 10-amino acid-long polymer) are identical, then the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matched or identical, then the two amino acid sequences are 90% identical.

[0031] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. A cell includes the primary subject cell and its progeny.

[0032] The term "transgene" refers to genetic material that has been or is to be artificially inserted into the genome of an animal, particularly a mammal, and more particularly a mammalian cell of a living animal.

[0033] The term "transgenic animal" refers to a non-human animal, usually a mammal, in which a non-endogenous (i.e., heterologous) nucleic acid sequence is present in some of its cells as an extrachromosomal element or stably integrated into the germline DNA (i.e., the genomic sequence of most or all of its cells), e.g., a transgenic mouse. The heterologous nucleic acid is introduced into the germline of such a transgenic animal, e.g., by genetic manipulation of the host animal's embryo or embryonic stem cells.

[0034] The term "humanized mouse" refers to a mouse that transgenicly expresses one or more human genes or chimeric genes that express at least a portion of a human protein.

[0035] As used herein, the phrases "under transcriptional control" or "operatively linked" mean that a promoter is in the correct location and orientation relative to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0036] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are well known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes self-replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and the like.

[0037] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be construed as including all the possible subranges specifically disclosed as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be construed as including specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0038] Transgenic mice and mouse cells Inhibitors against human c-KIT have therapeutic value. For example, anti-c-KIT antibodies can be used to deplete hematopoietic stem cells (HSCs) from a subject's bone marrow. Deletion of affected HSCs is useful for treating diseases or disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), thereby increasing the relative proportion of healthy HSCs.

[0039] Anti-c-KIT antibodies are well known in the art and / or commercially available, including, but not limited to, JSP191 (Jasper Therapeutics, Redwood City, CA), CDX-0158 (formerly KTN0158) or CDX-0159 (Celldex Therapeutics, Hampton, NJ), MGTA-117 (AB85) (Magenta Therapeutics, Cambridge, MA), CK6 (Magenta Therapeutics, Cambridge, MA), AB249 (Magenta Therapeutics, Cambridge, MA), and FSI-174 (Gilead, Foster City, CA).

[0040] In certain embodiments, the anti-c-KIT antibody binds to the extracellular region of c-KIT, i.e., amino acids 26-524 of SEQ ID NO: 1. Human c-KIT has eight isoforms: isoform 1 (NP_000213, SEQ ID NO: 1), isoform 2 (NP_001087241), isoform 3 (NP_001372213, SEQ ID NO: 2), isoform 4 (NP_001372214, SEQ ID NO: 3), isoform 5 (NP_001372215), isoform 6 (NP_001372217), isoform 7 (NP_001372219, SEQ ID NO: 4), and isoform 8 (NP_001372221).

[0041] These isoforms are highly homologous. However, four of them (isoform 1, isoform 3, isoform 4, and isoform 7, Table 1) contain the -GNNK- sequence (e.g., amino acid residues 510-514 of SEQ ID NO: 1), while the others do not. Isoform 1, isoform 3, isoform 4, and isoform 7 are referred to as "GNNK" isoforms.

[0042] [Table 1-1]

[0043] [Table 1-2]

[0044] [Table 1-3]

[0045] [Table 1-4]

[0046] We began by generating mouse cells expressing a chimeric c-KIT protein with a mouse signal peptide (mSP), a human extracellular portion (hEC), a mouse transmembrane domain (mTM), and a mouse intracellular portion (mIC). Initial studies showed that such a chimeric protein was able to bind to human SCF (hSCF) and mediate the proliferation and survival of transfected cells (Example 1). Because there was no indication that the human extracellular domain could mediate SCF signaling through the mouse transmembrane and intracellular domains, the chimeric protein was actually less effective than its human counterpart in mediating SCF signaling, which was surprising.

[0047] To generate transgenic cells with the hEC coding sequence integrated into the genome, we designed a cDNA containing an hEC-mTM-mIC portion with flanking sequences that allowed insertion of the cDNA into exon 2 of the mouse c-KIT genomic sequence. The coding portion of the mouse signal peptide terminates within exon 2 (Table 1, SEQ ID NO: 12). Therefore, such an insertion allows expression of an mSP-hEC-mTM-mIC fusion protein when the start codon of hEC is inserted in frame immediately after the last codon of mSP. In another surprising finding, such an insertion did not interfere with exon 2 splicing, and the fusion protein was expressed as designed. As a result, transgenic mouse cells and animals were successfully prepared.

[0048] According to one embodiment of the present disclosure, a transgenic mouse cell is provided comprising a nucleic acid encoding a chimeric c-KIT protein, the chimeric c-KIT protein comprising a mouse c-KIT signal peptide, a human c-KIT extracellular domain, a mouse c-KIT transmembrane domain, and a mouse c-KIT intracellular domain. In some embodiments, the chimeric c-KIT protein is capable of binding to human SCF. In some embodiments, the chimeric c-KIT protein is capable of mediating SCF signaling.

[0049] In some embodiments, the human c-KIT extracellular domain (hEC) comprises the extracellular domain of any of the known human c-KIT isoforms, such as isoform 1 through isoform 8, or a biological equivalent thereof. Biological equivalents of a reference sequence (e.g., SEQ ID NO: 7) include those having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the reference sequence.

[0050] Some human c-KIT isoforms contain the -GNNK- sequence (e.g., amino acid residues 510-514 of SEQ ID NO: 1) in their extracellular domains, while others do not. In preferred embodiments, hECs contain the extracellular domain of one of the "GNNK" isoforms (isoform 1, isoform 3, isoform 4, or isoform 7). The full protein sequences of isoform 1, isoform 3, isoform 4, and isoform 7 are provided in Table 1 as SEQ ID NOs: 1-4, and their extracellular domains are provided as SEQ ID NOs: 7-10.

[0051] In one embodiment, the c-KIT extracellular domain (hEC) comprises the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7. In one embodiment, the c-KIT extracellular domain (hEC) comprises the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8. In one embodiment, the c-KIT extracellular domain (hEC) comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9. In one embodiment, the c-KIT extracellular domain (hEC) comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10. An exemplary amino acid sequence of a chimeric protein is provided in SEQ ID NO: 11 (Table 1), which comprises the extracellular domain of human isoform 1.

[0052] In some embodiments, the c-KIT transmembrane domain (mTM) and intracellular domain (mIC) comprise the amino acid sequence of SEQ ID NO:6 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6.

[0053] In some embodiments, the chimeric c-KIT is encoded by a nucleic acid integrated into the genome of a transgenic cell. The nucleic acid may comprise an exogenous portion and an endogenous portion. As tested, a cDNA encoding the hEC-mTM-mIC portion of the chimeric c-KIT protein can be inserted into one of the exons of the endogenous mouse c-KIT gene.

[0054] In one embodiment, the insertion is at a locus within exon 2 of the mouse c-KIT gene. In a preferred embodiment, the cDNA encodes hEC-mTM-mIC, with the first codon of hEC inserted immediately after the last codon of the mouse signal peptide (SEQ ID NO: 5) (nucleotides 6-8 of SEQ ID NO: 12, which shows the exon 2 genomic sequence).

[0055] In an alternative embodiment, the insertion is at a locus within exon 2, but further downstream. This allows for the inclusion of several amino acids of the mouse extracellular domain in the expressed chimeric protein. However, the addition of a few mouse amino acids is not expected to affect the activity of the chimeric protein. Also, given the homology between mouse and human c-KIT proteins, if additional mouse extracellular amino acids are included, some of the human extracellular amino acids (N-terminal) can optionally be deleted from the cDNA to neutralize the effect.

[0056] In yet another alternative embodiment, the insertion is at a locus within exon 2, but further upstream from the end of the last codon of the signal peptide. To compensate for the partial truncation of the C-terminus of the mouse signal peptide, the cDNA can further contain the truncated portion to provide the entire expressed chimeric protein.

[0057] Similarly, in another embodiment, the insertion is at a locus within exon 1 upstream from the last codon of the signal peptide. To compensate for the partial truncation of the C-terminus of the mouse signal peptide, the cDNA can further contain the truncated portion to provide the entire expressed chimeric protein.

[0058] In some embodiments, the cDNA further comprises a stop codon. In some embodiments, the cDNA further comprises a poly(A) signal. In some embodiments, the cDNA further comprises a selectable marker, such as a coding sequence for a neomycin resistance protein or a fluorescent protein. Such markers can facilitate selection of successfully integrated cells. They can be excised from the genome using well-known techniques (e.g., Cre / loxP recombination) upon completion of selection. In some embodiments, the cDNA is codon-optimized. In some embodiments, the insertion is heterozygous. In some embodiments, the insertion is homozygous.

[0059] General methods for constructing recombinant DNA that can be introduced into target cells are well known to those skilled in the art, and the same compositions and methods of construction may be utilized to produce DNA useful herein. For example, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2nd ed., 1989) provides suitable construction methods. Recombinant DNA can be easily introduced into target cells (i.e., totipotent cells such as fertilized eggs) by methods well known in the art.

[0060] When target cell is a stem cell such as embryonic stem cell, transgenic mouse can be produced.Therefore, the present disclosure also provides a transgenic mouse having at least one transgenic cell as described herein.In some embodiments, the transgenic cell is heterozygous for chimeric protein.In some embodiments, the transgenic cell is homozygous.

[0061] Methods for using the transgenic cells or transgenic mice of the present disclosure are also provided. In one embodiment, a method for testing the activity of an inhibitor against human c-KIT is provided, involving contacting a candidate inhibitor with a transgenic mouse cell described herein and measuring c-KIT signaling in the cell. A method for testing the activity of an inhibitor against human c-KIT is provided, involving administering a candidate inhibitor to a transgenic mouse and measuring c-KIT signaling in the mouse. In some embodiments, the candidate inhibitor is an anti-c-KIT antibody.

[0062] c-KIT signaling can be measured by methods well known in the art. c-KIT is a cytokine receptor that binds to stem cell factor (SCF). This binding activates the intrinsic tyrosine kinase activity of c-KIT, which then phosphorylates and activates signaling molecules that propagate signals within the cell. Signaling through KIT plays a role in cell survival, proliferation, and differentiation. In some embodiments, inhibitor activity can be measured using cell proliferation or differentiation, as demonstrated in the accompanying experimental examples. [Example]

[0063] Example 1: Generation and testing of transgenic Ba / F3 cells This example examined the effect of exogenous c-KIT cDNA expression on the proliferation and survival of Ba / F3 cells.

[0064] Expression vectors containing (A) human c-KIT cDNA, (B) mouse c-KIT cDNA, (C) chimeric human / mouse c-KIT cDNA (mSP-hEC-mTM-mIC), or (D) mock control were constructed. The chimeric c-KIT cDNA encodes a chimeric c-KIT protein containing a mouse signal peptide (mSP), a human extracellular portion (hEC), a mouse transmembrane domain (mTM), and a mouse intracellular portion (mIC). Each expression vector also contained a sequence for expressing GFP and a neomycin resistance gene for cell sorting and selection (Figure 1). Each vector was transfected into Ba / F3 cells, and successfully transfected cells were collected.

[0065] Stably transfected Ba / F3 cells were starved overnight and stimulated with various concentrations of mouse or human stem cell factor (SCF) for 4 days. Cell survival was assessed by DAPI exclusion flow cytometry. Cell proliferation was assessed by EdU flow cytometry.

[0066] As shown in Figure 2, mouse SCF (mSCF) stimulation induced proliferation and survival of all vector-transfected mouse cells, demonstrating that both human and chimeric c-KIT proteins were capable of mediating mSCF binding and signaling. On the other hand, human SCF (hSCF) stimulation induced proliferation and survival only of Ba / F3 cells transfected with either human or chimeric c-KIT expression vectors. However, the chimeric c-KIT protein was observed to be less effective than its human counterpart in mediating SCF signaling.

[0067] Example 2. In vitro splicing assay It was proposed that the chimeric c-KIT cDNA be preferably inserted into the endogenous c-KIT locus of the mouse genome in target cells. However, it was suspected that such insertion would result in alteration of exon splicing regulatory sequences, causing disruption to the inserted cDNA. This example tested whether insertion of the chimeric cDNA into the c-KIT locus in the mouse genome would result in altered splicing.

[0068] The cDNA encoded a chimeric c-KIT protein (hEC-mTM-mIC) containing the human extracellular portion (hEC) and the mouse transmembrane (mTM) / intracellular portion (mIC) in addition to a stop codon and poly(A) sequence (Figure 3). A slightly different, codon-optimized version of the cDNA was also prepared. Homologous recombination was designed so that the cDNA was inserted in frame after exon 2 and the mouse signal peptide (mSP) of the mouse c-KIT gene. Thus, if exons 1 and 2 are correctly spliced, the transcript will contain the desired mSP-hEC-mTM-mIC chimeric protein.

[0069] The constructs were transfected into 3T3 mouse cells or embryonic stem (ES) cells. 24 hours after transfection, extracted RNA was reverse-transcribed and cDNA was amplified. Correct splicing in the transfected cells was confirmed by gel electrophoresis of the amplified PCR products.

[0070] Example 3. Generation and testing of transgenic mice Using a procedure similar to that of Example 2, this example prepared mouse ES cells with chimeric hEC-mTM-mIC cDNA stably integrated into the genome. Transgenic ES cell clones can be implanted into blastocyst-stage embryos for the generation of chimeras.

[0071] As shown in Figure 4A, the construct contained hEC-mTM-mIC plus a stop codon, poly(A), and a neomycin resistance gene (Neo). The hEC sequence was GNNK(+). The neomycin resistance gene was flanked by a pair of loxP sites. The neomycin resistance gene allowed for selection of transfected cells, which were then excised (Figure 4B). Insertion of the exogenous sequence was confirmed by PCR amplification using appropriate primers (as shown in Figure 4B).

[0072] To generate highly chimeric male mice carrying the recombinant gene locus, the transfected, integrated ES cell clones were placed into blastocysts. ES cells were derived from the inner cell mass of 3.5-day-old embryos (blastocyst stage). These cells are pluripotent and therefore can contribute to all cell lineages, including germ layers, when implanted into blastocyst-stage embryos. These blastocysts were implanted into pseudopregnant females and allowed to develop to term. The chimerism rate in the offspring was then assessed by comparison of coat color markers.

[0073] Of the 115 pups born, 23 (20%) were homozygous (10 males and 13 females), all with white patches on their black fur, and 65 (57%) were heterozygous (29 males and 36 females). Of these, 34 had white patches (15 males and 19 females), 31 were black (14 males and 17 females), and 27 (23%) were wild-type (17 males and 10 females), all black. No differences were observed between WT and c-KIT heterozygous mice with regard to cellularity, survival, and proportion of T / B cell precursors in the thymus and bone marrow.

[0074] Human c-Kit was detected in c-KIT heterozygous precursors using an anti-c-Kit antibody at levels similar to those of mouse c-Kit in WT mice. Similar proportions of lineage-negative cells, multiple precursors, and LSKs were observed in c-KIT heterozygous and WT mice.

[0075] The cell numbers were slightly lower in the thymus (Fig. 5A) and bone marrow (Fig. 5B) of c-KIT homozygous mice compared with those of WT mice. No differences were observed between WT and c-KIT homozygous mice in terms of survival rate and proportion of T / B cell precursors in the thymus and bone marrow (Fig. 5C).

[0076] Example 4. Testing of therapeutic agents in transgenic mice Stem cell factor (SCF) signaling via c-Kit (CD117) plays a key role in the differentiation and survival of mast cells. Inhibition of this pathway has the potential to treat mast cell-mediated disorders. In this example, we tested whether burquilimab, a humanized aglycosylated monoclonal antibody against CD117, can inhibit SCF signaling and deplete human mast cells in the transgenic mouse model prepared in Example 3.

[0077] Methods: As tested in Example 3, a mouse model expressed chimeric CD117 (hmCD117). Passive systemic anaphylaxis (PSA) was induced by IgE. The pharmacokinetics, pharmacodynamics, and effect of burikilimab on PSA response were evaluated in hmCD117 mice.

[0078] Results: Chimeric hmCD117 was responsive to mouse SCF and recognized by burikilimab. Treatment of hmCD117 mice with burikilimab (5, 10, and 25 mg / kg IV) caused a transient, moderate decrease in peripheral blood cell counts and transient depletion of CD117-expressing hematopoietic stem cells. The pharmacokinetic clearance of burikilimab in serum was dose-dependent in a nonlinear manner. Burikilimab treatment at 5 mg / kg, three times weekly for 3 weeks, followed by IgE-induced PSA challenge, resulted in a reduction in anaphylactic responses. Notably, treatment with a single 25 mg / kg dose of burikilimab 2 weeks before PSA challenge completely prevented anaphylactic responses, suggesting that a single high dose of burikilimab effectively blocked PSA responses in hmCD117 mice (Figure 6). * * *

[0079] The present disclosure should not be limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the disclosure; any compositions or methods that are functionally equivalent are within the scope of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the disclosed methods and compositions without departing from the spirit or scope of the disclosure. Therefore, the present disclosure is intended to cover the modifications and variations of the present disclosure provided they come within the scope of the appended claims and their equivalents.

[0080] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference herein.

Claims

1. Transgenic mouse cells comprising nucleic acids encoding a chimeric c-KIT protein, wherein the chimeric c-KIT protein comprises a mouse c-KIT signal peptide, a human c-KIT extracellular domain, a mouse c-KIT transmembrane domain, and a mouse c-KIT intracellular domain.

2. The transgenic mouse cell according to claim 1, wherein the human c-KIT extracellular domain contains a GNNK sequence.

3. The transgenic mouse cell according to claim 2, wherein the human c-KIT extracellular domain comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:

10.

4. The transgenic mouse cell according to claim 2, wherein the human c-KIT extracellular domain comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:

10.

5. The transgenic mouse cell according to claim 1, wherein the nucleic acid comprises an exogenous portion and an endogenous portion.

6. The transgenic mouse cell according to claim 5, wherein the exogenous portion of the nucleic acid includes coding sequences relating to the human c-KIT extracellular domain, the mouse c-KIT transmembrane domain, and the mouse c-KIT intracellular domain.

7. The transgenic mouse cell according to claim 6, wherein the exogenous portion is inserted into the mouse genome at a locus within exon 2 of the mouse c-KIT gene.

8. The transgenic mouse cell according to claim 7, wherein the gene locus is located 3' to the last codon of the endogenous mouse c-KIT signal peptide.

9. The transgenic mouse cell according to claim 6, wherein the exogenous portion further comprises a stop codon.

10. The transgenic mouse cells according to claim 7, wherein the insertion is homozygous.

11. The cells are transgenic mouse cells according to claim 1, which are present in transgenic mice in vivo.

12. A transgenic mouse comprising transgenic mouse cells according to any one of claims 1 to 11.

13. A method for testing the activity of an inhibitor against human c-KIT, comprising: contacting a candidate inhibitor with transgenic mouse cells described in any one of claims 1 to 11; and measuring c-KIT signaling in the cells.

14. A method for testing the activity of an inhibitor against human c-KIT, comprising administering a candidate inhibitor to a transgenic mouse according to claim 12, and measuring c-KIT signaling in the mouse.

15. The method according to claim 13, wherein the candidate inhibitor is an anti-c-KIT antibody.

16. The method according to claim 14, wherein the candidate inhibitor is an anti-c-KIT antibody.

17. The method according to claim 13, wherein the c-KIT signaling pathway is measured in conjunction with cell proliferation.

18. The method according to claim 14, wherein the c-KIT signaling is measured together with cell proliferation.

19. A method for preparing transgenic mouse cells, comprising introducing into target mouse cells a construct comprising (i) a coding sequence encoding a partial chimeric protein comprising a human c-KIT extracellular domain, a mouse c-KIT transmembrane domain, and a mouse c-KIT intracellular domain, and (ii) a flanking sequence that enables the recombination and incorporation of the coding sequence into a locus within the exon of the endogenous c-KIT gene in the mouse genome.

20. The method according to claim 19, wherein the exon is exon 2.

21. The method according to claim 19, wherein the transgenic mouse cells express a chimeric protein comprising a mouse c-KIT signal peptide, the human c-KIT extracellular domain, the mouse c-KIT transmembrane domain, and the mouse c-KIT intracellular domain.

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