Transgenic animals expressing chimeric c-kit proteins

Abstract

The present disclosure describes methods of producing a transgenic cell or mouse expressing a chimeric c-KIT protein capable of expression on a mouse cell and binding to mouse stem cell factor (SCF), the binding being inhibited by an anti-human c-KIT inhibitor. These transgenic cells and mice can be used to test candidate anti-human c-KIT inhibitors.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 371,489, filed on August 15, 2022, under 35 U.S.C. §119(e), the contents of which are hereby incorporated by reference in their entirety.

[0003] Reference to an electronic sequence listing

[0004] The contents of the electronic sequence listing (350546.xml, 18,701 bytes in size, and created on August 15, 2023) are incorporated herein by reference in their entirety. Background Art

[0005] Tyrosine-protein kinase KIT is also known as c-KIT, CD117 (cluster of differentiation 117) or SCFR (mast / stem cell growth factor receptor). C-KIT 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) and forms a dimer, thereby activating its intrinsic tyrosine kinase activity, which in turn phosphorylates and activates signal transduction molecules that propagate signals in cells.

[0006] Anti-c-KIT antibodies are being developed that can be used to inhibit c-KIT-mediated signaling, resulting in ablation of hematopoietic stem cells in a subject. Such ablation helps ensure the efficacy of subsequent hematopoietic stem cell engraftment. Animal models expressing human c-KIT protein can be used to facilitate the development of such antibodies. However, the introduction of cDNA encoding human c-KIT protein into animals may not be successful. For example, human c-KIT protein may not be expressed in the desired cells, correctly localized, or correctly post-translationally modified. In addition, human c-KIT protein may not trigger the correct downstream signaling in animal cells when activated.

[0007] The human c-KIT protein has an extracellular domain (ECD), a transmembrane domain, and an intracellular domain (ICD). The extracellular domain has five Ig-like domains (D1-D5) involved in ligand binding and homodimerization. Protein processing of c-KIT includes N-linked glycosylation and phosphorylation on tyrosine and serine residues. When SCF dimers bind to c-KIT, the binding induces c-KIT dimerization, conformational changes in both ECD and ICD, ICD trans-phosphorylation, and binding of various partners.

[0008] There are also significant challenges in the design of human-animal chimeric c-KIT proteins. For example, human and mouse c-KIT proteins share 90% sequence similarity. However, protein glycosylation and conformational changes of both the ECD and ICD required for activation may be impaired in chimeric proteins. In addition, it is unclear how chimeric proteins will function in cells with different endogenous c-KIT expression levels to induce cell proliferation and how they will respond to mouse SCF. Summary of the invention

[0009] The present disclosure describes methods for generating transgenic cells or mice expressing a chimeric c-KIT protein that can be expressed on mouse cells and bind 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.

[0010] According to one embodiment of the present disclosure, a transgenic mouse cell comprising a nucleic acid encoding a chimeric c-KIT protein is provided, 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.

[0011] In some embodiments, the human c-KIT extracellular domain comprises a GNNK sequence. In some embodiments, the human c-KIT extracellular domain comprises an amino acid sequence of SEQ ID NO: 7, 8, 9, or 10, or an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NO: 7, 8, 9, or 10. In some embodiments, the human c-KIT extracellular domain comprises an amino acid sequence of SEQ ID NO: 7, 8, 9, or 10.

[0012] 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 a coding sequence 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 mouse genome at a locus within exon 2 of the mouse c-KIT gene. In some embodiments, the locus is located 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 a transgenic mouse.

[0013] In another embodiment, a method of testing the activity of an inhibitor against human c-KIT is also provided, the method comprising contacting a candidate inhibitor with a transgenic mouse cell of the present disclosure and measuring c-KIT signaling in the cell.

[0014] Also provided is a transgenic mouse comprising the transgenic mouse cell of the present disclosure. Also provided is a method of testing the activity of an inhibitor against human c-KIT, the method comprising administering a candidate inhibitor to a transgenic mouse and measuring c-KIT signaling in the mouse.

[0015] In some embodiments, the candidate inhibitor is an anti-c-KIT antibody.In some embodiments, cell proliferation is used to measure c-KIT signaling.

[0016] Another embodiment provides a method for preparing a transgenic mouse cell, the method 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 to enable the coding sequence to be recombinantly integrated into a locus within an exon of an endogenous c-KIT gene in the mouse genome. In some embodiments, the exon is exon 2.

[0017] In some embodiments, the transgenic cell expresses 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 DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The structures of expression vectors tested for functional validation are exemplified.

[0019] Figure 2 Shown are stem cell factor (SCF)-mediated survival and proliferation in the presence of mouse or human SCF.

[0020] Figure 3 A schematic diagram of the c-Kit expression vector used for splicing validation is presented.

[0021] FIG. 4A to FIG. 4B The scheme of humanized c-Kit cDNA (hEC-mTM-mIC) (A) and in-frame insertion with the mouse signal peptide after removal of the neo sequence (B) is illustrated.

[0022] FIG. 5A to FIG. 5C Observations on c-Kit expression on T cell progenitors in the thymus (A), B cell progenitors in the bone marrow (B), and bone marrow HSPCs (C) in transgenic mice are presented.

[0023] Figure 6Briquilimab is shown to prevent IgE-induced PSA in hmCD117 mice. HmCD117 mice were treated with briquilimab (5 mg / kg, 3 times per week for three weeks) or bolus (25 mg / kg, two weeks) before sensitization with anti-DNP IgE and challenge with DNP-HSA 24 hours later. Data represent mean ± SEM. Statistical analysis was performed by comparing the group means from 0 min to 60 min. **p<0.01, Welch's t test. DETAILED DESCRIPTION

[0024] definition

[0025] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "antibody" is understood to mean one or more antibodies. Thus, the terms "a" (or "a"), "one or more", and "at least one" can be used interchangeably herein.

[0026] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptide", and refers to a molecule consisting of monomers (amino acids) linearly connected by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, and does not relate to the specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to one or more chains of two or more amino acids are included in the definition of "polypeptide", and the term "polypeptide" can be used in place of any of these terms, or can be used interchangeably with them. The term "polypeptide" is also intended to refer to the product of post-expression modification of the polypeptide, which post-expression modifications include but are not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage or modification by non-naturally occurring amino acids. The polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It can be produced in any way, including by chemical synthesis.

[0027] As used herein, the term "isolated" refers to molecules separated from other DNA or RNA present in the natural source of the macromolecule, respectively. 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 technology, or nucleic acids or peptides that are substantially free of chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acid" is intended to include nucleic acid fragments that are not naturally present as fragments and that will not be found in the natural state. The term "isolated" is also used herein to refer to cells or polypeptides separated from other cell proteins or tissues. The isolated polypeptide is intended to cover both purified polypeptides and recombinant polypeptides.

[0028] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The antibody can be a complete antibody and any antigen-binding fragment or single chain thereof. Therefore, the term "antibody" includes any protein or peptide containing molecule that contains at least a portion of an immunoglobulin molecule that has biological activity that binds to an antigen. Such examples include, but are not limited to, a heavy or light chain complementary determining region (CDR) or a 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.

[0029] As used herein, the term "antibody fragment" or "antigen binding fragment" is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of the structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. 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.

[0030] By "specific binding" or "specific for..." is generally meant that an antibody binds to an epitope through its antigen binding domain, and that binding requires a certain complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" an epitope when it binds to it through its antigen binding domain more easily than it binds 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 be more specific for a given epitope than antibody "B," or antibody "A" may be said to bind to epitope "C" with a higher specificity than it does to a related epitope "D."

[0031] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA or mRNA) used as a template for synthesizing other polymers and macromolecules with a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence and the resulting biological properties in a biological process. Therefore, in a cell or other biological system, if the transcription and translation of the mRNA corresponding to a gene produces a protein, the gene encodes the protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence table) and the non-coding strand (used as a template for transcription of a gene or cDNA) can be referred to as encoding a protein or other product of the gene or cDNA.

[0032] As used herein, "endogenous" refers to any material that originates from or is produced within an organism, cell, tissue, or system.

[0033] As used herein, the term "exogenous" refers to any material introduced from outside of or generated outside of an organism, cell, tissue, or system.

[0034] As used herein, the term "expansion" refers to an increase in number, such as an increase in the number of T cells. In one embodiment, the number of T cells expanded ex vivo is increased relative to the number initially present in the culture. In another embodiment, the number of T cells expanded ex vivo is increased relative to other cell types in the culture. As used herein, the term "ex vivo" refers to cells that have been removed from a living organism (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0035] The term "expression" as used herein is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0036] "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by a 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) incorporating a recombinant polynucleotide.

[0037] As used herein, "homologous" refers to the subunit sequence identity between two polymeric molecules, for example, between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When the subunit position in the two molecules is occupied by the same monomer subunit; for example, 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 direct 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 of ten subunits in length) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10 positions) are matched or homologous, then the two sequences are 90% homologous.

[0038] "Identity" as used herein refers to the subunit sequence identity between two polymeric molecules, in particular between two amino acid molecules, such as between two polypeptide molecules. When two amino acid sequences have the same residue at the same position; for example, if the position in each of the two polypeptide molecules is occupied by arginine, then they are identical at that position. The degree to which two amino acid sequences have the same residue at the same position in the identity or alignment is usually 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 polymer of ten amino acids in length) are identical, then the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10 positions) are matched or identical, then the two amino acid sequences are 90% identical.

[0039] As used herein, the term "transfection" or "transformation" or "transduction" refers to a process for transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. Cells include the primary subject cell and its progeny.

[0040] 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.

[0041] The term "transgenic animal" refers to a non-human animal, typically a mammal, that has a non-endogenous (i.e., heterologous) nucleic acid sequence present as an extrachromosomal element in a portion of its cells or stably integrated into its germline DNA (i.e., in the genomic sequence of most or all of its cells), such as a transgenic mouse. The heterologous nucleic acid is introduced into the germline of such a transgenic animal by genetic manipulation of, for example, an embryo or embryonic stem cell of the host animal.

[0042] The term "humanized mouse" refers to a mouse that transgenically expresses one or more human genes or a chimeric gene that expresses at least a portion of a human protein.

[0043] As used herein, the phrase "under transcriptional control" or "operably linked" means that the promoter is in the correct location and orientation relative to the polynucleotide to control initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0044] "Vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphipathic compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted as including non-plasmids and non-viral compounds that promote the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, etc.

[0045] Range: Throughout this disclosure, various aspects of the invention may be presented in range format. It should be understood that descriptions in range format are merely for convenience and brevity and should not be construed as hard limitations on the scope of the invention. Therefore, descriptions of ranges should be considered to specifically disclose all possible subranges and individual values ​​within the range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and individual numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies no matter how large the range is.

[0046] Transgenic mice and mouse cells

[0047] Inhibitors of human c-KIT have therapeutic value. For example, anti-c-KIT antibodies can be used to deplete hematopoietic stem cells (HSCs) from the bone marrow of a subject. Depletion of diseased HSCs can be used to treat diseases or conditions such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), thereby increasing the relative percentage of healthy HSCs.

[0048] Anti-c-KIT antibodies are 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).

[0049] In certain embodiments, the anti-c-KIT antibody binds to the extracellular region of c-KIT, amino acids 26-524 of SEQ ID NO: 1. Human c-KIT has eight isoforms, namely 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).

[0050] These isoforms are highly homologous. However, four of them (isoforms 1, 3, 4 and 7, Table 1) include a -GNNK- sequence (e.g., amino acid residues 510-514 of SEQ ID NO: 1), while the other isoforms do not. Isoforms 1, 3, 4 and 7 are referred to as "GNNK" isoforms.

[0051] Table 1. Sequences

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] The inventors set out to generate mouse cells expressing a chimeric c-KIT protein having a mouse signal peptide (mSP), a human extracellular portion (hEC), a mouse transmembrane domain (mTM), and a mouse intracellular portion (mIC). Initial tests showed that such chimeric proteins were able to bind human SCF (hSCF) and mediate proliferation and survival of transfected cells (Example 1). This was surprising, although the chimeric proteins were indeed not as effective as the human counterparts in mediating SCF signaling, as there was no evidence that the human extracellular domain would be able to conduct SCF signaling through the mouse transmembrane and intracellular domains.

[0058] In order to prepare transgenic cells with the hEC coding sequence integrated into the genome, the inventors designed a cDNA including a hEC-mTM-mIC portion with flanking sequences to enable the cDNA to be inserted into exon 2 of the mouse c-KIT genomic sequence. The mouse signal peptide coding portion ends within exon 2 (Table 1, SEQ ID NO: 12), so when the start codon of hEC is inserted in frame just after the last codon of mSP, this insertion enables the expression of the mSP-hEC-mTM-mIC fusion protein. In another surprising discovery, this insertion does not interrupt the splicing of exon 2, and the fusion protein is expressed as designed. Thus, transgenic mouse cells and animals were successfully prepared.

[0059] According to one embodiment of the present disclosure, a transgenic mouse cell comprising a nucleic acid encoding a chimeric c-KIT protein is provided, 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. 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.

[0060] In some embodiments, the human c-KIT extracellular domain (hEC) includes the extracellular domain of any known human c-KIT isoform (such as isoforms 1-8) or their biological equivalents. 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.

[0061] Some of the human c-KIT isoforms include a -GNNK- sequence in the extracellular domain (e.g., amino acid residues 510-514 of SEQ ID NO: 1), while other isoforms do not. In a preferred embodiment, the hEC includes the extracellular domain of one of the "GNNK" isoforms (isoforms 1, 3, 4, or 7). The complete protein sequences of isoforms 1, 3, 4, and 7 are provided in Table 1 as SEQ ID NOs: 1-4, and their extracellular domains are provided as SEQ ID NOs: 7-10.

[0062] 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.

[0063] In some embodiments, the c-KIT transmembrane domain (mTM) and the 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.

[0064] In some embodiments, the chimeric c-KIT is encoded by a nucleic acid that is integrated into the genome of the transgenic cell. The nucleic acid may include an exogenous portion and an endogenous portion. As tested, the 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.

[0065] 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 and the first codon of hEC is inserted immediately after the last codon of the mouse signal peptide (SEQ ID NO: 5) (nucleotides 6-8 of SEQ ID NO: 12 showing the exon 2 genomic sequence).

[0066] In an alternative embodiment, the insertion is at a locus within exon 2 but further downstream. This will allow some amino acids of the mouse extracellular domain to be included in the expressed chimeric protein. However, it is expected that the addition of small amounts of mouse amino acids will not affect the activity of the chimeric protein. In addition, given the homology between the mouse c-KIT protein and the human c-KIT protein, some human amino acids (N-terminal) may optionally be deleted from the cDNA to neutralize this effect when additional mouse extracellular amino acids are included.

[0067] In another alternative embodiment, the insertion is at a locus located within exon 2 but further upstream than 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 may also include a truncated portion so that the expressed chimeric protein is intact.

[0068] Likewise, in another embodiment, the insertion is at a locus upstream of the last codon of the signal peptide within exon 1. To compensate for the partial truncation of the C-terminus of the mouse signal peptide, the cDNA may also include a truncated portion, thereby rendering the expressed chimeric protein intact.

[0069] In some embodiments, cDNA also includes a stop codon. In some embodiments, cDNA also includes a poly (A) signal. In some embodiments, cDNA also includes a selection marker, such as a coding sequence of a neomycin resistance protein or a fluorescent protein. Such markers can be conducive to the selection of successfully integrated cells. When the selection is completed, known techniques (e.g., Cre-Lox recombination) can be used to excise them from the genome. In some embodiments, cDNA is codon optimized. In some embodiments, insertion is heterozygous. In some embodiments, insertion is homozygous.

[0070] The 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 construction methods can be used to produce DNA that can be used herein. For example, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2nd edition, 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.

[0071] If the target cell is a stem cell, such as an embryonic stem cell, a transgenic mouse can be generated. 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 the chimeric protein. In some embodiments, the transgenic cell is homozygous.

[0072] Methods of using the transgenic cells or mice disclosed herein are also provided. In one embodiment, a method of testing an inhibitor for activity against human c-KIT is provided, which requires contacting a candidate inhibitor with a transgenic mouse cell as described herein and measuring c-KIT signaling in the cell. A method of testing an inhibitor for activity against human c-KIT is also provided, which requires 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.

[0073] c-KIT signaling can be measured using methods known in the art. C-KIT is a cytokine receptor that binds to stem cell factor (SCF). Binding activates the intrinsic tyrosine kinase activity of c-KIT, which in turn phosphorylates and activates signal transduction molecules that propagate signals in cells. Signaling achieved by KIT plays a role in cell survival, proliferation, and differentiation. In some embodiments, inhibitor activity can be measured using cell proliferation or differentiation, as shown in the accompanying experimental examples.

[0074] Example

[0075] Example 1: Generation and testing of transgenic Ba / F3 cells

[0076] This example tests the effect of exogenous c-KIT cDNA expression on the proliferation and survival of Ba / F3 cells.

[0077] Expression vectors including (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 including 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 included a sequence for expressing GFP and a neomycin resistance gene ( Figure 1 ). Each vector was transfected into Ba / F3 cells, and the successfully transfected cells were harvested.

[0078] Stably transfected Ba / F3 cells were starved overnight and stimulated with various concentrations of mouse or human SCF (stem cell factor) for 4 days. Cell survival was assessed by DAPI exclusion flow cytometry. Cell proliferation was assessed by EdU flow cytometry.

[0079] like Figure 2 As shown, mouse SCF (mSCF) stimulation induced proliferation and survival of all vector-transfected mouse cells, demonstrating that both human and chimeric c-KIT proteins are capable of mediating mSCF binding and signaling. At the same time, human SCF (hSCF) stimulation induced proliferation and survival of Ba / F3 cells transfected with human or chimeric c-KIT expression vectors. However, it was observed that the chimeric c-KIT protein was not as effective as its human counterpart in mediating SCF signaling.

[0080] Example 2. In vitro splicing assay

[0081] It has been proposed that it is preferred to insert a chimeric c-KIT cDNA into the endogenous c-KIT locus of the mouse genome in the target cell. However, it is suspected that such insertion will result in modification of exonic splicing regulatory sequences, thereby causing disruption to the inserted cDNA. This example tests whether insertion of a chimeric cDNA into the c-KIT locus in the mouse genome will result in altered splicing.

[0082] The cDNA encodes a chimeric c-KIT protein (hEC-mTM-mIC) that includes the human extracellular portion (hEC) and the mouse transmembrane domain (mTM) / intracellular portion (mIC) in addition to a stop codon and poly(A) sequence ( Figure 3 ). A slightly different version of this cDNA was also prepared, which was codon optimized. Homologous recombination was designed so that the cDNA was inserted in frame into exon 2 of the mouse c-KIT gene and followed by the mouse signal peptide (mSP). Therefore, if exon 1 and exon 2 are correctly spliced, the transcript will include the desired mSP-hEC-mTM-mIC chimeric protein.

[0083] The constructs were transfected into 3T3 mouse cells or embryonic stem (ES) cells. 24 hours after transfection, the extracted RNA was reverse transcribed and cDNA was amplified. Gel electrophoresis using the amplified PCR products confirmed that correct splicing occurred in the transfected cells.

[0084] Example 3. Generation and testing of transgenic mice

[0085] Mouse ES cells with chimeric hEC-mTM-mIC cDNA stably integrated into their genome were prepared in this example using a procedure similar to that of Example 2. Transgenic ES cell clones can be implanted into blastocyst stage embryos to generate chimeras.

[0086] like Figure 4A As exemplified, in addition to hEC-mTM-mIC, the construct also includes a stop codon, poly(A), and a neomycin resistance gene (Neo). The hEC sequence is GNNK(+). The neomycin resistance gene is flanked by a pair of loxP sites. The neomycin resistance gene allows selection of transfected cells, which are then excised ( Figure 4B The insertion of the exogenous sequence was confirmed by PCR amplification using appropriate primers (e.g. Figure 4B exemplified in ).

[0087] In order to produce the highly mosaic male mice carrying the recombinant gene seat, ES cell clones are transfected and integrated in blastocysts. ES cells are derived from the inner cell mass of 3.5 day-old embryos (blastocyst stage). These cells are pluripotent, and therefore, when implanted in blastocyst stage embryos, can facilitate every kind of cell lineage, including germ layers. These blastocysts are implanted in pseudo-pregnant females, and are grown to full term. The mosaic rate in the offspring is then assessed by comparing the coat color markers.

[0088] Of the 115 pups born, 23 (20%) were homozygous (10 males and 13 females) and all had white spots on their black fur; 65 (57%) were heterozygous (29 males and 36 females); of these, 34 had white spots (15 males and 19 females), 31 were black (14 males and 17 females); and 27 (23%) were wild type (17 males and 10 females) and all were black. No differences were observed between WT and c-KIT heterozygous mice with respect to cellularity, viability, and T / B cell progenitor ratios in the thymus and bone marrow.

[0089] Human c-Kit was detected in c-KIT heterozygous progenitors using anti-c-Kit antibodies and at levels similar to mouse c-Kit on WT mice. Similar proportions of lineage-negative cells, multiple progenitors, and LSKs were observed in c-KIT heterozygous and WT mice.

[0090] Compared with WT mice, the thymus of c-KIT homozygous mice ( Figure 5A ) and bone marrow ( Figure 5B ) were slightly lower in number. Regarding the activity and T / B cell progenitor ratio in the thymus and bone marrow ( Figure 5C ), no difference was observed between WT and c-KIT homozygous mice.

[0091] Example 4. Testing of therapeutic agents in transgenic mice

[0092] Stem cell factor (SCF) signaling through c-Kit (CD117) plays a key role in mast cell differentiation and survival. Inhibition of this pathway has the potential to treat mast cell-mediated disorders. This example tests whether bricklimab, a humanized, non-glycosylated monoclonal antibody against CD117, can inhibit SCF signaling and deplete human mast cells in a transgenic mouse model as prepared in Example 3.

[0093] Methods: Mouse models expressing chimeric CD117 (hmCD117) were tested as in Example 3. Passive systemic anaphylaxis (PSA) was induced by IgE. The pharmacokinetics, pharmacodynamics, and effects on PSA responses of bricklimab were evaluated in hmCD117 mice.

[0094] Results: Chimeric hmCD117 was responsive to mouse SCF and recognized by bricklimab. Treatment with bricklimab (5 mg / kg, 10 mg / kg and 25 mg / kg, IV) in hmCD117 mice resulted in a transient moderate decrease in peripheral blood counts and a transient depletion of hematopoietic stem cells expressing CD117. The pharmacokinetic clearance of bricklimab in serum was dose-dependent in a nonlinear manner. Treatment with bricklimab at 5 mg / kg, 3 times per week for three weeks followed by an IgE-induced PSA challenge resulted in a reduction in allergic reactions. Notably, treatment with bricklimab at a single 25 mg / kg dose two weeks prior to PSA challenge completely prevented allergic reactions, indicating that a single high dose of bricklimab effectively blocked PSA responses in hmCD117 mice ( Figure 6 ).

[0095] ***

[0096] The scope of the present disclosure is not limited by the specific embodiments described, which are intended only to serve as illustrations of various aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations may be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is intended to encompass modifications and variations of the present disclosure as long as they fall within the scope of the appended claims and their equivalents.

[0097] 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.

Claims

1. A transgenic mouse cell comprising a nucleic acid 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 comprises a GNNK sequence.

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

5. A transgenic mouse cell according to any preceding claim, wherein the nucleic acid comprises an exogenous portion and an endogenous portion.

6. The transgenic mouse cell of claim 5, wherein the exogenous portion of the nucleic acid comprises the coding sequence of the human c-KIT extracellular domain, the mouse c-KIT transmembrane domain, and the mouse c-KIT intracellular domain.

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

8. The transgenic mouse cell of 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 of any one of claims 6 to 8, wherein the exogenous portion further comprises a stop codon.

10. The transgenic mouse cell of any one of claims 7 to 9, wherein the insertion is homozygous.

11. A transgenic mouse cell according to any preceding claim, wherein the cell is in a transgenic mouse.

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

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

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

15. The method of claim 13 or 14, wherein the candidate inhibitor is an anti-c-KIT antibody.

16. The method of any one of claims 13 to 15, wherein the c-KIT signaling is measured using cell proliferation.

17. A method for preparing a transgenic mouse cell, the method 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 to enable the coding sequence to be recombinantly integrated into a locus within an exon of an endogenous c-KIT gene in the mouse genome. The method of claim 17 , wherein the exon is exon 2.

19. The method of claim 17 or 18, wherein the transgenic 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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