阅读说明：本技术 具有人源化FC-γ受体的非人动物 (Non-human animals with humanized FC-gamma receptors ) 是由 A·J·莫菲 L·麦克唐纳 C·古雷尔 K·A·梅格尔 N·图 于 2015-04-08 设计创作，主要内容包括：本发明提供了经基因修饰的小鼠及用于制备和使用所述小鼠的方法和组合物,其中所述基因修饰包括FcγRI蛋白的人源化。(The present invention provides genetically modified mice, including humanization of Fc γ RI proteins, and methods and compositions for making and using the same.)

1. A nucleic acid encoding a functional fcyria chain comprising an extracellular portion of a human fcyria chain and an intracellular portion of a mouse fcyria chain.

2. The nucleic acid of claim 1, wherein the nucleic acid comprises exons 3, 4, and 5 of a human fcyri gene.

3. The nucleic acid of claim 1, wherein the nucleic acid further comprises one or more human 5' untranslated regions flanking human exon 1.

4. The nucleic acid of claim 1, wherein the extracellular portion of the human fcyri a chain comprises one or more of EC1, EC2, and EC 3.

5. The nucleic acid of claim 1, wherein the Fc γ RI α chain comprises an amino acid sequence having at least 90% identity to SEQ ID No. 5.

6. The nucleic acid of claim 1, wherein the FcyRI protein comprises an FcyRI a chain amino acid sequence identical to SEQ ID No. 5.

7. The nucleic acid of claim 1, wherein the nucleic acid comprises exon 6 of the mouse fcyri gene.

8. The nucleic acid of claim 1, wherein the intracellular portion of the mouse fcyri a chain comprises all or a portion of the mouse fcyri a chain cytoplasmic domain.

9. The nucleic acid of claim 1, wherein the nucleic acid is a vector.

10. The nucleic acid of claim 9, wherein the vector is a targeting vector.

11. A functional fcyri a chain comprising an extracellular portion of a human fcyri a chain and an intracellular portion of a mouse fcyri a chain.

12. The functional fcyri a chain of claim 11, wherein the extracellular portion of the human fcyri a chain comprises one or more of EC1, EC2, and EC 3.

13. The functional Fc γ RI α chain of claim 11, comprising an amino acid sequence having at least 90% identity to SEQ ID No. 5.

14. The functional fcyri a chain of claim 11, wherein the intracellular portion of the mouse fcyri a chain comprises all or part of the mouse fcyri a chain cytoplasmic domain.

15. The functional Fc γ RI α chain of claim 11, comprising the amino acid sequence of SEQ ID NO 5.

Background

Fc receptors (fcrs) are proteins that are present on the cell surface of the immune system and can perform multiple functions of the immune system in the body of a mammal. FcRs are present on a variety of cells in a variety of types and mediate a variety of immune functions, such as binding to antibodies attached to infected cells or invading pathogens, stimulation of phagocytes or cytotoxic cells by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC) to destroy microorganisms or infected cells.

ADCC is the process by which effector cells of the immune system lyse target cells to which antibodies bind. This process relies on prior exposure to foreign antigens or cells to generate an antibody response. ADCC can be mediated by effector cells, such as Natural Killer (NK) cells, by binding fcrs expressed on the surface of the effector cells to the Fc portion of the antibody, which in turn binds to a foreign antigen or cell. High affinity Fc γ R1 receptor signaling plays an important role in immune system regulation and effector cell function.

Disclosure of Invention

The present invention encompasses the recognition that: it is desirable to engineer non-human animals, such as mice, to express human or hybrid Fc γ RI proteins so that experiments can be performed on human immune effector responses that cannot be developed in humans.

The present invention also encompasses the recognition that: it is desirable to replace the endogenous mouse Fc γ RI gene with a humanized (human or hybrid) Fc γ RI gene.

In some embodiments, the invention provides a mouse that expresses an fcyri protein comprising an extracellular portion of a human fcyri a chain and an intracellular portion of a mouse fcyri a chain. In some embodiments, the extracellular portion of the human Fc γ RI α chain comprises an EC1 domain, an EC2 domain, an EC3 domain, or a combination thereof.

In some embodiments, the EC1 domain is encoded by an exon that is at least 50%, 70%, 85%, 90%, or 95% identical to exon 3 of SEQ ID No. 3.

In some embodiments, the EC2 domain is encoded by an exon that is at least 50%, 70%, 85%, 90%, or 95% identical to exon 4 of SEQ ID No. 3.

In some embodiments, the EC3 domain is encoded by an exon that is at least 50%, 70%, 85%, 90%, or 95% identical to exon 5 of SEQ ID No. 3.

In some embodiments, the invention provides a mouse that expresses an fcyri protein comprising an extracellular portion of a human fcyri a chain and an intracellular portion of a mouse fcyri a chain, wherein the mouse is not detectably expressing a full-length mouse fcyri a chain. In some embodiments, the intracellular portion of the fcyri α chain comprises all or part of the mouse fcyri α chain cytoplasmic domain. In some embodiments, the mouse further expresses an Fc γ RI α chain comprising all or a portion of a transmembrane domain of a mouse Fc γ RI α chain.

In some embodiments, the invention provides mice expressing an Fc γ RI α chain amino acid sequence having at least 70%, 85%, 90%, or 95% identity to SEQ ID No. 5. In some embodiments, the human or hybrid Fc γ RI protein is detectably expressed on monocytes, macrophages, neutrophils, dendritic cells, and/or combinations thereof.

In some embodiments, administration of murine granulocyte colony stimulating factor (mG-CSF) increases human fcyri protein levels. In some embodiments, administration of murine granulocyte colony stimulating factor (mG-CSF) does not increase mouse fcyri protein levels in monocytes, neutrophils, or dendritic cells.

In some embodiments, the invention provides a mouse that expresses an fcyri gene comprising one or more exons of a human fcyri gene operably linked to one or more exons of a mouse fcyri gene. In some embodiments, an exon of the human fcyri gene encodes one or more extracellular portions of a human fcyri protein. In some embodiments, an exon of the mouse fcyri gene encodes one or more intracellular portions of the mouse fcyri protein. In some embodiments, the exon of the human fcyri is selected from the group consisting of exons 3, 4, 5, and combinations thereof.

In some embodiments, the intracellular portion of the mouse fcyri is operably linked to one or more mouse intracellular signaling cascades.

In some embodiments, the invention provides a mouse expressing a human Fc γ RI, wherein germ line cells of the mouse lack a functional mouse Fc γ RI gene. In some embodiments, the invention provides a mouse expressing a human fcyri, wherein germ line cells of the mouse lack any mouse fcyri gene.

In some embodiments, the invention provides embryonic stem cells whose genome comprises an fcyri gene that encodes an extracellular portion of a human fcyri protein and an intracellular portion of a mouse fcyri protein. In some embodiments, the fcyri gene comprises exons 3, 4, and 5 of a human fcyri gene. In some embodiments, the fcyri gene further comprises one or more human 5' untranslated regions flanking human exon 1. In some embodiments, the extracellular portion of the human fcyri protein comprises one or more of EC1, EC2, and EC 3.

In some embodiments, the invention provides embryonic stem cells whose genome comprises an fcyri alpha chain amino acid sequence having at least 70%, 85%, 90%, or 95% identity to SEQ ID No. 5.

In some embodiments, the embryonic stem cell comprises an fcyri gene having amino acid residues of exon 6 of the mouse fcyri gene. In some embodiments, the intracellular portion of the mouse fcyri protein comprises all or part of the mouse fcyri protein cytoplasmic domain.

In some embodiments, the invention provides embryonic stem cells comprising a human fcyri gene, wherein the human fcyri gene is located at an endogenous fcyri locus that occurs in the genome of a mouse as it occurs in nature.

In some embodiments, the invention provides mouse embryos produced from the embryonic stem cells described herein.

In some embodiments, the invention provides the use of a mouse embryonic stem cell as described herein for making a transgenic mouse.

In some embodiments, a method of making a mouse expressing an fcyri protein comprising an extracellular portion of a human fcyri protein and an intracellular portion of a mouse fcyri protein is disclosed, the method comprising the steps of: (a) obtaining mouse embryonic stem cells; (b) replacing in an embryonic cell an endogenous mouse fcyri gene with a genomic fragment comprising a nucleic acid molecule encoding a portion of a human fcyri protein having a human extracellular region; and (c) producing a mouse using the embryonic cells of (b).

In some embodiments, the invention provides genomic fragments comprising a nucleic acid molecule encoding a portion of a human fcyri protein having a human extracellular region, and a nucleic acid molecule encoding an intracellular portion of a mouse fcyri protein.

As used in this application, the terms "about" and "approximately" are used as synonyms. Any number used in this application, whether modified by about/about, is intended to encompass any normal fluctuation recognized by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the invention will be apparent from the description which follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the following detailed description.

Drawings

The drawings included herein are for illustration purposes only and are not limiting.

Figure 1 shows genotyping of Fc γ RI mouse alleles for experimental mice and wild-type controls.

Figure 2 shows the relative copy number of the human Fc γ RI gene of experimental mice and wild-type controls.

FIG. 3 shows transformed data based on gene copy number from Het (+/-) mice with one copy of human Fc γ RI calibrated with an average Δ Ct.

Figure 4 shows Fc γ RI receptor expression in cells obtained from human donors.

Figure 5 shows a schematic of the human, mouse and humanized Fc γ RI genes (not drawn to scale).

Figure 6 shows a schematic (not to scale) of the MAID6074 cartridge preparation process.

Fig. 7 shows that mice with the humanized Fc γ RI gene exhibited normal cell frequency in spleen and blood.

Figure 8 shows a myeloid-lineage spleen cell population in mice with a humanized Fc γ RI gene.

Figure 9 shows the loss of murine Fc γ RI expression on macrophages from the spleen of mice with humanized Fc γ RI genes.

Figure 10 shows the acquisition of human fcyri expression on monocytes from the spleen of mice with humanized fcyri genes.

Figure 11 shows the gating strategy during FACS analysis of macrophages purified from the peritoneal cavity of mice with endogenous mouse Fc γ RI gene (MAID6074 WT) and humanized Fc γ RI gene homozygous mice (MAID6074 HO).

Figure 12 shows the expression of human Fc γ RI and mouse Fc γ RI in macrophages from the abdominal cavity of mice with endogenous mouse Fc γ RI gene (MAID6074 WT) and humanized Fc γ RI gene homozygous mice (MAID6074 HO).

Figure 13 shows gating strategies during FACS analysis of myeloid-derived macrophages from mice with endogenous mouse Fc γ RI gene (MAID6074 WT) and humanized Fc γ RI gene homozygous mice (MAID6074 HO).

Figure 14 shows the expression of human Fc γ RI and mouse Fc γ RI in bone marrow-derived macrophages from mice with endogenous mouse Fc γ RI gene (MAID6074 WT) and humanized Fc γ RI gene homozygous mice (MAID6074 HO).

Figure 15 shows a gating strategy during FACS analysis of bone marrow-derived macrophages from mice with endogenous mouse Fc γ RI gene (control 75/25) and humanized Fc γ RI gene-heterozygous mice (MAID6074 HET).

Figure 16 shows the expression of human Fc γ RI and mouse Fc γ RI in bone marrow-derived macrophages from mice bearing the endogenous mouse Fc γ RI gene (control 75/25) and humanized Fc γ RI gene-heterozygous mice (MAID6074 HET).

Figure 17 shows the myeloid blood cell population of MAID6074 HO mice 48 hours after treatment with PBS and compared to MAID6074 WT.

Fig. 18 shows the myeloid lineage blood cell population of MAID6074 HO mice 48 hours after treatment with mG-CSF and compared to MAID6074 WT mice.

Figure 19 shows the absence of human Fc γ RI expression in blood of MAID6074 WT and MAID6074 HO mice 48 hours after treatment with PBS.

Figure 20 shows human Fc γ RI expression in blood of MAID6074 HO mice 48 hours after treatment with mG-CSF and compared to MAID6074 WT mice.

Figure 21 shows the absence of murine Fc γ RI expression in the blood of MAID6074 WT and MAID6074 HO mice 48 hours after treatment with PBS.

Figure 22 shows murine Fc γ RI expression in blood of MAID6074 WT 48 hours after treatment with mG-CSF and compared to MAID6074 HO mice.

Figure 23 shows the myeloid splenocytes population in MAID6074 HO 48 hours after treatment with PBS and compared to MAID6074 WT mice.

Figure 24 shows the myeloid lineage splenocytes of MAID6074 HO mice 48 hours after treatment with mG-CSF and compared to MAID6074 WT mice.

Figure 25 shows the absence of human Fc γ RI expression in splenic monocytes of MAID6074 HO and 6074WT mice 48 hours after treatment with PBS.

Figure 26 shows human Fc γ RI expression in the spleen of MAID6074 HO mice 48 hours after treatment with mG-CSF and compared to MAID6074 WT mice.

Figure 27 shows murine Fc γ RI expression in the spleen of MAID6074 WT mice 48 hours after treatment with PBS and comparison to MAID6074 HO mice.

Figure 28 shows murine Fc γ RI expression in the spleen of MAID6074 WT mice 48 hours after treatment with mG-CSF and compared to MAID6074 HO mice.

Figure 29 shows a summary of human Fc γ RI expression in cell populations of MAID6074 WT and MAID6074 HO mice after treatment with PBS or meg-CSF.

Figure 30 shows the up-regulation of human Fc γ RI mRNA induced by mG-CSF (normalized to mHPRT1) in blood and spleen of MAID6074 HO mice.

Figure 31 shows the up-regulation of human Fc γ RI mRNA induced by mG-CSF in blood and spleen of MAID6074 HO mice.

Figure 32 shows a schematic and exemplary strategy for humanization of mouse Fc γ RI.

Definition of

The invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be defined by the appended claims.

Unless otherwise defined, all terms and phrases used herein include the meaning that the term and phrase has been accorded to such term and phrase as is known in the art, unless the context clearly dictates otherwise, or is otherwise clear from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the specific methods and materials are now described.

The term "about" as used herein for one or more values of interest refers to a value that approximates the referenced value. In certain embodiments, the term "about" or "approximately" refers to a range of values that deviates by 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less by a percentage in either direction (greater than or less than) of the referenced value, unless otherwise specified or unless otherwise evident from the context (except where such number would exceed 100% of one possible value).

As used herein, the term "biologically active" refers to the characteristic that any factor (agent) is active in a biological system in vitro or in vivo (e.g., in vivo). For example, a factor is considered to be biologically active if it has a biological effect in an organism when present in that organism. In particular embodiments, if a protein or polypeptide is biologically active, the portion of the protein or polypeptide having at least one biological activity of the protein or polypeptide is generally referred to as a "biologically active" portion.

As used herein, the term "comparable" means that two or more factors, entities, situations, sets of conditions, etc., may be different from each other but sufficiently similar to allow comparisons to be made therebetween, whereby a reasonable conclusion can be drawn based on the observed differences or similarities. One of ordinary skill in the art will understand from the context how much degree of identity is required to make two or more such factors, entities, situations, sets of conditions, etc. considered comparable in any given instance.

The term "conservative" when used herein to describe a conservative amino acid substitution, refers to the replacement of an amino acid residue with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of interest of the protein, e.g., the ability of a receptor to bind to a ligand. Examples of the group of amino acids having side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic hydroxyl side chains, such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains, such as aspartic acid and glutamic acid; and sulfur-containing side chains, such as cysteine and methionine. Conservative amino acid substitution groups include, for example, valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, glutamic acid/aspartic acid, and asparagine/glutamine. In some embodiments, a conservative amino acid substitution may be any natural residue in a protein substituted with alanine, as used, for example, for alanine scanning mutagenesis. In some embodiments, conservative substitutions are made with positive values in the PAM250 log-likelihood matrix as disclosed in: gonnet et al, (1992) analytical Matching of the entity Protein Sequence Database, Science 256:1443-45(Gonnet et al, 1992, Exhaustive Matching of Entire Protein Sequence databases, Science 256, page 1443 and 1445), which is hereby incorporated by reference. In some embodiments, the permutation is a moderately conservative permutation, wherein the permutation has a non-negative value in the PAM250 log-likelihood matrix.

As used herein, the term "disruption" refers to the result of a homologous recombination event with a DNA molecule (e.g., with an endogenous homologous sequence such as a gene or locus). In some embodiments, disruption may effect or indicate an insertion, deletion, substitution, missense mutation, or frameshift of a DNA sequence, or any combination thereof. Insertion may include insertion of an entire gene or gene fragment (e.g., exon) that may not originate from an endogenous sequence. In some embodiments, the disruption can increase the expression and/or activity of a gene or gene product (e.g., a protein encoded by a gene). In some embodiments, the disruption can reduce the expression and/or activity of the gene or gene product. In some embodiments, the disruption can alter the sequence of the gene or the encoded gene product (e.g., the encoded protein). In some embodiments, the disruption can truncate or fragment the gene or encoded gene product (e.g., the encoded protein). In some embodiments, the extendible gene or the encoded gene product is disrupted; in some such embodiments, disruption can effect assembly of the fusion protein. In some embodiments, the disruption can affect the level of the gene or gene product, but not the activity of the gene or gene product. In some embodiments, the disruption can affect the activity of the gene or gene product, but not the level of the gene or gene product. In some embodiments, the disruption may not have a significant effect on the level of the gene or gene product. In some embodiments, disruption may not have a significant effect on the activity of the gene or gene product. In some embodiments, the disruption may not have a significant effect on the level or activity of the gene or gene product.

As used herein, the phrase "endogenous locus" or "endogenous gene" refers to a genetic locus that is present in a parent or reference organism prior to introduction of a disruption, deletion, substitution, alteration, or modification as described herein. In some embodiments, the endogenous locus has a sequence that occurs in nature. In some embodiments, the endogenous locus is wild-type. In some embodiments, the reference organism is a wild-type organism. In some embodiments, the reference organism is an engineered organism. In some embodiments, the reference organism is a laboratory-bred organism (whether wild-type or engineered).

As used herein, the phrase "endogenous promoter" refers to a promoter that is naturally associated with an endogenous gene, for example, in a wild-type organism.

As used herein, the term "Fc γ RI protein" refers to a high affinity immunoglobulin Fc receptor comprising an alpha chain with three extracellular domains, one transmembrane domain, and one intracellular domain.

For example, representative nucleotide and amino acid sequences of the mouse and human Fc γ RI α genes are provided in table 3. One of skill will recognize upon reading this disclosure that one or more endogenous fcyri receptor genes (or all) in the genome can be replaced with one or more heterologous fcyri genes (e.g., polymorphic variants, subtypes, or mutants, genes from another species, etc.).

As used herein, "Fc γ RI-expressing cell" refers to a cell that is capable of expressing Fc γ RI. In some embodiments, the fcyri-expressing cell is capable of expressing an fcyri receptor on its surface. In some embodiments, the Fc γ RI receptor is expressed on the surface of a cell in an amount sufficient to mediate cell-to-cell interactions via Fc γ RI protein expressed on the cell surface. Exemplary Fc γ RI expressing cells include lymphocytes, bone marrow cells, macrophages, neutrophils, and Natural Killer (NK) cells. Fc γ RI expressing cells modulate immune cell interactions and thereby modulate immune responses to various foreign antigens or pathogens. In some embodiments, the non-human animals of the invention exhibit immune cell modulation via humanized Fc γ RI receptors expressed on the surface of one or more cells of the non-human animal. As used herein, the term "heterologous" refers to an agent or entity from a different source. For example, when used to refer to a polypeptide, gene, or gene product or is present in a particular cell or organism, the term clarifies that the relevant polypeptide, gene, or gene product: 1) manual transformation is carried out by a person; 2) introduction into a cell or organism (or a precursor thereof) by human hand (e.g., via genetic engineering); and/or 3) is not naturally produced by, or does not naturally occur in, the relevant cell or organism (e.g., the relevant cell type or organism type).

As used herein, the term "host cell" refers to a cell into which a heterologous (e.g., exogenous) nucleic acid or protein has been introduced. The skilled artisan, upon reading this disclosure, will understand that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Since certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In some embodiments, the host cell is or includes a prokaryotic or eukaryotic cell. In general, a host cell is any cell suitable for receiving and/or producing a heterologous nucleic acid or protein, regardless of the kingdom of life to which the cell is assigned. Exemplary cells include those of prokaryotes and eukaryotes (unicellular or multicellular), bacterial cells (e.g., strains of E.coli, Bacillus species (Bacillus spp.), Streptomyces species (Streptomyces spp.), and the like), mycobacteria, and the likeBacillus cells, fungal cells, yeast cells (e.g., saccharomyces cerevisiae (s. cerevisiae), schizosaccharomyces pombe (s. pombe), pichia pastoris (p. pastoris), pichia methanolica (p. methanolica), etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni (Trichoplusia ni), etc.), non-human animal cells, human cells, or cell fusions such as hybridomas or tetra-source hybridomas. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the group consisting of: CHO (e.g., CHO K1, DXB-11CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney cells (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60 (e.g., BHK21), Jurkat, Daudi, A431 (epidermis), CV-1, U937, 3T3, L cells, C127 cells, SP2/0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived therefrom. In some embodiments, the cells comprise one or more viral genes, such as retinal cells expressing viral genes (e.g., per.c 6)^TMA cell). In some embodiments, the host cell is or comprises an isolated cell. In some embodiments, the host cell is part of a tissue. In some embodiments, the host cell is part of an organism.

As used herein, the phrase "human fcyri gene" refers to a nucleotide sequence that encodes a fully human, substantially human, or humanized portion of an fcyri protein, depending on the context. In some embodiments, a "human fcyri" gene refers to a humanized fcyri gene that is distinct from a fully mouse-derived fcyri gene. In some embodiments, the human fcyri gene contains one or more substitutions, additions, deletions, or mutations. In some embodiments, the human Fc γ RI gene comprises Fc γ RIA (CD64A), Fc γ RIB (CD64B), Fc γ RIC (CD64C), or a combination thereof.

The phrase "human Fc γ RI protein" refers to a protein encoded by a fully human, substantially human, or humanized Fc γ RI gene, depending on the context. In some embodiments, a "human fcyri" protein refers to a humanized fcyri protein that is distinct from a fully murine fcyri protein. In some embodiments, the human fcyri protein comprises one or more amino acid substitutions, additions, deletions, or mutations. In some embodiments, the Fc γ RI protein comprises Fc γ RIA (CD64A), Fc γ RIB (CD64B), Fc γ RIC (CD64C), or a combination thereof.

The phrase "hybrid Fc γ RI gene" or "hybrid Fc γ RI protein" refers to an Fc γ RI gene or protein comprising Fc γ RI sequences of at least two different animal species. In some embodiments, the hybrid Fc γ RI gene comprises a portion of a human nucleic acid sequence and a portion of a mouse nucleic acid sequence. In some embodiments, the hybrid Fc γ RI protein comprises a portion of a human amino acid sequence and a portion of a mouse amino acid sequence.

The term "humanized" is used herein to refer to nucleic acids or proteins according to their well-known meaning in the art: their structure (i.e., nucleotide or amino acid sequence) includes portions that substantially or completely correspond to the structure of a particular gene or protein naturally occurring in the non-human animal, and also includes portions that differ from the portions found in the particular non-human gene or protein of interest but that more closely correspond to a comparable structure found in the corresponding human gene or protein. In some embodiments, a "humanized" gene is one that: the gene encodes a polypeptide having an amino acid sequence substantially identical to a human polypeptide (e.g., a human protein or portion thereof-e.g., a characteristic portion thereof). As just one example, in the case of a membrane receptor, a "humanized" gene may encode a polypeptide having an extracellular portion with an amino acid sequence that is identical to a human extracellular portion, and the remaining sequence that is identical to a non-human (e.g., mouse) polypeptide. In some embodiments, the humanized gene comprises at least a portion of the DNA sequence of a human gene. In a certain embodiment, the humanized gene comprises the entire DNA sequence of a human gene. In some embodiments, the humanized protein comprises a sequence having a portion that occurs in a human protein. In some embodiments, the humanized protein comprises the entire sequence of a human protein and is expressed from an endogenous locus of the non-human animal corresponding to a homolog or ortholog of the human gene.

The term "identity" as used herein with respect to sequence comparisons refers to identity as determined by a number of different algorithms known in the art that can be used to measure nucleotide and/or amino acid sequence identity. In some embodiments, identity as described herein is aligned using ClustalW v.1.83(slow mode) with an open gap penalty of 10.0, an extended gap penalty of 0.1 and using the Gonnet similarity Matrix (MACVECTOR)^TM10.0.2 MacVector, 2008).

As used herein, the term "intracellular signaling cascade" or "intracellular signaling" refers to the transmission of a signal from a cell surface to one or more intracellular targets. In some embodiments, intracellular signaling comprises a cellular physiological response elicited by binding of a target molecule (e.g., an immunoglobulin Fc region) to an extracellular component of an Fc γ R1 receptor.

As used herein, the term "isolated" refers to the following substances and/or entities: (1) has been separated from at least some of the components with which it was associated at the time of initial production (whether in nature and/or in the experimental setting), and/or (2) has been designed, produced, prepared and/or manufactured manually by a human. An isolated substance and/or entity may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% of the other components initially associated therewith. In some embodiments, the isolated factor has a purity of about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99%. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, one skilled in the art will appreciate that a substance may still be considered "isolated" or even "pure" after combination with certain other components such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percent separation or purity of the substance is calculated without the inclusion of such carriers or excipients. As just one example, in some embodiments, a biopolymer, such as a naturally occurring polypeptide or polynucleotide, is considered "isolated" when: a) not associated with some or all of the components with which it is associated in nature in its natural state, due to its origin and derivative source; b) substantially free of other polypeptides or nucleic acids of the same species as the species from which it is produced in nature; c) expressed by a cell or other expression system that is not the species from which it is produced in nature, or combined with components from the cell or other expression system. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system that is different from the cellular system in which it is produced in nature is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has undergone one or more purification techniques may be considered an "isolated" polypeptide as long as it has been separated from other components that a) naturally associate with the polypeptide; and/or b) binds to the polypeptide at the time of initial production of the polypeptide.

As used herein, the phrase "mouse Fc γ RI gene" refers to a gene comprising a nucleic acid molecule as set forth in SEQ ID NO. 1, or a nucleic acid molecule having substantial identity to a molecule as set forth in SEQ ID NO. 1.

As used herein, the phrase "mouse Fc γ RI protein" refers to a protein comprising the amino acid sequence set forth in SEQ ID NO.2, including proteins having substantial identity to the protein set forth in SEQ ID NO. 2.

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 vertebrate of the order ostomata, a bony fish, a cartilaginous fish (e.g., a shark or ray), an amphibian, a reptile, a mammal, and a bird. In some embodiments, the non-human mammal is a primate, goat, sheep, pig, dog, cow, or rodent. In some embodiments, the non-human animal is a rodent, such as a rat or a mouse.

As used herein in its broadest sense, the phrase "nucleic acid" refers to any compound and/or substance that is incorporated or can be incorporated into an oligonucleotide chain. In some embodiments, the nucleic acid is a compound and/or substance that is incorporated or can be incorporated into the oligonucleotide chain via a phosphodiester bond. As is clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, the nucleic acid is, comprises, or consists of one or more native nucleic acid residues. In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not employ a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of: one or more "peptide nucleic acids" known in the art and having peptide bonds in the backbone rather than phosphodiester bonds are considered to be within the scope of the present invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and/or 5' -N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, the nucleic acid is, comprises, or consists of: one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, the nucleic acid is, comprises, or consists of: one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxyadenosine, 8-oxoguanosine, O (6) -methylguanine, 2-thiocytidine, methylated bases, intercalating (intercalated) bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2 '-fluororibose, ribose, 2' -deoxyribose, arabinose, and hexose) compared to the sugars in the natural nucleic acid. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as an RNA or a protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of: isolation from natural sources, enzymatic synthesis (in vivo or in vitro) by complementary template-based polymerization, replication in recombinant cells or systems, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length. In some embodiments, the nucleic acid is single-stranded; in some embodiments, the nucleic acid is double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element that encodes a polypeptide, or is the complement of a sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

As used herein, the phrase "operably linked" refers to a contiguous state in which the components being described are in a relationship that allows them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Sequences that are "operably linked" include expression control sequences that are contiguous with the gene of interest, as well as expression control sequences that function in trans or at a distance to control the gene of interest. As used herein, the term "expression control sequence" refers to a polynucleotide sequence required to effect expression and processing of a coding sequence to which it is ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and sequences that enhance protein secretion when desired. The nature of such control sequences varies with the host organism. For example, in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence, while in eukaryotes, typically such control sequences include a promoter and a transcription termination sequence. The term "control sequence" is intended to include components whose presence is critical to expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

As used herein, the term "polypeptide" refers to any polymer chain of amino acids. In some embodiments, the polypeptide has a naturally occurring amino acid sequence. In some embodiments, the polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, the polypeptide has an engineered amino acid sequence, i.e., it is designed and/or produced by manual manipulation by a human.

As used herein, the term "recombinant" is intended to refer to a polypeptide (e.g., a signal-modulating protein as described herein) designed, engineered, prepared, expressed, produced or isolated by recombinant means, such as a polypeptide expressed using a recombinant expression vector transfected into a host cell, a polypeptide isolated from a combinatorial library of recombinant human polypeptides (Hoogenboom H.R., (1997) TIBTech.15:62-70(Hoogenboom H.R., 1997, Biotechnology trends, Vol.15, pp.62-70), AzzazyH., and Highsumt W.E., (2002) Clin. biochem.35: 425. v.425. and (Azzazy H. and Highsumt W.E., 2002, clinical biochemistry, Vol.35, pp.425. and J.V., LarrikJ.W.and W.2002, Gavido.J.V., Larrick J.2002, and Charnick J.145. 11. Chagno.145. Biotech., Vol.145. 145. J.145. and 35, Chagnomon.145. 371, Zogno.23, Biotech. H.E., Vol., 2000, immunology today, vol.21, p.371-; kellermann S-A, and Green L.L (2002) Current Opinion in Biotechnology 13: 593-; et al (2000) Immunology Today21: 364-370(Little M. et al, 2000, Today's Immunology, Vol. 21, p. 364-370)) or by any other means involving splicing of selected sequence elements to each other. In some embodiments, one or more of such selected sequence elements are naturally occurring. In some embodiments, one or more of such selected sequence elements are designed on a computer. In some embodiments, one or more such selected sequence elements are derived from mutagenesis (e.g., in vivo or in vitro mutagenesis) of known sequence elements (e.g., from natural or synthetic sources). For example, in some embodiments, a recombinant polypeptide consists of a sequence present in the genome of a source organism of interest (e.g., human, mouse, etc.). In some embodiments, the recombinant polypeptide has an amino acid sequence that results from mutagenesis (e.g., in vitro or in vivo mutagenesis, e.g., in a non-human animal), and thus the amino acid sequence of the recombinant polypeptide, while derived from and related to the polypeptide sequence, may not naturally occur within the genome of the non-human animal.

The term "replacement" is used herein to refer to a process by which a "replaced" nucleic acid sequence (e.g., a gene) present in a host locus (e.g., in a genome) is removed from the locus and a different "replacement" nucleic acid is located in its place. In some embodiments, the replaced nucleic acid sequence and the replacement nucleic acid sequence are equivalent to each other for reasons such as they are homologous to each other and/or comprise corresponding elements (e.g., protein coding elements, regulatory elements, etc.). In some embodiments, the replaced nucleic acid sequence comprises one or more of: promoters, enhancers, splice donor sites, splice acceptor sites, introns, exons, untranslated regions (UTRs); in some embodiments, the replacement nucleic acid sequence comprises one or more coding sequences. In some embodiments, the replacement nucleic acid sequence is a homolog of the replaced nucleic acid sequence. In some embodiments, the replacement nucleic acid sequence is an orthologue of the replaced sequence. In some embodiments, the replacement nucleic acid sequence is or comprises a human nucleic acid sequence. In some embodiments that encompass the case where the replacement nucleic acid sequence is or comprises a human nucleic acid sequence, the replaced nucleic acid sequence is or comprises a rodent sequence (e.g., a mouse sequence). The nucleic acid sequence so configured may comprise one or more regulatory sequences (which are part of the source nucleic acid sequence used to obtain the sequence so configured, e.g., a promoter, enhancer, 5 '-or 3' -untranslated region, etc.). For example, in various embodiments, the replacement is a replacement of the endogenous sequence with a heterologous sequence, such that the gene product is produced from the nucleic acid sequence (comprising the heterologous sequence) so configured, without expressing the endogenous sequence; the replacement is a replacement of the endogenous genomic sequence with a nucleic acid sequence encoding a protein having a similar function as the protein encoded by the endogenous sequence (e.g., the endogenous genomic sequence encodes an Fc γ RI protein, and the DNA fragment encodes one or more human Fc γ RI proteins). In various embodiments, an endogenous gene or fragment thereof is replaced with a corresponding human gene or fragment thereof. A corresponding human gene or fragment thereof is a human gene or fragment that is an ortholog of, or is structurally and/or functionally substantially similar or identical to, the endogenous gene or fragment thereof being replaced.

As used herein, the term "substantially" refers to a qualitative condition that exhibits a complete or nearly complete range or degree of a feature or characteristic of interest. One of ordinary skill in the biological arts will appreciate that few, if any, biological and chemical phenomena are complete and/or complete, or that absolute results are obtained or avoided. The term "substantially" is thus used herein to represent the lack of potential integrity inherent in many biological and chemical phenomena.

As used herein, the phrase "substantial homology" refers to a comparison between amino acid or nucleic acid sequences. As one of ordinary skill in the art will recognize, two sequences are generally considered "substantially homologous" if they contain homologous residues in corresponding positions. Homologous residues can be identical residues. Alternatively, homologous residues may be non-identical residues whose structural and/or functional characteristics will be suitably similar. For example, as is well known to those of ordinary skill in the art, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids, and/or as having "polar" or "non-polar" side chains. The replacement of one amino acid for another of the same type can generally be considered a "homologous" replacement. Typical amino acid classifications are summarized in tables 1 and 2.

TABLE 1

TABLE 2

As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences, and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such procedures are described in Altschul, et al, Basic local alignment search tool, J.mol.biol.,215(3): 403-; altschul, et al, Methods in Enzymology (Altschul et al, Methods in Enzymology); altschul, et al, "Gapped BLAST and PSI-BLAST: a new generation of protein database searches," Nucleic Acids Res.25:3389-3402,1997(Altschul et al, "gap BLAST and PSI-BLAST: New Generation protein database search program", "Nucleic Acids research", Vol.25, p.3389-3402, 1997); baxevanis, et al, Bioinformatics: A Practical Guide to the Analysis of Genesand Proteins, Wiley,1998(Baxevanis et al, Practical guidelines for Bioinformatics: Gene and protein Analysis, Wei Li Press, 1998); and Misener, et al, (eds.), Bioinformatics Methods and protocols (Methods in Molecular Biology, Vol.132), Humana Press,1999 (edited by Misener et al, Methods of Bioinformatics Methods (Methods of Molecular Biology, Vol.132), U.S. Pat. No. 1999). In addition to recognizing homologous sequences, the programs described above generally provide an indication of the degree of homology. In some embodiments, two sequences are considered substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are homologous within the relevant stretch of residues. In some embodiments, the relevant segment is the full sequence. In some embodiments, the segment of interest is at least 9, 10, 11, 12, 13, 14, 15, 16, 17, or more residues. In some embodiments, the relevant segment comprises contiguous residues along the entire sequence. In some embodiments, the relevant segment comprises non-contiguous residues along the entire sequence. In some embodiments, the segment of interest is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues.

As used herein, the phrase "substantial identity" refers to a comparison between amino acid or nucleic acid sequences. As one of ordinary skill in the art will recognize, two sequences are generally considered "substantially identical" if they contain identical residues in corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences, and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such procedures are described in Altschul, et al, Basic local alignment search tool, J.mol.biol.,215(3): 403-; altschul, et al, Methods in Enzymology (Altschul et al, Methods in Enzymology); altschul et al, Nucleic Acids Res.25:3389-3402,1997(Altschul et al, Nucleic Acids research, Vol.25, p.3389-3402, 1997); baxevanis et al, Bioinformatics: A Practical Guide to the analysis of Genes and Proteins, Wiley,1998(Baxevanis et al, Practical guidelines for Bioinformatics: Gene and protein analysis, Wei Li Press, 1998); and Misener, et al, (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol.132), Humana Press,1999 (edited by Misener et al, Methods of Bioinformatics Methods (Methods of Molecular Biology, Vol.132), U.S. Pat. No. 1999). In addition to identifying identical sequences, the above procedures typically provide an indication of the degree of identity. In some embodiments, two sequences are considered substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical within the relevant stretch of residues. In some embodiments, the relevant segment is the full sequence. In some embodiments, the segment of interest is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues.

As used herein, the phrase "targeting vector" or "targeting construct" refers to a polynucleotide molecule comprising a targeting region. The targeting region comprises a sequence that is identical or substantially identical to a sequence in the target cell, tissue or animal and integration of the targeting construct into a location within the genome of the cell, tissue or animal is achieved via homologous recombination. Also included are targeting regions targeted using site specific recombinase recognition sites (e.g., loxP or Frt sites). In some embodiments, the targeting constructs of the present invention further comprise nucleic acid sequences or genes of particular interest, selectable markers, control and/or regulatory sequences, and other nucleic acid sequences that allow for recombination mediated by exogenous addition of proteins that contribute to or facilitate recombination involving such sequences. In some embodiments, the targeting construct of the present invention further comprises, in whole or in part, a gene of interest, wherein the gene of interest is a heterologous gene encoding in whole or in part a protein having a function similar to the protein encoded by the endogenous sequence.

As used herein, the term "variant" refers to an entity that exhibits significant structural identity, as compared to a reference entity, but differs structurally from the reference entity in the presence or level of one or more chemical moieties. In many embodiments, the variant is also functionally different from its reference entity. Generally, whether a particular entity is properly considered a "variant" of a reference entity is based on the degree of structural identity to the reference entity. One skilled in the art will recognize that any biological or chemical reference entity has certain characteristic structural elements. Variants are, by definition, different chemical entities that share one or more of such characteristic structural elements. Small molecules may have a characteristic core structural element (e.g., a macrocyclic core) and/or one or more characteristic pendant moieties, to name a few, such that the variant of the small molecule is one that shares the core structural element and the characteristic pendant moieties, but differs in other pendant moieties and/or in the type of bond present within the core (e.g., whether a single or double bond, whether E or Z, etc.); a polypeptide may have characteristic sequence elements consisting of a plurality of amino acids having specified positions relative to each other in linear or three-dimensional space and/or causing a particular biological function; nucleic acids can have characteristic sequence elements composed of a plurality of nucleotide residues having specified positions relative to each other in linear or three-dimensional space. For example, a variant polypeptide can differ from a reference polypeptide due to one or more differences in amino acid sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, the variant polypeptide exhibits an overall sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% to the reference polypeptide. Alternatively or additionally, in some embodiments, the variant polypeptide does not share at least one characteristic sequence element with the reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, the variant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, the variant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, the variant polypeptide exhibits a reduced level of one or more biological activities as compared to the reference polypeptide. In many embodiments, a polypeptide of interest is considered a "variant" of a parent or reference polypeptide if the polypeptide of interest has the same amino acid sequence as the parent polypeptide except for a small number of sequence alterations at a particular position. Typically, less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variant are replaced compared to the parent. In some embodiments, the variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 replaced residues as compared to the parent. Typically, variants have few (e.g., less than 5, 4, 3, 2, or 1) functional residues substituted (i.e., residues involved in a particular biological activity). Furthermore, variants typically have no more than 5, 4, 3, 2, or 1 additions or deletions, and typically no additions or deletions, as compared to the parent. Furthermore, any addition or deletion is typically less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6 and generally less than about 5, about 4, about 3 or about 2 residues. In some embodiments, the parent or reference polypeptide is a naturally occurring polypeptide. One of ordinary skill in the art will appreciate that multiple variants of a particular polypeptide of interest may generally occur in nature, particularly when the polypeptide of interest is an infectious agent polypeptide.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid with which it is associated. In a certain embodiment, the vector is capable of extrachromosomal replication and/or expression of the nucleic acid to which it is linked in a host cell, such as a eukaryotic and/or prokaryotic cell. Vectors capable of directing the expression of an operably linked gene are referred to herein as "expression vectors".

As used herein, the term "wild-type" has its art-recognized meaning and refers to an entity having a structure and/or activity that occurs in nature in a "normal" (as opposed to mutated, pathological, altered, etc.) state or context. One of ordinary skill in the art will recognize that wild-type genes and polypeptides typically exist in a variety of different forms (e.g., alleles).

Various aspects of the invention are described in detail in the following sections. The use of these parts is not intended to limit the invention. Each section is applicable to any aspect of the invention. In this application, the use of "or" means "and/or" unless stated otherwise.

Detailed Description

The present invention provides, among other things, improved and/or engineered non-human animals having humanized genetic material encoding Fc γ RI receptors for experimenting with immune effector responses to humans or similar humans.

Fc receptor

Receptors (FcR) for the Fc (i.e., constant) region of immunoglobulins play an important role in the regulation of immune responses. FcR is present on helper cells of the host immune system, thereby facilitating disposal of foreign antigens to which the antibody binds. FcR also plays an important role in balancing the activation and suppression responses of immune system helper cells. FcR is involved in phagocytosis by macrophages, degranulation of mast cells, uptake of antibody-antigen complexes and regulation of immune responses, and other immune system processes.

In mice and humans, different fcrs, each specific for the immunoglobulin isotype present in the repertoire of antibodies expressed, are differentially expressed on the surface of different helper cells. For example, immunoglobulin g (IgG) antibodies mediate effector functions through IgG receptors (Fc γ R). Fc γ R has been classified into four groups: high affinity activation of Fc gamma RI (CD64), low affinity inhibition of Fc gamma RIIb (CD32b), low affinity activation of FcgRIIa/c (CD32a/c) and low affinity activation of Fc gamma RIII (CD 16). Although each group is present in both mice and humans, the number of isotypes (isoforms) and subtypes (subsets) of immune cells present in these groups is different. For example, Fc γ RIIA and Fc γ RIIIB are expressed on helper cells in humans, but are reported to be absent in mice. Furthermore, the affinity of different IgG isotypes (e.g., IgG 1) for each Fc γ R varies from mouse to human.

High affinity human Fc gamma RI

Human high affinity Fc γ RI (CD64) is at high affinity (usually Ka is about 10)^-8To 10^-9M) integral membrane glycoproteins that bind monomeric IgG-type antibodies. Upon binding of IgG, CD64 interacts with a helper chain called the common gamma chain (γ chain) that has an Immunoreceptor Tyrosine Activation Motif (ITAM) that triggers cell activation. In humans, it has been reported that CD64 is constitutively expressed on macrophages and monocytes, and expression can be induced on polymorphonuclear leukocytes by cytokines such as IFN γ and G-CSF.

Fc gamma RI sequence

Exemplary sequences of human, mouse and hybrid Fc γ RI are shown in table 3. For cDNA sequences, consecutive exons are separated by alternating underlined text. For protein sequences, the extracellular sequences are underlined. The reference sequence is exemplary; one skilled in the art can determine and compare sequence elements or degrees of identity to distinguish between additional mouse and human sequences.

TABLE 3

Humanized Fc gamma RI non-human animals

Non-human animals are provided that express a humanized Fc γ RI receptor protein on the surface of immune cells (e.g., bone marrow cells) of the non-human animal as a result of genetic modification of an endogenous locus encoding the Fc γ RI protein in the non-human animal. Suitable examples described herein include rodents, particularly mice.

The humanized endogenous fcyri gene in some embodiments comprises genetic material from a heterologous species (e.g., a human), wherein the fcyri protein encoded by the humanized endogenous fcyri gene comprises a portion encoded by the genetic material from the heterologous species. In some embodiments, the humanized endogenous fcyri genes of the present invention comprise genomic DNA of a heterologous species that corresponds to the extracellular portion of an fcyri protein expressed on the plasma membrane of a cell. Non-human animals, embryos, cells, and targeting constructs are also provided for making non-human animals, non-human embryos and cells comprising the humanized endogenous fcyri genes.

In some embodiments, the endogenous fcyri locus is deleted. In some embodiments, the endogenous fcyri locus is altered, wherein a portion of the endogenous fcyri locus is replaced with a heterologous sequence (e.g., a human fcyri sequence in whole or in part). In some embodiments, all or substantially all of the endogenous fcyri locus is replaced with a heterologous locus (e.g., a human fcyri locus). In some embodiments, a portion of the heterologous fcyri locus is inserted into an endogenous non-human fcyri locus. In some embodiments, the heterologous locus is a human locus.

The non-human animals of the invention comprise all or part of a human fcyri gene at an endogenous non-human fcyri locus. Thus, such non-human animals can be described as having a heterologous Fc γ RI gene. The replaced, inserted or modified endogenous Fc γ RI locus can be detected using a variety of methods including, for example, PCR, western blot, southern blot, Restriction Fragment Length Polymorphism (RFLP), or allele gain or loss assays.

In various embodiments, humanized Fc γ RI genes according to the invention include Fc γ RI genes having third, fourth, and fifth exons, each exon having a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identical to the third, fourth, and fifth exons found in the human Fc γ RI gene of SEQ ID No. 3.

In various embodiments, humanized Fc γ RI genes according to the invention include Fc γ RI genes having a nucleotide coding sequence (e.g., a cDNA sequence) that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the nucleotides found in SEQ ID No. 5.

In various embodiments, the humanized Fc γ RI protein produced by the non-human animals of the invention has an extracellular portion having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) identical to an extracellular portion of a human Fc γ RI protein found in table 3.

In various embodiments, the humanized Fc γ RI protein produced by the non-human animal of the invention has an extracellular portion having a sequence with at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to amino acid residues 18-288 as found in the human Fc γ RI protein of SEQ ID No. 4.

In various embodiments, a humanized Fc γ RI protein produced by a non-human animal of the invention has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence of a humanized Fc γ RI protein exemplified by SEQ id No. 5.

Compositions and methods are provided for making non-human animals that express humanized Fc γ RI proteins, including particular polymorphic forms or allelic variants (e.g., single amino acid differences), including compositions and methods for making non-human animals that express such proteins from human promoters and human regulatory sequences. In some embodiments, compositions and methods for making non-human animals that express such proteins from endogenous promoters and endogenous regulatory sequences are also provided. The method comprises inserting genetic material encoding a human Fc γ RI protein, in whole or in part, into the genome of a non-human animal at a precise location corresponding to an endogenous Fc γ RI gene, thereby forming a humanized Fc γ RI gene that expresses the Fc γ RI protein, in whole or in part, of human origin. In some embodiments, the method comprises inserting genomic DNA corresponding to exons 3-5 of a humanized gene encoding an Fc γ RI protein comprising a human portion comprising the amino acids encoded by the inserted exons.

In various embodiments, the humanized endogenous fcyri gene approach employs relatively minimal modification of the endogenous gene and elicits a native fcyri-mediated effector response in a non-human animal because the genomic sequence of the fcyri sequence is modified in a single fragment, thus maintaining normal function by inclusion of the necessary regulatory sequences. Thus, in such embodiments, the Fc γ RI gene modification does not affect other peripheral genes or other endogenous Fc γ RI genes. Furthermore, in various embodiments, the modification does not affect the assembly of a functional receptor on the cytoplasm, and normal effector function is maintained via binding to and subsequent signal transduction through the cytoplasmic portion of the receptor that is minimally affected, or not affected, by the modification.

Schematic representations (not to scale) of the endogenous murine fcyri gene and the humanized endogenous fcyri gene are provided in figure 5. As shown, genomic DNA comprising exons 3-5 of the human fcyri gene was inserted into the endogenous murine fcyri locus by targeting constructs. The genomic DNA comprises a portion of the gene encoding one or more extracellular domain regions (e.g., amino acid residues 28-362) of a human Fc γ RI protein that is involved in Fc binding.

Non-human animals (e.g., mice) having a humanized endogenous Fc γ RI gene can be prepared by any method known in the art. For example, a targeting vector can be prepared such that all or part of the human fcyri gene is introduced along with a selectable marker gene. Figure 5 shows a mouse genome comprising insertions of exons 1-5 of human Fc γ RI. As shown, the targeting construct comprises: a 5 'homology arm comprising a sequence located upstream of exon 1 of the endogenous murine fcyri gene, followed by a genomic DNA fragment comprising exons 1-5 of the human fcyri gene, a drug selection cassette (e.g., a neomycin resistance gene flanked by loxP sequences), and a 3' homology arm comprising a sequence located downstream of exon 6 of the endogenous murine fcyri gene. Following homologous recombination, exons 1-5 of the endogenous murine Fc γ RI gene and a portion of exon 6 of that gene are replaced with sequences contained in the targeting vector. Generating a humanized endogenous fcyri gene resulting in a cell or non-human animal that expresses a humanized fcyri protein comprising amino acids encoded by exons 1-5 of a human fcyri gene. The drug selection cassette may optionally be removed by subsequent addition of a recombinase (e.g., by Cre treatment).

In addition to mice having a humanized fcyri gene as described herein, other genetically modified non-human animals comprising a humanized fcyri gene are provided herein. In some embodiments, such non-human animals comprise a humanized fcyri gene operably linked to an endogenous fcyri promoter. In some embodiments, such non-human animals express a humanized fcyri protein from an endogenous locus, wherein the humanized fcyri protein comprises amino acid residues 16-290 of a human fcyri protein.

Such non-human animals may be selected from mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalos), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, macaques). For non-human animals for which suitable genetically-modifiable ES cells are not readily available, other methods can be used to prepare non-human animals comprising genetic modifications as described herein. Such methods include, for example, modifying the genome of a non-ES cell (e.g., a fibroblast or induced pluripotent cell) and transferring the modified genome to a suitable cell, e.g., an oocyte, using nuclear transfer, and then inoculating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo.

In some embodiments, the non-human animal of the invention is a mammal. In some embodiments, the non-human animal of the invention is a small mammal, e.g., belonging to the family of the diving rat superfamily (Dipodoidea) or the family of the mouse superfamily (Muroidea). In some embodiments, the genetically modified animal of the invention is a rodent. In some embodiments, the rodent of the invention is selected from a mouse, a rat, and a hamster. In some embodiments, the rodent of the invention is selected from the superfamily murinus. In some embodiments, the genetically modified animal of the invention is from a family selected from: mesomurine (calomycidae) (e.g., cricetulus griseus), Cricetidae (Cricetidae) (e.g., hamster, new world rat, vole), murine (Muridae) (true rat, gerbil, spiny rat, corolla), equine murine (Nesomyidae) (climbing rat, rock climbing rat, caucasian rat, equine island rat), spiny murine (platathomyidae) (e.g., spiny rat), and Spalacidae (Spalacidae) (e.g., mole, bamboo rat, and zokor). In some particular embodiments, the genetically modified rodent of the invention is selected from the group consisting of a mouse (muridae), an agromy, a spiny mouse, and a crow mouse. In some particular embodiments, the genetically modified mice of the invention are from members of the murine family. In a certain embodiment, the non-human animal of the invention is a rodent. In some particular embodiments, the rodent of the invention is selected from a mouse and a rat. In some embodiments, the non-human animal of the invention is a mouse.

In some embodiments, the non-human animal of the invention is a rodent of the C57BL strain selected from the group consisting of C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57 BL/Ola. In some particular embodiments, the mouse of the invention is a 129 strain selected from the following strains: 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129/SvJae, 129S6(129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al, 1999, Mammalian Genome 10:836(Festing et al, 1999, "Mammalian Genome", volume 10, page 836); Auerbach et al, 2000, techniques 29(5): 1028,1030, 1024 (erbach et al, 2000, biotechnology) 1032 (Biotech., 29, volume 5, page 1024, 1032)). In some particular embodiments, the genetically modified mice of the invention are hybrid strains of the 129 strain described above and the C57BL/6 strain described above. In some particular embodiments, the mouse of the invention is a hybrid strain of the 129 strain described above, or a hybrid strain of the BL/6 strain described above. In some particular embodiments, the 129 line of the hybrid lines described herein is the 129S6(129/SvEvTac) line. In some embodiments, the mouse of the invention is a BALB strain, e.g., a BALB/c strain. In some embodiments, the mouse of the invention is a hybrid strain of a BALB strain and another such strain.

In some embodiments, the non-human animal of the invention is a rat. In some particular embodiments, the rat of the invention is selected from the group consisting of Wistar rat, LEA strain, Sprague Dawley strain, Fischer strain, F344, F6 and Dark agenuti. In some particular embodiments, the rat lines described herein are two or more hybrid lines selected from the following lines: wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

Non-human animals with humanized Fc gamma RI genes

Fc γ R mutants and transgenic non-human animals (e.g., mice) have been reported, for example, by van de Winkel et al in U.S. patent No.6,111,166.

Such animals have been employed in assays to assess molecular aspects of Fc γ RI expression, function, and regulation. However, they are not without limitations and disadvantages. For example, the mice disclosed in the' 166 patent contain human Fc γ RI randomly inserted into their genomes, which (1) can unintentionally disrupt the expression and/or function of other genes, whether detected or not, and (2) can result in the expression of both fully human and fully mouse Fc γ RI, thereby complicating or interfering with the specific study of a single type of Fc γ R. In addition, the intracellular signaling region of the Fc γ RI α chain may be disrupted, or otherwise inconsistent with normal Fc γ RI in the mice disclosed in the' 116 patent, because the intracellular region of the Fc γ RI α chain involved in signaling is of human origin, not mouse origin.

The present invention provides a means to overcome these and other disadvantages. The invention provides, among other things, humanized Fc γ RI transgenes inserted at endogenous mouse loci to replace mouse Fc γ RI with human or hybrid Fc γ RI genes. In some embodiments, the hybrid Fc γ RI gene is inserted at an endogenous mouse locus, wherein the extracellular domain comprises a human sequence and the intracellular domain comprises a mouse sequence. In some embodiments, insertion of the hybrid Fc γ RI gene at the endogenous locus of the mouse Fc γ RI gene results in expression of the Fc γ RI protein on an immune cell that more closely approximates the human Fc γ RI protein distribution on a human immune cell than the human Fc γ RI protein expression distribution on a mouse immune cell that otherwise expresses the endogenous mouse Fc γ RI protein. In some embodiments, insertion of the hybrid fcyri gene at the endogenous locus of the mouse fcyri gene results in expression of the fcyri protein on immune cells, which is induced and regulated by appropriate signals and stimuli.

In some embodiments, Fc γ RI α chain-mediated MHC class II antigen presentation is functionally maintained in a mouse comprising a hybrid Fc γ RI protein having a humanized extracellular region and an intracellular region of a mouse Fc γ RI α chain. In some embodiments, intracellular processing of internalized Fc γ RI is maintained in mice with hybrid Fc γ RI proteins with mouse intracellular regions, as compared to mice with fully human Fc γ RI proteins or Fc γ RI proteins with non-murine intracellular regions.

The non-human animals of the invention provide improved in vivo systems and sources of biological material (e.g., cells) expressing human Fc γ RI that can be used in a variety of assays. In various embodiments, the non-human animals of the invention are used to develop therapeutic agents that target fcyri and/or modulate fcyri signaling and immune effector responses. In various embodiments, the mice of the invention are used to screen for and develop candidate therapeutic agents (e.g., antibodies) to which Fc γ RI binds. In various embodiments, the non-human animals of the invention are used to determine immune effector responses associated with a particular therapeutic antibody.

Genetically modified non-human animals that do not express the endogenous high affinity mouse Fc γ R gene can be used, for example: elucidating the different functions of the respective high affinity fcyr genes in immune responses, measuring the efficacy of therapeutic human antibodies via cell-mediated immunity (e.g., ADCC) to determine the role of fcyr in immune diseases or disorders, use as a model of immune diseases or disorders, generating antibodies against one or more fcyr proteins, and use as a breeding mate to generate other genetically modified mice of interest.

In one example, mice according to the invention can be used to determine the cytotoxic effect lost by such mice (as compared to wild-type mice) by administering to mice that do not express the high affinity fcyr gene an agent known to elicit fcyr-dependent cytotoxic effects in wild-type mice. In one embodiment, tumor cells are implanted in the mice of the invention and, after a subsequent period of time, antibodies specific for antigens expressed on the surface of the tumor cells are injected. The isotype of the antibody was known prior to injection and the animals were analyzed for impairment of Fc γ R dependent ADCC by comparison to ADCC observed in wild type animals.

In one aspect, mice lacking endogenous high affinity receptors can be combined (e.g., by breeding) with other immunodeficient mice to develop in vivo models of autoimmune disease. For example, Severe Combined Immunodeficiency (SCID) mice are routinely used in the art as model organisms for studying internal systems. The ability of the Scm mice to produce Tor B lymphocytes or activate some components of the complement system is impaired and not effective against infection, rejection of tumors, and rejection of transplants. The high affinity Fc γ R a-subunit gene deficient mice of the invention can be mated with SCID mice to ascertain cellular clearance in a host animal in response to administration of antibody therapeutics (e.g., anti-tumor antibodies), which will determine the role of ADCC and Complement Dependent Cytotoxicity (CDC) in tumor cell clearance in vivo.

In some embodiments, a genetically modified non-human animal is provided that comprises a replacement of an endogenous high affinity fcyr gene with a high affinity human fcyr gene. Such animals can be used to study the pharmacokinetics and Fc γ R mediated ADCC of fully human antibodies. In addition, the human Fc γ R gene has been shown to have disease-associated polymorphisms or allelic variants. Thus, genetically modified non-human animals comprising replacement of an endogenous high affinity Fc γ R gene by a particular allelic or polymorphic form of the human Fc γ R gene may be used to study human autoimmune disease and polymorphism-associated traits in animals. In some embodiments, the allelic form of the human fcyr gene correlates with enhanced efficacy of human IgG.

In some embodiments, the effect of a human high affinity fcyr polymorphism on the efficacy of a human antibody therapeutic is determined. In some embodiments, the anti-tumor antibody is administered to a first humanized mouse comprising a first polymorphism of human fcyr and is also administered to a second humanized mouse comprising a second polymorphism of human fcyr, wherein the first and second mice each comprise human tumor cells; and assessing the anti-tumor activity of the anti-tumor antibody in the first mouse and the second mouse.

In some embodiments, the treatment regimen is selected by the physician for treating a human having the first polymorphism or the second polymorphism and having a tumor corresponding to a human tumor cell based on an assessment of efficacy of the anti-tumor antibody in the first mouse and the second mouse.

The endogenous fcyri alpha-chain replacement method employs relatively minimal disruption of native fcyr-mediated signal transduction in animals. In various embodiments, the genomic sequence of the Fc γ R α -chain is replaced in a single fragment, thereby maintaining normal function by inclusion of essential regulatory sequences. Thus, in such embodiments, the Fc γ R α -chain modification does not impair other endogenous FcRs that are dependent on functional FcR γ -chain molecules. Furthermore, in various embodiments, the modification does not affect the assembly of functional receptor complexes involving the Fc γ R α -chain and the endogenous FcR γ -chain, which may be important for proper expression of some Fc γ R α -chains on the cell surface and for specific downstream signaling by the activating receptor. Since the FcR γ -chain is not deleted, in various embodiments, animals comprising a replacement of the endogenous fcyr α -chain gene with a human fcyr α -chain gene can treat normal effector functions from the antibody by allowing the Fc portion of the IgG immunoglobulin to bind to the human fcyr α -chain present on the surface of the helper cell.

The non-human animals of the invention express humanized Fc γ RI proteins and, thus, cells, cell lines, and cell cultures can be generated to serve as a source of humanized Fc γ RI for binding and functional assays (e.g., determining binding or function of Fc γ RI to potential therapeutic antibodies). In various embodiments, the humanized Fc γ RI protein expressed by the non-human animals described herein can comprise a variant amino acid sequence. Variant human Fc γ RI proteins have been reported with variations associated with ligand binding residues. In various embodiments, the non-human animals of the invention express humanized Fc γ RI protein variants. In various embodiments, the variant exhibits a polymorphism at an amino acid position associated with ligand binding. In various embodiments, the non-human animals of the invention are used to determine the immune effector response elicited by therapeutic antibodies through interaction with human fcyri polymorphic variants.

Cells from the non-human animals of the invention may be isolated and used at any time, or may be maintained in culture for multiple generations. In various embodiments, cells from a non-human animal of the invention are immortalized and maintained in culture for an indefinite period of time (e.g., continuous culture).

The non-human animals of the invention provide an improved in vivo system, thereby elucidating the mechanism of antibody-dependent cell-mediated cytotoxicity.

Embodiments of various aspects provided by the invention are also described in any of the following paragraphs.

1. A mouse that expresses an fcyri protein comprising an extracellular portion of a human fcyri a chain and an intracellular portion of a mouse fcyri a chain.

2. The mouse of paragraph 1, wherein the human fcyri α chain extracellular portion comprises an EC1 domain, an EC2 domain, an EC3 domain, or a combination thereof.

3. The mouse of paragraph 2, wherein the EC1 domain is encoded by an exon having at least 90% identity to exon 3 of SEQ ID NO. 3.

4. The mouse of paragraph 2, wherein the EC2 domain is encoded by an exon having at least 90% identity to exon 4 of SEQ ID NO 3.

5. The mouse of paragraph 2, wherein the EC3 domain is encoded by an exon having at least 90% identity to exon 5 of SEQ ID NO 3.

6. The mouse of paragraph 1, wherein the mouse is not detectably expressing a full-length mouse Fc γ RI α chain.

7. The mouse of paragraph 1, wherein the intracellular portion of the fcyri a chain comprises all or part of a mouse fcyri a chain cytoplasmic domain.

8. The mouse of paragraph 1, wherein the Fc γ RI protein further comprises all or a portion of a mouse Fc γ RI a chain transmembrane domain.

9. The mouse of paragraph 1, wherein the fcyri protein comprises an fcyri a chain amino acid sequence having at least 90% identity to SEQ ID No. 5.

10. The mouse of paragraph 1, wherein the Fc γ RI protein is expressed on monocytes, macrophages, neutrophils, or dendritic cells.

11. The mouse of paragraph 10, wherein the expression of the Fc γ RI protein is elevated upon administration of murine granulocyte colony stimulating factor (mG-CSF) to the mouse.

12. A mouse comprising an fcyri gene comprising at least one exon of a human fcyri gene encoding an extracellular portion of a human fcyri protein operably linked to at least one exon of a mouse fcyri gene encoding an intracellular portion of a mouse fcyri protein.

13. The mouse of paragraph 12, wherein the exon of the human fcyri gene is selected from exon 3, exon 4, and exon 5.

14. The mouse of paragraph 12, wherein the mouse does not express a functional mouse Fc γ RI gene.

15. An embryonic stem cell whose genome comprises an fcyri gene encoding an extracellular portion of a human fcyri protein and an intracellular portion of a mouse fcyri protein.

16. The cell of paragraph 15, wherein the fcyri gene comprises exon 3, exon 4, and exon 5 of a human fcyri gene.

17. The cell of paragraph 15, wherein the fcyri gene further comprises one or more human 5' untranslated regions flanking human exon 1.

18. The cell of paragraph 15, wherein the extracellular portion of the human fcyri protein comprises one or more of EC1, EC2, and EC 3.

19. The cell of paragraph 15, comprising an Fc γ RI α chain amino acid sequence having at least 90% identity to SEQ ID No. 5.

20. The cell of paragraph 15, wherein the fcyri gene comprises exon 6 of a mouse fcyri gene.

21. The cell of paragraph 15, wherein the intracellular portion of the mouse fcyri protein comprises all or a portion of the mouse fcyri protein cytoplasmic domain.

22. The cell of paragraph 15, wherein the fcyri gene is located at an endogenous fcyri locus.

23. A mouse embryo produced from the embryonic stem cell of paragraph 15.

24. A method of making a mouse that expresses an fcyri protein comprising an extracellular portion of a human fcyri protein and an intracellular portion of a mouse fcyri protein, the method comprising the steps of:

(a) obtaining a mouse embryonic stem cell according to paragraph 15; and

(b) generating a mouse using the embryonic cells of (a).

25. A method of making a mouse, the method comprising genetically engineering the mouse such that it expresses an fcyri protein comprising an extracellular portion of a human fcyri a chain and an intracellular portion of a mouse fcyri a chain.

26. The method of paragraph 25, wherein the mouse is genetically engineered such that the human fcyri a chain extracellular portion comprises an EC1 domain, an EC2 domain, an EC3 domain, or a combination thereof.

27. The method of paragraph 26, wherein the mouse is genetically engineered such that the EC1 domain is encoded by an exon having at least 90% identity to exon 3 of SEQ ID No. 3.

28. The method of paragraph 26, wherein the mouse is genetically engineered such that the EC2 domain is encoded by an exon having at least 90% identity to exon 4 of SEQ ID No. 3.

29. The method of paragraph 26, wherein the mouse is genetically engineered such that the EC3 domain is encoded by an exon having at least 90% identity to exon 5 of SEQ ID No. 3.

30. The method of paragraph 25, wherein the mouse is genetically engineered such that it does not detectably express a full-length mouse Fc γ RI α chain.

31. The method of paragraph 25, wherein the mouse is genetically engineered such that the intracellular portion of the fcyri a chain comprises all or part of a mouse fcyri a chain cytoplasmic domain.

32. The method of paragraph 25, wherein the mouse is genetically engineered such that the fcyri protein further comprises a mouse fcyri a chain transmembrane domain, in whole or in part.

33. The method of paragraph 25, wherein the mouse is genetically engineered such that the fcyri protein comprises an fcyri a chain amino acid sequence having at least 90% identity to SEQ ID No. 5.

34. The method of paragraph 25, wherein the mouse is genetically engineered such that the Fc γ RI protein is expressed on monocytes, macrophages, neutrophils, or dendritic cells.

35. The method of paragraph 34, wherein the mouse is genetically engineered such that the expression of the Fc γ RI protein is increased upon administration of murine granulocyte colony stimulating factor (mG-CSF) to the mouse.

36. A method of making a mouse genetically engineered to comprise in its genome an fcyri gene comprising at least one exon of a human fcyri gene encoding an extracellular portion of a human fcyri protein operably linked to at least one exon of a mouse fcyri gene encoding an intracellular portion of a mouse fcyri protein.

37. The method of paragraph 36, wherein the mouse is genetically engineered such that the exon of the human fcyri gene is selected from exon 3, exon 4, and exon 5.

38. The method of paragraph 36, wherein the mouse is genetically engineered such that the mouse does not express a functional mouse Fc γ RI gene.

Examples of the invention

The following examples are provided to describe to one of ordinary skill in the art how to make and use the methods and compositions of the present invention and are not intended to limit the scope of what the inventors regard as their invention. Unless otherwise indicated, temperatures are indicated in degrees celsius and pressures are at or near atmospheric.

Example 1 humanization of endogenous Fc γ RI Gene。

This example illustrates an exemplary method of humanizing an endogenous gene encoding a high affinity Fc γ RI in a non-human mammal such as a rodent (e.g., a mouse).

Construction of humanized Fc Gamma RI targeting vector (MAID6074)

Large Targeting Vectors (LTVECs) were constructed by replacing the mouse counterpart sequence on murine chromosome 3 with the human Fc γ receptor 1 gene (promoter region, signal plus ectodomain region).

Generation of BAC-based targeting vector (MAID6073)

Mouse BAC RP23-477p23 and human BAC CTD-2339o22 containing Fc γ RI gene were identified using blast and BAC terminal sequences based on NCBI and Ensemble databases.

The method of generating the targeting vector involves the step where the LTVEC contains the human sequence of the human fcyri gene from the (5 'distal) fcyri gene promoter (25kb) to the 3' proximal gene codon W290 (before its transmembrane domain (TM)), (followed by the mouse transmembrane domain and the rest of the gene).

First, the 5 'end of the human sequence from human BAC (CTD-2339o22) was removed by homologous recombination in bacteria, leaving the I-Ceu1 site and the 25kb promoter region of the Fc γ RI gene, the 3' end human sequence was removed using the loxp-bearing pgk-Neo cassette located in intron 5 of the Fc γ R1 gene (404 bp upstream of the TM domain), followed by the AsiS1 site.

Second, the mouse Fc γ RI gene (from its promoter (20kb) up to the transmembrane domain) was removed in mouse BAC RP23-477p23 by homologous recombination in bacteria using a Spec cassette flanked by the I-Ceu1 site and the AsiS1 site (8 amino acids of the EC3 domain of the human Fc γ RI sequence added before mouse TM (up to codon W290)).

Third, digestion and ligation were performed by I-Ceu1 site and AsiS1 site, generating LTVEC (MAID6073) containing human sequences from the (5 'distal) Fc γ RI gene promoter (25kb) to the 3' proximal end (before the transmembrane domain) at codon W290 of human Fc γ RI, followed by the mouse transmembrane domain and the rest of the gene.

Figure 32 shows a schematic and exemplary strategy for humanization of mouse Fc γ RI. MAID6074 is a cartridge removal version of MAID 6073. The connection sequence is shown in fig. 6.

Selection of targeted mouse ES cells

MAID6074 LTVEC was introduced into the mouse ES cell line F1H4 by electroporation.

Example 2 Generation of high affinity Fc γ RI humanized mice

This example illustrates the transformation and propagation of mice. The extracellular domain of hFcgR1 (MAID6073) LTVEC was introduced into parental F1H4 mouse Embryonic Stem (ES) cells by electroporation. Colonies surviving after G418 drug selection were picked and screened for homologous recombination of human Fc γ RI sequences into the Fc γ RI mouse locus. Eight clones were identified with appropriate modifications and were heterozygous for the extracellular domain of human Fc γ RI, one of which was clone 6073F-D2. All of these clones contained the neo cassette.

Mu.g of Cre plasmid was introduced into clone 6073F-D2 by electroporation to remove the neo cassette. Colonies were picked and colonies without neo cassette were then screened. In the clones determined to have removed the neo cassette, ES clones 6074B-A1 and 6074B-A10 were microinjected using the VelociMouse method.

Male and female (XY female) F0VelociMice were generated from clones 6074B-A1 and 6074B-A10. These F0 mice were mated to each other in clonal and non-clonal pairs. F1 mice were generated, which showed human Fc γ RI heterozygote and homozygote (as well as wild-type). F1 mice appeared normal, and the ratio of Hom: Het: WT mice followed the predicted 1:2:1 Mendelian ratio. Human Fc γ RI-transferred wild-type and homozygous male and female groups were studied.

Example 3 characterization of high affinity Fc γ RI humanized mice

This example illustrates the characteristic expression of high affinity fcyri proteins on the surface of cells taken from non-human animals engineered to contain a humanized fcyri gene construct as described in example 1 at the endogenous fcyri locus.

Genotype characterization of high affinity Fc γ RI humanized mice is shown in fig. 1-3. No transcripts of mouse Fc γ RI were detected in mice homozygous for the humanized Fc γ RI.

The results of the phenotypic characterization are shown in fig. 4 and fig. 7-16.

Example 4 high affinity Fc γ RI humanized mice treated with murine granulocyte colony stimulating factor (mG-CSF) Phenotypic characterization

MAID6074 (hFc. gamma.RI) HO mice treated with murine G-CSF (mG-CSF) were phenotyped and compared to Phosphate Buffered Saline (PBS) controls. Mice were examined 48 hours after subcutaneous injection. Data comparing 6-7 week old MAID6074 WT mice treated with PBS (n-2) or mG-CSF (n-2) to MAID6074 HO mice treated with PBS (n-2) or mG-CSF (n-3) is shown. The results were similar for 16-17 week old MAID6074 WT mice treated with PBS (n-1) or mG-CSF (n-1) compared to MAID6074 HO mice treated with PBS (n-1) or mG-CSF (n-1). Baseline (PBS) expression and mG-CSF-induced expression of mouse Fc γ RI or hybrid Fc γ RI in monocytes, macrophages, neutrophils, and dendritic cells in the blood and spleen of MAID6074 WT and MAID6074 HO mice are shown in fig. 17-31.

Untreated MAID6074 HO mice expressed Fc γ RI (CD64) mRNA in blood, but no protein was detected by FACS. G-CSF (48 hours) induced elevation of Fc γ RI (CD64) mRNA in blood and spleen, and protein was detected by FACS.

Example 5 phenotypic characterization of mice expressing humanized high and Low affinity Fc gamma receptorsMice expressing humanized high and low affinity Fc γ receptors were generated by using standard breeding techniques. Specifically, mice expressing humanized high affinity Fc γ RI produced as described in examples 1 and 2 were hybridized with mice expressing humanized low affinity Fc γ RIIa, Fc γ RIIb, Fc γ RIIc, Fc γ RIIIa and Fc γ RIIIb produced as described in examples 1-6 and figures 1-6 of U.S. patent application publication No.2014/0154701, which is incorporated herein by reference. The resulting mice were mated to obtain homozygotes.

Humanized high-and low-affinity Fc γ R mice were phenotyped after treatment with murine G-CSF (mG-CSF) or Phosphate Buffered Saline (PBS) controls. PBS or mG-CSF (62. mu.g subcutaneously, single dose) was administered to mice by subcutaneous injection. After 48 hours, blood and spleen were collected from the treated mice and phenotypically characterized by FACS as described in example 4. The cell surface phenotype of the humanized high-and low-affinity Fc γ R mice is similar to that observed in the high-affinity Fc γ R humanized mice. Also, similar to the high affinity Fc γ receptor humanized mice, mice in which both the high and low affinity Fc γ receptors are humanized show increased expression of human Fc γ RI in monocytes, macrophages and neutrophils in the blood and spleen. In summary, humanization of low affinity Fc γ receptors in Fc γ RI humanized mice did not produce significant phenotypic changes.

Equivalent embodiments

Having thus described several aspects of at least one embodiment of this invention, those of ordinary skill in the art will appreciate that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only, and the invention is described in detail by the claims which follow.

Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

As used herein in the specification and in the claims, the terms "a" and "an" are to be understood as including the plural referents unless the content clearly dictates otherwise. Claims or descriptions that include "or," "or" between one or more members of a group are deemed to be satisfied if one, more than one, or all of the group members are present, employed, or relevant to a given product or process, unless stated to the contrary or apparent from the context. The invention includes embodiments in which exactly one member of a group is present, employed, or associated with a given product or process. The invention also includes embodiments in which more than one or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Moreover, it is to be understood that the invention encompasses all such variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., of one or more of the listed claims is introduced into another claim (or any other claim where relevant) that is dependent on the same base claim, unless otherwise indicated or unless it is clearly foreseen by those of ordinary skill in the art that a contradiction or inconsistency would arise. When elements are provided in an enumerated form, such as in a Markush group (Markush group) or similar format, it is to be understood that each sub-group of the elements is also disclosed, and that any element can be removed from the group. It will be understood that, in general, when the invention or aspects of the invention are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist of, or consist essentially of, such elements, features, etc. For the sake of simplicity, those embodiments are not specifically detailed in every case herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, whether or not the specific exclusion item is recited in the specification.

One skilled in the art will recognize that values obtained in the assays or other processes described herein can have typical standard deviations or errors.