Immunodeficiency FCGR1 knockout mouse model

Abstract

The present disclosure provides, in some aspects, immunodeficient NOD-Fcgr1 antibodies that can be used to measure the pharmacokinetics and activity of human IgG antibodies, to generate human IgG antibodies, and to model human disease treatments. null Some aspects of the present disclosure provide a mouse Fcγ receptor 1 null (Fcgr1 null ) allele and mouse interleukin-2 receptor gamma null (IL-2Rγ null ) allele.

Description

[Technical field]

[0001] (Government License Rights) This invention was made with Government support under AI132963 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0002] (Related Applications) This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 323,773, filed March 25, 2022, which is incorporated by reference in its entirety herein.

[0003] (Reference to Electronic Sequence Listing) The contents of the electronic sequence listing (J022770112WO00-SEQ-EMB.xml; size: 25,766 bytes; creation date: March 23, 2023) are incorporated by reference in their entirety herein. [Background technology]

[0004] (background) Fragment crystallizable (Fc) receptors are cell surface proteins that bind immunoglobulin (Ig) antibodies and internalize them into cells. The internalized Ig antibodies can either be recycled to the cell's surface to re-enter circulation or be transported to lysosomes. The Ig antibodies transported to lysosomes are degraded into peptides that are loaded onto major histocompatibility complex class I (MHC-I) and major histocompatibility complex class II (MHC-II) proteins and presented as antigens on the cell surface. These antigens regulate immune cell signaling. Mice have five types of Fc receptors. These are Fcγ receptor I (FcγRI), Fcγ receptor IIB (FcγRIIB), Fcγ receptor III (FcγRIII), Fcγ receptor IV (FcγRIV), and fetal Fc receptor (FcRn) (Bruhns P., Blood, 2012;119(24):5640-5649). FcγRI, FcγRIII, FcγRIV, and FcRn activate immune signaling, while FcγRIIB inhibits immune signaling. Dendritic cells (DCs) express FcγRI, FcγRIIB, FcγRIII, and FcγRIV; mouse Ly6C lo Monocytes, macrophages, and neutrophils express FcγRIV; B cells express FcγRIIB; natural killer cells and natural killer T cells express FcγRIII. During the prenatal period, neutrophils, splenic monocytes, B cells, vascular endothelial cells, and epithelial cells in the intestine express FcRn. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Bruhns P., Blood (2012) 119(24):5640~5649 Summary of the Invention [Means for solving the problem]

[0006] (overview) Some aspects of the present disclosure relate to mouse Fcγ receptor 1 null (Fcgr1 null ) allele and mouse interleukin-2 receptor gamma null (IL-2Rγ null ) allele.

[0007] In some embodiments, the mouse has a non-obese diabetic (NOD) genetic background (eg, NOD / ShiLtJ).

[0008] In some embodiments, the mouse has an IL-2Rγ null Homozygous for the Fcgr1 allele null Being homozygous for the allele.

[0009] In some embodiments, the mouse is scid Further includes alleles.

[0010] In some embodiments, the mouse has a Prkdc scid Being homozygous for the allele.

[0011] In some embodiments, the mouse is Rag1 null Further includes alleles.

[0012] In some embodiments, the mouse has a Rag1 null Being homozygous for the allele.

[0013] Some aspects of the present disclosure relate to mouse Fcγ receptor 1 null (Fcgr1 null ) allele, the mouse is immunodeficient and is scid Il2rg tm1Wjl / SzJ background included.

[0014] In some embodiments, the mouse lacks murine T cells, B cells, and / or natural killer (NK) cells.

[0015] In some embodiments, macrophage and / or dendritic cell function in the mouse is defective.

[0016] In some embodiments, the immunodeficient mouse is transplanted with human hematopoietic stem cells (HSCs).

[0017] In some embodiments, the immunodeficient mice are engrafted with human peripheral blood mononuclear cells (PBMCs).

[0018] In some embodiments, the immunodeficient mouse is engrafted with human diseased cells.

[0019] In some embodiments, the human diseased cell is a human tumor cell.

[0020] In some embodiments, the human tumor cells are human cancer cells.

[0021] In some embodiments, the mouse Fcgr1 null The allele comprises a deletion in the region from exon 3 to exon 6 compared to the unmodified endogenous mouse Fcgr1 gene. null The allele contains a deletion of exons 3 to 6.

[0022] In some embodiments, the immunodeficient mouse further comprises a human IL-15 transgene. In some embodiments, the human IL-15 transgene is integrated into the genome of the mouse.

[0023] Another aspect of the disclosure provides a method comprising administering an antibody to an immunodeficient mouse described herein.

[0024] In some embodiments, the antibody is a monoclonal antibody.

[0025] In some embodiments, the antibody is a human or humanized antibody.

[0026] In some embodiments, the antibody is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0027] In some embodiments, the antibody is an immune checkpoint inhibitor (ICI) antibody. In some embodiments, the ICI antibody is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-CTLA-4 antibody.

[0028] In some embodiments, the method further comprises assaying a biological sample from the mouse for a therapeutic effect of the antibody.

[0029] In some embodiments, the therapeutic effect is decreased human diseased cell proliferation as compared to a control.

[0030] In some embodiments, the method further comprises assaying a biological sample from the mouse for circulating levels of the antibody.

[0031] In some embodiments, circulating levels of the antibody are at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold higher compared to a control.

[0032] In some embodiments, the assaying step is at least 21 days, at least 28 days, or at least 35 days after administration of the antibody.

[0033] Some aspects of the present disclosure provide a method comprising administering human cells to the immunodeficient mouse described herein.In some embodiments, the human cells are HSCs.In other embodiments, the human cells are human PBMCs.In some embodiments, the human cells are human diseased cells.In some embodiments, the human diseased cells are human tumor cells.In some embodiments, the human tumor cells are human cancer cells.

[0034] Another aspect of the present disclosure provides a guide RNA comprising any one of the sequences set forth in SEQ ID NOs: 2 to 5.

[0035] Yet another aspect of the disclosure provides a method of making an immunodeficient mouse as described herein, comprising: introducing one or more guide RNAs and a Cas protein into a mouse embryo (e.g., having any one of the immunodeficient genetic backgrounds described herein); implanting the mouse embryo into a pseudopregnant female mouse; collecting F1 mice born from the pseudopregnant female; and breeding the F1 mice.

[0036] In some embodiments, the one or more guide RNAs bind to a region upstream of exon 3 and / or downstream of exon 6 of the mouse Fcgr1 gene (optionally, a guide RNA comprising any one of the sequences of SEQ ID NOs: 2 to 5).

[0037] In some embodiments, the mouse embryo is an immunodeficient mouse embryo, optionally having a NOD genetic background, and optionally (a) IL-2Rγ null allele, and (b) Prkdc scid Allele or Rag1 null Contains one or more of the alleles. [Brief description of the drawings]

[0038] [Figure 1-1] FIG. 1. Upstream mouse Fcgr1 deletion allele targeting strategy. [Figure 1-2] Same as above.

[0039] [Figure 2-1] FIG. 2: Downstream mouse Fcgr1 deletion allele targeting strategy. [Figure 2-2] Same as above.

[0040] [Diagram 3] FIG. 3 shows the mouse Fcgr1 deletion allele sequencing strategy.

[0041] [Figure 4]4 shows mouse Fcgr1 deletion allele predicted protein translation. The deletion of exon 3 to exon 6 of Fcgr1 is predicted to encode an Fcgr1 protein with early truncation after amino acid 26.

[0042] [Diagram 5] Figure 5 shows the results of the founder lineage for NSG-Fcgr1 null mice. "Wild" is wild type, "het" is Fcgr1 null heterozygous mice, and "hom" is Fcgr1 null homozygous mice. The bold text indicates NSG-Fcgr1 null mice that are heterozygous for targeted deletion of exon 3 to exon 6 from Fcgr1.

[0043] [Figure 6] Figure 6 shows the results of the establishment lineage for NSG-Fcgr1nullTg(Hu-IL15) mice. "Wild" is wild type, "het" is Fcgr1null heterozygous mice, and "hom" is Fcgr1null homozygous mice. Bold text indicates live-born NSG-Fcgr1nullTg(Hu-IL15) mice heterozygous for targeted deletion of exon 3 to exon 6 from Fcgr1.

[0044] [Figure 7] FIG. 7. Increased persistence of circulating trastuzumab (Herceptin®) IgG antibodies in NSG-Fcgr1null mice compared to NSG™ mice.

[0045] [Figure 8]Figure 8 is a pharmacokinetic analysis of trastuzumab IgG antibody in NSG™ and NSG-Fcgr1null (NSG™ Fcgr1 KO) mice. "SD" is standard deviation, "CV" is variation, "AUC" is area under the curve, "λz" is estimated terminal phase elimination rate constant, "Vss" is steady state distribution volume, "CL" is trastuzumab clearance, and "Vd" is distribution volume.

[0046] [Figure 9] FIG. 9. Increased persistence of circulating pembrolizumab (Keytruda®) IgG antibodies in NSG-Fcgr1null mice compared to NSG™ mice.

[0047] [Figure 10] FIG. 10 is a pharmacokinetic analysis of pembrolizumab IgG antibody in NSG™ and NSG-Fcgr1null (NSG™ Fcgr1 KO) mice.

[0048] [Figure 11] FIG. 11. Increased human IgG antibody levels following transplantation of NSG-Fcgr1null mice with CD34+ umbilical cord hematopoietic stem cells (HSCs) compared to NSG™ mice transplanted with CD34+ HSCs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] (Detailed Description) Human immunoglobulins (IgG) have been widely used as therapeutic antibodies. However, mouse models for testing the pharmacokinetics and efficacy of novel antibodies are limited. This is in part because human IgG antibodies bind with high affinity to the mouse FcγR1 receptor (encoded by the Fcgr1 gene), resulting in rapid clearance of the antibodies, which in turn greatly reduces their half-life. For example, the immunodeficient NSG™ mouse model (i.e., NOD.Cg-Prkdc scid Il2rgtm1Wjl Human IgG administered to mice (i.e., mice with IgG serotype 1 / SzJ) is cleared much more rapidly than human IgG administered to humans. This rapid clearance rate limits the use of immunodeficient mouse models to evaluate the therapeutic efficacy of human IgG monoclonal antibody (mAb) drugs against, for example, patient-derived xenografts and other transplanted human cells (e.g., tumor cells).

[0050] The present disclosure relates in some aspects to an immunodeficient mouse model that does not express FcγR1 (e.g., an immunodeficient Fcgr1 gene knockout, e.g., NSG-Fcgr1 null The present invention advances the field by providing a genetically engineered mouse model that is able to express a novel gene ... null The -Tg(Hu-IL15) mouse model has the added advantage of generating human natural killer (NK) cells to induce antibody-dependent cell-mediated cytotoxicity (ADCC) in response to human IgG.

[0051] The data provided herein demonstrate that, in some cases, immunodeficiency Fcgr1 null We show an unexpected 20- to 30-fold increase in the circulating half-life of human IgG antibodies administered to mice. nullMice develop approximately 20-fold higher circulating human IgG antibody levels compared to controls after transplantation with human hematopoietic stem cells (HSCs). Thus, the present disclosure provides the art with an immunodeficient mouse model that can be used to evaluate the pharmacokinetics and efficacy of human monoclonal antibody therapy (more precisely, human monoclonal antibody therapy, e.g., particularly for human cancer and other diseases).

[0052] (immunodeficiency Fcgr1 null Mouse model) In some aspects, the present disclosure provides an immunodeficient mouse with knocked-out Fc receptor (FcR). Thus, the immunodeficient mouse provided herein comprises an FcR "null" allele (an allele that codes for a non-functional product). Immunoglobulin Fc receptor is a membrane molecule expressed by several hematopoietic cell populations that binds to the Fc region of immunoglobulin (Ig). Non-limiting examples of Fc receptors that can be knocked out in immunodeficient mouse strains include Fcγ receptor (FcγR / FcgR), Fcε receptor (FcεR, FceR), Fcα receptor (FcαR, FcaR), Fcμ receptor (FcμR, FcmR), fetal Fc receptor (FcRn), or combinations thereof.

[0053] In some embodiments, Fcγ receptor is knocked out in immunodeficient mice.Non-limiting examples of Fcγ receptor include Fcgr1-encoded Fcγ receptor (CD64), Fcgr2-encoded Fcγ receptor, Fcgr3-encoded Fcγ receptor (CD16), Fcgr3a-encoded Fcγ receptor (CD32a), Fcgr3b-encoded Fcγ receptor (CD16b), Fcgr3c-encoded Fcγ receptor (CD16c), and Fcgr4-encoded Fcγ receptor.Fcγ receptor binds to the Fc region of immunoglobulin gamma (IgG) molecule.For example, Fcγ receptor binds to IgG-coated molecules, such as opsonized pathogens and immune complexes. Cross-linking between Fcγ receptors and IgG results in the internalization and degradation of the IgG-coated molecules. Thus, in some aspects, the present disclosure provides mice with reduced internalization and degradation of IgG. Fcγ receptors bind IgG via its crystallizable (Fc) region and internalize IgG into cells. Once internalized, both the receptor and IgG are delivered to and degraded by lysosomes. This degradation of IgG prevents the IgG from being recycled back into the systemic circulation. Portions of the degraded IgG molecules are then loaded onto major histocompatibility complex class 1 (MHC-I) and major histocompatibility complex class II (MHC-II) molecules for presentation as antigens on the cell surface.

[0054] In some embodiments, the Fcgr1 gene (e.g., Ensembl# ENSMUSG00000015947; GenBank NM_010186) is knocked out in an immunodeficient mouse. Such mice are referred to herein as Fcgr1 null Mouse Fcgr1 null Mice were cloned with detectable levels of mouse FcγR1 protein (e.g., GenBank NP_034316.1). [ka] [ka] does not generate

[0055] The mouse Fcgr1 gene can be knocked out by deleting or introducing modifications (e.g., mutations) at any position in the gene sequence or regulatory gene sequence (e.g., promoter, 5'UTR, enhancer, insulator, 3'UTR), provided that the resulting sequence does not code for a functional protein. In some embodiments, the mouse Fcgr1 gene is knocked out in the gene sequence. The gene sequence can be any sequence that is translated into messenger RNA. In some embodiments, the gene sequence is an exon, an intron, a combination of exons, a combination of introns, or a combination of exons and introns.

[0056] Mouse Fcgr1 has six exons, designated exon 1 to exon 6. In some embodiments, the immunodeficient mouse model provided herein has a deletion or modification in an exon of the endogenous Fcgr1 gene. For example, the mouse may have a deletion or modification in any one or more of exon 1, exon 2, exon 3, exon 4, exon 5, and exon 6 of the endogenous Fcgr1 gene. In some embodiments, the immunodeficient mouse model has a deletion or modification in exon 3 to exon 6 of the endogenous Fcgr1 gene. In some embodiments, the immunodeficient mouse model has a deletion of exon 3 to exon 6 of the endogenous Fcgr1 gene.

[0057] In some embodiments, the immunodeficiency Fcgr1 nullThe mouse further comprises a human interleukin (IL-15) transgene. A transgene is a nucleic acid whose sequence originates from a species different from the species that comprises it (e.g., a human transgene in a mouse). The transgene can be expressed from a nucleic acid in a cell (e.g., in the mouse) or can be integrated into the genome of the mouse. Integration of the transgene (e.g., encoding human IL-15) can be by any of the genome editing methods described herein. The transgene can be integrated into the genome of the mouse at the site of the mouse's orthologous gene (e.g., human IL-15 integrated into the mouse IL-15 gene) or at another site in the genome of the mouse that does not disrupt mouse function (e.g., a safe harbor locus, e.g., Rosa26).

[0058] In some embodiments, the immunodeficiency Fcgr1 null Mice transgenically express human interleukin-15 (Tg(hu-IL15)). Human interleukin-15 (IL-15) is a cytokine that plays a major role in the development of inflammation and protective immune responses against microbial invaders and parasites by regulating immune cells of both the innate and adaptive immune systems. IL-15 plays an important role in the development, differentiation, and survival of natural killer (NK) cells, which protect against viral infections, bacterial infections, and tumor cells. NK cells require IL-15 for their in vivo development and maintenance. NK cells are important in antibody-dependent cellular cytotoxicity (ADCC). ADCC is an immune mechanism in which effector cells bearing Fc receptors bind to and kill antibody-coated target cells expressing tumor- or pathogen-derived antigens on their surface. Thus, immunodeficient Fcgr1 mice transgenically expressing human interleukin-15 were developed. null The mice (Tg(hu-IL15)) are immunodeficient Fcgr1 mice that do not express IL-15. null Compared to mice, they have increased NK cell development and maintenance as well as ADCC activity.

[0059] (Mouse model) For brevity, reference will be made herein to "mouse" and "mouse model" (e.g., a surrogate for the human condition). It should be understood that these terms may be used interchangeably throughout the specification to include "rodent" and "rodent model," including mice, rats and other rodent species, unless otherwise indicated.

[0060] It should also be understood that the standard genetic nomenclature used herein provides a unique identification for various rodent strains, and the strain symbol conveys basic information regarding the type of strain or stock used and the genetic content of the strain. Rules for symbolizing strains and stocks have been published by the International Committee on Standardized Genetic Nomenclature for Mice. These rules are available online at the Mouse Genome Database (MGD; informatics.jax.org) and have been published in print (Lyon et al., 1996). Strain symbols typically include a Laboratory Registration Code (Lab Code). The first lab code attached to the strain symbol identifies and credits the creator of the strain. The lab code at the end of the strain symbol indicates the current source for obtaining mice of that strain. Different lab codes attached to the same strain distinguish substrains and alert the user that genetic diversity may exist among the various substrains. Lab codes are assigned by a central registry to ensure that each one is unique. The registry is maintained at the Institute for Laboratory Animal Research (ILAR) at the National Academy of Sciences, Washington, DC. Lab codes are available electronically on the ILAR website (nas.edu / cls / ilarhome.nsf).See also Davisson MT, Genetic and Phenotypic Definition of Laboratory Mice and Rats / What Constitutions an Acceptable Genetic-Phenotypic Definition, National Research Council (US) International Committee of the Institute for Laboratory Animal Research, Washington (DC): National Academies Press (US); 1999.

[0061] The mouse model of disease can be modified to allow disease evaluation. Any system (e.g., immune system, respiratory system, nervous system, or circulatory system), organ (e.g., blood, heart, blood vessel, spleen, thymus, lymph node, or lung), tissue (e.g., epithelial tissue, connective tissue, muscle tissue, and nervous tissue), or cell type (e.g., lymphocyte or macrophage) can be modified, either independently or in combination, to allow disease to be studied in the model provided herein.

[0062] Three conventional methods used for the production of genomically modified mice (e.g., knockout mice, transgenic mice) include DNA microinjection (Gordon and Ruddle, Science, 1981:214:1244-124, incorporated herein by reference), embryonic stem cell mediated gene transfer (Gossler et al., Proc. Natl. Acad. Sci., 1986, 83:9065-9069, incorporated herein by reference), and retrovirus mediated gene transfer (Jaenisch, Proc. Natl. Acad. Sci., 1976, 73:1260-1264, incorporated herein by reference), any of which may be used as provided herein. For example, genome editing methods using clustered regularly interspaced palindromic repeats (CRISPR / Cas) nucleases, transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs) are described elsewhere herein.

[0063] Following delivery of the nucleic acid into a fertilized embryo (e.g., a one-cell embryo (e.g., a zygote) or a multicellular embryo (e.g., a post-zygote developmental stage (e.g., a blastocyst)), the fertilized embryo is introduced into a pseudopregnant female, which then gives birth to offspring. The presence or absence of nucleic acid encoding human FcRn and / or a chimeric IgG antibody can be confirmed, for example, using a number of genotyping methods (e.g., sequencing and / or genomic PCR).

[0064] New mouse models can also be made by crossing parental lines, as described in the examples herein.Using various available mutants, knockouts, knock-ins, transgenics, Cre-lox, Tet induction system and other mouse lines, multiple mutations and transgenes can be combined to make new mouse models.Multiple mouse lines can be crossed together to make double, triple, or even quadruple or more multiple mutant / transgenic mice.

[0065] In some embodiments, parent mice are crossed to generate F1 mice.Parent mice can be, for example, homozygous, heterozygous, hemizygous, or homozygous null at a particular allele.Homozygous describes the genotype of two identical alleles at a given locus, heterozygous describes the genotype of two different alleles at a locus, hemizygous describes the genotype of only one copy of a particular gene in an otherwise diploid organism, and homozygous null refers to an otherwise diploid organism in which both copies of that gene are missing.

[0066] (immunodeficient mouse model) In some embodiments, Fcγ receptor 1 (null) Fcgr1 null Allele and mouse interleukin-2 receptor gamma null (IL-2Rγ null Provided herein is an immune-deficient mouse model comprising an allele that is a mutant (e.g., Fcgr1) relative to a wild-type gene. null In some embodiments, the immune-deficient mouse model is a mouse model that contains one copy of a gene (e.g., Fcgr1) that contains Fcgr1 null Alleles and IL-2Rγ null In some embodiments, the immunodeficient mouse comprises an IL-2Rγ allele and a transgene encoding human IL-15. null In some embodiments, the immunodeficient mouse is homozygous for the Fcgr1 allele. null In some embodiments, the immunodeficient mouse is homozygous for the IL-2Rγ allele. null Homozygous for the Fcgr1 allele null Homozygous for the allele. Homozygous means that the immunodeficient mouse has two copies of the allele (e.g., Fcgr1 null Allele, IL-2Rγ null This means that one has a specific allele.

[0067] In some embodiments, the immunodeficient mouse is an interleukin-2 receptor gamma null (IL-2Rγ null ) allele. null The allele is a null mutation in the gene encoding the interleukin 2 receptor gamma chain (IL2Rγ, which is homologous to IL2RG in humans), which blocks natural killer (NK) cell differentiation, thereby removing the obstacle that prevents efficient engraftment of primary human cells (Cao et al., 1995; Greiner et al., 1998; and Shultz et al., 2005, each of which is incorporated herein by reference).

[0068] In some embodiments, Fcgr1 null Alleles and IL-2Rγ null Immunodeficient mice containing the Prkdc allele scid The Prkdc allele is also included. scid The mutation is a loss-of-function (null) mutation in the mouse homolog of the human PRKDC gene - this mutation essentially eliminates adaptive immunity (see, e.g., (Blunt et al., 1995; Greiner, Hesselton, and Shultz, 1998), each of which is incorporated herein by reference). In some embodiments, the immunodeficient Fcgr1 null -IL-2Rγ null Mice, Prkdc scid Being homozygous for the allele.

[0069] In some embodiments, Fcgr1 null Alleles and IL-2Rγ null Immunodeficient mice containing the Rag1 allele null The Rag1 allele null The mutation renders the mouse defective in B cells and T cells. null -IL-2Rγ null Mice are Rag1 null Being homozygous for the allele.

[0070] As known in the art, immunodeficient mice have immune deficiencies due to impaired or destroyed immune system, such as specific deficiencies in MHC class I, specific deficiencies in MHC class II, or both, deficiencies in B cells or T cells, or both, natural killer (NK) cell deficiencies, myeloid deficiencies (e.g., deficiencies in granulocytes and / or monocytes), macrophage deficiencies, dendritic cell deficiencies, and knockdown of genes related to cytokines, cytokine receptors, TLR receptors, and various transducers and transcription factors of signal transduction pathways.Immune-deficient mouse models include single gene mutation models, such as nude mouse (nu) strains and severe combined immunodeficiency (scid) strains, non-obese diabetic (NOD) strains, RAG (recombinant activating gene) strains with targeted gene deletions, and various hybrids made by crossing double and triple mutant mouse strains with additional deficiencies in innate and adaptive immunity.

[0071] An impaired immune system can be measured by any method known in the art, including but not limited to: generation of mature immune cells (e.g., B cells, T cells, dendritic cells, macrophages, natural killer cells), defective endogenous cytokine signaling, limited resistance to infection, and reduced survival. In some embodiments, the immunodeficient mice lack mature mouse T cells, lack mature mouse B cells, lack functional natural killer cells, and are deficient in endogenous (e.g., mouse) cytokine signaling. Mature T cells develop in the thymus and are released into other tissues, including blood, spleen, and lymphatic system. Mature B cells express pathogen-specific antibodies on their surface. Functional natural killer cells recognize and kill previously unexposed malignant and virally transformed cells. Endogenous (e.g., mouse) cytokine signaling is important in maintaining homeostasis and depends on cytokines to regulate immune, nervous, and endocrine system functions. Defective endogenous (e.g., mouse) cytokine signaling means that the level of cytokine signaling is not sufficient to maintain immune system homeostasis compared to a non-deficient endogenous immune system. The deficiency of mature cells (e.g., T cells or B cells), the deficiency of functional cells (e.g., natural killer cells), the defective cytokine signaling, or some combination thereof, can be 10%-99%, 5%-95%, 20%-90%, 30%-80%, 40%-70%, or 50%-60% reduction compared to a non-immunodeficient mouse. The deficiency in mature cells (e.g., T cells or B cells), the deficiency in functional cells (e.g., natural killer cells), defective cytokine signaling, or some combination thereof, can be a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% reduction compared to non-immunodeficient mice.

[0072] Deficiency of mature or functional cells (e.g., T cells, B cells, NK cells) can be assessed by any method known in the art, including, but not limited to: flow cytometry; quantitative PCR (qPCR) of T cell markers (e.g., CD3, CD8, CD4, CD25, CD127, CD152), B cell markers (e.g., CD19, IgM, BCAP), and NK cells (e.g., CD224, CD122, NK11, NKp46, Ly49, CD11b, CD49b); immunofluorescence, and ELISA. Defective cytokine signaling (e.g., mouse cytokine signaling) can be assessed by any method known in the art, including but not limited to: flow cytometry, qPCR of cytokines (e.g., IL-2, IL-7, IL-15, IFNγ, IL-4, IL-5, IL-9, IL-13, IL-25, IL-17A, IL-17F, IL-22, TNFα, IL-12, CCL3, GM-CSF, IL-6, IL-10, TGFβ, IL18, IL-21), immunofluorescence, and ELISA.

[0073] The immunodeficient mouse may express any human cytokine or combination of human cytokines that increases the effectiveness of the immunodeficient mouse as an animal model (e.g., of human IgG antibody pharmacokinetics or activity, of human IgG antibody production, of human disease). Cytokines are proteins or peptides that regulate the activity of individual cells or tissues (e.g., other human cells, mouse cells). Non-limiting examples of types of human cytokines that may be expressed in human fibrosis models include hematopoietic cytokines, lymphokines, monokines, interferons, and chemokines.

[0074] In some embodiments, the immunodeficient mouse expresses human hematopoietic cytokines. Human hematopoietic cytokines are extracellular proteins and extracellular peptides that stimulate hematopoietic cells (e.g., hematopoietic stem cells) to develop into differentiated blood cells (e.g., neutrophils, basophils, eosinophils, macrophages). Non-limiting examples of human hematopoietic cytokines include interleukin 3 (IL-3), granulocyte / macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), thrombopoietin (TPO), IL-11, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), IL-5, IL-6, IL-2, IL-7, IL-4, IL-17, and IL-15.

[0075] In some embodiments, the immunodeficient mice described herein express 1-20, 2-19, 3-18, 4-17, 5-16, 6-15, 7-14, 8-13, 9-12, or 10-11 human cytokines (e.g., human hematopoietic cytokines). In some embodiments, the immunodeficient mice described herein express 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more human cytokines. In some embodiments, the human cytokines expressed in the immunodeficient mice are involved in the persistence and development of human antibodies.

[0076] The human cytokine can be expressed in the immunodeficient mouse by any method known in the art, including but not limited to: from a transgene in the immunodeficient mouse and from a virus (e.g., lentivirus, adenovirus, adeno-associated virus) that contains a sequence encoding the human cytokine. A transgene is a gene that is transferred from one organism (e.g., a human) to another organism (e.g., a mouse). In some embodiments, the human cytokine is expressed from a transgene. The transgene in the immunodeficient mouse can be inserted into the genome of the immunodeficient mouse or can be encoded in a vector that expresses the transgene. Non-limiting examples of naturally and transgenic immunodeficient mouse models include the following mouse strains: · nude (nu) [Flanagan SP, Genet Res, 1966;8:295-309; and Nehls M et al., Nature, 1994;372:103-7]; · Scid (scid) [Bosma GC et al., Nature, 1983;301:527-30; Mosier DE et al., Nature, 1988;335:256-9; and Greiner DL et al., Stem Cells, 1998;16:166-77]; · NOD [Kikutani H et al., Adv Immunol, 1992;51:285-322; and Anderson MS et al., Ann Rev Immunol, 2005;23:447-85]; · RAG1 and RAG2 (rag) [Mombaerts P et al., Cell, 1992;68:869-77; Shinkai U et al., Cell, 1992;68:855-67]; · NOD-scid [Greiner DL et al., 1998; Shultz LD et al., J Immunol, 1995;154:180-91; Melkus MW et al., Nature Med, 2006;12:1316-22; and Denton PW et al., PLoS Med, 2008;4(12):e357]; ·IL2rgnull[DiSanto JP et al., Proc Natl Acad Sci USA, 1995;92:377~81]; · B2mnull [Christianson SW et al., J Immunol, 1997;158:3578~86]; · NOD-scid IL2rγnull [Shultz LD et al., Nat Rev Immunol, 2007;7:118-30; Ito M et al., Blood, 2002;100:3175-82; Ishikawa I et al., Blood, 2005;106:1565-73; and Macchiarini F et al., J Exp Med, 2005;202:1307-11]; · NOD-scid B2mnull [Shultz et al., 2007; Shultz LD et al., Transplantation, 2003;76:1036-42; Islas-Ohlmayer MA et al., J Virol, 2004;78:13891-900; and Macchiarini et al., 2005]; HLA transgenic mice [Grusby MJ et al., Proc Natl Acad Sci USA, 1993;90(9):3913-7; and Roy CJ et al., Infect Immun, 2005;73(4):2452-60]. See e.g., Belizario JE, The Open Immunology Journal, 2009;2:79-85 ·NOG mice (NOD.cg-Prkdc scid Il2rg tm1Sug ) [Shultz LD et al., Nat Rev Immunol, 2007;7:118-30]; and ·BRG mice (BALB / c; 129S4- Rag2 tm1.1Flv ) [Shultz LD et al., Nat Rev Immunol, 2007;7:118~30].

[0077] In some embodiments, an immunodeficient mouse model having a non-obese diabetic (NOD) mouse genotype is provided herein. NOD mice (e.g., Jackson Labs Stock No. 001976, NOD-ShiLtJ NOD mice are a polygenic mouse model of autoimmune (e.g., type 1) diabetes characterized by hyperglycemia and insulitis (leukocyte infiltration of islet cells). The NOD mouse is hypoinsulinemic and hyperglucagonemic, indicating selective destruction of islet β cells. A major component of diabetes susceptibility in NOD mice is the unique MHC haplotype. NOD mice also exhibit multiple abnormal immune phenotypes, including defective antigen-presenting cell immunoregulatory function, defective regulation of the T lymphocyte repertoire, defective NK cell function, defective cytokine production from macrophages (Fan et al., 2004), and impaired wound healing. NOD mice also lack the hemolytic complement C5. NOD mice are also severely deaf. Various mutations causing immune deficiencies, targeted mutations in cytokine genes, as well as transgenes affecting immune function have been backcrossed onto the NOD inbred background.

[0078] In some aspects of the present disclosure, the immunodeficient mice provided herein based on a NOD background are NOD-Cg.-Prkdc scid IL2rg tm1wJl / SzJ(NSG(trademark)), NOD.Cg-Rag1 tm1Mom Il2rg tm1Wjl / SzJ(NRG), and NOD.Cg-Prkdc scid Il2rg tm1Sug / ShiJic (NOG). Other immunodeficient mouse strains are contemplated herein.

[0079] In some embodiments, the immunodeficiency mouse model based on a NOD background is NOD-Cg.-Prkdc scid IL2rg tm1wJl / SzJ (NSG™) genetic background. NSG™ mice (e.g., Jackson Labs stock number: #005557) are immunodeficient mice lacking mouse T cells, mouse B cells, and mouse NK cells, are defective in multiple cytokine signaling pathways, and have numerous defects in innate immunity (see, e.g., Shultz, Ishikawa, and Greiner, 2007; Shultz et al., 2005; and Shultz et al., 1995, each of which is incorporated herein by reference). NSG™ mice, derived from the NOD mouse strain NOD / ShiLtJ (see, e.g., Makino et al., 1980, which is incorporated herein by reference), are immunodeficient mice lacking mouse T cells, mouse B cells, and mouse NK cells, are defective in multiple cytokine signaling pathways, and have numerous defects in innate immunity (see, e.g., Shultz, Ishikawa, and Greiner, 2007; Shultz et al., 2005; and Shultz et al., 1995, each of which is incorporated herein by reference). NSG™ mice are derived from the NOD mouse strain NOD / ShiLtJ (see, e.g., Makino et al., 1980, which is incorporated herein by reference), and are characterized by the expression of Prkdc. scid mutation (also called the "severe combined immunodeficiency" mutation or "scid" mutation) and Il2rg tm1Wjl This Il2rg tm1Wjl The mutation is a null mutation in the gene encoding the interleukin 2 receptor gamma chain (IL2Rγ, homologous to IL2RG in humans), which blocks NK cell differentiation, thereby removing the obstacle that prevents efficient engraftment of primary human cells (Cao et al., 1995; Greiner et al., 1998; and Shultz et al., 2005, each of which is incorporated herein by reference).

[0080] In some embodiments, the immunodeficient mouse model has the NRG genotype. NRG mice (e.g., Jackson Labs stock number 007799) are severely immunodeficient. These mice are bred with a targeted knockout mutation in recombination activating gene 1 (Rag1) and a complete null allele of the IL2 receptor common gamma chain (IL2rg null ), which contains two mutations in the NOD / ShiLtJ genetic background. The severe immune deficiency of NRG is due to the human CD34 +It allows for the efficient humanization of the mice by transplantation of hematopoietic stem cells (HSCs) and patient-derived xenografts (PDXs). Immunodeficient NRG mice are more resistant to irradiation and genotoxic drugs than mice with a scid mutation in the DNA repair enzyme Prkdc.

[0081] In some embodiments, the immunodeficient mouse model is NOG mouse. NOG mouse (Ito M et al., Blood, 2002) is a severe combined immunodeficient (scid) mouse established by combining NOD / scid mouse with IL-2 receptor-gamma chain knockout (IL2rγKO) mouse (Ohbo K. et al., Blood, 1996). NOG mouse lacks T cells and B cells, lacks natural killer (NK) cells, shows reduced dendritic cell function and reduced macrophage function, and lacks complement activity.

[0082] In some embodiments, the immunodeficient mouse model has NCG genotype. NCG mice (e.g., Charles River stock number 572) are generated by sequential CRISPR / Cas9 editing of Prkdc locus and Il2rg locus in NOD / Nju mice to generate coisogenic mice for the NOD / Nju. The NOD / Nju carries a mutation in Sirpa (SIRPα) gene that allows transplantation of foreign hematopoietic stem cells. The Prkdc knockout causes a SCID-like phenotype that lacks proper T cell and B cell formation. The knockout of Il2rg gene further aggravates this SCID-like phenotype, and also causes a concomitant decrease in NK cell production.

[0083] In some embodiments, immunodeficient mice that are deficient in MHC class I, immunodeficient mice that are deficient in MHC class II, or immunodeficient mice that are deficient in MHC class I and MHC class II are provided herein. MHC class I and / or MHC class II deficient mice do not express the same level of MHC class I protein (e.g., α-microglobulin and β2-microglobulin (B2M)) and / or MHC class II protein (e.g., α chain and β chain) as non-immunodeficient (e.g., MHC class I / II wild type) mice, and do not have the same level of MHC class I protein activity and / or MHC class II protein activity as non-immunodeficient (e.g., MHC class I / II wild type) mice. In some embodiments, the expression or activity of MHC class I protein and / or MHC class II protein is reduced (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more) compared to non-immunodeficient mice.

[0084] Immunodeficient mice deficient in MHC class I, immunodeficient mice deficient in MHC class II, and immunodeficient mice deficient in MHC class I and MHC class II are described in International Publication No. WO2018 / 209344, the contents of which are incorporated herein by reference.

[0085] NSG-SGM3 mice are derived from the NSG mouse NOD.Cg-Prkdc scid Il2rg tm1WjlTg(CMV-IL3,CSF2,KITLG)1Eav / MloySzJ (Jackson Laboratory stock number 013062). The transgenic NSG-SGM3 mice express three human cytokines: human interleukin 3 (IL-3), human granulocyte / macrophage colony-stimulating factor 2 (GM-CSF), and human stem cell factor (SCF). NSG-SGM3 mice combine the characteristics of the highly immunodeficient NSG mice with the expression of the human cytokines IL-3, GM-CSF, and SCF, which support stable engraftment of myeloid lineage and regulatory T cells.

[0086] In some embodiments, NSG mice transgenically express human IL15. NSG-IL-15 mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl Tg(IL15)1Sz / SzJ (Jackson Laboratory stock number 030890)) expresses human IL15 and has been mated with highly immunodeficient NOD scid gamma (NSG) mice. Expression of human IL15, in some embodiments, enhances the development of human NK cells in immunodeficient mice transplanted with human stem cells.

[0087] Thus, the transgenic mice described herein are Fcγ receptor 1 (null) Fcgr1 null Allele and mouse interleukin-2 receptor gamma null (IL-2Rγ null ) allele. In some embodiments, the transgenic mice described herein are generated by knocking out the Fcgr1 gene in NSG-IL-15 mice.

[0088] In some embodiments, the immunodeficient mice are NSG™ mice with an Fcgr1 knockout (NSG-Fcgr1 null In some embodiments, NSG-Fcgr1 nullMice transgenically express the gene encoding the human interleukin-15 protein (NSG-Fcgr1 null Tg(Hu-IL15).NSG-Fcgr1 null Tg(Hu-IL15) can express human IL-15 from anywhere in the mouse genome. null Tg(Hu-IL15) mice are generated by knocking out Fcgr1 in NSG-Tg(Hu-IL15) mice as described in US Patent Application No. 16 / 637621, which is incorporated herein by reference.

[0089] (humanized mouse model) In some embodiments, a humanized immunodeficient mouse model and a method for making the model are provided herein. An immunodeficient mouse transplanted with functional human cells and / or functional human tissues is called a "humanized mouse". As used herein, the terms "humanized mouse", "humanized immune deficient mouse", "humanized immune deficient mouse" and their plural versions are used interchangeably to refer to an immunodeficient mouse that is humanized by transplantation of functional human cells and / or functional human tissues. For example, the mouse model can be transplanted with human hematopoietic stem cells (HSCs) (e.g., CD34+HSCs) and / or human peripheral blood mononuclear cells (PMBCs). In some embodiments, the mouse model is transplanted with human tissues (e.g., islets, liver, skin, and / or solid or blood cancers). In other embodiments, mouse models can be genetically modified so that endogenous mouse genes are converted to human homologs (see, e.g., Pearson et al., Curr Protoc Immunol., 2008, Chapter:Unit-15.21).

[0090] Humanized mice are generated by starting with immunodeficient mice (e.g., 2-week-old, 3-week-old, 4-week-old, 5-week-old, 6-week-old, 7-week-old, 8-week-old, 9-week-old, 10-week-old or older immunodeficient mice) and, if necessary, depleting and / or suppressing (e.g., chemically or by irradiation) any remaining mouse immune cells. That is, successful survival of the human immune system in an immunodeficient mouse may require suppression of the mouse's immune system to prevent GVHD (graft-versus-host disease) rejection. After the immune system of the immunodeficient mouse has been successfully suppressed, the mouse is transplanted with human cells (e.g., HSCs and / or PBMCs). As used herein, "transplanting" refers to the process of human cells migrating and incorporating in vivo into existing tissues of interest. With respect to humanized immunodeficient mice, the transplanted human cells repopulate the mouse with a functional human hematopoietic system and / or human immune system.

[0091] In some embodiments, the human cells (e.g., HSCs or PMBCs) transplanted for humanization are human leukocyte antigen (HLA) compatible with the human cells (e.g., human cancer cells) of the mouse model. HLA compatibility refers to cells that express the same major histocompatibility complex (MHC) genes. Transplanting HLA-compatible human xenografts and human immune cells into mice, for example, reduces or prevents the immunogenicity of the human immune cells. In some embodiments, the humanized mice provided in the present disclosure are transplanted with human PMBCs or human HSCs that are HLA compatible with PDXs or human cancer cell lines.

[0092] In some embodiments, the human cells (e.g., HSCs or PMBCs) transplanted for humanization are not HLA-compatible with the human cells (e.g., human cancer cells) of the mouse model. That is, in some embodiments, the humanized mice provided in the present disclosure are transplanted with human PMBCs or human HSCs that are not HLA-compatible with either the PDX or the human cancer cell line.

[0093] (Bone marrow destruction) As described above, in some embodiments, the immunodeficient mouse is treated to deplete and / or suppress any remaining mouse immune cells (e.g., chemically or by irradiation). In some embodiments, the immunodeficient mouse is treated only chemically or only by irradiation. In other embodiments, the immunodeficient mouse is treated both chemically and by irradiation.

[0094] In some embodiments, the immunodeficient mice are administered a myeloablative agent (i.e., a chemical that suppresses or depletes mouse immune cells). Examples of myeloablative agents include busulfan, dimethyl mileran, melphalan, and thiotepa.

[0095] In some embodiments, immunodeficient mice are irradiated prior to transplantation of human cells (e.g., human HSCs and / or PMBCs). Irradiation of immunodeficient mice destroys mouse immune cells in the peripheral blood, spleen, and bone marrow, which is thought to facilitate transplantation of human cells (e.g., human HSCs and / or PMBCs), as well as proliferation of other immune cells (e.g., by increasing human cell survival factors). Irradiation also shortens the time it takes to accumulate the number of human immune cells required to "humanize" the mouse model.

[0096] For immunodeficient mice (e.g., NSG™ mice), this preparation is typically accomplished through whole-body gamma irradiation. The irradiator can vary in size depending on its intended use. Animals are typically irradiated for a short period of time (less than 15 minutes). The amount of time spent in the irradiator varies depending on the radioisotope decay chart, the dose required, and the ionizing energy source (i.e., X-rays vs. gamma rays, for which a cesium or cobalt source is required).

[0097] Myeloablative radiation doses are typically between 700 cGy and 1300 cGy, although in some embodiments lower doses may be used, such as between 1 cGy and 100 cGy (eg, about 2 cGy, 5 cGy, or 10 cGy), or between 300 cGy and 700 cGy.

[0098] By way of example, a mouse may be irradiated with 100 cGy of X-rays (or 75 cGy to 125 cGy of X-rays). In some embodiments, the dose is about 1 cGy, 2 cGy, 3 cGy, 4 cGy, 5 cGy, 10 cGy, 20 cGy, 100 cGy, 125 cGy, 150 cGy, 175 cGy, 200 cGy, 300 cGy, 400 cGy, 500 cGy, 600 cGy, 700 cGy, 800 cGy, 900 cGy, 1000 cGy, 1100 cGy, 1200 cGy, or 1300 cGy, or between any two doses described herein (e.g., 100 cGy to 300 cGy, 200 cGy to 500 cGy, 600 cGy to 1000 cGy, or 700 cGy to 1300 cGy). In some embodiments, the immunodeficient mice are irradiated about 15 minutes, 30 minutes, 45 minutes, 1 hour, or more prior to transplantation of the human HSCs and / or human PMBCs. In some embodiments, the immunodeficient mice are transplanted with the human HSCs and / or human PMBCs 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, or 18 days after irradiation.

[0099] (Transplantation (humanization)) As described above, in some embodiments, irradiated immunodeficient mice are transplanted with HSCs and / or PBMCs, and the mice are humanized. Transplantation refers to the process of human cells migrating and incorporating in vivo into existing tissues of interest. The PBMCs can be transplanted after irradiation and prior to transplantation of human diseased cells (e.g., human cancer cells), after irradiation and simultaneously with transplantation of human diseased cells, or after irradiation and subsequent to transplantation of human diseased cells.

[0100] (PBMC) Peripheral blood mononuclear cells (PBMCs) are peripheral blood cells with round nuclei. These mononuclear blood cells recirculate between tissues and blood and are important components in the immune system that fights infection and adapts to invaders. There are two major types of mononuclear cells: lymphocytes and monocytes. The lymphocyte population of PBMCs typically includes T cells, B cells, and NK cells.

[0101] PBMCs can be isolated, for example, from a whole blood sample (e.g., a Ficoll gradient). In some embodiments, PBMCs from a subject (e.g., a human subject) with a current or previous diagnosis of cancer or an autoimmune disease can be used.

[0102] (HSC) Hematopoietic stem cells (HSCs) are stem cells that give rise to other blood cells during a process called hematopoiesis. Hematopoietic stem cells give rise to various types of blood cells of lineages called myeloid and lymphoid. Both the myeloid and lymphoid lineages are involved in dendritic cell formation. Myeloid cells include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes-platelets. Lymphoid cells include T cells, B cells, natural killer cells, and innate lymphocytes.

[0103] Methods for transplanting HSCs and / or PBMCs into immunodeficient mice to generate humanized mouse models include, but are not limited to, intraperitoneal or intravenous injections (Shultz et al., J Immunol, 2015, 174:6477-6489; Pearson et al., Curr Protoc Immunol., 2008;15-21; Kim et al., AIDS Res Hum Retrovirus, 2016, 32(2):194-2020; Yamaguchi et al., Cell & Mol Immunol, 2018, 15:953-962). In some embodiments, mice are injected with 1.0×10 6 pieces~3.0×10 7 In some embodiments, mice are transplanted with 1×10 HSCs and / or PBMCs. 7In some embodiments, the mouse is transplanted with less than 1×10 HSCs and / or PBMCs. 6 Less than 5 x 10 6 In some embodiments, the mouse is transplanted with about 2×10 HSCs and / or PBMCs. 6 pcs or 1×10 6 In some embodiments, the mouse is transplanted with 25,000 to 100,000 HSCs (e.g., 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000 or more HSCs).

[0104] For example, a mouse has a resolution of 1.0×10 6 pieces, 1.1×10 6 pieces, 1.2×10 6 pieces, 1.3×10 6 pieces, 1.4×10 6 pieces, 1.5×10 6 pieces, 1.6×10 6 pieces, 1.7×10 6 pieces, 1.8×10 6 pieces, 1.9×10 6 pieces, 2.0×10 6 pieces, 2.5×10 6 pieces, 3.0×10 6 10 or more HSCs and / or PBMCs. In some embodiments, mice may be transplanted with 1.0×10 6 pieces~1.1×10 6 pieces, 1.0×10 6 pieces~1.2×10 6 pieces, 1.0×10 6 pieces~1.3×10 6 pieces, 1.0×10 6 pieces~1.4×10 6 pieces, 1.0×10 6 pieces~1.5×10 6 pieces, 1.0×10 6 pieces~1.6×10 6pieces, 1.0×10 6 pieces~1.7×10 6 pieces, 1.0×10 6 pieces~1.8×10 6 pieces, 1.0×10 6 pieces~1.9×10 6 pieces, 1.0×10 6 pieces~2.0×10 6 pieces, 1.0×10 6 pieces~2.25×10 6 pieces, 1.0×10 6 pieces~2.5×10 6 pieces, 1.0×10 6 pieces~2.75×10 6 pieces, 1.0×10 6 pieces~3.0×10 6 pieces, 1.1×10 6 pieces~1.2×10 6 pieces, 1.1×10 6 pieces~1.3×10 6 pieces, 1.1×10 6 pieces~1.4×10 6 pieces, 1.1×10 6 pieces~1.5×10 6 pieces, 1.1×10 6 pieces~1.6×10 6 pieces, 1.1×10 6 pieces~1.7×10 6 pieces, 1.1×10 6 pieces~1.8×10 6 pieces, 1.1×10 6 pieces~1.9×10 6 pieces, 1.1×10 6 pieces~2.0×10 6 pieces, 1.1×10 6 pieces~2.25×10 6 pieces, 1.1×10 6 pieces~2.5×10 6 pieces, 1.1×10 6 pieces~2.75×10 6 pieces, 1.1×10 6 pieces~3.0×10 6 pieces, 1.2×10 6 pieces~1.3×10 6 pieces, 1.2×10 6 pieces~1.4×10 6 pieces, 1.2×10 6 pieces~1.5×106 pieces, 1.2×10 6 pieces ~ 1.6×10 6 pieces, 1.2×10 6 pieces ~ 1.7×10 6 pieces, 1.2×10 6 pieces ~ 1.8×10 6 pieces, 1.2×10 6 pieces ~ 1.9×10 6 pieces, 1.2×10 6 pieces ~ 2.0×10 6 pieces, 1.2×10 6 pieces ~ 2.25×10 6 pieces, 1.2×10 6 pieces ~ 2.5×10 6 pieces, 1.2×10 6 pieces ~ 2.75×10 6 pieces, 1.2×10 6 pieces ~ 3.0×10 6 pieces, 1.3×10 6 pieces ~ 1.4×10 6 pieces, 1.3×10 6 pieces ~ 1.5×10 6 pieces, 1.3×10 6 pieces ~ 1.6×10 6 pieces, 1.3×10 6 pieces ~ 1.7×10 6 pieces, 1.3×10 6 pieces ~ 1.8×10 6 pieces, 1.3×10 6 pieces ~ 1.9×10 6 pieces, 1.3×10 6 pieces ~ 2.0×10 6 pieces, 1.3×10 6 pieces ~ 2.25×10 6 pieces, 1.3×10 6 pieces ~ 2.5×10 6 pieces, 1.3×10 6 pieces ~ 2.75×10 6 pieces, 1.3×10 6 pieces ~ 3.0×10 6 pieces, 1.4×10 6 pieces ~ 1.5×10 6 pieces, 1.4×10 6 pieces ~ 1.6×10 6 pieces, 1.4×10 6 pieces ~ 1.7×10 6 pieces, 1.4×10 6individuals to 1.8×10 6 individuals, 1.4×10 6 individuals to 1.9×10 6 individuals, 1.4×10 6 individuals to 2.0×10 6 individuals, 1.4×10 6 individuals to 2.25×10 6 individuals, 1.4×10 6 individuals to 2.5×10 6 individuals, 1.4×10 6 individuals to 2.75×10 6 individuals, 1.4×10 6 individuals to 3.0×10 6 individuals, 1.5×10 6 individuals to 1.6×10 6 individuals, 1.5×10 6 individuals to 1.7×10 6 individuals, 1.5×10 6 individuals to 1.8×10 6 individuals, 1.5×10 6 individuals to 1.9×10 6 individuals, 1.5×10 6 individuals to 2.0×10 6 individuals, 1.5×10 6 individuals to 2.25×10 6 individuals, 1.5×10 6 individuals to 2.5×10 6 individuals, 1.5×10 6 individuals to 2.75×10 6 individuals, 1.5×10 6 individuals to 3.0×10 6 individuals, 1.6×10 6 individuals to 1.7×10 6 individuals, 1.6×10 6 individuals to 1.8×10 6 individuals, 1.6×10 6 individuals to 1.9×10 6 individuals, 1.6×10 6 individuals to 2.0×10 6 individuals, 1.6×10 6 individuals to 2.25×10 6 individuals, 1.6×10 6 individuals to 2.5×10 6 individuals, 1.6×10 6 individuals to 2.75×10 6 individuals, 1.6×10 6 individuals to 3.0×10 6 individuals, 1.7×106 to 1.8×10 6 to 1.7×10 6 to 1.9×10 6 to 1.7×10 6 to 2.0×10 6 to 1.7×10 6 to 2.25×10 6 to 1.7×10 6 to 2.5×10 6 to 1.7×10 6 to 2.75×10 6 to 1.7×10 6 to 3.0×10 6 to 1.8×10 6 to 1.9×10 6 to 1.8×10 6 to 2.0×10 6 to 1.8×10 6 to 2.25×10 6 to 1.8×10 6 to 2.5×10 6 to 1.8×10 6 to 2.75×10 6 to 1.8×10 6 to 3.0×10 6 to 1.9×10 6 to 2.0×10 6 to 1.9×10 6 to 2.25×10 6 to 1.9×10 6 to 2.5×10 6 to 1.9×10 6 to 2.75×10 6 to 1.9×10 6 to 3.0×10 6 to 2.0×10 6 to 2.25×10 6 to 2.0×10 6 to 2.5×10 6 to 2.0×10 6 to 2.75×10 6 to 2.0×10 6 to 3.0×10 6 to 2.25×10 6 to 2.5×10 6 to 2.25×10 6 to 2.75×106 pieces, 2.25×10 6 pieces~3.0×10 6 pieces, 2.5×10 6 pieces~2.75×10 6 pieces, 2.5×10 6 pieces~3.0×10 6 pieces or 2.75 x 10 6 pieces~3.0×10 6 HSCs and / or PBMCs are transplanted.

[0105] In some embodiments, the mouse is 7 A dose of less than 1 x 10 PBMCs (e.g., 1 x 10 6 pieces~approx. 5×10 6 PBMCs, approximately 2 x 10 6 PBMCs, or approximately 1 x 10 6 Each mouse is transplanted with 10 PBMCs.

[0106] As described herein, in some embodiments, transplantation with HSCs and / or PBMCs results in transgenic mice that contain more human CD45+ cells in their peripheral blood (e.g., a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40% or more increase) compared to humanized NSG control mice or humanized NSG-Tg(Hu-IL15) control mice. In embodiments, the transgenic mice contain significantly more human NK cells (e.g., a 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more increase measured as a percentage of CD45+ cells) in the peripheral blood compared to humanized NSG control mice or humanized NSG-Tg(Hu-IL15) control mice.

[0107] In some embodiments, transplantation with PBMCs results in transgenic mice that contain more human myeloid cells in their peripheral blood (e.g., an increase of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2% or more measured as a percentage of CD45+ cells) compared to humanized NSG control mice or humanized NSG-Tg(Hu-IL15) control mice.

[0108] In some embodiments, the transgenic mice contain more activated human T cells compared to humanized NSG control mice or humanized NSG-Tg(Hu-IL15) control mice. In some embodiments, the activated T cells are present in the spleen of the transgenic mice. The term "T cell activation" refers to T cell activation mechanisms that may differ slightly among various types of T cells. However, the "two-signal model" in CD4+ T cells is applicable to most types of T cells. More specifically, activation of CD4+ T cells typically occurs through engagement of the T cell receptor and CD28 on the T cell surface with antigen-presenting molecules encoded by major histocompatibility complexes, respectively, on the surface of antigen-presenting cells (APCs), and with their bound antigenic peptides and B7 family members. Both cell-cell contacts are generally required for the generation of an effective immune response. For example, in the absence of CD28 costimulation, T cell receptor signaling alone can result in T cell anergy. Further signaling pathways downstream from both CD28 and the T cell receptor include many further proteins known to those of skill in the art. Activation of T cells can be determined by cytokine release and / or cell proliferation, particularly proliferation of T cells.

[0109] (Method of use) (Pharmacokinetics of human antibodies) The present disclosure relates in some aspects to immunodeficiency Fcgr1 nullA method is provided for administering an antibody to a mouse and assaying the level of the antibody. The antibody can be a monoclonal or polyclonal antibody. A monoclonal antibody is an antibody produced by a single clone of a cell or cell line and consists of identical antibody molecules. A polyclonal antibody is an antibody produced by various B cell lineages and contains a group of immunoglobulin molecules that react against a specific antigen.

[0110] In some embodiments, the antibody is a human antibody or a humanized antibody. A human antibody is an antibody that contains only human antibody sequences. A humanized antibody is an antibody that contains sequences from a non-human species. In some embodiments, the sequences of a humanized antibody are modified to more closely align with antibodies generated in humans.

[0111] In some embodiments, the antibody is an immune checkpoint inhibitor (ICI) antibody. Immune checkpoint is a regulator of the immune system that prevents the immune system from attacking cells indiscriminately. The immune checkpoint prevents immune cells from attacking specific cells. Immune checkpoint inhibitor (ICI) is a molecule that inhibits the immune checkpoint, thus allowing the immune system to attack specific cells (e.g., cancer cells). ICI can be any ICI known in the art. Non-limiting examples of immune checkpoint inhibitors include programmed cell death 1 (PD-1), cytotoxic T lymphocyte protein 4 (CTLA4), adenosine A2A receptor (A2AR), B7-H3, B7-H4, B and T lymphocyte attenuator (BTLA), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3 (LAG3), nicotinamide adenine dinucleotide phosphate oxidase isoform 2 (NOX2), T cell immunoglobulin and mucin domain 3 (TIM3), V domain Ig suppressor of T cell activation (VISTA), and sialic acid-binding immunoglobulin-type lectin 7 (SIGLEC7).

[0112] In some embodiments, the ICI antibody is an anti-PD-1 antibody. PD-1 (also known as CD279) is a protein on the surface of T cells and B cells that inhibits the immune system's response to human body cells. PD-1 inhibits the immune system by increasing apoptosis of antigen-specific T cells in lymph nodes and decreasing apoptosis in regulatory T cells. Human anti-PD-1 antibodies can agonize (increase) or antagonize (decrease) the activity of PD-1. Antibodies that agonize the activity of PD-1 are useful for further inhibiting the immune system's response to human body cells, and antibodies that agonize the activity of PD-1 can be useful in treating diseases in which the immune system is overactive (e.g., autoimmune diseases). Antibodies that antagonize the activity of PD-1 are useful for enabling the immune system to attack cells of the human body, and may be useful in treating diseases (e.g., cancer) in which cells of the human body are not undergoing apoptosis or are undergoing apoptosis at a reduced rate compared to a control. The anti-PD-1 antibody may be any anti-PD-1 antibody known in the art. Non-limiting examples of anti-PD-1 antibodies include pembrolizumab (Keytruda®), nivolumab (Opdivo®), pidilizumab (CT-011), toripalimab (JS-001), cemiplimab (Libtayo®), dostallimab (Jemperli®), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), and INCMGA00012 (MGA012).

[0113] In some embodiments, the ICI antibody is an anti-PD-L1 antibody. PD-L1 (also known as CD274 and B7-H1) is a transmembrane protein that binds to PD-1. Binding of PD-L1 to PD-1 generates an inhibitory signal that reduces the proliferation of antigen-specific T cells in lymph nodes, and simultaneously reduces apoptosis in regulatory T cells. Anti-PD-L1 antibodies can agonize (increase) or antagonize (decrease) the activity of PD-L1. Antibodies that agonize the activity of PD-L1 are useful for further inhibiting the immune system's response to cells of the human body, and antibodies that agonize the activity of PD-L1 can be useful in treating diseases in which the immune system is overactive (e.g., autoimmune diseases). Antibodies that antagonize the activity of PD-L1 are useful for enabling the immune system to attack cells of the human body, and may be useful in treating diseases (e.g., cancer) in which cells of the human body are not undergoing apoptosis or are undergoing apoptosis at a reduced rate compared to a control. The anti-PD-L1 antibody may be any anti-PD-L1 antibody known in the art. Non-limiting examples of anti-PD-L1 antibodies include atezolizumab (Tecentriq®), avelumab (Bavencio®), durvalumab (Imfinzi®), and KN035.

[0114] In some embodiments, the ICI is an anti-CTLA-4 antibody. CTLA-4 (also known as CD152) is constitutively expressed on regulatory T cells and downregulates immune responses. Anti-CTLA-4 antibodies can agonize (increase) or antagonize (decrease) the activity of CTLA-4. Antibodies that agonize the activity of CTLA-4 are useful for decreasing immune activity, and antibodies that agonize the activity of CTLA-4 can be useful in treating diseases in which the immune system is overactive (e.g., autoimmune diseases). Antibodies that antagonize the activity of CTLA-4 are useful for allowing the immune system to attack cells of the human body, and antibodies that antagonize the activity of CTLA-4 can be useful in treating diseases in which the cells of the human body are not undergoing apoptosis or are undergoing apoptosis at a reduced rate compared to controls (e.g., cancer). The anti-CTLA-4 antibody can be any anti-CTLA-4 antibody known in the art. Non-limiting examples of anti-CTLA-4 antibodies include ipilimumab (Yervoy®), L3D10, and tremelimumab (CP-675,206).

[0115] In some embodiments, the antibody is a human immunoglobulin (Ig) antibody. The human Ig antibody can be an immunoglobulin gamma (IgG), an immunoglobulin alpha (IgA), an immunoglobulin beta (IgB), an immunoglobulin epsilon (IgE), or an immunoglobulin mu (IgM) antibody. In some embodiments, the human Ig antibody is an IgG antibody. The IgG antibody can be any IgG antibody described herein.

[0116] In some embodiments, the immunodeficiency Fcgr1 nullThe mice are non-obese diabetic (NOD) immunodeficient mice. Human IgG has a significantly reduced half-life in NOD mice compared to other immunodeficient mouse strains, including CB17-scid and BALB / c-nude (Li et al., Molecular Cancer Therapeutics, 2019;18(4):780-787). This reduced half-life in NOD mice is due to an abnormality in the NOD FcγR1 (FcγRI) receptor (also known as CD64). The NOD strain background expresses the "d" allele at its Fcgr1 locus, which encodes an abnormally hyperactive FcgR1 receptor with a remarkably high affinity for human IgG (Gavin et al., Immunogenetics, 2000;51(3):206-211). This increased affinity was found to be due to a gain-of-function mutation in the NOD mouse strain Fcgr1 gene (Gavin et al., EMBO Journal, 1998;17(14):3850-3857).

[0117] Any mouse with a similar gain-of-function mutation in the knocked-out Fcgr1 gene (or its ortholog) is similarly useful in the methods provided herein for studying the pharmacokinetics and activity of human IgG antibodies, because without the hyperactive FcgR1 receptor with significant affinity for human IgG antibodies, the mouse will more closely recapitulate the recycling and activity of human IgG antibodies.

[0118] The pharmacokinetics of an antibody can include any pharmacokinetic parameters that are assayed in the art. Non-limiting examples of pharmacokinetic parameters include antibody distribution (e.g., in tissues, across blood vessels, across barriers, in cell types), half-life, excretion route (e.g., kidney, liver), administration route (e.g., intravenous, subcutaneous, intramuscular), and bioavailability (e.g., metabolism in mice, solubility in blood, ability to reach target cells).

[0119] In some embodiments, antibody half-life is assayed. Antibody half-life is the length of time for the concentration of an antibody to decrease by half (e.g., in mice). As presented herein, the half-life of a human IgG antibody is measured in immunodeficient Fcgr1 null In mice, half-life is longer than that in its wild-type counterpart or immunodeficient counterpart.Half-life can be studied by any method known in the art.Non-limiting methods for studying antibody half-life include enzyme-linked immunosorbent assay (ELISA), Western blot, and surface plasmon resonance (SPR).

[0120] In some embodiments, the half-life of the antibody is increased 2-100 times, 5-95 times, 10-90 times, 15-85 times, 20-80 times, 25-75 times, 30-70 times, 35-65 times, 40-60 times, or 45-55 times compared to a control. In some embodiments, the half-life of the antibody is increased 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold or more compared to a control. The control can be, for example, a mouse expressing functional mouse FcγR1 (e.g., a wild-type mouse or an immunodeficient mouse).

[0121] In some embodiments, circulating levels of an antibody are assayed in biological samples obtained from mice administered the antibody. The circulating level of an antibody is the amount of the antibody present in the circulation of a mouse at a given time (e.g., after administration of the antibody). The circulating level of an antibody can be compared to a control (e.g., Fcgr1) to study the pharmacokinetics of the antibody over time. null The mice can be compared to those without the allele.

[0122] The biological sample can be any biological sample obtained from the mouse. Non-limiting examples of biological samples include blood, serum, plasma, lymph, cerebrospinal fluid, urine, feces, and tissue. The biological sample can be obtained from the mouse by any method known in the art. Non-limiting methods of obtaining biological samples include intravenous withdrawal, intraarterial withdrawal, urine collection, spinal tap, lymphatic drainage, fecal collection, and biopsy.

[0123] Circulating levels of an antibody (e.g., to assay half-life) may be assayed multiple times over a given period of time. In some embodiments, the levels of an antibody are assayed 2-25 times, 3-24 times, 4-23 times, 5-22 times, 6-21 times, 7-20 times, 8-19 times, 9-18 times, 10-17 times, 11-16 times, 12-15 times, or 13-14 times. In some embodiments, the levels of an antibody are assayed 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 times or more. In some embodiments, the level of antibody is assayed multiple times over 1 day to 30 weeks, 3 days to 29 weeks, 7 days to 28 weeks, 1.5 weeks to 27 weeks, 2 weeks to 26 weeks, 2.5 weeks to 25 weeks, 3 weeks to 24 weeks, 3.5 weeks to 23 weeks, 4 weeks to 22 weeks, 4.5 weeks to 21 weeks, 5 weeks to 20 weeks, 6 weeks to 19 weeks, 7 weeks to 18 weeks, 8 weeks to 17 weeks, 9 weeks to 16 weeks, 10 weeks to 15 weeks, 11 weeks to 14 weeks, or 12 weeks to 13 weeks. In some embodiments, the levels of antibodies are measured at 1 day, 3 days, 7 days, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 4.5 weeks, 5 weeks, 5.5 weeks, 6 weeks, 6.5 weeks, 7 weeks, 7.5 weeks, 8 weeks, 8.5 weeks, 9 weeks, 9.5 weeks, 10 weeks, 10.5 weeks, 11 weeks, 11.5 weeks, 12 weeks, 12.5 weeks, 13 weeks, 13.5 weeks, 14 weeks, 14.5 weeks, 15 weeks, 15.5 weeks, 16 weeks, 16.5 weeks, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 In some embodiments, the assay is repeated multiple times over a period of 10.5 weeks, 17 weeks, 17.5 weeks, 18 weeks, 18.5 weeks, 19 weeks, 19.5 weeks, 20 weeks, 20.5 weeks, 21 weeks, 21.5 weeks, 22 weeks, 22.5 weeks, 23 weeks, 23.5 weeks, 24 weeks, 24.5 weeks, 25 weeks, 25.5 weeks, 26 weeks, 26.5 weeks, 27 weeks, 27.5 weeks, 28 weeks, 28.5 weeks, 29 weeks, 29.5 weeks, or 30 weeks or longer.

[0124] In some embodiments, the human antibody is an IgG antibody. The human IgG antibody can be any human IgG antibody known in the art. Non-limiting examples of human IgG antibodies include immunoglobulin 1 (IgG1), immunoglobulin 2 (IgG2), immunoglobulin 3 (IgG3), and immunoglobulin 4 (IgG4). In some embodiments, the human IgG antibody is an IgG1 antibody, an IgG4 antibody, or a combination thereof.

[0125] More than one antibody (e.g., human IgG antibody) inhibits the immunodeficiency of Fcgr1 null In some embodiments, 1 to 20 antibodies, 2 to 19 antibodies, 3 to 18 antibodies, 4 to 17 antibodies, 5 to 16 antibodies, 6 to 15 antibodies, 7 to 14 antibodies, 8 to 13 antibodies, 9 to 12 antibodies, or 10 to 11 antibodies may be administered to an immunodeficient Fcgr1 mouse. null In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty or more antibodies are administered to an immunodeficient Fcgr1 mouse. null Administered to mice.

[0126] In some embodiments, the antibody is a therapeutic human IgG antibody. A therapeutic human IgG antibody is an antibody that is known to treat or prevent a human disorder or is believed to treat or prevent a human disorder. A therapeutic human IgG antibody can be any human IgG antibody known in the art (e.g., IgG1, IgG2, IgG3, IgG4). Non-limiting examples of therapeutic human IgG antibodies include trastuzumab (Herceptin®), pembrolizumab (Keytruda®), adaluminab (Humira®), alemtuzumab (Lemtrada®), alirocumab (Praluent®), basiliximab (Simulect®), belimumab (Benlysta®), bevacizumab (Amtrada®), and pembrolizumab (Keytruda®). vastin®), canakinumab (Ilaris®), cetuximab (Erbitux®), daclizumab (Zinbryta®), daratumumab (Darzalex®), denosumab (Prolia®), dinutuximab (Unituxin®), eculizumab (Soliris®), elotuzumab (Empliciti®), evolocumab (Repat Other notables include: golimumab (Simponi®), infliximab (Remicade®), ipilimumab (Yervoy®), ixekizumab (Taltz®), mepolizumab (Nucala®), natalizumab (Tysabri®), necitumumab (Portrazza®), nivolumab (Opdivo®), obiltoxaximab (Anthim®), rifampinib (Renzo ... trademark), obinutuzumab (Gazyva®), ofatumumab (Arzerra®), olaratumab (Lartruvo®), omalizumab (Xolair®), palivizumab (Synagis®), panitumumab (Vectibix®), pertuzumab (Perjeta®), ramucirumab (Cyramza®), ranibizumab (Lucentis®),Raxibacumab (Abthrax®), reslizumab (Cinqair®), rituximab (Rituxan®), secukinumab (Cosentyx®), siltuximab (Sylvant®), tocilizumab (Actemra®), atlizumab (RoActemra®), tositumomab (Bexxar®), ustekinumab (Stelara®), vedolizumab (Entyvio®), L3D10, Tremelli These include tumab (CP-675,206), atezolizumab (Tecentriq®), avelumab (Bavencio®), durvalumab (Imfinzi®), KN035, pidilizumab (CT-011), toripalimab (JS-001), cemiplimab (Libtayo®), dostallimab (Jemperli®), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317) and INCMGA00012 (MGA012).

[0127] Immunodeficiency Fcgr1 null The route of administration of the antibody to the mouse is not limited. Non-limiting examples of administration routes include subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intraperitoneal injection, intracranial injection, intraventricular injection, inhalation, and ingestion.

[0128] (antibody generation) One of the limitations of mice with hyperactive Fcγ receptors (FcgR) (e.g., NSG mice) is that these mice do not generate sufficient human IgG antibodies after transplantation of human immune cells to model the human immune response. null Mice were engrafted with human hematopoietic stem cells (HSCs) and / or human peripheral blood mononuclear cells (PMBCs) and were immunodeficient, Fcgr1 null A method for measuring the level of human IgG antibodies in mice is provided. nullThe mouse can be any mouse described herein. In some embodiments, the mouse is transplanted with HSCs. In some embodiments, the mouse is transplanted with PMBCs. In some embodiments, the mouse is transplanted with HSCs and PMBCs.

[0129] HSCs and PMBCs are cells that give rise to other blood cells in a process known as hematopoiesis. Transplanting a mouse with human HSCs and / or human PMBCs allows the non-human animal to generate human blood cells. If the mouse generates human blood cells, it may also generate human antibodies (e.g., human IgG). This is especially true if the mouse is immunodeficient prior to transplantation of human HSCs. Immune deficiency is discussed in more detail in the "Mouse Models" section below. Thus, immunodeficient Fcgr1 null Engrafting mice with human HSCs and / or human PMBCs allows the mice to model human immune behavior.

[0130] An antigen can be administered to the non-human animal transplanted with human HSCs to induce a human immune response. The antigen can be any antigen that generates an immune response in humans. Non-limiting examples of such antigens include human tumor cells, bacterial cells, viruses, and fungal cells.

[0131] Human HSCs and human PMBCs are described above.

[0132] Immunodeficient Fcgr1 mice engrafted with human HSCs and / or human PMBCs null The half-life and circulating levels of any human antibody in mice can be measured by any of the methods provided herein, at any frequency and for any period of time provided herein.

[0133] In some embodiments, circulating levels of human antibodies are increased 2-fold to 50-fold, 5-fold to 45-fold, 10-fold to 40-fold, 15-fold to 35-fold, 20-fold to 35-fold, or 25-fold to 35-fold compared to a control. In some embodiments, the circulating levels of human IgG antibodies are increased by 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, or 50-fold or more compared to a control. The control can be a wild-type Fcgr1 mouse or an immunodeficient Fcgr1 mouse not transplanted with HSCs and / or PMBCs. null It may be a mouse.

[0134] (Disease Treatment) Human antibodies are used to treat and prevent a wide variety of diseases, including cancer, rheumatoid arthritis (RA), Crohn's disease (CD), multiple sclerosis (MS), heterozygous familial hypercholesterolemia (HeFH) and homozygous familial hypercholesterolemia (HoFH), graft rejection, systemic lupus erythematosus (SLE), paroxysmal nocturnal hemoglobinuria, asthma, macular degeneration, psoriasis vulgaris (PP), psoriatic arthritis (PA), and ulcerative colitis (UC). Identifying novel human therapeutic antibodies and novel diseases that are treated or prevented by human therapeutic antibodies is an important area of ​​research. The present disclosure provides, in some embodiments, a method for the identification of human therapeutic antibodies that can be used to treat or prevent human diseases, including immunodeficiency Fcgr1 with human disease. null Mice were administered therapeutic and / or putative therapeutic antibodies and their Fcgr1 null Measuring disease growth or survival in mice is provided.

[0135] In some embodiments, the immunodeficiency Fcgr1 nullThe mouse is transplanted with human diseased cells. The transplantation can be by any method provided herein. The human diseased cells are cells derived from humans and associated with the disease (e.g., patient-derived xenografts or human cell lines). The association with the disease can be confirmed by any method known in the art. Non-limiting examples of confirming whether a human cell is associated with a disease are: detecting or measuring whether the cell expresses a mutant gene or mutant protein associated with the disease (e.g., BRCA1, p53, etc.), detecting increased proliferation in the human cell compared to a control, detecting decreased apoptosis in the human cell compared to a control, and detecting increased mobility in the human cell compared to a control. The control can be human cells (e.g., wild-type human cells) from the same human or the same cell population that are known not to be associated with the disease.

[0136] Human diseased cells can be derived from any human disease known in the art.Non-limiting examples of human diseases include cancer (e.g., breast cancer, lung cancer, colorectal cancer, melanoma), autoimmune disorders (e.g., RA, CD, MS, SLE, UC, PP, PA), genetic diseases (e.g., HeFH, HoFH, cystic fibrosis, Down's syndrome, Duchenne muscular dystrophy), and infectious diseases (e.g., coronavirus disease 19 (COVID-19), hepatitis B virus, rabies virus, respiratory syncytial virus, Clostridium tetani, Clostridium botulinum, vaccinia virus, echovirus, enterovirus).

[0137] In some embodiments, the human disease is cancer, and the human affected cell is a human tumor cell. A human tumor cell is a cell that constitutes a tumor or is associated with a tumor. A tumor is an abnormal growth or accumulation of cells, and the tumor can be either malignant or benign. The human cancer can be any cancer known in the art.Non-limiting examples of human cancers include adenoid cystic carcinoma, adrenal tumors, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telegiectasia, Beckwith-Wiedemann syndrome, cholangiocarcinoma, Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer, brain stem glioma, brain cancer, breast cancer, Carney complex, cervical cancer, and uterine cancer. cancer), colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, ependymoma, esophageal cancer, Ewing's sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), germ cell tumors, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell carcinoma, hereditary mixed polyposis syndrome syndrome), hereditary pancreatitis, hereditary papillary renal carcinoma, HIV / AIDS-related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumors, cancer of the larynx and hypopharynx, leukemia, Li-Fraumeni syndrome, liver cancer, lung cancer, lymphoma, Lynch syndrome, mastocytosis, medullablastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH-associated polyposis, myelodysplastic syndrome, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumors of the digestive tract, neuroendocrine tumors of the lung, neuroendocrine tumors of the pancreas, neurofibroma These include: neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral cavity and oropharynx cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary tumors, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, non-melanoma skin cancer, small intestine cancer, gastric cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, uterine cancer, vaginal cancer, von Hippel-Lindau syndrome, vulvar cancer, Waldenström macroglobulinemia, Werner syndrome, Wilms tumor, and xeroderma pigmentosum.

[0138] In some embodiments, the human cancer is immunodeficiency Fcgr1null The human cancer cells are generated in mice by transplanting human cancer cells into the mice.The human cancer cells can be transplanted by any method provided herein.The human cancer cells can be patient-derived xenografts (PDX) or cultured human cancer cells.

[0139] The human IgG antibody can be any human IgG antibody provided herein. In some embodiments, the human IgG antibody is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, or a combination thereof.

[0140] Disease growth can be any aspect of the pathology of the disease.Non-limiting examples of disease growth include the growth of human diseased cells compared to control, the survival of human diseased cells compared to control, the motility of human diseased cells compared to control, and the division of human diseased cells compared to control.The control can be human cells (e.g., wild-type human cells) from the same human or the same cell population that are known not to be associated with disease.

[0141] Disease growth and / or survival can be measured by any method known in the art.The method of measuring disease growth depends on the disease being studied.Non-limiting methods of measuring disease growth include immunostaining, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and imaging (e.g., magnetic resonance imaging, positron emission tomography, computed tomography, X-ray imaging).

[0142] In some embodiments, the immunodeficiency Fcgr1 nullDisease growth and / or survival in mice is reduced by 1% to 200%, 5% to 195%, 10% to 190%, 15% to 185%, 20% to 180%, 25% to 175%, 30% to 170%, 35% to 165%, 40% to 160%, 45% to 155%, 50% to 150%, 55% to 145%, 60% to 140%, 65% to 135%, 70% to 130%, 75% to 125%, 80% to 120%, 85% to 115%, 90% to 110%, or 95% to 105% compared to controls. null Disease growth and / or survival in mice is reduced by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200% compared to controls. Controls include wild-type Fcgr1 mice, immunodeficient Fcgr1 mice not injected with human IgG antibodies, null Fcgr1 before treatment with mouse or its human IgG antibody null It may be a mouse.

[0143] Disease growth and / or survival can be measured multiple times over a given period of time. In some embodiments, disease growth and / or survival is measured 2-25 times, 3-24 times, 4-23 times, 5-22 times, 6-21 times, 7-20 times, 8-19 times, 9-18 times, 10-17 times, 11-16 times, 12-15 times, or 13-14 times. In some embodiments, disease growth and / or survival is measured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more times. In some embodiments, disease growth and / or survival is measured multiple times over 1 day to 30 weeks, 3 days to 29 weeks, 7 days to 28 weeks, 1.5 weeks to 27 weeks, 2 weeks to 26 weeks, 2.5 weeks to 25 weeks, 3 weeks to 24 weeks, 3.5 weeks to 23 weeks, 4 weeks to 22 weeks, 4.5 weeks to 21 weeks, 5 weeks to 20 weeks, 6 weeks to 19 weeks, 7 weeks to 18 weeks, 8 weeks to 17 weeks, 9 weeks to 16 weeks, 10 weeks to 15 weeks, 11 weeks to 14 weeks, or 12 weeks to 13 weeks. In some embodiments, disease growth and / or survival is measured at 1 day, 3 days, 7 days, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 4.5 weeks, 5 weeks, 5.5 weeks, 6 weeks, 6.5 weeks, 7 weeks, 7.5 weeks, 8 weeks, 8.5 weeks, 9 weeks, 9.5 weeks, 10 weeks, 10.5 weeks, 11 weeks, 11.5 weeks, 12 weeks, 12.5 weeks, 13 weeks, 13.5 weeks, 14 weeks, 14.5 weeks, 15 weeks, 15.5 weeks, 16 weeks The method may be performed multiple times over a period of 16.5 weeks, 17 weeks, 17.5 weeks, 18 weeks, 18.5 weeks, 19 weeks, 19.5 weeks, 20 weeks, 20.5 weeks, 21 weeks, 21.5 weeks, 22 weeks, 22.5 weeks, 23 weeks, 23.5 weeks, 24 weeks, 24.5 weeks, 25 weeks, 25.5 weeks, 26 weeks, 26.5 weeks, 27 weeks, 27.5 weeks, 28 weeks, 28.5 weeks, 29 weeks, 29.5 weeks, or 30 weeks or more.

[0144] Nucleic Acids: Manipulation and Delivery The mice described herein contain a nucleic acid encoding human interleukin 15 (IL-15) and Fcgr1 null In some embodiments, the mouse comprises a transgene encoding human IL-15 integrated into its genome, and a nucleic acid comprising an Fcgr1 allele. null Includes alleles.

[0145] The nucleic acids provided herein are engineered in some embodiments. Engineered nucleic acids are nucleic acids that do not occur in nature (e.g., at least two nucleotides covalently linked together, in some cases including phosphodiester bonds, referred to as phosphodiester backbones). Engineered nucleic acids include recombinant and synthetic nucleic acids. Recombinant nucleic acids are molecules that are constructed by linking nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids, or combinations thereof) from two different organisms (e.g., human and mouse). Synthetic nucleic acids are molecules that are amplified or synthesized by chemical or other means. Synthetic nucleic acids include those that have been chemically or otherwise modified but can base pair (bind) with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include molecules that result from any of the above replications.

[0146] An engineered nucleic acid can comprise DNA (e.g., genomic DNA, cDNA, or a combination of genomic DNA and cDNA), RNA, or a hybrid molecule (e.g., the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides (e.g., artificial or natural), and any combination of two or more bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine).

[0147] In some embodiments, the nucleic acid is complementary DNA (cDNA). cDNA is synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by reverse transcriptase.

[0148] The engineered nucleic acids of the present disclosure can be made using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, the nucleic acids are made using GIBSON ASSEMBLY® Cloning (see, e.g., Gibson, DG et al., Nature Methods, 343-345, 2009; and Gibson, DG et al., Nature Methods, 901-903, 2010, each of which is incorporated by reference herein). GIBSON ASSEMBLY® typically uses three enzyme activities in a single tube reaction: a 5' exonuclease, a 3' extension activity of a DNA polymerase, and a DNA ligase activity. The 5' exonuclease activity chews back the 5' terminal sequence, exposing the complementary sequence for annealing. The polymerase activity then fills the gaps in the annealed domains. DNA ligase then seals the nicks and covalently joins the DNA fragments together. The overlapping sequences of the adjacent fragments are much longer than those used in Golden Gate Assembly, thus resulting in a higher percentage of correct assembly. Other methods of generating engineered nucleic acids can be used in accordance with the present disclosure.

[0149] A gene is a distinct nucleotide sequence, the order of which determines the order of monomers in a polynucleotide or polypeptide. A gene typically encodes a protein. A gene may be endogenous (naturally occurring in a host organism) or exogenous (naturally transferred to a host organism or transferred through genetic engineering). An allele is one of two or more alternative forms of a gene that arise by mutation and are found at the same locus on a chromosome. In some embodiments, a gene includes a promoter sequence, a coding region (e.g., exons), a non-coding region (e.g., introns), and a regulatory region (also called a regulatory sequence).

[0150] A mouse containing a human gene is considered to contain a human transgene. A transgene is a gene that is exogenous to the host organism. That is, a transgene is a gene that is transferred to the host organism naturally or through genetic engineering. A transgene does not naturally occur in the host organism (the organism that contains the transgene, e.g., a mouse).

[0151] A promoter is a nucleotide sequence (e.g., ATG) to which RNA polymerase binds and initiates transcription. A promoter is typically located immediately upstream (at its 5' end) from the transcription initiation site. In some embodiments, the promoter is an endogenous promoter. An endogenous promoter is a promoter that is naturally present in the host animal.

[0152] An open reading frame is a contiguous series of codons beginning with a start codon (e.g., ATG) and ending with a stop codon (e.g., TAA, TAG, or TGA) that encodes a polypeptide (e.g., a protein). An open reading frame is operably linked to a promoter if the promoter controls transcription of the open reading frame.

[0153] Exons are regions of genes that code for amino acids. Introns (and other non-coding DNA) are regions of genes that do not code for amino acids.

[0154] The nucleotide sequence encoding the product (e.g., a protein) has a length of 200 base pairs (bp) to 100 kilobases (kb) in some embodiments. The nucleotide sequence has a length of at least 10 kb in some embodiments. For example, the nucleotide sequence may have a length of at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, or at least 35 kb. In some embodiments, the nucleotide sequence has a length of 10 kb to 100 kb, 10 kb to 75 kb, 10 kb to 50 kb, 10 kb to 30 kb, 20 kb to 100 kb, 20 kb to 75 kb, 20 kb to 50 kb, 20 kb to 30 kb, 30 kb to 100 kb, 30 kb to 75 kb, or 30 kb to 50 kb.

[0155] Any one of the nucleic acids provided herein may have a length of 200bp to 500kb, 200bp to 250kb, or 200bp to 100kb. The nucleic acid, in some embodiments, has a length of at least 10kb. For example, the nucleic acid may have a length of at least 15kb, at least 20kb, at least 25kb, at least 30kb, at least 35kb, at least 50kb, at least 100kb, at least 200kb, at least 300kb, at least 400kb, or at least 500kb. In some embodiments, the nucleic acid has a length of 10kb to 500kb, 20kb to 400kb, 10kb to 300kb, 10kb to 200kb, or 10kb to 100kb. In some embodiments, the nucleic acid has a length of 10 kb to 100 kb, 10 kb to 75 kb, 10 kb to 50 kb, 10 kb to 30 kb, 20 kb to 100 kb, 20 kb to 75 kb, 20 kb to 50 kb, 20 kb to 30 kb, 30 kb to 100 kb, 30 kb to 75 kb, or 30 kb to 50 kb. The nucleic acid may be circular or linear.

[0156] The nucleic acids described herein include modifications in some embodiments. Modification, in terms of nucleic acid, is any manipulation of the nucleic acid compared to the corresponding wild-type nucleic acid (e.g., naturally occurring nucleic acid). Thus, genomic modification is any manipulation of the nucleic acid in the genome (e.g., in the coding region, non-coding region, and / or regulatory region) compared to the corresponding wild-type nucleic acid in the genome (e.g., naturally occurring (unmodified) nucleic acid). Non-limiting examples of nucleic acid (e.g., genomic) modifications include deletions, insertions, "indels" (deletions and insertions), and substitutions (e.g., point mutations). In some embodiments, deletions, insertions, indels, or other modifications in a gene result in frameshift mutations, such that the gene no longer codes for a functional product (e.g., a protein). Modifications also include chemical modifications, such as chemical modifications of at least one nucleic acid base. Methods of nucleic acid modification (e.g., methods that result in gene inactivation) are known and include, but are not limited to, RNA interference, chemical modification, and gene editing (e.g., using recombinases or other programmable nuclease systems, such as CRISPR / Cas, TALEN, and / or ZFN).

[0157] Loss-of-function mutation, as known in the art, produces a gene product with little or no function. Null mutation, which is one type of loss-of-function mutation, produces a gene product with no function. In some embodiments, the inactivating allele is a null allele. Other examples of loss-of-function mutation include missense mutation and frameshift mutation.

[0158] A nucleic acid (e.g., an allele or alleles of a gene) can be altered such that the nucleic acid does not produce detectable levels of a functional gene product (e.g., a functional protein). Thus, an inactivated allele is an allele that does not produce detectable levels of a functional gene product (e.g., a functional protein). A detectable level of protein is any level of protein that is detected using a standard detection assay (e.g., flow cytometry and / or ELISA). In some embodiments, an inactivated allele is not transcribed. In some embodiments, an inactivated allele does not encode a functional protein.

[0159] Vectors used for delivery of nucleic acids include minicircles, plasmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes. However, it should be understood that a vector may not be necessary. For example, a circularized or linear nucleic acid can be delivered to an embryo without its vector backbone. The vector backbone is small (approximately 4 kb), but the donor DNA that can be circularized can range from, for example, >100 bp to 50 kb.

[0160] Methods for delivering nucleic acids into mouse embryos (e.g., mice) for the generation of transgenic mice include electroporation (see, e.g., Wang W et al., J Genet Genomics, 2016;43(5):319-27; WO2016 / 054032; and WO2017 / 124086, each of which is incorporated by reference herein), DNA microinjection (see, e.g., Gordon and Ruddle, Science, 1981:214:1244-124, each of which is incorporated by reference herein), embryonic stem cell-mediated gene transfer (see, e.g., Gossler et al., Pr , 1986;83:9065-9069, herein incorporated by reference), and retroviral-mediated gene transfer (see, e.g., Jaenisch, Proc. Natl. Acad. Sci., 1976;73:1260-1264, herein incorporated by reference), any of which may be used as provided herein. (Genome editing)

[0161] The present application contemplates the use of various gene editing techniques, for example, using engineered nucleic acids to knock out a target gene (e.g., Fcgr1) or to introduce a nucleic acid into the genome of a mouse (e.g., to generate a transgenic mouse). null The mouse may be generated by any gene editing technique known in the art. null The mouse further contains a human interleukin-15 transgene integrated into its genome.

[0162] The engineered nucleic acid (such as, for example, guide RNA, donor polynucleotide, and other nucleic acid coding sequences) can be introduced into the genome of an embryo or cell (e.g., stem cell) using any suitable method. The present application contemplates the use of various gene editing techniques, for example, to delete nucleic acid from the genome of an embryo or cell to create a knockout mouse, or to introduce nucleic acid into the genome of an embryo or cell to create a transgenic mouse. Non-limiting examples include programmable nuclease-based systems, such as clustered regularly interspaced short palindromic repeats (CRISPR) systems, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). See, e.g., Carroll D, Genetics., 2011;188(4):773-782; Joung JK et al., Nat Rev Mol Cell Biol., 2013;14(1):49-55; and Gaj T et al., Trends Biotechnol., 2013 Jul;31(7):397-405, each of which is incorporated by reference herein.

[0163] In some embodiments, the CRISPR system is used to edit the genome of mouse (e.g., mouse) embryos provided herein.See, for example, Harms DW et al., Curr Protoc Hum Genet., 2014;83:15.7.1-15.7.27; and Inui M et al., Sci Rep., 2014;4:5396 (each of which is incorporated herein by reference).For example, Cas9 mRNA or Cas9 protein, one or more guide RNAs (gRNAs), and / or donor nucleic acid can be directly delivered (e.g., injected or electroporated) into one-cell (zygote) stage or later stage mouse embryos to promote homology-directed repair (HDR), for example, to delete nucleic acid sequences from genome, or to introduce engineered nucleic acid (e.g., donor nucleic acid) into genome.

[0164] The CRISPR / Cas system is a naturally occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided DNA targeting platform for gene editing. Engineered CRISPR systems contain two main components: a guide RNA (gRNA) and a CRISPR-associated endonuclease (e.g., a Cas protein). The gRNA is a short synthetic RNA composed of a scaffold sequence for nuclease binding and a user-defined nucleotide spacer (e.g., about 15-25 nucleotides, or about 20 nucleotides) that defines the genomic target (e.g., gene) to be modified. Thus, one can change the genomic target of the Cas protein by simply changing the target sequence present in the gRNA. In some embodiments, the Cas9 endonuclease is derived from Streptococcus pyogenes (NGG PAM) or Staphylococcus aureus (NNGRRT or NNGRR(N) PAM), although other Cas9 homologs, Cas9 orthologs, and / or Cas9 variants (e.g., evolved versions of Cas9) may be used as provided herein. Further non-limiting examples of RNA-guided nucleases that can be used as provided herein include Cpf1 (TTN PAM); SpCas9 D1135E variant (NGG (reduced NAG binding) PAM); SpCas9 VRER variant (NGCG PAM); SpCas9 EQR variant (NGAG PAM); SpCas9 VQR variant (NGAN or NGNG PAM); Cas9 from Neisseria meningitidis (NM) (NNNNGATT PAM); Cas9 from Streptococcus thermophilus (ST) (NNAGAAW PAM); and Cas9 from Treponema denticola (TD) (NAAAAC). In some embodiments, the CRISPR-associated endonuclease is selected from Cas9, Cpf1, C2c1, and C2c3. In some embodiments, the Cas nuclease is Cas9.

[0165] Guide RNA comprises at least a spacer sequence that hybridizes (binds) to target nucleic acid sequence and a CRISPR repeat sequence that binds to its endonuclease and guides its endonuclease to its target nucleic acid sequence.As understood by those skilled in the art, each gRNA is designed to comprise a spacer sequence that is complementary to its genome target sequence.See, for example, Jinek et al., Science, 2012;337:816-821 and Deltcheva et al., Nature, 2011;471:602-607 (each of which is incorporated herein by reference).

[0166] In some embodiments, the RNA-guided nuclease and the gRNA are complexed to form a ribonucleoprotein (RNP) prior to delivery to the embryo.

[0167] The concentration of the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease may vary. In some embodiments, the concentration is 100ng / μl to 1000ng / μl. For example, the concentration may be 100ng / μl, 150ng / μl, 200ng / μl, 250ng / μl, 300ng / μl, 350ng / μl, 400ng / μl, 450ng / μl, 500ng / μl, 550ng / μl, 600ng / μl, 650ng / μl, 700ng / μl, 750ng / μl, 800ng / μl, 850ng / μl, 900ng / μl, 950ng / μl, or 1000ng / μl. In some embodiments, the concentration is between 100 ng / μl and 500 ng / μl, or between 200 ng / μl and 500 ng / μl.

[0168] The concentration of gRNA can also be varied. In some embodiments, the concentration is 200ng / μl to 2000ng / μl. For example, the concentration can be 200ng / μl, 300ng / μl, 400ng / μl, 500ng / μl, 600ng / μl, 700ng / μl, 800ng / μl, 900ng / μl, 1000ng / μl, 1100ng / μl, 1200ng / μl, 1300ng / μl, 1400ng / μl, 1500ng / μl, 1600ng / μl, 1700ng / μl, 1700ng / μl, 1900ng / μl, or 2000ng / μl. In some embodiments, the concentration is 500ng / μl to 1000ng / μl. In some embodiments, the concentration is 100ng / μl to 1000ng / μl. For example, the concentration can be 100ng / μl, 150ng / μl, 200ng / μl, 250ng / μl, 300ng / μl, 350ng / μl, 400ng / μl, 450ng / μl, 500ng / μl, 550ng / μl, 600ng / μl, 650ng / μl, 700ng / μl, 750ng / μl, 800ng / μl, 850ng / μl, 900ng / μl, 950ng / μl, or 1000ng / μl.

[0169] In some embodiments, the ratio of the concentration of the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease to the concentration of the gRNA is 2: 1. In other embodiments, the ratio of the concentration of the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease to the concentration of the gRNA is 1: 1.

[0170] Donor nucleic acid typically comprises a sequence of interest flanked by homology arms. Homology arms are ssDNA regions that are homologous to genomic DNA regions located in genomic loci. One homology arm is located on the left (5') side of the genomic region of interest (where the sequence of interest is inserted) (left homology arm), and another homology arm is located on the right (3') side of the genomic region of interest (right homology arm). These homology arms allow homologous recombination between the ssDNA donor and genomic locus, resulting in the insertion of the sequence of interest into the genomic locus of interest (e.g., via CRISPR / Cas9-mediated homology-directed repair (HDR)).

[0171] The homology arms can vary in length. For example, each homology arm (left and right homology arms) can have a length of 20 to 1000 nucleotide bases. In some embodiments, each homology arm has a length of 20 to 200 nucleotide bases, 20 to 300 nucleotide bases, 20 to 400 nucleotide bases, 20 to 500 nucleotide bases, 20 to 600 nucleotide bases, 20 to 700 nucleotide bases, 20 to 800 nucleotide bases, or 20 to 900 nucleotide bases. In some embodiments, each homology arm has a length of 20 nucleotide bases, 30 nucleotide bases, 40 nucleotide bases, 50 nucleotide bases, 60 nucleotide bases, 70 nucleotide bases, 80 nucleotide bases, 90 nucleotide bases, 100 nucleotide bases, 150 nucleotide bases, 200 nucleotide bases, 250 nucleotide bases, 300 nucleotide bases, 350 nucleotide bases, 400 nucleotide bases, 450 nucleotide bases, 500 nucleotide bases, 550 nucleotide bases, 600 nucleotide bases, 650 nucleotide bases, 700 nucleotide bases, 750 nucleotide bases, 800 nucleotide bases, 850 nucleotide bases, 900 nucleotide bases, 950 nucleotide bases, or 1000 nucleotide bases. In some embodiments, the length of one homology arm is different from the length of the other homology arm. For example, one homology arm can have a length of 20 nucleotide bases, and the other homology arm can have a length of 50 nucleotide bases. In some embodiments, the donor DNA is single-stranded. In some embodiments, the donor DNA is double stranded. In some embodiments, the donor DNA is modified, for example, via phosphorothiolation. Other modifications can be made. EXAMPLES

[0172] Example 1 NSG-Fcgr1 null Mice and NSG-Fcgr1null Generation of Tg(Hu-IL15) mice CRISPR-Cas9 genome editing was used to delete exons 3 to 6 from the mouse Fcgr1 gene in NSG™ mice (JAX line number 005557) and NSG™ mice transgenically expressing human IL15 (NSG-Tg(Hu-IL15)) (JAX line number 030890). The deletion of exons 3 to 6 is predicted to result in an approximately 8500 base pair deletion. The mouse Fcgr1 locus encodes a 404 amino acid protein-encoding transcript (Fcgr1-201) and a 50 amino acid nonsense-mediated decay transcript (Fcgr1-202). CRISPR-Cas9 gene primers were designed upstream (Figure 1) and downstream (Figure 2) of the targeted exons 3 to 6. See Table 1 below. Table 1. CRISPS-Cas9 genome editing targeting strategy to delete exon 3 to exon 6 of the Fcgr1 gene in NSG™ and NSG-Tg(Hu-IL15) mice. [Table 1-1] [Table 1-2]

[0173] The risk of off-target genome editing for the CRISPR-Cas9 primers was evaluated. If the CRISPR-Cas9 primers have a non-canonical protospacer adjacent motif (PAM) linked to them, the risk of off-target genome editing is considered high if the score is <20. All four primers have a non-canonical linked one, and their risk of off-target editing is <3.5, so the risk of off-target genome editing is minimal.

[0174] A deletion of exon 3 through exon 6 of mouse Fcgr1 is predicted to result in a mutant allele, which can be detected by sequencing in the forward direction starting upstream of the targeted exon 3 and / or in the reverse direction downstream of exon 6 (Figure 3). This deletion is also predicted to result in a premature truncation after amino acid 26 of the transcript encoding the Fcgr1-201 protein (Figure 4).

[0175] Fcgr1 deletion from exon 3 to exon 6 (Fcgr1 null We generated 19 founder lines of NSG™ mice heterozygous for the Fcgr1 exon 3 to exon 6 deletion (Fcgr1 null Six established lines of NSG-Tg(Hu-IL15) mice heterozygous for IL15 were generated (Figure 6).

[0176] Example 2 NSG-Fcgr1 null Decreased clearance of human IgG1 antibodies in mice Clearance of the therapeutic human immunoglobulin 1 (IgG1) antibody trastuzumab (Herceptin®) was assessed in NSG™ mice and NSG-Fcgr1 null Trastuzumab is approved for early breast cancer that is human epidermal growth factor receptor 2 (HER2+). null Groups of mice and groups of five NSG™ control mice were injected intravenously with 200 μg of trastuzumab. In a 25 g mouse, this is a dose of approximately 8 mg / kg. Mice were bled 1, 2, 7, 14, 21, 28, and 35 days after trastuzumab injection and serum was frozen. The percentage of the initial trastuzumab dose remaining was determined by enzyme-linked immunosorbent assay (ELISA). Trastuzumab was rapidly cleared in NSG™ mice (1% remaining 21 days after injection), whereas NSG-Fcgr1 mice were rapidly cleared (Figure 1). null There was decreased clearance and increased persistence of trastuzumab in mice (approximately 40% remaining 35 days after injection) (FIG. 7).

[0177] Two separate programs (PKAnalix2019 and R_Studio) were used to analyze NSG™ mice and NSG-Fcgr1 null The pharmacokinetics of trastuzumab clearance in mice was calculated. NSG-Fcgr1 null The half-life of trastuzumab in mice was approximately 24 days compared to approximately 5 days in NSG™ mice (Figure 8). Thus, knocking out the Fcgr1 gene in NSG™ mice resulted in a five-fold increase in the half-life of trastuzumab in the circulation. This increased half-life is similar to the half-life of trastuzumab in humans (Leveque et al., Curr Clin Pharmacol., (2008), 3(1):51-55).

[0178] Example 3. NSG-Fcgr1 null Decreased clearance of human IgG4 antibodies in mice One of the most efficient approaches for the activation of antitumor immunity is the blockade of immune checkpoints. Tumors use immune checkpoints to suppress antitumor immune responses. Blockade of immune checkpoint proteins (e.g., programmed cell death protein 1 (PD-1)) has presented broad and diverse opportunities to enhance antitumor immunity with the ability to generate durable clinical responses. Pembrolizumab (Keytruda®) is one of the most widely used immune checkpoint blockade monoclonal antibodies (mAbs). Pembrolizumab is a humanized IgG4-κ mAb that recognizes human PD-1. PD-1 inhibits the activation of CD4 + T cells, CD8 + T cells, activated CD4 + Controllability T(T reg) cells, B cells, and natural killer (NK) cells. PD-1 is also constitutively expressed on tumor-infiltrating lymphocytes (TILs) in various tumor types, reflecting an exhausted T cell state. PD-1 binds to two ligands: PD-1 ligand 1 (PD-LI; also known as B7-H1) and PD-L2 (B7-DC). PD-L1 is widely expressed on healthy (non-cancerous) and malignant (cancerous) cells, while PD-L2 is expressed primarily on antigen-presenting cells. Binding of PD-L1 to PD-1 results in inhibition of T cell activation and effector function mediated by recruitment of tyrosine phosphatases to the immune synapse that disrupt T cell receptor signaling. Mounting evidence has shown that PD-L1 expression is normally upregulated in many different types of human cancers, including melanoma, lung tumors, and ovarian tumors.

[0179] NSG™ mice were implanted with human tumor cell lines and patient-derived tumor xenografts, and then Treatment with multiple injections of the human IgG4 therapeutic antibody pembrolizumab (Keytruda®) at 5 mg / kg to 10 mg / kg every 5 days results in slower tumor growth in some, but not all, experiments. The reduced efficacy of pembrolizumab against human tumor therapy in NSG™ mice may be related to the rapid clearance of this human IgG4 antibody due to increased activity of its mouse Fcgr1 receptor.

[0180] To test this hypothesis, we assessed the clearance of pembrolizumab in NSG™ mice and NSG-Fcgr1 mice. null The results were compared between five NSG-Fcgr1 mice and five NSG-Fcgr1 mice. nullGroups of mice and groups of five NSG™ control mice were injected intravenously with 25 μg pembrolizumab. In a 25 g mouse, this is a dose of approximately 1 mg / kg. Mice were bled 1-35 days after pembrolizumab injection and serum was frozen. The percentage of the initial pembrolizumab dose remaining was determined by enzyme-linked immunosorbent assay (ELISA). Pembrolizumab was rapidly cleared in NSG™ mice (>1% remaining by day 6 post-injection), whereas NSG-Fcgr1 mice were rapidly cleared. null There was decreased clearance and increased persistence of pembrolizumab in mice (approximately 30% remaining 35 days after injection) (FIG. 9).

[0181] Two separate programs (PKAnalix2019 and R_Studio) were used to analyze NSG™ mice and NSG-Fcgr1 null The pharmacokinetics of pembrolizumab clearance in mice was calculated. NSG-Fcgr1 null The half-life of pembrolizumab in mice is approximately 19 days, compared to only about 0.6 days in NSG™ mice (Figure 10). Thus, knocking out the Fcgr1 gene in NSG™ mice resulted in a 30-fold increase in the half-life of pembrolizumab in circulation. This increased half-life is similar to the half-life of pembrolizumab in humans (Centanni et al., Clinical Pharmacokinetics, (2019), 58:85-857).

[0182] Example 4. NSG-Fcgr1 null Increased circulating human IgG antibody levels in mice Attempts to generate human IgG antibodies following immunization of NSG™ mice engrafted with human CD34+ hematopoietic stem cells (HSCs) have been limited by the short half-life of circulating human IgG. NSG™ mice and NSG-Fcgr1 null Mice were transplanted with human umbilical cord HSCs, and human IgG levels were measured in plasma 21 weeks after transplantation. nullMice had 20-fold higher circulating human IgG levels compared to NSG™ mice (Figure 11). null This is consistent with the increased persistence of human IgG therapeutic antibodies (trastuzumab and pembrolizumab) in the circulation of mice compared to NSG™ mice.

[0183] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which may include the entirety of that document. The indefinite articles "a" and "an," as used herein in the specification and claims, should be understood to mean "at least one," unless clearly indicated to the contrary.

[0184] It is also to be understood that, unless expressly indicated to the contrary, in any method claimed in this document that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0185] In the claims and the above specification, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0186] The terms "about" and "substantially" preceding a numerical value mean ±10% of the stated numerical value.

[0187] When a range of values ​​is provided, each value between the upper and lower limits of the range is specifically contemplated and described herein.

Claims

1. Mouse Fcγ receptor 1 null (Fcgr1 ｎｕｌｌ ) Allele and mouse interleukin-2 receptor γ null (IL-2Rγ ｎｕｌｌ Immunodeficient mice containing alleles.

2. An immunodeficient mouse according to claim 1, comprising a non-obesity diabetes mellitus (NOD) genetic background.

3. The aforementioned IL-2Rγ ｎｕｌｌ Regarding the allele, it is homozygous and the aforementioned Fcgr1 ｎｕｌｌ The immunodeficient mouse according to claim 1, wherein the allele is homozygous.

4. Prkdc ｓｃｉｄ An immunodeficient mouse according to any one of claims 1 to 3, further comprising an allele and / or a Rag1 null allele.

5. The aforementioned Prkdc ｓｃｉｄ The immunodeficient mouse according to claim 4, wherein it is homozygous for the allele and / or the Rag1 null allele.

6. Mouse lacking T cells, B cells, and / or natural killer (NK) cells, and / or The aforementioned mice exhibit a deficiency in the function of macrophages and / or dendritic cells. An immunodeficient mouse according to any one of claims 1 to 3.

7. An immunodeficient mouse according to any one of claims 1 to 3, which has been transplanted with human hematopoietic stem cells (HSCs) or human peripheral blood mononuclear cells (PBMCs).

8. An immunodeficient mouse according to any one of claims 1 to 3, which has been transplanted with human diseased cells.

9. The immunodeficient mouse according to claim 8, wherein the human diseased cells are human tumor cells.

10. The immunodeficient mouse according to claim 9, wherein the human tumor cells are human cancer cells.

11. The mouse Fcgr1 ｎｕｌｌ The immunodeficient mouse according to any one of claims 1 to 3, wherein the allele contains a deletion in the region of exon 3 to exon 6 compared to the unmodified endogenous mouse Fcgr1 gene.

12. An immunodeficient mouse according to any one of claims 1 to 3, further comprising a human IL-15 transgene.

13. A method comprising the step of administering an antibody to an immunodeficient mouse according to any one of claims 1 to 3.

14. The method according to claim 13, further comprising the step of assaying a biological sample derived from the mouse for the therapeutic effect of the antibody and / or the circulating level of the antibody.

15. A method comprising the step of administering human cells to an immunodeficient mouse according to any one of claims 1 to 3.

16. The method according to claim 15, wherein the human cells are selected from human hematopoietic stem cells (HSCs), human peripheral blood mononuclear cells (PBMCs), and human diseased cells.

17. Mouse Fcγ receptor 1 null (Fcgr1 ｎｕｌｌ ) immunodeficient mice containing the allele, NOD.Cg-Prkdc ｓｃｉｄ Il2rg ｔｍ１Ｗｊｌ / SzJ background, immunodeficient mice.

18. A guide RNA containing one of the sequences from sequence numbers 2 to 5.

19. A method for producing an immunodeficient mouse according to any one of claims 1 to 3, A step of introducing one or more guide RNAs and Cas proteins into a mouse embryo; The process of transplanting the mouse embryo into a pseudo-pregnant female mouse; A step of collecting F1 mice born from the pseudo-pregnant female; and The process of breeding the F1 mice. Methods that include...

20. The method according to claim 19, wherein the one or more guide RNAs bind to the region upstream of exon 3 and / or downstream of exon 6 of the mouse Fcgr1 gene, and are optionally the guide RNAs described in claim 19.

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