Immunodeficiency FCGR1 knockout mouse model

Abstract

In some aspects, the disclosure provides an immunodeficient NOD-Fcgr1null mouse that can be used to measure the pharmacokinetics and activity of human IgG antibodies, to produce human IgG antibodies, and to model human disease treatment.

Description

[0001] Government licensing rights

[0002] This invention was made with government support under Grant AI132963 from the National Institutes of Health. The government has certain rights in this invention.

[0003] Related Applications

[0004] Pursuant to 35 USC §119(e), this application claims the benefit of U.S. Provisional Application No. 63 / 323,773, filed on March 25, 2022, the entire contents of which are incorporated herein by reference.

[0005] References to Electronic Sequence Listings

[0006] The contents of the electronic sequence listing (J022770112WO00-SEQ-EMB.xml; size: 25,766 bytes; and creation date: March 23, 2023) are incorporated herein by reference in their entirety. Background Art

[0007] Crystallizable fragment (Fc) receptor is a cell surface protein that binds immunoglobulin (Ig) antibody and internalizes Ig antibody into cells. Internalized Ig antibody can be recycled to the cell surface to re-enter the circulation or transported to lysosome. The Ig antibody transported to lysosome is degraded into peptides, which are loaded onto major histocompatibility class I (MHC-I) and major histocompatibility class II (MHC-II) proteins to present as antigens on the cell surface. These antigens regulate immune cell signaling.

[0008] 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 neonatal Fc receptor (FcRn) (Bruhns P., Blood, 2012; 119 (24): 5640-5649). FcγRI, FcγRIII, FcγRIV, and FcRn activate immune signaling, and 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; and natural killer cells and natural killer T cells express FcγRIII. During the prenatal period, neutrophils, spleen monocytes, B cells, vascular endothelial cells, and epithelial cells in the intestine express FcRn. Summary of the invention

[0009] Some aspects of the present disclosure provide an immunodeficient mouse comprising a mouse Fcγ receptor 1 null (Fcgr1 null ) allele and mice with interleukin-2 receptor γ null (IL-2Rγ null ) allele.

[0010] In some embodiments, the mouse has a non-obese diabetic (NOD) genetic background (eg, NOD / ShiLtJ).

[0011] In some embodiments, the mouse is resistant to IL-2Rγ null The allele is homozygous and for Fcgr1 null The alleles are homozygous.

[0012] In some embodiments, the mouse further comprises Prkdc scid Allele.

[0013] In some embodiments, the mouse is for Prkdc scid The alleles are homozygous.

[0014] In some embodiments, the mouse further comprises Rag1 null Allele.

[0015] In some embodiments, the mouse is for Rag1 null The alleles are homozygous.

[0016] Some aspects of the present disclosure provide an immunodeficient mouse comprising a mouse Fcγ receptor 1 null (Fcgr1 null ) allele, wherein the mouse comprises NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ background.

[0017] In some embodiments, the mouse lacks mouse T cells, B cells, and / or Natural Killer (NK) cells.

[0018] In some embodiments, macrophage and / or dendritic cell function is defective in the mouse.

[0019] In some embodiments, the immunodeficient mice are transplanted with human hematopoietic stem cells (HSCs).

[0020] In some embodiments, the immunodeficient mice are transplanted with human peripheral blood mononuclear cells (PBMCs).

[0021] In some embodiments, the immunodeficient mice are transplanted with human diseased cells.

[0022] In some embodiments, the human diseased cells are human tumor cells.

[0023] In some embodiments, the human tumor cell is a human cancer cell.

[0024] In some embodiments, mouse Fcgr1 null The allele comprises a deletion in the region of exons 3-6 relative to the endogenous unmodified mouse Fcgr1 gene. In some embodiments, the mouse Fcgr1 null The allele contains a deletion of exons 3-6.

[0025] 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.

[0026] Other aspects of the present disclosure provide a method comprising administering an antibody to an immunodeficient mouse as described herein.

[0027] In some embodiments, the antibody is a monoclonal antibody.

[0028] In some embodiments, the antibody is a human or humanized antibody.

[0029] In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

[0030] 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.

[0031] In some embodiments, the method further comprises determining the therapeutic effect of the antibody in a biological sample from the mouse.

[0032] In some embodiments, the therapeutic effect is a decrease in the growth of human diseased cells relative to a control.

[0033] In some embodiments, the method further comprises measuring circulating levels of the antibody in a biological sample from the mouse.

[0034] In some embodiments, the circulating level of the antibody is at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold greater relative to a control.

[0035] In some embodiments, the assay is at least 21 days, at least 28 days, or at least 35 days after administration of the antibody.

[0036] Some aspects of the present disclosure provide a method comprising administering a human cell to an immunodeficient mouse as described herein. In some embodiments, the human cell is an HSC. In other embodiments, the human cell is a human PBMC. In some embodiments, the human cell is a human diseased cell. In some embodiments, the human diseased cell is a human tumor cell. In some embodiments, the human tumor cell is a human cancer cell.

[0037] Other aspects of the present disclosure provide a guide RNA comprising the sequence of any one of SEQ ID NOs: 2-5.

[0038] Still other aspects of the present disclosure provide a method for producing the immunodeficient mice described herein, comprising introducing one or more guide RNAs and Cas proteins into mouse embryos (e.g., having any of the immunodeficient genetic backgrounds described herein); transplanting the mouse embryos into pseudopregnant female mice; collecting F1 mice born from the pseudopregnant females; and breeding the F1 mice.

[0039] In some embodiments, one or more guide RNAs bind to a region upstream of exon 3 and / or downstream of exon 6 of the mouse Fcgr1 gene, and optionally the guide RNA comprises a sequence of any one of SEQ ID NOs: 2-5.

[0040] In some embodiments, the mouse embryo is an immunodeficient mouse embryo, optionally having a NOD genetic background, and optionally comprising (a) IL-2Rγ null Alleles and (b) Prkdc scid Allele or Rag1 null One or more of the alleles. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 . Upstream targeting strategy for mouse Fcgr1 deletion alleles.

[0042] Figure 2 . Downstream mouse Fcgr1 deletion allele targeting strategy.

[0043] Figure 3 . Sequencing strategy for mouse Fcgr1 deletion alleles.

[0044] Figure 4 Predicted protein translation of mouse Fcgr1 deletion alleles. The deletion of Fcgr1 exons 3-6 is predicted to encode an Fcgr1 protein with an early truncation after amino acid 26.

[0045] Figure 5 .NSG-Fcgr1 nullFounder results of mice. "wild" is wild type, "het" is Fcgr1 null Heterozygous mice, while "hom" is Fcgr1 null Homozygous mice. Bold text indicates NSG-Fcgr1 heterozygous only for the targeted deletion of exons 3-6 of Fcgr1 null Mouse.

[0046] Figure 6 .NSG-Fcgr1 null (Hu-IL15) mouse founder results. "wild" is wild type, "het" is Fcgr1 null Heterozygous mice, while "hom" is Fcgr1 null Homozygous mice. Bold text indicates live-born NSG-Fcgr1 heterozygous for targeted deletion of exons 3-6 of Fcgr1 null Tg(Hu-IL15) mice.

[0047] Figure 7 .With NSG TM Compared with mice, NSG-Fcgr1 null Trastuzumab in mouse circulation IgG antibodies increase survival.

[0048] Figure 8 .NSG TM and NSG-Fcgr1 null (NSG TM Pharmacokinetic analysis of trastuzumab IgG antibody in Fcgr1KO) mice. "SD" is standard deviation, "CV" is variability, "AUC" is area under the curve, "λ.z" is the estimated terminal elimination rate constant, "Vss" is the steady-state distribution volume, "CL" is the trastuzumab clearance, and "Vd" is the distribution volume.

[0049] Fig. 9 .With NSG TM Compared with mice, NSG-Fcgr1 null Pembrolizumab in mouse circulation IgG antibodies increase survival.

[0050] Fig.10 .NSG TM and NSG-Fcgr1 null (NSG TM Pharmacokinetic analysis of pembrolizumab IgG antibody in Fcgr1 KO) mice.

[0051] Fig.11 .With transplantation of CD34 + NSG of HSCTM Compared with mice, NSG-Fcgr1 null Mouse transplantation CD34 + The levels of human IgG antibodies increased after umbilical cord hematopoietic stem cells (HSC) were generated. DETAILED DESCRIPTION

[0052] Human immunoglobulins (IgG) are widely used as therapeutic antibodies. However, mouse models for testing the pharmacokinetics and efficacy of new antibodies are limited. This is partly because human IgG antibodies bind to the mouse FcγR1 receptor (encoded by the Fcgr1 gene) with high affinity, resulting in rapid clearance of the antibody, which in turn greatly reduces the half-life of the antibody. For example, in immunodeficient NSG TM Mouse model (i.e. NOD.Cg-Prkdc scid Iq tm1Wjl Human IgG administered by immunodeficiency mouse models (i.e., IgG1 / IgG2) is cleared much faster than those administered to humans. This rapid clearance rate limits the use of immunodeficient mouse models for evaluating 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).

[0053] In some aspects, the present disclosure provides an immunodeficient mouse model that does not express FcγR1 (e.g., an immunodeficient Fcgr1 gene knockout, such as NSG-Fcgr1 null ) to advance the field. The generation of this genetically engineered mouse model involves a complex set of experimental procedures, including the design and implementation of gene editing, as well as a carefully planned breeding program. TM Background strain and NSG containing human interleukin-15 (IL-15) transgene TM In the background strain (i.e., NSG-Tg(Hu-IL15)), CRISPR / Cas9 gene editing was used to edit out (delete) exons 3-6 of the endogenous mouse Fcgr1 gene. NSG-Fcgr1 null The Hu-Tg(Hu-IL15) mouse model has the additional advantage of generating human natural killer (NK) cells to induce antibody-dependent cell-mediated cytotoxicity (ADCC) in response to human IgG.

[0054] The data presented herein show that, in some cases, administration of immunodeficient Fcgr1 null The circulating half-life of human IgG antibodies in mice was unexpectedly increased by about 20-fold to about 30-fold. nullMice produce approximately 20-fold higher levels of circulating human IgG antibodies after transplantation of human hematopoietic stem cells (HSCs). Thus, the present disclosure provides an immunodeficient mouse model for the field that can be used to more accurately assess the pharmacokinetics and efficacy of human monoclonal antibody therapies, for example, particularly for human cancers and other diseases.

[0055] Immunodeficiency Fcgr1 null Mouse Model

[0056] In some aspects, the present disclosure provides immunodeficient mice in which Fc receptors (FcR) are knocked out. Therefore, the immunodeficient mice provided herein include FcR "null" alleles-alleles encoding non-functional products. Immunoglobulin Fc receptors are membrane molecules expressed by several hematopoietic stem cell populations, which bind to the Fc region of immunoglobulins (Ig). Non-limiting examples of Fc receptors that can be knocked out in immunodeficient mouse strains include: Fcγ receptors (FcγR / FcgR), Fcε receptors (FcεR, FceR), Fcα receptors (FcαR, FcaR), Fcμ receptors (FcμR, FcmR), neonatal Fc receptors (FcRn) or combinations thereof.

[0057] In some embodiments, Fcγ receptors are knocked out in immunodeficient mice. Non-limiting examples of Fcγ receptors include those encoded by Fcgr1 (CD64), Fcgr2, Fcgr3 (CD16), Fcgr3a (CD32a), Fcgr3b (CD16b), Fcgr3c (CD16c) and Fcgr4. Fcγ receptors bind to the Fc region of immunoglobulin gamma (IgG) molecules. For example, Fcγ receptors bind to molecules coated with IgG, such as opsonized pathogens and immune complexes. The crosslinking between Fcγ receptors and IgG leads to internalization and degradation of molecules coated with IgG. Therefore, in some aspects, the present disclosure provides mice with reduced IgG internalization and degradation. Fcγ receptors bind to IgG and internalize IgG into cells through the crystallizable (Fc) region. Once internalized, both the receptor and IgG are delivered to and degraded by lysosomes. This degradation of IgG prevents it from recycling into the systemic circulation. Part of the degraded IgG molecules are then loaded onto major histocompatibility complex class I (MHC-I) and major histocompatibility complex class II (MHC-II) molecules for presentation as antigens on the cell surface.

[0058] In some embodiments, the Fcgr1 gene is knocked out in an immunodeficient mouse (e.g., Ensembl #ENSMUSG00000015947; GenBank NM_010186). Such mice may be referred to herein as Fcgr1 null Mouse. Fcgr1 nullMice do not produce detectable levels of mouse FcγR1 protein (eg, GenBank NP_034316.1).

[0059]

[0060]

[0061] The mouse Fcgr1 gene can be knocked out by deleting or introducing a modification (e.g., mutation) 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 encode 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.

[0062] Mouse Fcgr1 has 6 exons, represented as exon 1-exon 6. In some embodiments, the immunodeficient mouse model provided herein has a deletion or modification in the exons of the endogenous Fcgr1 gene. For example, the mouse can have a deletion or modification in any one or more of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 of the endogenous Fcgr1 gene. In some embodiments, the immunodeficient mouse model has a deletion or modification in exons 3-6 of the endogenous Fcgr1 gene. In some embodiments, the immunodeficient mouse model has a deletion of exons 3-6 of the endogenous Fcgr1 gene.

[0063] In some embodiments, the immunodeficiency Fcgr1 null Mouse also includes human interleukin (IL-15) transgenic. Transgenic is a nucleic acid (e.g., human transgenic in mouse) whose sequence is derived from species different from the species containing it. Transgenic can be expressed from nucleic acids in cells (e.g., in mice) or integrated into the genome of mice. Integration of transgenic (e.g., encoding human IL-15) can be carried out by any genome editing method described herein. Transgenic can be integrated into the genome of mice at the site of the orthologous gene of mice (e.g., integrated into the human IL-15 in mouse IL-15 gene) or integrated into another site in the mouse genome that does not destroy mouse function (e.g., safe harbor locus, such as Rosa26).

[0064] In some embodiments, the immunodeficiency Fcgr1 nullMice 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 inflammatory and protective immune responses to microbial invaders and parasites by regulating immune cells of the innate and adaptive immune systems. IL-15 plays a vital role in the development, differentiation and survival of natural killer (NK) cells that defend against viral infections, bacterial infections and tumor cells. NK cells require IL-15 for their development and maintenance in vivo. NK cells play a key role in antibody-dependent cellular cytotoxicity (ADCC). ADCC is an immune mechanism by which effector cells bearing Fc receptors bind to and kill antibody-coated target cells expressing tumor-derived or pathogen-derived antigens on their surface. Therefore, immunodeficient Fcgr1 cells that do not express IL-15 are more likely to be involved in the development, differentiation and survival of natural killer (NK) cells. null Mice transgenically expressing human interleukin-15 are immunodeficient in Fcgr1 null Mice (Tg(hu-IL15)) have increased NK cell development and maintenance and ADCC activity.

[0065] Mouse Model

[0066] Herein, for simplicity, reference is made to "mouse" and "mouse models" (e.g., surrogates for human conditions). It should be understood that, unless otherwise indicated, these terms are used interchangeably throughout the specification to encompass "rodents" and "rodent models," including mice, rats, and other rodent species.

[0067] It should also be understood that the standard genetic nomenclature used herein provides unique identification for different rodent strains, and the strain symbol conveys basic information about the type of strain or stock used and the genetic content of the strain. The International Committee on Standardized Genetic Nomenclature of Mouse has promulgated rules for symbolic naming of strains and stock. These rules can be obtained online from the Mouse Genome Database (MGD; informatics.jax.org) and published in printed copies (Lyon et al., 1996). The strain symbol usually includes a laboratory registration code (laboratory code). The first laboratory code attached to the strain symbol identifies and authenticates the founder of the strain. The laboratory code at the end of the strain symbol indicates the current source for obtaining the strain mouse. The different laboratory codes attached to the same strain symbol distinguish sub-strains and warn users that there may be genetic differences between different sub-strains. The laboratory code is assigned from the central registry to ensure that each is unique. The registry is kept at the Institute of Laboratory Animals (ILAR) of the National Academy of Sciences in Washington, D.C. The laboratory code can be obtained electronically on the website of ILAR (nas.edu / cls / ilarhome.nsf). See also Davisson MT, Genetic and Phenotypic Definition of Laboratory Mice and Rats / What Constitutes an Acceptable Genetic-Phenotypic Definition, National Research Council (US). Washington (DC): National Academies Press (US); 1999.

[0068] The mouse model of disease can be changed to enable the disease to be assessed. Any system (e.g., immune, respiratory, neural or circulatory), organ (e.g., blood, heart, blood vessel, spleen, thymus, lymph node or lung), tissue (e.g., epithelial, connective, muscle and neural) or cell type (e.g., lymphocyte or macrophage) can be changed independently or in combination to enable the disease to be studied in the model provided herein.

[0069] Three conventional methods for generating genome-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 can be used as provided herein. Genome editing methods using, for example, clustered regularly interspaced palindromic repeats (CRISPR / Cas) nucleases, transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs) are described elsewhere herein.

[0070] After delivering the nucleic acid to a fertilized embryo (e.g., a one-cell embryo (e.g., a zygote) or a multicellular embryo (e.g., a developmental stage after the zygote, such as a blastocyst), the fertilized embryo is transferred to a pseudopregnant female, which then produces offspring. The presence or absence of nucleic acid encoding human FcRn and / or chimeric IgG antibodies can be confirmed, for example, using a variety of genotyping methods (e.g., sequencing and / or genomic PCR).

[0071] As described in the examples herein, new mouse models can also be created by breeding parental lines. Utilizing various available mutants, knockouts, knock-ins, transgenics, Cre-lox, Tet induction systems and other mouse strains, multiple mutations and transgenics can be combined to produce new mouse models. Multiple mouse strains can be bred together to produce double, triple or even quadruple and higher multiple mutant / transgenic mice.

[0072] In some embodiments, breeding parental mice to produce F1 mice.Parental mice can be for example homozygous, heterozygous, hemizygous or homozygous invalid at specific allele place.Homozygous describes the genotype of two identical alleles at a given locus, heterozygous describes the genotype of two different alleles at the locus, hemizygous describes the genotype that is only composed of a single copy of a specific gene in other diploid organisms, and homozygous invalid refers to other diploid organisms that two copies of a gene are missing.

[0073] Immunodeficient mouse model

[0074] In some embodiments, provided herein are Fcγ receptor 1 (null) Fcgr1 null Alleles and mice null for interleukin-2 receptor gamma (IL-2Rγ null) allele. The allele is a variant of the wild-type gene (Fcgr1 null ) gene (e.g., Fcgr1). In some embodiments, the immunodeficient mouse model comprises a copy of a gene (e.g., Fcgr1) that is regulated by the Fcgr1 gene. null Allele, IL-2Rγ null Alleles and a transgene encoding human IL-15. In some embodiments, the immunodeficient mice are resistant to IL-2Rγ null In some embodiments, the immunodeficient mouse is homozygous for Fcgr1 null In some embodiments, the immunodeficient mouse is homozygous for IL-2Rγ null The allele is homozygous and for Fcgr1 null The allele is homozygous. Homozygous means that the immunodeficient mouse has two copies of the allele (e.g., Fcgr1 null Allele, IL-2Rγ null allele).

[0075] In some embodiments, the immunodeficient mouse comprises an interleukin-2 receptor gamma null (IL-2Rγ null ) allele. IL-2Rγ null The allele is a null mutation in the gene encoding the interleukin 2 receptor gamma chain (IL2Rγ, homologous to IL2RG in humans) that blocks natural killer (NK) cell differentiation, thereby eliminating the barrier 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).

[0076] In some embodiments, the immunodeficient mouse comprises Fcgr1 null Alleles and IL-2Rγ null alleles, also containing Prkdc scid Allele. Prkdc scid The mutation is a loss-of-function (null) mutation in the mouse homolog of the human PRKDC gene - this mutation essentially abolishes adaptive immunity (see, e.g., (Blunt et al., 1995; Greiner, Hesselton, & Shultz, 1998), each of which is incorporated herein by reference). In some embodiments, the immunodeficient Fcgr1 null -IL-2Rγ null Prkdc scid The alleles are homozygous.

[0077] In some embodiments, comprising Fcgr1 nullAlleles and IL-2Rγ null Immunodeficient mice also contain Rag1 null Allele. Rag1 null The mutation confers B and T cell deficiency on the mouse. In some embodiments, the immunodeficiency Fcgr1 null -IL-2Rγ null Mouse Rag1 null The alleles are homozygous.

[0078] As is known in the art, immunodeficient mice have an impaired or destroyed immune system, such as specific defects in MHC class I, MHC class II, or both, B cell or T cell defects, or both, natural killer (NK) cell defects, bone marrow defects (e.g., defects in granulocytes and / or monocytes), macrophage defects, dendritic cell defects, and immune deficiencies caused by gene knockdown of cytokines, cytokine receptors, TLR receptors, and various transducers and transcription factors of signaling pathways. Immunodeficient mouse models include single gene mutation models, such as nude (nu) strains and severe combined immunodeficiency (scid) strains, non-obese diabetic (NOD) strains, RAG (recombination activation gene) strains with targeted gene deletions, and various hybrids derived from crosses of double and triple mutant mouse strains with additional innate and adaptive immune deficiencies.

[0079] The impaired immune system can be measured by any method known in the art, including but not limited to: the production of mature immune cells (e.g., B cells, T cells, dendritic cells, macrophages, natural killer cells), defective endogenous cytokine signaling, limited infection resistance and reduced survival. In some embodiments, immunodeficient mice lack mature mouse T cells, lack mature mouse B cells, lack functional natural killer cells, and are endogenous (e.g., mouse) cytokine signaling defects. Mature T cells develop in the thymus and are released to 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 malignant and virally transformed cells without prior exposure. Endogenous (e.g., mouse) cytokine signaling is important in maintaining homeostasis and relies 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 insufficient to maintain immune system homeostasis compared to a non-defective endogenous immune system. Lack of mature cells (e.g., T cells or B cells), functional cells (e.g., natural killer cells), defective cytokine signaling, or some combination thereof, can be reduced by 10-99%, 5%-95%, 20%-90%, 30%-80%, 40%-70%, or 50%-60% compared to non-immunodeficient mice. Lack of mature cells (e.g., T cells or B cells), functional cells (e.g., natural killer cells), defective cytokine signaling, or some combination thereof, can be reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to non-immunodeficient mice.

[0080] The lack 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 for 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.

[0081] Immunodeficient mice can express any human cytokine or combination of human cytokines that increases the efficacy of immunodeficient mice as animal models (e.g., human IgG antibody pharmacokinetics or activity, human IgG antibody production, animal models of human diseases). 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 human cytokine types that can be expressed in human fibrosis models include: hematopoietic cytokines, lymphokines, monokines, interferons, and chemokines.

[0082] In some embodiments, immunodeficient mice express human hematopoietic cytokines. Human hematopoietic cytokines are extracellular proteins and 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.

[0083] In some embodiments, 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 kinds of human cytokines (e.g., human hematopoietic cytokines). In some embodiments, 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 kinds or more kinds of human cytokines. In some embodiments, the human cytokines expressed in immunodeficient mice participate in the persistence and development of human antibodies.

[0084] Human cytokines can be expressed in immunodeficient mice by any method known in the art, including but not limited to: from a transgenic in an immunodeficient mouse and from a virus (e.g., slow virus, adenovirus, adeno-associated virus) expressing a sequence encoding a human cytokine. A transgenic is a gene transferred from an organism (e.g., a human) to another organism (e.g., a mouse). In some embodiments, human cytokines are expressed by a transgenic. The transgenic in an immunodeficient mouse can be inserted into the genome of the immunodeficient mouse or encoded in a vector expressing the transgenic.

[0085] Non-limiting examples of spontaneous and transgenic immunodeficient mouse models include the following mouse strains:

[0086] Nude (nu) [Flanagan SP. Genet Res 1966; 8: 295-309; and Nehls M et al., Nature 1994; 372: 103-7];

[0087] 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];

[0088] NOD [Kikutani H et al., Adv Immunol 1992; 51: 285-322; and Anderson MS et al., Ann Rev Immunol 2005; 23: 447-85];

[0089] ·RAG1 and RAG2 (rag) [Mombaerts P et al., Cell 1992; 68: 869-77; Shinkai U et al., Cell 1992; 68: 855-67];

[0090] 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];

[0091] ·IL2rgnull [DiSanto JP et al., Proc Natl Acad Sci USA 1995;92:377-81];

[0092] ·B2mnull[Christianson SW et al., J Immunol 1997;158:3578-86];

[0093] 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];

[0094] 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];

[0095] 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;

[0096] NOG mice (NOD.cg-Prkdc scid Iq tm1Sug ) [Shultz LD et al., Nat Rev Immunol 2007;7:118-30]; and

[0097] BRG mice (BALB / c; 129S4-Rag2 tm1.1Flv ) [Shultz LD et al., Nat Rev Immunol 2007;7:118-30].

[0098] In some embodiments, provided herein is an immunodeficient mouse model having a non-obese diabetic (NOD) mouse genotype. NOD mice (e.g., Jackson Labs Stock #001976, NOD-Shi LtJ ) is a polygenic mouse model of autoimmune (e.g., type 1) diabetes characterized by hyperglycemia and insulitis, leukocyte infiltration of islet cells. NOD mice are hypoinsulinemia and hyperglucagonemia, indicating selective destruction of pancreatic beta cells. The main component of diabetes susceptibility in NOD mice is a unique MHC haplotype. NOD mice also show a variety of abnormal immune phenotypes, including defects in antigen presenting cell immunoregulatory function, defects in T lymphocyte reservoir regulation, defects in NK cell function, defects in cytokine production of macrophages (Fan et al., 2004), and impaired wound healing. They also lack hemolytic complement, C5. NOD mice are also severely hearing-impaired. Multiple mutations that cause immunodeficiency, targeted mutations in cytokine genes, and transgenics that affect immune function have been backcrossed into the NOD inbred strain background.

[0099] In some aspects of the present disclosure, the immunodeficient mice provided herein based on the NOD background have a gene selected from NOD-Cg.-Prkdc scid IL tm1wJl / SzJ(NSG TM )、NOD.Cg-Rag1 tm1Mom Iq tm1Wjl / SzJ(NRG) and NOD.Cg-Prkdc scid Iq tm1Sug / ShiJic(NOG) genetic background. Other immunodeficient mouse strains were considered in this paper.

[0100] In some embodiments, the NOD background-based immunodeficient mouse model has NOD-Cg.-Prkdc scid IL tm1wJl / SzJ(NSG TM )Genetic background. TMThe mouse (e.g., Jackson Labs Stock No.: #005557) is an immunodeficient mouse that lacks mature T cells, B cells, and NK cells, is defective in multiple cytokine signaling pathways, and has many defects in innate immunity (see, e.g., Shultz, Ishikawa, & Greiner, 2007; Shultz et al., 2005; and Shultz et al., 1995, each of which is incorporated herein by reference). NSG derived from the NOD mouse strain NOD / ShiLtJ (see, e.g., Makino et al., 1980, which is incorporated herein by reference) TM Mouse Prkdc scid mutation (also called the "severe combined immunodeficiency" mutation or "scid" mutation) and Il2rg tm1Wjl Targeted mutation. Il2rg tm1Wjl The mutation is a null mutation in the gene encoding the interleukin 2 receptor gamma chain (IL2Rγ, homologous to human IL2RG) that blocks NK cell differentiation, thereby eliminating the barrier 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).

[0101] In some embodiments, the immunodeficient mouse model has the NRG genotype. The NRG mouse (e.g., JacksonLabs Stock #007799) is extremely immunodeficient. The mouse contains two mutations on the NOD / ShiLtJ genetic background; a targeted knockout mutation in the recombination activation gene 1 (Rag1) and a complete null allele of the IL2 receptor common gamma chain (IL2rg null The extreme immunodeficiency of NRG allows for the efficient engraftment of human CD34 + Hematopoietic stem cells (HSCs) and patient-derived xenografts (PDXs) are used to humanize mice. Compared with mice with a scid mutation in the DNA repair enzyme Prkdc, immunodeficient NRG mice are more resistant to irradiation and genotoxic drugs.

[0102] In some embodiments, the immunodeficient mouse model is a NOG mouse. NOG mice (Ito M et al., Blood 2002) are extremely severe combined immunodeficient (scid) mice, which are established by combining NOD / scid mice and IL-2 receptor-γ chain knockout (IL2rγKO) mice (Ohbo K. et al., Blood 1996). NOG mice lack T and B cells, lack natural killer (NK) cells, show reduced dendritic cell function and reduced macrophage function, and lack complement activity.

[0103] In some embodiments, the immunodeficient mouse model has an NCG genotype. NCG mice (e.g., Charles River Stock #572) are generated by sequential CRISPR / Cas9 editing of the Prkdc and Il2rg loci in NOD / Nju mice, thereby generating mice that are homologous to NOD / Nju. NOD / Nju carries a mutation in the Sirpα (SIRPα) gene that allows transplantation of foreign hematopoietic stem cells. Prkdc knockout produces a SCID-like phenotype lacking proper T cell and B cell formation. Knockout of the Il2rg gene further exacerbates the SCID-like phenotype while additionally leading to a reduction in NK cell production.

[0104] In some embodiments, provided herein are immunodeficient mouse models of MHC class I, MHC class II, or MHC class I and MHC class II defects. MHC class I and / or MHC class II defective mice do not express the same level of MHC class I proteins (e.g., α-microglobulin and β2-microglobulin (B2M)) and / or MHC class II proteins (e.g., α chain and β chain) as non-immune-deficient (e.g., MHC class I / II wild-type) mice or do not have the same level of MHC class I and / or MHC class II protein activity. In some embodiments, relative to non-immune-deficient mice, the expression or activity of MHC class I and / or MHC class II proteins is reduced (e.g., reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more).

[0105] MHC class I, MHC class II, and immunodeficient mice deficient in MHC class I and MHC class II are described in International Publication No. WO 2018 / 209344, the contents of which are incorporated herein by reference.

[0106] NSG-SGM3 mice are NSG-derived mice NOD.Cg-Prkdc scid Iq tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav / MloySzJ (Jackson Laboratory Stock No:013062). 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 mouse with the expression of the human cytokines IL-3, GM-CSF, and SCF that support stable engraftment of myeloid lineages and regulatory T cell populations.

[0107] In some embodiments, the NSG mouse transgenic expresses human IL15. NSG-IL-15 mouse, NOD.Cg-Prkdc scid Iq tm1Wjl Tg(IL15)1Sz / SzJ (Jackson Laboratory Stock No: 030890), expressing human IL15 and combined with highly immunodeficient NOD scidγ (NSG) mice. In some embodiments, expression of human IL15 enhances the development of human NK cells in immunodeficient mice transplanted with human stem cells.

[0108] Therefore, the transgenic mice described herein can be bred to contain Fcγ receptor 1 (null) Fcgr1 null Alleles and mouse interleukin-2 receptor γ null (IL-2Rγ null In some embodiments, the transgenic mice described herein are generated by knocking out the Fcgr1 gene in NSG-IL-15 mice.

[0109] In some embodiments, the immunodeficient mouse is an NSG with Fcrg1 knockout TM Mouse (NSG-Fcgr1 null ). In some embodiments, NSG-Fcgr1 null Mice transgenic for expressing the gene encoding human interleukin-15 protein (NSG-Fcgr1 null Tg(Hu-IL15)). NSG-Fcgr1 null Tg(Hu-IL15) can express human IL-15 from any position in the mouse genome. In some embodiments, NSG-Fcgr1 is generated by knocking out Fcgr1 in NSG-Tg(Hu-IL15) mice described in U.S. Application No. 16 / 637621. null Tg(Hu-IL15) mice, which application is incorporated herein by reference.

[0110] Humanized Mouse Model

[0111] In some embodiments, humanized immunodeficient mouse models and methods for producing the models are provided herein. Immunodeficient mice transplanted with functional human cells and / or tissues are referred to as "humanized mice". As used herein, the terms "humanized mice", "humanized immunodeficient mice", "humanized immunodeficient mice" and their plural forms are used interchangeably to refer to immunodeficient mice humanized by implanting functional human cells and / or tissues. For example, the mouse model can be transplanted with human hematopoietic stem cells (HSC) (e.g., CD34+HSC) and / or human peripheral blood mononuclear cells (PMBC). In some embodiments, the mouse model is transplanted with human tissues, such as pancreatic islets, liver, skin and / or solid or blood cancers. In other embodiments, the mouse model can be genetically modified so that endogenous mouse genes are converted into human homologs (see, e.g., Pearson et al., Curr Protoc Immunol., 2008, Chapter: Unit-15.21).

[0112] By starting from immunodeficient mice (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks of immunodeficient mice), and if necessary, exhausting and / or suppressing any remaining mouse immune cells (e.g., chemically or with irradiation) to produce humanized mice. In other words, the successful survival of the human immune system in immunodeficient mice may require suppression of the immune system of mice to prevent GVHD (graft versus host disease) rejection. After the immune system of immunodeficient mice is fully suppressed, mice are transplanted with human cells (e.g., HSC and / or PBMC). As used herein, "transplantation" refers to the process in which human cells migrate to and merge into existing target tissues in vivo. With regard to humanized immunodeficient mice, implanted human cells reimplant functional human hematopoietic system and / or human immune system in mice.

[0113] In some embodiments, the human cells (e.g., HSC or PMBC) for humanized implantation are matched with the human cells (e.g., human cancer cells) human leukocyte antigen (HLA) of the mouse model. HLA matching refers to cells expressing the same major histocompatibility complex (MHC) genes. For example, the immunogenicity of human immune cells is reduced or prevented by implanting HLA-matched human xenografts and human immune cells to mice. In some embodiments, the humanized mice provided in the present disclosure are transplanted with human PMBC or human HSC that are HLA-matched with PDX or human cancer cell lines.

[0114] In some embodiments, the human cells (e.g., HSC or PMBC) used for humanized implantation are not HLA-matched 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 people PMBC or people HSC that are not HLA-matched with PDX or human cancer cell lines.

[0115] Myeloablation

[0116] As described above, in some embodiments, immunodeficient mice are treated to deplete and / or suppress any remaining murine immune cells (e.g., chemically and / or with irradiation). In some embodiments, immunodeficient mice are treated only chemically or only with irradiation. In other embodiments, immunodeficient mice are treated chemically and with irradiation.

[0117] In some embodiments, a myeloablative agent, a chemical agent that suppresses or depletes murine immune cells, is administered to an immunodeficient mouse. Examples of myeloablative agents include busulfan, dimethylmelane, melphalan, and thiotepa.

[0118] In some embodiments, immunodeficient mice are irradiated before transplanting human cells (such as people HSC and / or PMBC).It is believed that the irradiation of immunodeficient mice destroys the mouse immune cells in peripheral blood, spleen and bone marrow, which promotes the implantation (for example, by increasing human cell survival factor) and the amplification of other immune cells of human cells (such as people HSC and / or PMBC).Irradiation also shortens the time spent by accumulating the human immune cells of required number so that mouse model "humanization" is spent.

[0119] For immunodeficient mice (such as NSG TM In the case of mice, this preparation is usually accomplished by whole-body gamma irradiation. The size of the irradiator can vary 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 graph, the amount of irradiation required, and the ionizing energy source (i.e., X-rays versus gamma rays, which require a cesium or cobalt source).

[0120] Myeloablative irradiation doses are typically 700 to 1300 cGy, although in some embodiments, lower doses, such as 1-100 cGy (eg, about 2, 5, or 10 cGy) or 300-700 cGy, may be used.

[0121] As an example, 100cGy X-ray (or 75cGy-125cGy X-ray) irradiation mice can be used. In some embodiments, dosage is about 1, 2, 3, 4, 5, 10, 20, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 or 1300cGy, or between any two dosages as described herein, such as 100-300cGy, 200-500cGy, 600-1000cGy or 700-1300cGy. In some embodiments, immunodeficient mice are irradiated for about 15 minutes, 30 minutes, 45 minutes, 1 hour or longer before transplanting people's HSC and / or PMBC. In some embodiments, immunodeficient mice are transplanted with human HSCs and / or PMBCs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days after irradiation.

[0122] Transplantation (humanization)

[0123] As described above, in some embodiments, irradiated immunodeficient mice are transplanted with HSC and / or PBMC to humanize the mice. Transplantation refers to the process in which human cells migrate to and merge into an existing target tissue in vivo. PBMC can be implanted after irradiation and before human diseased cells (e.g., human cancer cells) are transplanted, after irradiation and simultaneously with human diseased cells, or after irradiation and after human diseased cells are transplanted.

[0124] PBMC

[0125] Peripheral blood mononuclear cells (PBMC) are peripheral blood cells with round nuclei. These mononuclear blood cells recirculate between tissues and blood, and are key components in the immune system to fight infection and adapt to invaders. There are two main types of mononuclear cells, lymphocytes and monocytes. The lymphocyte colony of PBMC generally includes T cells, B cells and NK cells.

[0126] For example, PBMCs can be isolated from a whole blood sample (eg, Ficoll gradient). In some embodiments, PBMCs from a subject (eg, a human subject) currently or previously diagnosed with cancer or an autoimmune disease can be used.

[0127] HSC

[0128] Hematopoietic stem cells (HSC) are stem cells that produce other blood cells in a process called hematopoiesis. Hematopoietic stem cells produce different types of blood cells in the lines called myeloid and lymphoid. Both myeloid and lymphoid lineages are involved in dendritic cell formation. Myeloid cells include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes and megakaryocytes to platelets. Lymphoid cells include T cells, B cells, natural killer cells and innate lymphocytes.

[0129] Methods for transplanting HSC and / or PBMC into immunodeficient mice to generate humanized mouse models include, but are not limited to, intraperitoneal or intravenous injection (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; Yaguchi et al., Cell & Mol Immunol, 2018, 15: 953-962). In some embodiments, mice are transplanted with 1.0×10 6 -3.0×10 7 In some embodiments, the mouse is transplanted with less than 1×10 7 In some embodiments, the mouse is transplanted with less than 1×10 6 About 5×10 6 In some embodiments, the mouse is transplanted with about 2×10 6 or 1×10 6 In some embodiments, the mouse is transplanted with 25,000-100,000 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).

[0130] For example, mice can be transplanted with 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×106 , 1.9×10 6 , 2.0×10 6 , 2.5×10 6 , 3.0×10 6 or more HSCs and / or PBMCs. In some embodiments, mice are transplanted with 1.0-1.1×10 6 1.0-1.2×10 6 1.0-1.3×10 6 1.0-1.4×10 6 1.0-1.5×10 6 1.0-1.6×10 6 1.0-1.7×10 6 1.0-1.8×10 6 1.0-1.9×10 6 1.0-2.0×10 6 1.0-2.25×10 6 1.0-2.5×10 6 1.0-2.75×10 6 1.0-3.0×10 6 , 1.1-1.2×10 6 , 1.1-1.3×10 6 1.1-1.4×10 6 1.1-1.5×10 6 1.1-1.6×10 6 , 1.1-1.7×10 6 1.1-1.8×10 6 1.1-1.9×10 6 1.1-2.0×10 6 1.1-2.25×10 6 1.1-2.5×10 6 1.1-2.75×10 6 1.1-3.0×10 6 , 1.2-1.3×10 6 , 1.2-1.4×10 6 1.2-1.5×10 6 1.2-1.6×10 6 , 1.2-1.7×10 6 1.2-1.8×10 6 , 1.2-1.9×10 6 1.2-2.0×10 6 1.2-2.25×10 6 1.2-2.5×106 、1.2-2.75×10 6 、1.2-3.0×10 6 、1.3-1.4×10 6 、1.3-1.5×10 6 、1.3-1.6×10 6 、1.3-1.7×10 6 、1.3-1.8×10 6 、1.3-1.9×10 6 、1.3-2.0×10 6 、1.3-2.25×10 6 、1.3-2.5×10 6 、1.3-2.75×10 6 、1.3-3.0×10 6 、1.4-1.5×10 6 、1.4-1.6×10 6 、1.4-1.7×10 6 、1.4-1.8×10 6 、1.4-1.9×10 6 、1.4-2.0×10 6 、1.4-2.25×10 6 、1.4-2.5×10 6 、1.4-2.75×10 6 、1.4-3.0×10 6 、1.5-1.6×10 6 、1.5-1.7×10 6 、1.5-1.8×10 6 、1.5-1.9×10 6 、1.5-2.0×10 6 、1.5-2.25×10 6 、1.5-2.5×10 6 、1.5-2.75×10 6 、1.5-3.0×10 6 、1.6-1.7×10 6 、1.6-1.8×10 6 、1.6-1.9×10 6 、1.6-2.0×10 6 、1.6-2.25×10 6 、1.6-2.5×10 6 、1.6-2.75×10 6 、1.6-3.0×10 6 、1.7-1.8×106 , 1.7-1.9×10 6 1.7-2.0×10 6 1.7-2.25×10 6 1.7-2.5×10 6 1.7-2.75×10 6 1.7-3.0×10 6 , 1.8-1.9×10 6 1.8-2.0×10 6 1.8-2.25×10 6 , 1.8-2.5×10 6 1.8-2.75×10 6 1.8-3.0×10 6 , 1.9-2.0×10 6 , 1.9-2.25×10 6 , 1.9-2.5×10 6 1.9-2.75×10 6 、1.9-3.0×10 6 2.0-2.25×10 6 2.0-2.5×10 6 2.0-2.75×10 6 , 2.0-3.0×10 6 2.25-2.5×10 6 2.25-2.75×10 6 2.25-3.0×10 6 2.5-2.75×10 6 , 2.5-3.0×10 6 or 2.75-3.0×10 6 HSCs and / or PBMCs.

[0131] In some embodiments, the mouse is transplanted with less than 1×10 7 PBMCs (e.g., 1×10 6 About 5×10 6 PBMCs, about 2×10 6 PBMCs, or about 1 × 10 6 PBMC) dose.

[0132] As described herein, in some embodiments, transplantation of HSC and / or PBMC produces transgenic mice that contain more human CD45+ cells in the peripheral blood relative to humanized NSG or NSG-Tg (Hu-IL15) control mice (e.g., an increase of 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). In embodiments, the transgenic mice contain significantly more human NK cells in the peripheral blood relative to humanized NSG or NSG-Tg (Hu-IL15) control mice (e.g., an increase of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more, measured as a percentage of CD45+ cells).

[0133] In some embodiments, transplantation of PBMCs produces transgenic mice that contain more human myeloid cells in the peripheral blood relative to humanized NSG or NSG-Tg (Hu-IL15) control mice (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).

[0134] In some embodiments, the transgenic mice contain more activated human T cells relative to humanized NSG or NSG-Tg (Hu-IL15) control mice. In some embodiments, activated T cells are present in the spleen of transgenic mice. The term "T cell activation" refers to the activation mechanism of T cells, which can vary slightly between different types of T cells. However, the "dual signal model" in CD4+T cells is applicable to most types of T cells. In more detail, the activation of CD4+T cells is usually carried out by the T cell receptor and CD28 on the surface of the T cell, which are both antigen presenting molecules encoded by major histocompatibility and the B7 family members on the surface of the antigenic peptide and antigen presenting cell (APC) bound thereto, respectively. Two cell-cell contacts are usually required for the generation of an effective immune response. For example, in the absence of CD28 costimulation, a single T cell receptor signaling can lead to T cell anergy. Further signaling pathways downstream of CD28 and T cell receptors involve many further proteins known to those skilled in the art. The activation of T cells can be determined by cytokine release and / or cell proliferation, particularly the proliferation of T cells.

[0135] How to use

[0136] Human Antibody Pharmacokinetics

[0137] In some aspects, the present disclosure provides for the provision of immunodeficiency Fcgr1 null Method for administering antibodies to mice and determining the level of antibodies. Antibodies can be monoclonal antibodies or polyclonal antibodies. Monoclonal antibodies are antibodies produced by a single clone of a cell or cell line and are composed of identical antibody molecules. Polyclonal antibodies are antibodies produced by different B cell lineages and include a collection of immunoglobulin molecules that react with a specific antigen.

[0138] 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 non-human species. In some embodiments, the sequence of a humanized antibody has been modified to more closely align with antibodies produced in the human body.

[0139] In some embodiments, the antibody is an immune checkpoint inhibitor (ICI) antibody. Immune checkpoints are regulators of the immune system that prevent the immune system from attacking cells indiscriminately. Immune checkpoints prevent immune cells from attacking certain cells. Immune checkpoint inhibitors (ICIs) are molecules that inhibit immune checkpoints and thus allow the immune system to attack certain cells (e.g., cancer cells). ICIs can be any ICI known in the art. Non-limiting examples of immune checkpoint inhibitors include programmed death 1 (PD-1), cytotoxic T lymphocyte-associated 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 domain and mucin domain 3 (TIM3), V-domain Ig inhibitor of T cell activation (VISTA), and sialic acid-binding immunoglobulin-type lectin 7 (SIGLEC7).

[0140] 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 cells. PD-1 inhibits immune responses by increasing apoptosis of antigen-specific T cells in lymph nodes and by reducing apoptosis of regulatory T cells. Human anti-PD-1 antibodies can excite (increase) or antagonize (reduce) the activity of PD-1. Antibodies that excite PD-1 activity can be used to further inhibit the immune system's response to human cells, and can be used to treat diseases in which the immune system is overactive (e.g., autoimmune diseases). Antibodies that antagonize PD-1 activity can be used to allow the immune system to attack human cells, and can be used to treat diseases in which human cells do not undergo apoptosis or undergo apoptosis at a reduced rate compared to controls (e.g., cancer). The anti-PD-1 antibody can be any anti-PD-1 antibody known in the art. Non-limiting examples of anti-PD-1 antibodies include: Pembrolizumab Nivolumab Pidilimab (CT-011), Torelizumab (JS-001), Simizumab Dosilimab Canrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), and INCMGA00012 (MGA012).

[0141] 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. The binding of PD-L1 to PD-1 produces an inhibitory signal that reduces the proliferation of antigen-specific T cells in the lymph nodes while reducing the apoptosis of regulatory T cells. Anti-PD-L1 antibodies can excite (increase) or antagonize (reduce) the activity of PD-L1. Antibodies that excite PD-L1 activity can be used to further suppress the immune system's response to human cells, and can be used to treat diseases in which the immune system is overactive (e.g., autoimmune diseases). Antibodies that antagonize PD-L1 activity can be used to allow the immune system to attack human cells, and can be used to treat diseases in which human cells do not undergo apoptosis or undergo apoptosis at a reduced rate compared to controls (e.g., cancer). The anti-PD-L1 antibody can be any anti-PD-L1 antibody known in the art. Non-limiting examples of anti-PD-L1 antibodies include: Atezolizumab Avelumab Durvalumab and KN035.

[0142] In some embodiments, 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 excite (increase) or antagonize (reduce) the activity of CTLA-4. Antibodies that excite CTLA-4 activity can be used to reduce immune activity and can be used to treat diseases in which the immune system is overactive (e.g., autoimmune diseases). Antibodies that antagonize CTLA-4 activity can be used to allow the immune system to attack cells of the human body and can be used to treat diseases in which human cells do not undergo apoptosis or undergo apoptosis at a reduced rate compared to a control (e.g., cancer). Anti-CTLA-4 antibodies can be any anti-CTLA-4 antibodies known in the art. Non-limiting examples of anti-CTLA-4 antibodies include: Ipilimumab L3D10 and tremelimumab (CP-675,206).

[0143] In some embodiments, the antibody is a human immunoglobulin (Ig) antibody. The human Ig antibody can be an immunoglobulin gamma (IgG), immunoglobulin alpha (IgA), immunoglobulin beta (IgB), immunoglobulin epsilon (IgE) or immunoglobulin μ (IgM) antibody. In some embodiments, the human Ig antibody is an IgG antibody. The IgG antibody can be any IgG antibody described herein.

[0144] In some embodiments, the immunodeficiency Fcgr1 null The mice were non-obese diabetic (NOD) immunodeficient mice. Human IgG has a greatly shortened 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 shortened 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 the Fcgr1 locus encoding an abnormally active FcgR1 receptor that has an abnormally 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 Fcgr1 gene of the NOD mouse strain (Gavin et al., EMBO Journal, 1998; 17(14): 3850-3857).

[0145] Any mouse with a similar gain-of-function mutation in the knocked-out Fcgr1 gene (or its ortholog) can similarly be used in the methods provided herein to study human IgG antibody pharmacokinetics and activity because, in the absence of an aberrantly active Fcgr1 receptor with unusual affinity for human IgG antibodies, the mouse more faithfully recapitulates human IgG antibody recycling and activity.

[0146] Antibody pharmacokinetics can include any pharmacokinetic parameter determined 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, elimination pathways (e.g., kidney, liver), administration routes (e.g., intravenous, subcutaneous, intramuscular) and bioavailability (e.g., metabolism in mice, solubility in blood, ability to reach target cells).

[0147] In some embodiments, the antibody half-life is determined. The antibody half-life is the length of time it takes for the antibody concentration (e.g., in mice) to decrease by half. As provided herein, the half-life of a human IgG antibody in an immunodeficient Fcgr1 mouse compared to its wild-type or immunodeficient counterpart is null The half-life of antibodies is longer in mice. The half-life can be studied by any method known in the art. Non-limiting methods for studying the half-life of antibodies include: enzyme-linked immunosorbent assay (ELISA), Western blot and surface plasmon resonance (SPR).

[0148] In some embodiments, the half-life of the antibody is increased by 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 relative to the control. In some embodiments, the half-life of the antibody is increased by 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 relative to the control. The control can be, for example, a mouse expressing functional mouse FcγR1 (e.g., a wild-type or immunodeficient mouse).

[0149] In some embodiments, the circulating level of the antibody is determined in a biological sample obtained from a mouse to which the antibody is administered. The circulating level of the antibody is the amount of the antibody present in the circulation of the mouse over a given period of time (e.g., after administration of the antibody). The circulating level of the antibody can be compared to a control (e.g., a mouse that does not contain Fcgr1 null allele) to study the pharmacokinetics of the antibody over time.

[0150] The biological sample can be any biological sample obtained from a 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 a mouse by any method known in the art. Non-limiting methods for obtaining a biological sample include: intravenous extraction, intra-arterial extraction, urine collection, spinal puncture, lymphatic drainage, feces collection, and biopsy.

[0151] The circulating level of the antibody can be determined multiple times over a given period of time (e.g., for determining half-life). In some embodiments, the antibody level is determined 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 antibody level is determined 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, antibody levels are measured multiple times over 1 day-30 weeks, 3 days-29 weeks, 7 days-28 weeks, 1.5 weeks-27 weeks, 2 weeks-26 weeks, 2.5 weeks-25 weeks, 3 weeks-24 weeks, 3.5 weeks-23 weeks, 4 weeks-22 weeks, 4.5 weeks-21 weeks, 5 weeks-20 weeks, 6 weeks-19 weeks, 7 weeks-18 weeks, 8 weeks-17 weeks, 9 weeks-16 weeks, 10 weeks-15 weeks, 11 weeks-14 weeks, or 12-13 weeks. In some embodiments, in 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, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks Antibody levels may be measured multiple times over 6.5 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.

[0152] 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.

[0153] More than one antibody (e.g., human IgG antibody) can be administered to the immunodeficient Fcgr1 null In some embodiments, 1-20 antibodies, 2-19 antibodies, 3-18 antibodies, 4-17 antibodies, 5-16 antibodies, 6-15 antibodies, 7-14 antibodies, 8-13 antibodies, 9-12 antibodies, or 10-11 antibodies are administered to an immunodeficient Fcgr1 mouse. null In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more antibodies are administered to an immunodeficient Fcgr1 mouse. null Mouse.

[0154] In some embodiments, the antibody is a therapeutic human IgG antibody. A therapeutic human IgG antibody is an antibody known or believed to treat or prevent a human condition. The 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 Pembrolizumab Adalumab Alemtuzumab Alirocumab Basiliximab Belimumab Bevacizumab Canakinumab Cetuximab Daclizumab Daratumumab Denosumab Deutuximab Eculizumab Elotuzumab Elotumab Golimumab Infliximab Ipilimumab Ixekizumab Mepolizumab Natalizumab Nexituzumab Nivolumab Otuximab Obinutuzumab Ofatumumab Olaratumab Omalizumab Palivizumab Panitumumab Pertuzumab Ramucirumab Ranibizumab Raciliximab Reslizumab Rituximab Secukinumab Siltuximab Tocilizumab Atezolizumab Tositumomab Ustekinumab Vedolizumab L3D10, Tremelimumab (CP-675,206), Atezolizumab Avelumab Durvalumab KN035, pidilizumab (CT-011), toripalimab (JS-001), cemiprilimab Dotalimumab camrizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317) and IMCMG A00012 (MGA012).

[0155] Targeting immunodeficiency Fcgr1 null The administration route of the antibody of the mouse is not limited. Non-limiting examples of the administration route include: subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intraperitoneal injection, intracranial injection, intraventricular injection, inhalation and ingestion.

[0156] Antibody production

[0157] One limitation of mice with overactive Fcγ receptors (FcgR), such as NSG mice, is that these mice do not produce sufficient human IgG antibodies after transplantation of human immune cells to mimic human immune responses. null Mice transplanted with human hematopoietic stem cells (HSC) and / or peripheral blood mononuclear cells (PMBC) and measured immunodeficiency of Fcgr1 null Levels of human IgG antibodies in mice. Fcgr1 nullThe mouse can be any mouse described herein. In some embodiments, the mouse is transplanted with HSC. In some embodiments, the mouse is transplanted with PMBC. In some embodiments, the mouse is transplanted with HSC and PMBC.

[0158] HSCs and PMBCs are cells that give rise to other blood cells in a process called hematopoiesis. Transplantation of mice with human HSCs and / or PMBCs allows non-human animals to produce human blood cells. When mice produce human blood cells, the mice can also produce human antibodies (e.g., human IgG). This is especially true when the mice are immunodeficient prior to transplantation of human HSCs. Immunodeficiency is discussed in more detail in the "Mouse Model" section below. Thus, immunodeficiency in Fcgr1 null Transplantation of mice with human HSCs and / or PMBCs enables mice to mimic human immune behavior.

[0159] Antigens can be administered to non-human animals transplanted with HSCs to stimulate a human immune response. The antigen can be any antigen that produces an immune response in humans. Non-limiting examples of such antigens include: human tumor cells, bacterial cells, viruses, and fungal cells.

[0160] Human HSCs and PMBCs were described previously.

[0161] Transplantation of human HSCs and / or PMBCs into immunodeficient Fcgr1 null The half-life and circulating levels of any human antibody in mice can be measured by any of the methods provided herein and using any frequency and duration.

[0162] In some embodiments, circulating levels of the human antibody 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 relative to a control. In some embodiments, the circulating level of human IgG antibodies is 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 relative to a control. The control can be a wild-type Fcgr1 mouse or an immunodeficient Fcgr1 mouse that has not been transplanted with HSCs and / or PMBCs. null Mouse

[0163] Disease treatment

[0164] 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 and homozygous familial hypercholesterolemia (HeFH, HoFH), transplant rejection, systemic lupus erythematosus (SLE), paroxysmal nocturnal hemoglobinuria, asthma, macular degeneration, plaque psoriasis (PP), psoriatic arthritis (PA), and ulcerative colitis (UC). Identifying new human therapeutic antibodies and new diseases treated or prevented by human therapeutic antibodies is a key research area. In some embodiments, the present disclosure provides for the delivery of Fcgr1 antibodies to immunodeficient patients with human diseases. null Administration of therapeutic antibodies and / or putative therapeutic antibodies and measurement of Fcgr1 null Disease proliferation or survival in mice.

[0165] In some embodiments, the immunodeficiency Fcgr1 null Mice can be transplanted with human diseased cells. Transplantation can be performed by any method provided herein. Human diseased cells are cells derived from humans (e.g., xenografts or human cell lines derived from patients) and associated with diseases. Can be confirmed to be associated with diseases by any method known in the art. Non-limiting examples of confirming whether human cells are associated with diseases are: detecting or measuring whether cells express mutant genes or proteins (e.g., BRCA1, p53, etc.) associated with diseases, detecting increased proliferation in human cells compared to controls, detecting reduced apoptosis in human cells compared to controls, and detecting increased mobility in human cells compared to controls. Controls can be human cells (e.g., wild-type human cells) derived from the same people or the same cell population known to be unrelated to diseases.

[0166] 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 disorders (e.g., HeFH, HoFH, cystic fibrosis, Down syndrome, Duchenne muscular dystrophy), and infectious diseases (e.g., Coronavirus 19 (COVID-19), Hepatitis B virus, rabies virus, respiratory syncytial virus, Clostridium tetani, Clostridium botulinum, vaccinia virus, echovirus, enterovirus).

[0167] In some embodiments, the human disease is cancer, and the human diseased cells are human tumor cells. Human tumor cells are cells that constitute or are associated with a tumor. A tumor is an abnormal growth or accumulation of cells and can be malignant or benign. 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-distal expansion, Beckwith-Wiedeman syndrome, bile duct cancer, Birt-Hogg-Dube syndrome, bladder cancer, bone cancer, brain stem glioma, brain cancer, breast cancer, Carney complex, cervical cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, ventricular Tubuloma, esophageal cancer, Ewing's sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumors (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, hereditary pancreatitis, hereditary papillary renal cancer, HIV / AIDS-related cancers, juvenile polyposis syndrome, kidney cancer, Lacrimal gland tumors, laryngeal and hypopharyngeal cancer, leukemia, Li-Fraumeni syndrome, liver cancer, lung cancer, lymphoma, Lynch syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH-associated polyposis, myelodysplastic syndrome, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, gastrointestinal neuroendocrine tumors, lung neuroendocrine tumors, pancreatic neuroendocrine tumors, neuroendocrine tumors type 1 fibromatosis, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary gland tumors, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, non-melanoma skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymoma and thymic cancer, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Waldenstrom macroglobulinemia, Werner syndrome, Wilms tumor, and xeroderma pigmentosum.

[0168] In some embodiments, by immunodeficiency of Fcgr1 null Human cancer can be generated by transplanting human cancer cells into mice. Human cancer cells can be transplanted by any method provided herein. Human cancer cells can be patient-derived xenografts (PDX) or cultured human cancer cells.

[0169] 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.

[0170] Disease proliferation can be any aspect of disease pathology. Non-limiting examples of disease proliferation include: growth of human diseased cells relative to a control, survival of human diseased cells, mobility of human diseased cells, and division of human diseased cells. A control can be a human cell (e.g., a wild-type human cell) derived from the same person or the same cell population known to be unrelated to the disease.

[0171] Disease proliferation and / or survival can be measured by any method known in the art. The method for measuring disease proliferation depends on the disease under study. Non-limiting methods for measuring disease proliferation include: immunostaining, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and imaging (e.g., magnetic resonance imaging, positron emission tomography, computed tomography, X-ray imaging).

[0172] In some embodiments, the immunodeficiency Fcgr1 null Disease proliferation and / or survival in mice is reduced by 1%-200%, 5%-195%, 10%-190%, 15%-185%, 20%-180%, 25%-175%, 30%-170%, 35%-165%, 40%-160%, 45%-155%, 50%-150%, 55%-145%, 60%-140%, 65%-135%, 70%-130%, 75%-125%, 80%-120%, 85%-115%, 90%-110%, or 95%-105% compared to controls. In some embodiments, the immunodeficient Fcgr1 null Disease proliferation 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 a control. The control can be a wild type Fcgr1 mouse, an immunodeficient Fcgr1 mouse not injected with human IgG antibody, or null Fcgr1 before mouse or human IgG antibody treatment null Mouse.

[0173] Disease proliferation and / or survival can be measured multiple times within a given time period. In some embodiments, disease proliferation and / or survival are 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 proliferation and / or survival are measured 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, or 25 times or more. In some embodiments, disease proliferation and / or survival is measured multiple times over 1 day-30 weeks, 3 days-29 weeks, 7 days-28 weeks, 1.5 weeks-27 weeks, 2 weeks-26 weeks, 2.5 weeks-25 weeks, 3 weeks-24 weeks, 3.5 weeks-23 weeks, 4 weeks-22 weeks, 4.5 weeks-21 weeks, 5 weeks-20 weeks, 6 weeks-19 weeks, 7 weeks-18 weeks, 8 weeks-17 weeks, 9 weeks-16 weeks, 10 weeks-15 weeks, 11 weeks-14 weeks, or 12-13 weeks. In some embodiments, in 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, Weeks, 15, 16, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5 or 30 weeks or more.

[0174] Nucleic Acids: Engineering and Delivery

[0175] The mice described herein comprise a nucleic acid encoding human interleukin-15 (IL-15) and a Fcgr1 null In some embodiments, the mouse comprises a transgene encoding human IL-15 integrated into its genome and comprises Fcgr1 null .

[0176] In some embodiments, nucleic acid provided herein is engineered. Engineered nucleic acid is a nucleic acid that does not exist in nature (e.g., at least two nucleotides are covalently linked together, and in some cases, contain a phosphodiester bond, referred to as a phosphodiester backbone). Engineered nucleic acid includes recombinant nucleic acid and synthetic nucleic acid. Recombinant nucleic acid is a molecule constructed by joining nucleic acids (e.g., isolated nucleic acid, synthetic nucleic acid, or a combination thereof) from two different organisms (e.g., humans and mice). Synthetic nucleic acid is a molecule amplified or synthesized by chemical or other means. Synthetic nucleic acid includes chemically modified or otherwise modified but can be paired (combined) with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include those molecules obtained by any of the aforementioned replications.

[0177] The engineered nucleic acid can comprise DNA (e.g., genomic DNA, cDNA, or a combination of genomic DNA and cDNA), RNA, or a hybrid molecule, for example, wherein 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.

[0178] In some embodiments, the nucleic acid is complementary DNA (cDNA).cDNA is synthesized from a single-stranded RNA (eg, messenger RNA (mRNA) or micro RNA (miRNA)) template in a reaction catalyzed by reverse transcriptase.

[0179] The engineered nucleic acids disclosed herein can be produced 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 produced using GIBSON Clonal generation (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 herein by reference). Three enzyme activities are usually used in a single tube reaction: 5' exonuclease, 3' extension activity of DNA polymerase and DNA ligase activity. The 5' exonuclease activity cuts back the 5' end sequence and exposes the complementary sequence for annealing. The polymerase activity then fills the gap on the annealing domain. The DNA ligase then seals the nick and covalently connects the DNA fragments together. The overlapping sequences of adjacent fragments are much longer than those used in Golden Gate Assembly, thus resulting in a higher percentage of correct assembly. Other methods for producing engineered nucleic acids can be used according to the present disclosure.

[0180] Gene is different nucleotide sequences, and its order determines the order of monomers in polynucleotides or polypeptides.Gene usually encodes protein.Gene can be endogenous (naturally present in host organism) or exogenous (naturally or by genetic engineering transfer to host organism).Allele is produced by mutation and one of two or more optional forms of gene found at the same locus on chromosome.In some embodiments, gene includes promoter sequence, coding region (such as exon), non-coding region (such as intron) and regulatory region (also referred to as regulatory sequence).

[0181] Mice containing human genes are considered to contain human transgenes. Transgenes are genes that are exogenous to the host organism. That is, transgenes are genes that have been transferred to the host organism naturally or by genetic engineering. Transgenes do not naturally occur in the host organism (organism containing the transgene, such as a mouse).

[0182] A promoter is a nucleotide sequence (e.g., ATG) to which RNA polymerase binds to initiate transcription. A promoter is typically located directly upstream (5' end) of the transcription start site. In some embodiments, a promoter is an endogenous promoter. An endogenous promoter is a promoter that occurs naturally in the host animal.

[0183] An open reading frame is a continuous stretch of codons that begins with a start codon (e.g., ATG) and ends with a stop codon (e.g., TAA, TAG, or TGA) and encodes a polypeptide, such as a protein. An open reading frame is operably linked to a promoter if the promoter regulates the transcription of the open reading frame.

[0184] Exons are regions of genes that code for amino acids. Introns (and other noncoding DNA) are regions of genes that do not code for amino acids.

[0185] In some embodiments, the nucleotide sequence of the encoding product (e.g., 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 10kb. For example, the nucleotide sequence can have a length of at least 15kb, at least 20kb, at least 25kb, at least 30kb, or at least 35kb. In some embodiments, the nucleotide sequence has a length of 10 to 100kb, 10 to 75kb, 10 to 50kb, 10 to 30kb, 20 to 100kb, 20 to 75kb, 20 to 50kb, 20 to 30kb, 30 to 100kb, 30 to 75kb, or 30 to 50kb.

[0186] Arbitrary nucleic acid provided herein can have a length of 200bp to 500kb, 200bp to 250kb or 200bp to 100kb. In some embodiments, the nucleic acid has a length of at least 10kb. For example, the nucleic acid can 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 10 to 500kb, 20 to 400kb, 10 to 300kb, 10 to 200kb or 10 to 100kb. In some embodiments, the nucleic acid has a length of 10 to 100 kb, 10 to 75 kb, 10 to 50 kb, 10 to 30 kb, 20 to 100 kb, 20 to 75 kb, 20 to 50 kb, 20 to 30 kb, 30 to 100 kb, 30 to 75 kb, or 30 to 50 kb. The nucleic acid can be circular or linear.

[0187] In some embodiments, nucleic acid as described herein includes modification. Modification of nucleic acid is any operation on nucleic acid relative to corresponding wild-type nucleic acid (e.g., naturally occurring nucleic acid). Therefore, genomic modification is any operation on nucleic acid in genome (e.g., coding region, non-coding region and / or regulatory region) relative to corresponding wild-type nucleic acid in genome (e.g., naturally occurring (unmodified) nucleic acid). Non-limiting examples of nucleic acid (e.g., genome) modification include deletion, insertion, "indel" (deletion and insertion) and substitution (e.g., point mutation). In some embodiments, deletion, insertion, indel or other modification in gene leads to frameshift mutation, so that gene no longer encodes functional product (e.g., protein). Modification also includes chemical modification, such as chemical modification of at least one nucleobase. Methods of nucleic acid modification, such as those leading to gene inactivation, are known, and include but are not limited to RNA interference, chemical modification and gene editing (e.g., using recombinase or other programmable nuclease systems, such as CRISPR / Cas, TALEN and / or ZFN).

[0188] As known in the art, loss-of-function mutations result in gene products with little or no function. Null mutations are a type of loss-of-function mutation that results in a gene product without function. In some embodiments, the inactivated allele is a null allele. Other examples of loss-of-function mutations include missense mutations and frameshift mutations.

[0189] Nucleic acid, such as one or more alleles of a gene, can be modified so that it does not produce a functional gene product (e.g., a functional protein) of a detectable level. Therefore, an inactivated allele is an allele that does not produce a functional gene product (e.g., a functional protein) of a detectable level. The detectable level of protein is any protein level detected using a standard protein detection assay (e.g., flow cytometry and / or ELISA). In some embodiments, the inactivated allele is not transcribed. In some embodiments, the inactivated allele does not encode a functional protein.

[0190] Carriers for delivering nucleic acids include minicircles, plasmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes. However, it should be appreciated that carriers may not be needed. For example, circularized or linearized nucleic acids can be delivered to embryos without their carrier backbones. The carrier backbone is small (~4kb), and the donor DNA to be circularized can be in the range of, for example, >100bp to 50kb.

[0191] Methods for delivering nucleic acids to mouse embryos (e.g., mice) to generate transgenic mice include, but are not limited to, electroporation (see, e.g., Wang W et al., J Genet Genomics 2016; WO 2016 / 054032; and WO 2017 / 124086, each of which is incorporated herein by reference), DNA microinjection (see, e.g., Gordon and Ruddle, Science 1981:214:1244-124, incorporated herein by reference), embryonic stem cell-mediated gene transfer (see, e.g., Gossler et al., Proc. Natl. Acad. Sci. 1986;83:9065-9069, incorporated herein by reference), and retrovirus-mediated gene transfer (see, e.g., Jaenisch, Proc. Natl. Acad. Sci. 1976;73:1260-1264, incorporated herein by reference), any of which can be used as provided herein.

[0192] Genome Editing Methods

[0193] The present application contemplates the use of a variety of gene editing techniques using engineered nucleic acids, for example, to knock out a target gene (e.g., Fcgr1) or to introduce nucleic acids into the genome of a mouse (e.g., to generate a transgenic mouse). null Mice can be generated by any gene editing technique known in the art. In some embodiments, the immunodeficient Fcgr1 nul The mice also contained a human interleukin-15 transgene integrated into their genome.

[0194] Any suitable method can be used to introduce engineered nucleic acids (such as guide RNA, donor polynucleotides and other nucleic acid coding sequences) into the genome of an embryo or cell (such as a stem cell). The present application contemplates the use of a variety of gene editing techniques to, for example, delete nucleic acids from the genome of an embryo or cell to produce a knockout mouse or introduce nucleic acids into the genome of an embryo or cell to produce a transgenic mouse. Non-limiting examples include systems based on programmable nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR) systems, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). See, for example, 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 July; 31 (7): 397-405, each of which is incorporated herein by reference.

[0195] In some embodiments, the CRISPR system is used to edit the genome of a mouse (e.g., mouse) embryo provided herein. See, e.g., 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 protein, one or more guide RNAs (gRNAs) and / or donor nucleic acids can be delivered (e.g., injected or electroporated) directly to mouse embryos at the single cell (zygote) stage or subsequent stages to promote homology-directed repair (HDR), e.g., to delete a nucleic acid sequence from the genome or to introduce an engineered nucleic acid (e.g., a donor nucleic acid) into the genome.

[0196] The CRISPR / Cas system is a naturally occurring defense mechanism in prokaryotes that has been redirected to serve as an RNA-guided DNA targeting platform for gene editing. The engineered CRISPR system contains two main components: guide RNA (gRNA) and CRISPR-related nucleases (e.g., Cas proteins). gRNA is a short synthetic RNA consisting of a scaffold sequence for nuclease binding and a user-defined nucleotide spacer (e.g., 15-25 nucleotides, or 20 nucleotides) that defines the genomic target to be modified (e.g., gene). Therefore, the genomic target of the Cas protein can be changed by simply changing the target sequence present in the gRNA. In some embodiments, the Cas9 endonuclease is from Streptococcus pyogenes (NGG PAM) or Staphylococcus aureus (NNGRRT or NNGRR (N) PAM), although other Cas9 homologs, orthologs and / or variants (e.g., evolved forms of Cas9) can be used, as provided herein. Other non-limiting examples of RNA-guided nucleases that can be used as provided herein include Cpf1 (TTN PAM); SpCas9D1135E variant (NGG (reduced NAG binding) PAM; SpCas9VRER variant (NGCG PAM); SpCas9EQR variant (NGAG PAM); SpCas9VQR variant (NGAN or NGNG PAM); Neisseria meningitidis (NM) Cas9 (NNNNGATT PAM); Streptococcus thermophilus (ST) Cas9 (NNAGAAW PAM); and Treponema denticola (TD) Cas9 (NAAAAC). In some embodiments, the CRISPR-associated nuclease is selected from Cas9, Cpf1, C2c1, and C2c3. In some embodiments, the Cas nuclease is Cas9.

[0197] The guide RNA comprises at least a spacer sequence that hybridizes (binds) to a target nucleic acid sequence and a CRISPR repeat sequence that binds to an endonuclease and guides the endonuclease to the target nucleic acid sequence. As understood by those of ordinary skill in the art, each gRNA is designed to include a spacer sequence that is complementary to its genomic 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.

[0198] In some embodiments, the RNA-guided nuclease and gRNA are complexed to form a ribonucleoprotein (RNP) prior to delivery to the embryo.

[0199] The concentration of the RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease can vary. In some embodiments, the concentration is 100 ng / μl to 1000 ng / μl. For example, the concentration can be 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 ng / μl. In some embodiments, the concentration is 100 ng / μl to 500 ng / μl or 200 ng / μl to 500 ng / μl.

[0200] The concentration of gRNA can also vary. In some embodiments, the concentration is 200 ng / μl to 2000 ng / μl. For example, the concentration can be 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1700, 1900 or 2000 ng / μl. In some embodiments, the concentration is 500 ng / μl to 1000 ng / μl. In some embodiments, the concentration is 100 ng / μl to 1000 ng / μl. For example, the concentration can be 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 ng / μl.

[0201] In some embodiments, the ratio of the concentration of RNA-guided nuclease or nucleic acid encoding RNA-guided nuclease to the concentration of gRNA is 2: 1. In other embodiments, the ratio of the concentration of RNA-guided nuclease or nucleic acid encoding RNA-guided nuclease to the concentration of gRNA is 1:1.

[0202] Donor nucleic acid generally includes the target sequence of side joint homology arms.Homology arms are regions of ssDNA homologous to the regions of the genomic DNA located in the genomic locus.One homology arm is located at the left (5') side (left homology arm) of the target genomic region (wherein the target sequence is introduced), and another homology arm is located at the right (3') side (right homology arm) of the target genomic region.These homology arms enable homologous recombination to be carried out between ssDNA donor and genomic locus, thereby causing the target sequence to be inserted into the target genomic locus (for example, homology directed repair (HDR) mediated by CRISPR / Cas9).

[0203] In some embodiments, each homology arm has a length of 20 to 200, 20 to 300, 20 to 400, 20 to 500, 20 to 600, 20 to 700, 20 to 800 or 20 to 900 nucleotide bases. In some embodiments, each homology arm has a length of 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 nucleotide bases. In some embodiments, the length of a homology arm is different from the length of another homology arm. For example, one homology arm can have a length of 20 nucleotide bases, and another 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, by thiophosphorylation. Other modifications can be performed.

[0204] Example

[0205] Example 1. Generation of NSG-Fcgr1 null and NSG-Fcgr1 null Tg(Hu-IL15) mice.

[0206] Using CRISPR-Cas9 genome editing to extract NSG TM Mice (JAX strain number: 005557) and NSG transgenic for expressing human IL15 TM The mouse Fcgr1 gene in NSG-Tg (Hu-IL15) (JAX strain number: 030890) mice deleted exons 3-6. The deletion of exons 3-6 is expected to produce an approximately 8500 base pair deletion. The mouse Fcgr1 locus encodes a 404 amino acid protein-coding transcript (Fcgr1-201) and a 50 amino acid nonsense-mediated decay transcript (Fcgr1-202). Upstream of the targeted exon 3-6 ( Figure 1 ) and downstream ( Figure 2 ) Design CRISPR-Cas9 gene primers. See Table 1 below.

[0207] Table 1. Deleting NSG TM CRISPS-Cas9 genome editing targeting strategy for exons 3-6 of the Fcgr1 gene in Hu-IL15 and NSG-Tg (Hu-IL15) mice

[0208]

[0209]

[0210] The off-target genome editing risk of CRISPR-Cas9 primers was evaluated. When the CRISPR-Cas9 primer had a linked non-canonical protospacer adjacent motif (PAM), the off-target genome editing risk was considered high when the score was <20. All 4 primers had minimal off-target genome editing risk because they had linked, non-canonical, and their off-target editing risk scores were <3.5.

[0211] Deletion of mouse Fcgr1 exons 3-6 is expected to generate mutant alleles, which can be detected by sequencing from the targeted exon 3 upstream in the forward direction and / or exon 6 downstream in the reverse direction ( Figure 3 This deletion is also expected to produce an early truncation of the Fcgr1-201 protein encoding transcript after amino acid 26 ( Figure 4 ).

[0212] 19 Fcgr1 exon 3-6 deletions were generated (Fcgr1 null ) Heterozygous founder NSG TM Mouse ( Figure 5 Six Fcgr1 exon 3-6 deletions were generated (Fcgr1 null ) heterozygous founder NSG Tg(Hu-IL15) mice ( Figure 6 ).

[0213] Example 2. NSG-Fcgr1 null Reduced clearance of human IgG1 antibodies in mice.

[0214] In NSG TM and NSG-Fcgr1 null The therapeutic human immunoglobulin 1 (IgG1) antibody trastuzumab was compared in mice Trastuzumab is approved for early breast cancer that is HER2+. null and 5 NSG TM Groups of control mice were injected intravenously with 200 μg of trastuzumab. In a 25 g mouse, this is a dose of about 8 mg / kg. At 1, 2, 7, 14, 21, 28, and 35 days after the trastuzumab injection, the mice were bled and the serum was frozen. The remaining percentage of the initial trastuzumab dose was determined by enzyme-linked immunosorbent assay (ELISA). Although trastuzumab was not effective in NSG TM mice (1% remaining on day 21 after injection), but in NSG-Fcgr1 nullThe clearance of trastuzumab was reduced and the persistence was increased in mice (about 40% remaining on day 35 after injection) ( Figure 7 ).

[0215] Calculation of NSG using two different programs (PKAnalix2019 and R_Studio) TM and NSG-Fcgr1 null Pharmacokinetics of trastuzumab clearance in mice. Trastuzumab is expressed in NSG-Fcgr1 null The half-life in mice is approximately 24 days, similar to that of NSG TM In mice, about 5 days compared with ( Figure 8 ). Therefore, knocking out NSG TM The Fcgr1 gene in mice causes a 5-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 ( et al., Curr Clin Pharmacol., (2008), 3(1):51-55).

[0216] Example 3. NSG-Fcgr1 null Reduced clearance of human IgG4 antibodies in mice.

[0217] One of the most effective ways to activate anti-tumor immunity is to block immune checkpoints. Tumors exploit immune checkpoints to suppress anti-tumor immune responses. Blocking immune checkpoint proteins, such as programmed cell death protein 1 (PD-1), offers broad and diverse opportunities to enhance anti-tumor immunity and has the potential to produce durable clinical responses. It is one of the most widely used immune checkpoint blockade monoclonal antibodies (mAb). Pembrolizumab is a humanized IgG4κ mAb that recognizes human PD-1. PD-1 is expressed in activated CD4 + T cells, CD8 + T cells, activated CD4 + Regulatory T(T reg) cells, B cells, and natural killer (NK) cells. PD-1 is also constitutively expressed on tumor-infiltrating lymphocytes (TILs) in a variety of 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) cells and malignant (cancerous) cells, while PD-L2 is primarily expressed on antigen-presenting cells. Binding of PD-L1 to PD-1 results in T cell activation and inhibition of effector function mediated by recruitment of tyrosine phosphatases to the immune synapse, which disrupts T cell receptor signaling. A large body of evidence has demonstrated that PD-L1 expression is commonly upregulated in many different human cancer types, including melanoma, lung, and ovarian tumors.

[0218] NSG TM Mice were transplanted with human tumor cell lines and patient-derived tumor xenografts and then given multiple injections of the human IgG4 therapeutic antibody pembrolizumab at 5-10 mg / kg every 5 days Treatment with pembrolizumab resulted in slower tumor growth in some but not all experiments. TM The reduced efficacy of human tumor treatment in mice may be related to the rapid clearance of this human IgG4 antibody due to increased mouse Fcgr1 receptor activity.

[0219] To test this hypothesis, in NSG TM and NSG-Fcgr1 null The clearance of pembrolizumab was compared in 5 NSG-Fcgr1 mice. null and 5 NSG TM Groups of control mice were injected intravenously with 25 μg of pembrolizumab. In a 25 g mouse, this is a dose of approximately 1 mg / kg. At 1-35 days after the pembrolizumab injection, the mice were bled and the serum was frozen. The remaining percentage of the initial pembrolizumab dose was determined by enzyme-linked immunosorbent assay (ELISA). Although pembrolizumab was not effective in NSG TM mice (>1% remaining on day 6 after injection), but in NSG-Fcgr1 null In mice, the clearance of pembrolizumab was reduced and the persistence was increased (about 30% remaining on day 35 after injection) ( Fig. 9 ).

[0220] Calculation of NSG using two different programs (PKAnalix2019 and R_Studio) TM and NSG-Fcgr1 null Pharmacokinetics of pembrolizumab clearance in mice. TM In mice, pembrolizumab was only about 0.6 days in NSG-Fcgr1null The half-life in mice is approximately 19 days ( Fig.10 ). Therefore, knocking out NSG TM The Fcgr1 gene in mice results in a 30-fold increase in the half-life of pembrolizumab in the circulation. This increased half-life is similar to the half-life of pembrolizumab in humans (Centanni et al., Clinical Pharmacokinetics (2019), 58: 85-857).

[0221] Example 4. NSG-Fcgr1 null Increased levels of circulating human IgG antibodies in mice.

[0222] In NSG transplanted with human CD34+ hematopoietic stem cells (HSCs) TM Attempts to generate human IgG antibodies after mouse immunization are limited by the short half-life of circulating human IgG. TM and NSG-Fcgr1 null Mice were transplanted with human umbilical cord HSCs, and plasma levels of human IgG were measured 21 weeks after transplantation. TM Compared with mice, NSG-Fcgr1 null Mice have 20-fold higher circulating human IgG levels ( Fig.11 ), which is consistent with NSG-Fcgr1 null Persistence of circulating human IgG therapeutic antibodies (trastuzumab and pembrolizumab) in mice compared with NSG TM The increase was consistent in mice.

[0223] All references, patents, and patent applications disclosed herein are hereby incorporated by reference with respect to the subject matter of each citation, which in some cases may be incorporated in its entirety.

[0224] Unless expressly indicated to the contrary, the indefinite articles "a" and "an" as used herein, as in the specification and claims, should be understood to mean "at least one".

[0225] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described unless explicitly stated to the contrary.

[0226] In the claims and the foregoing specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "including," etc. shall be understood as open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the U.S. Patent Office Manual of Patent Examining Procedures.

[0227] The terms "about" and "substantially" preceding a numerical value mean ±10% of the stated numerical value.

[0228] Where a range of values ​​is provided, every value between the upper and lower end of that range is specifically contemplated and described herein.

Claims

1. An immunodeficient mouse comprising a mouse Fcγ receptor 1 null (Fcgr1 null ) allele and mice with interleukin-2 receptor γ null (IL-2Rγ null ) allele.

2. The immunodeficient mouse of claim 1, wherein the mouse comprises a non-obese diabetic (NOD) genetic background.

3. The immunodeficient mouse according to claim 1 or 2, wherein the mouse is resistant to the IL-2Rγ null The allele is homozygous and for the Fcgr1 null The alleles are homozygous.

4. according to the immunodeficient mouse described in any one of claims 1-3, wherein said mouse also comprises Prkdc scid Allele.

5. The immunodeficient mouse according to claim 4, wherein the mouse is scid The alleles are homozygous.

6. The immunodeficient mouse according to any one of claims 1 to 3, wherein the mouse further comprises Rag1 null Allele.

7. The immunodeficient mouse according to claim 6, wherein the mouse is resistant to the Rag1 null The alleles are homozygous.

8. The immunodeficient mouse of any one of the preceding claims, wherein the mouse lacks mouse T cells, B cells and / or Natural Killer (NK) cells.

9. The immunodeficient mouse of any one of the preceding claims, wherein macrophage and / or dendritic cell function in the mouse is defective.

10. The immunodeficient mouse of any one of the preceding claims, transplanted with human hematopoietic stem cells (HSCs).

11. The immunodeficient mouse according to any one of the preceding claims transplanted with human peripheral blood mononuclear cells (PBMC).

12. An immunodeficient mouse according to any one of the preceding claims transplanted with human diseased cells.

13. The immunodeficient mouse of claim 12, wherein the human diseased cells are human tumor cells.

14. The immunodeficient mouse of claim 12, wherein the human tumor cells are human cancer cells.

15. The immunodeficient mouse of any of the preceding claims, wherein the mouse Fcgr1 null The allele contains a deletion in the region of exons 3-6.

16. The immunodeficient mouse according to claim 15, wherein the mouse Fcgr1 null The allele contains a deletion of exons 3-6.

17. The immunodeficient mouse of any one of the preceding claims, further comprising a human IL-15 transgene.

18. The immunodeficient mouse of claim 17, wherein the human IL-15 transgene is integrated into the genome of the mouse.

19. A method comprising administering to an immunodeficient mouse according to any one of the preceding claims an antibody.

20. The method of claim 19, wherein the antibody is a monoclonal antibody.

21. The method of claim 19 or 20, wherein the antibody is a human or humanized antibody.

22. The method according to any one of the preceding claims, wherein the antibody is an IgGl, IgG2, IgG3 or IgG4 antibody.

23. The method of any one of the preceding claims, wherein the antibody is an immune checkpoint inhibitor (ICI) antibody.

24. The method of claim 23, wherein the ICI antibody is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-CTLA-4 antibody.

25. The method of any of the preceding claims, further comprising assaying a biological sample from the mouse for the therapeutic effect of the antibody.

26. The method of claim 25, wherein the therapeutic effect is a reduction in growth of human diseased cells relative to a control.

27. The method of any of the preceding claims, further comprising determining circulating levels of the antibody in a biological sample from the mouse.

28. The method of claim 27, wherein the circulating level of the antibody is at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold higher relative to a control.

29. The method of any one of claims 25-28, wherein the determining is at least 21 days, at least 28 days, or at least 35 days after administration of the antibody.

30. A method comprising administering human cells to an immunodeficient mouse according to any one of claims 1-9.

31. The method of claim 30, wherein the human cell is a human hematopoietic stem cell (HSC).

32. The method of claim 30, wherein the human cells are human peripheral blood mononuclear cells (PBMCs).

33. The method of any one of claims 30-32, wherein the human cell is a human diseased cell.

34. The method of claim 33, wherein the human diseased cells are human tumor cells.

35. The method of claim 34, wherein the human tumor cell is a human cancer cell.

36. An immunodeficient mouse comprising a mouse Fcγ receptor 1 null (Fcgr1 null ) allele, wherein the mouse comprises NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ background.

37. The immunodeficient mouse of claim 36, wherein the mouse is resistant to Fcgr1 null The alleles are homozygous.

38. The immunodeficient mouse of claim 36 or 37, wherein the mouse Fcgr1 is null The allele contains a deletion in the region of exons 3-6.

39. The immunodeficient mouse according to claim 38, wherein the mouse Fcgr1 null The allele contains a deletion of exons 3-5.

40. The immunodeficient mouse of any one of claims 36-39, further comprising a human IL-15 transgene.

41. The immunodeficient mouse of claim 40, wherein the human IL-15 transgene is integrated into the genome of the mouse.

42. The immunodeficient mouse of any one of claims 36-41 transplanted with human hematopoietic stem cells (HSC).

43. The immunodeficient mouse of any one of claims 36-41 transplanted with human peripheral blood mononuclear cells (PBMC).

44. The immunodeficient mouse of any one of claims 36-43 transplanted with human diseased cells.

45. The immunodeficient mouse of claim 44, wherein the human diseased cells are human tumor cells.

46. ​​The immunodeficient mouse of claim 45, wherein the human tumor cells are human cancer cells.

47. A guide RNA comprising the sequence of any one of SEQ ID NOs: 2-5.

48. A method for producing an immunodeficient mouse according to any one of the preceding claims, comprising introducing one or more guide RNAs and Cas proteins into mouse embryos; transplanting the mouse embryos into pseudopregnant female mice; collecting F1 mice born from the pseudopregnant females; and breeding the F1 mice.

49. The method of claim 48, wherein 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 the guide RNA of claim 48.

50. according to the method for claim 48 or 49, wherein said mouse embryo is an immunodeficient mouse embryo, optionally has NOD genetic background, and optionally comprises (a) IL-2Rγ null Alleles and (b) Prkdc scid Allele or Rag1 null One or more alleles.

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