WO2018086583A1 - Animal non humain génétiquement modifié, doté d'un gène lag-3 humain ou chimérique - Google Patents

Animal non humain génétiquement modifié, doté d'un gène lag-3 humain ou chimérique Download PDF

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WO2018086583A1
WO2018086583A1 PCT/CN2017/110435 CN2017110435W WO2018086583A1 WO 2018086583 A1 WO2018086583 A1 WO 2018086583A1 CN 2017110435 W CN2017110435 W CN 2017110435W WO 2018086583 A1 WO2018086583 A1 WO 2018086583A1
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lag
animal
human
exon
sequence
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PCT/CN2017/110435
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Yuelei SHEN
Yanan GUO
Chaoshe GUO
yang BAI
Jiawei Yao
Lei Zhao
Rui Huang
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Beijing Biocytogen Co., Ltd
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Priority claimed from CN201711103773.2A external-priority patent/CN108070614B/zh
Publication of WO2018086583A1 publication Critical patent/WO2018086583A1/fr
Priority to US16/409,683 priority Critical patent/US10925264B2/en

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    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) Lymphocyte Activation Gene 3 (LAG-3) , and methods of use thereof.
  • human or chimeric e.g., humanized Lymphocyte Activation Gene 3 (LAG-3)
  • LAG-3 Lymphocyte Activation Gene 3
  • the immune system has developed multiple mechanisms to prevent deleterious activation of T cells.
  • One such mechanism is the intricate balance between positive and negative costimulatory signals delivered to T cells.
  • Targeting the inhibitory pathways for the immune system is considered to be a potential approach for the treatment of various diseases, e.g., cancers, and autoimmune diseases.
  • the animal model can express human LAG-3 or chimeric LAG-3 (e.g., humanized LAG-3) protein in its body. It can be used in the studies on the function of LAG-3 gene, and can be used in the screening and evaluation of anti-human LAG-3 antibodies.
  • the animal models prepared by the methods described herein can be used in drug screening, pharmacodynamics studies, treatments for immune-related diseases (e.g., autoimmune disease) , and cancer therapy for human LAG-3 target sites; in addition, they can be used to facilitate the development and design of new drugs, and save time and cost.
  • this disclosure provides a powerful tool for studying the function of LAG-3 protein and screening for cancer drugs.
  • the disclosure also provides LAG-3 gene knockout mice.
  • the mice described in the present disclosure can be mated with the mice containing other human or chimeric genes (e.g., chimeric CTLA-4, chimeric PD-1, or other immunomodulatory factors) , so as to obtain a mouse expressing two or more human or chimeric proteins.
  • the mice can also, e.g., be used for screening antibodies in the case of a combined use of drugs, as well as evaluating the efficacy of the combination therapy.
  • the disclosure relates to genetically-modified, non-human animals whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric Lymphocyte Activation Gene 3 (LAG-3) .
  • the sequence encoding the human or chimeric LAG-3 is operably linked to an endogenous regulatory element at the endogenous LAG-3 gene locus in the at least one chromosome.
  • the sequence encoding a human or chimeric LAG-3 comprises a sequence encoding an amino acid sequence that is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human LAG-3 (NP_002277.4 (SEQ ID NO: 27) ) .
  • the sequence encoding a human or chimeric LAG-3 comprises a sequence encoding an amino acid sequence that is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 31.
  • the sequence encoding a human or chimeric LAG-3 comprises a sequence encoding an amino acid sequence that corresponds to amino acids 25-166 of SEQ ID NO: 27.
  • the animal is a mammal, e.g., a monkey, a rodent or a mouse. In some embodiments, the animal is a C57BL/6 mouse. In some embodiments, the animal does not express endogenous LAG-3. In some embodiments, the animal has one or more cells expressing human or chimeric LAG-3. In some embodiments, the animal has one or more cells expressing human or chimeric LAG-3, and the expressed human or chimeric LAG-3 can bind to or interact with human protein MHC II (Major Histocompatibility Complex Class II) , human CD3, human L-selectin or human galectin-3.
  • human protein MHC II Major Histocompatibility Complex Class II
  • the animal has one or more cells expressing human or chimeric LAG-3, and the expressed human or chimeric LAG-3 can bind to or interact with endogenous MHC II, endogenous CD3, endogenous L-selectin or endogenous galectin-3.
  • the disclosure relates to genetically-modified, non-human animals, wherein the genome of the animals comprises a replacement, at an endogenous LAG-3 gene locus, of a sequence encoding a region of endogenous LAG-3 with a sequence encoding a corresponding region of human LAG-3.
  • the sequence encoding the corresponding region of human LAG-3 is operably linked to an endogenous regulatory element at the endogenous LAG-3 locus, and one or more cells of the animal expresses a chimeric LAG-3.
  • the animal does not express endogenous LAG-3.
  • the region of endogenous LAG-3 is the extracellular region of LAG-3.
  • the animal has one or more cells expressing a chimeric LAG-3 having an extracellular region, a transmembrane region, and a cytoplasmic region, wherein the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%identical to the extracellular region of human LAG-3.
  • the extracellular region of the chimeric LAG-3 has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human LAG-3.
  • the animal is a mouse, and the sequence encoding the region of endogenous LAG-3 is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7 and/or exon 8 of the endogenous mouse LAG-3 gene (e.g., exon 2, exon 3, or part of exon 2 and exon 3) .
  • the animal is heterozygous with respect to the replacement at the endogenous LAG-3 gene locus.
  • the animal is homozygous with respect to the replacement at the endogenous LAG-3 gene locus.
  • the disclosure relates to methods for making a genetically-modified, non-human animal, including: replacing in at least one cell of the animal, at an endogenous LAG-3 gene locus, a sequence encoding a region of an endogenous LAG-3 with a sequence encoding a corresponding region of human LAG-3.
  • the sequence encoding the corresponding region of human LAG-3 comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7 and/or exon 8 of a human LAG-3 gene.
  • the sequence encoding the corresponding region of LAG-3 comprises exon 2 and exon 3 of a human LAG-3 gene, and/or a part of exon 2 and/or exon 3 of a human LAG-3 gene.
  • the sequence encoding the corresponding region of human LAG-3 encodes amino acids 25-166 of SEQ ID NO: 27.
  • the region is located within the extracellular region of LAG-3.
  • the animal is a mouse, and the sequence encoding the region of the endogenous LAG-3 locus is exon 2 and exon 3 of mouse LAG-3 gene.
  • the disclosure relates to non-human animals comprising at least one cell comprising a nucleotide sequence encoding a chimeric LAG-3 polypeptide, wherein the chimeric LAG-3 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human LAG-3, wherein the animal expresses the chimeric LAG-3.
  • the chimeric LAG-3 polypeptide has at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human LAG-3 extracellular region.
  • the chimeric LAG-3 polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 25-166 of SEQ ID NO: 27.
  • the nucleotide sequence is operably linked to an endogenous LAG-3 regulatory element of the animal.
  • the chimeric LAG-3 polypeptide comprises an endogenous LAG-3 transmembrane region and/or an endogenous LAG-3 cytoplasmic region.
  • the nucleotide sequence is integrated to an endogenous LAG-3 gene locus of the animal.
  • the chimeric LAG-3 has at least one mouse LAG-3 activity (e.g., interacting with mouse MHC II, inhibiting mouse T-cell immune responses, binding to mouse CD3, L-selectin or galectin-3) and/or at least one human LAG-3 activity (e.g., interacting with human MHC II, inhibiting human T-cell immune responses, binding to human CD3, L-selectin or galectin-3) .
  • the disclosure relates to methods of making a genetically-modified mouse cell that expresses a chimeric LAG-3, the method including: replacing, at an endogenous mouse LAG-3 gene locus, a nucleotide sequence encoding a region of mouse LAG-3 with a nucleotide sequence encoding a corresponding region of human LAG-3, thereby generating a genetically-modified mouse cell that includes a nucleotide sequence that encodes the chimeric LAG-3, wherein the mouse cell expresses the chimeric LAG-3.
  • the chimeric LAG-3 comprises an extracellular region of mouse LAG-3 comprising a mouse signal peptide sequence, an extracellular region of human LAG-3, a transmembrane and/or a cytoplasmic region of a mouse LAG-3.
  • the nucleotide sequence encoding the chimeric LAG-3 is operably linked to an endogenous LAG-3 regulatory region, e.g., promoter.
  • the animals further comprise a sequence encoding an additional human or chimeric protein.
  • the additional human or chimeric protein is programmed cell death protein 1 (PD-1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) , B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , TNF Receptor Superfamily Member 9 (4-1BB) , CD27, CD28, CD47, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , CD27, Glucocorticoid-Induced TNFR-Related Protein (GITR) , or TNF Receptor Superfamily Member 4 (TNFRSF4 or OX40) .
  • PD-1BB cytotoxic T-lymphocyte-associated protein 4
  • TIM-3 T-cell immunoglobulin and mucin-domain containing-3
  • the animal or mouse further comprises a sequence encoding an additional human or chimeric protein.
  • the additional human or chimeric protein is programmed cell death protein 1 (PD-1) , CTLA-4, TIM-3, BTLA, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, CD27, GITR, or OX40.
  • the disclosure relates to methods of determining effectiveness of an anti-LAG-3 antibody for the treatment of cancer, including: administering the anti-LAG-3 antibody to the animal as described herein, wherein the animal has a tumor, and determining the inhibitory effects of the anti-LAG-3 antibody to the tumor.
  • the animal comprises one or more cells (e.g., tumor cells, antigen presenting cells, dendritic cells, mononuclear phagocytes, endothelial cells, thymic epithelial cells, and B cells) that express MHC II, CD3, L-selectin or galectin-3.
  • the tumor comprises one or more cancer cells that are injected into the animal.
  • determining the inhibitory effects of the anti-LAG-3 antibody to the tumor involves measuring the tumor volume in the animal.
  • the tumor cells are melanoma cells, non-small cell lung carcinoma (NSCLC) cells, small cell lung cancer (SCLC) cells, bladder cancer cells, breast cancer cells, and/or prostate cancer cells (e.g., metastatic hormone-refractory prostate cancer) .
  • the disclosure relates to methods of determining effectiveness of an anti-LAG-3 antibody for the treatment of various immune-related disorders, e.g., autoimmune diseases (e.g., psoriasis) .
  • autoimmune diseases e.g., psoriasis
  • the disclosure relates to methods of determining effectiveness of an anti-LAG-3 antibody and an additional therapeutic agent for the treatment of a tumor, including administering the anti-LAG-3 antibody and the additional therapeutic agent to the animal as described herein, wherein the animal has a tumor, and determining the inhibitory effects on the tumor.
  • the animal further comprises a sequence encoding a human or chimeric programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) .
  • the additional therapeutic agent is an anti-PD-1 antibody.
  • the additional therapeutic agent is an anti-CTLA-4 antibody.
  • the animal comprises one or more cells (e.g., tumor cells, antigen presenting cells, dendritic cells, mononuclear phagocytes, endothelial cells, thymic epithelial cells, and B cells) that express MHC II, CD3, L-selectin or galectin-3.
  • the tumor comprises one or more tumor cells that express PD-L1 or PD-L2.
  • the tumor comprises one or more tumor cells that express CD80 or CD86.
  • the tumor is caused by injection of one or more cancer cells into the animal.
  • determining the inhibitory effects of the treatment involves measuring the tumor volume in the animal.
  • the tumor comprises melanoma cells, non-small cell lung carcinoma (NSCLC) cells, small cell lung cancer (SCLC) cells, bladder cancer cells, breast cancer cells, and/or prostate cancer cells (e.g., metastatic hormone-refractory prostate cancer cells) .
  • NSCLC non-small cell lung carcinoma
  • SCLC small cell lung cancer
  • bladder cancer cells bladder cancer cells
  • breast cancer cells and/or prostate cancer cells (e.g., metastatic hormone-refractory prostate cancer cells) .
  • the disclosure relates to proteins comprising an amino acid sequence, wherein the amino acid sequence is one of the following: (a) an amino acid sequence set forth in SEQ ID NO: 31; (b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 31; (c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 31; (d) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 31 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and (e) an amino acid sequence that comprises a substitution, a deletion and /or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 31.
  • cells comprising the proteins disclosed herein.
  • provided herein are animals having the proteins disclosed herein.
  • the disclosure relates to nucleic acids comprising a nucleotide sequence, wherein the nucleotide sequence is one of the following: (a) a sequence that encodes the protein as described herein; (b) SEQ ID NO: 29; (c) SEQ ID NO: 30; (d) a sequence that is at least 90%identical to SEQ ID NO: 29 or SEQ ID NO: 30; (e) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 29; and (f) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 30.
  • cells comprising the nucleic acids disclosed herein.
  • provided herein are animals having the nucleic acids disclosed herein.
  • the disclosure relates to a targeting vector, including a) a DNA fragment homologous to the 5’end of a region to be altered (5’arm) , which is selected from the LAG-3 gene genomic DNAs in the length of 100 to 10,000 nucleotides; b) a desired/donor DNA sequence encoding a donor region; and c) a second DNA fragment homologous to the 3’end of the region to be altered (3’arm) , which is selected from the LAG-3 gene genomic DNAs in the length of 100 to 10,000 nucleotides.
  • a) the DNA fragment homologous to the 5’end of a region to be altered (5’arm/receptor) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000072.6; c) the DNA fragment homologous to the 3’end of the region to be altered (3’arm/receptor) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000072.6.
  • a) the DNA fragment homologous to the 5’end of a region to be altered (5’arm/receptor) is selected from the nucleotides from the position 124911766 to the position 124910898 of the NCBI accession number NC_000072.6; c) the DNA fragment homologous to the 3’end of the region to be altered (3’arm/receptor) is selected from the nucleotides from the position 124910116 to the position 124908702 of the NCBI accession number NC_000072.6.
  • a length of the selected genomic nucleotide sequence is about 1.2 kb, 1.5 kb or 1 kb. In some embodiments, the length is about 869 bp or 1415 bp. In some embodiments, the region to be altered is exon 2, exon 3, and/or part of exon 2 and exon 3 of LAG-3 gene.
  • sequence of the 5’arm is shown in SEQ ID NO: 32. In some embodiments, the sequence of the 3’arm is shown in SEQ ID NO: 38.
  • the targeting vector further includes a selectable gene marker.
  • the target region is derived from human. In some embodiments, the target region is a part or entirety of the nucleotide sequence of a humanized LAG-3. In some embodiments, the nucleotide sequence is shown as one or more of the first exon, the second exon, the third exon, the fourth exon, the fifth exon, the sixth exon, the seventh exon, and the eighth exon of the DNA sequence of the human LAG-3.
  • the nucleotide sequence of the human LAG-3 encodes the human LAG-3 protein with the NCBI accession number NP_002277.4 (SEQ ID NO: 27) .
  • the disclosure also relates to a cell including the targeting vector as described herein.
  • the disclosure relates to an sgRNA sequence for constructing a humanized animal model, wherein the sgRNA sequence targets the LAG-3 gene, the sgRNA is unique on the target sequence of the LAG-3 gene to be altered, and meets the sequence arrangement rule of 5’-NNN (20) -NGG3’or 5’-CCN-N (20) -3’.
  • the targeting site of the sgRNA in the mouse LAG-3 gene is located on exon 2 or exon 3 of the mouse LAG-3 gene.
  • the disclosure relates to an sgRNA sequence for constructing a humanized animal model, wherein an upstream sequence thereof is shown as SEQ ID NO: 15, and a downstream sequence thereof is shown as SEQ ID NO: 17, and the sgRNA sequence recognizes a 5’targeting site.
  • the disclosure also relates to an sgRNA sequence for constructing a humanized animal model, wherein an upstream sequence thereof is shown as SEQ ID NO: 16, which is obtained by adding TAGG to the 5’end of SEQ ID NO: 15; a downstream sequence thereof is shown as SEQ ID NO: 18, which is obtained by adding AAAC to the 5’end of SEQ ID NO: 17, and the sgRNA sequence recognizes a 5’targeting site.
  • the disclosure also relates to an sgRNA sequence for constructing a humanized animal model, wherein an upstream sequence thereof is shown as SEQ ID NO: 19, and a downstream sequence thereof is shown as SEQ ID NO: 21, and the sgRNA sequence recognizes a 3’targeting site.
  • the disclosure further relates to an sgRNA sequence for constructing a humanized animal model, wherein an upstream sequence thereof is shown as SEQ ID NO: 20, which is obtained by adding TAGG to the 5’end of SEQ ID NO: 19; a downstream sequence thereof is shown as SEQ ID NO: 22, which is obtained by adding AAAC to the 5’end of SEQ ID NO: 21, and the sgRNA sequence recognizes a 3’targeting site.
  • the disclosure relates to a construct including the sgRNA sequence as described herein.
  • the disclosure also relates to a cell comprising the construct as described herein.
  • the disclosure relates to a non-human mammalian cell, comprising the targeting vector as described herein, and one or more in vitro transcripts of the sgRNA construct.
  • the cell includes Cas9 mRNA or an in vitro transcript thereof.
  • the genes in the cell are heterozygous. In some embodiments, the genes in the cell are homozygous.
  • the non-human mammalian cell is a mouse cell.
  • the cell is a fertilized egg cell.
  • the cell is a germ cell.
  • the cell is a blastocyst.
  • the cell is a lymphocyte (e.g., a B-cell or a T-cell) .
  • the disclosure relates to methods for establishing a LAG-3 gene humanized animal model.
  • the methods include the steps of:
  • step (d) identifying the germline transmission in the offspring genetically modified humanized non-human mammal of the pregnant female in step (c) .
  • the establishment of a humanized animal model of LAG-3 gene using a gene editing technique is based on CRISPR /Cas9.
  • the non-human mammal is mouse. In some embodiments, the mouse is a C57BL/6 mouse. In some embodiments, the non-human mammal in step (c) is a female with false pregnancy.
  • the disclosure also relates to a method for establishing a genetically-modified non-human animal expressing two human or chimeric (e.g., humanized) genes.
  • the method includes the steps of
  • step (b) mating the LAG-3 gene genetically modified humanized mouse obtained in step (a) with another humanized mouse, and then screening to obtain a double humanized mouse model.
  • step (b) the LAG-3 gene genetically modified humanized mouse obtained in step (a) is mated with a PD-1 or CTLA-4 humanized mouse to obtain a LAG-3 and PD-1 double humanized mouse model or a LAG-3 and CTLA-4 double humanized mouse model.
  • the disclosure also relates to non-human mammal generated through the methods as described herein.
  • the genome thereof contains human gene (s) .
  • the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.
  • the non-human mammal expresses a protein encoded by a humanized LAG-3 gene.
  • the disclosure also relates to an offspring of the non-human mammal.
  • the disclosure relates to a tumor bearing non-human mammal model, characterized in that the non-human mammal model is obtained through the method as described herein.
  • the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.
  • the disclosure also relates to a cell or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal.
  • the disclosure further relates to the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal.
  • the disclosure relates to a tumor tissue derived from the non-human mammal or an offspring thereof when it bears a tumor, or the tumor bearing non-human mammal.
  • the disclosure relates to a LAG-3 amino acid sequence of a humanized animal, wherein the amino acid sequence is selected from the group consisting of:
  • nucleic acid sequence an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is able to hybridize to a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 31 under a low stringency condition;
  • amino acid sequence having a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%with the amino acid sequence shown in SEQ ID NO: 31;
  • amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 31 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid;
  • the disclosure also relates to a LAG-3 nucleotide sequence of a humanized animal, wherein the nucleotide sequence is selected from the group consisting of:
  • nucleotide sequence that encodes the LAG-3 amino acid sequence of a humanized animal as described herein;
  • CDS coding DNA sequence
  • nucleotide sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 30 or SEQ ID NO: 29 under a low stringency condition;
  • nucleotide sequence that has a homology of at least 90%with the nucleotide sequence as shown in SEQ ID NO: 29 or SEQ ID NO: 30;
  • nucleotide sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%with the amino acid sequence shown in SEQ ID NO: 31;
  • nucleotide sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%with the amino acid sequence shown in SEQ ID NO: 31;
  • nucleotide sequence that encodes an amino acid sequence, wherein the amino acid sequence is different from the amino acid sequence shown in SEQ ID NO: 31 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid;
  • nucleotide sequence that encodes an amino acid sequence, wherein the amino acid sequence comprises a substitution, a deletion and /or insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acids to the amino acid sequence shown in SEQ ID NO: 31.
  • the disclosure further relates to a LAG-3 genomic DNA sequence of a humanized mouse, a DNA sequence obtained by a reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence; a construct expressing the amino acid sequence thereof; a cell comprising the construct thereof; a tissue comprising the cell thereof.
  • the disclosure further relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the method as described herein in the development of a product related to an immunization processes of human cells, the manufacture of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.
  • the disclosure also relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the method as described herein in the production and utilization of an animal experimental disease model of an immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and /or a therapeutic strategy.
  • the disclosure further relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the methods as described herein, in the screening, verifying, evaluating or studying the LAG-3 gene function, human LAG-3 antibodies, the drugs or efficacies for human LAG-3 targeting sites, and the drugs for immune-related diseases and antitumor drugs.
  • FIG. 1A is a graph showing activity testing results for sgRNA1-sgRNA7 (Con is a negative control; PC is a positive control; and blank is a blank control) .
  • FIG. 1B is a graph showing activity testing results for sgRNA8-sgRNA14 (Con is a negative control; and PC is a positive control) .
  • FIG. 2 is a schematic diagram showing pT7-sgRNA plasmid map.
  • FIG. 3A is a schematic diagram showing comparison of human and mouse LAG-3 genes.
  • FIG. 3B is a schematic diagram showing a humanized LAG-3 mouse gene map.
  • FIG. 3C is a schematic diagram showing a mouse LAG-3 gene targeting strategy.
  • FIG. 4 shows pClon-4G-LAG plasmid digestion result (CK is undigested plasmid) .
  • FIG. 5 shows PCR identification result of samples collected from tails of F0 generation mice (M is the Marker; WT is wildtype; mice labeled with No. 1, 2 and 3 are positive) .
  • FIG. 6 shows PCR identification result of samples collected from tails of F1 generation mice (WT is wildtype; + is positive control; mice labeled with F1-1, F1-2, F1-3, F1-4, F1-5 are all positive) .
  • FIG. 7 shows Southern blot results for F1 generation mice by P1 and P2 probes (WT is wildtype) . The results show that the mice labeled with F1-2, F1-3, and F1-4 had no random insertion.
  • FIGS. 8A-8I are results of flow cytometry analysis for C57BL/6 mice and LAG-3 humanized mice.
  • the anti-mouse CD3 antibody was used to stimulate the T cells in the spleens. Then the cells were labeled with anti-mouse LAG-3 antibody (mLAG3 PE; FIGS. 8A-8C) , anti-human LAG-3 antibody (hLAG3 Alexa Fluor 647; FIGS. 8D-8F) , or anti-human LAG-3 antibody (hLAG3 APC; FIGS. 8G-8I) and analyzed using flow cytometry. Compared to the control group (FIGS.
  • cells expressing humanized LAG-3 protein can be detected in the spleens of humanized LAG-3 F1 heterozygous mouse; whereas in the spleens of C57BL/6 mice, no cell expressing humanized LAG-3 protein was detected.
  • FIG. 9A-9I are results of flow cytometry analysis of C57BL/6 mice and homozygous humanized LAG-3 mice.
  • the anti-mouse CD3 antibody was used to stimulate the T cells in the spleens. Then the cells were labeled with anti-mouse LAG-3 antibody (mLAG3 PE; FIGS. 9A-9C) , anti-human LAG-3 antibody (hLAG3 APC; FIGS. 9D-9F) , or anti-human LAG-3 antibody (hLAG3 Alexa Fluor 647; FIGS. 9G-9I) , and analyzed using flow cytometry. Cells expressing humanized LAG-3 protein can be detected in the spleens of humanized LAG-3 F1 homozygotes (FIGS. 9F, 9I) .
  • FIG. 10 shows PCR analysis results, wherein WT is wildtype C57BL/6 mouse; mice labeled No. 1, 2, and 3 were LAG-3 knockout mice.
  • FIG. 11 shows results from experiments where mouse colon cancer cells MC38 were injected into B-hLAG-3 mice, followed by studies on antitumor efficacy of three anti-human LAG-3 antibodies (Ab-A, Ab-B, and Ab-C, 10mg/kg) . There was no significant difference in the average weight between the G1 control group and the G2-G4 treatment groups.
  • FIG. 12 shows results from experiments where mouse colon cancer cells MC38 were injected into B-hLAG-3 mice, followed by studies on antitumor efficacy of three anti-human LAG-3 antibodies (Ab-A, Ab-B, and Ab-C, 10mg/kg) . While the decease of tumor size in G2 and G3 groups were not significant, the tumor size in G4 group (treated with Ab-C) was significantly smaller compared to the G1 control group.
  • FIG. 13 shows PCR detection results.
  • + is a known humanized LAG-3 homozygous mouse, -is wildtype control, WT is wildtype, “mut” indicates humanized LAG-3.
  • FIGS. 13C, 13D -/-is a humanized PD-1 homozygous mouse, +/-is humanized PD-1 heterozygous mouse, WT is wildtype, “mut” indicates humanized PD-1.
  • FIGS. 13A and 13B show that the mice numbered 3020 to 3023 were humanized LAG-3 homozygous mice.
  • FIGS. 13C and 13D show that the mice numbered 3019 to 3027 were humanized PD-1 homozygous mice.
  • FIGS. 13A-13D show that the four mice numbered 3020 to 3023 are double humanized mice that are homozygous for both humanized PD-1 and humanized LAG-3.
  • FIGS. 14A-14F are results of flow cytometry analysis for C57BL/6 mice and double humanized LAG-3/PD-1 homozygous mice.
  • Anti-mouse CD3 antibody was used to stimulate T cell activation in the spleens of the mice.
  • the cells were then stained with either (1) mouse LAG-3 antibody (mLAG-3 PE) and anti-mTCR ⁇ antibody (mTcR ⁇ PerCP) (FIGS. 14A, 14B, 14C) , or (2) human LAG-3 antibody (hLAG-3 APC) and anti-mTCR ⁇ antibody (mTcR ⁇ PerCP) (FIGS. 14D, 14E, 14F) .
  • FIGS. 15A-15F are results of flow cytometry analysis for C57BL/6 mice and double humanized LAG-3/PD-1 homozygous mice.
  • Anti-mouse CD3 antibody was used to stimulate T cell activation in the spleens of the mice.
  • the cells were then stained with either mouse PD-1 antibody (mPD-1 PE) and anti-mTCR ⁇ antibody (mTcR ⁇ PerCP) (FIGS. 15A, 15B, 15C) , or human PD-1 antibody (hPD-1 FITC) and anti-mTCR ⁇ antibody (mTcR ⁇ PerCP) (FIGS. 15D, 15E, 15F) .
  • FIGS. 16A-16D are PCR results identifying double humanized LAG-3/CTLA-4 homozygous mice.
  • + is humanized CTLA-4 homozygous mouse, -is wildtype, WT is wildtype, “mut” indicates humanized CTLA-4.
  • + is LAG-3 heterozygous mouse, -is wildtype, WT is wildtype, “mut” indicates humanized LAG-3.
  • FIGS. 16A and 16B show that the mice numbered 1106 to 1117 were humanized CTLA-4 homozygous mice.
  • FIGS. 16C and 16D show that the mice numbered 1106 to 1117 were humanized LAG-3 homozygous mice.
  • FIGS. 16A-16D show that the twelve mice numbered 1106 to 1117 were double humanized mice that are homozygous for both humanized CTLA-4 and humanized LAG-3.
  • FIG. 17 Mouse colon cancer cells MC38 were injected into double humanized homozygous LAG-3/PD-1 mice. Antitumor efficacy studies were performed with Keytruda (anti-human PD-1 antibody) , and Keytruda in combination with anti-human LAG-3 antibody (Ab-C) . There was no significant difference in the average weight between the G1 control group and the G2-G3 treatment groups.
  • FIG. 18 Mouse colon cancer cells MC38 were injected into double humanized homozygous LAG-3/PD-1 mice. Antitumor efficacy studies were performed with Keytruda, and Keytruda in combination with anti-human LAG-3 antibody (Ab-C) . There was no significant difference in the percentage of body weight change among the different groups.
  • FIG. 19 Mouse colon cancer cells MC38 were injected into double humanized homozygous LAG-3/PD-1 mice. Antitumor efficacy studies were performed with Keytruda, and Keytruda in combination with anti-human LAG-3 antibody (Ab-C) . The average volumes of tumors in the G2-G3 treatment groups were significantly smaller than the G1 control group. Furthermore, the tumor size in mice treated with the combination of Keytruda and Ab-C is significantly smaller than the tumor size in mice treated with Keytruda alone.
  • Ab-C anti-human LAG-3 antibody
  • FIG. 20 is a schematic diagram of a targeting strategy for embryonic stem cells.
  • FIG. 21 shows the alignment between mouse LAG-3 amino acid sequence (NP_032505.1; SEQ ID NO: 25) and human LAG-3 amino acid sequence (NP_002277.4; SEQ ID NO: 27) by NCBI Basic Local Alignment Search Tool (BLAST) .
  • BLAST NCBI Basic Local Alignment Search Tool
  • This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) Lymphocyte Activation Gene 3 (LAG-3) , and methods of use thereof.
  • human or chimeric e.g., humanized Lymphocyte Activation Gene 3 (LAG-3)
  • LAG-3 Lymphocyte Activation Gene 3
  • LAG-3 (CD223) is a co-inhibitory receptor of T cells. Expression of LAG-3 has been reported in activated CD4+ and CD8+ effector T cells, CD4+Foxp3+ Treg, Tr1 cells, B cells, plasmacytoid DCs, and NK cells. LAG-3 associates with CD3 in the TCR complex and crosslinking of LAG-3 together with CD3 negatively regulates signal transduction leading to reduced T cell proliferation and cytokine production. LAG-3 deficient OVA-specific CD4+ T cells show uncontrolled expansion upon immunization with their cognate antigen (Workman, Creg J., and Dario AA Vignali.
  • LAG ⁇ 3 The CD4 ⁇ related molecule, LAG ⁇ 3 (CD223) , regulates the expansion of activated T cells.
  • European journal of immunology 33.4 (2003) : 970-979) increased proliferation of LAG-3 deficient donor T cells causes more severe acute GVHD.
  • LAG-3 expression is induced by T cell activation and, like in CD4+ T cells, blockade of LAG-3 improves cytotoxic T cell (CTL) proliferation and effector function.
  • CTL cytotoxic T cell
  • loss of LAG-3 reduced the suppressive function of Tregs, while forced expression of LAG-3 conferred effector T cells with suppressive capacity.
  • LAG-3 thus plays an important role in dampening immune responses by functionally contributing to immune suppression by regulatory T cells. (Nicole Joller and Vijay K. Kuchroo, “Tim-3, Lag-3, and TIGIT, ” Curr Top Microbiol Immunol. 2017 Sep 13. )
  • the LAG-3 antibody has great application values, e.g., as a tumor immunotherapy or a treatment for autoimmune disease (e.g., psoriasis and plaque psoriasis) .
  • a tumor immunotherapy e.g., a tumor immunotherapy or a treatment for autoimmune disease (e.g., psoriasis and plaque psoriasis) .
  • a treatment for autoimmune disease e.g., psoriasis and plaque psoriasis
  • the present disclosure provides humanized LAG-3 genetically modified animal models and methods of establishing such animal models.
  • Experimental animal models are an indispensable research tool for studying the etiology, pathogenesis of the disease, as well as the development of prevention and control techniques and therapeutic drugs for the disease.
  • Common experimental animals include mice, rats, guinea pigs, hamsters, rabbits, dogs, monkeys, pigs, fish and so on.
  • human and animal genes and protein sequences there are many differences between human and animal genes and protein sequences, and many human proteins cannot bind to the animal’s homologous proteins to produce biological activity, leading to that the results of many clinical trials do not match the results obtained from animal experiments.
  • a large number of clinical studies are in urgent need of better animal models.
  • the use of human cells or genes to replace or substitute an animal’s endogenous similar cells or genes to establish a biological system or disease model closer to human, and establish the humanized experimental animal models (humanized animal model) has provided an important tool for new clinical approaches or means.
  • the genetically engineered animal model that is, the use of genetic manipulation techniques, the use of human normal or mutant genes to replace animal homologous genes, can be used to establish the genetically modified animal models that are closer to human gene systems.
  • the humanized animal models have various important applications. For example, due to the presence of human or humanized genes, the animals can express or express in part of the proteins with human functions, so as to greatly reduce the differences in clinical trials between humans and animals, and provide the possibility of drug screening at animal levels.
  • LAG-3 (Lymphocyte Activation Gene 3)
  • LAG-3 (also known as CD223) is an immunoglobulin superfamily member composed of four extracellular Ig-like domains and a type I transmembrane domain.
  • Major histocompatibility complex class II (MHC-II) is a ligand for LAG-3; additional ligands including L-selectin and galectin-3 have also been identified.
  • LAG-3+ tumor- infiltrating lymphocytes (TILs) have been reported in melanoma, colon, pancreatic, breast, lung, hematopoietic, and head and neck cancer patients, in association with aggressive clinical features. (Nicole Joller and Vijay K.
  • LAG-3 gene locus has eight exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 (FIG. 3A) .
  • the LAG-3 protein also has an extracellular region, a transmembrane region, and a cytoplasmic region, and the signal peptide is located at the extracellular region of LAG-3.
  • the nucleotide sequence for human LAG-3 mRNA is NM_002286.5 (SEQ ID NO: 26)
  • amino acid sequence for human LAG-3 is NP_002277.4 (SEQ ID NO: 27) .
  • the location for each exon and each region in human LAG-3 nucleotide sequence and amino acid sequence is listed below:
  • LAG-3 gene locus has eight exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 (FIG. 3A) .
  • the LAG-3 protein also has an extracellular region, a transmembrane region, and a cytoplasmic region, and the signal peptide is located at the extracellular region of LAG-3.
  • the nucleotide sequence for mouse LAG-3 cDNA is NM_008479.2 (SEQ ID NO: 24)
  • the amino acid sequence for mouse LAG-3 is NP_032505.1 (SEQ ID NO: 25) .
  • the location for each exon and each region in the mouse LAG-3 nucleotide sequence and amino acid sequence is listed below:
  • the mouse LAG-3 gene (Gene ID: 16768) is located in Chromosome 11 of the mouse genome, which is located from 124904359-124912434 of NC_000072.6 (GRCm38. p4 (GCF_000001635.24) ) .
  • the approximate locations of 5’-UTR, 3’-URT, exons and introns are listed below (LAG-3 has a reverse orientation on the chromosome) .
  • the 5’-UTR is from 124, 911, 705 to 124, 911, 352, exon 1 is from 124911351 to 124911294, the first intron is from 124, 911, 293 to 124, 910, 912, exon 2 is from 124, 910, 911 to 124, 910, 764, the second intron is from 124, 910, 763 to 124, 910, 397, exon 3 is from 124, 910, 396 to 124, 910, 104, the third intron is from 124, 910, 103 to 124, 909, 490, exon 4 is from 124, 909, 489 to 124, 909, 226, the fourth intron is from 124, 909, 225 to 124, 908, 653, exon 5 is from 24, 908, 652 to 124, 908, 377, the fifth intron is from 124, 908, 376 to 124, 905, 493, exon 6 is from 124, 905, 492 to 124
  • FIG. 22 shows the alignment between mouse LAG-3 amino acid sequence (NP_032505.1; SEQ ID NO: 25) and human LAG-3 amino acid sequence (NP_002277.4; SEQ ID NO: 27) .
  • NP_032505.1 mouse LAG-3 amino acid sequence
  • NP_002277.4 human LAG-3 amino acid sequence
  • LAG-3 genes, proteins, and locus of the other species are also known in the art.
  • the gene ID for LAG-3 in Rattus norvegicus is 297596
  • the gene ID for LAG-3 in Macaca mulatta (Rhesus monkey) is 713737
  • the gene ID for LAG-3 in Sus scrofa (pig) is 100125962.
  • the relevant information for these genes can be found, e.g., intron sequences, exon sequences, amino acid residues of these proteins
  • NCBI database e.g., NCBI database.
  • the present disclosure provides human or chimeric (e.g., humanized) LAG-3 nucleotide sequence and/or amino acid sequences.
  • the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region are replaced by the corresponding human sequence.
  • a “region” or “portion” of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region are replaced by the corresponding human sequence.
  • region or “portion” can refer to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, or 400 nucleotides, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, or 150 amino acid residues.
  • the “region” or “portion” can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%identical to exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, signal peptide, extracellular region, transmembrane region, or cytoplasmic region.
  • a region, a portion, or the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 are replaced by the human exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 (e.g., exon 2 and exon 3) sequence.
  • the present disclosure also provides a chimeric (e.g., humanized) LAG-3 nucleotide sequence and/or amino acid sequences, wherein in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%of the sequence are identical to or derived from mouse LAG-3 mRNA sequence (e.g., SEQ ID NO: 24) , or mouse LAG-3 amino acid sequence (e.g., SEQ ID NO: 25) ; and in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
  • sequence encoding amino acids 25-162 of mouse LAG-3 (SEQ ID NO: 25) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human LAG-3 (e.g., amino acids 25-166 of human LAG-3 (SEQ ID NO: 27) .
  • the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse LAG-3 promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.
  • a promotor or regulatory element e.g., an endogenous mouse LAG-3 promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.
  • the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from a portion of or the entire mouse LAG-3 nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or NM_008479.2 (SEQ ID NO: 24)) .
  • a portion e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides
  • NM_008479.2 SEQ ID NO: 24
  • the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire mouse LAG-3 nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or NM_008479.2 (SEQ ID NO: 24) ) .
  • a portion e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides
  • the entire mouse LAG-3 nucleotide sequence e.g., exon 1, exon 2, exon 3, exon 4, ex
  • the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from a portion of or the entire human LAG-3 nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or NM_002286.5 (SEQ ID NO: 26) ) .
  • a portion e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides
  • NM_002286.5 SEQ ID NO: 26
  • the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire human LAG-3 nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or NM_002286.5 (SEQ ID NO: 26) ) .
  • a portion e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides
  • the entire human LAG-3 nucleotide sequence e.g., exon 1, exon 2, exon 3, exon 4, exon
  • the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from a portion of or the entire mouse LAG-3 amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 or NP_032505.1 (SEQ ID NO: 25) ) .
  • a portion e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues
  • NP_032505.1 SEQ ID NO: 25
  • the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as a portion of or the entire mouse LAG-3 amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or NP_032505.1 (SEQ ID NO: 25) ) .
  • a portion e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues
  • the entire mouse LAG-3 amino acid sequence e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or NP
  • the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from a portion of or the entire human LAG-3 amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or NP_002277.4 (SEQ ID NO: 27) ) .
  • a portion e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues
  • NP_002277.4 SEQ ID NO: 27
  • the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as a portion of or the entire human LAG-3 amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or NP_002277.4 (SEQ ID NO: 27) ) .
  • a portion e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues
  • the entire human LAG-3 amino acid sequence e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or
  • the present disclosure also provides a humanized LAG-3 mouse amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:
  • nucleic acid sequence an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is able to hybridize to a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 31 under a low stringency condition;
  • amino acid sequence having a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 31;
  • amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 31 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid;
  • the present disclosure also relates to a LAG-3 nucleotide (e.g., DNA or RNA) sequence, wherein the nucleotide sequence can be selected from the group consisting of:
  • nucleotide sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 29 or SEQ ID NO: 30 under a low stringency condition;
  • nucleotide sequence that has a homology of at least 90%or at least 90%identical to the nucleotide sequence as shown in SEQ ID NO: 29 or SEQ ID NO: 30;
  • nucleotide sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 31;
  • nucleotide sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%with, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 31;
  • nucleotide sequence that encodes an amino acid sequence, wherein the amino acid sequence is different from the amino acid sequence shown in SEQ ID NO: 31 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid;
  • nucleotide sequence that encodes an amino acid sequence, wherein the amino acid sequence comprises a substitution, a deletion and /or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 31.
  • the present disclosure further relates to a LAG-3 genomic DNA sequence of a humanized mouse.
  • the DNA sequence is obtained by a reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence homologous to the sequence shown in SEQ ID NO: 29 or SEQ ID NO: 30.
  • the disclosure also provides an amino acid sequence that has a homology of at least 90%with, or at least 90%identical to the sequence shown in SEQ ID NO: 31, and has protein activity.
  • the homology with the sequence shown in SEQ ID NO: 31 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.
  • the foregoing homology is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or at least about 59%.
  • the percentage identity with the sequence shown in SEQ ID NO: 31 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or at least about 59%.
  • the disclosure also provides a nucleotide sequence that has a homology of at least 90%, or at least 90%identical to the sequence shown in SEQ ID NO: 30, and encodes a polypeptide that has protein activity.
  • the homology with the sequence shown in SEQ ID NO: 30 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.
  • the foregoing homology is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or at least about 59%.
  • the percentage identity with the sequence shown in SEQ ID NO: 30 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or at least about 59%.
  • the disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any amino acid sequence as described herein.
  • the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein.
  • the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, or 500 nucleotides.
  • the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, or 150 amino acid residues.
  • the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.
  • the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.
  • the sequences are aligned for optimal comparison purposes (e. g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) .
  • the length of a reference sequence aligned for comparison purposes is at least 80%of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • percent homology is often used to mean “sequence similarity. " The percentage of identical residues (percent identity) and the percentage of residues conserved with similar physicochemical properties (percent similarity) , e.g. leucine and isoleucine, are both used to "quantify the homology” . Residues conserved with similar physicochemical properties are well known in the art. The percent homology, in many cases, is higher than the percent identity.
  • Cells, tissues, and animals are also provided that comprise the nucleotide sequences as described herein, as well as cells, tissues, and animals (e.g., mouse) that express human or chimeric (e.g., humanized) LAG-3 from an endogenous non-human LAG-3 locus.
  • the term “genetically-modified non-human animal” refers to a non-human animal having exogenous DNA in at least one chromosome of the animal’s genome.
  • at least one or more cells e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%of cells of the genetically-modified non-human animal have the exogenous DNA in its genome.
  • the cell having exogenous DNA can be various kinds of cells, e.g., an endogenous cell, a somatic cell, an immune cell, a T cell, a B cell, a germ cell, a blastocyst, or an endogenous tumor cell.
  • genetically-modified non-human animals comprise a modified endogenous LAG-3 locus that comprises an exogenous sequence (e.g., a human sequence) , e.g., a replacement of one or more non-human sequences with one or more human sequences.
  • the animals are generally able to pass the modification to progeny, i.e., through germline transmission.
  • chimeric gene or “chimeric nucleic acid” refers to a gene or a nucleic acid, wherein two or more portions of the gene or the nucleic acid are from different species, or at least one of the sequences of the gene or the nucleic acid does not correspond to the wildtype nucleic acid in the animal.
  • the chimeric gene or chimeric nucleic acid has at least one portion of the sequence that is derived from two or more different sources, e.g., sequences encoding different proteins or sequences encoding the same (or homologous) protein of two or more different species.
  • the chimeric gene or the chimeric nucleic acid is a humanized gene or humanized nucleic acid.
  • chimeric protein or “chimeric polypeptide” refers to a protein or a polypeptide, wherein two or more portions of the protein or the polypeptide are from different species, or at least one of the sequences of the protein or the polypeptide does not correspond to wildtype amino acid sequence in the animal.
  • the chimeric protein or the chimeric polypeptide has at least one portion of the sequence that is derived from two or more different sources, e.g., same (or homologous) proteins of different species.
  • the chimeric protein or the chimeric polypeptide is a humanized protein or a humanized polypeptide.
  • the chimeric gene or the chimeric nucleic acid is a humanized LAG-3 gene or a humanized LAG-3 nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human LAG-3 gene, at least one or more portions of the gene or the nucleic acid is from a non-human LAG-3 gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes a LAG-3 protein.
  • the encoded LAG-3 protein is functional or has at least one activity of the human LAG-3 protein or the non-human LAG-3 protein, e.g., binding to human or non-human MHC II, CD3, L-selectin or galectin-3, and/or inhibiting immune responses.
  • the chimeric protein or the chimeric polypeptide is a humanized LAG-3 protein or a humanized LAG-3 polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a human LAG-3 protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human LAG-3 protein.
  • the humanized LAG-3 protein or the humanized LAG-3 polypeptide is functional or has at least one activity of the human LAG-3 protein or the non-human LAG-3 protein
  • the genetically modified non-human animal can be various animals, e.g., a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo) , deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey) .
  • a mouse, rat, rabbit, pig, bovine e.g., cow, bull, buffalo
  • deer sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey)
  • suitable genetically modifiable ES cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification.
  • Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo.
  • a suitable cell e.g., an oocyte
  • gestating the modified cell e.g., the modified oocyte
  • the animal is a mammal, e. g., of the superfamily Dipodoidea or Muroidea.
  • the genetically modified animal is a rodent.
  • the rodent can be selected from a mouse, a rat, and a hamster.
  • the rodent is selected from the superfamily Muroidea.
  • the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters) , Cricetidae (e.g., hamster, New World rats and mice, voles) , Muridae (true mice and rats, gerbils, spiny mice, crested rats) , Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice) , Platacanthomyidae (e.g., spiny dormice) , and Spalacidae (e.g., mole rates, bamboo rats, and zokors) .
  • Calomyscidae e.g., mouse-like hamsters
  • Cricetidae e.g., hamster, New World rats and mice, voles
  • Muridae true mice and rats, gerbils, spiny mice, crested rats
  • the genetically modified rodent is selected from a true mouse or rat (family Muridae) , a gerbil, a spiny mouse, and a crested rat.
  • the non-human animal is a mouse.
  • the animal is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola.
  • a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola.
  • the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm) , 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac) , 129S7, 129S8, 129T1, 129T2.
  • a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm) , 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac) , 129S7, 129S8, 129T1, 129T2.
  • the genetically modified mouse is a mix of the 129 strain and the C57BL/6 strain.
  • the mouse is a mix of the 129 strains, or a mix of the BL/6 strains.
  • the mouse is a BALB strain, e.g., BALB/c strain.
  • the mouse is a mix of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50%BALB/c-50%12954/Sv; or 50%C57BL/6-50%129) .
  • a hybrid line e.g., 50%BALB/c-50%12954/Sv; or 50%C57BL/6-50%129
  • the animal is a rat.
  • the rat can be selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti.
  • the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.
  • the animal can have one or more other genetic modifications, and/or other modifications, that are suitable for the particular purpose for which the humanized LAG-3 animal is made.
  • suitable mice for maintaining a xenograft e.g., a human cancer or tumor
  • mice for maintaining a xenograft can have one or more modifications that compromise, inactivate, or destroy the immune system of the non-human animal in whole or in part.
  • Compromise, inactivation, or destruction of the immune system of the non-human animal can include, for example, destruction of hematopoietic cells and/or immune cells by chemical means (e.g., administering a toxin) , physical means (e.g., irradiating the animal) , and/or genetic modification (e.g., knocking out one or more genes) .
  • Non-limiting examples of such mice include, e.g., NOD mice, SCID mice, NOD/SCID mice, IL2R ⁇ knockout mice, NOD/SCID/ ⁇ cnull mice (Ito, M.
  • a genetically modified mouse can include a humanization of at least a portion of an endogenous non-human LAG-3 locus, and further comprises a modification that compromises, inactivates, or destroys the immune system (or one or more cell types of the immune system) of the non-human animal in whole or in part.
  • modification is, e.g., selected from the group consisting of a modification that results in NOD mice, SCID mice, NOD/SCID mice, IL-2R ⁇ knockout mice, NOD/SCID/ ⁇ c null mice, nude mice, Rag1 and/or Rag2 knockout mice, and a combination thereof.
  • NOD mice SCID mice, NOD/SCID mice, IL-2R ⁇ knockout mice, NOD/SCID/ ⁇ c null mice, nude mice, Rag1 and/or Rag2 knockout mice, and a combination thereof.
  • the mouse can include a replacement of all or part of mature LAG-3 coding sequence with human mature LAG-3 coding sequence.
  • genetically modified non-human animals that comprise a modification of an endogenous non-human LAG-3 locus.
  • the modification can comprise a human nucleic acid sequence encoding at least a portion of a mature LAG-3 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the mature LAG-3 protein sequence) .
  • genetically modified cells are also provided that can comprise the modifications described herein (e.g., ES cells, somatic cells)
  • the genetically modified non-human animals comprise the modification of the endogenous LAG-3 locus in the germline of the animal.
  • Genetically modified animals can express a human LAG-3 and/or a chimeric (e.g., humanized) LAG-3 from endogenous mouse loci, wherein the endogenous mouse LAG-3 gene has been replaced with a human LAG-3 gene and/or a nucleotide sequence that encodes a region of human LAG-3 sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70&, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the human LAG-3 sequence.
  • an endogenous non-human LAG-3 locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a mature LAG-3 protein.
  • the genetically modified mice express the human LAG-3 and/or chimeric LAG-3 (e.g., humanized LAG-3) from endogenous loci that are under control of mouse promoters and/or mouse regulatory elements.
  • the replacement (s) at the endogenous mouse loci provide non-human animals that express human LAG-3 or chimeric LAG-3 (e.g., humanized LAG-3) in appropriate cell types and in a manner that does not result in the potential pathologies observed in some other transgenic mice known in the art.
  • the human LAG-3 or the chimeric LAG-3 (e.g., humanized LAG-3) expressed in animal can maintain one or more functions of the wildtype mouse or human LAG-3 in the animal.
  • human or non-human MHC II can bind to the expressed LAG-3 and downregulate immune response, e.g., downregulate immune response by at least 10%, 20%, 30%, 40%, or 50%.
  • the animal does not express endogenous LAG-3.
  • endogenous LAG-3 refers to LAG-3 protein that is expressed from an endogenous LAG-3 nucleotide sequence of the genetically modified non-human animal (e.g., mouse) before the genetic modification.
  • the genome of the animal can comprise a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human LAG-3 (NP_002277.4) (SEQ ID NO: 27) .
  • the genome comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 31.
  • the genome of the genetically modified animal can comprise a replacement at an endogenous LAG-3 gene locus of a sequence encoding a region of endogenous LAG-3 with a sequence encoding a corresponding region of human LAG-3.
  • the sequence that is replaced is any sequence within the endogenous LAG-3 gene locus, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, 5’-UTR, 3’UTR, the first intron, the second intron, and the third intron, the fourth intron, the fifth intron, the sixth intron, the seventh intron etc.
  • the sequence that is replaced is within the regulatory region of the endogenous LAG-3 gene.
  • the sequence that is replaced is exon 2 and exon 3, or part thereof of an endogenous mouse LAG-3 gene locus.
  • the genetically modified animal can have one or more cells expressing a human or chimeric LAG-3 (e.g., humanized LAG-3) having an extracellular region and a cytoplasmic region, wherein the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the extracellular region of human LAG-3.
  • the extracellular region of the humanized LAG-3 has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids (e.g., contiguously or non-contiguously) that are identical to human LAG-3.
  • human LAG-3 and non-human LAG-3 e.g., mouse LAG-3 sequences
  • antibodies that bind to human LAG-3 will not necessarily have the same binding affinity with mouse LAG-3 or have the same effects to mouse LAG-3. Therefore, the genetically modified animal having a human or a humanized extracellular region can be used to better evaluate the effects of anti-human LAG-3 antibodies in an animal model.
  • the genome of the genetically modified animal comprises a sequence encoding an amino acid sequence that corresponds to part or the entire sequence of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 of human LAG-3, part or the entire sequence of extracellular region of human LAG-3 (with or without signal peptide) , or part or the entire sequence of amino acids 25-166 of SEQ ID NO: 27.
  • the non-human animal can have, at an endogenous LAG-3 gene locus, a nucleotide sequence encoding a chimeric human/non-human LAG-3 polypeptide, wherein a human portion of the chimeric human/non-human LAG-3 polypeptide comprises a portion of human LAG-3 extracellular domain, and wherein the animal expresses a functional LAG-3 on a surface of a cell of the animal.
  • the human portion of the chimeric human/non-human LAG-3 polypeptide can comprise a portion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 of human LAG-3.
  • the human portion of the chimeric human/non-human LAG-3 polypeptide can comprise a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 25-166 of SEQ ID NO: 27.
  • the non-human portion of the chimeric human/non-human LAG-3 polypeptide comprises transmembrane and/or cytoplasmic regions of an endogenous non-human LAG-3 polypeptide.
  • a LAG-3 ligand e.g., MHC II
  • a human or humanized transmembrane and/or cytoplasmic regions may not function properly in non-human animal cells.
  • a few extracellular amino acids that are close to the transmembrane region of LAG-3 are also derived from endogenous sequence.
  • the genetically modified animal can be heterozygous with respect to the replacement at the endogenous LAG-3 locus, or homozygous with respect to the replacement at the endogenous LAG-3 locus.
  • the humanized LAG-3 locus lacks a human LAG-3 5’-UTR.
  • the humanized LAG-3 locus comprises a rodent (e.g., mouse) 5’-UTR.
  • the humanization comprises a human 3’-UTR.
  • mouse and human LAG-3 genes appear to be similarly regulated based on the similarity of their 5’-flanking sequence.
  • humanized LAG-3 mice that comprise a replacement at an endogenous mouse LAG-3 locus, which retain mouse regulatory elements but comprise a humanization of LAG-3 encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for human LAG-3 are grossly normal.
  • the present disclosure further relates to a non-human mammal generated through the methods as described herein.
  • the genome thereof contains human gene (s) .
  • the non-human mammal is a rodent, and preferably, the non-human mammal is a mouse.
  • the non-human mammal expresses a protein encoded by a humanized LAG-3 gene.
  • the present disclosure also relates to a tumor bearing non-human mammal model, characterized in that the non-human mammal model is obtained through the methods as described herein.
  • the non-human mammal is a rodent (e.g., a mouse) .
  • the present disclosure further relates to a cell or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; and the tumor tissue derived from the non-human mammal or an offspring thereof when it bears a tumor, or the tumor bearing non-human mammal.
  • the present disclosure also provides non-human mammals produced by any of the methods described herein.
  • a non-human mammal is provided; and the genetically modified animal contains the DNA encoding human or humanized LAG-3 in the genome of the animal.
  • the non-human mammal comprises the genetic construct as described herein (e.g., gene construct as shown in FIG. 3) .
  • a non-human mammal expressing human or humanized LAG-3 is provided.
  • the tissue-specific expression of human or humanized LAG-3 protein is provided.
  • the expression of human or humanized LAG-3 in a genetically modified animal is controllable, as by the addition of a specific inducer or repressor substance.
  • Non-human mammals can be any non-human animal known in the art and which can be used in the methods as described herein.
  • Preferred non-human mammals are mammals, (e.g., rodents) .
  • the non-human mammal is a mouse.
  • the present disclosure also relates to the progeny produced by the non-human mammal provided by the present disclosure mated with the same or other genotypes.
  • the present disclosure also provides a cell line or primary cell culture derived from the non-human mammal or a progeny thereof.
  • a model based on cell culture can be prepared, for example, by the following methods.
  • Cell cultures can be obtained by way of isolation from a non-human mammal, alternatively cell can be obtained from the cell culture established using the same constructs and the standard cell transfection techniques.
  • the integration of genetic constructs containing DNA sequences encoding human LAG-3 protein can be detected by a variety of methods.
  • RNA quantification approaches using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, and in situ hybridization
  • protein level including histochemistry, immunoblot analysis and in vitro binding studies
  • RT-PCR reverse transcriptase polymerase chain reaction
  • protein level including histochemistry, immunoblot analysis and in vitro binding studies
  • the expression level of the gene of interest can be quantified by ELISA techniques well known to those skilled in the art.
  • Many standard analysis methods can be used to complete quantitative measurements. For example, transcription levels can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis (RNAdot) analysis. Immunohistochemical staining, flow cytometry, Western blot analysis can also be used to assess the presence of human LAG-3 protein.
  • the present disclosure relates to a targeting vector, comprising: a) a DNA fragment homologous to the 5’end of a region to be altered (5’arm) , which is selected from the LAG-3 gene genomic DNAs in the length of 100 to 10,000 nucleotides; b) a desired/donor DNA sequence encoding a donor region; and c) a second DNA fragment homologous to the 3’end of the region to be altered (3’arm) , which is selected from the LAG-3 gene genomic DNAs in the length of 100 to 10,000 nucleotides.
  • a) the DNA fragment homologous to the 5’end of a conversion region to be altered (5’arm) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000072.6; c) the DNA fragment homologous to the 3’end of the region to be altered (3’arm) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000072.6.
  • a) the DNA fragment homologous to the 5’end of a region to be altered (5’arm) is selected from the nucleotides from the position 124911766 to the position 124910898 of the NCBI accession number NC_000072.6; c) the DNA fragment homologous to the 3’end of the region to be altered (3’arm) is selected from the nucleotides from the position 124910116 to the position 124908702 of the NCBI accession number NC_000072.6.
  • the length of the selected genomic nucleotide sequence in the targeting vector can be about 1.2 kb, about 1.5 kb, or about 1 kb. In some embodiments, the length is about 869 bp or about 1415 bp.
  • the region to be altered is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 of LAG-3 gene (e.g., exon 2 and/or exon 3 of LAG-3 gene) .
  • the targeting vector can further include a selected gene marker.
  • sequence of the 5’arm is shown in SEQ ID NO: 32; and the sequence of the 3’arm is shown in SEQ ID NO: 38.
  • the target region is derived from human (e.g., 6773206-6773988 of NC_000012.12) .
  • the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human LAG-3, preferably the nucleotide sequence is shown as a first exon, a second exon, a third exon, a fourth exon, a fifth exon, a sixth exon, a seventh exon and/or an eighth exon of the DNA sequence of the human LAG-3.
  • the nucleotide sequence of the humanized LAG-3 encodes the humanized LAG-3 protein with the NCBI accession number NP_002277.4 (SEQ ID NO: 27) .
  • the disclosure also relates to a cell comprising the targeting vectors as described above.
  • the disclosure also relates to an sgRNA sequence for constructing a humanized animal model, wherein the sgRNA sequence targets the LAG-3 gene, the sgRNA is unique on the target sequence of the LAG-3 gene to be altered, and meets the sequence arrangement rule of 5’-NNN (20) -NGG3’or 5’-CCN-N (20) -3’; and in some embodiments, the targeting site of the sgRNA in the mouse LAG-3 gene is located on the exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or exon 8 of the mouse LAG-3 gene (e.g., exon 2 and/or exon 3 of the mouse LAG-3 gene) .
  • the targeting site of the sgRNA in the mouse LAG-3 gene is located on the exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or exon 8 of the mouse LAG-3 gene (e.g., exon 2 and/or exon 3 of the mouse LAG-3 gene) .
  • an upstream sequence thereof is shown as SEQ ID NO: 15, and a downstream sequence thereof is shown as SEQ ID NO: 17, and the sgRNA sequence recognizes a 5’targeting site.
  • the forward oligonucleotide sequence is obtained by adding TAGG to the 5’end of SEQ ID NO: 15; and the reverse oligonucleotide sequence is obtained by adding AAAC to the 5’end of SEQ ID NO: 17.
  • the disclosure provides an sgRNA sequence for constructing a humanized animal model, wherein an upstream sequence thereof is shown as SEQ ID NO: 19, and a downstream sequence thereof is shown as SEQ ID NO: 21, and the sgRNA sequence recognizes a 3’targeting site.
  • the forward oligonucleotide sequence is obtained by adding TAGG to the 5’end of SEQ ID NO: 19; and the reverse oligonucleotide sequence is obtained by adding AAAC to the 5’end of SEQ ID NO: 21.
  • the disclosure relates to a construct including the sgRNA sequence, and/or a cell including the construct.
  • the present disclosure further relates to a non-human mammalian cell, having any one of the foregoing targeting vectors, and one or more in vitro transcripts of the sgRNA construct as described herein.
  • the cell includes Cas9 mRNA or an in vitro transcript thereof.
  • the genes in the cell are heterozygous. In some embodiments, the genes in the cell are homozygous.
  • the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell.
  • Genetically modified animals can be made by several techniques that are known in the art, including, e.g., nonhomologous end-joining (NHEJ) , homologous recombination (HR) , zinc finger nucleases (ZFNs) , transcription activator-like effector-based nucleases (TALEN) , and the clustered regularly interspaced short palindromic repeats (CRISPR) -Cas system.
  • NHEJ nonhomologous end-joining
  • HR homologous recombination
  • ZFNs zinc finger nucleases
  • TALEN transcription activator-like effector-based nucleases
  • CRISPR clustered regularly interspaced short palindromic repeats
  • homologous recombination is used.
  • CRISPR-Cas9 genome editing is used to generate genetically modified animals.
  • genome editing techniques are known in the art, and is described, e.g., in Yin et al., "Delivery technologies for genome editing, " Nature Reviews Drug Discovery 16.6 (2017) : 387-399, which is incorporated by reference in its entirety.
  • Many other methods are also provided and can be used in genome editing, e.g., micro- injecting a genetically modified nucleus into an enucleated oocyte, and fusing an enucleated oocyte with another genetically modified cell.
  • the disclosure provides replacing in at least one cell of the animal, at an endogenous LAG-3 gene locus, a sequence encoding a region of an endogenous LAG-3 with a sequence encoding a corresponding region of human or chimeric LAG-3.
  • the replacement occurs in a germ cell, a somatic cell, a blastocyst, or a fibroblast, etc.
  • the nucleus of a somatic cell or the fibroblast can be inserted into an enucleated oocyte.
  • FIG. 3C shows a humanization strategy for a mouse LAG-3 locus.
  • the targeting strategy involves a vector comprising the 5’end homologous arm, human LAG-3 gene fragment, 3’homologous arm.
  • the process can involve replacing endogenous LAG-3 sequence with human sequence by homologous recombination.
  • the cleavage at the upstream and the downstream of the target site e.g., by zinc finger nucleases, TALEN or CRISPR
  • the homologous recombination is used to replace endogenous LAG-3 sequence with human LAG-3 sequence.
  • the methods for making a genetically modified, humanized animal can include the step of replacing at an endogenous LAG-3 locus (or site) , a nucleic acid encoding a sequence encoding a region of endogenous LAG-3 with a sequence encoding a corresponding region of human LAG-3.
  • the sequence can include a region (e.g., a part or the entire region) of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8, of a human LAG-3 gene.
  • the sequence includes a region of exon 2 and exon 3 of a human LAG-3 gene (e.g., amino acids 25-166 of SEQ ID NO: 27) .
  • the region is located within the extracellular region of LAG-3.
  • the endogenous LAG-3 locus is exon 2 and exon 3 of mouse LAG-3.
  • the methods of modifying a LAG-3 locus of a mouse to express a chimeric human/mouse LAG-3 peptide can include the steps of replacing at the endogenous mouse LAG-3 locus a nucleotide sequence encoding a mouse LAG-3 with a nucleotide sequence encoding a human LAG-3, thereby generating a sequence encoding a chimeric human/mouse LAG-3.
  • the nucleotide sequence encoding the chimeric human/mouse LAG-3 can include a first nucleotide sequence encoding an extracellular region of mouse LAG-3 (with or without the mouse signal peptide sequence) ; a second nucleotide sequence encoding an extracellular region of human LAG-3; a third nucleotide sequence encoding a transmembrane and a cytoplasmic region of a mouse LAG-3.
  • the nucleotide sequences as described herein do not overlap with each other (e.g., the first nucleotide sequence, the second nucleotide sequence, and/or the third nucleotide sequence do not overlap) .
  • the amino acid sequences as described herein do not overlap with each other.
  • the present disclosure further provides a method for establishing a LAG-3 gene humanized animal model, involving the following steps:
  • step (d) identifying the germline transmission in the offspring genetically modified humanized non-human mammal of the pregnant female in step (c) .
  • the non-human mammal in the foregoing method is a mouse (e.g., a C57BL/6 mouse) .
  • the non-human mammal in step (c) is a female with pseudopregnancy (or false pregnancy) .
  • the fertilized eggs for the methods described above are C57BL/6 fertilized eggs.
  • Other fertilized eggs that can also be used in the methods as described herein include, but are not limited to, FVB/N fertilized eggs, BALB/c fertilized eggs, DBA/1 fertilized eggs and DBA/2 fertilized eggs.
  • Fertilized eggs can come from any non-human animal, e.g., any non-human animal as described herein.
  • the fertilized egg cells are derived from rodents.
  • the genetic construct can be introduced into a fertilized egg by microinjection of DNA. For example, by way of culturing a fertilized egg after microinjection, a cultured fertilized egg can be transferred to a false pregnant non-human animal, which then gives birth of a non-human mammal, so as to generate the non-human mammal mentioned in the method described above.
  • the transgene with human regulatory elements expresses in a manner that is unphysiological or otherwise unsatisfactory, and can be actually detrimental to the animal.
  • the disclosure demonstrates that a replacement with human sequence at an endogenous locus under control of endogenous regulatory elements provides a physiologically appropriate expression pattern and level that results in a useful humanized animal whose physiology with respect to the replaced gene are meaningful and appropriate in the context of the humanized animal's physiology.
  • Genetically modified animals that express human or humanized LAG-3 protein provide a variety of uses that include, but are not limited to, developing therapeutics for human diseases and disorders, and assessing the efficacy of these human therapeutics in the animal models.
  • genetically modified animals are provided that express human or humanized LAG-3, which are useful for testing agents that can decrease or block the interaction between LAG-3 and MHC II or the interaction between LAG-3 and other ligands (e.g., L-selectin, galectin-3) , testing whether an agent can increase or decrease the immune response, and/or determining whether an agent is an LAG-3 agonist or antagonist.
  • the genetically modified animals can be, e.g., an animal model of a human disease, e.g., the disease is induced genetically (aknock-in or knockout) .
  • the genetically modified non-human animals further comprise an impaired immune system, e.g., a non-human animal genetically modified to sustain or maintain a human xenograft, e.g., a human solid tumor or a blood cell tumor (e.g., a lymphocyte tumor, e.g., a B or T cell tumor) .
  • an impaired immune system e.g., a non-human animal genetically modified to sustain or maintain a human xenograft, e.g., a human solid tumor or a blood cell tumor (e.g., a lymphocyte tumor, e.g., a B or T cell tumor) .
  • the genetically modified animals can be used for determining effectiveness of an anti-LAG-3 antibody for the treatment of cancer.
  • the methods involving administering the anti-LAG-3 antibody to the animal as described herein, wherein the animal has a tumor; and determining the inhibitory effects of the anti-LAG-3 antibody to the tumor.
  • the inhibitor effects that can be determined include, e.g., a decrease of tumor size or tumor volume, a decrease of tumor growth, a reduction of the increase rate of tumor volume in a subject (e.g., as compared to the rate of increase in tumor volume in the same subject prior to treatment or in another subject without such treatment) , a decrease in the risk of developing a metastasis or the risk of developing one or more additional metastasis, an increase of survival rate, and an increase of life expectancy, etc.
  • the tumor volume in a subject can be determined by various methods, e.g., as determined by direct measurement, MRI or CT.
  • the tumor comprises one or more tumor cells that express MHC II (Hemon, Patrice, et al. "MHC class II engagement by its ligand LAG-3 (CD223) contributes to melanoma resistance to apoptosis. " The Journal of Immunology 186.9 (2011) : 5173-5183) .
  • the tumor comprises one or more cancer cells (e.g., human or mouse cancer cells) that are injected into the animal.
  • the anti-LAG-3 antibody or anti-MHC II antibody prevents MHC II from binding to LAG-3.
  • the anti-LAG-3 antibody or anti-MHC II antibody does not prevent MHC II from binding to LAG-3.
  • the genetically modified animals can be used for determining whether an anti-LAG-3 antibody is an LAG-3 agonist or antagonist.
  • the methods as described herein are also designed to determine the effects of the agent (e.g., anti-LAG-3 antibodies) on LAG-3, e.g., whether the agent can stimulate T cells or inhibit T cells, whether the agent can upregulate the immune response or downregulate immune response.
  • the genetically modified animals can be used for determining the effective dosage of a therapeutic agent for treating a disease in the subject, e.g., cancer, or autoimmune diseases.
  • the inhibitory effects on tumors can also be determined by methods known in the art, e.g., measuring the tumor volume in the animal, and/or determining tumor (volume) inhibition rate (TGI TV ) .
  • the anti-LAG-3 antibody is designed for treating various cancers.
  • cancer refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • tumor refers to cancerous cells, e.g., a mass of cancerous cells.
  • Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • the agents described herein are designed for treating or diagnosing a carcinoma in a subject.
  • carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
  • the cancer is renal carcinoma or melanoma.
  • Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • carcinosarcomas e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • an “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • the term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.
  • the anti-LAG-3 antibody is designed for the treating melanoma, non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , bladder cancer, breast cancer, and/or prostate cancer (e.g., metastatic hormone-refractory prostate cancer) .
  • NSCLC non-small cell lung carcinoma
  • SCLC small cell lung cancer
  • bladder cancer e.g., breast cancer
  • prostate cancer e.g., metastatic hormone-refractory prostate cancer
  • the present disclosure also relates to the use of the animal model generated through the methods as described herein in the development of a product related to an immunization processes of human cells, the manufacturing of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.
  • the disclosure provides the use of the animal model generated through the methods as described herein in the production and utilization of an animal experimental disease model of an immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and /or a therapeutic strategy.
  • the disclosure also relates to the use of the animal model generated through the methods as described herein in the screening, verifying, evaluating or studying the LAG-3 gene function, human LAG-3 antibodies, drugs for human LAG-3 targeting sites, the drugs or efficacies for human LAG-3 targeting sites, the drugs for immune-related diseases and antitumor drugs.
  • the present disclosure further relates to methods for generating genetically modified animal model with two or more human or chimeric genes.
  • the animal can comprise a human or chimeric LAG-3 gene and a sequence encoding an additional human or chimeric protein.
  • the additional human or chimeric protein can be programmed cell death protein 1 (PD-1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) , B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , TNF Receptor Superfamily Member 9 (4-1BB) , CD27, CD28, CD47, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , CD27, Glucocorticoid-Induced TNFR-Related Protein (GITR) , or TNF Receptor Superfamily Member 4 (TNFRSF4; or OX40) .
  • PD-1BB cytotoxic T-lymphocyte-associated protein 4
  • TIM-3 T-cell immunoglobulin and mucin-domain containing-3
  • BTLA B And T Lymphocyte Associated
  • PD-L1 Programmed
  • the methods of generating genetically modified animal model with two or more human or chimeric genes can include the following steps:
  • the genetically modified animal in step (b) of the method, can be mated with a genetically modified non-human animal with human or chimeric PD-1, CTLA-4, TIM-3, BTLA, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, CD27, GITR, or OX40.
  • a genetically modified non-human animal with human or chimeric PD-1, CTLA-4, TIM-3, BTLA, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, CD27, GITR, or OX40.
  • the LAG-3 humanization is directly performed on a genetically modified animal having a human or chimeric PD-1, CTLA-4, BTLA, TIM-3, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, CD27, GITR, or OX40 gene.
  • the genetically modified animal model with two or more human or humanized genes can be used for determining effectiveness of a combination therapy that targets two or more of these proteins, e.g., an anti-LAG-3 antibody and an additional therapeutic agent for the treatment of cancer.
  • the methods include administering the anti-LAG-3 antibody and the additional therapeutic agent to the animal, wherein the animal has a tumor; and determining the inhibitory effects of the combined treatment to the tumor.
  • the animal further comprises a sequence encoding a human or humanized programmed cell death protein 1 (PD-1) , or a sequence encoding a human or humanized CTLA4.
  • the additional therapeutic agent is an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, avelumab, durvalumab, atezolizumab) or an CTLA-4 antibody.
  • the tumor comprises one or more tumor cells that express MHC II, CD80, CD86, PD-L1 or PD-L2.
  • the combination treatment is designed for treating various cancer as described herein, e.g., melanoma, non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , bladder cancer, and/or prostate cancer (e. g., metastatic hormone-refractory prostate cancer) .
  • NSCLC non-small cell lung carcinoma
  • SCLC small cell lung cancer
  • prostate cancer e. g., metastatic hormone-refractory prostate cancer
  • the methods described herein can be used to evaluate the combination treatment with some other methods.
  • the methods of treating a cancer that can be used alone or in combination with methods described herein, include, e.g., treating the subject with chemotherapy, e.g., campothecin, doxorubicin, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, adriamycin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, bleomycin, plicomycin, mitomycin, etoposide, verampil, podophyllotoxin, tamoxifen, taxol, transplatinum, 5-flurouracil, vincristin, vinblastin, and/or methotrexate.
  • the methods can include performing surgery on the subject to remove at least a portion of the subject to remove at least
  • Ambion TM in vitro transcription kit was purchased from Ambion. Catalog number is AM1354.
  • E. coli TOP10 competent cells were purchased from the Tiangen Biotech (Beijing) Co. Catalog number is CB104-02.
  • Kanamycin was purchased from Amresco. Catalog number is 0408.
  • Cas9 mRNA was obtained from SIGMA. Catalog number is CAS9MRNA-1EA.
  • AIO kit was obtained from Beijing Biocytogen Co., Ltd. Catalog number is BCG-DX-004.
  • UCA kit was obtained from Beijing Biocytogen Co., Ltd. Catalog number is BCG-DX-001.
  • C57BL/6 mice were purchased from the China Food and Drugs Research Institute National Rodent Experimental Animal Center.
  • B-hPD-1 mice, B-hCTLA-4 mice were obtained from Beijing Biocytogen Co., Ltd.
  • Mouse colon cancer cell line MC38 was purchased from Shanghai Enzyme Research Biotechnology Co., Ltd.
  • Anti-mouse CD3 antibody was obtained from BD. Catalog number is 563123.
  • PE labeled anti-mouse CD223 (LAG-3) antibody was obtained from Biolegend. Catalog number is 125208.
  • Monoclonal anti-human LAG-3 antibody 3DS223H (hLAG3 APC) was obtained from eBioscience. Catalog number is 17-2239-42.
  • PE labeled anti-mouse CD279 (PD-1) antibody was obtained from Biolegend. Catalog number is 109104.
  • FITC labeled anti-human CD279 (PD-1) antibody (hPD-1 FITC) was obtained from Biolegend. Catalog number is 329904.
  • the Flow Cytometer was purchased from BD Biosciences; model FACS Calibur TM .
  • EXAMPLE 1 Construction of pT7-LAG-5 and pT7-LAG-12
  • the target sequence determines the targeting specificity of small guide RNA (sgRNA) and the efficiency of Cas9 cleavage at the target gene. Therefore, target sequence selection is important for sgRNA vector construction.
  • sgRNA small guide RNA
  • the 5’-terminal targeting sites (sgRNA1 to sgRNA7) and the 3’-terminal targeting sites (sgRNA8 to sgRNA14) were designed and synthesized.
  • the 5’-terminal targeting sites and the 3’-terminal targeting sites are located on exon 2 and exon 3 of mouse LAG-3 gene respectively, and the targeting site sequences on LAG-3 for each sgRNA is as follows:
  • sgRNA-1 targeting sequence
  • sgRNA-2 targeting sequence
  • sgRNA-4 targeting sequence
  • sgRNA-5 targeting sequence
  • sgRNA-6 targeting sequence
  • sgRNA-7 targeting sequence
  • sgRNA-8 targeting sequence
  • sgRNA-9 targeting sequence
  • sgRNA-10 targeting sequence
  • sgRNA-11 targeting sequence
  • sgRNA-12 targeting sequence
  • sgRNA-13 targeting sequence
  • sgRNA-14 targeting sequence
  • the UCA kit was used to detect the activities of sgRNAs (FIGS. 1A and 1B) , sgRNA9 was not detected due to the incorrect sequence. The results show that the guide sgRNAs have different activities. Two of them (sgRNA5 and sgRNA12) were selected for follow-up experiments. TAGG was added to the 5’end to obtain a forward oligonucleotide sequence, and its complementary strand was added with AAAC to obtain a reverse oligonucleotide sequence. After annealing, they were respectively digested by restriction enzyme (BbsI) and ligated to pT7-sgRNA plasmid to obtain the expression vectors pT7-LAG-5 and pT7-LAG-12.
  • BbsI restriction enzyme
  • the ligation reaction was carried out at room temperature for 10 to 30 minutes.
  • the ligation product was then transferred to 30 ⁇ L of TOP10 competent cells.
  • the cells were then plated on a petri dish with Kanamycin, and then cultured at 37 °C for at least 12 hours and then two clones were selected and added to LB medium with Kanamycin (5 ml) , and then cultured at 37 °C at 250 rpm for at least 12 hours.
  • Randomly selected clones were sequenced, so as to verify their sequences.
  • the correct expression vectors pT7-LAG-5 and pT7-LAG-12 were selected for subsequent experiments.
  • PT7-sgRNA vector map is shown in FIG. 2.
  • the plasmid backbone was obtained from Takara (Catalog No. 3299) .
  • the DNA fragment containing T7 promoter and sgRNA scaffold was synthesized by a plasmid synthesis company, and linked to the backbone vector by restriction enzyme digestion (EcoRI and BamHI) and ligation.
  • the target plasmid was confirmed by the sequencing results.
  • the DNA fragment containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 23) :
  • a partial coding sequence of the mouse LAG-3 gene (Gene ID: 16768) from exon 2 and exon 3 (based on the transcript of NCBI accession number NM_008479.2 ⁇ NP_032505.1 whose mRNA sequence is shown in SEQ ID NO: 24, and the corresponding protein sequence is shown in SEQ ID NO: 25) was replaced with a corresponding coding sequence of human homologous LAG-3 gene (Gene ID: 3902) (based on the transcript of NCBI accession number NM_002286.5 ⁇ NP_002277.4, whose mRNA sequence was shown in SEQ ID NO: 26, and the corresponding protein sequence is shown in SEQ ID NO: 27) .
  • the comparison between the mouse LAG-3 and human LAG-3 is shown in FIG. 3A, and the finally obtained humanized LAG-3 gene is shown in FIG. 3B, the humanized mouse LAG-3 gene DNA sequence (chimeric LAG-3 gene DNA) is shown in SEQ ID NO: 28.
  • SEQ ID NO: 28 lists only the portion of DNA sequence involved in the modification, wherein the italicized underlined region is the human LAG-3 gene sequence fragment.
  • the coding region sequence, mRNA sequence and the encoded protein sequence thereof of the modified humanized LAG-3 are respectively shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31.
  • a targeting strategy involving a vector comprising the 5’end homologous arm, human LAG-3 gene fragment, 3’homologous arm as shown in FIG. 3C is also developed.
  • the process is as follows:
  • Upstream primer (SEQ ID NO: 33) :
  • Human DNA fragment (783 bp) (SEQ ID NO: 35) is the nucleotide sequence from positions 6773206 to 6773988 of the NCBI accession number NC_000012.12.
  • the upstream primer (SEQ ID NO: 36) is:
  • the downstream primer (SEQ ID NO: 37) is:
  • Upstream primer (SEQ ID NO: 39) :
  • C57BL/6 mouse DNA is used as the template to carry out PCR amplification for the 5’-terminal homologous arm fragment and the 3’-terminal homologous arm fragment.
  • Human DNA is used as the template to carry out PCR amplification for the DNA fragment, and the AIO kit is used to ligate the fragments to the pClon-4G plasmid provided by the kit, so as to obtain the vector pClon-4G-LAG.
  • the pre-mixed Cas9 mRNA, pClon-4G-LAG plasmid and in vitro transcription products of pT7-LAG-5 , pT7-LAG-12 plasmids were injected into the cytoplasm or nucleus of mouse fertilized eggs (C57BL/6 background) with a microinjection instrument (using Ambion in vitro transcription kit to carry out the transcription according to the method provided in the product instruction) .
  • the embryo microinjection was carried out according to the method described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2003.
  • the injected fertilized eggs were then transferred to a culture medium for a short time culture, and then was transplanted into the oviduct of the recipient mouse to produce the genetically modified humanized mice (F0 generation) .
  • the mice population was further expanded by cross-mating and self-mating to establish stable mouse lines.
  • the humanized mouse was named as B-hLAG-3 mouse.
  • PCR analysis was performed for mouse tail genomic DNA of F0 generation mice.
  • the primers for PCR-1 were located on the left side of the 5’homologous arm, the primers for PCR-4 were located on the right side of the 3’homologous arm; in addition, the primers for PCR-2 and PCR-3 were located on the humanized fragment, which are shown below:
  • PCR-1 (SEQ ID NO: 41) : 5’-agcattcacacagggtggggaattt-3’
  • PCR-2 (SEQ ID NO: 42) : 5’-ggcgtgagggtcgctgattg-3’
  • PCR-4 (SEQ ID NO: 44) : 5’-aggagtccacttggcaatgagcaaa-3’
  • the recombinant vector has the correct insertion, there should be one PCR band from 5’terminus primers and one PCR band from the 3’terminus primers.
  • the length of the 5’terminus product should be 2049 bp, and the length of the 3’terminus product should be 2206 bp.
  • F1 generation mice were obtained by cross-mating F0 generation mice with C57BL/6 mice. PCR was performed for four F1 generation mice with the same primers under the same conditions used for F0 verification. The results showed that all five F1 generation mice were positive (FIG. 6) .
  • mice Five mice were further examined by Southern blotting to determine whether they had a random insertion.
  • the genomic DNA was extracted from the mouse tail, and BamHI was used to digest the genomic DNA. The digestion products were transferred to membrane and hybridized.
  • the probes P1 and P2 were located respectively outside of the 5’homologous arm, and on the 3’homologous arm.
  • the primers for probe synthesis are as follows:
  • P1-F (SEQ ID NO: 45) : 5’-ggccacttatcatcacttgccc-3’
  • P1-R (SEQ ID NO: 46) : 5’-ggtggtaaaggggcctaggag-3’
  • P2-F (SEQ ID NO: 47) : 5’-cagggagagcaatggctaggg-3’
  • P2-R (SEQ ID NO: 48) : 5’-gctgaaaaactcatagaaatggggc-3’
  • the wildtype C57BL/6 mice would have a 6.3kb band and a 10.2kb band for probe P1 and probe P2 respectively.
  • the genetically engineered homozygous mice should have a 16.6kb band for either P1 or P2.
  • the genetically engineered heterozygous mice should have a 6.3kb band and a 16.6kb band (P1 probe) , or a 10.2kb and a 16.6kb band (P2 probe) . No other band should be present.
  • F1-2, F1-3, and F1-4 showed the expected bands for both the P1 probe and the P2 probe, with no other band, and without any random insertion.
  • a humanized heterozygous F1 generation mouse was selected for this experiment. Wildtype C57BL/6 mice used as the control. 7.5 ⁇ g of mouse anti-CD3 antibody was injected intraperitoneally to the mice. The spleens were collected 24 hours after the injection, and the spleen samples were grinded. The ground samples were then passed through 70 ⁇ m cell mesh. The filtered cell suspensions were centrifuged and the supernatants were discarded. Erythrocyte lysis solution was added to the sample, which was lysed for 5 min and neutralized with PBS solution. The solution was centrifuged again and the supernatants were discarded. The cells were washed once with PBS.
  • the cells were then stained with (1) an anti-mTCR ⁇ antibody and an anti-mouse LAG-3 antibody (mLAG3 PE; FIGS. 8A-8C) ; (2) an anti-mTCR ⁇ antibody and an anti-human LAG-3 antibody (hLAG3 Alexa Fluor 647; FIGS 8D-8F) ; or (3) an anti-mTCR ⁇ antibody and an anti-human LAG-3 antibody (hLAG3 APC; FIGS. 8G-8I) .
  • the stained samples were washed in PBS once and analyzed by flow cytometry.
  • FIGS. 8A-8I show that cells expressing humanized LAG-3 proteins were detected by anti-human LAG-3 antibodies in the spleens of humanized LAG-3 mice (FIGS. 8F and 8I) . Cells expressing humanized LAG-3 proteins were not detected in the spleens of C57BL/6 mice (FIGS. 8D, 8E, 8G, and 8H) .
  • heterozygous humanized LAG-3 mice F1 were mated with each other, producing homozygous humanized LAG-3 mice (B-hLAG-3) .
  • B-hLAG-3 mice One homozygous B-hLAG-3 mouse (6 weeks) and two wildtype C57BL/6 mice were selected for flow cytometry analysis (FIGS. 9A-9I) .
  • mice CD3 antibody 7.5 ⁇ g was injected intraperitoneally to the homozygous B-hLAG-3 mouse and the control mice.
  • the spleens were collected 24 hours after the injection, and the spleen samples were grinded.
  • the ground samples were then passed through 70 ⁇ m cell mesh.
  • the filtered cell suspensions were centrifuged and the supernatants were discarded.
  • Erythrocyte lysis solution was added to the sample, which was lysed for 5 min and neutralized with PBS solution. The solution was centrifuged again and the supernatants were discarded. The cells were washed once with PBS.
  • the cells were then stained with (1) an anti-mTCR ⁇ antibody and an anti-mouse LAG-3 antibody (mLAG3 PE; FIGS. 9A-9C) ; (2) an anti-mTCR ⁇ antibody and an anti-human LAG-3 antibody (hLAG-3 APC; FIGS. 9D-9F) ; or (3) an anti-mTCR ⁇ antibody and an anti-human LAG-3 antibody (hLAG3 Alexa Fluor 647; FIGS 9G-9I) .
  • mLAG3 PE an anti-mTCR ⁇ antibody and an anti-mouse LAG-3 antibody
  • hLAG-3 APC an anti-mTCR ⁇ antibody and an anti-human LAG-3 antibody
  • an anti-mTCR ⁇ antibody and an anti-human LAG-3 antibody hLAG3 Alexa Fluor 647; FIGS 9G-9I
  • FIGS. 9A-9I show that cells expressing humanized LAG-3 were detected with anti-human LAG-3 antibody in the spleens of homozygous B-hLAG-3 mice stimulated with CD3 antibody (FIGS. 9F, 9I) .
  • No cell expressing humanized LAG-3 was detected in the spleens of C57BL/6 mice, either stimulated with CD3 antibody (FIGS. 9E and 9H) , or without CD3 antibody stimulation (FIGS. 9D and 9G) .
  • the PRC reaction systems and conditions are listed in Table 5 and Table 6. Under these conditions, the wild type mice should have only one PCR band at approximately 1494bp. The homozygous knockout mice should have only one band at approximately 750 bp. The results are shown in FIG. 10. The results confirmed successful generation of LAG-3 knockout mice.
  • B-hLAG-3 homozygous mice (4-6 weeks) were subcutaneously injected with mouse colon cancer cell MC38.
  • the treatment groups were randomly selected for treatment with anti-human LAG-3 antibodies (Ab-A, Ab-B, Ab-C) at 10 mg/kg; the control group was injected with an equal volume of blank solvent.
  • the antibodies and control were administered through intraperitoneal injection once every three days for a total of three times.
  • the tumor volume was measured twice a week and the body weight of the mice was weighed as well. Euthanasia was performed when the tumor volume of the mouse reached 3000 mm 3 .
  • Table 7 shows results for this experiment, including the tumor volumes at the day of grouping (Day 0) , 10 days after grouping (Day 10) , and at the end of the experiment (Day 17) ; the survival rate of the mice; the number of tumor-free mice (non-existence of tumor) ; the Tumor Growth Inhibition value (TGI TV ) .
  • the animals in each group were healthy, with normal weight increase over the course of the experiment.
  • the body weights of the treatment group mice and the control group mice were not significantly different from each other, indicating that the antibodies were well tolerated and safe to use without obvious toxicity (FIG. 11) .
  • the tumors in the control group mice continued growing during the experimental period (FIG. 12) .
  • Tumor sizes in the treatment groups G2 (Ab-A) and G3 (Ab-B) were not significantly different from the control group G1, while the tumor sizes in group G4 (Ab-C) mice were significantly smaller as compared to the control group mice (FIG. 12) .
  • the humanized LAG-3 mouse model could also be used to screen for potential modulators of the LAG-3 signaling pathway, to evaluate the efficacy and toxicity of these modulators, and to design treatment regimens using these modulators.
  • the humanized LAG-3 mouse model could additionally be used to evaluate and predict the efficacy and toxicity of anti-human LAG-3 antibodies, and the potential treatment results of drug candidates targeting human LAG-3.
  • mice containing the humanized LAG-3 gene can also be used to prepare an animal model with double-humanized or multi-humanized genes.
  • the fertilized eggs used in the microinjection and embryo transfer process can be selected from the fertilized eggs of other genetically modified mice or the fertilized eggs of B-hLAG-3 mice, so as to obtain double-or multiple-gene modified mouse models.
  • the B-hLAG-3 animal model homozygote or heterozygote can be mated with other genetically modified homozygous or heterozygous animal models, and the progeny is then screened; according to the Mendelian law, there is a chance to obtain the double-gene or multiple-gene modified heterozygous animal models, and then the obtained heterozygous can be mated with each other to finally obtain the double-gene or multiple-gene modified homozygotes.
  • the double humanized LAG-3/PD-1 mice were obtained by mating B-hLAG-3 mice with humanized PD-1 mice. The progeny mice were screened and further mated to obtain double humanized LAG-3/PD-1 mice.
  • FIGS. 13A-13D The results for a number of double humanized LAG-3/PD-1 mice are shown in FIGS. 13A-13D, wherein FIGS. 13A and 13B show that the mice numbered 3020-3023 were homozygous for humanized LAG-3, FIGS. 13C and 13D show that the mice numbered 3019-3027 were homozygous for humanized PD-1.
  • the combined results show that the four mice numbered 3020-3023 were homozygous for both humanized LAG-3 and humanized PD-1.
  • the double humanized LAG-3/PD-1 mice were further analyzed for protein expression using flow cytometry.
  • One homozygous double humanized LAG-3/PD-1 mice (6-7 weeks) and two wildtype C57BL/6 mice were selected for the analysis.
  • 7.5 ⁇ g of anti-mouse CD3 antibody was injected intraperitoneally to the mice.
  • the spleens were collected 24 hours after the injection, and the spleen samples were grinded.
  • the ground samples were then passed through 70 ⁇ m cell mesh.
  • the filtered cell suspensions were centrifuged and the supernatants were discarded. Erythrocyte lysis solution was added to the sample, which was lysed for 5 min and neutralized with PBS solution. The solution was centrifuged again and the supernatants were discarded.
  • the cells were washed once with PBS.
  • the cells were then stained with either (1) an anti-mouse LAG-3 antibody (mLAG3 PE) and an anti-mTcR ⁇ antibody (mTcR ⁇ PerCP) (FIGS. 14A-14C) , or (2) an anti-human LAG-3 antibody (hLAG3 APC) and an anti-mTcR ⁇ antibody (mTcR ⁇ PerCP) (FIGS. 14D-14F) .
  • the cells were also stained with (1) an anti-mouse PD-1 antibody (mPD-1 PE) and an anti-mTcR ⁇ antibody (mTcR ⁇ PerCP) (FIGS. 15A-15C) , or (2) an anti-human PD-1 antibody (hPD-1 FITC) and anti-mTcR ⁇ antibody (mTcR ⁇ PerCP) (FIGS. 15D-15E) .
  • results in FIGS. 14A-14F show that cells expressing humanized LAG-3 proteins were detected by anti-human LAG-3 antibodies in the spleens of homozygous double humanized LAG-3/PD-1 mice. No cells expressing humanized LAG-3 proteins were detected in the spleens of C57BL/6 mice with or without anti-CD3 antibody stimulation.
  • FIGS. 15A-15F show that cells expressing humanized PD-1 proteins were detected by anti-human PD-1 antibodies in the spleens of homozygous double humanized LAG-3/PD-1 mice. No cells expressing humanized PD-1 proteins were detected in the spleens of C57BL/6 mice either stimulated with or without anti-CD3 antibody stimulation.
  • mice were also performed to generate double humanized LAG-3/CTLA-4 mice. Since the mouse LAG-3 gene and CTLA-4 gene are located on different chromosomes (chromosome 6 and chromosome 1 respectively) , the double humanized LAG-3/CTLA-4 mice were obtained by mating B-hLAG-3 mice with humanized CTLA-4 mice (e.g. B-hCTLA-4, mice with humanized CTLA-4 gene) . The progeny mice were screened and further mated to eventually obtain double humanized LAG-3/CTLA-4 mice.
  • humanized CTLA-4 mice e.g. B-hCTLA-4, mice with humanized CTLA-4 gene
  • FIGS. 16A-16D The results for a number of double humanized LAG-3/CTLA-4 mice are shown in FIGS. 16A-16D, wherein FIGS. 16A and 16B show that the mice numbered 1106-1117 were homozygous for humanized CTLA-4, FIGS. 16C and 16D show that the mice numbered 1106-1117 were homozygous for humanized LAG-3. These results show that the twelve mice numbered 1106-1117 were all homozygous for both humanized LAG-3 and humanized CTLA-4.
  • mice Homozygous double humanized LAG-3/PD-1 mice (4-8 weeks) were subcutaneously injected with mouse colon cancer cell MC38. When the tumor volume grew to about 100 mm 3 , the mice were randomly divided to a control group and two treatment groups. The treatment groups were randomly selected for treatment with Keytruda (0.1 mg/kg) or with both of Keytruda (0.1 mg/kg) and Ab-C (10mg/kg) . The control group was injected with an equal volume of blank solvent. The tumor volume and the body weight were measured twice a week. Euthanasia was performed when the tumor volume of the mouse reached 3000 mm 3 . Table 12 provides more details on the method of administration.
  • FIG. 17 and FIG. 18 show that the mice in the control group and the treatment groups had no significant difference in body weight or body weight percentage change.
  • FIG. 19 shows that the tumors in control group mice (G1) kept growing during the experimental period, while tumor sizes in the treatment group mice (G2 and G3) decreased, indicating that tumor growth were inhibited.
  • Table 13 shows the tumor volumes at the day of grouping (Day 0) , 17 days after grouping (Day 17) , and at the end of the experiment (day 24) ; the survival rate of the mice; the number of tumor-free mice (non-existence of tumor) ; the Tumor Growth Inhibition value (TGI TV ) ; and the statistical differences (P value) in mouse body weight and tumor volume between the treatment and control groups.
  • the average tumor size of the control group (G1) was 2038 ⁇ 423mm 3 ; the average tumor size in the treatment group treated with Keytruda alone (G2) was 1234 ⁇ 142mm 3 ; and the average tumor size in the treatment group treated with both Keytruda and Ab-C (G3) was 771 ⁇ 164mm 3 .
  • the combination treatment group (G3) had significantly smaller tumor size as compared to G2.
  • the TGI TV values were 42.3%and 66.8%for group G2 and G3 respectively, indicating that the efficacy of using a combination of these two antibodies is better than that of using only one antibody.
  • EXAMPLE 10 Methods Based On Embryonic Stem Cell Technologies
  • non-human mammals described herein can also be prepared through other gene editing systems and approaches, including but not limited to: gene homologous recombination techniques based on embryonic stem cells (ES) , zinc finger nuclease (ZFN) techniques, transcriptional activator-like effector factor nuclease (TALEN) technique, homing endonuclease (megakable base ribozyme) , or other techniques.
  • ES embryonic stem cells
  • ZFN zinc finger nuclease
  • TALEN transcriptional activator-like effector factor nuclease
  • homing endonuclease homing endonuclease (megakable base ribozyme) , or other techniques.
  • the conventional ES cell gene homologous recombination technique is used as an example to describe how to obtain LAG-3 gene humanized mice by other methods.
  • a gene editing strategy and methods are shown in FIG. 20, which also shows the design of the recombinant vector. Since the objective is to replace exon 2 and exon 3 of the mouse LAG-3 gene in whole or in part with the corresponding human LAG-3 gene fragment, a recombinant vector that contains a 5’homologous arm (5068 bp) , a 3’homologous arm (5180bp) and a humanized gene fragment (783 bp) is also designed.
  • the vector can also contain a resistance gene for positive clone screening, such as neomycin phosphotransferase coding sequence Neo.
  • a resistance gene for positive clone screening such as neomycin phosphotransferase coding sequence Neo.
  • two site-specific recombination systems in the same orientation such as Frt or LoxP, can be added.
  • a coding gene with a negative screening marker such as the diphtheria toxin A subunit coding gene (DTA)
  • DTA diphtheria toxin A subunit coding gene
  • the recombinant vector with correct sequence can be next transfected into mouse embryonic stem cells, such as C57BL/6 mouse embryonic stem cells, and then the recombinant vector can be screened by positive clone screening gene.
  • the cells transfected with the recombinant vector are next screened by using the positive clone marker gene, and Southern Blot technique can be used for DNA recombination identification.
  • the positive clonal cells black mice
  • the isolated blastocysts white mice
  • the resulting chimeric blastocysts formed following the injection are transferred to the culture medium for a short time culture and then transplanted into the fallopian tubes of the recipient mice (white mice) to produce F0 generation chimeric mice (black and white) .
  • the F0 generation chimeric mice with correct gene recombination are then selected by extracting the mouse tail genome and detecting by PCR for subsequent breeding and identification.
  • the F1 generation mice are obtained by mating the F0 generation chimeric mice with wildtype mice.
  • Stable gene recombination positive F1 heterozygous mice are selected by extracting rat tail genome and PCR detection. Next, the F1 heterozygous mice are mated to each other to obtain genetically recombinant positive F2 generation homozygous mice. In addition, the F1 heterozygous mice can also be mated with Flp or Cre mice to remove the positive clone screening marker gene (neo, etc. ) , and then the LAG-3 gene humanized homozygous mice can be obtained by mating these mice with each other. The methods of genotyping and phenotypic detection of the obtained F1 heterozygous mice or F2 homozygous mice are similar to those used in Example 5 described above.

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Abstract

L'invention concerne un animal non humain génétiquement modifié exprimant un gène 3 d'activation des lymphocytes (LAG-3) humain ou chimérique (humanisé, par exemple), ainsi qu'une méthode permettant de déterminer l'efficacité d'un anticorps anti-LAG-3 pour le traitement du cancer ou d'une tumeur par administration à l'animal de l'anticorps anti-LAG-3.
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US11234420B2 (en) 2016-06-28 2022-02-01 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Method for constructing PD-1 gene modified humanized animal model and use thereof
US10980222B2 (en) 2016-12-23 2021-04-20 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Genetically modified non-human animal with human or chimeric CD27
US11154040B2 (en) 2016-12-30 2021-10-26 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Genetically modified non-human animal with human or chimeric CD137
US11723348B2 (en) 2017-03-31 2023-08-15 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Genetically modified mice expressing humanized CD47
US10918095B2 (en) 2017-03-31 2021-02-16 Beijing Biocytogen Co., Ltd Genetically modified mice expressing humanized CD47
US10973212B2 (en) 2017-03-31 2021-04-13 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Genetically modified non-human animal with human or chimeric SIRPa
US11497198B2 (en) 2017-06-19 2022-11-15 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Genetically modified mice expressing humanized CD40
US10945419B2 (en) 2017-06-19 2021-03-16 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Genetically modified non-human animal with human or chimeric GITR
US11350614B2 (en) 2017-06-19 2022-06-07 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Genetically modified non-human animal with human or chimeric CD28
US11272695B2 (en) 2017-10-13 2022-03-15 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Genetically modified non-human animal with human or chimeric PD-1
US10945420B2 (en) 2017-12-12 2021-03-16 BiocytogenPharmaceuticals (Beijing) Co., Ltd. Genetically modified non-human animal with human or chimeric CD3e
US11154041B2 (en) 2018-10-12 2021-10-26 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Genetically modified non-human animal with human or chimeric genes
CN111304247A (zh) * 2018-12-20 2020-06-19 百奥赛图江苏基因生物技术有限公司 人源化lag-3基因改造动物模型的制备方法及应用
US11564381B2 (en) 2018-12-20 2023-01-31 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Genetically modified non-human animal with human or chimeric LAG3
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