CD8A and / or CD8B gene modified non-human animal

Abstract

The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) CD8A or human or chimeric CD8B protein and methods of use thereof.

Description

Technical Field

[0001] The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) CD8A and / or human or chimeric CD8B protein and methods of using the same. Background Art

[0002] Traditional drug research and development typically uses in vitro screening methods. However, these screening methods cannot provide the in vivo environment (such as the tumor microenvironment, stromal cells, extracellular matrix components, and immune cell interactions), resulting in a relatively high failure rate in drug development. In addition, due to the differences between humans and animals, the test results obtained from in vivo pharmacological tests using conventional laboratory animals may not reflect the true disease state and the interactions at the target site, leading to significant differences between the results of many clinical trials and those of animal experiments.

[0003] Therefore, the development of humanized animal models suitable for human drug screening and evaluation will significantly improve the efficiency of new drug development and reduce the cost of drug research and development. Summary of the Invention

[0004] The present application provides an animal model having a human or chimeric CD8A and / or human or chimeric CD8B protein. This animal model can express a human or chimeric CD8A protein and / or a human or chimeric CD8B (e.g., humanized CD8A and / or CD8B) protein. It can be used for the study of the functions of the CD8A and / or CD8B genes and also for the screening and evaluation of modulators of the CD8A and / or CD8B signaling pathways (e.g., therapeutic agents targeting human CD8A and / or CD8B, such as anti-human CD8A antibodies, anti-human CD8B antibodies, anti-human CD8A and CD8B antibodies, or nucleic acid drugs, ADCs, PDCs, and / or polypeptide drugs targeting human CD8A and / or CD8B). In addition, the animal model prepared by the method described in the present application can be used for drug screening, pharmacodynamic studies, and the treatment of diseases (such as cancer, inflammation, or immune diseases) at the human CD8A and / or CD8B target sites; this animal model can also be used to facilitate new drug development and design, saving time and cost. In summary, the present invention provides a powerful tool for studying the functions of the CD8A and / or CD8B proteins and a platform for screening related drugs.

[0005] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same. The genome of the non-human animal comprises at least one chromosome, and the chromosome comprises a nucleotide sequence encoding a human or chimeric CD8 molecule (CD8) protein. In some embodiments, the chromosome comprises a nucleotide sequence encoding a human or chimeric CD8A molecule (CD8A) protein. In some embodiments, the chimeric CD8A protein comprises a human or humanized extracellular region, a non-human animal endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the amino acid sequence of the human or chimeric CD8A protein comprises SEQ ID NO: 2, positions 1-175 of SEQ ID NO: 2, positions 1-182 of SEQ ID NO: 2, or positions 22-182 of SEQ ID NO: 2, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to SEQ ID NO: 2, positions 1-175 of SEQ ID NO: 2, positions 1-182 of SEQ ID NO: 2, or positions 22-182 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the chimeric CD8A protein comprises SEQ ID NO: 7, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to SEQ ID NO: 7. In some embodiments, the chromosome comprises a nucleotide sequence encoding a human or chimeric CD8B molecule (CD8B) protein. In some embodiments, the chimeric CD8B protein comprises a human or humanized extracellular region, a non-human animal endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the amino acid sequence of the human or chimeric CD8B protein comprises SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9, or positions 22-170 of SEQ ID NO: 9; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9, or positions 22-170 of SEQ ID NO: 9. In some embodiments, the amino acid sequence of the chimeric CD8B protein comprises SEQ ID NO: 12, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to SEQ ID NO: 12.

[0006] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises replacing the nucleotide sequence of the corresponding region of endogenous CD8 with the nucleotide sequence of human or chimeric CD8 at the endogenous CD8 locus. In some embodiments, the nucleotide sequence encoding human or chimeric CD8 protein or the nucleotide sequence of human or chimeric CD8 is operably linked to the endogenous regulatory elements of the endogenous CD8 locus. In some embodiments, the endogenous CD8 protein of the non-human animal is not expressed or has a reduced expression level compared to CD8 in wild-type animals. In some embodiments, the modified CD8 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous locus being replaced. In some embodiments, the nucleotide sequence of human or chimeric CD8 comprises the nucleotide sequence of human or chimeric CD8A, and the nucleotide sequence of human or chimeric CD8A comprises all or part of the extracellular region encoding human CD8A protein, preferably comprises the nucleotide sequence encoding human or chimeric CD8A protein. In some embodiments, the nucleotide sequence of human or chimeric CD8A comprises the nucleotide sequence encoding SEQ ID NO: 2, positions 1-175 of SEQ ID NO: 2, positions 1-182 of SEQ ID NO: 2, or positions 22-182 of SEQ ID NO: 2. In some embodiments, the nucleotide sequence of human or chimeric CD8A comprises a portion of exon 4 to a portion of exon 7 of human CD8A, or comprises a portion of exon 5 to a portion of exon 7 of human CD8A. In some embodiments, the nucleotide sequence of human or chimeric CD8A comprises SEQ ID NO: 5, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence of chimeric CD8 comprises the nucleotide sequence of endogenous CD8A of the non-human animal, preferably comprises the nucleotide sequence encoding the cytoplasmic region and / or transmembrane region of non-human animal CD8A, preferably comprises a portion of exon 3 to the whole of exon 5 of non-human animal CD8A, and preferably further comprises a portion of exon 1. In some embodiments, the nucleotide sequence of the corresponding region of endogenous CD8 comprises the nucleotide sequence of the corresponding region of endogenous CD8A; the nucleotide sequence of the corresponding region of endogenous CD8A comprises all or part of the sequence encoding the extracellular region; preferably comprises the nucleotide sequence encoding SEQ ID NO: 1, positions 1-187 of SEQ ID NO: 1, positions 1-196 of SEQ ID NO: 1, or positions 28-196 of SEQ ID NO: 1; and further preferably comprises all or part of exon 1 to a portion of exon 3 of non-human animal CD8A.In some embodiments, the nucleotide sequence of the human or chimeric CD8 comprises the nucleotide sequence of human or chimeric CD8B, and the nucleotide sequence of the human or chimeric CD8B comprises all or part of the nucleotide sequence encoding the extracellular region of the human CD8B protein, preferably comprising the nucleotide sequence encoding the human or chimeric CD8B protein. In some embodiments, the nucleotide sequence of the human or chimeric CD8B comprises the nucleotide sequence encoding SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9, or positions 22-170 of SEQ ID NO: 9. In some embodiments, the nucleotide sequence of the human or chimeric CD8B comprises the part of exon 1 to the part of exon 4 of human CD8B, or comprises the part of exon 2 to the part of exon 4 of human CD8B. In some embodiments, the nucleotide sequence of the human or chimeric CD8B comprises SEQ ID NO: 10, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 10. In some embodiments, the nucleotide sequence of the chimeric CD8 comprises the nucleotide sequence of the endogenous CD8B of a non-human animal, preferably including the nucleotide sequence encoding the cytoplasmic region and / or transmembrane region of the non-human animal CD8B, preferably including the part of exon 4 to all of exon 6 of the non-human animal CD8B, and preferably further including part of exon 1. In some embodiments, the nucleotide sequence of the corresponding region of the endogenous CD8 includes the nucleotide sequence of the corresponding region of the endogenous CD8B; the nucleotide sequence of the corresponding region of the endogenous CD8B includes all or part of the sequence encoding the extracellular region; preferably includes the nucleotide sequence encoding SEQ ID NO: 8, positions 1-172 of SEQ ID NO: 8, positions 1-175 of SEQ ID NO: 8, or positions 22-175 of SEQ ID NO: 8; more preferably includes the part of exon 1 to the part of exon 4 of the non-human animal CD8B, or includes the part of exon 2 to the part of exon 4 of the non-human animal CD8B. In some embodiments, the construction method includes replacing the corresponding region of the endogenous non-human animal with a nucleotide sequence comprising the nucleotide sequence encoding the human or chimeric CD8A protein and the human or chimeric CD8B protein. In some embodiments, the construction method includes replacing the corresponding region of the endogenous non-human animal with a nucleotide sequence encoding, from N to C, in sequence, the signal peptide of human CD8B, all or part of the extracellular region of human CD8B, the transmembrane region of the endogenous CD8B of the non-human animal, the cytoplasmic region of the endogenous CD8B of the non-human animal, the signal peptide of human CD8A, and all or part of the extracellular region of human CD8A. In some embodiments, the construction method includes replacing the corresponding region of the endogenous non-human animal with a nucleotide sequence comprising the nucleotide sequence of human CD8A and the donor sequence of human CD8B.In some embodiments, the donor sequence sequentially includes, from 5' to 3', a portion of exon 1 to a portion of exon 4 of human CD8B, a portion of exon 4 of non-human animal endogenous CD8B to a portion of exon 1 of CD8A, and a portion of exon 4 to a portion of exon 7 of human CD8A. In some embodiments, the portion of exon 1 to exon 4 of human CD8B contains SEQ ID NO: 10, or contains a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 10. In some embodiments, the portion of exon 4 of non-human animal endogenous CD8B to exon 1 of CD8A includes the nucleotide sequence from positions 71309434 to 71350536 of NCBI accession number NC_000072.7. In some embodiments, the nucleotide sequence of the portion of exon 4 to exon 7 of human CD8A includes SEQ ID NO: 5, or contains a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the corresponding non-human animal endogenous region includes the nucleotide sequence encoding CD8A protein and CD8B protein in non-human animals. In some embodiments, the corresponding non-human animal endogenous region includes the nucleotide sequence encoding the extracellular region of CD8A protein and the extracellular region of CD8B protein in non-human animals. In some embodiments, the corresponding non-human animal endogenous region includes the nucleotide sequence encoding the signal peptide and extracellular region of CD8B protein and CD8A in non-human animals. In some embodiments, a portion of exon 1 of non-human animal endogenous CD8B to exon 3 of CD8A. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. Preferably, the non-human animal is a mouse or a rat. In some embodiments, the mRNA transcribed from the modified CD8A gene in the genome of the non-human animal contains SEQ ID NO: 6, or contains a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the mRNA transcribed from the modified CD8B gene in the genome of the non-human animal contains SEQ ID NO: 11, or contains a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 11.In some embodiments, the non-human animal further comprises a nucleotide sequence encoding a human or chimeric protein encoded by other genes, and the human or chimeric protein is selected from at least one of TRBC1, TRBC2, HLA-A, B2M, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0007] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same. The genome of the non-human animal comprises at least one chromosome, and the chromosome comprises a nucleotide sequence encoding a human or chimeric CD8A molecule (CD8A) protein. In some embodiments, the chimeric CD8A protein is a humanized protein. In some embodiments, the nucleotide sequence encoding the human or chimeric CD8A protein can be CDS, cDNA or genomic DNA. In some embodiments, the nucleotide sequence encoding the human or chimeric CD8A protein is regulated by endogenous regulatory elements (such as promoters and / or UTRs, and the UTRs are preferably 5' UTRs and / or 3' UTRs) to express the human or chimeric CD8A protein. In some embodiments, the nucleotide sequence encoding the human or chimeric CD8A protein is operably linked to endogenous regulatory elements (such as promoters and / or UTRs, and the UTRs are preferably 5' UTRs and / or 3' UTRs) of the endogenous CD8A locus of at least one chromosome. In some embodiments, the chimeric CD8A protein comprises all or part of the nucleotide sequence of the extracellular region of human CD8A. In some embodiments, the chimeric CD8A protein comprises a human or humanized signal peptide. In some embodiments, the chimeric CD8A protein comprises a human or humanized extracellular region, an endogenous transmembrane region of the non-human animal and an endogenous cytoplasmic region. In some embodiments, the chimeric CD8A protein comprises a human or humanized signal peptide, a human or humanized extracellular region, an endogenous transmembrane region of the non-human animal and an endogenous cytoplasmic region. In some embodiments, the amino acid sequence of the chimeric CD8A protein comprises an amino acid sequence that is identical to at least 50 to 235 consecutive amino acids of the human CD8A protein, such as at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 151, 152, 160, 161, 170, 175, 180, 182, 183, 185, 190, 200, 210 or 235 consecutive amino acids. In some embodiments, the amino acid sequence of the human or chimeric CD8A protein comprises SEQ ID NO: 2, positions 1-175 of SEQ ID NO: 2, positions 1-182 of SEQ ID NO: 2 or positions 22-182 of SEQ ID NO: 2, or comprises an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 2, positions 1-175 of SEQ ID NO: 2, positions 1-182 of SEQ ID NO: 2 or positions 22-182 of SEQ ID NO: 2. In some embodiments, the chimeric CD8A protein comprises a part of the human CD8A protein and a part of the CD8A protein of the non-human animal.In some embodiments, the portion of the human CD8A protein comprises amino acids 1-175 of SEQ ID NO: 2, and the portion of the non-human animal CD8A protein comprises amino acids 188-247 of SEQ ID NO: 1. In some embodiments, the portion of the human CD8A protein comprises amino acids 1-182 of SEQ ID NO: 2, and the portion of the non-human animal CD8A protein comprises amino acids 197-247 of SEQ ID NO: 1. In some embodiments, the portion of the human CD8A protein comprises amino acids 22-182 of SEQ ID NO: 2, and the portion of the non-human animal CD8A protein comprises amino acids 1-27 and 197-247 of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the human or chimeric CD8A protein comprises SEQ ID NO: 7, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 7. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the non-human animal is a mouse or a rat. In some embodiments, the non-human animal is a mouse. In some embodiments, the endogenous CD8A protein of the non-human animal is not expressed or has a reduced expression level compared to CD8A in a wild-type animal. In some embodiments, one or more cells of the non-human animal express a human or chimeric CD8A protein.

[0008] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises, at the endogenous CD8A locus, a nucleotide sequence encoding a human or chimeric CD8A protein replacing the corresponding region of endogenous CD8A, or a nucleotide sequence of human CD8A replacing the nucleotide sequence encoding the corresponding region of endogenous CD8A. In some embodiments, the nucleotide sequence encoding a human or chimeric CD8A protein or the nucleotide sequence of human CD8A is operably linked to an endogenous regulatory element (such as a promoter and / or UTR, preferably the 5'UTR and / or 3'UTR) of the endogenous CD8A locus. In some embodiments, the nucleotide sequence encoding a human or chimeric CD8A protein or the nucleotide sequence of human CD8A is regulated by an endogenous regulatory element (such as a promoter and / or UTR, preferably the 5'UTR and / or 3'UTR), and one or more cells of the non-human animal express a human or humanized CD8A protein. In some embodiments, the endogenous CD8A protein of the non-human animal is not expressed or has a reduced expression level compared to CD8A in a wild-type animal. In some embodiments, the nucleotide sequence of human CD8A can be CDS, cDNA or genomic DNA. In some embodiments, the nucleotide sequence of human CD8A comprises at least 5 bp to 23792 bp of contiguous nucleotides of the human CD8A gene, such as at least 5, 50, 100, 200, 300, 400, 455, 456, 457, 460, 480, 482, 483, 485, 500, 524, 525, 526, 545, 546, 547, 550, 600, 700, 800, 900, 1000, 1100,

[0009] 1400, 1403, 1404, 1500, 2000, 2500, 3000, 3100, 3177, 3200, 3500, 4000, 5000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 20000, 23000 or 23792 bp of contiguous nucleotides. In some embodiments, the nucleotide sequence of the human CD8A comprises all or part of the extracellular region encoding the human CD8A protein. In some embodiments, the nucleotide sequence of the human CD8A comprises all or part of the signal peptide encoding the human. In some embodiments, the nucleotide sequence of the human CD8A comprises the nucleotide sequence encoding SEQ ID NO: 2, positions 1-175 of SEQ ID NO: 2, positions 1-182 of SEQ ID NO: 2, or positions 22-182 of SEQ ID NO: 2. In some embodiments, the nucleotide sequence of the human CD8A comprises the nucleotide sequence encoding a human or chimeric CD8A protein. In some embodiments, the nucleotide sequence of the human CD8A comprises the portion of exon 4 to the portion of exon 7 of human CD8A. In some embodiments, the nucleotide sequence of the human CD8A comprises the portion of exon 5 to the portion of exon 7 of human CD8A. In some embodiments, the portion of exon 5 of the human CD8A comprises the nucleotide sequence encoding the extracellular region. In some embodiments, the portion of exon 5 of the human CD8A comprises a contiguous nucleotide sequence of at least 5 bp to 354 bp of exon 5, such as at least 5, 10, 15, 20, 30, 40, 50, 100, 110, 120, 130, 135, 140, 150, 200, 300, 310, 312, 313, 314, 315, 320, 330, 339, 340, 341 or 354 bp of contiguous nucleotide sequence. In some embodiments, the portion of exon 4 of the human CD8A comprises a contiguous nucleotide sequence of at least 5 bp to 319 bp of exon 4, such as at least 5, 10, 15, 48, 49, 50, 100, 110, 120, 130, 135, 140, 150, 200, 300, 310 or 319 bp of contiguous nucleotide sequence. In some embodiments, the portion of exon 4 of the human CD8A comprises the nucleotide sequence of the coding region. In some embodiments, the portion of exon 4 of the human CD8A comprises the nucleotides from the start codon to the last nucleotide of exon 4. In some embodiments, the portion of exon 7 of the human CD8A comprises a contiguous nucleotide sequence of at least 5 bp to 111 bp of exon 7, such as at least 5, 10, 11, 12, 15, 31, 32, 33, 35, 40, 45, 48, 49, 50, 100, 110 or 111 bp of contiguous nucleotide sequence.In some embodiments, the portion of exon 7 of human CD8A comprises the nucleotide sequence of the coding region. In some embodiments, the portion of exon 7 of human CD8A comprises the nucleotide sequence encoding the extracellular region. In some embodiments, the nucleotide sequence of human CD8A comprises SEQ ID NO: 5, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence of the corresponding region of endogenous CD8A comprises all or part of the sequence encoding the extracellular region. In some embodiments, the nucleotide sequence of the corresponding region of endogenous CD8A comprises all or part of the encoding endogenous signal peptide. In some embodiments, the nucleotide sequence of the corresponding region of endogenous CD8A comprises the nucleotide sequence encoding the endogenous CD8A protein. In some embodiments, the nucleotide sequence of the corresponding region of endogenous CD8A comprises the nucleotide sequence encoding SEQ ID NO: 1, positions 1-187 of SEQ ID NO: 1, positions 1-196 of SEQ ID NO: 1 or positions 28-196 of SEQ ID NO: 1. In some embodiments, the nucleotide sequence of the corresponding region of endogenous CD8A comprises all or part of exon 1 to part of exon 3 of non-human animal CD8A. In some embodiments, the portion of exon 1 of non-human animal CD8A comprises a continuous nucleotide sequence of at least 5 bp to 571 bp of exon 1, such as at least 5, 10, 15, 50, 100, 110, 120, 130, 135, 140, 150, 200, 300, 336, 337, 338, 350, 363, 364, 365, 400, 444, 445, 446, 450, 500, 570 or 571 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 1 of non-human animal CD8A comprises the nucleotide sequence of the coding region. In some embodiments, the portion of exon 1 of non-human animal CD8A comprises from the start codon to the last nucleotide of exon 1. In some embodiments, the portion of exon 3 of non-human animal CD8A comprises a continuous nucleotide sequence of at least 5 bp to 111 bp of exon 3, such as at least 5, 10, 11, 12, 15, 20, 30, 35, 37, 38, 39, 40, 50, 100, 110 or 111 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 3 of non-human animal CD8A comprises the nucleotide sequence of the coding region. In some embodiments, the portion of exon 3 of non-human animal CD8A comprises the nucleotide sequence encoding the extracellular region.In some embodiments, one or more cells of the non-human animal express a chimeric CD8A, which comprises an extracellular region, a transmembrane region, and a cytoplasmic region, and the amino acid sequence identity of the extracellular region with the extracellular region of human CD8A is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99.5%. In some embodiments, the extracellular region is identical to at least 10, 20, 30, 40, 50, 60, 70, 80, 100, or 100 consecutive amino acids of the extracellular region of human CD8A. In some embodiments, the nucleotide sequence encoding the human or chimeric CD8A protein comprises a portion from exon 4 to exon 7 or a portion from exon 5 to exon 7 of the human CD8A gene. In some embodiments, the nucleotide sequence encoding the human or chimeric CD8A protein comprises a portion of exon 4, all of exons 5-6, and a portion of exon 7 of the human CD8A gene (preferably also comprising intron 4 and / or intron 6), and preferably, the nucleotide sequence encoding the human or chimeric CD8A protein comprises at least 500-800, 800-1000, or 1000-1400 bp of nucleotides of a portion of exon 4, all of exons 5-6, and a portion of exon 7 of the human CD8A gene. In some embodiments, the nucleotide sequence of the corresponding region of the endogenous CD8A comprises all or a portion of exon 1 to a portion of exon 3 of the CD8A gene of the non-human animal (such as a mouse). In some embodiments, the nucleotide sequence of the corresponding region of the endogenous CD8A comprises all or a portion of exon 1, all of exon 2, and a portion of exon 3 of the CD8A gene of the non-human animal (such as a mouse) (preferably also including intron 1 and / or intron 2). In some embodiments, the modified CD8A gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous locus being replaced. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the non-human animal is a rat or a mouse. In some embodiments, the mRNA transcribed from the modified gene in the genome of the non-human animal comprises SEQ ID NO: 6, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity with the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by another gene, and the human or chimeric protein includes, but is not limited to, at least one of CD8B, TRBC1, TRBC2, HLA-A, B2M, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the human or chimeric protein encoded by the other gene is a human or chimeric CD8B protein. In some embodiments, the amino acid sequence of the human or chimeric CD8B protein comprises SEQ ID NO: 9 or SEQ ID NO: 12.

[0010] In one aspect, the present invention provides a non-human animal comprising at least one cell encoding a nucleotide sequence of a human or chimeric CD8A protein, wherein the human or chimeric CD8A protein comprises at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 151, 152, 160, 161, 170, 175, 180, 182, 183, 185, 190, 200, 210 or 235 consecutive amino acid sequences that are identical to the corresponding region of a human. The non-human animal expresses the human or chimeric CD8A protein. In some embodiments, the chimeric CD8A protein comprises at least 50 consecutive amino acids of the extracellular region of human CD8A. In some embodiments, the human or chimeric CD8A protein comprises the amino acids of SEQ ID NO: 2, positions 1-175 of SEQ ID NO: 2, positions 1-182 of SEQ ID NO: 2, or positions 22-182 of SEQ ID NO: 2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acids of SEQ ID NO: 2, positions 1-175 of SEQ ID NO: 2, positions 1-182 of SEQ ID NO: 2, or positions 22-182 of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the human or chimeric CD8A protein is operably linked to an endogenous CD8A regulatory element (such as a promoter and / or UTR, preferably a 5'UTR and / or 3'UTR). In some embodiments, the nucleotide sequence encoding the corresponding region of the human or chimeric CD8A can be integrated into the endogenous CD8A locus of the non-human animal. In some embodiments, the human or chimeric CD8A protein has at least one non-human animal (such as a mouse) CD8A activity and / or human CD8A activity.

[0011] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, in which, in at least one cell of the non-human animal, at the endogenous CD8A locus of the non-human animal, the nucleotide sequence encoding the endogenous CD8A region is replaced by the nucleotide sequence encoding the corresponding region of human CD8A. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8A may be CDS, cDNA or genomic DNA. In some embodiments, the endogenous CD8A protein of the non-human animal is not expressed or has a reduced expression level compared to CD8A in a wild-type animal. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8A comprises a portion from exon 4 to exon 7 or a portion from exon 5 to exon 7 of the human CD8A gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8A comprises a portion of exon 4, all of exons 5-6 and a portion of exon 7 of the human CD8A gene (preferably further comprising intron 4 and / or intron 6), preferably, the nucleotide sequence encoding the corresponding region of human CD8A comprises at least 500-800, 800-1000 or 1000-1400 nucleotides from a portion of exon 4 to exon 7 of the human CD8A gene. In some embodiments, the amino acid sequence of the corresponding region of human CD8A comprises SEQ ID NO: 2, positions 1-175 of SEQ ID NO: 2, positions 1-182 of SEQ ID NO: 2 or positions 22-182 of SEQ ID NO: 2, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% amino acid identity with SEQ ID NO: 2, positions 1-175 of SEQ ID NO: 2, positions 1-182 of SEQ ID NO: 2 or positions 22-182 of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the endogenous CD8A region is the endogenous CD8A extracellular region, the endogenous transmembrane region and / or the endogenous cytoplasmic region of the non-human animal. In some embodiments, the amino acid sequence of the corresponding region of human CD8A comprises SEQ ID NO: 2 or 7, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% amino acid identity with the amino acid sequence shown in SEQ ID NO: 2 or 7. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8A comprises SEQ ID NO: 5, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% nucleotide identity with the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous CD8A region comprises all or a portion of exon 1 to a portion of exon 3 of the CD8A gene of a non-human animal (such as a mouse).In some embodiments, the nucleotide sequence encoding the endogenous CD8A region comprises all or part of exon 1 of the CD8A gene of a non-human animal (such as a mouse), all of exon 2, and part of exon 3 (preferably also including intron 1 and / or intron 2). In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8A is operably linked to an endogenous CD8A regulatory element (such as a promoter and / or UTR, preferably the 5'UTR and / or 3'UTR). In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the non-human animal is a mouse or a rat.

[0012] In one aspect, the present invention provides a method for constructing a non-human animal cell expressing a human or chimeric CD8A gene, the construction method comprising replacing the nucleotide sequence encoding the endogenous CD8A region with the nucleotide sequence encoding the corresponding region of human CD8A at the endogenous CD8A locus of a non-human animal (such as a mouse), thereby generating a genetically modified non-human animal cell, and the non-human animal cell expressing a human or chimeric CD8A protein. In some embodiments, the chimeric CD8A protein comprises a human or humanized extracellular region, a non-human animal endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8A comprises a portion from exon 4 to exon 7 or a portion from exon 5 to exon 7 of the human CD8A gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8A comprises a portion of exon 4, all of exons 5-6, and a portion of exon 7 of the human CD8A gene (preferably also comprising intron 4 and / or intron 6). Preferably, the nucleotide sequence encoding the corresponding region of human CD8A comprises at least 500-800, 800-1000, or 1000-1400 nucleotides of a portion of exon 4, all of exons 5-6, and a portion of exon 7 of the human CD8A gene. In some embodiments, the amino acid sequence of the corresponding region of human CD8A comprises SEQ ID NO: 2, positions 1-175 of SEQ ID NO: 2, positions 1-182 of SEQ ID NO: 2, or positions 22-182 of SEQ ID NO: 2, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the amino acid sequences shown in SEQ ID NO: 2, positions 1-175 of SEQ ID NO: 2, positions 1-182 of SEQ ID NO: 2, or positions 22-182 of SEQ ID NO: 2. In some embodiments, the endogenous CD8A region is an endogenous CD8A extracellular region, a non-human animal endogenous transmembrane region, and / or an endogenous cytoplasmic region. In some embodiments, the nucleotide sequence encoding the endogenous CD8A region comprises all or a portion of exon 1 to a portion of exon 3 of the CD8A gene of a non-human animal (such as a mouse). In some embodiments, the nucleotide sequence encoding the endogenous CD8A region comprises all or a portion of exon 1, all of exon 2, and a portion of exon 3 of the CD8A gene of a non-human animal (such as a mouse) (preferably also including intron 1 and / or intron 2). In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8A is operably linked to regulatory elements of endogenous CD8A (such as a promoter and / or UTR, and the UTR is preferably a 5'UTR and / or a 3'UTR). In some embodiments, the non-human animal is a mouse.In some embodiments, the non-human animal further comprises nucleotide sequences encoding human or chimeric proteins encoded by other genes, and the human or chimeric proteins include, but are not limited to, at least one of CD8B, TRBC1, TRBC2, HLA-A, B2M, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0013] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the endogenous CD8A protein in the non-human animal with a nucleotide sequence encoding a human or chimeric CD8A protein. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the endogenous CD8A protein in the non-human animal with a nucleotide sequence encoding SEQ ID NO: 2 or 7. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the endogenous SEQ ID NO: 1 in the non-human animal with a nucleotide sequence encoding SEQ ID NO: 2 or 7. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the extracellular region of human CD8A with the nucleotide sequence encoding the extracellular region of endogenous CD8A in the non-human animal. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the extracellular region and signal peptide of human CD8A with the nucleotide sequence encoding the extracellular region and signal peptide of endogenous CD8A in the non-human animal. In some embodiments, the construction method comprises replacing the corresponding region of endogenous CD8A in the non-human animal with a nucleotide sequence encoding positions 1-175, positions 1-182 or positions 22-182 of SEQ ID NO: 2. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding positions 1-187 of endogenous SEQ ID NO: 1 in the non-human animal with a nucleotide sequence encoding positions 1-175 of SEQ ID NO: 2. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding positions 28-196 of endogenous SEQ ID NO: 1 in the non-human animal with a nucleotide sequence encoding positions 22-182 of SEQ ID NO: 2. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding positions 1-196 of endogenous SEQ ID NO: 1 in the non-human animal with a nucleotide sequence encoding positions 1-182 of SEQ ID NO: 2.

[0014] In some embodiments, the construction method includes replacing the nucleotide sequence of the endogenous CD8A gene of a non-human animal with the nucleotide sequence of human CD8A. In some embodiments, the construction method includes replacing the portion from exon 4 to exon 7 of the human CD8A gene with the nucleotide sequence encoding positions 1-187 of SEQ ID NO: 1, positions 1-196 of SEQ ID NO: 1, or positions 28-196 of SEQ ID NO: 1 in the non-human animal. In some embodiments, the construction method includes replacing the portion from exon 5 to exon 7 of the human CD8A gene with the nucleotide sequence encoding positions 28-196 of SEQ ID NO: 1 in the non-human animal. In some embodiments, the construction method includes replacing the portion from exon 4 to exon 7 of the human CD8A gene with all or part of exon 1 to part of exon 3 of the nucleotide sequence of the endogenous CD8A gene of a non-human animal. In some embodiments, the construction method includes replacing the portion from exon 5 to exon 7 of the human CD8A gene with all or part of exon 1 to part of exon 3 of the nucleotide sequence of the endogenous CD8A gene of a non-human animal. In some embodiments, the construction method includes replacing the nucleotide sequence encoding positions 1-187 of SEQ ID NO: 1 in the non-human animal with SEQ ID NO: 5. In some embodiments, the construction method includes replacing the nucleotide sequence encoding SEQ ID NO: 1 in the non-human animal with SEQ ID NO: 6. In some embodiments, the construction method includes replacing all or part of exon 1 to part of exon 3 of the nucleotide sequence of the endogenous non-human animal exon with SEQ ID NO: 5.

[0015] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same. The genome of the non-human animal comprises at least one chromosome, and the chromosome comprises a nucleotide sequence encoding a human or chimeric CD8B molecule (CD8B) protein. In some embodiments, the chimeric CD8B protein is a humanized protein. In some embodiments, the nucleotide sequence encoding the human or chimeric CD8B protein can be CDS, cDNA or genomic DNA. In some embodiments, the nucleotide sequence encoding the human or chimeric CD8B protein is regulated by endogenous regulatory elements (such as promoters and / or UTRs, and the UTR is preferably 5'UTR and / or 3'UTR) to express the human or chimeric CD8B protein. In some embodiments, the nucleotide sequence encoding the human or chimeric CD8B protein is operably linked to endogenous regulatory elements (such as promoters and / or UTRs, such as 5'UTR and / or 3'UTR) of the endogenous CD8B locus of at least one chromosome. In some embodiments, the chimeric CD8B protein comprises all or part of the nucleotide sequence of the extracellular region of human CD8B. In some embodiments, the chimeric CD8B protein comprises a human or humanized signal peptide. In some embodiments, the chimeric CD8B protein does not comprise a human or humanized signal peptide. In some embodiments, the chimeric CD8B protein comprises a human or humanized extracellular region, an endogenous transmembrane region of the non-human animal, and an endogenous cytoplasmic region. In some embodiments, the chimeric CD8B protein comprises a human or humanized signal peptide, a human or humanized extracellular region, an endogenous transmembrane region of the non-human animal, and an endogenous cytoplasmic region. In some embodiments, the amino acid sequence of the chimeric CD8B protein comprises an amino acid sequence that is identical to at least 50 to 210 consecutive amino acids of the human CD8B protein, such as at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 140, 145, 147, 148, 149, 150, 160, 169, 170, 175, 180, 185, 190, 200 or 210 consecutive amino acids, etc. In some embodiments, the amino acid sequence of the human or chimeric CD8B protein comprises SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9 or positions 22-170 of SEQ ID NO: 9, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9 or positions 22-170 of SEQ ID NO: 9. In some embodiments, the chimeric CD8B protein comprises a portion of the human CD8B protein and a portion of the non-human animal CD8B protein.In some embodiments, the portion of the human CD8B protein comprises positions 1-169 of SEQ ID NO: 9, and the portion of the non-human animal CD8B protein comprises positions 173-213 of SEQ ID NO: 8. In some embodiments, the portion of the human CD8B protein comprises positions 1-170 of SEQ ID NO: 9, and the portion of the non-human animal CD8B protein comprises positions 176-213 of SEQ ID NO: 8. In some embodiments, the portion of the human CD8B protein comprises positions 22-170 of SEQ ID NO: 9, and the portion of the non-human animal CD8B protein comprises positions 1-21 and 176-213 of SEQ ID NO: 8. In some embodiments, the amino acid sequence of the chimeric CD8B protein comprises SEQ ID NO: 12, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 12. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the non-human animal is a mouse or a rat. In some embodiments, the non-human animal is a mouse. In some embodiments, the endogenous CD8B protein of the non-human animal is not expressed or has a reduced expression level compared to CD8B in wild-type animals. In some embodiments, one or more cells of the non-human animal express a human or chimeric CD8B protein.

[0016] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises, at the endogenous CD8B locus, a nucleotide sequence of human CD8B replacing the nucleotide sequence of the corresponding region of endogenous CD8B, or a nucleotide sequence encoding a human or chimeric CD8B protein replacing the nucleotide sequence encoding the corresponding region of endogenous CD8B. In some embodiments, the nucleotide sequence encoding a human or chimeric CD8B protein or the nucleotide sequence of human CD8B is operably linked to an endogenous regulatory element (such as a promoter and / or UTR, preferably the 5'UTR and / or 3'UTR) of the endogenous CD8B locus. In some embodiments, the nucleotide sequence encoding a human or chimeric CD8B protein or the nucleotide sequence of human CD8B is regulated by an endogenous regulatory element (such as a promoter and / or UTR, preferably the 5'UTR and / or 3'UTR), and one or more cells of the animal express a human or humanized CD8B protein. In some embodiments, the endogenous CD8B protein of the non-human animal is not expressed or has a reduced expression level compared to CD8B in a wild-type animal. In some embodiments, the nucleotide sequence of human CD8B can be CDS, cDNA or genomic DNA. In some embodiments, the nucleotide sequence of human CD8B comprises at least 5 bp to 46518 bp of continuous nucleotides of the human CD8B gene, such as at least 5, 50, 100, 200, 300, 400, 445, 446, 447, 448, 450, 500, 505, 506, 507, 508, 509, 510, 511, 550, 600, 700, 800, 900, 1000, 1100, 1500, 2000, 2500, 3000, 3100, 3200, 3500, 4000, 4500, 4700, 4794, 4795, 4800, 5000, 10000, 15000, 15105, 15106, 15107, 11000, 12000, 13000, 14000, 15000, 16000, 20000, 23000, 30000, 35000, 40000, 45000, 46000 or 46518 bp of continuous nucleotides. In some embodiments, the nucleotide sequence of human CD8B encodes all or part of the extracellular region of the human CD8B protein. In some embodiments, the nucleotide sequence of human CD8B comprises all or part of the nucleotide sequence encoding the human signal peptide. In some embodiments, the nucleotide sequence of human CD8B comprises the nucleotide sequence encoding SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9 or positions 22-170 of SEQ ID NO: 9. In some embodiments, the nucleotide sequence of human CD8B comprises a nucleotide sequence encoding a human or chimeric CD8B protein.In some embodiments, the nucleotide sequence of the human CD8B comprises a portion from exon 1 to exon 4 of human CD8B. In some embodiments, the nucleotide sequence of the human CD8B comprises a portion from exon 2 to exon 4 of human CD8B. In some embodiments, the portion of exon 2 of the human CD8B comprises a continuous nucleotide sequence of at least 5 bp to 360 bp of exon 2, such as a continuous nucleotide sequence of at least 5, 10, 20, 30, 40, 50, 60, 100, 200, 300, 330, 340, 350, or 360 bp. In some embodiments, the portion of exon 2 of the human CD8B comprises a nucleotide sequence encoding an extracellular region. In some embodiments, the portion of exon 1 of the human CD8B comprises a continuous nucleotide sequence of at least 5 bp to 64 bp of exon 1, such as a continuous nucleotide sequence of at least 5, 10, 15, 20, 30, 40, 42, 43, 44, 45, 50, 55, 60, or 64 bp. In some embodiments, the portion of exon 1 of the human CD8B comprises a nucleotide sequence of the coding region. In some embodiments, the portion of exon 1 of the human CD8B comprises the nucleotide sequence from the start codon to the last nucleotide of exon 1. In some embodiments, the portion of exon 4 of the human CD8B comprises a continuous nucleotide sequence of at least 5 bp to 90 bp of exon 4, such as a continuous nucleotide sequence of at least 5, 10, 12, 13, 14, 15, 16, 17, 18, 20, 30, 40, 45, 50, 60, 70, 80, or 90 bp. In some embodiments, the portion of exon 4 of the human CD8B comprises a nucleotide sequence of the coding region. In some embodiments, the portion of exon 4 of the human CD8B comprises a nucleotide sequence encoding an extracellular region. In some embodiments, the portion of exon 4 of the human CD8B comprises a nucleotide sequence encoding a hinge region. In some embodiments, the nucleotide sequence of the human CD8B comprises SEQ ID NO: 10, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 10. In some embodiments, the nucleotide sequence of the corresponding region of the endogenous CD8B comprises all or part of the sequence encoding an extracellular region. In some embodiments, the nucleotide sequence of the corresponding region of the endogenous CD8B comprises all or part of the encoding sequence of the endogenous signal peptide. In some embodiments, the nucleotide sequence of the corresponding region of the endogenous CD8B comprises the nucleotide sequence encoding the endogenous CD8B protein. In some embodiments, the nucleotide sequence of the corresponding region of the endogenous CD8B comprises the nucleotide sequence encoding SEQ ID NO: 8, positions 1-172 of SEQ ID NO: 8, positions 1-175 of SEQ ID NO: 8, or positions 22-175 of SEQ ID NO: 8.In some embodiments, the nucleotide sequence of the corresponding region of the endogenous CD8B comprises a portion from exon 1 to exon 4 of the non-human animal CD8B. In some embodiments, the nucleotide sequence of the corresponding region of the endogenous CD8B comprises a portion from exon 2 to exon 4 of the non-human animal CD8B. In some embodiments, the portion of exon 2 of the non-human animal CD8B comprises a nucleotide sequence encoding an extracellular region. In some embodiments, the portion of exon 2 of the non-human animal CD8B comprises a continuous nucleotide sequence of at least 5 bp to 363 bp of exon 2, such as a continuous nucleotide sequence of at least 5, 10, 20, 30, 40, 50, 60, 100, 200, 300, 330, 340, 342, 343, 345, 350 or 363 bp. In some embodiments, the portion of exon 1 of the non-human animal CD8B comprises a continuous nucleotide sequence of at least 5 bp to 112 bp of exon 1, such as a continuous nucleotide sequence of at least 5, 10, 15, 20, 30, 40, 42, 43, 44, 45, 50, 100, 110 or 112 bp. In some embodiments, the portion of exon 1 of the non-human animal CD8B comprises a nucleotide sequence of the coding region. In some embodiments, the portion of exon 1 of the non-human animal CD8B comprises the nucleotide sequence from the start codon to the last nucleotide of exon 1. In some embodiments, the portion of exon 4 of the non-human animal CD8B comprises a continuous nucleotide sequence of at least 5 bp to 90 bp of exon 4, such as a continuous nucleotide sequence of at least 5, 10, 12, 13, 14, 15, 20, 22, 23, 24, 25, 30, 40, 45, 50, 60, 70, 80 or 90 bp. In some embodiments, the portion of exon 4 of the non-human animal CD8B comprises a nucleotide sequence of the coding region. In some embodiments, the portion of exon 4 of the non-human animal CD8B comprises a nucleotide sequence encoding an extracellular region. In some embodiments, the portion of exon 4 of the non-human animal CD8B comprises a nucleotide sequence encoding a hinge region. In some embodiments, one or more cells of the non-human animal express a chimeric CD8B, which comprises an extracellular region, a transmembrane region and a cytoplasmic region, and the amino acid sequence identity of the extracellular region with the extracellular region of human CD8B is at least 50%, 60%, 70%, 80%, 90%, 95% or 99.5%. In some embodiments, the extracellular region is identical to at least 10, 20, 30, 40, 50, 60, 70, 80, 10 or 100 consecutive amino acids of the extracellular region of human CD8B. In some embodiments, the nucleotide sequence encoding the human or chimeric CD8B protein comprises a portion from exon 1 to exon 4 or a portion from exon 2 to exon 4 of the human CD8B gene.In some embodiments, the nucleotide sequence encoding a human or chimeric CD8B protein comprises a portion of exon 1 of the human CD8B gene, all of exons 2-3, and a portion of exon 4 (preferably also including intron 1 and / or intron 3). Preferably, the nucleotide sequence encoding a human or chimeric CD8B protein comprises at least 500-1000, 1000-10000, or 10000-15000 bp of nucleotides of a portion of exon 1 of the human CD8B gene, all of exons 2-3, and a portion of exon 4. In some embodiments, the nucleotide sequence of human CD8B comprises all or a portion of the extracellular region encoding the human CD8B protein, preferably comprising the nucleotide sequence encoding a human or chimeric CD8B protein. In some embodiments, the nucleotide sequence of human CD8B encodes the nucleotide sequence of SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9, or positions 22-170 of SEQ ID NO: 9. In some embodiments, the nucleotide sequence of human CD8B comprises a portion of exon 1 to a portion of exon 4 or a portion of exon 2 to a portion of exon 4 of human CD8B. In some embodiments, the nucleotide sequence of human CD8B comprises SEQ ID NO: 10, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 10. In some embodiments, the nucleotide sequence of the corresponding region of endogenous CD8B comprises a portion of exon 1 of the CD8B gene of a non-human animal (such as a mouse), all of exons 2-3, and a portion of exon 4 (preferably also including intron 1 and / or intron 3). In some embodiments, the modified CD8B gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous locus being replaced. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the non-human animal is a mouse or a rat. In some embodiments, the mRNA transcribed from the modified gene in the genome of the non-human animal comprises SEQ ID NO: 11, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 11. In some embodiments, the non-human animal further comprises the nucleotide sequence of a human or chimeric protein encoded by another gene, and the human or chimeric protein includes, but is not limited to, at least one of CD8A, TRBC1, TRBC2, HLA-A, B2M, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the amino acid sequence of the human or chimeric CD8A protein comprises SEQ ID NO: 2 or SEQ ID NO: 7.

[0017] In one aspect, the present invention provides a non-human animal comprising at least one cell encoding a nucleotide sequence of a human or chimeric CD8B protein, wherein the human or chimeric CD8B protein comprises at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 140, 145, 148, 149, 150, 160, 169, 170, 175, 180, 185, 190, 200 or 210 consecutive amino acid sequences identical to the corresponding human region, and the non-human animal expresses the human or chimeric CD8B protein. In some embodiments, the chimeric CD8B protein comprises at least 50 consecutive amino acids of the extracellular region of human CD8B. In some embodiments, the human or chimeric CD8B protein comprises SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9 or positions 22-170 of SEQ ID NO: 9, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% amino acid identity to SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9 or positions 22-170 of SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the human or chimeric CD8B protein is operably linked to an endogenous CD8B regulatory element (such as a promoter and / or UTR, and the UTR is preferably a 5'UTR and / or a 3'UTR). In some embodiments, the nucleotide sequence encoding the human or chimeric CD8B protein is integrated into the endogenous CD8B locus of the non-human animal. In some embodiments, the human or chimeric CD8B protein has at least one non-human animal (such as a mouse) CD8B activity and / or human CD8B activity.

[0018] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same. In at least one cell of the non-human animal, at the endogenous CD8B locus of the non-human animal, the nucleotide sequence encoding the endogenous CD8B region is replaced with the nucleotide sequence encoding the corresponding region of human CD8B. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8B may be CDS, cDNA or genomic DNA. In some embodiments, the endogenous CD8B protein of the non-human animal is not expressed or the expression level is reduced compared to CD8B in a wild-type animal. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8B comprises a portion of exon 1 to a portion of exon 4 or a portion of exon 2 to a portion of exon 4 of the human CD8B gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8B comprises a portion of exon 1, all of exons 2-3, and a portion of exon 4 of the human CD8B gene (preferably also comprising intron 1 and / or intron 3). Preferably, the nucleotide sequence encoding the corresponding region of human CD8B comprises a portion of exon 1, all of exons 2-3, and a portion of exon 4 of the human CD8B gene at least 500-1000, 1000-10000 or 10000-15000 nucleotides. In some embodiments, the amino acid sequence of the corresponding region of human CD8B comprises SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9 or positions 22-170 of SEQ ID NO: 9, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% amino acid identity to SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9 or positions 22-170 of SEQ ID NO: 9. In some embodiments, the endogenous CD8B region is the endogenous CD8B extracellular region, the endogenous transmembrane region and / or the endogenous cytoplasmic region. In some embodiments, the amino acid sequence of the corresponding region of human CD8B comprises SEQ ID NO: 9 or 12, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% amino acid identity to the amino acid sequence shown in SEQ ID NO: 9 or 12. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8B comprises SEQ ID NO: 10, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% nucleotide identity to the nucleotide sequence shown in SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the endogenous CD8B region comprises a portion of exon 1 to a portion of exon 4 or a portion of exon 2 to a portion of exon 4 of the CD8B gene of a non-human animal (such as a mouse).In some embodiments, the nucleotide sequence encoding the endogenous CD8B region comprises a portion of exon 1 of the CD8B gene of a non-human animal (such as a mouse), all of exons 2-3, and a portion of exon 4. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8B is operably linked to an endogenous CD8B regulatory element (such as a promoter and / or UTR, preferably the 5'UTR and / or 3'UTR). In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the non-human animal is a mouse or a rat.

[0019] In one aspect, the present invention provides a method for constructing a non-human animal cell expressing a human or chimeric CD8B gene, the construction method comprising replacing the nucleotide sequence encoding the endogenous CD8B region with the nucleotide sequence encoding the corresponding region of human CD8B at the endogenous CD8B locus of a non-human animal (such as a mouse), to generate a genetically modified non-human animal cell, and the non-human animal cell expressing a human or chimeric CD8B protein. In some embodiments, the chimeric CD8B protein comprises a human or humanized extracellular region, a non-human animal endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8B comprises a portion of exon 1 to a portion of exon 4 or a portion of exon 2 to a portion of exon 4 of the human CD8B gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8B comprises a portion of exon 1, all of exons 2-3, and a portion of exon 4 of the human CD8B gene (preferably further comprising intron 1 and / or intron 3). Preferably, the nucleotide sequence encoding the corresponding region of human CD8B comprises at least 500-1000, 1000-10000, or 10000-15000 nucleotides of a portion of exon 1, all of exons 2-3, and a portion of exon 4 of the human CD8B gene. In some embodiments, the amino acid sequence of the corresponding region of human CD8B comprises SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9, or positions 22-170 of SEQ ID NO: 9, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the amino acid sequences shown in SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9, or positions 22-170 of SEQ ID NO: 9. In some embodiments, the encoded endogenous CD8B region is an endogenous CD8B extracellular region, an endogenous transmembrane region, and / or an endogenous cytoplasmic region. In some embodiments, the nucleotide sequence encoding the endogenous CD8B region comprises a portion of exon 1 to a portion of exon 4 or a portion of exon 2 to a portion of exon 4 of the CD8B gene of a non-human animal (such as a mouse). In some embodiments, the nucleotide sequence encoding the endogenous CD8B region comprises a portion of exon 1, all of exons 2-3, and a portion of exon 4 of the CD8B gene of a non-human animal (such as a mouse) (preferably further comprising intron 1 and / or intron 3). In some embodiments, the nucleotide sequence encoding the corresponding region of human CD8B is operably linked to a regulatory element of endogenous CD8B (such as a promoter and / or UTR, and the UTR is preferably a 5'UTR and / or a 3'UTR). In some embodiments, the non-human animal is a mouse.In some embodiments, the non-human animal further comprises nucleotide sequences encoding other human or chimeric proteins, and the human or chimeric proteins include, but are not limited to, at least one of CD8A, TRBC1, TRBC2, HLA-A, B2M, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0020] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the endogenous CD8B protein in the non-human animal with a nucleotide sequence encoding a human or chimeric CD8B protein. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the endogenous CD8B protein in the non-human animal with a nucleotide sequence encoding SEQ ID NO: 9 or 12. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the endogenous SEQ ID NO: 8 in the non-human animal with a nucleotide sequence encoding SEQ ID NO: 9 or 12. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the extracellular region of human CD8B with the nucleotide sequence encoding the extracellular region of CD8B in the non-human animal. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the extracellular region and signal peptide of human CD8B with the nucleotide sequence encoding the extracellular region and signal peptide of CD8B in the non-human animal. In some embodiments, the construction method comprises replacing the corresponding region of endogenous CD8B in the non-human animal with a nucleotide sequence encoding positions 1-169, positions 1-170 or positions 22-170 of SEQ ID NO: 9. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding positions 1-172 of endogenous SEQ ID NO: 8 in the non-human animal with a nucleotide sequence encoding positions 1-169 of SEQ ID NO: 9. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding positions 22-175 of endogenous SEQ ID NO: 8 in the non-human animal with a nucleotide sequence encoding positions 22-170 of SEQ ID NO: 9. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding positions 1-175 of endogenous SEQ ID NO: 8 in the non-human animal with a nucleotide sequence encoding positions 1-170 of SEQ ID NO: 9.

[0021] In some embodiments, the construction method includes replacing the nucleotide sequence of the endogenous CD8B gene in a non-human animal with the nucleotide sequence of human CD8B. In some embodiments, the construction method includes replacing the partial nucleotide sequence encoding positions 1-172 of SEQ ID NO: 8, positions 1-175 of SEQ ID NO: 8, or positions 22-175 of SEQ ID NO: 8 in the endogenous gene of a non-human animal with the partial nucleotide sequence from exon 1 to exon 4 of the human CD8B gene. In some embodiments, the construction method includes replacing the partial nucleotide sequence encoding positions 22-175 of SEQ ID NO: 8 in the endogenous gene of a non-human animal with the partial nucleotide sequence from exon 2 to exon 4 of the human CD8B gene. In some embodiments, the construction method includes replacing the partial nucleotide sequence from exon 1 to exon 4 of the endogenous CD8B gene in a non-human animal with the partial nucleotide sequence from exon 1 to exon 4 of the human CD8B gene. In some embodiments, the construction method includes replacing the partial nucleotide sequence from exon 2 to exon 4 of the endogenous CD8B gene in a non-human animal with the partial nucleotide sequence from exon 2 to exon 4 of the human CD8B gene. In some embodiments, the construction method includes replacing the partial nucleotide sequence encoding positions 1-172 of SEQ ID NO: 8 in the endogenous gene of a non-human animal with SEQ ID NO: 10. In some embodiments, the construction method includes replacing the nucleotide sequence encoding SEQ ID NO: 8 in the endogenous gene of a non-human animal with SEQ ID NO: 11. In some embodiments, the construction method includes replacing the partial nucleotide sequence from exon 1 to exon 4 of the endogenous exon in a non-human animal with SEQ ID NO: 10.

[0022] In some embodiments, the construction method includes replacing the corresponding endogenous region in a non-human animal with a nucleotide sequence encoding a human or chimeric CD8A protein and a human or chimeric CD8B protein. In some embodiments, the construction method includes replacing the corresponding endogenous region in a non-human animal with a donor sequence encoding the extracellular region of human CD8A and the extracellular region of human CD8B. In some embodiments, the donor sequence includes a nucleotide sequence encoding, from N to C, all or part of the extracellular region of human CD8B, the transmembrane region of endogenous CD8B of the non-human animal, the cytoplasmic region of endogenous CD8B of the non-human animal, and all or part of the extracellular region of human CD8A. In some embodiments, the construction method includes replacing the corresponding endogenous region in a non-human animal with a donor sequence encoding the signal peptide of human CD8A, the extracellular region of human CD8A, the signal peptide of human CD8B, and the extracellular region of human CD8B. In some embodiments, the donor sequence includes a nucleotide sequence encoding, from N to C, the signal peptide of human CD8B, all or part of the extracellular region of human CD8B, the transmembrane region of endogenous CD8B of the non-human animal, the cytoplasmic region of endogenous CD8B of the non-human animal, the signal peptide of endogenous CD8A of the non-human animal, and all or part of the extracellular region of human CD8A. In some embodiments, the corresponding endogenous region in the non-human animal includes a nucleotide sequence encoding the extracellular region of endogenous CD8B of the non-human animal and the extracellular region of endogenous CD8A of the non-human animal. In some embodiments, the corresponding endogenous region in the non-human animal includes a nucleotide sequence encoding the signal peptide of endogenous CD8B of the non-human animal, the extracellular region of endogenous CD8B, the signal peptide of endogenous CD8A of the non-human animal, and the extracellular region of endogenous CD8A. In some embodiments, the corresponding endogenous region in the non-human animal includes a nucleotide sequence encoding the extracellular region of endogenous CD8B of the non-human animal to the extracellular region of endogenous CD8A of the non-human animal. In some embodiments, the corresponding endogenous region in the non-human animal includes a nucleotide sequence encoding the signal peptide of endogenous CD8B of the non-human animal to the extracellular region of endogenous CD8A of the non-human animal. In some embodiments, the donor sequence is a chimeric sequence of human and non-human animals. In some embodiments, the regions to be replaced are the CD8A protein and CD8B protein of the non-human animal.

[0023] In some embodiments, the construction method includes replacing the corresponding endogenous region in a non-human animal with a donor sequence containing the nucleotide sequence of human CD8A and the nucleotide sequence of human CD8B. In some embodiments, the donor sequence includes, in order from 5' to 3', a part of exon 1 to a part of exon 4 of human CD8B (preferably SEQ ID NO: 10), a part of exon 4 of non-human animal endogenous CD8B to a part of exon 1 of CD8A, and a part of exon 4 to a part of exon 7 of human CD8A (preferably SEQ ID NO: 5). In some embodiments, the donor sequence includes, in order from 5' to 3', a part of exon 2 to a part of exon 4 of human CD8B (preferably the nucleotide sequence encoding the extracellular region), a part of exon 4 of non-human animal endogenous CD8B to a part of exon 1 of CD8A, and a part of exon 4 to a part of exon 7 of human CD8A (preferably SEQ ID NO: 5). In some embodiments, the donor sequence includes, in order from 5' to 3', a part of exon 2 to a part of exon 4 of human CD8B (preferably the nucleotide sequence encoding the extracellular region), a part of exon 4 of non-human animal endogenous CD8B to a part of exon 1 of CD8A, and a part of exon 5 to a part of exon 7 of human CD8A (preferably the nucleotide sequence encoding the extracellular region). In some embodiments, the donor sequence includes, in order from 5' to 3', a part of exon 1 to a part of exon 4 of human CD8B (preferably SEQ ID NO: 10), a part of exon 4 of non-human animal endogenous CD8B to a part of exon 1 of CD8A, and a part of exon 5 to a part of exon 7 of human CD8A (preferably the nucleotide sequence encoding the extracellular region). In some embodiments, the part of exon 4 of non-human animal endogenous CD8B to a part of exon 1 of CD8A is preferably the nucleotide sequence at positions 71309434 to 71350536 of NCBI accession number NC_000072.7. In some embodiments, the corresponding endogenous region in the non-human animal includes a part of exon 1 of non-human animal endogenous CD8B to a part of exon 3 of CD8A. In some embodiments, the corresponding endogenous region in the non-human animal includes a part of exon 2 of non-human animal endogenous CD8B to a part of exon 3 of CD8A.

[0024] In one aspect, the present invention provides an application of the non-human animal or the non-human animal obtained by the described construction method, and the application includes: A) an application in the development of products related to immune processes associated with CD8A and / or CD8B involving human cells; B) an application as a model system related to CD8A and / or CD8B in pharmacological, immunological, microbiological, and medical research; C) an application involving the production and utilization of animal experimental disease models for etiological research related to CD8A and / or CD8B and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) an application in the in vivo screening, efficacy detection, evaluation of efficacy, verification, or evaluation of human CD8A and / or CD8B signaling pathway regulators; or, E) an application in studying the functions of CD8A and / or CD8B genes, studying the drugs and drug effects targeting human CD8A and / or CD8B target sites, and studying drugs for cancers, immune diseases, or inflammation related to CD8A and / or CD8B.

[0025] In one aspect, the present invention provides a method for determining the effectiveness or toxicity of a therapeutic agent in treating a disease, and the method includes: 1) administering the therapeutic agent to the non-human animal or the non-human animal obtained by the described construction method; 2) determining the effect of the therapeutic agent on the disease or the non-human animal. In some embodiments, the therapeutic agent is a therapeutic agent targeting CD8A and / or CD8B, such as anti-human CD8A antibody, anti-human CD8B antibody, anti-human CD8A and CD8B antibody, or, a nucleic acid drug, ADC, PDC, and / or polypeptide drug targeting CD8A and / or CD8B. In some embodiments, the therapeutic agent further includes an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to TRBC1, TRBC2, HLA-A, B2M, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the disease includes cancer, immune disease, or inflammation.

[0026] In some embodiments, the cancer is a solid tumor or a hematological tumor, such as breast cancer, lymphoma, digestive tract cancer, gastrointestinal cancer, genital cancer, endocrine cancer, head and neck cancer, liver cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, or lung cancer.

[0027] In some embodiments, the immune diseases include but are not limited to GVHD (graft-versus-host disease), psoriasis, allergy, asthma, atopic dermatitis, myocarditis, nephritis, hepatitis (preferably non-alcoholic steatohepatitis), systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or nerve disorders, etc. In some embodiments, the immune diseases are psoriasis, AIDS, atopic dermatitis, transplant rejection, asthma, rheumatoid arthritis or multiple sclerosis. In some embodiments, the inflammation described in the present application includes acute inflammation and chronic inflammation. Specifically, the inflammation includes but is not limited to degenerative inflammation, exudative inflammation (such as serous inflammation, fibrin inflammation, suppurative inflammation, hemorrhagic inflammation, necrotic inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (such as tuberculosis, syphilis, leprosy or lymphogranuloma). In some embodiments, the inflammation described in the present application includes infection, and the infection refers to a local tissue and systemic inflammatory response caused by bacteria, viruses, fungi, parasites and / or other pathogens invading the human body. In some embodiments, the inflammation is ankylosing spondylitis, Crohn's disease, hepatitis or inflammatory bowel disease (IBD).

[0028] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating cancer, the method comprising: 1) administering the therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal has cancer; 2) determining the inhibitory effect of the therapeutic agent on cancer. In some embodiments, the cancer is a tumor, and the inhibitory effect of the therapeutic agent on the tumor is determined by measuring the tumor volume of the animal. In some embodiments, the cancer comprises injecting one or more cancer cells into the non-human animal body.

[0029] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent targeting CD8A and / or CD8B and an additional therapeutic agent in treating cancer, the method comprising: 1) administering the therapeutic agent targeting CD8A and / or CD8B and the additional therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal has a tumor; 2) determining the inhibitory effect of the therapeutic agent targeting CD8A and / or CD8B and the additional therapeutic agent on the tumor. In some embodiments, the tumor comprises injecting one or more cancer cells into the non-human animal body. In some embodiments, the inhibitory effect of the therapeutic agent on the tumor is determined by measuring the tumor volume of the animal.

[0030] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating an immune disease, the method comprising: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal has an immune disease; 2) determining the therapeutic effect of the therapeutic agent on the immune disease.

[0031] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating inflammation, the method comprising: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal has inflammation; 2) determining the effectiveness of the therapeutic agent in treating inflammation.

[0032] In one aspect, the present invention provides a method for determining the toxicity of a therapeutic agent, the method comprising: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method; 2) determining the effect of the therapeutic agent on the non-human animal. In some embodiments, determining the effect of the therapeutic agent on the animal involves measuring the change in body weight of the animal and / or a blood test. In some embodiments, the blood test includes, but is not limited to, red blood cell count, hematocrit, and / or hemoglobin content.

[0033] In one aspect, the present invention provides a humanized CD8A protein, the humanized CD8A protein comprising all or part of the human CD8A protein. In some embodiments, the humanized CD8A protein comprises a human or humanized extracellular region, a non-human animal endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the amino acid sequence of the humanized CD8A protein comprises SEQ ID NO: 2, positions 1-175 of SEQ ID NO: 2, positions 1-182 of SEQ ID NO: 2, or positions 22-182 of SEQ ID NO: 2, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to SEQ ID NO: 2, positions 1-175 of SEQ ID NO: 2, positions 1-182 of SEQ ID NO: 2, or positions 22-182 of SEQ ID NO: 2. In some embodiments, the humanized CD8A protein comprises SEQ ID NO: 7, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the amino acid sequence shown in SEQ ID NO: 7.

[0034] In one aspect, the present invention provides a humanized CD8B protein, which comprises all or part of the human CD8B protein. In some embodiments, the humanized CD8B protein comprises a human or humanized extracellular region, a non-human animal endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the amino acid sequence of the humanized CD8B protein comprises SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9, or positions 22-170 of SEQ ID NO: 9; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9, or positions 22-170 of SEQ ID NO: 9. In some embodiments, the humanized CD8B protein comprises SEQ ID NO: 12, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the amino acid sequence shown in SEQ ID NO: 12.

[0035] In one aspect, the present invention provides a humanized CD8A gene, which encodes the humanized CD8A protein. In some embodiments, the humanized CD8A gene comprises a portion from exon 4 to exon 7 of the human CD8A gene, and the humanized CD8A gene further comprises all or part of exon 1, a portion of exon 3, and / or all of exons 4-5 of the non-human animal CD8A gene. In some embodiments, the humanized CD8A gene comprises a portion from exon 5 to exon 7 of the human CD8A gene, and the humanized CD8A gene further comprises all or part of exon 1, a portion of exon 3, and / or all of exons 4-5 of the non-human animal CD8A gene. In some embodiments, the humanized CD8A gene comprises SEQ ID NO: 4, 5, 6, 21, or 23, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 4, 5, 6, 21, or 23.

[0036] In one aspect, the present invention provides a humanized CD8B gene, and the humanized CD8B gene encodes the above-mentioned humanized CD8B protein. In some embodiments, the humanized CD8B gene comprises a portion of exon 1 to a portion of exon 4 of the human CD8B gene, and the humanized CD8B gene further comprises a portion of exon 1, a portion of exon 4, and / or all of exons 5-6 of the non-human animal CD8B gene. In some embodiments, the humanized CD8B gene comprises a portion of exon 2 to a portion of exon 4 of the human CD8B gene, and the humanized CD8B gene further comprises all of exon 1, a portion of exon 2, a portion of exon 4, and / or all of exons 5-6 of the non-human animal CD8B gene. In some embodiments, the humanized CD8B gene comprises SEQ ID NO: 3, 10, 11, 13, 14, 20 or 22, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 3, 10, 11, 13, 14, 20 or 22.

[0037] In one aspect, the present invention provides a cell, tissue or organ, which comprises the above-mentioned humanized CD8A protein, the above-mentioned humanized CD8B protein, the above-mentioned humanized CD8A gene or the above-mentioned humanized CD8B gene.

[0038] In one aspect, the present invention provides a non-human animal genome, the non-human animal genome comprising at least one chromosome, the chromosome comprising a nucleotide sequence encoding a human or chimeric CD8A protein and / or a human or chimeric CD8B protein. In some embodiments, the chromosome comprises a human or chimeric CD8A gene and / or a human or chimeric CD8B gene. In some embodiments, the chromosome comprises a nucleotide sequence encoding a human CD8A protein and / or a human CD8B protein. In some embodiments, the chromosome comprises a nucleotide sequence encoding the extracellular region of the human CD8A protein. In some embodiments, the chromosome comprises a nucleotide sequence encoding the extracellular region of the human CD8B protein. In some embodiments, the chromosome comprises a portion from exon 4 to exon 7 or a portion from exon 5 to exon 7 of the human CD8A gene. In some embodiments, the chromosome comprises a portion from exon 1 to exon 4 or a portion from exon 2 to exon 4 of the human CD8B gene. In some embodiments, the CD8A and / or CD8B of the endogenous genome of the non-human animal is replaced. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 1, positions 1-187 of SEQ ID NO: 1, positions 1-196 of SEQ ID NO: 1, or positions 28-196 of SEQ ID NO: 1 in the endogenous genome of the non-human animal is replaced. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 8, positions 1-172 of SEQ ID NO: 8, positions 1-175 of SEQ ID NO: 8, or positions 22-175 of SEQ ID NO: 8 in the endogenous genome of the non-human animal is replaced. In some embodiments, all or part of exon 1 to a portion of exon 3 of the endogenous CD8A gene of the non-human animal is replaced. In some embodiments, a portion of exon 1 to exon 4 of the endogenous CD8B gene of the non-human animal is replaced. In some embodiments, a portion of exon 2 to exon 4 of the endogenous CD8B gene of the non-human animal is replaced. In some embodiments, the chromosome comprises a nucleotide sequence encoding a human or chimeric CD8A protein or a nucleotide sequence of human CD8A to replace the corresponding portion of the endogenous chromosome of the non-human animal. In some embodiments, the chromosome comprises a nucleotide sequence encoding a human or chimeric CD8B protein or a nucleotide sequence of human CD8B to replace the corresponding portion of the endogenous chromosome of the non-human animal.

[0039] In one aspect, the present invention provides a cell, tissue or organ comprising the above non-human animal genome.

[0040] In one aspect, the present invention provides an animal model, the animal model comprising the humanized CD8A protein or humanized CD8B protein or humanized CD8A gene or humanized CD8B gene.

[0041] The term "all or part" in the present invention, "all" refers to the whole, and "part" refers to a local part in the whole, or some individuals that make up the whole.

[0042] The term "locus" in the present invention, in a broad sense, represents the position occupied by a gene on a chromosome, and in a narrow sense, represents a DNA fragment on a certain gene, which can be either a gene or a part of a gene or a gene regulatory region, etc. For example, the "CD8A locus" described above includes any DNA fragment of exons 1-5 of the CD8A gene and its regulatory region. Another example, the "CD8B locus" described above includes any DNA fragment of exons 1-6 of the CD8B gene and its regulatory region.

[0043] The term "part of exon XX" in the present invention means that a continuous or spaced sequence of several, dozens or hundreds of nucleotides is identical to the entire exon nucleotide sequence.

[0044] The term "exon XX to exon XXX" or "exon XX-XXX" or "the whole of exon XX to the whole of exon XXX" or "the whole of exon XX to the whole of exon XXX" in the present invention refers to including exons and the introns between them.

[0045] The term "part of exon x to part of exon xx" or "the whole of exon x to part of exon xx" or "part of exon x - part of exon xx" in the present invention includes all or part of the exons and the introns between them. For example, "part of exon 1 to part of exon 4" includes part of exon 1, the whole of intron 1, the whole of exon 2, the whole of intron 2, the whole of exon 3, the whole of intron 3, and part of exon 4. Another example, "part of exon 4 to part of exon 7" includes part of exon 4, the whole of intron 4, the whole of exon 5, the whole of intron 5, the whole of exon 6, the whole of intron 6, and part of exon 7.

[0046] The term "intron xx" in the present application refers to the intron between two exons. For example, intron 1 is the intron between exon 1 and exon 2.

[0047] The term "comprising" or "including" in the present invention is an open-ended writing method, including the specified components or steps described, as well as other specified components or steps that will not be substantially affected. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the described sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still has the same or similar activity as the original sequence.

[0048] As used in this invention, the term "and / or" encompasses all combinations of the items connected by this term, and each combination should be regarded as having been separately listed in this application. For example, "A and / or B" includes "A", "A and B", and "B". Another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0049] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. This application describes the methods and materials for this invention; other suitable methods and materials known in the art can be used. The materials, methods, and examples are merely exemplary and not restrictive. All publications, patent applications, patents, sequences, database entries, and other references mentioned in this application are hereby incorporated by reference in their entirety. In case of conflict, the present specification (including definitions) shall prevail.

[0050] Those skilled in the art can readily discern other aspects and advantages of this application from the following detailed description.

[0051] CD8A

[0052] In the human genome, the CD8A gene (NCBI Gene ID: 925, UniProt ID: P01732, located at positions 86784605 to 86808396 on chromosome 2, NC_000002.12) contains 9 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and exon 9. The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM_001145873.1 and its encoded protein NP_001139345.1 (SEQ ID NO: 2) are shown in Table 1.

[0053] Table 1

[0054]

[0055] In the mouse genome, the CD8A gene (NCBI Gene ID: 12525, UniProt ID: P01731, located at positions 71350411 to 71356155 on chromosome 6, NC_000072.7) contains 5 exons, namely exon 1, exon 2, exon 3, exon 4, and exon 5. The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM_001081110.2 and its encoded protein NP_001074579.1 (SEQ ID NO: 1) are shown in Table 2.

[0056] Table 2

[0057]

[0058] CD8B

[0059] In the human genome, the CD8B gene (NCBI Gene ID: 926, UniProt ID: P10966, located at positions 86815369 to 86861886 on chromosome 2, NC_000002.12) contains 6 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, and exon 6. The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM_004931.5 and its encoded protein NP_004922.1 (SEQ ID NO: 9) are shown in Table 3.

[0060] Table 3

[0061]

[0062] In the mouse genome, the CD8B gene (NCBI Gene ID: 12526, UniProt ID: P10300, located at positions 71299772 to 71314476 on chromosome 6, NC_000072.7) contains 6 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, and exon 6. The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM_009858.3 and its encoded protein NP_033988.1 (SEQ ID NO: 8) are shown in Table 4.

[0063] Table 4

[0064]

[0065] The CD8A and CD8B genes, proteins, and gene loci of other species in the art are also known. For example, in Rattus norvegicus (rat), Macaca mulatta (rhesus monkey), Canis lupus familiaris (dog), and Sus scrofa (pig), the relevant information of these genes (such as intron sequences, exon sequences, and amino acid sequences) can all be found in NCBI, and the entire content is incorporated herein by reference.

[0066] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be disregarded for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced to achieve optimal alignment of the two sequences. For example, the comparison of sequences and the determination of the percent identity between two sequences can be done using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0067] The percentage of conserved residues with similar physicochemical properties (percent homology), such as leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (such as aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (such as threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the percent homology is higher than the percent identity.

[0068] vector

[0069] The present invention provides a targeting vector, and the targeting vector comprises a donor region. In some embodiments, the donor region comprises the nucleotide sequence of human CD8A, and / or the nucleotide sequence of human CD8B. In some embodiments, the donor region comprises the nucleotide sequence encoding the extracellular region of human CD8A, and / or the nucleotide sequence encoding the extracellular region of human CD8B. In some embodiments, the donor region comprises the nucleotide sequence encoding the signal peptide of human CD8A, and / or the nucleotide sequence encoding the signal peptide of human CD8B. In some embodiments, the donor region comprises the nucleotide sequence encoding the extracellular region and signal peptide of human CD8A, and / or the nucleotide sequence encoding the extracellular region and signal peptide of human CD8B. In some embodiments, the donor region comprises the nucleotide sequence encoding SEQ ID NO: 2, positions 1-175 of SEQ ID NO: 2, positions 1-182 of SEQ ID NO: 2, or positions 22-182 of SEQ ID NO: 2, and the donor region comprises the nucleotide sequence encoding SEQ ID NO: 9, positions 1-169 of SEQ ID NO: 9, positions 1-170 of SEQ ID NO: 9, or positions 22-170 of SEQ ID NO: 9. In some embodiments, the donor region comprises SEQ ID NO: 5 and SEQ ID NO: 10.

[0070] In some embodiments, the targeting vector comprises: a) a DNA fragment (5'-arm or 5'-homologous arm) homologous to the 5'-end of the region to be modified, which is selected from the genomic DNA of the CD8B gene and has a length of 100 to 10,000 nucleotides; b) a donor region; and c) a DNA fragment (3'-arm or 3'-homologous arm) homologous to the 3'-end of the region to be modified, which is selected from the genomic DNA of the CD8A gene and has a length of 100 to 10,000 nucleotides.

[0071] In some embodiments, a) the DNA fragment homologous to the 5'-end of the region to be modified is selected from nucleotide sequences having at least 90% homology with NCBI accession number NC_000072.7; c) the DNA fragment homologous to the 3'-end of the region to be modified is selected from nucleotide sequences having at least 90% homology with NCBI accession number NC_000072.7.

[0072] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed about 0.8 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 15 kb, 16 kb, 18 kb, 19 kb, or 20 kb.

[0073] In some embodiments, the region to be modified is located on the CD8A and CD8B genes of a non-human animal. In some embodiments, the region to be modified is located on exons 1 to 5 of the CD8A gene and exons 1 to 6 of the CD8B gene of a non-human animal.

[0074] In some embodiments, the 5'-arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 3. In some embodiments, the 3'-arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 4.

[0075] In some embodiments, the targeting vector further comprises one or more marker genes (or resistance genes). For example, a positive selection marker gene or a negative selection marker gene. In some embodiments, the resistance gene for positive clone selection is the neomycin phosphotransferase coding sequence Neo or the hygromycin resistance gene sequence HygR. Preferably, the targeting vector further comprises two directly repeated Frt3 recombination sites flanking the marker gene. In some embodiments, the coding gene for the negative selection marker is the coding gene for the diphtheria toxin A subunit (DTA).

[0076] The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the CD8A and CD8B genes, and the sgRNA is unique on the target sequence of the gene to be modified and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'. In some embodiments, the targeting sites of the sgRNA in the CD8A and CD8B genes of a non-human animal (such as a mouse) are located on exons 1 to 5 of the CD8A gene and exons 1 to 6 of the CD8B gene of the non-human animal.

[0077] In some embodiments, the present application relates to a plasmid construct (such as pT7-sgRNA) comprising an sgRNA sequence and / or a cell comprising the construct.

[0078] The present invention also relates to a cell comprising the targeting vector or the sgRNA vector as described above.

[0079] In addition, the present invention also provides a non-human mammalian cell having any one of the above targeting vectors and one or more in vitro transcripts of the construct described in the present application. In some embodiments, the cell comprises Cas9 mRNA or its in vitro transcript.

[0080] In some embodiments, the genes in the cell are heterozygous. In some embodiments, the genes in the cell are homozygous.

[0081] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is an embryonic stem cell.

[0082] Genetically modified non-human animal

[0083] As used herein, the term "genetically modified non-human animal" or "genetically engineered non-human animal" refers to a non-human animal in which at least one chromosome in the non-human animal genome has exogenous DNA. In some embodiments, at least one or more cells, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in the genetically modified non-human animal, have exogenous DNA. The cells having exogenous DNA can be various cells, e.g., somatic cells, immune cells (such as T cells, B cells, NK cells, antigen-presenting cells, macrophages, dendritic cells), germ cells, blastocysts or tumor cells. In some embodiments, there is provided a genetically modified non-human animal, wherein the non-human animal comprises an endogenous CD8A and CD8B locus that is modified to comprise an exogenous sequence (e.g., a human sequence), e.g., by replacing one or more non-human sequences with one or more human sequences, or inserting one or more human and / or non-human sequences. The non-human animal is generally capable of transmitting the genetic modification to its offspring through the germ line.

[0084] As used herein, the term "chimeric (x) gene" or "chimeric (x) nucleic acid" refers to a gene or nucleic acid in which two or more portions of the gene or nucleic acid are from different species, or in which at least one sequence of the gene or nucleic acid is different from the nucleic acid in a wild-type animal. In some embodiments, the chimeric (x) gene or chimeric (x) nucleic acid has at least a portion of the sequence having two or more different species origins, e.g., sequences encoding different proteins or sequences encoding the same (or homologous) proteins of two or more different species. In some embodiments, the chimeric (x) gene or chimeric (x) nucleic acid refers to a humanized (x) gene or humanized (x) nucleic acid.

[0085] As used herein, the term "chimeric (x) protein" or "chimeric (x) polypeptide" refers to a protein or polypeptide in which two or more portions of the polypeptide or protein are from different species, or in which at least one sequence of the protein or polypeptide is different from the amino acid sequence in a wild-type animal. In some embodiments, at least a portion of the sequence of the chimeric (x) protein or chimeric (x) polypeptide has two or more different species origins, e.g., the same (or homologous) proteins of different species. In some embodiments, the chimeric (x) protein or chimeric (x) polypeptide refers to a humanized (x) protein or humanized (x) polypeptide.

[0086] As used herein, the "humanized (x) protein" or "humanized (x) polypeptide" refers to a protein or polypeptide in which at least a portion of the protein or polypeptide is derived from a human protein or human polypeptide. In some embodiments, the humanized (x) protein or humanized (x) polypeptide refers to a human protein or polypeptide.

[0087] As used herein, the "humanized (x) nucleic acid" or "humanized (x) gene" refers to a nucleic acid or gene in which at least a portion of the nucleic acid or gene is derived from a human. In some embodiments, the nucleic acid or gene in the humanized (x) nucleic acid or humanized (x) gene is entirely derived from a human. In some embodiments, the humanized (x) nucleic acid or humanized (x) gene refers to a humanized exon, which may be a human exon or a chimeric exon.

[0088] In some embodiments, the chimeric CD8A gene or chimeric CD8A nucleic acid is a humanized CD8A gene or humanized CD8A nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from the human CD8A gene, and at least a portion of the gene or nucleic acid is derived from a non-human CD8A gene. In some embodiments, the gene or nucleic acid comprises a sequence encoding a CD8A protein. The encoded CD8A protein has at least one activity of a human CD8A protein or a non-human animal CD8A protein.

[0089] In some embodiments, the chimeric CD8B gene or chimeric CD8B nucleic acid is a humanized CD8B gene or humanized CD8B nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from the human CD8B gene, and at least a portion of the gene or nucleic acid is derived from a non-human CD8B gene. In some embodiments, the gene or nucleic acid comprises a sequence encoding a CD8B protein. The encoded CD8B protein has at least one activity of a human CD8B protein or a non-human animal CD8B protein.

[0090] In some embodiments, the chimeric CD8A protein or chimeric CD8A polypeptide is a humanized CD8A protein or humanized CD8A polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a human CD8A protein, and at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a non-human animal CD8A protein. The humanized CD8A protein or humanized CD8A polypeptide is functional or has at least one activity of a human CD8A protein or a non-human animal CD8A protein.

[0091] In some embodiments, the chimeric CD8B protein or chimeric CD8B polypeptide is a humanized CD8B protein or humanized CD8B polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or polypeptide are from a human CD8B protein, and at least one or more portions of the amino acid sequence of the protein or polypeptide are from a non-human animal CD8B protein. The humanized CD8B protein or humanized CD8B polypeptide is functional or has at least one activity of a human CD8B protein or a non-human animal CD8B protein.

[0092] In some embodiments, the extracellular domain of CD8A is human or humanized. In some embodiments, the signal peptide of CD8A is human or humanized. In some embodiments, the cytoplasmic domain of CD8A is human or humanized. In some embodiments, the transmembrane domain of CD8A is human or humanized.

[0093] In some embodiments, the extracellular domain of CD8B is human or humanized. In some embodiments, the signal peptide of CD8B is human or humanized. In some embodiments, the cytoplasmic domain of CD8B is human or humanized. In some embodiments, the transmembrane domain of CD8B is human or humanized.

[0094] The genetically modified non-human animal includes modification of the endogenous non-human animal CD8A and / or CD8B gene locus. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of the mature CD8A and / or CD8B protein (e.g., having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of the mature CD8A and / or CD8B protein). Cells (e.g., ES cells, somatic cells) that may comprise the genetic modification described in the present application are provided in the present invention. In some embodiments, the genetically modified non-human animal includes modification of the endogenous CD8A and / or CD8B gene locus in the non-human animal.

[0095] The genetically modified non-human animal can express human CD8A and / or chimeric (e.g., humanized) CD8A at the endogenous locus in a non-human animal (such as a mouse), wherein the endogenous CD8A gene in the non-human animal (such as a mouse) has been replaced or inserted with a gene of human CD8A and / or a nucleotide sequence encoding a human CD8A sequence region or a nucleotide sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identity to the human CD8A sequence. In some embodiments, the endogenous non-human animal CD8A locus is modified with all or part of the human nucleic acid sequence encoding the mature CD8A protein.

[0096] Genetically modified non-human animals can express human CD8B and / or chimeric (e.g., humanized) CD8B at an endogenous locus in the non-human animal (such as a mouse), wherein the endogenous CD8B gene in the non-human animal (such as a mouse) has been replaced or inserted with a gene of human CD8B and / or a nucleotide sequence encoding a human CD8B sequence region or a nucleotide sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identity to the human CD8B sequence. In some embodiments, the endogenous non-human animal CD8B locus is modified with all or part of the nucleic acid sequence of a human encoding a mature CD8B protein.

[0097] In some embodiments, the genetically modified non-human animal (such as a mouse) can express human CD8A and / or chimeric CD8A (e.g., humanized CD8A) under the control of non-human animal (such as a mouse) regulatory elements (such as a promoter and / or UTR, preferably a 5'UTR and / or a 3'UTR). Insertion or replacement at the endogenous locus in the non-human animal (such as a mouse) provides a non-human animal that expresses human CD8A or chimeric CD8A (e.g., humanized CD8A) in appropriate cells and in a manner that does not result in potential pathologies observed in some other transgenic non-human animals (such as a mouse) known in the art. The human CD8A or chimeric CD8A (e.g., humanized CD8A) expressed in the non-human animal can maintain one or more functions of a wild-type non-human animal (such as a mouse) or human CD8A in the non-human animal. In addition, in some embodiments, the non-human animal does not express endogenous CD8A. In some embodiments, the level of endogenous CD8A expression in the non-human animal is reduced compared to the level of CD8A expression in a wild-type animal. As used herein, the term "endogenous CD8A" refers to the CD8A protein expressed from the endogenous CD8A nucleotide sequence of a non-human animal (e.g., a mouse) prior to any genetic modification.

[0098] In some embodiments, a genetically modified non - human animal (such as a mouse) can express human CD8B and / or chimeric CD8B (e.g., humanized CD8B) under the control of non - human animal (such as a mouse) regulatory elements (such as promoters and / or UTRs, preferably 5'UTR and / or 3'UTR). Insertion or replacement at the endogenous locus in the non - human animal (such as a mouse) provides a non - human animal that expresses human CD8B or chimeric CD8B (e.g., humanized CD8B) in appropriate cells and in a manner that does not result in potential pathologies observed in some other transgenic non - human animals (such as mice) known in the art. The human CD8B or chimeric CD8B (e.g., humanized CD8B) expressed in the non - human animal can maintain one or more functions of the wild - type non - human animal (such as a mouse) or human CD8B in the non - human animal. In addition, in some embodiments, the non - human animal does not express endogenous CD8B. In some embodiments, the level of endogenous CD8B expression in the non - human animal is reduced compared to the CD8B expression level in the wild - type animal. As used in the present invention, the term "endogenous CD8B" refers to the CD8B protein expressed from the endogenous CD8B nucleotide sequence of a non - human animal (e.g., a mouse) prior to any genetic modification.

[0099] A genetically modified non - human animal can have one or more cells that express human or chimeric CD8A (e.g., humanized CD8A), which has a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region from the N - terminus to the C - terminus. In many cases, the human CD8A and non - human CD8A (e.g., mouse CD8A) sequences are different, so an antibody that binds to human CD8A may not have the same affinity for or the same effect on non - human CD8A. Thus, a genetically modified non - human animal having a human or humanized extracellular region can be used to better evaluate the effect of therapeutic agents targeting CD8A in an animal model.

[0100] A genetically modified non - human animal can have one or more cells that express human or chimeric CD8B (e.g., humanized CD8B), which has a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region from the N - terminus to the C - terminus. In many cases, the human CD8B and non - human CD8B (e.g., mouse CD8B) sequences are different, so an antibody that binds to human CD8B may not have the same affinity for or the same effect on non - human CD8B. Thus, a genetically modified non - human animal having a human or humanized extracellular region can be used to better evaluate the effect of therapeutic agents targeting CD8B in an animal model.

[0101] In some embodiments, the entire signal peptide, the entire transmembrane region, and the entire cytoplasmic region of the humanized CD8A described in the present application are derived from the endogenous CD8A sequence.

[0102] In some embodiments, the entire signal peptide, the entire transmembrane region, and the entire cytoplasmic region of the humanized CD8B of the present application are derived from the endogenous CD8B sequence.

[0103] In some embodiments, the humanized CD8A locus contains the 5'UTR of the human CD8A gene. In some embodiments, the humanized CD8A locus contains the endogenous 5'UTR of a non-human animal (e.g., mouse). In some embodiments, the humanized CD8A locus contains the 3'UTR of the human CD8A gene. In some embodiments, the humanized CD8A locus contains the endogenous 3'UTR of a non-human animal (e.g., mouse). In appropriate cases, it can be reasonably assumed that based on the similarity of the 5' flanking sequences of the mouse and human CD8A genes, they seem to be regulated similarly. As shown in the present application, humanized CD8A mice with insertions or replacements in the endogenous mouse CD8A locus, which retain mouse regulatory elements but contain humanized CD8A coding sequences, do not exhibit pathological phenomena. Both gene-modified mice that are heterozygous or homozygous for humanized CD8A are normal.

[0104] In some embodiments, the humanized CD8B locus contains the 5'UTR of the human CD8B gene. In some embodiments, the humanized CD8B locus contains the endogenous 5'UTR of a non-human animal (e.g., mouse). In some embodiments, the humanized CD8B locus contains the 3'UTR of the human CD8B gene. In some embodiments, the humanized CD8B locus contains the endogenous 3'UTR of a non-human animal (e.g., mouse). In appropriate cases, it can be reasonably assumed that based on the similarity of the 5' flanking sequences of the mouse and human CD8B genes, they seem to be regulated similarly. As shown in the present application, humanized CD8B mice with insertions or replacements in the endogenous mouse CD8B locus, which retain mouse regulatory elements but contain humanized CD8B coding sequences, do not exhibit pathological phenomena. Both gene-modified mice that are heterozygous or homozygous for humanized CD8B are normal.

[0105] The genetically modified non-human animals can be various animals, such as, for example, mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, water buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys). For non-human animals for which it is not easy to obtain suitable genetically modifiable embryonic stem cells (ES), other methods are used to construct non-human animals containing genetic modifications. Such methods include, for example, modifying the genome of non-ES cells (e.g., fibroblasts or induced pluripotent stem cells) and using nuclear transfer to transfer the modified genome to a suitable cell, such as an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under appropriate conditions to form an embryo. The construction methods described above are known in the art and are described in “A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition),” Cold Spring Harbor Laboratory Press, 2006”, the entire content of which is incorporated herein by reference.

[0106] In one aspect, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. The rodent can be selected from mice, rats, and hamsters. In some embodiments, the rodent is selected from the Muridae family. In some embodiments, the genetically modified animal is selected from the families Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (mountain mice, rock mice, tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rats, bamboo rats, and zokors). In some embodiments, the genetically modified rodent is selected from mice or rats (Muroidea), gerbils, spiny mice, and crested rats. In some embodiments, the genetically modified mouse is from a member of the Muridae family. In some embodiments, the animal is a rodent. In some embodiments, the rodent is selected from mice and rats. In some embodiments, the non-human animal is a mouse.

[0107] In some embodiments, the non-human animal can be an immunodeficient non-human mammal. For example, immunodeficient rodents, immunodeficient rabbits, immunodeficient pigs, immunodeficient monkeys, etc. In some embodiments, the non-human animal is a mouse of the C57BL strain, and the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 10, C57BL10ScSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the mouse is of the 129 strain selected from 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, 129 / Sv-ter / +. These mice are described in, for example, Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10:836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant content of which is incorporated herein by reference in its entirety. In some embodiments, the genetically modified mouse is a hybrid of the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a hybrid of the 129 strain, or a hybrid of the C57BL / 6 strain. In some embodiments, the mouse is of the BALB strain, such as the BALB / c strain. In some embodiments, the mouse is a hybrid of the 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). In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse having a strain of BALB / c, BALB / cHeAn, BALB / cJ, BALB / cRl, BALB / cWt, C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st, or CBA / H. In some embodiments, the non-human animal is a rat. The rat can be selected from Wistar rats, LEA strain, Sprague-Dawley strain, Fischer strain, F344, F6, and Dark Agouti.In some embodiments, the rat strain is a hybrid species of two or more strains selected from Wistar, LEA, Sprague-Dawley, Fischer, F344, F6, and Dark Agouti.

[0108] The non-human animal can have one or more other genetic modifications and / or other modifications that are suitable for a particular purpose of preparing a humanized animal. For example, a suitable mouse for maintaining xenografts (such as human cancers or tumors) can have one or more modifications that impair, inactivate, or disrupt all or part of the immune system of the non-human animal. Impairment, inactivation, or disruption of the immune system of the non-human animal can include, for example, by chemical means (such as administration of toxins), physical means (such as irradiating the animal), and / or genetic modification (such as knocking out one or more genes). Non-limiting examples of such mice include, for example, NOD mice, SCID mice, NOD / SCID mice, IL2Rγ knockout mice, NOD / SCID / γcnull mice (Ito, M. et al., NOD / SCID / γcnull mouse: an excellent recipient mouse model for engraftment of human cells, Blood 100(9):3175 - 3182, 2002), nude mice, and Rag1 and / or Rag2 knockout mice. These mice can optionally be irradiated or otherwise treated to destroy one or more immune cell types. Thus, in some embodiments, there is provided a genetically modified mouse that can include humanization of at least a portion of the endogenous non-human CD8A and / or CD8B locus and also includes a modification that impairs, inactivates, or partially disrupts the immune system (or one or more cell types of the immune system) of the non-human animal. In some embodiments, the type of mouse modification is selected from NOD mice, SCID mice, NOD / SCID mice, IL-2Rγ knockout mice, NOD / SCID / γc null mice, nude mice, Rag1 and / or Rag2 knockout mice, NOD Prkdc scid IL-2Rγ null mice, NOD Rag1 - / - IL2rg - / - (NRG) mice, Rag2 - / - IL2rg - / - (RG) mice and combinations thereof. These transgenic animals are described, for example, in US10820580B2, which is incorporated herein by reference in its entirety. In some embodiments, the mouse can include replacement of all or part of the endogenous mature CD8A and CD8B coding sequences of the mouse with all or part of the human mature CD8A and CD8B coding sequences, respectively.

[0109] The present invention further relates to a non-human mammal or its offspring produced by the above method. In some embodiments, its genome contains human genes.

[0110] In some embodiments, the non-human mammal is a rodent, preferably, the non-human mammal is a mouse.

[0111] In some embodiments, the non-human mammal expresses a protein encoded by humanized CD8A and CD8B genes.

[0112] In addition, the present invention also provides a non-human mammal model carrying a tumor, and the non-human mammal model is obtained by the method described in the present application. In some embodiments, the non-human mammal is a rodent (such as a mouse).

[0113] The present invention also provides a cell or cell line, or a primary cell culture, derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, which is derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, a tissue, an organ or its culture derived from a non-human mammal or its offspring. When carrying a tumor, it is derived from a tumor tissue of a non-human mammal or its offspring or a non-human mammal carrying a tumor.

[0114] The present invention provides a non-human mammal produced by any of the methods described in the present application. In some embodiments, a non-human mammal, a genetically modified non-human animal is provided, and the genome of the genetically modified non-human animal contains DNA of human or humanized CD8A and CD8B.

[0115] In some embodiments, the non-human mammal includes the gene construct described in the present application. In some embodiments, a non-human mammal expressing human or humanized CD8A and CD8B proteins is provided. In some embodiments, a tissue specifically expressing human or humanized CD8A and CD8B proteins is provided.

[0116] In some embodiments, the expression of the non-human animal or human or humanized CD8A and CD8B proteins is controllable. Such as by adding a specific inducer or repressor. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0117] The non-human mammal can be any non-human animal known in the art and can be used in the method described in the present application. The preferred non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

[0118] Perform genetic, molecular, and behavioral analyses on the non-human mammals described above. The present invention provides offspring produced by mating non-human mammals of the same genotype or other genotypes.

[0119] The present invention provides a cell line or primary cell culture derived from a non-human mammal or its offspring. For example, a cell culture-based model can be prepared by the following method. The cell culture can be obtained by isolation from a non-human mammal, or cells can be obtained from a cell culture established using the same construct and standard cell transfection techniques. Integration of a genetic construct containing a DNA sequence encoding human CD8A and CD8B proteins can be detected by various methods.

[0120] There are many analytical methods available for detecting exogenous DNA, including methods at the nucleic acid level (including the use of reverse transcription-polymerase chain reaction (RT-PCR), Southern Blot, and in situ hybridization) and methods at the protein level (including histochemical analysis, immunoblot analysis, and in vitro binding studies). In addition, the expression level of the target gene can be quantified by ELISA methods well-known to those skilled in the art. Many standard analytical methods can be used to complete quantitative detection. For example, RT-PCR and hybridization methods can be used to detect the transcriptional level, including ribonuclease protection assay, Southern Blot, RNA dot blot analysis (RNAdot). Immunohistochemical staining, flow cytometry, and Western blot can also be used to detect the presence of human or humanized CD8A and CD8B proteins.

[0121] Method for constructing a genetically modified non-human animal

[0122] Genetically modified non-human animals can be prepared by several techniques known in the art, including gene targeting techniques using embryonic stem cells, homologous recombination techniques, CRISPR / Cas9 techniques, zinc finger nuclease techniques, transcription activator-like effector nuclease techniques, homing endonucleases, or other molecular biology techniques. In some embodiments, homologous recombination techniques are preferably used. In some embodiments, CRISPR / Cas9 gene editing techniques can be used to construct genetically modified non-human animals. In some embodiments, CRISPR-Cas9 gene editing techniques are used to generate genetically modified non-human animals. Many of these genome editing techniques are known in the art and are described in Yin et al., "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6 (2017): 387-399, which is incorporated herein by reference. The present invention also provides many other methods for genome editing, for example, microinjecting genetically modified non-human animal cells into enucleated oocytes and fusing the enucleated oocytes with another genetically modified non-human animal cell.

[0123] In some embodiments, the nucleotide sequence encoding the endogenous CD8A and / or CD8B regions in the endogenous genome of at least one cell of the non-human animal is replaced with the nucleotide sequence encoding the corresponding regions of human CD8A and / or CD8B. In some embodiments, the expression level of the endogenous CD8A and / or CD8B protein in the non-human animal is reduced or absent compared to the wild type. In some embodiments, the replacement occurs in cells such as germ cells, somatic cells, blastocysts, or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0124] Preferably, the non-human animal further comprises other genetic modifications. More preferably, the other genes include, but are not limited to, at least one of TRBC1, TRBC2, HLA-A, B2M, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4.

[0125] Preferably, the human or humanized CD8A and / or CD8B gene and / or other genes are homozygous for the endogenous modified (preferably replaced or inserted) locus.

[0126] Preferably, the human or humanized CD8A and / or CD8B gene and / or other genes are heterozygous for the endogenous modified (preferably replaced or inserted) locus.

[0127] Preferably, the non-human animal can be selected from any non-human animal such as rodents, pigs, rabbits, monkeys, etc. that can be genetically edited to prepare a humanized gene.

[0128] Preferably, the non-human animal is a non-human mammal. More preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0129] Therefore, the present invention provides a method for constructing a non-human animal with humanized CD8A and / or CD8B genes, wherein the non-human animal expresses human or humanized CD8A and / or CD8B proteins in vivo, and / or, the genome of the non-human animal contains a part of the human CD8A and / or CD8B genes or humanized CD8A and / or CD8B genes.

[0130] In some embodiments, the method for modifying the CD8A locus of a mouse to express a chimeric human / mouse CD8A polypeptide may include replacing the nucleotide sequence encoding mouse CD8A at the endogenous mouse CD8A locus with a nucleotide sequence encoding human CD8A, thereby generating a sequence encoding a chimeric human / mouse CD8A. In some embodiments, the method may include inserting a nucleotide sequence encoding a chimeric human / mouse CD8A at the endogenous mouse CD8A locus, thereby generating a sequence encoding a chimeric human / mouse CD8A.

[0131] In some embodiments, the method for modifying the CD8B locus of a mouse to express a chimeric human / mouse CD8B polypeptide may include replacing the nucleotide sequence encoding mouse CD8B at the endogenous mouse CD8B locus with a nucleotide sequence encoding human CD8B, thereby generating a sequence encoding a chimeric human / mouse CD8B. In some embodiments, the method may include inserting a nucleotide sequence encoding a chimeric human / mouse CD8B at the endogenous mouse CD8B locus, thereby generating a sequence encoding a chimeric human / mouse CD8B.

[0132] The present invention also provides a method for establishing a humanized animal model of CD8A and CD8B genes, comprising the following steps:

[0133] (a) Providing cells (such as fertilized egg cells) based on the method described in the present application;

[0134] (b) Culturing the cells, preferably culturing the cells in a liquid medium;

[0135] (c) Transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, and allowing the cells to develop in the uterus of the female non-human mammal;

[0136] (d) Identify germline transmission in the offspring of the genetically modified humanized non-human mammal of the pregnant female identified in step (c).

[0137] In some embodiments, the non-human mammal in the above method is a mouse (such as a C57BL / 6 mouse).

[0138] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0139] In some embodiments, the fertilized egg used in the above method is a C57BL / 6 fertilized egg. Other fertilized eggs that can also be used in the method described in this application include but are not limited to FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0140] The fertilized egg can be from any non-human animal, such as any non-human animal described in this application. In some embodiments, the fertilized egg cells are derived from rodents. The gene construct can introduce DNA into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, and then the pseudopregnant non-human animal gives birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0141] In some embodiments, the method for preparing a genetically modified non-human animal includes modifying the coding frames of the CD8A and CD8B genes of the non-human animal. For example, by replacing the nucleic acid sequences (such as CDS, genomic DNA, or cDNA sequences) encoding the endogenous CD8A and CD8B regions with nucleotide sequences encoding the corresponding regions of human CD8A and CD8B under the control of the endogenous regulatory elements (such as promoters and / or UTRs, and the UTR is preferably 5'UTR and / or 3'UTR) of the CD8A and CD8B genes of the non-human animal. For example, one or more functional region sequences of the CD8A and CD8B genes of the non-human animal can be knocked out or inserted with sequences, so that the endogenous CD8A and CD8B proteins of the non-human animal cannot be expressed or the expression level is reduced.

[0142] In some embodiments, the method for preparing a genetically modified non-human animal includes inserting nucleotide sequences encoding human or humanized CD8A and CD8B proteins and / or auxiliary sequences after the endogenous regulatory elements of the CD8A and CD8B genes of the non-human animal. In some embodiments, the auxiliary sequence can be a stop codon, so that the humanized animal model of the CD8A and CD8B genes can express human or humanized CD8A and CD8B proteins in vivo, but does not express the CD8A and CD8B proteins of the non-human animal. In some embodiments, the auxiliary sequence includes WPRE (WHP post-transcriptional response element), loxP, STOP, and / or polyA.

[0143] In some embodiments, the construction method includes obtaining by using the above-mentioned vector. In some embodiments, the vector is a targeting vector and / or an sgRNA vector. In some embodiments, the construction method includes introducing the targeting vector into a non-human animal or its cells. In some embodiments, the construction method includes introducing the targeting vector and the sgRNA vector into a non-human animal or its cells.

[0144] In some embodiments, a method for preparing a genetically modified non-human animal includes:

[0145] (1) Providing a plasmid (preferably the above-mentioned vector) containing human CD8A and CD8B gene fragments, with the 5'-homologous arm and 3'-homologous arm flanking the plasmid, wherein the 5'-homologous arm and 3'-homologous arm target endogenous CD8A and CD8B;

[0146] (2) Providing one or more guide RNAs (sgRNAs) targeting endogenous CD8A and CD8B genes;

[0147] (3) Modifying the genome of a fertilized egg or embryonic stem cell by using the plasmid of step (1), the sgRNA of step (2), and Cas9;

[0148] (4) Transplanting the fertilized egg obtained in step (3) into the fallopian tube of a pseudopregnant female mouse, or transplanting the embryonic stem cell obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the fallopian tube of a pseudopregnant female mouse to produce offspring mice that functionally express humanized CD8A and CD8B proteins;

[0149] (5) Mating the offspring mice obtained in step (4) to obtain homozygous mice.

[0150] In some embodiments, the fertilized egg is modified by CRISPR with sgRNAs targeting the 5'-terminal targeting site and 3'-terminal targeting site.

[0151] In some embodiments, the sequences encoding humanized CD8A and CD8B proteins are operably linked to endogenous regulatory elements (such as promoters and / or UTRs, and the UTR is preferably 5'UTR and / or 3'UTR) at the endogenous CD8A and CD8B gene loci.

[0152] In some embodiments, the genetically modified non-human animal does not express endogenous CD8A and CD8B proteins.

[0153] In some embodiments, a method for preparing a genetically modified non-human animal includes:

[0154] (1) Provide a plasmid (such as the above vector) containing human or chimeric CD8A and CD8B gene fragments, the plasmid flanked by 5' homologous arms and 3' homologous arms, wherein the 5' homologous arms and 3' homologous arms target endogenous CD8A and CD8B;

[0155] (2) Provide one or more guide RNAs (sgRNAs) targeting endogenous CD8A and CD8B genes;

[0156] (3) Modify the genome of a fertilized egg or embryonic stem cell by inserting the human or chimeric CD8A and CD8B gene fragments into the genome.

[0157] In some embodiments, the nucleotide sequences encoding CD8A and / or CD8B proteins endogenously in the non-human animal are deleted. In some embodiments, all or part of exon 1 to part of exon 3 of the endogenous CD8A gene in the non-human animal are deleted. In some embodiments, part of exon 1 to part of exon 4 of the endogenous CD8B gene in the non-human animal are deleted. In some embodiments, part of exon 2 to part of exon 4 of the endogenous CD8B gene in the non-human animal are deleted.

[0158] Use of the gene-modified non-human animal

[0159] Replacing a non-human animal gene with a homologous or orthologous human gene or human sequence or inserting a homologous or orthologous human gene or human sequence into a non-human animal at the endogenous non-human animal locus and under the control of endogenous regulatory elements (such as promoters and / or UTRs, the UTRs preferably 5' UTR and / or 3' UTR) can generate non-human animals with qualities and characteristics that may be significantly different from typical knockout plus transgenic animals. In typical knockout plus transgenic animals, the endogenous gene locus is removed or disrupted, and a fully human transgene is inserted into the genome of the animal and may be randomly integrated into the genome. Generally, the location of the integrated transgene is unknown; the expression of human proteins is measured by transcription of human genes and / or protein assays and / or functional assays. In human transgenes, the upstream and / or downstream of the human sequence provides appropriate support for the expression and / or regulation of the transgene.

[0160] A gene-modified animal model expressing human or humanized CD8A and / or CD8B proteins, for example, in a physiologically appropriate manner, provides a variety of uses, including but not limited to developing treatment methods for human diseases and disorders, and evaluating the toxicity and / or efficacy of these treatment methods for human diseases and disorders in an animal model.

[0161] The present invention also provides the use of a non-human animal modified with the above CD8A and / or CD8B genes, and a non-human animal obtained by any of the above construction methods.

[0162] In some embodiments, the application comprises:

[0163] A) Application in the development of products related to immune processes associated with CD8A and / or CD8B involving human cells;

[0164] B) Application as a model system related to CD8A and / or CD8B in pharmacological, immunological, microbiological, and medical research;

[0165] C) Application involving the production and utilization of animal experimental disease models for etiological research related to CD8A and / or CD8B and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0166] D) Application in the screening, pharmacodynamic detection, evaluation of efficacy, verification, or assessment of human CD8A and / or CD8B signaling pathway modulators in vivo; or,

[0167] E) Application in studying the functions of CD8A and / or CD8B genes, studying the drugs and pharmacodynamics targeting human CD8A and / or CD8B target sites, and studying drugs for inflammatory and immune diseases related to CD8A and / or CD8B.

[0168] The present invention provides a non-human animal expressing human or humanized CD8A and / or CD8B proteins, which can be used for the screening of human CD8A and / or CD8B-specific therapeutic agents. The therapeutic agents can reduce or block the interaction between the CD8A and CD8A receptor complex or the CD8B and CD8B receptor complex, test whether the therapeutic agent can increase or decrease the immune response, and / or determine whether the therapeutic agent is an agonist or antagonist of CD8A and / or CD8B. In some embodiments, the non-human animal is a human disease animal model. For example, the disease is genetically induced (knock-in or knock-out). In different embodiments, the genetically modified non-human animal also comprises a damaged immune system, such as genetically modified human xenograft tissues, including human solid tumors (e.g., breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer, digestive tract cancer, gastrointestinal cancer, genital cancer, breast cancer, endocrine cancer, head and neck cancer, liver cancer, or lung cancer) or hematological tumors (e.g., lymphoma, preferably B or T cell tumors).

[0169] In some embodiments, the genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (therapeutic agents targeting CD8A and / or CD8B, such as anti-human CD8A antibodies, anti-human CD8B antibodies, anti-human CD8A and CD8B antibodies, or nucleic acid drugs, ADCs, PDCs, and / or polypeptide drugs targeting CD8A and / or CD8B) in treating various immune diseases. In some embodiments, the immune diseases include but are not limited to GVHD (graft-versus-host disease), psoriasis, allergy, asthma, atopic dermatitis, myocarditis, nephritis, hepatitis (preferably non-alcoholic steatohepatitis), systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorders, etc. In some embodiments, the immune diseases are psoriasis, AIDS, transplant rejection, asthma, atopic dermatitis, rheumatoid arthritis, or multiple sclerosis.

[0170] In some embodiments, the genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (therapeutic agents targeting CD8A and / or CD8B, such as anti-human CD8A antibodies, anti-human CD8B antibodies, anti-human CD8A and CD8B antibodies, or nucleic acid drugs, ADCs, PDCs, and / or polypeptide drugs targeting CD8A and / or CD8B) in treating cancer. In some embodiments, the method involves administering a therapeutic agent to a non-human animal as described in the present application, wherein the non-human animal has cancer or a tumor; and determining the inhibitory effect of the therapeutic agent on the cancer or tumor. The inhibitory effects that can be determined include, for example, a reduction in tumor size or tumor volume, a reduction in tumor growth, a decrease in the rate of increase in tumor volume in a subject (e.g., compared to the rate of increase in tumor volume in the same subject before treatment or in another subject not receiving such treatment), a reduction in the risk of metastasis or the risk of developing one or more additional metastases, an increase in survival rate, and an extension of life expectancy, etc. The tumor volume of the subject can be determined by various methods, such as by direct measurement, MRI, or CT. In some embodiments, the antibody can directly target the expression of CD8A and / or CD8B.

[0171] In some embodiments, the tumor contains one or more cancer cells (e.g., human or mouse cancer cells) injected into the animal. In some embodiments, the therapeutic agent activates the CD8A and / or CD8B signaling pathway. In some embodiments, the therapeutic agent does not activate the CD8A and / or CD8B signaling pathway.

[0172] In some embodiments, genetically modified non-human animals can be used to determine whether a therapeutic agent is an agonist or antagonist of CD8A and / or CD8B. In some embodiments, the methods described herein are also designed to determine the effect of a therapeutic agent (a therapeutic agent targeting CD8A and / or CD8B, such as, an anti-human CD8A antibody, an anti-human CD8B antibody, an anti-human CD8A and CD8B antibody, or a nucleic acid drug, ADC, PDC, and / or polypeptide drug targeting CD8A and / or CD8B) on CD8A and / or CD8B, for example, whether the therapeutic agent can upregulate or downregulate the immune response, and / or whether the therapeutic agent can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cell cytotoxicity (ADCC). In some embodiments, genetically modified non-human animals can be used to determine the effective dose of a therapeutic agent to treat a disease, such as cancer, in a subject.

[0173] The inhibitory effect on tumors can also be determined by methods known in the art, for example, measuring the tumor volume in an animal, and / or determining the tumor (volume) growth inhibition rate (TGI TV ). The tumor growth inhibition rate can be calculated using the formula TGI TV (%) = (1 – T Vt / T Vc ) × 100, where T Vt and T Vc are the average tumor volumes (or weights) of the treatment group and the control group.

[0174] In some embodiments, therapeutic agents (therapeutic agents targeting CD8A and / or CD8B, such as anti-human CD8A antibodies, anti-human CD8B antibodies, anti-human CD8A and CD8B antibodies, or nucleic acid drugs, ADCs, PDCs, and / or polypeptide drugs targeting CD8A and / or CD8B) are designed for the treatment of various cancers. As used herein, the term "cancer" refers to cells with the ability of autonomous growth, that is, an abnormal state or condition characterized by the growth of rapidly proliferating cells. This term is intended to include all types of cancerous growths or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or the stage of invasiveness. The term "tumor" as used in this application refers to cancer cells, such as a mass of cancer cells. Cancers that can be treated or diagnosed using the methods described in this application include malignancies of various organ systems, such as malignancies affecting the lung, breast, thyroid, lymph, gastrointestinal, and genitourinary tracts, as well as adenocarcinomas, which include malignancies such as most colon cancers, renal cell carcinomas, prostate cancers, and / or testicular tumors, non-small cell lung cancers, cancers, and cancers. In some embodiments, the therapeutic agents described in this application are designed for the treatment or diagnosis of cancer in a subject. The term "carcinoma" is well recognized and refers to malignancies of epithelial or endocrine tissues, including carcinomas of the respiratory system, gastrointestinal system, genitourinary system, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. In some embodiments, the cancer is renal cancer or melanoma. Exemplary carcinomas include carcinomas formed from cervical, lung, prostate, breast, head and neck, colon, and ovarian tissues. The term also includes carcinosarcomas, for example, malignant tumors composed of carcinoma tissue and sarcoma tissue. "Adenocarcinoma" refers to cancers that originate from glandular tissue or in which tumor cells form recognizable glandular structures. The term "sarcoma" is well recognized and refers to malignant tumors of mesenchymal origin.

[0175] In some embodiments, the cancers described in the present application are lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, cancer, bladder cancer, glioma, cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. In some embodiments, the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia. In some embodiments, the lymphoma is selected from Hodgkin lymphoma and non-Hodgkin lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, and T-cell lymphoma, as well as Waldenstrom macroglobulinemia. In some embodiments, the sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In a specific embodiment, the tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer, or cancer. In some embodiments, the cancer is a solid tumor or a hematological tumor. In some embodiments, the cancer is lymphoma, ovarian cancer, endometrial cancer, melanoma, kidney cancer, digestive tract cancer, gastrointestinal cancer, genital cancer, breast cancer, endocrine cancer, head and neck cancer, liver cancer, or lung cancer.

[0176] In some embodiments, the therapeutic agent (a therapeutic agent targeting CD8A and / or CD8B, such as an anti-human CD8A antibody, an anti-human CD8B antibody, an anti-human CD8A and CD8B antibody, or a nucleic acid drug, ADC, PDC, and / or polypeptide drug targeting CD8A and / or CD8B) is designed to treat various inflammations, such as viral inflammations. In some embodiments, the inflammations described in the present application include acute inflammation and chronic inflammation. Specifically, the inflammation includes, but is not limited to, degenerative inflammation, exudative inflammation (such as serous inflammation, fibrinous inflammation, suppurative inflammation, hemorrhagic inflammation, necrotic inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (such as tuberculosis, syphilis, leprosy, or lymphogranuloma). In some embodiments, the inflammations described in the present application include infections, and the infection refers to a local tissue and systemic inflammatory response caused by bacteria, viruses, fungi, parasites, and / or other pathogens invading the human body. In some embodiments, the inflammation is ankylosing spondylitis, Crohn's disease, hepatitis, or inflammatory bowel disease (IBD).

[0177] The present invention also provides a method for detecting the toxicity of a therapeutic agent (a therapeutic agent targeting CD8A and / or CD8B, such as an anti-human CD8A antibody, an anti-human CD8B antibody, an anti-human CD8A and CD8B antibody, or a nucleic acid drug, ADC, PDC, and / or polypeptide drug targeting CD8A and / or CD8B). The method includes administering the therapeutic agent to the aforementioned non-human animal and evaluating the weight change and / or blood test of the animal. In some embodiments, the blood test includes, but is not limited to, red blood cell count, hematocrit, and / or hemoglobin content. In some embodiments, the antibody can reduce red blood cells (RBC), hematocrit, or hemoglobin by 20%, 30%, 40%, or more than 50%. In some embodiments, the weight of the animal is at least 5%, 10%, 20%, 30%, or 40% less than that of the control group (such as the average weight of the animals not treated with the therapeutic agent).

[0178] The present invention also provides an animal model constructed by the method described in the present application for developing products related to the human cellular immune process, manufacturing human antibodies, or as a model system for pharmacological, immunological, microbiological, and medical research.

[0179] In some embodiments, there is provided an animal model generated by the method described in the present application for producing and utilizing an animal experimental disease model of the immune process of human cells, studying pathogens, or formulating new diagnostic strategies and / or treatment strategies.

[0180] The present invention also provides an animal model generated by the method described in the present application for screening, validating, evaluating, or studying the gene function of CD8A or CD8B, drugs targeting human CD8A or CD8B target sites (such as anti-human CD8A antibodies, anti-human CD8B antibodies, anti-human CD8A and CD8B antibodies, or nucleic acid drugs, ADCs, PDCs, and / or polypeptide drugs targeting CD8A and / or CD8B), or the effectiveness, drugs for treating immune diseases or inflammation, and anti-tumor drugs.

[0181] In some embodiments, the present application provides a method for verifying the in vivo efficacy of TCR-T, CAR-T, and / or other immunotherapies (e.g., adoptive T cell transfer therapy). For example, the method includes transplanting human tumor cells into the non-human animals described in the present application and applying human CAR-T to the animals with human tumor cells. The effectiveness of CAR-T treatment can be determined and evaluated. In some embodiments, the non-human animals are selected from CD8A and / or CD8B gene humanized non-human animals prepared by the method described in the present application, CD8A and / or CD8B gene humanized non-human animals described in the present application, dual or multiple gene humanized non-human animals (or their offspring) generated by the method described in the present application, non-human animals expressing human or humanized CD8A and / or CD8B proteins, or the tumor or inflammatory or immune disease animal models described in the present application. In some embodiments, TCR-T, CAR-T, and / or other immunotherapies can treat CD8A and / or CD8B-related diseases described in the present application. In some embodiments, TCR-T, CAR-T, and / or other immunotherapies provide an evaluation method for treating CD8A and / or CD8B-related diseases described in the present application.

[0182] Non-human animal models of two or more human or chimeric genes

[0183] The present invention also provides a method for generating a genetically modified animal model having two or more human or chimeric genes. The animal may contain the sequences of human or chimeric CD8A and / or CD8B genes and additional human or chimeric genes. In some embodiments, the non-human animal includes human or humanized CD8A and / or CD8B genes.

[0184] In some embodiments, the additional chimeric genes include, but are not limited to, at least one gene-modified non-human animal of TRBC1, TRBC2, HLA-A, B2M, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the above-mentioned non-human animals also express at least one of human or humanized TRBC1, TRBC2, HLA-A, B2M, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 proteins.

[0185] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric genes, and the construction method includes:

[0186] (1) Providing a non-human animal obtained by the above construction method;

[0187] (ii) Crossbreed, in vitro fertilize or directly perform gene editing on the non-human animal provided in step (i), and conduct screening to obtain a multi-gene modified non-human animal.

[0188] In some embodiments, the other gene-modified non-human animals include, but are not limited to, non-human animals with humanization of one or a combination of two or more of the genes TRBC1, TRBC2, HLA-A, B2M, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0189] In some embodiments, humanization is directly performed on a non-human animal having one or more gene modifications of human or chimeric TRBC1, TRBC2, HLA-A, B2M, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0190] Since these proteins may be involved in different mechanisms, combination therapies targeting two or more of these proteins may be a more effective treatment method. In fact, many related clinical trials are underway and showing good results. The multi-gene modified non-human animal model can be used to determine the effectiveness of combination therapies targeting two or more proteins. For example, a therapeutic agent targeting CD8A and / or CD8B, and an additional therapeutic agent for treating cancer or immune diseases or inflammation. The method includes administering to the non-human animal a therapeutic agent targeting CD8A and / or CD8B and an additional therapeutic agent, where the non-human animal has a tumor or immune disease or inflammation, and determining the effect of the combination treatment on the inflammation or tumor or immune disease. In some embodiments, the additional therapeutic agent is an antibody specifically binding to TRBC1, TRBC2, HLA-A, B2M, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), or an anti-PD-L1 antibody. In some embodiments, the above-mentioned non-human animal further includes a sequence encoding human or humanized PD-1, a sequence encoding human or humanized PD-L1, or a sequence encoding human or humanized CTLA-4. In some embodiments, the above-mentioned tumor includes one or more tumor cells expressing PD-L1 and / or PD-L2.

[0191] In some embodiments, the combination therapy is used to treat various cancers described in the present application, such as breast cancer, lymphoma, digestive tract cancer, gastrointestinal cancer, genital cancer, endocrine cancer, head and neck cancer, liver cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer or lung cancer. In some embodiments, the combination therapy is designed to treat immune diseases described in the present application, such as psoriasis, AIDS, atopic dermatitis, transplant rejection, asthma, rheumatoid arthritis or multiple sclerosis.

[0192] In some embodiments, the methods described in the present application can be used to evaluate combination therapy with some other methods. Methods for treating cancer that can be used alone or in combination with the methods described in the present application include, for example, treating a subject with chemotherapy, such as camptothecin, doxorubicin, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, adriamycin, ifosfamide, melphalan, chlorambucil, bleomycin, nitrosourea, dacarbazine, daunorubicin, bleomycin, plicamycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, transplatin, 5-fluorouracil, vincristine, vinblastine and / or methotrexate. Alternatively, in addition, the method can include performing surgery on the subject to remove at least a portion of the cancer, such as removing a part or all of the tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0193] Figure 1 : Comparison diagram of mouse CD8A gene and human CD8A gene (not to scale);

[0194] Figure 2 : Comparison diagram of mouse CD8B and human CD8B genes (not to scale);

[0195] Figure 3 : Schematic diagram of humanized modification of mouse CD8A and CD8B genes (not to scale);

[0196] Figure 4 : Schematic diagram of CD8A and CD8B gene targeting strategies and targeting vector design (not to scale);

[0197] Figure 5 : PCR identification results of F1 generation of CD8A and CD8B double-gene humanized mice, where WT is the wild-type control and H2O is the water control;

[0198] Figure 6 : ELISPOT results, where + / + is wild-type C57BL / 6 mice, H / H is CD8A and CD8B double-gene humanized homozygous mice, NC1 and NC2 are negative control groups, G1-G8 are treatment groups, and PC1 and PC2 are positive control groups. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0199] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention.

[0200] Materials and Methods

[0201] In each of the following embodiments, the equipment and materials were obtained from the several companies indicated below:

[0202] C57BL / 6 mice were purchased from the National Rodent Laboratory Animal Seed Center, China National Institute for Food and Drug Control;

[0203] Purified anti-mouse CD16 / 32 Antibody was purchased from Biolegend, catalog number: 101302;

[0204] Zombie NIR TM Fixable Viability Kit was purchased from Biolegend, catalog number: 423106;

[0205] Brilliant Violet 510 TM anti-mouse CD45 Antibody was purchased from Biolegend, catalog number: 103138;

[0206] Brilliant Violet 421 TM anti-mouse CD3 Antibody was purchased from Biolegend, catalog number: 100228;

[0207] Brilliant Violet 785 TM anti-mouse CD4 Antibody was purchased from Biolegend, catalog number: 100453;

[0208] PE anti-mouse CD8a Antibody was purchased from Biolegend, catalog number: 100708;

[0209] Alexa 700 anti-human CD8a Antibody was purchased from Biolegend, catalog number: 300920;

[0210] BV605 Mouse Anti-Human CD8b was purchased from BD, with the product number: 742392.

[0211] Example 1 CD8A and CD8B Bigenic Humanized Mice

[0212] In this example, non-human animals (such as mice) were modified so that the non-human animals contained nucleotide sequences encoding human CD8A protein and human CD8B protein, and the genetically modified non-human animals could express human or humanized CD8A protein and human or humanized CD8B protein.

[0213] Mouse CD8A gene (Gene ID: 12525, located at positions 71350411 to 71356155 of chromosome 6 NC_000072.7, based on transcript NM_001081110.2 and its encoded protein NP_001074579.1 (SEQ ID NO: 1)) and human CD8A gene (Gene ID: 925, located at positions 86784605 to 86808396 of chromosome 2 NC_000002.12, based on transcript NM_001145873.1 and its encoded protein NP_001139345.1 (SEQ ID NO: 2)), the comparison diagram of mouse CD8A gene and human CD8A gene is as Figure 1 shown.

[0214] Mouse CD8B gene (Gene ID: 12526, located at positions 71299772 to 71314476 of chromosome 6 NC_000072.7, based on transcript NM_009858.3 and its encoded protein NP_033988.1 (SEQ ID NO: 8)) and human CD8B gene (Gene ID: 926, located at positions 86815369 to 86861886 of chromosome 2 NC_000002.12, based on transcript NM_004931.5 and its encoded protein NP_004922.1 (SEQ ID NO: 9)), the comparison diagram of mouse CD8B and human CD8B gene is as Figure 2 shown.

[0215] To achieve the object of the present invention, nucleotide sequences encoding human CD8A and CD8B proteins can be introduced into the endogenous CD8A and CD8B gene loci of mice, such that the mice express human or humanized CD8A and human or humanized CD8B proteins. Specifically, all or part of exon 1 to part of exon 3 of the mouse is replaced with a sequence comprising part of exon 4 to part of exon 7 of the human CD8A gene; part of exon 1 to part of exon 4 of the mouse is replaced with a sequence comprising part of exon 1 to part of exon 4 of the human CD8B gene, thereby realizing the humanization of the CD8A and CD8B gene loci of the mouse. The schematic diagram of the humanized CD8A and CD8B gene loci is as shown in Figure 3 shown below.

[0216] To achieve the object of the present invention, a targeting strategy schematic diagram as shown in Figure 4 is constructed. The targeting vector contains an upstream homologous arm sequence of the mouse CD8B gene and a downstream homologous arm sequence of the CD8A gene, as well as fragment A containing nucleotide sequences encoding human CD8A and CD8B proteins. Among them, the upstream homologous arm sequence (5'-homologous arm, SEQ ID NO: 3) is the same as the nucleotide sequence from position 71294752 to 71299840 of NCBI accession number NC_000072.7, and the downstream homologous arm sequence (3'-homologous arm, SEQ ID NO: 4) is the same as the nucleotide sequence from position 71351754 to 71357294 of NCBI accession number NC_000072.7. Fragment A, from 5'-3', is as follows: fragment 1 containing part of human CD8B (SEQ ID NO: 10, the sequence after removing the Neo cassette, the same as the nucleotide sequence from position 86846760 to 86861865 of NCBI accession number NC_000002.12), fragment 2 containing part of mouse CD8B and part of mouse CD8A (the nucleotide sequence from position 71309434 to 71350536 of NCBI accession number NC_000072.7), and fragment 3 containing part of human CD8A (SEQ ID NO: 5, the same as the nucleotide sequence from position 86789423 to 86790825 of NCBI accession number NC_000002.12). Among them, the connection design between fragment 1 and fragment 2 is (SEQ ID NO: 20), where the "C" in the sequence is the last nucleotide of fragment 1, and the "C" in the sequence " CTTA " is the first nucleotide of fragment 2; the connection design between fragment 2 and fragment 3 is (SEQ ID NO: 21), where the sequence The last "C" in ATGG is the last nucleotide of fragment 2, and the "A" in the sequence (SEQ ID NO: 22), where the "G" in the sequence is the last nucleotide at the 3'-end of the mouse sequence connected to the upstream of fragment A, and the "A" in the sequence ATGC is the first nucleotide of fragment A; the connection of fragment A to the downstream with the mouse is designed as (SEQ ID NO: 23), where the last "G" in the sequence is the last nucleotide of fragment A, and the first "G" in the sequence GGAT is the first nucleotide at the 5'-end of the mouse sequence connected to the downstream of fragment A.

[0221] The targeting vector also includes a resistance gene for positive clone screening, namely the hygromycin coding sequence HygR, and two site-specific recombination systems Frt3 recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a HygR cassette. Among them, the connection of the upstream of the HygR cassette to human CD8B is designed as (SEQ ID NO: 13), where the last "T" in the sequence TCGAT is the last nucleotide at the connection of human CD8B to the upstream of the HygR cassette, and the "G" in the sequence is the first nucleotide of the HygR cassette; the connection of the downstream of the HygR cassette to human CD8B is designed as (SEQ ID NO: 14), where the last "C" in the sequence is the last nucleotide of the HygR cassette, and the "A" in the sequence ATCCT is the first nucleotide at the connection of human CD8B to the downstream of the HygR cassette. In addition, a coding gene with a negative selection marker (the coding gene of diphtheria toxin A subunit (DTA)) is constructed downstream of the 3'-homologous arm of the targeting vector. The mRNA sequence transcribed from the modified humanized mouse CD8A gene is as shown in SEQ ID NO: 6, and the protein sequence expressed is as shown in SEQ ID NO: 7. The mRNA sequence transcribed from the humanized mouse CD8B gene is as shown in SEQ ID NO: 11, and the protein sequence expressed is as shown in SEQ ID NO: 12.

[0224] The construction of the targeting vector can be carried out by conventional methods, such as enzymatic digestion and ligation. After the constructed targeting vector is preliminarily verified by enzymatic digestion, it is then sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is transfected into embryonic stem cells of C57BL / 6 mice by electroporation, and the obtained cells are screened using a positive clone selection marker gene to screen out the correct positive clone cells. The correctly screened positive clone cells (black mice) are introduced into the isolated blastocysts (white mice) according to the techniques known in the art. The obtained chimeric blastocysts are transferred to a culture medium for short-term culture and then transplanted into the oviduct of a recipient female mouse (white mouse) to produce F0 generation chimeric mice (black and white). The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then the F1 generation heterozygous mice are interbred to obtain F2 generation homozygous mice. The positive mice can also be mated with Flp tool mice to remove the positive clone selection marker gene, and then homozygous mice with humanized CD8 genes can be obtained by interbreeding.

[0225] The somatic cell genotypes of F1 generation mice can be identified by PCR. The PCR primers are shown in Table 5. Among them, CD8-L-GT-F and CD8-L-GT-R are used to detect the mRNA expression of the humanized CD8B fragment, and CD8-R-GT-F and CD8-R-GT-R are used to detect the mRNA expression of the humanized CD8A fragment. Exemplary results are as Figure 5 shown, Figure 5 and the 3 mice numbered F1-01, F1-02, and F1-03 in it are all positive mice.

[0226] Table 5 PCR Detection Primer Sequences and Recombinant Fragment Sizes

[0227]

[0228] The expression of humanized CD8A and humanized CD8B proteins in mice is confirmed by flow cytometry. Specifically, the peripheral blood and spleen tissues of 5-week-old male wild-type C57BL / 6 mice (+ / +) and 5-week-old male homozygous mice with humanized CD8A and CD8B genes (H / H) are selected, and the leukocyte labeling antibody Brilliant Violet 510 TM anti-mouse CD45Antibody(mCD45), Purified anti-mouse CD16 / 32Antibody, BrilliantViolet421 TM anti-mouse CD3 Antibody, Brilliant Violet 785 TManti-mouse CD4 Antibody, PE anti-mouse CD8a Antibody, Alexa 700 anti-human CD8a Antibody, BV605 Mouse Anti-Human CD8b and Zombie NIR TM After identification and staining with Fixable Viability Kit, flow cytometry was performed.

[0229] The detection results are shown in Table 6. The expression of murine CD8A protein was detected only in the spleen, peripheral blood and lymph nodes of wild-type C57BL / 6 mice using specific anti-mouse CD8A antibody, and the expression of humanized CD8A and humanized CD8B proteins was detected only in the spleen, peripheral blood and lymph nodes of CD8A and CD8B double-gene humanized homozygous mice using specific anti-human antibodies, indicating that the humanized CD8A and humanized CD8B proteins can be normally expressed in the CD8A and CD8B double-gene humanized homozygous mice constructed by the method of this application.

[0230] Table 6 Flow cytometry detection results

[0231]

[0232] The expression and function of humanized CD8A and humanized CD8B proteins in CD8A and CD8B double-gene humanized mice can also be verified by ELISPOT.

[0233] OVA peptide 257–264 is a restricted epitope peptide of ovalbumin (OVA) presented by MHC class I molecule H-2Kb. OVA peptide 257–264 The amino acid sequence is shown in SEQ ID NO: 19.

[0234] For example, 2 randomly selected female wild-type C57BL / 6 mice (8-9 weeks old, + / +) and 3 female CD8A and CD8B double-gene humanized homozygous mice (8-9 weeks old, H / H) were used. On day 0 and day 7, all mice were immunized by intraperitoneal injection of 0.5 mg OVA (Simga, A5503-25MG) and 50 μg poly(I:C) (InvivoGen, tlrl-pic). On day 14, all mice were euthanized, spleen tissues were collected, and splenocytes were obtained for enzyme-linked immunospot assay (ELISPOT). Specifically, spleen tissues of wild-type C57BL / 6 mice (+ / +) and CD8A and CD8B double-gene humanized homozygous mice (H / H) were ground with a 40-μm cell strainer to obtain splenocytes, which were diluted to different concentrations and placed in 96-well plates. According to Table 7, they were divided into a negative control group (NC1 and NC2), a treatment group (G1, G2, G3, G4, G5, G6, G7, and G8), and a positive control group (PC1 and PC2), and stimulants were added. Specifically, mixtures of OVA peptides 257–264 and poly(I:C) at different concentrations were added to the treatment group, an equal volume of medium was added to the negative control group, and an equal volume of Cell Activation Cocktail (Biolegend, 42330) was added to the positive control group. After the splenocytes and stimulants were co-incubated at 37 °C and 5% CO2 for 24 h, IFN-γ was detected with an enzyme-linked immunosorbent assay (ELISA) reader, and the number of spot-forming units (one spot-forming unit represents one active IFN-γ-secreting CD8+ T cell) was counted. The results are shown as Figure 6 .

[0235] Table 7 Grouping of splenocytes for ELISPOT

[0236]

[0237]

[0238] When CD8A and CD8B proteins function normally, after stimulation with OVA peptides 257–264 , MHC class I molecule H-2Kb will present the antigen to CD8+ T cells, activate the intracellular signaling pathway, and secrete IFN-γ. As shown in Figure 6 , compared with wild-type C57BL / 6 mice (+ / +), there was no significant difference in the number of spot-forming units in CD8A and CD8B double-gene humanized homozygous mice (H / H), indicating that CD8A and CD8B double-gene humanized homozygous mice (H / H) have normal T cell immunogenicity similar to that of wild-type C57BL / 6 mice (+ / +). This shows that in the CD8A and CD8B double-gene humanized mice prepared by this method, the humanized CD8A and CD8B proteins are normally expressed and function normally.

[0239] Example 2 Pharmacodynamic Model

[0240] The CD8A and CD8B dual-gene humanized mice prepared by this method can be used to evaluate the in vivo safety and in vivo pharmacodynamics of regulators targeting human CD8A and / or CD8B in tumor diseases.

[0241] For example, take homozygous CD8A and CD8B dual-gene humanized mice and subcutaneously inoculate them with MC38 cells. Wait until the tumor volume grows to about 100 mm 3 Then, according to the tumor volume, divide them into a control group or a treatment group. In the treatment group, randomly select drugs targeting human CD8A and / or human CD8B, and inject an equal volume of physiological saline in the control group. Regularly measure the tumor volume and weigh the mice. The in vivo safety and in vivo pharmacodynamics of the compound can be effectively evaluated by comparing the changes in mouse body weight and tumor size.

[0242] Example 3 Preparation of Multi-Gene Humanized Mice

[0243] The CD8A and CD8B dual-gene humanized mice prepared by this method can also be used to prepare a multi-gene humanized mouse model. For example, in Example 1 above, the embryonic stem cells used for microinjection can be selected from mice containing at least one gene modification of TRBC1, TRBC2, HLA-A, B2M, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. Alternatively, based on the CD8A and CD8B dual-gene humanized mice, a multi-gene humanized mouse model can be obtained by separating mouse ES embryonic stem cells and gene recombination targeting technology. The homozygous or heterozygous CD8A and CD8B mice obtained by this method can also be mated with other gene-modified mice, and their offspring can be screened. According to Mendelian inheritance, there is a certain probability of obtaining multi-gene humanized mice with humanized CD8A and CD8B dual genes and other gene modifications. Then, mating the heterozygotes with each other can obtain homozygotes with multi-gene modifications.

[0244] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0245] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any suitable way. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0246] In addition, any combination can be made among various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A method for constructing a genetically modified non-human animal, characterized in that: The genome of the non-human animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric CD8 molecule (CD8) protein.

2. The construction method according to claim 1, characterized in that: The chromosome comprises a nucleotide sequence encoding a human or chimeric CD8A molecule (CD8A) protein; Preferably, the chimeric CD8A protein comprises a human or humanized extracellular region, a non-human animal endogenous transmembrane region, and an endogenous cytoplasmic region; Further preferably, the amino acid sequence of the human or chimeric CD8A protein comprises SEQ ID NO: 2, SEQ ID NO: 2 positions 1-175, SEQ ID NO: 2 positions 1-182, or SEQ ID NO: 2 positions 22-182, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 2, SEQ ID NO: 2 positions 1-175, SEQ ID NO: 2 positions 1-182, or SEQ ID NO: 2 positions 22-182; More preferably, the amino acid sequence of the chimeric CD8A protein comprises SEQ ID NO: 7, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

7.

3. The construction method according to claim 1 or 2, characterized in that: The chromosome comprises a nucleotide sequence encoding a human or chimeric CD8B molecule (CD8B) protein; Preferably, the chimeric CD8B protein comprises a human or humanized extracellular region, a non-human animal endogenous transmembrane region, and an endogenous cytoplasmic region; Further preferably, the amino acid sequence of the human or chimeric CD8B protein comprises SEQ ID NO: 9, SEQ ID NO: 9 positions 1-169, SEQ ID NO: 9 positions 1-170 or SEQ ID NO: 9 positions 22-170; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 9, SEQ ID NO: 9 positions 1-169, SEQ ID NO: 9 positions 1-170 or SEQ ID NO: 9 positions 22-170; More preferably, the amino acid sequence of the chimeric CD8B protein comprises SEQ ID NO: 12, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

12.

4. A method for constructing a genetically modified non-human animal, characterized in that: The genome of the non-human animal comprises a nucleotide sequence at the endogenous CD8 locus, in which the nucleotide sequence of the corresponding region of the endogenous CD8 is replaced with the nucleotide sequence of human or chimeric CD8.

5. The construction method according to any one of claims 1 to 4, characterized in that: The nucleotide sequence encoding the human or chimeric CD8 protein or the nucleotide sequence of the human or chimeric CD8 is operably linked to an endogenous regulatory element of an endogenous CD8 locus; Preferably, the endogenous CD8 protein of the non-human animal is not expressed or is expressed at a reduced level compared to CD8 in wild-type animals; Preferably, the modified CD8 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

6. The construction method according to claim 4 or 5, characterized in that: The nucleotide sequence of human or chimeric CD8 comprises the nucleotide sequence of human or chimeric CD8A, and the nucleotide sequence of human or chimeric CD8A includes all or part of the extracellular region encoding human CD8A protein, preferably includes the nucleotide sequence encoding human or chimeric CD8A protein; Preferably, the nucleotide sequence of human or chimeric CD8A comprises the nucleotide sequence encoding SEQ ID NO: 2, SEQ ID NO: 2 positions 1-175, SEQ ID NO: 2 positions 1-182, or SEQ ID NO: 2 positions 22-182; Preferably, the nucleotide sequence of the human or chimeric CD8A comprises a portion of exon 4 to a portion of exon 7 of human CD8A, or comprises a portion of exon 5 to a portion of exon 7 of human CD8A; Further preferably, the nucleotide sequence of the human or chimeric CD8A comprises SEQ ID NO: 5, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO:

5.

7. The construction method according to any one of claims 4 to 6, characterized in that: The nucleotide sequence of the chimeric CD8 comprises the nucleotide sequence of endogenous CD8A of a non-human animal, preferably includes a nucleotide sequence encoding the cytoplasmic region and / or transmembrane region of CD8A of a non-human animal, preferably includes part of exon 3 to all of exon 5 of CD8A of a non-human animal, and preferably also includes part of exon 1.

8. The construction method according to any one of claims 4 to 7, characterized in that: The nucleotide sequence of the corresponding region of endogenous CD8 includes the nucleotide sequence of the corresponding region of endogenous CD8A; the nucleotide sequence of the corresponding region of endogenous CD8A contains all or part of the sequence encoding the extracellular region; preferably contains the nucleotide sequence encoding SEQ ID NO: 1, SEQ ID NO: 1 positions 1-187, SEQ ID NO: 1 positions 1-196 or SEQ ID NO: 1 positions 28-196; further preferably contains all or part of exon 1 to exon 3 of non-human animal CD8A.

9. The construction method according to any one of claims 4 to 8, characterized in that: The nucleotide sequence of human or chimeric CD8 comprises the nucleotide sequence of human or chimeric CD8B, and the nucleotide sequence of human or chimeric CD8B comprises all or part of the nucleotide sequence encoding the extracellular region of human CD8B protein, preferably comprises the nucleotide sequence encoding human or chimeric CD8B protein; Preferably, the nucleotide sequence of the human or chimeric CD8B comprises the nucleotide sequence encoding SEQ ID NO: 9, SEQ ID NO: 9 positions 1-169, SEQ ID NO: 9 positions 1-170, or SEQ ID NO: 9 positions 22-170; Preferably, the nucleotide sequence of the human or chimeric CD8B comprises a portion of exon 1 to a portion of exon 4 of human CD8B, or comprises a portion of exon 2 to a portion of exon 4 of human CD8B; Further preferably, the nucleotide sequence of the human or chimeric CD8B comprises SEQ ID NO: 10, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the nucleotide sequence shown in SEQ ID NO:

10.

10. The construction method according to any one of claims 4 to 9, characterized in that: The nucleotide sequence of the chimeric CD8 comprises the nucleotide sequence of endogenous CD8B of a non-human animal, preferably including a nucleotide sequence encoding the cytoplasmic region and / or transmembrane region of CD8B of a non-human animal, preferably including part of exon 4 to all of exon 6 of CD8B of a non-human animal, and preferably also including part of exon 1.

11. The construction method according to any one of claims 4 to 10, characterized in that: The nucleotide sequence of the corresponding region of endogenous CD8 includes the nucleotide sequence of the corresponding region of endogenous CD8B; the nucleotide sequence of the corresponding region of endogenous CD8B contains all or part of the sequence encoding the extracellular region; preferably contains the nucleotide sequence encoding SEQ ID NO: 8, SEQ ID NO: 8 positions 1-172, SEQ ID NO: 8 positions 1-175 or SEQ ID NO: 8 positions 22-175; further preferably contains part of exon 1 to part of exon 4 of non-human animal CD8B, or contains part of exon 2 to part of exon 4 of non-human animal CD8B.

12. The construction method according to any one of claims 1 to 11, characterized in that: The construction method comprises replacing the corresponding endogenous regions of non-human animals with nucleotide sequences encoding human or chimeric CD8A protein and human or chimeric CD8B protein; Preferably, the construction method comprises replacing the corresponding endogenous regions of non-human animals with a nucleotide sequence encoding, from N to C, the human CD8B signal peptide, all or part of the human CD8B extracellular region, the non-human animal endogenous CD8B transmembrane region, the non-human animal endogenous CD8B cytoplasmic region, the human CD8A signal peptide and all or part of the human CD8A extracellular region; Further preferably, the construction method comprises replacing the corresponding endogenous regions of the non-human animal with the nucleotide sequence comprising human CD8A and the donor sequence of human CD8B; Preferably, the donor sequence includes, from 5' to 3', a portion of human CD8B exon 1 to a portion of exon 4, a portion of non-human animal endogenous CD8B exon 4 to a portion of CD8A exon 1, and a portion of human CD8A exon 4 to a portion of exon 7; Further preferably, the portion of human CD8B exon 1 to exon 4 comprises SEQ ID NO: 10, or comprises a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 10 ... 99% or 99.5% of the nucleotide sequence; Further preferably, the portion of exon 4 of endogenous CD8B of a non-human animal to the portion of exon 1 of CD8A includes the nucleotide sequence from position 71309434 to position 71350536 with NCBI accession number NC_000072.7; Further preferably, the nucleotide sequence of part of human CD8A exon 4 to part of exon 7 includes SEQ ID NO:5, or comprising a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% of the nucleotide sequence.

13. The construction method according to claim 12, characterized in that: The non-human animal endogenous corresponding region includes the non-human animal endogenous nucleotide sequence encoding CD8A protein and CD8B protein; Preferably, the non-human animal endogenous corresponding region includes the non-human animal endogenous coding CD8A protein extracellular region and The nucleotide sequence of the extracellular region of the CD8B protein; Further preferably, the non-human animal endogenous corresponding region includes the nucleotide sequence of the signal peptide and extracellular region encoding the CD8B protein and CD8A endogenously in the non-human animal; Preferably, the non-human animal endogenous CD8B exon 1 portion to CD8A exon 3 portion.

14. The construction method according to any one of claims 1 to 13, characterized in that: The non-human animal is a mammal, For example, monkeys or rodents, preferably, the non-human animal is a mouse or a rat; Preferably, the mRNA transcribed from the modified CD8A gene in the genome of the non-human animal comprises SEQ ID NO: 6, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 6; Preferably, the mRNA transcribed from the modified CD8B gene in the genome of the non-human animal comprises SEQ ID NO: 11, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO:

11.

15. The construction method according to any one of claims 1 to 14, characterized in that: The non-human animal also includes nucleotide sequences of human or chimeric proteins encoded by other genes, wherein the human or chimeric proteins are selected from TRBC1, TRBC2, HLA-A, B2M, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or At least one of CTLA4.

16. A use of a non-human animal obtained by the construction method according to any one of claims 1 to 15, characterized in that: The application includes: A) Application in the development of products involving CD8A and / or CD8B-related immune processes in human cells; B) Application as a model system related to CD8A and / or CD8B in pharmacology, immunology, microbiology and medical research; C) Applications involving the production and use of animal experimental disease models for the study of etiology associated with CD8A and / or CD8B and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human CD8A and / or CD8B signaling pathway modulators; or, E) Study the function of CD8A and / or CD8B genes, study the drugs and drug efficacy targeting human CD8A and / or CD8B target sites, and study the application of CD8A and / or CD8B related cancer, immune disease or inflammation drugs.

17. A method for determining the effectiveness or toxicity of a therapeutic agent in treating a disease, characterized in that: The method comprises: 1) administering a therapeutic agent to a non-human animal obtained by the construction method according to any one of claims 1 to 15; 2) Determining the effect of therapeutic agents on disease or non-human animals; Preferably, the therapeutic agent is a therapeutic agent targeting CD8A and / or CD8B, such as an anti-CD8A antibody, an anti-CD8B antibody, an anti-CD8A and CD8B antibody, or a nucleic acid drug, ADC, PDC and / or polypeptide drug targeting CD8A and / or CD8B; Further preferably, the therapeutic agent further comprises an additional therapeutic agent, such as an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody; Preferably, the disease comprises cancer; Further preferably, the cancer is a solid tumor or a blood tumor, such as lymphoma, ovarian cancer, endometrial cancer, melanoma, renal cancer, digestive tract cancer, gastrointestinal cancer, genital cancer, breast cancer, endocrine cancer, head and neck cancer, liver cancer or lung cancer.

18. A humanized CD8A protein, characterized in that: The humanized CD8A protein comprises a human or humanized extracellular region, a non-human animal endogenous transmembrane region and an endogenous cytoplasmic region; Preferably, the amino acid sequence of the humanized CD8A protein comprises SEQ ID NO: 2, SEQ ID NO: 2 positions 1-175, SEQ ID NO: 2 positions 1-182 or SEQ ID NO: 2 positions 22-182, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 2, SEQ ID NO: 2 positions 1-175, SEQ ID NO: 2 positions 1-182 or SEQ ID NO: 2 positions 22-182; Further preferably, the amino acid sequence of the humanized CD8A protein comprises SEQ ID NO: 7, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

7.

19. A humanized CD8B protein, characterized in that: The humanized CD8B protein comprises a human or humanized extracellular region, a non-human animal endogenous transmembrane region and an endogenous cytoplasmic region; preferably, the amino acid sequence of the humanized CD8B protein comprises SEQ ID NO: 9, SEQ ID NO: 9 positions 1-169, SEQ ID NO: 9 positions 1-170 or SEQ ID NO: 9 positions 22-170; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 9, SEQ ID NO: 9 positions 1-169, SEQ ID NO: 9 positions 1-170 or SEQ ID NO: 9 positions 22-170; Further preferably, the amino acid sequence of the humanized CD8B protein comprises SEQ ID NO: 12, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

12.

20. A humanized CD8A gene, characterized in that: The humanized CD8A gene encodes the humanized CD8A protein according to claim 18; preferably, the humanized CD8A gene comprises SEQ ID NO: 4, 5, 6, 21 or 23, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO: 4, 5, 6, 21 or 23.

21. A humanized CD8B gene, characterized in that: The humanized CD8B gene encodes the humanized CD8B protein according to claim 19; Preferably, the humanized CD8B gene comprises SEQ ID NO: 3, 10, 11, 13, 14, 20 or 22, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO: 3, 10, 11, 13, 14, 20 or 22.

22. A cell, tissue or organ, characterized in that: The cells, tissues or organs contain the humanized CD8A protein of claim 18 or the humanized CD8B protein of claim 19 or the humanized CD8A gene of claim 20 or the humanized CD8B gene of claim 21.

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