Non-human animals having a humanized programmed cell death 1 gene
A gene and cell technology, applied in the field of non-human animals with humanized programmed cell death 1 gene, can solve problems such as downregulation
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Embodiment 1
[0213] Example 1 Humanization of Endogenous Programmed Cell Death 1 (Pdcd1) Gene
[0214] This example illustrates an exemplary method for humanizing the endogenous Pdcd1 gene encoding programmed cell death protein 1 (PD-1 ) in a non-human mammal, such as a rodent (eg, mouse). The methods described in this example can be used to humanize the endogenous Pdcd1 gene of a non-human animal using any human sequence or combination of human sequences (or sequence fragments) as described. In this example, ~883 bp of exon 3 comprising exon 2, intron 2 and the first 71 bp of the human PDCD1 gene presented in GenBank accession number: NM_005018.2 (SEQ ID NO: 23) was used Human DNA fragments were used to humanize the endogenous Pdcd1 gene. The targeting vector used to humanize the genetic material encoding the extracellular N-terminal IgV region of the endogenous Pdcd1 gene was used technology (see, eg, US Patent No. 6,586,251 and Valenzuela et al., 2003, Nature Biotech. 21(6):652-659; i...
Embodiment 2
[0221] Example 2 Expression of humanized PD-1 on activated T cells
[0222] This example demonstrates that a non-human animal (eg, a rodent) modified to contain a humanized Pdcd1 gene according to Example 1 expresses a humanized PD-1 protein on the surface of activated lymphocytes. In this example, activated T cells from mice heterozygous for the humanization of the endogenous Pdcd1 gene as described in Example 1 were stained with an anti-PD-1 antibody to determine the role of PD-1 in segregating Expression in stimulated T cells from wild-type and humanized mice.
[0223] Briefly, spleens were harvested from wild-type mice and mice heterozygous humanized for the endogenous Pdcd1 gene as described in Example 1, and processed into single cell suspensions by mechanical dissociation. Cells were washed in culture medium (RPMI, supplemented with 10% FBS) and washed at 1×10 6 / mL resuspension, and plate 200 μL (200,000 cells) in a 96-well plate. Cells in selected cells were stimul...
Embodiment 3
[0225] Example 3 In vivo efficacy of PD-1 regulators
[0226] This example demonstrates that non-human animals (e.g., rodents) modified to contain a humanized Pdcd1 gene according to Example 1 can be used in in vivo assays to screen for PD-1 modulators (e.g., anti-PD-1 antibodies) and Various properties are determined, for example, inhibition of tumor growth and / or killing of tumor cells. In this example, several anti-PD-1 antibodies were screened in humanized heterozygous mice against the endogenous Pdcd1 gene as described in Example 1 to determine the optimal antibody dose and Extent of Anti-PD-1 Antibody Mediated Tumor Cell Killing.
[0227] Briefly, mice were evenly divided according to body weight into five treatment or control groups for Study 1 (n=5 / group), and eight treatment or control groups for Study 2 (n=5 / group) and five treatment groups or control groups for study 3 (n=7 / group). On day zero, mice were anesthetized by isoflurane inhalation, and then a suspensio...
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