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Non-human animals having humanized cluster of differentiation 47 gene

A gene, alpha gene technology, applied in the field of non-human animals with humanized differentiation cluster 47 gene

Active Publication Date: 2017-09-26
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This grand challenge is partly due to the ability of cancer cells to evade the surveillance mechanisms of the innate and adaptive immune systems, partly as a result of inhibition of phagocytic clearance

Method used

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  • Non-human animals having humanized cluster of differentiation 47 gene
  • Non-human animals having humanized cluster of differentiation 47 gene
  • Non-human animals having humanized cluster of differentiation 47 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0214] Example 1. Humanization of the Endogenous Cluster of Differentiation 47 (CD47) Gene

[0215] This example illustrates exemplary methods for humanizing an endogenous gene encoding cluster of differentiation 47 (CD47) in a non-human mammal, such as a rodent (eg, mouse). The methods described in this example can be used to humanize the endogenous CD47 gene of a non-human animal using any human sequence or combination of human sequences (or sequence fragments) as described. In this example, the human CD47 gene present in bacterial artificial chromosome (BAC) clone RP11-69A17 was used to humanize the endogenous CD47 gene in mice.

[0216] use technology to construct a targeting vector for humanizing the genetic material encoding the extracellular N-terminal IgV domain and five transmembrane domains of the endogenous CD47 gene (see, e.g., U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expr...

Embodiment 2

[0223] Example 2. Expression of humanized CD47 polypeptide by mouse erythrocytes.

[0224] This example demonstrates that non-human animals (e.g., rodents) modified to contain a humanized CD47 gene according to Example 1 can be used to screen for CD47 modulators (e.g., anti-CD47 antibodies) and to determine various properties such as drug Kinetic and safety properties. In this example, multiple anti-CD47 antibodies were screened on mouse red blood cells (RBC) isolated from rodents prepared according to Example 1 expressing a humanized CD47 polypeptide as described herein.

[0225] Briefly, 2 mL of whole blood (n=2) from humanized CD47 mice was transferred to a 15 mL tube and centrifuged at 200 x g for 10 min at 4°C. Aspirate the plasma and buffy coat, then add 15 mL of PBS and mix the cells gently. Centrifuge the mixture again at 200 x g for five minutes at 4 °C. Aspirate the supernatant and wash the cells two more times. Resuspend the pelleted RBCs in a final volume of 10...

Embodiment 3

[0231] Example 3. Hemagglutination of mouse erythrocytes expressing humanized CD47 polypeptide.

[0232] This example also demonstrates that non-human animals (e.g., rodents) modified to contain a humanized CD47 gene according to Example 1 can be used in various assays (e.g., hemagglutination assays) to screen for modulators of CD47 ( For example, anti-CD47 antibody) and determine various properties such as pharmacokinetic and safety profiles. In this example, several anti-CD47 antibodies were screened on mouse red blood cells (RBC) expressing a humanized CD47 polypeptide as described herein to determine the concentration of the antibody that promotes hemagglutination.

[0233] Briefly, RBCs (n=2) from wild-type and humanized CD47 mice were prepared as described in Example 2. Twenty (20) μL of anti-CD47 antibody (5-fold serial dilution) was added to wells 1-12 of a 96-well V-bottom plate, followed by 80 μL of 0.5% mouse RBCs to all wells of the plate. Tap the plate to mix an...

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Abstract

Non-human animals, and methods and compositions for making and using the same, are provided, wherein said non-human animals comprise a humanization of an endogenous cluster of differentiation (CD) gene, in particular a humanization of a CD47 gene. Said non-human animals may be described, in some embodiments, as having a genetic modification to an endogenous CD47 gene so that said non-human animals express a CD47 polypeptide that includes a human portion and a non-human portion (e.g., a murine portion).

Description

[0001] Cross references to related patent applications [0002] This application claims priority to US Patent Application Serial No. 62 / 087,992, filed December 5, 2014, which is hereby incorporated by reference in its entirety. [0003] The sequence listing is incorporated by reference [0004] The sequence listing is an ASCII text file named 32584_10108WO01_SequenceListing.txt, 96.0 KB in size, created on November 23, 2015, submitted to the US Patent and Trademark Office via EFS-Web, and incorporated herein by reference. Background technique [0005] Cancer treatments can be divided into four main categories: chemotherapy / radiation, hormone therapy, targeted therapy, and immunotherapy. Medical research and development has focused on targeted therapies and has seen major improvements, but cancer remains a major challenge for patients and the global healthcare industry. This grand challenge is partly due to the ability of cancer cells to evade the surveillance mechanisms of t...

Claims

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Application Information

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IPC IPC(8): A01K67/027C12N5/10C07K14/705A61K49/00
CPCA01K67/0278A61K49/0008C07K14/70503A01K2207/15A01K2217/05A01K2227/105A01K2267/03C07K14/70596G01N33/5088A01K2217/072A01K2217/15A01K2267/0381G01N33/5011G01N33/5014G01N33/5055G01N2333/70596C07K16/2803C07K14/4703C07K2319/00C07K2319/02A01K2267/0331A61P35/00A01K2207/12A01K2217/052C07K14/705C12N15/8509
Inventor C·居雷尔E·约费A·穆希卡G·瑟斯顿
Owner REGENERON PHARM INC
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