Non-human animals expressing humanized CD3 complex

Patent metadata

Field	Value
Publication number	US-12419282-B2
Application number	US-202418442444-A
Country	US
Kind code	B2
Filing date	Feb 15, 2024
Priority date	Nov 24, 2014
Publication date	Sep 23, 2025
Grant date	Sep 23, 2025

Abstract

Non-human animals, expressing humanized CD3 proteins are provided. Non-human animals, e.g., rodents, genetically modified to comprise in their genome humanized CD3 proteins are also provided. Additionally, provided are methods and compositions of making such non-human animals, as well as methods of using said non-human animals.

First claim

What is claimed is:

1. An isolated rodent cell comprising:
at an endogenous CD3ε locus, a nucleic acid sequence encoding a functional chimeric human/rodent CD3ε protein that comprises an extracellular domain of a human CD3ε protein,
at an endogenous CD3δ locus, a nucleic acid sequence encoding a functional chimeric human/rodent CD3δ protein that comprises an extracellular domain of a human CD3δ protein, and
at an endogenous CD3γ locus, a nucleic acid sequence encoding a functional chimeric human/rodent CD3γ protein that comprises an extracellular domain of a human CD3γ protein.

2. The isolated rodent cell of claim 1 , wherein the isolated rodent cell is an isolated rodent embryonic stem cell.

3. The isolated rodent cell of claim 1 , wherein the isolated rodent cell is an isolated rodent T cell.

4. The isolated rodent cell of claim 3 , wherein the isolated rodent T cell comprises, on its surface, a functional chimeric human/rodent CD3 complex that (a) comprises the functional chimeric human/rodent CD3ε protein, the functional chimeric human/rodent CD3δ protein, and the functional chimeric human/rodent CD3γ protein and (b) is complexed with an endogenous T-cell receptor.

5. The isolated rodent cell of claim 1 , wherein the isolated rodent cell is heterozygous for the modified endogenous CD3 loci.

6. The isolated rodent cell of claim 1 , wherein the isolated rodent cell is homozygous for the modified endogenous CD3 loci.

7. The isolated rodent cell of claim 1 , wherein the isolated rodent cell comprises a replacement of
(i) a genomic nucleic acid sequence encoding the endogenous CD3ε protein with the nucleic acid sequence encoding the functional chimeric human/rodent CD3ε protein,
(ii) a genomic nucleic acid sequence encoding the endogenous CD3δ protein with the nucleic acid sequence encoding the functional chimeric human/rodent CD3δ protein, and
(iii) a genomic nucleic acid sequence encoding the endogenous CD3γ protein with the nucleic acid sequence encoding the functional chimeric human/rodent CD3γ protein.

8. The isolated rodent cell of claim 1 , wherein the functional chimeric human/rodent CD3ε protein comprises the sequence of SEQ ID NO:33, the functional chimeric human/rodent CD3δ protein comprises the sequence of SEQ ID NO:34, and the functional chimeric human/rodent CD3γ protein comprises the sequence of SEQ ID NO:35.

9. The isolated rodent cell of claim 1 , wherein the isolated rodent cell is an isolated rat cell.

10. The isolated rodent cell of claim 1 , wherein the isolated rodent cell is an isolated mouse cell.

11. The isolated rodent cell of claim 10 , wherein the functional chimeric human/mouse CD3ε protein comprises the sequence of SEQ ID NO:24, the functional chimeric human/mouse CD3δ protein comprises the sequence of SEQ ID NO:25, and the functional chimeric human/mouse CD3γ protein comprises the sequence of SEQ ID NO:26.

12. The isolated rodent cell of claim 1 , wherein the isolated rodent cell further comprises a nucleic acid comprising a nucleic acid sequence encoding an antigen of interest.

13. The isolated rodent cell of claim 12 , wherein the antigen of interest is:
(a) a tumor associated antigen,
(b) an infectious disease antigen, or
(c) a bacterial antigen.

14. The isolated rodent cell of claim 13 , wherein:
(a) the antigen of interest is a tumor associated antigen selected from a group consisting of ALK, BAGE proteins, BIRC5 (survivin), BIRC7, CA9, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD27, CD30, CD33, CD38, CD40, CD44, CD52, CD56, CD79, CDK4, CEACAM3, CEACAM5, CLEC12A, EGFR, EGFR variant III, ERBB2 (HER2), ERBB3, ERBB4, EPCAM, EPHA2, EPHA3, FCRL5, FLT3, FOLR1, GAGE proteins, GD2, GD3, GPNMB, GM3, GPR112, IL3RA, KIT, KRAS, LGR5, EBV-derived LMP2, L1CAM, MAGE proteins, MLANA, MSLN, MUC1, MUC2, MUC3, MUC4, MUC5, MUC16, MUM1, ANKRD30A, NY-ESO1 (CTAG1B), OX40, PAP, PAX3, PAX5, PLAC1, PRLR, PMEL, PRAME, PSMA (FOLH1), RAGE proteins, RET, RGS5, ROR1, SART1, SART3, SLAMF7, SLC39A6 (LIV1), STEAP1, STEAP2, TERT, TMPRSS2, Thompson-nouvelle antigen, TNFRSF17, TYR, UPK3A, VTCN1, and WT1;
(b) the antigen of interest is an infectious disease antigen, and the infectious disease antigen is:
(i) a viral antigen;
(ii) a viral antigen selected from a group consisting of HIV, hepatitis A, hepatitis B, hepatitis C, herpes virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, ebola virus, and arboviral encephalitis virus antigen; and/or
(iii) a herpes virus antigen, wherein the herpes virus is selected from the group consisting of HSV-1, HSV-2, CMV, HAV-6, VZV, and Epstein Barr virus; or

(c) the antigen of interest is a bacterial antigen selected from a group consisting of chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumococci, meningococci, gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospira, and Lyme disease bacterial antigen.

15. A method of making an isolated rodent cell, the method comprising modifying the genome of an isolated rodent cell to comprise:
at an endogenous CD3ε locus, a nucleic acid sequence encoding a functional chimeric human/rodent CD3ε protein that comprises an extracellular domain of a human CD3ε protein,
at an endogenous CD3δ locus, a nucleic acid sequence encoding a functional chimeric human/rodent CD3δ protein that comprises an extracellular domain of a human CD3δ protein,
at an endogenous CD3γ locus, a nucleic acid sequence encoding a functional chimeric human/rodent CD3γ protein that comprises an extracellular domain of a human CD3γ protein, and
a nucleic acid comprising a nucleic acid sequence encoding an antigen of interest.

16. A method of making a genetically modified rodent, the method comprising propagating the genetically modified rodent from the isolated rodent cell of claim 2 .

17. A genetically modified rodent made according to the method of claim 16 .

18. A method of screening an antigen-binding protein for T-cell activating activity, wherein the antigen-binding protein binds human CD3 and an antigen of interest, the method comprising:
(a) administering the antigen-binding protein to the genetically modified rodent of claim 17 , and
(b) determining if the antigen-binding protein is efficacious in activating T cells of the genetically modified rodent.

19. The method of claim 18 , wherein the T cells of the genetically modified rodent comprise, on their surface, a functional chimeric human/rodent CD3 complex that (a) comprises the functional chimeric human/rodent CD3ε protein, the functional chimeric human/rodent CD3δ protein, and the functional chimeric human/rodent CD3γ protein and (b) is complexed with an endogenous T-cell receptor.

20. The method of claim 18 , wherein:
(a) the antigen-binding protein is an antibody;
(b) the antigen-binding protein is a bispecific antibody; and/or
(c) the antigen-binding protein is capable of recognizing monkey CD3 protein.

Assignees

• Regeneron Pharma

Inventors

• Olson Kara L
• Smith Eric
• Lai Ka-Man Venus
• Murphy Andrew J
• Thurston Gavin
• Guo Dayong

Classifications

• A01K2267/03

  Animal model, e.g. for test or diseases · CPC title

• A01K2267/01

  Animal expressing industrially exogenous proteins · CPC title

• A01K2217/075

  inducing loss of function, i.e. knock out · CPC title

• A01K2207/15

  Humanized animals · CPC title

• A01K2207/12

  Animals modified by administration of exogenous cells · CPC title