Murine expression of a human IgA lambda locus

• US 6,998,514 B2
• Filed: 12/13/2000
• Issued: 02/14/2006
• Est. Priority Date: 11/03/1998
• Status: Expired due to Term

• Current Assignee: The Babraham Institute
• Original Assignee: The Babraham Institute
• Inventors: Bruggemann, Marianne
• Primary Examiner(s): WEHBE, ANNE MARIE SABRINA
• Application Number:
  US09/734,613
• Publication Number:
  US 20020088016A1
• Time in Patent Office: 1,889 Days
• Field of Search: 800/18, 800/21, 800/22, 435/325, 435/320.1
• US Class Current: 800/18
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 2267/0381 Animal model for diseases of the hematopoietic system
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic

Abstract

In humans, approximately 60% of expressed immunoglobulin light chains are of the Kappa type and 40% of the Lambda type. In mice, there is almost no expression from the Lambda locus and over 95% of light chains are of Kappa type. The present invention discloses, among other things, transgenic mice carrying most of the human Ig Lambda light chain locus in their genome. The resulting mice express light chains with Kappa/Lambda ratio similar to the human ratio. Breeding of HuIg Lamda mice to Kappa-deficient mice also is described, as well as the generation of human monoclonal antibodies from transgenic mice with human Ig Lambda locus.

Claims

1. A transgenic mouse comprising in its genome a yeast artificial chromosome (YAC), wherein the YAC contains at least a majority of the human Ig Vλ

genes of cluster A and all the human Ig Jλ

-Cλ

segments in germline configuration, wherein the proportion of the κ and

λ

light chains expressed by said transgenic mouse resembles that found in humans, and exhibits relative proportions of <

60% κ

light chains and >

40% λ

light chains, wherein at least one endogenous κ

light chain locus of the transgenic mouse is not disrupted.

2. The transgenic mouse according to claim 1, wherein the YAC is about 410 Kb, and wherein the YAC containing human Ig segments shows high expression and is able to compete with the endogenous mouse κ

locus.

3. The transgenic mouse according to claim 1, wherein one of the endogenous Igκ

loci of the mouse is disrupted, and wherein the YAC containing human Ig segments shows high expression.

4. The transgenic mouse according to claim 1, comprising a 380 Kb region of the human immunoglobulin (Ig) λ

light (L) chain locus in germline configuration wherein the 380 Kb region resides on a yeast artificial chromosome (YAC) that accommodates the most proximal V (variable gene) λ

cluster, wherein the 380 Kb regions has 15 V λ

genes and all J λ

-C λ

segments with the 3′

region, wherein the 3′

region includes a downstream enhancer.

5. The transgenic mouse according to claim 1, wherein the mouse includes a HuIgλ

YAC that accommodates a 380 Kb region of the human λ

light chain locus in authentic configuration with all Vλ

genes of cluster A, the Jλ

-Cλ

segments and the 3′

enhancer.

6. The transgenic mouse according to claim 5, wherein the HuIgλ

YAC is shown in FIG. 1.

7. A method for producing a transgenic mouse according to claim 1, comprising:

(a) introducing a HuIgλ

YAC into murine embryonic stem cells, wherein the HuIgλ

YAC accommodates a 380 Kb region of the human λ

light chain locus in germline configuration with all Vλ

genes of cluster A, the Jλ

-Cλ

segments, and a downstream enhancer at the 3′

region; and

(b) deriving a transgenic mouse from the cells of step (a) by blastocyte injection to form a chimeric animal and then breeding the chimeric mouse to obtain a transgenic mouse.

8. The method of claim 7, wherein the HuIgλ

YAC is about 410 Kb.

9. The method according to claim 7, wherein two copies of the neomycin resistance gene (NEO^r) are site-specifically integrated into the ampicillin gene on the left (centromeric) YAC arm in order to permit selection.

10. The method according to claim 7, wherein YAC-containing yeast cells are fused with HM-1 embryonic stem (ES) cells and G418 resistance colonies are picked and analysed 2-3 weeks after protoplast fusion.

11. The method according to claim 7, wherein ES cells containing a complete HuIgλ

YAC copy are used for blastocyte injection to produce a chimeric animal.

12. The method according to claim 11, wherein breeding of a chimeric animal with a Balb/c mouse results in germline transmission.

13. The method according to claim 12, wherein the germline transmission establishes lines of transgenic mice, wherein at least one endogenous κ

light chain locus of the transgenic mouse is not disrupted.

14. A transgenic mouse comprising in its genome a yeast artificial chromosome (YAC), wherein the YAC contains at least a majority of the human Ig Vλ

genes of cluster A and all the human Ig Jλ

-Cλ

segments in germline configuration, and expressing human λ

light chain locus genes and endogenous κ

light chain locus genes, wherein the expression of the human λ

locus is equal to or greater than that of the endogenous κ

light chain locus, and wherein at least one endogenous κ

light chain locus of the transgenic mouse is not disrupted.

15. The transgenic mouse according to claim 14, wherein the mouse further comprises a human κ

light chain locus and wherein expression of the human λ

light chain locus is equal to or greater than that of the human κ

light chain locus.

16. The transgenic mouse according to claim 14, wherein the λ

locus has been bred to homozygosity.

17. The transgenic mouse according to claim 14, wherein the rearranged variable genes in the λ

locus are subject to somatic hypermutation.

18. The transgenic mouse according to claim 14, wherein the mouse comprises a yeast artificial chromosome (YAC) of greater than 100 Kb which contains at least a majority of the human Vλ

genes proximal to the Jλ

-Cλ

cluster in germline configuration.

19. The transgenic mouse according to claim 18, wherein the YAC includes a 380 Kb region of the human Igλ

locus in authentic configuration with at least a majority of the Vλ

genes of cluster A, Jλ

-Cλ

segments and a 3′

enhancer.

20. The transgenic mouse according to claim 18, wherein the transgenic mouse comprises variable, joining and constant genes of the human λ

light chain locus as a transgenic locus on a YAC, wherein B cells of said mouse rearranges said λ

light chain genes and the mouse expresses serum immunoglobulins containing human λ

light chains.

21. The transgenic mouse according to claim 18, wherein the λ

locus is rearranged with similar efficiency as endogenous mouse κ and

at the same time as or before the endogenous κ

locus.

22. The transgenic mouse according to claim 18, wherein one of the the endogenous κ

loci is silenced, and the mouse expresses serum immunoglobulins containing human λ

light chains.

23. The transgenic mouse according to claim 18, further comprising human heavy chain genes as a second transgenic locus integrated on a separate YAC, wherein the mouse expresses serum immunoglobulin molecules containing combinations of human heavy and λ

light chains.

24. The transgenic mouse according to claim 23, wherein the second transgenic locus carries a diversity of human heavy chain constant region genes and includes μ

, δ and

y genes.

25. The transgenic mouse according to claim 24, wherein the heavy chain transgenic locus carries a diversity of human heavy chain constant region genes and includes μ

, δ and

y genes, wherein the heavy chain constant regions genes are in authentic germline configuration.

26. The transgenic mouse according to claim 18, further comprising human κ

light chain genes as a second transgenic light chain locus integrated on a separate YAC, wherein the mouse expresses serum immunoglobulin molecules containing human κ and

λ

light chains.

27. The transgenic mouse according to claim 18, further comprising human heavy chain genes as a second transgenic locus and human κ

light chain genes as a third transgenic locus, wherein the mouse expresses serum immunoglobulin molecules containing human heavy chains in combination with at least one of human κ

or λ

light chains.

28. The transgenic mouse according to claim 18, wherein expression of the endogenous mouse heavy and/or light chain loci are prevented and the transgenic mouse expresses serum immunoglobulin containing human heavy and/or light chains, wherein at least one endogenous κ

light chain locus of the transgenic mouse is not disrupted, and wherein the transgenic mouse is deficient in production of mouse immunoglobulin.