Suppression of endogenous immunoglobulin expression

• US 20060117398A1
• Filed: 10/21/2005
• Published: 06/01/2006
• Est. Priority Date: 10/22/2004
• Status: Abandoned Application

• Current Assignee: Therapeutic Human Polyclonals, Inc., (Roche Holding AG)
• Original Assignee: Therapeutic Human Polyclonals, Inc., (Roche Holding AG)
• Inventors: Buelow, Roland, Platzer, Josef
• Application Number:
  US11/255,820
• Publication Number:
  US 20060117398A1
• Time in Patent Office: Days
• US Class Current: 800/14
• 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 2227/105 Murine
• CPC Class Codes: A01K 2227/107 Rabbit
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C07K 14/005 from viruses
• 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 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2770/32022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00

Abstract

The invention provides a novel approach for the suppression of endogenous antibody expression in non-human transgenic animals genetically engineered to express one or several human or humanized immunoglobulin transloci. Endogenous immunoglobulin expression in transgenic non-human animals is suppressed by selective expression of a suicide gene like a toxin only in B-cells expressing endogenous immunoglobulin but not in B-cells expressing human(ized) immunoglobulins. This method allows the dominant expression of transloci coding for humanized or human antibodies in the blood, milk and eggs of transgenic animals.

Claims

1. A method for selective suppression of endogenous immunoglobulin production in B-cells of a non-human transgenic animal carrying an exogenous immunoglobulin translocus, comprising selectively expressing at least one suicide gene in B-cells producing an endogenous immunoglobulin of said non-human transgenic animal, but not in B cells producing an exogenous immunoglobulin, whereby B cells producing the endogenous immunoglobulin are depleted, and production of the endogenous immunoglobulin is suppressed, without suppressing the production of said exogenous immunoglobulin.

2. The method of claim 1 wherein said exogenous immunoglobulin is a human(ized) immunoglobulin heavy and/or light chain sequence.

3. The method of claim 2 wherein said suicide gene, introduced into the B-cells of said non-human transgenic animal, is under the control of a B-cell specific promoter and is flanked by recombination sequences.

4. The method of claim 3 wherein said human(ized) immunoglobulin chain translocus is introduced into the B-cells of said non-human transgenic animal as part of an expression construct, additionally encoding a recombinase recognizing said recombination sequences, wherein expression of said suicide gene is inactivated through expression of said recombinase in B-cells expressing said human(ized) immunoglobulin translocus.

5. The method of claim 1 wherein said suicide gene is selected from the group consisting of bacterial, fungal, insecticidal and plant toxins.

6. The method of claim 1 wherein said suicide gene is a diphtheria toxin chain A.

7. The method of claim 1 wherein said suicide gene is a prodrug converting enzyme.

8. The method of claim 7 wherein the prodrug converting enzyme is of non-mammalian origin.

9. The method of claim 8 wherein said prodrug converting enzyme of non-mammalian origin is selected from the group consisting of viral thymidine kinase (TK), bacterial cytosine deaminase (CD), bacterial carboxypeptidase G2 (CPG2), purine nucleotide phosphorylase (PNP), thymidine phosphorylase (TP), nitroreductase (NR), D-amino acid oxidase (DAAO), xanthine-guanine phosphoribosyl transferease (XGPRT), penicillin-G amidase (PGA), β

-lactamase, multiple drug activation enzyme (MDAE), β

-galactosidase (β

-Gal), horseradish peroxidase (HRP) and deoxyribonucleotide kinase (DRNK).

10. The method of claim 7 wherein the prodrug converting enzyme is of human origin.

11. The method of claim 10 wherein the prodrug converting enzyme of human origin is selected from the group consisting of deoxycytidine kinase (dCK), carboxyesterases (CEs), carboxypeptidase A (CPA), β

-glucuronidase (-Glu), and cytochrome P450 (CYP).

12. The method of claim 4 wherein said recombinase is selected from the group consisting of a Cre, Cre-like, Flp, φ

C31, λ

integrase, phage R4 recombinase, TP901-1 recombinase, a prokaryotic transposase, a eukaryotic transposase, a viral retrotransposase, a Drosophila copia-like retrotransposase and a non-viral retrotransposase.

13. The method of claim 12 wherein said transposase or retrotransposase is selected from the group consisting of Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn9, Tn10, Tn30, Tn101, Tn501, Tn903, Tn1000, Tn1681, Tn2901, Drosophila mariner, sleeping beauty transposase, Drosophila P element, maize Ac, Ds, Mp, Spm, En, dotted, Mu, I, L1, Tol2 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1) and Minos.

14. The method of claim 1 wherein said non-human transgenic animal substantially stops antibody diversification by gene rearrangement early in life.

15. The method of claim 14 wherein said non-human transgenic animal substantially stops antibody diversification within the first month of its life.

16. The method of claim 1 wherein said non-human transgenic animal is selected from the group consisting of rodents, rabbits, birds, cows, pigs, sheep, goats and horses.

17. The method of claim 16 wherein said rodent is a mouse or a rat.

18. A transgenic expression construct comprising a first transgene further comprising a human or humanized immunoglobulin heavy and/or light chain translocus, a self-cleaving peptide and a recombinase.

21. The transgenic expression construct of claim 18, 19 or 20 wherein said recombinase is a site specific recombinase selected from the group consisting of a Cre, Cre-like, Flp, φ

C31, λ

integrase, phage R4 and TP901-1 recombinase.

22. The transgenic expression construct of claim 18, 19 or 20 wherein said recombinase is either a prokaryotic or a eukaryotic transposase.

23. The transgenic expression construct of claim 22 wherein said recombinase is either a viral, Drosophila copia-like or non-viral retrotransposon.

24. The transgenic expression construct of claim 23 wherein said retrotransposon is selected from the group consisting of Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn9, Tn10, Tn30, Tn101, Tn501, Tn903, Tn1000, Tn1681, Tn2901, Drosophila mariner, sleeping beauty transposase, Drosophila P element, maize Ac, Ds, Mp, Spm, En, dotted, Mu, I, L1, Tol2 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1) and Minos.

27. The transgenic expression construct of claims 18 or 20 wherein said self-cleaving peptide is obtained from viral 2A/2B or 2A-like/2B sequences.

28. The transgenic expression construct of claim 27 wherein said virus is selected from the group consisting of the picornaviridae virus family, the equine rhinitis A (ERAV) virus family, the picornavirus-like insect virus family or from the type C rotavirus family.

29. The transgenic expression construct of claim 27 wherein said virus is selected from the group consisting of the foot and mouth disease virus (FMDV), the equine rhinitis A (ERAV) virus, or the Thosea asigna virus (TaV).

32. A non-human transgenic animal expressing the transgenic expression constructs of claims 18, 19 or 20.

33. The non-human transgenic animal of claim 32 that generates antibody diversity substantially by gene conversion.

34. The non-human transgenic animal of claim 32 that is selected from the group consisting of rodents, rabbits, birds including chickens, turkeys, ducks and geese.

35. The non-human transgenic animal of claim 34 that is either a mouse or a rat.

19. A transgenic expression construct comprising a second transgene further comprising a suicide gene that is under the control of a B-cell specific promoter, and is flanked by recombination sites recognized by a recombinase.

25. The transgenic expression construct of claims 19 or 20 wherein said recombination sites are selected from a group consisting of a lox P site, FRT site, a bacterial genomic recombination site and a phage recombination site.

26. The transgenic expression construct of claim 25 wherein said bacterial genomic recombination site is attB and said phage recombination site is an attP or a pseudo-attP or a pseudo-attB site.

30. The transgenic expression construct of claims 19 or 20 wherein said suicide gene is specifically expressed in B-cells using a promoter/enhancer selected from the group consisting of CD19, CD20, CD21, CD22, CD23, CD24, CD40, CD72, Blimp-1, CD79b, mb-1, tyrosine kinase blk, VpreB, immunoglobulin kappa light chain, immunoglobulin lambda-light chain and immunoglobulin J-chain or modifications thereof.

31. The transgenic expression construct of claim 30 wherein said B-cell specific promoter/enhancer is the kappa light chain gene promoter or modifications thereof.

20. A transgenic expression construct comprising:

a first transgene further comprising a human or humanized immunoglobulin heavy and/or light chain locus, a self-cleaving peptide and a recombinase, and,

a second transgene further comprising a suicide gene that is under the control of a B-cell specific promoter, and is flanked by recombination sites recognized by said recombinase.