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Expression of xenogenous (human) immunoglobulins in cloned, transgenic ungulates

a technology of human immunoglobulin and ungulate, which is applied in the field of genetically modified ungulate, can solve the problems of insufficient human antibody availability for therapeutic and prophylactic use, demand exceeding supply, and routine shortfalls of human antibody availability

Inactive Publication Date: 2006-06-01
KYOWA HAKKO KIRIN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This means that there is an inherent limitation in the amount of human antibody available for therapeutic and prophylactic usage.
Already, the demand exceeds the supply and severe shortfalls in availability have been routine.
While human Ig has been expressed in mice, it is unpredictable whether human Ig will be fractionally rearranged and expressed in bovines, or other ungulates, because of differences in antibody gene structure, antibody production mechanism, and B cell function.
In addition, it would also have been unpredictable whether a bovine would be able to survive, i.e., elicit its normal immune functions, in the absence of its endogenous Ig or with interference from human antibodies.
For example, it is not certain if bovine B cells expressing human Ig would correctly migrate to the illeal Peyer's Patch in bovines because this does not happen in humans.

Method used

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  • Expression of xenogenous (human) immunoglobulins in cloned, transgenic ungulates
  • Expression of xenogenous (human) immunoglobulins in cloned, transgenic ungulates
  • Expression of xenogenous (human) immunoglobulins in cloned, transgenic ungulates

Examples

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example 1

Bovine IgM Knock Out

[0150] The following procedures were used to generate bovine fibroblast cell lines in which one allele of the immunoglobulin heavy chain (mu) locus is disrupted by homologous recombination. A DNA construct for effecting an IgM knockout was generated by the removal of exons 1-4 of the Mu locus (corresponds to IgM heavy chain gene) which were replaced with a copy of a neomycin resistance gene. Using this construct, neomycin resistant cell lines have been obtained which were successfully used in nuclear transfer procedures, and blastocysts from these cell lines have been implanted into recipient cows. Additionally, some of these blastocysts were tested to confirm that targeted insertion occurred appropriately in the mu locus using PCR procedures. Blastocysts resulting from nuclear transfer procedures from several of the cell lines obtained indicated that heterozygous IgM-KO fetuses were in gestation. Additionally, both male and female cell lines that comprise a sin...

example 2

Introduction of HAC

[0176] Additional experiments were carried out to demonstrate that immunoglobulin heavy chain (mu) and lambda light chain may be produced by a bovine host, either alone or in combination. In addition, these experiments demonstrated that the immunoglobulin chains were rearranged and that polyclonal sera was obtained. In these procedures, immunoglobulin-expressing genes were introduced into bovine fibroblasts using human artificial chromosomes. The fibroblasts were then utilized for nuclear transfer, and fetuses were obtained and analyzed for antibody production. These procedures and results are described in more detail below.

[0177] HAC Constructs The human artificial chromosomes (HACs) were constructed using a previously described chromosome-cloning system (Kuroiwa et al., Nature Biotech. 18: 1086-1090, 2000). Briefly, for the construction of ΔHAC, the previously reported human chromosome 22 fragment (hChr22) containing a loxP sequence integrated at the HCF2 locu...

example 3

Transgenic Ungulates Producing Xenogenous Antibodies and Reduced Amounts of Endogenous Antibodies

[0230] Transgenic ungulates expressing a xenogenous antibody and having a reduced level of expression of endogenous antibodies may also be generated. By increasing the number of functional xenogenous immunoglobulin heavy or light chain genes relative to the number of functional endogenous heavy or light chain genes, the percentage of B cells expressing xenogenous antibodies should increase.

[0231] To generate these transgenic ungulates, ΔHAC or ΔΔHAC transgenic ungulates may be mated with transgenic ungulates containing a mutation in one or both alleles of an endogenous immunoglobulin chain (e.g., a mu heavy chain or a lambda or kappa light chain). If desired, the resulting transgenic ungulates may be mated with (i) transgenic ungulates containing a mutation in one or both alleles of an endogenous alpha-(1,3)-galactosyltransferase, prion, and / or J chain nucleic acid or (ii) transgenic u...

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Abstract

The present invention relates to the production of a transgenic bovine which comprises a genetic modification that results in inactivation and loss of expression of its endogenous antibodies, and the expression of xenogenous antibodies, preferably human antibodies. This is effected by inactivation of the IgM heavy chain expression and, optionally, by inactivation of the Ig light chain expression, and by the further introduction of an artificial chromosome which results in the expression of non-bovine antibodies, preferably human antibodies.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional of U.S. application Ser. No. 09 / 988,115, filed Nov. 16, 2001, which claims the benefit of the filing date of U.S. provisional patent application 60 / 311,625, filed Aug. 9, 2001 and U.S. provisional patent application 60 / 256,458, filed Dec. 20, 2000, and is a continuation-in-part of U.S. utility application Ser. No. 09 / 714,185, filed Nov. 17, 2000, which claims the benefit of the filing date of U.S. provisional patent application 60 / 166,410, filed Nov. 19, 1999.FIELD OF THE INVENTION [0002] The present invention is a genetically modified ungulate that contains either part or all of a xenogenous antibody gene locus, which undergoes rearrangement and expresses a diverse population of antibody molecules. In particular, the xenogenous antibody gene may be of human origin. In addition, the present invention provides for an ungulate in which expression of the endogenous antibody genes is either reduced or elimin...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027C12N5/06C12N9/10C12N15/85
CPCA01K67/0273A01K67/0276A01K67/0278A01K2207/15A01K2217/00A01K2217/05A01K2217/075A01K2227/101A01K2267/01C12N9/1051C12N15/8509C12N2800/30
Inventor ROBL, JAMESGOLDSBY, RICHARDFERGUSON, STACYKUROIWA, YOSHIMITOMIZUKA, KAZUMAISHIDA, ISAOOSBORNE, BARBARA
Owner KYOWA HAKKO KIRIN CO LTD
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