Production of humanized antibodies in transgenic animals

• US 7,129,084 B2
• Filed: 08/03/2001
• Issued: 10/31/2006
• Est. Priority Date: 08/03/2000
• Status: Expired due to Term

• Current Assignee: Therapeutic Human Polyclonals, Inc., (Roche Holding AG)
• Original Assignee: Therapeutic Human Polyclonals, Inc., (Roche Holding AG)
• Inventors: Platzer, Josef, Buelow, Roland, van Schooten, Wim, Buelow, Jens-Ulrich
• Primary Examiner(s): WEHBE, ANNE MARIE SABRINA
• Application Number:
  US09/921,819
• Publication Number:
  US 20030017534A1
• Time in Patent Office: 1,915 Days
• Field of Search: 435/320.1, 435/455, 435/325
• US Class Current: 435/320.1
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2217/20 Animal model comprising regulated expression system
• CPC Class Codes: A01K 2227/107 Rabbit
• CPC Class Codes: A01K 2227/30 Bird
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0273 Cloned vertebrates
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• 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 16/082 Hepadnaviridae, e.g. hepatitis B virus
• CPC Class Codes: C07K 16/1271 from Micrococcaceae (F), e.g. Staphylococcus
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/734 Complement-dependent cytotoxicity [CDC]
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Abstract

This invention relates to humanized antibodies and antibody preparations produced from transgenic non-human animals. The non-human animals are genetically engineered to contain one or more humanized immunoglobulin loci which are capable of undergoing gene rearrangement and gene conversion in the transgenic non-human animals to produce diversified humanized immunoglobulins. The present invention further relates to novel sequences, recombination vectors and transgenic vectors useful for making these transgenic animals. The humanized antibodies of the present invention have minimal immunogenicity to humans and are appropriate for use in the therapeutic treatment of human subjects.

Claims

1. A transgenic vector comprising a humanized Immunoglobulin (Ig) locus, wherein

(a) said humanized Ig locus comprises multiple Ig gene segments, including multiple variable (V) gene segments, multiple J gene segments, and one or more constant region gene segments,

(b) at least one of said gene segments is a functional V gene segment encoding a human V region amino acid sequence,

(c) said V gene segments are separated only by non-coding, non-human sequences derived from a non-human animal that generates antibody diversity primarily through gene conversion and/or hypermutation,

(d) at least one of said functional V gene segments encoding a human V region amino acid sequence is placed downstream of the other V gene segments, and

(e) said Ig gene segments are juxtaposed in an unrearranged, partially rearranged or fully rearranged configuration, and

wherein, as a result of structural features (a)–

(e), said humanized Ig locus is capable of undergoing gene conversion and producing a repertoire of humanized immunoglobulins with V region amino acid sequences encoded by segments of more than one V region gene, in said non-human animal.

2. The transgenic vector of claim 1, wherein said non-human animal is rabbit, pig, chicken, sheep or cow.

3. The transgenic vector of claim 1, wherein said humanized Ig locus is a heavy chain locus and comprises at least two human V gene segments, at least one D gene segment, at least two J gene segments and one or more constant region segments.

4. The transgenic vector of claim 1, wherein said humanized Ig locus is a light chain locus and comprises at least two V gene segments, at least two J gene segments, and one or more constant region gene segments.

5. The transgenic vector of claim 3, wherein the non-human animal is a rabbit, and at least one of said non-coding sequences comprises the sequence of SEQ ID NO:

10.

6. The transgenic vector of claim 3, wherein said constant region gene segment is a human heavy chain constant region gene segment.

7. The transgenic vector of claim 6, wherein said human heavy chain constant region gene segment is a Cγ

.

8. The transgenic vector of claim 6, comprising about 10–

100 V gene segments and at least two human V gene segment, wherein said human V gene segment is placed downstream to said 10–

100 V gene segments.

9. The transgenic vector of claim 8, wherein said V gene segments are selected from V gene segments at the 3′

V-region of said non-human animal and human V gene segments.

10. The transgenic vector of claim 4, wherein said constant region gene segment is a human light chain constant region gene segment.

11. The transgenic vector of claim 10, wherein said human light chain constant region gene segment is Cλ

or Cκ

.

12. The transgenic vector of claim 10, comprising about 10–

100 V gene segments and at least two human V gene segment, wherein said human V gene segment is placed downstream to said 10–

100 V gene segments.

13. The transgenic vector of claim 12, wherein said V gene segments are selected from V gene segments at the 3′

V-region of said non-human animal and human V gene segments.

14. The transgenic vector of claim 10, wherein said human V gene segment is placed immediately 5′

to a J gene segment in a rearranged configuration.

15. A method of making a transgenic vector comprising a humanized Immunoglobulin (Ig) locus capable of producing a functional repertoire of humanized antibodies in a non-human animal generating antibody diversity primarily through gene conversion and/or hypermutation, comprising:

(i) obtaining a DNA fragment comprising an Ig locus or a portion therefor from said non-human animal, which comprises at least two V gene segments, at least one J gene segment and at least one constant region gene segment, and non-coding sequences; and

(ii) integrating by homologous recombination at least one functional human V gene segment encoding a human V region amino acid sequence, into said DNA fragment of step (i) to produce a humanized Ig locus,

wherein the V gene segments present are separated only by non-coding, non-human sequences derived from said non-human animal that generates antibody diversity primarily through gene conversion and/or hypermutation, and at least one of the functional V gene segments encoding a human V region amino acid sequence is placed downstream of the other V gene segments, and

wherein said Ig gene segments are juxtaposed in an unrearranged, partially rearranged or fully rearranged configuration, as to permit gene rearrangement and gene conversion of said humanized Ig locus and the production of a functional repertoire of humanized antibodies with V region amino acid sequences encoded by more than one V region gene segment, in said non-human animal.

16. The method of claim 15, wherein the integration of said human Ig gene segment by homologous recombination, replaces an Ig gene segment in said Ig locus or said portion thereof from said non-human animal.

17. The method of claim 16, wherein the homologous recombination is achieved in a bacterial cell, a yeast cell, or a non-human animal cell.

18. The method of claim 16, wherein the human Ig gene segment is provided on a recombination vector, and is linked to a 5′

nucleotide sequence and a 3′

nucleotide sequence which are homologous to the 5′ and

3′

flanking sequences of said Ig gene segment from the non-human animal.