NON-HUMAN MAMMALS FOR THE PRODUCTION OF CHIMERIC ANTIBODIES

• US 20110236378A1
• Filed: 09/30/2009
• Published: 09/29/2011
• Est. Priority Date: 09/30/2008
• Status: Active Grant

• Current Assignee: Ablexis LLC
• Original Assignee: Ablexis LLC
• Inventors: Green, Larry, Shizuya, Hiroaki
• Granted Patent: US 9,346,873 B2
• Time in Patent Office: Days
• US Class Current: 424/133.1
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/052 inducing gain of function
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2217/15 Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• 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 16/283 against Fc-receptors, e.g. CD16, CD32, CD64 CD23 C07K16/2851
• CPC Class Codes: C07K 16/462 Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/522 CH1 domain
• CPC Class Codes: C07K 2317/524 CH2 domain
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2317/53 Hinge
• CPC Class Codes: C07K 2317/56 : variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 : Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C12N 15/8509 : for producing genetically modified animals, e.g. transgenic

Abstract

The invention provides knock-in non-human cells and mammals having a genome encoding chimeric antibodies and methods of producing knock-in cells and mammals. Certain aspects of the invention include chimeric antibodies, humanized antibodies, pharmaceutical compositions and kits. Certain aspects of the invention also relate to diagnostic and treatment methods using the antibodies of the invention.

Claims

1-161. -161. (canceled)

162. A non-human mammalian cell having a genome comprising (1) a human VH gene segment operably linked to (2) a portion of an Ig heavy chain locus, said portion comprising all or a part of non-human gene segments downstream of JH and a human CH1 domain, wherein said cell comprises a genome encoding a chimeric Ig heavy chain.

165. A chimeric antibody produced by the cell according to claim 162.

166. A method of producing the cell according to claim 162 comprising the steps of:

producing one or more constructs comprising a human VH gene segment and a portion of an Ig heavy chain locus, said portion comprising all or a part of non-human gene segments downstream of JH and a human CH1 domain; and

introducing said one or more constructs into a non-human mammalian cell and integrating said human VH gene segment and said portion into the genome of said cell,

wherein said cell comprises a genome encoding a chimeric Ig heavy chain.

163. A non-human mammalian cell having a genome comprising (1) one or more human VH gene segments operably linked to (2) a portion of an Ig heavy chain locus, said portion comprising all or a part of non-human gene segments downstream of JH, wherein the one or more human VH gene segments are introduced upstream of the endogenous constant region, and wherein said cell comprises a genome encoding a chimeric Ig heavy chain.

164. A non-human mammalian cell comprising a human V region operably linked to an endogenous non-human constant region, wherein the endogenous Ig heavy chain locus comprises one or more endogenous VH, DH or JH gene segments.

167. A method of introducing a DNA sequence comprising one or more human VH gene segments into an endogenous Ig heavy chain locus of a non-human mammalian cell, said method comprising the steps of:

introducing a first loxP site into the endogenous Ig heavy chain locus upstream of endogenous DNA sequences encoding constant region domain segments via homologous recombination,

introducing a DNA construct comprising a second loxP site and one or more human VH gene segments, and expressing a CRE recombinase, thereby recombining said one or more human VH gene segments into the endogenous Ig heavy chain locus.

168. A non-human mammalian cell having a genome comprising a transgene encoding an Ig light chain locus, or a portion thereof, wherein all or part of said transgene is derived from DNA synthesized in vitro, and wherein said transgene comprises a human variable region.

169. A method of producing a non-human mammalian cell having a genome comprising a transgene encoding an Ig light chain locus, or a portion thereof, wherein all or part of said transgene is derived from DNA synthesized in vitro, and wherein said transgene comprises a human variable region comprising the steps of:

producing a first construct in vitro, wherein said first construct comprises an Ig light chain locus, or a portion thereof, wherein said Ig light chain locus comprises a human variable region; and

introducing said first construct into a non-human mammalian cell and integrating said Ig light chain locus into the genome of said cell, wherein the genome of said cell comprises a transgene encoding an Ig light chain, or a portion thereof, wherein said Ig light chain comprises a human variable region.

170. A non-human mammal having a genome comprising (1) a human VH gene segment operably linked to (2) a portion of an Ig heavy chain locus, said portion comprising all or a part of non-human gene segments downstream of JH and a human CH1 domain, wherein said mammal is capable of producing a chimeric Ig heavy chain.

176. A chimeric antibody produced by the non-human mammal according to claim 170.

177. A method of producing a chimeric antibody which specifically binds to a target antigen comprising immunizing the non-human mammal according to claim 170 with said target antigen and recovering said antibody.

178. A method of detecting a target antigen comprising detecting the antibody according to claim 177 with a secondary detection agent which recognizes a portion of said antibody.

179. A pharmaceutical composition comprising the antibody according to claim 177 and a pharmaceutically acceptable carrier.

180. A kit comprising the antibody according to claim 177 and instructions for use of said antibody.

182. A method of producing the mammal according to claim 170 comprising the steps of:

producing one or more constructs comprising a human VH gene segment and a portion of an Ig heavy chain locus, said portion comprising all or a part of non-human gene segments downstream of JH and a human CH1 domain;

introducing said one or more constructs into a non-human mammalian cell and integrating said human VH gene segment and said portion into the genome of said cell; and

generating a non-human mammal capable of producing a chimeric Ig heavy chain from said cell.

185. A method of producing an antibody display library, comprising:

providing a first non-human mammal according to claim 170 and a second non-human mammal according to claim 172;

recovering polynucleotide sequences encoding human Ig heavy chain variable regions and human Ig light chain variable regions from said first and second non-human mammals, respectively; and

producing an antibody display library comprising said human Ig heavy chain variable regions and human Ig light chain variable regions.

186. An antibody display library produced according to claim 185.

171. A non-human mammal having a genome comprising (1) one or more human VH gene segments operably linked to (2) a portion of an Ig heavy chain locus, said portion comprising all or a part of non-human gene segments downstream of JH, wherein the one or more human VH gene segments are introduced upstream of the endogenous constant region, and wherein said mammal comprises a genome encoding a chimeric Ig heavy chain.

172. A non-human mammal having a genome comprising a transgene encoding an Ig light chain locus, or a portion thereof, wherein said transgene is all or in part derived from DNA synthesized in vitro, wherein said Ig light chain locus comprises a human variable region, and wherein said mammal is capable of producing the Ig light chain, or a portion thereof.

183. A method of producing the mammal according to claim 172 comprising the steps of:

producing a first construct in vitro, wherein said first construct comprises an Ig light chain locus, or a portion thereof, wherein said Ig light chain locus comprises a human variable region;

introducing said first construct into a non-human mammalian cell and integrating said Ig light chain locus into the genome of said cell; and

generating from the cell a non-human mammal capable of producing the Ig light chain, or a portion thereof.

173. A chimeric antibody, or antigen-binding fragment thereof, comprising a human VH gene segment and a portion comprising all or a part of a non-human Ig heavy chain constant region.

174. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 173 and a pharmaceutically acceptable carrier.

175. A kit comprising the antibody according to claim 173 and instructions for use of said antibody.

181. A nucleic acid molecule comprising a polynucleotide sequence encoding an Ig heavy chain variable region of the antibody according to claim 173.

184. A method of producing a non-human mammal having a genome comprising (1) a human VH gene segment operably linked to (2) a portion of an Ig heavy chain locus, said portion comprising all or a part of non-human gene segments downstream of JH and a human CH1 domain, wherein said mammal is capable of producing a chimeric Ig heavy chain, and (3) a human Ig light chain locus, or a portion thereof, said method comprising the steps of:

breeding a first non-human mammal comprising a chimeric Ig heavy chain locus, wherein said Ig heavy chain locus comprises (1) a human VH gene segment and (2) a portion of an Ig heavy chain locus comprising all or a part of non-human gene segments downstream of JH and a human CH1 domain, with a second non-human mammal comprising an Ig light chain locus, or a portion thereof, wherein said Ig light chain locus comprises a human variable region; and

selecting offspring having a genome comprising said chimeric Ig heavy chain locus and said Ig light chain locus.

187. A method of producing an antibody display library, comprising:

providing a non-human mammal having a genome comprising (1) a human VH gene segment operably linked to (2) a portion of an Ig heavy chain locus, said portion comprising all or a part of non-human gene segments downstream of JH and a human CH1 domain, wherein said mammal is capable of producing a chimeric Ig heavy chain, and (3) a human Ig light chain locus, or a portion thereof;

recovering polynucleotide sequences encoding human Ig heavy chain variable regions and human Ig light chain variable regions from said non-human mammal; and

producing an antibody display library comprising said human Ig heavy chain variable regions and human Ig light chain variable regions.

188. An antibody display library produced according to claim 187.