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EP-4215043-B1 - ANIMAL MODELS AND THERAPEUTIC MOLECULES

EP4215043B1EP 4215043 B1EP4215043 B1EP 4215043B1EP-4215043-B1

Inventors

  • BRADLEY, ALLAN
  • LEE, E-CHIANG
  • LIANG, Qi
  • WANG, WEI

Dates

Publication Date
20260506
Application Date
20100707

Claims (15)

  1. A rodent whose genome comprises: (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host rodent constant region; wherein the rodent genome is modified to prevent expression of the native (fully host species specific) antibodies in the rodent by inversion of all of the rodent VDJ region; and wherein the rodent is able to produce a repertoire of chimaeric heavy chains, having a rodent constant region and a human variable region.
  2. The rodent of claim 1, whose genome further comprises: (b) one or more human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host rodent kappa constant region and/or one or more human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host rodent lambda constant region; wherein the rodent is able to produce a repertoire of chimaeric antibodies, or chimaeric light or heavy chains, having a rodent constant region and a human variable region.
  3. The rodent of claim 2, wherein the rodent genome is modified by inversion of all or part of the rodent VJ region.
  4. A rodent whose genome comprises: (a) a plurality of human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host rodent kappa constant region and/or one a plurality of human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host rodent lambda constant region; wherein the rodent genome is modified to prevent expression of the native (fully host species specific) antibodies in the rodent by inversion of all of the rodent VJ region; wherein the rodent is able to produce a repertoire of chimaeric antibodies, or chimaeric light chains, having a rodent constant region and a human variable region.
  5. The rodent of claim 4, whose genome further comprises: (a) one or more human IgH V regions, one or more human D regions and one or more human J regions upstream of the host rodent constant region; wherein the rodent is able to produce a repertoire of chimaeric antibodies, or chimaeric heavy chains, having a rodent constant region and a human variable region.
  6. A rodent cell whose genome comprises: (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host rodent constant region; wherein the rodent genome is modified to prevent expression of the native (fully host species specific) antibodies in the rodent by inversion of all of the rodent VDJ region, or VJ region.
  7. The rodent cell of claim 6, whose genome further comprises: (b) one or more human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host rodent kappa constant region and/or one or more human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host rodent lambda constant region.
  8. The rodent cell of claim 7, wherein the rodent cell genome is modified by inversion of all or part of the rodent VJ region.
  9. A rodent cell whose genome comprises: (a) a plurality of human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host rodent kappa constant region and/or one a plurality of human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host rodent lambda constant region; wherein the rodent cell genome is modified to prevent expression of the native (fully host species specific) antibodies in the rodent by inversion of all of the rodent VJ region.
  10. The rodent cell of claim 9, whose genome further comprises: (a) one or more human IgH V regions, one or more human D regions and one or more human J regions upstream of the host rodent constant region.
  11. A method for producing a chimaeric antibody or antibody chain specific to a desired antigen, the method comprising immunizing a rodent according to any one of claims 1-5 with the antigen and recovering the antibody or antibody chain.
  12. A method for producing a fully humanised antibody, the method comprising immunizing a rodent according to any one of claims 1-5 with an antigen, and then replacing the rodent constant region of the chimaeric antibody specifically reactive with the antigen with a human constant region, optionally by engineering of the nucleic acid encoding the antibody, optionally wherein the antibody is a monoclonal antibody.
  13. A method of making a pharmaceutical composition, the method comprising producing an antibody according to the method of claim 12 and further comprising combining the antibody with a pharmaceutically acceptable carrier or other excipient to produce the composition.
  14. A method of producing a chimeric or fully humanised antibody or antibody chain, the method comprising carrying out the method according to claim 11 or claim 12, and expressing the antibody or antibody chain.
  15. A method of making a pharmaceutical composition, the method comprising carrying out the method of producing a chimeric or fully humanised antibody or antibody chain according to claim 14, and further comprising formulating the antibody or antibody chain with a pharmaceutically acceptable carrier or other excipient.

Description

Background The present disclosure relates inter alia to rodents and cells that are engineered to contain exogenous DNA, such as human immunoglobulin gene DNA, their use in medicine and the study of disease, methods for production of rodents and cells, and antibodies and antibody chains produced by such rodents and derivatives thereof. In order to get around the problems of humanizing antibodies a number of companies set out to generate mice with human immune systems. The strategy used was to knockout the heavy and light chain loci in ES cells and complement these genetic lesions with transgenes designed to express the human heavy and light chain genes. Although fully human antibodies could be generated, these models have several major limitations: (i) The size of the heavy and light chain loci (each several Mb) made it impossible to introduce the entire loci into these models. As a result the transgenic lines recovered had a very limited repertoire of V-regions, most of the constant regions were missing and important distant enhancer regions were not included in the transgenes.(ii) The very low efficiency of generating the large insert transgenic lines and the complexity and time required to cross each of these into the heavy and light chain knockout strains and make them homozygous again, restricted the number of transgenic lines which could be analysed for optimal expression.(iii) Individual antibody affinities rarely reached those which could be obtained from intact (non-transgenic) animals. WO2007117410 discloses chimaeric constructs for expressing chimaeric antibodies. WO2010039900 discloses knock in cells and mammals having a genome encoding chimaeric antibodies. The present disclosure provides, inter alia, a process for the generation in rodents of antibodies that comprise a human Ig variable region, and further provides rodent models for the generation of such antibodies. Summary of the Invention The invention is set out in claims 1-15. Any reference to non-human mammals herein is to rodents. Embodiments directed to methods for the construction of chimeric human heavy and light chain loci in a non-human mammal are not encompassed by the wording of the claims but are considered as useful for understanding the invention. Figures Figures 1 - 8 show an iterative process for insertion of a series of human BACs into a mouse Ig locus.Figures 9 - 18 show in more detail the process of figures 1-8 for the IgH and kappa locusFigures 19 and 20 show the principles behind antibody generation in chimaeric mice.Figure 21 shows a possible insertion site for the human DNA in a mouse chromosome.Figures 22 - 26 disclose an alternative iterative process for insertion of a series of human BACs into a mouse Ig locus.Figure 27 - 29 illustrate a mechanism for inversion of the host VDJ regionFigure 30 illustrates proof of principle for insertion of a plasmid using an RMCE approach.Figure 31 illustrates sequential RMCE - Integration into Landing PadFigure 32 illustrates confirmation of Successful Insertion into Landing PadFigure 33 illustrates PCR Confirmation of 3' End CuringFigure 34 illustrates insertion of BAC#1 and PCR Diagnostics General Description All nucleotide coordinates for the mouse are from NCBI m37, April 2007 ENSEMBL Release 55.37h for the mouse C57BL/6J strain. Human nucleotides are from GRCh37, Feb 2009 ENSEMBL Release 55.37 and rat from RGSC 3.4 Dec 2004 ENSEMBL release 55.34w. Disclosed herein are methods for the construction of chimaeric human heavy and light chain loci in a rodent, for example a mouse. Reference to work in mice herein is by way of example only, and reference to mice is taken to include reference to all rodents unless otherwise apparent from the disclosure, with mice being preferred as the rodent. In one aspect the disclosure relates to a non-human mammal whose genome comprises: (a) a plurality of human IgH V regions, one or more human D regions and one or more human J regions upstream of the host non-human mammal constant region; and(b) optionally one or more human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host non-human mammal kappa constant region and/or one or more human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host non-human mammal lambda constant region; wherein the non-human mammal is able to produce a repertoire of chimaeric antibodies or antibody chains having a non-human mammal constant region and a human variable region. In a further aspect the disclosure relates to a non-human mammal whose genome comprises: (a) a plurality of human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of the host non-human mammal kappa constant region and/or a plurality of human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of the host non-human mammal lambda constant region; and(b) optiona