High-throughput mouse model for optimizing antibody affinities

1. A cell comprising an engineered IgH locus in which the 3′-most V H segment of the IgH locus comprises a non-native V H segment sequence; and

the engineered IgH locus does not comprise a functional IGCR1 sequence within the nucleic acid sequence separating the 3′ end of the 3′-most V H segment of the IgH locus and the 5′ end of a D H segment of the IgH locus.

2. The cell of claim 1 , wherein the non-native V H segment is a human V H segment.

3. The cell of claim 1 , wherein the non-native V H segment is a human V H segment from a known antibody in need of improvement of affinity or specificity.

4. The cell of claim 3 , wherein the human V H segment is IGHV1-2*02, IGVH1-46 or IGHV1-69.

5. The cell of claim 1 , further comprising:

(a) a 3′ recombinase site being located 3′ of the one or more J H segments;

and a passenger cassette being located at the position of a deleted native 3′-most V H segment, the passenger cassette comprising, from 5′ to 3′:

a 5′ recombinase site

an inverted passenger VDJ exon and/or a cassette targeting sequence; and

a maturation-compatible V H segment;

wherein the recombinase sites are inverted with respect to each other; or

(b) a 3′ recombinase site being located 3′ of the one or more J H segments;

and a passenger cassette being located at the position of a deleted native 3′-most V H segment, the passenger cassette comprising, from 5′ to 3′:

a 5′ to 3′-oriented passenger VDJ exon and/or a cassette targeting sequence;

a 5′ recombinase site; and

a maturation-compatible V H segment;

wherein the recombinase sites are in the same orientation.

6. The cell of claim 5 , wherein the recombinase site is a LoxP site and the cell further comprises a locus encoding cre recombinase.

7. The cell of claim 6 , wherein the locus encoding cre recombinase is under the control of a promoter which is not active in immature B cells and is active in peripheral B cells.

8. The cell of claim 7 , wherein the promoter is the CD21 promoter.

9. A genetically engineered non-human mammal comprising the cell of claim 1 .

10. A chimeric genetically engineered non-human mammal comprising two populations of cells,

a first population comprising cells which are V(D)J recombination-defective; and

a second population comprising cells of claim 1 .

11. A method of making an optimized antibody from a known antibody, the method comprising the steps of:

injecting a mouse blastocyst with a cell of claim 1 , wherein the cell is a mouse embryonic stem cell, and wherein the non-native V H segment comprises the V H segment of the known antibody;

implanting the mouse blastocyst into a female mouse under conditions suitable to allow maturation of the blastocyst into a genetically engineered mouse;

isolating

1) An optimized antibody comprising the non-native V H segment; or

2) a cell producing an optimized antibody comprising the non-native V H segment from the genetically engineered mouse.

12. A method of producing B lymphocytes comprising V H (D)J H rearrangements with a VH segment from a known monoclonal antibody, the method comprising the steps of:

injecting engineered mouse embryonic stem cells which are cells according to claim 1 and wherein the non-native V H segment comprises the V H segment of the known monoclonal antibody, into a blastocyst of a mouse which is incapable of forming mature B cells, thereby creating a chimeric mouse that produces B lymphocytes comprising V H (D)J H rearrangements comprising the V H segment from the known monoclonal antibody.

13. A method of identifying a candidate antigen as an antigen that activates a B cell population comprising a V H segment of interest, the method comprising:

immunizing a mammal of claim 9 , engineered such that a majority of the mammal's peripheral B cells express the V H segment of interest, with the antigen;

measuring B cell activation in the mammal; and

identifying the candidate antigen as an activator of a B cell population comprising the V H segment of interest if the B cell activation in the mammal is increased relative to a reference level.

14. The cell of claim 1 , wherein the engineered IgH locus comprises a non-functional IGCR1 sequence comprising mutated CBE sequences; the engineered IgH locus comprises a non-functional IGCR1 sequence in which CBE sequences of the IGCR1 sequence have been deleted; or the IGCR1 sequence has been deleted from the engineered IgH locus.

15. The cell of claim 1 , wherein the cell is a murine cell.

16. The cell of claim 15 , wherein the cell is a murine stem cell or murine embryonic stem cell.

17. The cell of claim 1 , wherein the cell is a stem cell or embryonic stem cell.

18. The cell of claim 1 , wherein the IgH locus further comprises at least one of the following:

one or more non-native D H segments;

one native D H segment;

one or more non-native J H segments, the

a human J H 2 J H segment

one native J H segment;

a murine IgH locus sequence;

a human IgH locus sequence; and

a humanized IgH locus sequence.

19. The cell of claim 1 , wherein the J H locus has been replaced by human D and J H cassette or a cassette with an assembled human DJ H .

20. The cell of claim 1 , wherein the cell is heterozygous for the engineered IgH locus of claim 1 and the other IgH locus has been engineered to be inactive, wherein the cell will express an IgH chain only from the engineered IgH locus of claim 1 .

21. The cell of claim 1 , further comprising at least one of the following:

an IgL locus with human sequence;

a humanized IgL locus;

a human IgL locus;

an IgL locus with one V L segment;

an IgL locus with one J L segment;

a human rearranged V L J L at the IgL kappa or lambda locus;

a human rearranged V L J L at the murine IgL kappa or lambda locus; and

an IgL locus encoding IGκV1 or VRC01 IgL.

22. The cell of claim 1 , further comprising a mutation capable of activating, inactivating or modifying genes leading to increased GC antibody maturation responses.