KR-102964498-B1 - Transgenic chickens that produce human antibodies

Abstract

A transgenic chicken having a genome containing a modified immunoglobulin heavy chain (IgH) locus is provided. The locus lacks the entire continuous endogenous chicken V-D-J region and includes multiple upstream sequence pseudogenes based on human VH segments, human D clusters, human J segments, and human VH sequences. The modified IgH locus undergoes V(D)J recombination in the chicken, and the chicken produces antibodies with various immunoglobulin heavy chains.

Inventors

• 레이튼, 필립 에이.
• 하리만, 윌리엄 돈

Assignees

• 크리스탈 바이오사이언스 주식회사

Dates

Publication Date

20260512

Application Date

20190305

Priority Date

20180321

Claims (20)

1. As a transgenic chicken containing a genome containing a modified endogenous immunoglobulin heavy chain (IgH) locus, (a) The entire adjacent endogenous chicken VDJ area is missing; (b) (i) Immunoglobulin heavy chain gene promoter; (ii) a germline human VH segment comprising coding sequences for variable domains including FR1, CDR1, FR2, CDR2, and FR3 sequences; (iii) Human D cluster; (iv) single human J segment; (v) Multiple sequences encoding invariant regions; and comprising a plurality of human germline VH pseudogenes in an operable linkage of the structure FR1-CDR1-FR2-CDR2-FR3, each comprising: (vi) FR1, FR2, and FR3 sequences substantially identical to the functional human VH segment of (b)(ii) located upstream of the germline human VH segment of (b)(ii); and CDR1 and CDR2 sequences different for each pseudogene, respectively, and The altered IgH locus undergoes V(D)J recombination in chickens; Multiple germline human VH pseudogenes donate nucleotide sequences to germline human VH segments by genetic transformation following V(D)J recombination; Producing human antibodies containing various immunoglobulin heavy chains, Transgenic chicken containing a genome containing a modified endogenous immunoglobulin heavy chain (IgH) locus.
2. A transgenic chicken according to claim 1, characterized in that the CDR1 and CDR2 sequences of (b) (vi) encode the CDR1 and CDR2 of different human VH segments in the same family as the functional human VH segment of (b) (ii).
3. A transgenic chicken according to claim 1 or 2, characterized in that the entire adjacent endogenous chicken VDJ region of (a) is in the range of length 10-12 kb.
4. A transgenic chicken according to claim 1, characterized in that (b) the promoter of (i) is a chicken immunoglobulin heavy chain gene promoter.
5. A transgenic chicken according to claim 1, characterized in that (b) the germline human VH segment of (ii) and the CDR1 and CDR2 sequences of (vi) are derived from the VH3 family, VH1 family, or VH4 family.
6. A transgenic chicken according to claim 1, characterized in that any codon for cysteine in the D cluster is modified to code for a different amino acid.
7. A transgenic chicken according to claim 6, characterized in that any codon for cysteine in cluster D is mutated to encode tyrosine or tryptophan.
8. A transgenic chicken according to claim 1, characterized in that the intervening sequence of the D cluster is derived from a chicken.
9. A transgenic chicken according to claim 1, characterized in that (b) the plurality of pseudogenes of (v) comprises at least 10 pseudogenes.
10. A transgenic chicken according to claim 1, characterized in that the pseudogene of (b) (v) is reversed with respect to the germline human VH segment of (b) (ii).
11. A transgenic chicken according to claim 1, characterized in that (b) a plurality of sequences encoding the invariant region of (v) are endogenous to the chicken.
12. A transgenic chicken according to claim 1, characterized in that the transcript of the modified immunoglobulin heavy chain (IgH) locus comprises an intron connecting the 3' end of a copy of the J coding sequence to the 5' end of a copy of the invariant region coding sequence of (b) (v).
13. A transgenic chicken according to claim 1, characterized in that the chicken is homozygous for a modified IgH locus.
14. A transgenic chicken according to claim 1, characterized in that the chicken is heterozygous for the modified IgH locus and the IgH locus on the homologous chromosome is knocked out.
15. A transgenic chicken according to claim 1, characterized in that the chicken is heterozygous for the modified IgH locus and the IgH locus on the homologous chromosome is wild-type.
16. A transgenic chicken according to claim 14, characterized in that the chicken is heterozygous for the modified IgH locus and the IgH locus on the homologous chromosome lacks its entire adjacent endogenous chicken VDJ region.
17. A transgenic chicken according to claim 14, characterized in that the chicken is heterozygous for the modified IgH locus and lacks a J region at the IgH locus on the homologous chromosome.
18. A transgenic chicken according to claim 1, characterized in that the antibodies produced by the chicken are diversified in the heavy chain CDR1, CDR2, and CDR3 regions.
19. delete
20. B cells derived from the transgenic chicken of Article 1.

Description

Transgenic chickens that produce human antibodies This application claims the benefit of U.S. provisional application serial number 62 / 646,319 filed on March 21, 2018, which is incorporated herein by reference. As in all higher vertebrates, a critical checkpoint in B cell development in chickens is intraframe V(D)J rearrangement, which leads to the expression of functional B cell receptor complexes on the cell surface (1-3). The rearrangement process in chickens uses the same recombination signal sequences and enzymes as in mammals to recombine V, D, and J genes into functional V regions (4-8). The main difference in chickens is that there are only monogenetic V and monogenetic J genes in both light and heavy chain loci, while the heavy chain contains clusters of very similar D segments (5,8). Therefore, compared to the diverse V, D, and J genes in humans, the rearrangement process generates little sequence diversity in the early B cell repertoire. Incomplete junctions and exonucleolytic chewing backs at VJ and VDJ junctions can produce some diversity, but since chicken B cells do not express TdT (9), there is no N-addition in CDR-H3 and immunoglobulin diversity generally produced by gene rearrangement processes is minimized (5,10,11). To produce a diverse repertoire, chickens utilize a gene transformation process in which upstream pseudogenes at light and heavy chain loci act as sequence donors to mutate the expressed functional V (8,11-13). Since these pseudogenes do not contain promoter or recombination signal sequences, they cannot be expressed on their own; however, their sequences are incorporated into a single functional V as segments of varying lengths. Multiple redundant gene transformations from different pseudogenes in the pool lead to a highly diverse naive repertoire. In addition to gene transformation, non-template somatic hypermutations also contribute to repertoire diversity (14-16). Despite the limitations of V(D)J rearrangements in chickens, CDR-H3 exhibits length and sequence diversity similar to mammals (1,17). In V (D) J rearrangement, the function of chicken D may be more related to providing internal CDR-H3 disulfide bridges for the stabilization of antigen-binding loops, as most D encodes a single cysteine residue and DD binding encodes a paired cysteine (17). Variation in chicken CDR-H3 is derived from gene conversion/somatic hypermutation rather than the rearrangement process itself. The present disclosure provides a transgenic chicken that produces human antibodies. Some aspects of the present invention are best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, in accordance with general practice, the various functions of the drawings are not expanded. In practice, the sizes of the various functions are expanded or reduced at will for clarity. The drawings include the following figures.Figure 1 provides a diagram of the pre-rearranged human VH segment in chickens. Scale diagram of SynVH-C (pre-rearranged) and SynVH-SD (rearranged) transgenes and heavy chain knockout (IgH-J KO). Human sequences are shown in red, and chickens in blue. In the top row, the SynVH-C transgene consists of a pre-rearranged human V region (hVDJ) with an upstream array of human pseudogenes. The human V region splices downstream chicken invariant regions (only Cmu is shown). The chicken germline V and D genes and pseudogene arrays are located in the upstream sequence of the human V gene. The exact mapping of the chicken pseudogenes is not shown (indicated in parentheses), but the distance to the functional chicken V is accurate. The loxP and attR sites remaining from the insertion event are shown. The intermediate line SynVH-SD transgene contains a single human germline VH3-23 gene, a single JH6 gene, and 24 unique human D genes. All intervening sequences and recombination sites originate from chicken heavy chain loci (indicated in blue). The upstream sequence of the human germline V gene contains an array of human-based pseudogenes. The upstream sequences of the chicken germline V and D genes are deleted, but the chicken pseudogenes are still present. Bottom line, structure of the chicken heavy chain knockout (21). In this study, the genotypes of the transgenic chickens were SynVH-C or SD "knockout"/IgH knockout. A single chicken JH gene was replaced with a promoter-free neo gene. The attP site adjacent to the neo gene is removed via Cre-lox recombination after the insertion of the SynVH-C and SynVH-SD constructs to remove selectable markers and plasmid backbone sequences. Figure 2 provides a diagram of signal peptide changes observed in transgenic chickens and the alignment of germline signal peptide sequences. The SynVH-SD signal peptide undergoes changes to become less hydrophobic and more hydrophobic than SynVH-C. A. Diagram of signal peptide changes observed in transgenic chickens. Signal peptides can be modified into ch