Species-Specific Chromosome Engineering Greatly Improves Fully Human Polyclonal Antibody Production Profile in Cattle

Abstract

Large-scale production of fully human IgG (hIgG) or human polyclonal antibodies (hpAbs) by transgenic animals could be useful for human therapy. However, production level of hpAbs in transgenic animals is generally very low, probably due to the fact that evolutionarily unique interspecies-incompatible genomic sequences between human and non-human host species may impede high production of fully hIgG in the non-human environment. To address this issue, we performed species-specific human artificial chromosome (HAC) engineering and tested these engineered HAC in cattle. Our previous study has demonstrated that site-specific genomic chimerization of pre-B cell receptor/B cell receptor (pre-BCR/BCR) components on HAC vectors significantly improves human IgG expression in cattle where the endogenous bovine immunoglobulin genes were knocked out. In this report, hIgG1 class switch regulatory elements were subjected to site-specific genomic chimerization on HAC vectors to further enhance hIgG expression and improve hIgG subclass distribution in cattle. These species-specific modifications in a chromosome scale resulted in much higher production levels of fully hIgG of up to 15 g/L in sera or plasma, the highest ever reported for a transgenic animal system. Transchromosomic (Tc) cattle containing engineered HAC vectors generated hpAbs with high titers against human-origin antigens following immunization. This study clearly demonstrates that species-specific sequence differences in pre-BCR/BCR components and IgG1 class switch regulatory elements between human and bovine are indeed functionally distinct across the two species, and therefore, are responsible for low production of fully hIgG in our early versions of Tc cattle. The high production levels of fully hIgG with hIgG1 subclass dominancy in a large farm animal species achieved here is an important milestone towards broad therapeutic applications of hpAbs.

Fig 1. Comparison of IgM in κHAC, KcHACΔ and cKSL-HACΔ vectors.

Overall structure of the κHAC, KcHACΔ and cKSL-HACΔ vectors were illustrated at the upper paneland their IgM structures were illustrated at the lower panel. The KcHACΔ vector is a derivative of the original κHAC where part of the hIGHM gene constant region, the CH1 through TM domains, is bovinized with the bovine-origin sequence. Because of this modification, the KcHACΔ vector expresses the cIgM(CH1) protein on pre-B/B cell surface. The previously reported cKSL-HACΔ vector is composed of three different hChr fragments, hChr14 (14D), hChr2 and hChr22 containing the entire hIGL and surrogate light chain (hVPREB1 and hIGLL1) loci. In this vector, part of the hIGHM gene constant region, the CH2 through TM domains, is bovinized to express the cIgM(CH2) protein. SLC, surrogate light chain; LC, light chain.

Fig 2. Construction of the KcHACΔ.

(A) Construction of CH1D vector. 14D1 was constructed as previously described [7]. Subsequently, the cIgM(CH1) or cIgM(CH2) bovinization generated CH1D2 or CH2D4, which was used for the KcHACΔ or cKSL-HACΔ vector construction [7], respectively. (B) Construction of the KcHACΔ vector in chicken DT40 cells. (C) cIgM(CH1) modification for the CH1D construction. The bovinization vector pCH1CAGzeo(R)DT(F) comprises 7.4 kb and 1.7 kb human genomic DNA as a long and short arm, respectively, 6 kb of the bovine IGHM constant region genomic DNA covering the CH1 through TM2 domains where the floxed, CAG promoter-driven zeo gene cassette was integrated between the CH4 and TM1 intron, and the DT-A gene. After the homologous recombination, part of the hIGHM constant region, the CH1 through TM2 domains, was bovinized on the CH1D.

Fig 3. PCR genotyping for KcHACΔ construction.

(A) Extensive genomic PCR for genotyping of the KcHACΔ vector. Location of each genomic PCR primer pair is depicted in relation to the KcHACΔ vector structure. (B) Occurrence of the chromosome inversion on the CH1D. A recombination between the loxP at the RNR2 locus and the lox511 at the deletion junction site AP391156/AP512355 caused the inversion that also reconstituted the CAG promoter-driven GFP gene, leading to the higher GFP expression than the PGK promoter-driven GFP gene from the cKSL-HACΔ vector. The inversion was confirmed by genomic PCR, STOPpuro-F2R2 and GFP2 x CAGpuro-F3.

Fig 4. Construction of the isHAC and isKcHACΔ vectors.

A flow of the isHAC and isKcHACΔ vector construction are illustrated. The bovinizing vector pCC1BAC-isHAC is a BAC-based one (backbone is pCC1BAC vector), consisting of 10.5 kb and 2 kb of genomic DNA as a long and short arm, respectively, 9.7 kb of the bovine genomic DNA covering the bovine I_γ1-S_γ1 and its surrounding region to replace the human corresponding 6.8 kb of I_γ1-S_γ1 region, the chicken β-actin promoter-driven neo gene flanked by FRT sequence and the DT-A gene. After the targeted bovinization, the neo cassette is removed by FLP introduction.

Fig 5. Construction of the istHAC vector.

A flow of the istHAC vector construction is illustrated. The attP sequence is integrated at 5’ side of the hI_γ1 exon 1 and 3’ side of the hIGHG1 TM2 by the targeting vectors phI_γ1FRTCAGattPhisDDT and ph_γ1TMNeoattPDT, respectively. Then, the replacement vector pBAC-istHAC is introduced with the ΦC31 recombinase to bring about the attP/attB recombination to replace the flanked region. The successful replacement causes the CAG promoter-driven DsRed gene to be reconstituted to provide red fluorescence for sorting. Finally, the DsRed cassette is removed by the FLP expression.

Fig 6. Analysis of constructed isHAC vector.

(A) Extensive genomic PCR for genotyping of the isHAC vector. Location of each genomic PCR primer pair is depicted in relation to the isHAC vector structure. (B) CGH analysis among three different CHO clones containing the isHAC vector. DNA from isC1-133 was used as a reference. There was no apparent structural difference in the isHAC among the three cell lines.

Fig 7. Analysis of constructed isKcHACΔ vector.

(A) Extensive genomic PCR for genotyping of the isKcHACΔ vector. Location of each genomic PCR primer pair is depicted in relation to the isKcHACΔ vector structure. (B) CGH analysis among three different CHO clones containing the isKcHACΔ vector. DNA from isKCDC15-8 was used as a reference. There was no apparent structural difference in the isKcHACΔ among the three cell lines.

Fig 8. Analysis of constructed istHAC vector.

(A) Extensive genomic PCR for genotyping of the istHAC vector. Location of each genomic PCR primer pair is depicted in relation to the istHAC vector structure. (B) CGH analysis among three different CHO clones containing the istHAC vector. DNA from istC1-6 was used as a reference. There was no apparent structural difference in the istHAC among the three cell lines.

Fig 9. Characterization of isHAC/TKO, istHAC/TKO and isKcHACΔ/TKO cattle by PBMC expression profile.

Representative flow cytometry analysis of PBMCs from a series of HAC/TKO calves at newborn stage. For IgM detection, anti-hIgM or anti-bIgM antibody was used. From left to right panels, PBMCs were stained for IgM alone, IgM/bCD21, IgM/bIgλ, IgM/bIgκ and IgM/hIgκ. Each red number represents percentages of cells in Q1 (IgM alone) or Q2 (IgM/bCD21, IgM/bIgλ, IgM/bIgκ and IgM/hIgκ).

Fig 10. Characterization of κHAC/DKO, cKSL-HACΔ/DKO and KcHAC/DKO cattle by PBMC expression profile and serum IgG profile.

(A) Representative flow cytometry analysis of PBMCs from a series of HAC/DKO calves at newborn stage. For IgM detection, anti-hIgM or anti-bIgM antibody was used. From left to right panels, PBMCs were stained for IgM alone, IgM/bCD21, IgM/bIgλ, IgM/bIgκ and IgM/hIgκ. Each red number represents percentages of cells in Q1 (IgM alone) or Q2 (IgM/bCD21, IgM/bIgλ, IgM/bIgκ and IgM/hIgκ). NA: not applicable (because, at that time, the anti-bIgκ antibody was not available). (B) Box-whisker plots of serum concentrations of total hIgG (g/L)(top left), fully hIgG/hIgκ (g/L)(top right), serum fully hIgG/hIgκ (%)/total hIgG (bottom right) and hIgG1/hIgG2 ratio (bottom left) in a series of HAC/DKO cattle at 5–6 months of age. F, cKSL-HACΔ/DKO (n = 33); G, KcHAC/DKO (n = 12); H, κHAC/DKO (n = 8). Dots represent outliers. The value of calf 468 were indicated with arrows.

Fig 11. Characterization of isHAC/TKO, istHAC/TKO and isKcHACΔ/TKO cattle by serum IgG profile.

(A) Box-whisker plots of serum concentrations of total hIgG (g/L) in a series of HAC/TKO and HAC/DKO cattle at 6 months of age. A, cKSL-HACΔ/TKO (n = 14); B, isHAC/TKO (n = 12); C, istHAC/TKO (n = 13); D, KcHACΔ/TKO (n = 20); E, isKcHACΔ/TKO (n = 17); F, cKSL-HACΔ/DKO (n = 43); G, KcHAC/DKO (n = 25); H, κHAC/DKO (n = 8). Dots represent outliers. The value of calf 468 was indicated with an arrow. (B) Box-whisker plots of serum concentrations of fully hIgG/hIgκ (g/L) in a series of HAC/TKO and HAC/DKO cattle at 6 months of age. A, cKSL-HACΔ/TKO (n = 13); B, isHAC/TKO (n = 12); C, istHAC/TKO (n = 13); D, KcHACΔ/TKO (n = 19); E, isKcHACΔ/TKO (n = 16); F, cKSL-HACΔ/DKO (n = 42); G, KcHAC/DKO (n = 21); H, κHAC/DKO (n = 8). Dots represent outliers. The value of calf 468 was indicated with an arrow. (C) Box-whisker plots of serum fully hIgG/hIgκ (%)/total hIgG in a series of HAC/TKO and HAC/DKO cattle at 6 months of age. A, cKSL-HACΔ/TKO (n = 13); B, isHAC/TKO (n = 12); C, istHAC/TKO (n = 13); D, KcHACΔ/TKO (n = 19); E, isKcHACΔ/TKO (n = 16); F, cKSL-HACΔ/DKO (n = 42); G, KcHAC/DKO (n = 21); H, κHAC/DKO (n = 3). Dots represent outliers. (D) Box-whisker plots of hIgG1/hIgG2 ratio in a series of HAC/TKO and HAC/DKO cattle at 6 months of age. A, cKSL-HACΔ/TKO (n = 14); B, isHAC/TKO (n = 12); C, istHAC/TKO (n = 13); D, KcHACΔ/TKO (n = 19); E, isKcHACΔ/TKO (n = 17); F, cKSL-HACΔ/DKO (n = 42); G, KcHAC/DKO (n = 12). Dots represent outliers. P values calculated for multiple comparison were shown in supplemental tables (S1, S2 and S3 Table).

Fig 12. Generation of trans-class switched bovine IgG.

Upper panel shows normal class switch recombination between human Sμ and human Sγ1 on the same chromosome (cis-reaction) in human or Tc bovine, which results in the expression unit of normal fully human γ1 heavy chain. The lower panel of the figure illustrates the trans-class switch recombination. Human Sμ on the HAC and bovine Sγ1 on the bovine chromosome 21 can make class switch recombination (trans-reaction), which results in the generation of expression unit with human VDJ connected to bovine gamma1 constant region.

Fig 13. Trans-class switched bovine IgG levels in a series of Tc cattle.

(A) The ratio of t-bIgG to total IgG (human IgG and t-bIgG) in percentage. The number of Tc cattle evaluated at each time points was noted on top of each bar in brackets. (B) Serum t-bIgG concentration in Tc bovines. The serum t-bIgG concentrations of Tc bovines with TKO background were evaluated at ages of 5–6 months and at 11–12 months. The number of Tc bovines evaluated at each time points was noted on top of the each bar in brackets.

Fig 14. Anti-human carcinoma human IgG response in a series of Tc cattle.

A series of HAC/TKO cattle received two times vaccinations (V2) of human oral squamous cell carcinoma and antigen-specific antibody generation was evaluated. Mean fluorescence intensity (MFI) of tumor cells stained with the sera from pre- and post-immunized cattle were plotted against a series of serum dilutions. Left panels (A, C, E) show the MFI with anti-hIgG antibodies (measuring total hIgG); right panels (B, D, F) show the MFI with anti-hIgκ antibodies (measuring fully hIgG/hIgκ). (A) and (B): KcHACD/TKO. (C) and (D): istHAC/TKO. (E) and (F): isKcHACD/TKO. Closed triangle and closed circle in the plot show MFI with sera from pre- and post- immunized cattle, respectively.

Fig 15. The proposed model of interspecies-incompatibilities at two levels in Tc bovine B cells.

One is at protein-protein interaction, such as pre-BCR/BCR structure (e.g. pairing between human IgM and bovine surrogate/orthodox light chain, interaction between human IgM/IgG1 and bovine Ig-α/β). The other one is at DNA-protein interaction, such as between human I_γ1-S_γ1 DNA sequence and bovine cytokine/activator-induced bovine DNA binding proteins. The first interspecies-incompatibility is addressed by KcHAC(Δ) and cKSL-HACΔ (also by isHAC, istHAC and isKcHACΔ). The second interspecies-incompatibility is addressed by isHAC, istHAC and isKcHACΔ.