Human membrane cofactor protein (CD46) controls complement activation and when expressed sufficiently as a transgene protects xenografts against complement‐mediated rejection, as shown here using non‐immunosuppressed baboons and heterotopic CD46 transgenic pig kidney xenografts. This report is of a carefully engineered transgene that enables high‐level CD46 expression. A novel CD46 minigene was validated by transfection and production of a transgenic pig line. Pig lymphocytes were tested for resistance to antibody and complement‐mediated lysis, transgenic tissues were characterized for CD46 expression, and kidneys were transplanted to baboons without immunosuppression. Absorption of anti‐Galα(1,3)Gal epitope (anti‐GAL) serum antibodies was measured. Transgenic pigs expressed high levels of CD46 in all tissues, especially vascular endothelium, with stable expression through three generations that was readily monitored by flow cytometry of transgenic peripheral blood mononuclear cells (PBMC). Transgenic PBMC pre‐sensitized with antibody were highly resistant to human complement‐mediated lysis which readily lysed normal pig PBMC. Normal pig kidneys transplanted without cold ischemia into non‐immunosuppressed adult baboons survived a median of 3.5 h (n = 7) whereas transgenic grafts (n = 9), harvested at ∼24‐h intervals, were either macroscopically normal (at 29, 48 and 68 h) or showed limited macroscopic damage (median > 50 h). Microscopic assessment of transplanted transgenic kidneys showed only focal tubular infarcts with viable renal tissue elsewhere, no endothelial swelling or polymorph adherence and infiltration by lymphocytes beginning at 3 days. Coagulopathy was not a feature of the histology in four kidneys not rejected and assessed at 48 h or later after transplantation. Baboon anti‐GAL serum antibody titers were high before transplantation and, in one extensively analyzed recipient, reduced ∼8‐fold within 5.5 h. The data demonstrate that a single CD46 transgene controls hyperacute kidney graft rejection in untreated baboons despite the presence of antibody and complement deposition. The expression levels, tissue distribution and in vitro functional tests indicate highly efficient CD46 function, controlling both classical and alternative pathway complement activation, which suggests it might be the complement regulator of choice to protect xenografts.
Under the influence of testosterone, rat seminal vesicles synthesise large amounts of a tissue specific protein, S. Recombinant X clones have been isolated containing overlapping sequences covering a 27.5 kilo base region of the rat genome within which the gene for protein S is located. Recombinant plasmids bearing cDNA sequences for protein S were constructed in pBR328. One (pcS2) contains a 690 nucleotide insert and is probably full length. Detailed restriction maps of the S-gene are presented and the structure was confirmed by analysis of R-loops and heteroduplexes. The S-gene covers a 2 kbp region of the genome and consists of a 5' intron (490 bp) separating a leading exon (120 bp) containing the 5' untranslated region from a central exon (310 bp) containing most of the coding sequence and part of the 3' untranslated region.
Background Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance. Methods An intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (dbBiTES, 300 kDa). dbBiTEs were tested in vitro by EM, flow cytometry and cell cytoxicity and in vivo by PET tumor imaging and redirected T-cell therapy. Results The interlocked hinge regions are compatible with a structural model that fits the electron micrographs of 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES exhibit high in vitro cytotoxicity, high in vivo tumor targeting as demonstrated by PET imaging, and redirected dbBiTE coated T-cells (1 microgram/10 million cells) that kill CEA + target cells in vivo in CEA transgenic mice. Conclusion dbBiTE redirected T-cell therapy is a promising, efficient approach for targeting and killing cancer cells. Electronic supplementary material The online version of this article (10.1186/s12885-019-6056-8) contains supplementary material, which is available to authorized users.
Testosterone controls the synthesis of seminal vesicle protein F in male rats by regulating the cellular concentration of its mRNA (mRNAF). Phage lambda recombinants have been isolated containing the complete F gene. In addition plasmids have been constructed containing cDNAF sequences some of which are probably full-length (approximately 700 bp). Detailed restriction mapping shows that the F gene is 1.7 kbp long and contains approximately 1.0 kbp of intervening sequence arranged in at least two introns (420 bp and 600 bp). Part of cDNAF has been sequenced showing that the terminal 125 bp of the 3' untranslated region of mRNAF has substantial (greater than 70%) sequence homology with the 3' end of the mRNA coding for another androgen-dependent seminal vesicle protein (protein S). The cloned F gene has been detected in liver and seminal vesicle DNA along with an homologous but structurally different gene. The hormonal control of mRNAF was examined with cDNAF. A pronounced (approximately 3000-fold) differential response to testosterone was observed.
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