A truncated diphtheria toxin based recombinant porcine CTLA-4 fusion toxin.

Targeted cell therapies are possible through the generation of recombinant fusion proteins that combine a toxin, such as diphtheria toxin (DT), with an antibody or other molecule that confers specificity. Upon binding of the fusion protein to the cell of interest, the diphtheria toxin is internalized which results in protein synthesis inhibition and subsequent cell death. We have recently expressed and purified the recombinant soluble porcine CTLA-4 both with and without N-glycosylation in yeast Pichia pastoris for in vivo use in our preclinical swine model. The glycosylated and non-N-glycosylated versions of this recombinant protein each bind to a porcine CD80 expressing B-cell lymphoma line (LCL13271) with equal affinity (K(D)=13 nM). In this study we have linked each of the glycosylated and non-N-glycosylated soluble porcine CTLA-4 proteins to the truncated diphtheria toxin DT390 through genetic engineering yielding three versions of the porcine CTLA-4 fusion toxins: 1) monovalent glycosylated soluble porcine CTLA-4 fusion toxin; 2) monovalent non-N-glycosylated soluble porcine CTLA-4 fusion toxin and 3) bivalent non-N-glycosylated soluble porcine CTLA-4 fusion toxin. Protein synthesis inhibition analysis demonstrated that while all three fusion toxins are capable of inhibiting protein synthesis in vitro, the non-N-glycosylated porcine CTLA-4 isoforms function most efficiently. Binding analysis using flow cytometry of the porcine CTLA-4 fusion toxins to LCL13271 cells also demonstrated that the non-N-glycosylated porcine CTLA-4 isoforms bind to these cells with higher affinity compared to the glycosylated fusion toxin. The monovalent non-N-glycosylated porcine CTLA-4 fusion toxin was tested in vivo. NSG (NOD/SCID IL-2 receptor γ(-)/(-)) mice were injected with porcine CD80(+) LCL13271 tumor cells. All animals succumbed to tumors and those treated with the monovalent non-N-glycosylated porcine CTLA-4 fusion toxin survived longer based on a symptomatic scoring system compared to the untreated controls. This recombinant protein may therefore provide a novel approach for in vivo depletion of porcine antigen presenting cells (APCs) for studies investigating the induction of transplantation tolerance, autoimmune disease and cancer treatment.

Expression and characterization of recombinant soluble porcine CD3 ectodomain molecules: mapping the epitope of an anti-porcine CD3 monoclonal antibody 898H2-6-15.

The porcine CD3 specific monoclonal antibody 898H2-6-15 has been used in allo- and xeno-transplantation studies as a porcine CD3 marker and as an effective T cell depletion reagent when conjugated to the diphtheria toxin mutant, CRM9. A recombinant anti-porcine CD3 immuntoxin was recently developed using single-chain variable fragments (scFv) derived from 898H2-6-15. In this study, using published sequence data, we have expressed the porcine CD3 ectodomain molecules in E. coli through inclusion body isolation and in vitro refolding approach. The expressed and refolded porcine CD3 ectodomain molecules include CD3ε, CD3γ, CD3δ, CD3εγ heterodimer, CD3εδ heterodimer, CD3εγ single-chain fusion protein and CD3εδ single-chain fusion protein. These refolded porcine CD3 ectodomain molecules were purified with a strong anion exchange resin Poros 50HQ. ELISA analysis demonstrated that only the porcine CD3εγ ectodomain single-chain fusion protein can bind to the porcine CD3 specific monoclonal antibody 898H2-6-15. The availability of this porcine CD3εγ ectodomain single-chain fusion protein will allow screening for affinity matured variants of scFv derived from 898H2-6-15 to improve the recombinant anti-porcine CD3 immunotoxin. Porcine CD3εγ ectodomain single-chain fusion protein will also be a very useful reagent to study the soluble phase interaction between porcine CD3εγ and porcine CD3 antibodies such as 898H2-6-15.

Expression and purification of soluble porcine CTLA-4 in yeast Pichia pastoris.

Co-stimulation blockade can be used to modulate the immune response for induction of organ transplantation tolerance, treatment of autoimmune disease as well as cancer treatment. Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4), also known as CD152, is an important co-stimulatory molecule which serves as a negative regulator for T cell proliferation and differentiation. CTLA-4/CD28-CD80/CD86 pathway is a critical co-stimulatory pathway for adaptive immune response. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for CD80 and CD86. MGH MHC-defined miniature swine provide a unique large animal model useful for preclinical studies of transplantation tolerance and immune regulation. In this study, we have expressed the codon-optimized soluble porcine CTLA-4 in the yeast Pichia pastoris system. The secreted porcine CTLA-4 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50HQ. Glycosylation analysis using PNGase F demonstrated the N-linked glycosylation on P. pastoris expressed soluble porcine CTLA-4. To improve the expression level and facilitate the downstream purification we mutated the two potential N-linked glycosylation sites with non-polarized alanines by site-directed mutagenesis. Removal of the two N-glycosylation sites significantly improved the production level from ∼2 to ∼8mg/L. Biotinylated glycosylated and non-N-glycosylated soluble porcine CTLA-4 both bind to a porcine CD80-expressing B-cell lymphoma cell line (K(D)=13nM) and competitively inhibit the binding of an anti-CD80 monoclonal antibody. The availability of soluble porcine CTLA-4, especially the non-N-glycosylated CTLA-4, will provide a very valuable tool for assessing co-stimulatory blockade treatment for translational studies in the clinically relevant porcine model.

Expression and purification of non-N-glycosylated porcine interleukin 3 in yeast Pichia pastoris.

Yeast Pichia pastoris has been widely utilized to express heterologous recombinant proteins. P. pastoris expressed recombinant porcine interleukin 3 (IL3) has been used for porcine stem cell mobilization in allo-hematopoietic cell transplantation models and pig-to-primate xeno-hematopoietic cell transplantation models in our lab for many years. Since the yeast glycosylation mechanism is not exactly the same as those of other mammalian cells, P. pastoris expressed high-mannose glycoprotein porcine IL3 has been shown to result in a decreased serum half-life. Previously this was avoided by separation of the non-glycosylated porcine IL3 from the mixture of expressed glycosylated and non-glycosylated porcine IL3. However, this process was very inefficient and lead to a poor yield following purification. To overcome this problem, we engineered a non-N-glycosylated version of porcine IL3 by replacing the four potential N-glycosylation sites with four alanines. The codon-optimized non-N-glycosylated porcine IL3 gene was synthesized and expressed in P. pastoris. The expressed non-N-glycosylated porcine IL3 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50 HQ. In vivo mobilization studies performed in our research facility demonstrated that the non-N-glycosylated porcine IL3 still keeps the original stem cell mobilization function.

Expression and purification of soluble murine CD40L monomers and polymers in yeast Pichia pastoris.

The anti-murine CD40L monoclonal antibody MR1 has been widely used in immunology research to block the CD40-CD40L interaction for induction of transplantation tolerance and to abrogate autoimmune diseases. The availability of recombinant CD40L with high binding capacity for MR1 would provide a valuable immunologic research tool. In this study, we constructed the single chain murine soluble CD40L monomer, dimer, trimer and successfully expressed them in yeast Pichia pastoris under the control of the alcohol oxidase promoter. The secreted single chain murine soluble CD40L monomers, dimers, and trimers were initially enriched through histidine tag capture by Ni-Sepharose 6 fast flow resin and further purified on a cation exchange resin. Purity reached more than 95% for the monomer and dimer forms and more than 90% for the trimer. Protein yield following purification was 16 mg/L for the monomer and dimer, and 8 mg/L for the trimer. ELISA analysis demonstrated that the CD40L dimers and trimers correctly folded in conformations exposing the MR1 antigenic determinant.