ABSTRACT
KEY MESSAGE: A simple and versatile ternary vector system that utilizes improved accessory plasmids for rapid maize transformation is described. This system facilitates high-throughput vector construction and plant transformation. The super binary plasmid pSB1 is a mainstay of maize transformation. However, the large size of the base vector makes it challenging to clone, the process of co-integration is cumbersome and inefficient, and some Agrobacterium strains are known to give rise to spontaneous mutants resistant to tetracycline. These limitations present substantial barriers to high throughput vector construction. Here we describe a smaller, simpler and versatile ternary vector system for maize transformation that utilizes improved accessory plasmids requiring no co-integration step. In addition, the newly described accessory plasmids have restored virulence genes found to be defective in pSB1, as well as added virulence genes. Testing of different configurations of the accessory plasmids in combination with T-DNA binary vector as ternary vectors nearly doubles both the raw transformation frequency and the number of transformation events of usable quality in difficult-to-transform maize inbreds. The newly described ternary vectors enabled the development of a rapid maize transformation method for elite inbreds. This vector system facilitated screening different origins of replication on the accessory plasmid and T-DNA vector, and four combinations were identified that have high (86-103%) raw transformation frequency in an elite maize inbred.
Subject(s)
Genetic Vectors , Transformation, Genetic , Zea mays/genetics , Agrobacterium tumefaciens/genetics , DNA, Bacterial , DNA, Plant , Plasmids , Replication OriginABSTRACT
The CTLA4-Ig therapeutics abatacept and belatacept inhibit CD28-mediated T cell activation by binding CD80 (B7-1) and CD86 (B7-2) co-stimulatory ligands. Both compounds preferentially bind CD80, yet CD86 has been implicated as the dominant co-stimulatory ligand. Using directed evolution methods, novel CTLA4-Ig variants were created with selective CD86 binding affinity, a property that confers increased immunosuppressive potency and potentially improved efficacy and safety profiles. Relative to abatacept (wild-type CTLA4-Ig), ASP2408 and ASP2409 have 83-fold and 220-fold enhanced binding affinity to CD86 while retaining 1.5-fold and 5.6-fold enhanced binding affinity to CD80, respectively. Improvements in CD86 binding affinity correlates with increased immunosuppressive potencyin vitroandin vivo Our results highlight the power of directed evolution methods to obtain non-intuitive protein engineering solutions and represent the first examples of CD86-selective CTLA4-Ig compounds that have entered clinical trials.
Subject(s)
Abatacept/genetics , Abatacept/pharmacology , B7-2 Antigen/metabolism , Directed Molecular Evolution , Immunoconjugates/metabolism , Immunoconjugates/pharmacology , Immunosuppressive Agents/metabolism , Immunosuppressive Agents/pharmacology , Abatacept/chemistry , Abatacept/metabolism , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , Female , Humans , Immunoconjugates/chemistry , Immunosuppressive Agents/chemistry , Ligands , Mice , Models, Molecular , Protein Conformation , Substrate SpecificityABSTRACT
Type I IFNs are unusually pleiotropic cytokines that bind to a single heterodimeric receptor and have potent antiviral, antiproliferative, and immune modulatory activities. The diverse effects of the type I IFNs are of differential therapeutic importance; in cancer therapy, an enhanced antiproliferative effect may be beneficial, whereas in the therapy of viral infections (such as hepatitis B and hepatitis C), the antiproliferative effects lead to dose limiting bone marrow suppression. Studies have shown that various members of the natural IFN-alpha family and engineered variants, such as IFN-con1, vary in the ratios between various IFN-mediated cellular activities. We used DNA shuffling to explore and confirm the hypothesis that one could simultaneously increase the antiviral and Th1-inducing activity and decrease the antiproliferative activity. We report IFN-alpha hybrids wherein the ratio of antiviral:antiproliferative and Th1-inducing: antiproliferative potencies are markedly increased with respsect to IFN-con1 (75- and 80-fold, respectively). A four-residue motif that overlaps with the IFNAR1 binding site and is derived by cross breeding with a pseudogene contributes significantly to this phenotype. These IFN-alphas have an activity profile that may result in an improved therapeutic index and, consequently, better clinical efficacy for the treatment of chronic viral diseases such as hepatitis B virus, human papilloma virus, HIV, or chronic hepatitis C.