Conditional PD-1/PD-L1 Probody Therapeutics Induce Comparable Antitumor Immunity but Reduced Systemic Toxicity Compared with Traditional Anti-PD-1/PD-L1 Agents.

Immune-checkpoint blockade has revolutionized cancer treatment. However, most patients do not respond to single-agent therapy. Combining checkpoint inhibitors with other immune-stimulating agents increases both efficacy and toxicity due to systemic T-cell activation.

ASP2408 and ASP2409, novel CTLA4-Ig variants with CD86-selective ligand binding activity and improved immunosuppressive potency, created by directed evolution.

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.

Directed molecular evolution improves the immunogenicity and protective efficacy of a Venezuelan equine encephalitis virus DNA vaccine.

We employed directed molecular evolution to improve the cross-reactivity and immunogenicity of the Venezuelan equine encephalitis virus (VEEV) envelope glycoproteins. The DNA encoding the E1 and E2 proteins from VEEV subtypes IA/B and IE, Mucambo virus (MUCV), and eastern and western equine encephalitis viruses (EEEV and WEEV) were recombined in vitro to create libraries of chimeric genes expressing variant envelope proteins. ELISAs specific for all five parent viruses were used in high-throughput screening to identify those recombinant DNAs that demonstrated cross-reactivity to VEEV, MUCV, EEEV, and WEEV after administration as plasmid vaccines in mice.

DNA shuffling and screening strategies for improving vaccine efficacy.

The efficacy of vaccines can be improved by increasing their immunogenicity, broadening their crossprotective range, as well as by developing immunomodulators that can be coadministered with the vaccine antigen. One technology that can be applied to each of these aspects of vaccine development is MolecularBreeding directed molecular evolution. Essentially, this technology is used to evolve genes in vitro through an iterative process consisting of recombinant generation followed by selection of the desired recombinants.

Yeast Mn2+ transporter, Smf1p, is regulated by ubiquitin-dependent vacuolar protein sorting.

Conditional cdc1(Ts) mutants of S. cerevisiae arrest with a phenotype similar to that exhibited by Mn(2+)-depleted cells. Sequence similarity between Cdc1p and a class of Mn(2+)-dependent phosphoesterases, as well as the observation that conditional cdc1(Ts) growth can be ameliorated by Mn(2+) supplement, suggests that Cdc1p activity is sensitive to intracellular Mn(2+) levels.

Effects of a gerF (lgt) mutation on the germination of spores of Bacillus subtilis.

One of the proteins of the membrane-bound receptors that recognize individual nutrients that trigger germination of spores of Bacillus subtilis contains the recognition sequence for diacylglycerol addition to a cysteine residue near the protein's N terminus. B. subtilis spores lacking the gerF (lgt) gene that codes for prelipoprotein diacylglycerol transferase exhibited significantly slowed germination in response to nutrient germinants as found previously, but germination of gerF spores with a mixture of Ca2+ and dipicolinic acid or with dodecylamine was normal, as was the spontaneous germination of gerF spores lacking all nutrient germinant receptors.

Mechanisms of induction of germination of Bacillus subtilis spores by high pressure.

Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid.