Abstract: Disclosed herein are novel Pichia pastoris strains for expression of exogenous proteins with substantially homogeneous N-glycans. The strains are genetically engineered to include a mutant OCH1 allele which is transcribed into an mRNA coding for a mutant OCH1 gene product (i.e., ?-1,6-mannosyltransferase, or “OCH1 protein”). The mutant OCH1 protein contains a catalytic domain substantially identical to that of the wild type OCH1 protein, but lacks an N-terminal sequence necessary to target the OCH1 protein to the Golgi apparatus. The strains disclosed herein are robust, stable, and transformable, and the mutant OCH1 allele and the ability to produce substantially homogeneous N-glycans are maintained for generations after rounds of freezing and thawing and after subsequent transformations.

Abstract: This invention relates to engineered CH2 domain molecules containing amino acids in the framework regions that confer enhanced stability and/or solubility. In particular, the invention provides engineered CH2 domain molecules containing amino acid residues that differ from a wild type CH2 domain or a template CH2 domain molecule within one or more framework regions and that result in improved stability and/or solubility.

Abstract: Disclosed herein are novel Pichia pastoris strains for expression of exogenous proteins with substantially homogeneous N-glycans. The strains are genetically engineered to include a mutant OCH1 allele which is transcribed into an mRNA coding for a mutant OCH1 gene product (i.e., ?-1,6-mannosyltransferase, or “OCH1 protein”). The mutant OCH1 protein contains a catalytic domain substantially identical to that of the wild type OCH1 protein, but lacks an N-terminal sequence necessary to target the OCH1 protein to the Golgi apparatus. The strains disclosed herein are robust, stable, and transformable, and the mutant OCH1 allele and the ability to produce substantially homogeneous N-glycans are maintained for generations after rounds of freezing and thawing and after subsequent transformations.

Abstract: Disclosed herein are novel Pichia pastoris strains for expression of exogenous proteins with substantially homogeneous N-glycans. The strains are genetically engineered to include a mutant OCH1 allele which is transcribed into an mRNA coding for a mutant OCH1 gene product (i.e., ?-1,6-mannosyltransferase, or “OCH1 protein”). The mutant OCH1 protein contains a catalytic domain substantially identical to that of the wild type OCH1 protein, but lacks an N-terminal sequence necessary to target the OCH1 protein to the Golgi apparatus. The strains disclosed herein are robust, stable, and transformable, and the mutant OCH1 allele and the ability to produce substantially homogeneous N-glycans are maintained for generations after rounds of freezing and thawing and after subsequent transformations.

Abstract: The present disclosure is directed to a modified isolated immunoglobulin CH2 domain that specifically binds to an extracellular region of an EphA2 receptor, wherein the amino acid sequence of the modified immunoglobulin CH2 domain includes at least one amino acid substitution, addition or deletion in comparison to a wild type immunoglobulin CH2 domain amino acid sequence, wherein the wild type immunoglobulin CH2 domain amino acid sequence includes SEQ ID NO:1 or SEQ ID NO:2. Heterologous immunoconjugates including fusion proteins and pharmaceutical compositions including the modified isolated immunoglobulin CH2 domain are also disclosed. In addition, methods of treating a disease associated with EphA2 overexpression and methods for killing a target cell expressing EphA2 receptors using the modified isolated immunoglobulin CH2 domain are provided.

Abstract: Disclosed herein are novel Pichia pastoris strains for expression of exogenous proteins with substantially homogeneous N-glycans. The strains are genetically engineered to include a mutant OCH1 allele which is transcribed into an mRNA coding for a mutant OCH1 gene product (i.e., ?-1,6-mannosyltransferase, or “OCH1 protein”). The mutant OCH1protein contains a catalytic domain substantially identical to that of the wild type OCH1 protein, but lacks an N-terminal sequence necessary to target the OCH1 protein to the Golgi apparatus. The strains disclosed herein are robust, stable, and transformable, and the mutant OCH1 allele and the ability to produce substantially homogeneous N-glycans are maintained for generations after rounds of freezing and thawing and after subsequent transformations.

Abstract: Disclosed herein are novel Pichia pastoris strains for expression of exogenous proteins with substantially homogeneous N-glycans. The strains are genetically engineered to include a mutant OCH1 allele which is transcribed into an mRNA coding for a mutant OCH1 gene product (i.e., ?-1,6-mannosyltransferase, or “OCH1 protein”). The mutant OCH1 protein contains a catalytic domain substantially identical to that of the wild type OCH1 protein, but lacks an N-terminal sequence necessary to target the OCH1 protein to the Golgi apparatus. The strains disclosed herein are robust, stable, and transformable, and the mutant OCH1 allele and the ability to produce substantially homogeneous N-glycans are maintained for generations after rounds of freezing and thawing and after subsequent transformations.

Abstract: Disclosed herein are novel Pichia pastoris strains for expression of exogenous proteins with substantially homogeneous N-glycans. The strains are genetically engineered to include a mutant OCH1 allele which is transcribed into an mRNA coding for a mutant OCH1 gene product (i.e., ?-1,6-mannosyltransferase, or “OCH1 protein”). The mutant OCH1 protein contains a catalytic domain substantially identical to that of the wild type OCH1 protein, but lacks an N-terminal sequence necessary to target the OCH1 protein to the Golgi apparatus. The strains disclosed herein are robust, stable, and transformable, and the mutant OCH1 allele and the ability to produce substantially homogeneous N-glycans are maintained for generations after rounds of freezing and thawing and after subsequent transformations.

Abstract: The present invention provides modified fibronectin type III (Fn3) molecules, and nucleic acid molecules encoding the modified Fn3 molecules. Also provided are methods of preparing these molecules, and kits to perform the methods.

Abstract: Disclosed herein are novel Pichia pastoris strains for expression of exogenous proteins with substantially homogeneous N-glycans. The strains are genetically engineered to include a mutant OCH1 allele which is transcribed into an mRNA coding for a mutant OCH1 gene product (i.e., ?-1,6-mannosyltransferase, or “OCH1 protein”). The mutant OCH1 protein contains a catalytic domain substantially identical to that of the wild type OCH1 protein, but lacks an N-terminal sequence necessary to target the OCH1 protein to the Golgi apparatus. The strains disclosed herein are robust, stable, and transformable, and the mutant OCH1 allele and the ability to produce substantially homogeneous N-glycans are maintained for generations after rounds of freezing and thawing and after subsequent transformations.

Abstract: The present invention provides a fibronectin type III (Fn3) molecule, wherein the Fn3 contains a stabilizing mutation. The present invention also provides Fn3 polypeptide monobodies, nucleic acid molecules encoding monobodies, and variegated nucleic acid libraries encoding such monobodies. Also provided are methods of preparing a Fn3 polypeptide monobody, and kits to perform the methods.

Abstract: This disclosure relates to novel Pichia pastoris display systems, e.g., display systems featuring the Pichia pastoris strains (such as SuperMan5) with substantially homogeneous N-glycans displayed on cell surface proteins.

Abstract: The present application relates to modified T cell epitopes derived from fungal ribotoxins, including ?-sarcin, clavin, gigantin, mitogillin, and restrictocin, as well as modified ribotoxin molecules comprising one or more of the modified epitopes. The modified ribotoxin molecules inhibit protein synthesis, like the wild type ribotoxins, but exhibit reduced immunogenicity as compared to the corresponding wild type ribotoxin. Another aspect relates to a fusion protein which comprises a modified ribotoxin fused or conjugated or otherwise linked to a targeting molecule that is effective for binding a target of interest. Another aspect relates to the use of the modified ribotoxin or fusion protein for treating or managing a disease or condition.

Abstract: The present invention provides a fibronectin type III (Fn3) molecule, wherein the Fn3 contains a stabilizing mutation. The present invention also provides Fn3 polypeptide monobodies, nucleic acid molecules encoding monobodies, and variegated nucleic acid libraries encoding such monobodies. Also provided are methods of preparing a Fn3 polypeptide monobody, and kits to perform the methods.

Abstract: This disclosure features fusion proteins comprising a base protein linked to or incorporated in a CH2 scaffold of IgG. The CH2 scaffold can derive from the macaque CH2 domain of IgG. The fusion proteins can effectively bind a single or multiple targets, and can be engineered to regulate effector functions as desired. The fusion proteins can have an increased serum half-life, solubility, stability, protease resistance, and/or expression as compared to the scaffolds alone and/or as compared to the base protein alone. This disclosure also features fusion proteins comprising a base protein, a CH2 scaffold and a discrete polyethylene glycol (dPEG) linked to the scaffold via a serine, tyrosine, cysteine, lysine, or a glycosylation site of the scaffold. This disclosure additionally features scaffolds linked to a discrete polyethylene glycol (dPEG) via a serine, tyrosine, cysteine, or lysine of the scaffolds or a glycosylation site of the scaffold.

Abstract: This disclosure features fusion proteins comprising a base protein linked to or incorporated in a CH2 scaffold of IgG. The CH2 scaffold can derive from the macaque CH2 domain of IgG. The fusion proteins can effectively bind a single or multiple targets, and can be engineered to regulate effector functions as desired. The fusion proteins can have an increased serum half-life, solubility, stability, protease resistance, and/or expression as compared to the scaffolds alone and/or as compared to the base protein alone. This disclosure also features fusion proteins comprising a base protein, a CH2 scaffold and a discrete polyethylene glycol (dPEG) linked to the scaffold via a serine, tyrosine, cysteine, lysine, or a glycosylation site of the scaffold. This disclosure additionally features scaffolds linked to a discrete polyethylene glycol (dPEG) via a serine, tyrosine, cysteine, or lysine of the scaffolds or a glycosylation site of the scaffold.

Abstract: Disclosed herein are novel Pichia pastoris strains for expression of exogenous proteins with substantially homogeneous N-glycans. The strains are genetically engineered to include a mutant OCH1 allele which is transcribed into an mRNA coding for a mutant OCH1 gene product (i.e., ?-1,6-mannosyltransferase, or “OCH1 protein”). The mutant OCH1protein contains a catalytic domain substantially identical to that of the wild type OCH1 protein, but lacks an N-terminal sequence necessary to target the OCH1 protein to the Golgi apparatus. The strains disclosed herein are robust, stable, and transformable, and the mutant OCH1 allele and the ability to produce substantially homogeneous N-glycans are maintained for generations after rounds of freezing and thawing and after subsequent transformations.

Abstract: Disclosed herein are novel Pichia pastoris strains for expression of exogenous proteins with substantially homogeneous N-glycans. The strains are genetically engineered to include a mutant OCH1 allele which is transcribed into an mRNA coding for a mutant OCH1 gene product (i.e., ?-1,6-mannosyltransferase, or “OCH1 protein”). The mutant OCH1 protein contains a catalytic domain substantially identical to that of the wild type OCH1 protein, but lacks an N-terminal sequence necessary to target the OCH1 protein to the Golgi apparatus. The strains disclosed herein are robust, stable, and transformable, and the mutant OCH1 allele and the ability to produce substantially homogeneous N-glycans are maintained for generations after rounds of freezing and thawing and after subsequent transformations.

Abstract: The present application relates to modified T cell epitopes derived from fungal ribotoxins, including a-sarcin, clavin, gigantin, mitogillin, and restrictocin, as well as modified ribotoxin molecules comprising one or more of the modified epitopes. The modified ribotoxin molecules inhibit protein synthesis, like the wild type ribotoxins, but exhibit reduced immunogenicity as compared to the corresponding wild type ribotoxin. Another aspect relates to a fusion protein which comprises a modified ribotoxin fused or conjugated or otherwise linked to a targeting molecule that is effective for binding a target of interest. Another aspect relates to the use of the modified ribotoxin or fusion protein for treating or managing a disease or condition.

Abstract: Viable Gram-negative bacteria or components thereof comprising outer membranes that substantially lack a ligand, such as Lipid A or 6-acyl lipidpolysaccharide, that acts as an agonist of TLR4/MD-2. The bacteria may comprise reduced activity of arabinose-5-phosphate isomerases and one or more suppressor mutations, for example in a transporter thereby increasing the transporter's capacity to transport lipid IVA or in membrane protein YhjD. One or more genes (e.g., lpxL, lpxM, pagP, lpxP, and/or eptA) may be substantially deleted and/or one or more enzymes (e.g., LpxL, LpxM, PagP, LpxP, and/or EptA) may be substantially inactive. The bacteria may be competent to take up extracellular DNA, may be donor bacteria, or may be members of a library. The present invention also features methods of creating and utilizing such bacteria.