Abstract: Provided are ciliary neurotrophic factor receptor (CNTFR) ligands. In certain aspects, a CNTFR ligand of the present disclosure exhibits increased affinity for CNTFR relative to the corresponding wild-type CNTFR ligand. In certain aspects, a CNTFR ligand of the present disclosure results in reduced binding affinity of glycoprotein 130 (gp130), leukemia inhibitory factor receptor (LIFR), or both, for a complex including the CNTFR ligand and CNTFR, relative to the binding affinity for a complex including the corresponding wild-type CNTFR ligand and CNTFR. In certain aspects, a CNTFR ligand of the present disclosure has both of the aforementioned properties. Also provided are pharmaceutical compositions including the CNTFR ligands, as well as methods of using the CNTFR ligands.

Abstract: Compositions and methods are provided for alleviating cancer in a mammal by administering a therapeutic dose of a pharmaceutical composition that inhibits activity of AXL, MER or Tyro3 protein activity, for example by competitive or non-competitive inhibition of the binding interaction between AXL, MER or Tyro3 and its ligand GAS6.

Abstract: Provided are soluble leukemia inhibitory factor receptor (LIFR) polypeptides, soluble glycoprotein 130 (gp130) polypeptides, and soluble fusion proteins and dimers including such polypeptides. The soluble polypeptides bind to leukemia inhibitory factor (LIF). In certain aspects, the soluble polypeptides exhibit increased binding affinity for LIF relative to the corresponding wild-type polypeptides. Also provided are nucleic acids encoding such soluble polypeptides, expression vectors including such nucleic acids, and cells including such nucleic acids and/or expression vectors. Methods of using the soluble polypeptides, including methods of inhibiting LIF activity in an individual in need thereof (e.g., to treat cancer), are also provided.

Abstract: The present invention provides a method of treating cancer with a combination of IL-2 and an integrin-binding-Fc fusion protein. The methods of the invention can be applied to a broad range of cancer types.

Abstract: High-throughput methods for screening large populations of variant proteins are provided. The methods utilize large-scale arrays of microcapillaries, where each microcapillary comprises a solution containing a variant protein, an immobilized target molecule, and a reporter element. Immobilized target molecules may include any molecule of interest, including proteins, nucleic acids, carbohydrates, and other biomolecules. The association of a variant protein with a molecular target is assessed by measuring a signal from the reporter element. The contents of microcapillaries identified in the assays as containing variant proteins of interest can be isolated, and cells expressing the variant proteins of interest can be characterized. Also provided are systems for performing the disclosed screening methods.

Abstract: The present disclosure presents a general approach to engineering existing protein-protein interactions through domain addition and evolution. The disclosure teaches the creation of novel fusion proteins that include knottin peptides where a portion of the knottin peptide is replaced with a sequence that has been created for binding to a particular target. Such fusion proteins can also be bispecific or multi specific in that they can bind to and/or inhibit two or more receptors or receptor ligands. Knottins may be fused with an existing ligand (or receptor) as a general platform tor increasing the affinity of a ligand-receptor interaction or for creating a multi specific protein. In addition, the fusion proteins may comprise a knottin peptide fused to another protein where the other protein facilitates proper expression and folding of the knottin.

Abstract: The present invention provides a method of treating cancer with an integrin-binding-Fc fusion protein alone or in combination with IL-2 and/or an immune stimulant (i.e., an immune checkpoint stimulator), and/or an immune checkpoint inhibitor. The invention also provides composition for use in such methods.

Abstract: Engineered peptides that bind with high affinity (low equilibrium dissociation constant (Kd)) to the cell surface receptors of fibronectin (?5?1) or vitronectin (?v?3 and ?v?5 integrins) are disclosed as useful as imaging tissue. These peptides are based on a molecular scaffold into which a subsequence containing the RGD integrin-binding motif has been inserted. The subsequence (RGD mimic) comprises about 9-13 amino acids, and the RGD contained within the subsequence can be flanked by a variety of amino acids, the sequence of which was determined by sequential rounds of selection (in vitro evolution). The molecular scaffold is preferably based on a knottin, e.g., EETI (Trypsin inhibitor 2 (Trypsin inhibitor II) (EETI-II) [Ecballium elaterium (Jumping cucumber)], AgRP (Agouti-related protein), and Agatoxin IVB, which peptides have a rigidly defined three-dimensional conformation. It is demonstrated that EETI tolerates mutations in other loops and that the present peptides may be used as imaging agents.

Abstract: The present invention provides a method of treating cancer with a combination of IL-2 (e.g., extended-PK IL-2), an integrin-binding-Fc fusion protein, and a cancer vaccine. The methods of the invention can be used to treat a broad range of cancer types.

Abstract: Provided are knottin-drug conjugates. The conjugates include a knottin peptide that includes an engineered loop that binds to a target on a cancer cell surface, and a drug (e.g., a nucleoside drug) conjugated to the knottin peptide through a linker. Also provided are pharmaceutical compositions and kits that include the knottin-drug conjugates, as well as methods of using the knottin-drug conjugates, e.g., for therapeutic purposes.

Abstract: The present disclosure presents a general approach to engineering existing protein-protein interactions through domain addition and evolution. The disclosure teaches the creation of novel fusion proteins that include knottin peptides where a portion of the knottin peptide is replaced with a sequence that has been created for binding to a particular target. Such fusion proteins can also be bispecific or multi specific in that they can bind to and/or inhibit two or more receptors or receptor ligands. Knottins may be fused with an existing ligand (or receptor) as a general platform for increasing the affinity of a ligand-receptor interaction or for creating a multi specific protein. In addition, the fusion proteins may comprise a knottin peptide fused to another protein where the other protein facilitates proper expression and folding of the knottin.

Abstract: Microcavity arrays and methods for quantitative biochemical and biophysical analysis of populations of biological variants. Examples include high-throughput analysis of cells and protein products use a range of fluorescent assays, including binding-affinity measurement and time-resolved enzyme assays. Laser-based extraction of microcavity contents.

Abstract: Provided are knottin-drug conjugates. The conjugates include a knottin peptide that includes an engineered loop that binds to a target on a cancer cell surface, and a drug (e.g., a nucleoside drug) conjugated to the knottin peptide through a linker. Also provided are pharmaceutical compositions and kits that include the knottin-drug conjugates, as well as methods of using the knottin-drug conjugates, e.g., for therapeutic purposes.

Abstract: Disclosed are knottin peptides containing non-natural amino acids so that they can be formed by chemical conjugation into two or more knottin monomers. The knottin monomers comprise a non-natural amino acid such as an aminooxy residue within the polypeptide sequence. The exemplified dimers were produced by oxime formation between two aldehyde groups present on a polyether linker and an aminooxy functional group that was site-specifically incorporated the knottin. Knottins variants based on EETI (Ecballium elaterium trypsin inhibitor) and AgRP (Agouti-related protein) were engineered to contain integrin-binding loops. These dimers were shown to have increased binding strength to integrins on U87MG tumor cells, achieving significant increases in inhibition of cell adhesion and proliferation. Also disclosed are knottin monomers comprising an aminooxy residue; these may be conjugated to molecules such as doxorubicin.

Abstract: The present invention provides a method of treating cancer with an integrin-binding-Fc fusion protein alone or in combination with IL-2 and/or an immune stimulant (i.e., an immune checkpoint stimulator), and/or an immune checkpoint inhibitor. The invention also provides composition for use in such methods.

Abstract: High-throughput methods for screening large populations of variant proteins are provided. The methods utilize large-scale arrays of microcapillaries, where each microcapillary comprises a solution containing a variant protein, an immobilized target molecule, and a reporter element. Immobilized target molecules may include any molecule of interest, including proteins, nucleic acids, carbohydrates, and other biomolecules. The association of a variant protein with a molecular target is assessed by measuring a signal from the reporter element. The contents of microcapillaries identified in the assays as containing variant proteins of interest can be isolated, and cells expressing the variant proteins of interest can be characterized. Also provided are systems for performing the disclosed screening methods.

Abstract: Microcavity arrays and methods for quantitative biochemical and biophysical analysis of populations of biological variants. Examples include high-throughput analysis of cells and protein products use a range of fluorescent assays, including binding-affinity measurement and time-resolved enzyme assays. Laser-based extraction of microcavity contents.

Abstract: Provided are agents that specifically bind a ligand of ciliary neurotrophic factor receptor (CNTFR). In certain aspects, an agent of the present disclosure is a soluble CNTFR polypeptide. The soluble CNTFR polypeptide may have an altered (e.g., reduced) binding affinity for one or more ligand-CNTFR complex subunits, an altered (e.g., increased) binding affinity for one or more CNTFR ligands, or any combination thereof. Compositions that include the agents of the present disclosure are also provided, as are methods of using the agents (e.g., for treating a cell proliferative disorder) and methods of identifying an individual as having a cell proliferative disorder associated with CNTFR signaling.

Abstract: The present invention provides a method of treating cancer with a combination of IL-2 and an integrin-binding-Fc fusion protein. The methods of the invention can be applied to a broad range of cancer types.

Abstract: Provided are ciliary neurotrophic factor receptor (CNTFR) ligands. In certain aspects, a CNTFR ligand of the present disclosure exhibits increased affinity for CNTFR relative to the corresponding wild-type CNTFR ligand. In certain aspects, a CNTFR ligand of the present disclosure results in reduced binding affinity of glycoprotein 130 (gp130), leukemia inhibitory factor receptor (LIFR), or both, for a complex including the CNTFR ligand and CNTFR, relative to the binding affinity for a complex including the corresponding wild-type CNTFR ligand and CNTFR. In certain aspects, a CNTFR ligand of the present disclosure has both of the aforementioned properties. Also provided are pharmaceutical compositions including the CNTFR ligands, as well as methods of using the CNTFR ligands.