Kalobios Pharmaceuticals, Inc.

Kalobios, Inc.

Other

Leland Stanford Junior University

Robert F Balint

Jeng-Horng Her

Christopher Robert Bebbington

John P Cooke

Mitchell Wayne Mutz

Geoffrey T Yarranton

Helen M Blau

Peter Flynn

Kenneth Luehrsen

Thomas S Wehrman

Methods and compositions for detecting molecular interactions, particularly protein-protein interactions, using at least two inactive, weakly-complementing &#x3b2;-lactamase fragments are provided. The invention allows detection of such interactions in eukaryotic and mammalian cells, in situ or in vitro. Detection of molecular interactions in mammalian cells is not limited to the nuclear compartment, but can be accomplished in the cytoplasm, cell surface, organelles, or between these entities. Methods provided utilize novel compositions comprising fusion proteins between molecules of interest and inactive, weakly-complementing &#x3b2;-lactamase fragments. Association of the molecules of interest brings the corresponding complementary &#x3b2;-lactamase fragments into close enough proximity for complementation to occur and &#x3b2;-lactamase activity to be observed. The invention is useful in the study of protein-protein interactions, functional genomics, agonist and antagonist screening and drug discovery.

Detection of molecular interactions by &#x3b2;-lactamase reporter fragment complementation

The present invention provides methods of making antibodies having the binding specificity of a reference antibody. Antibodies generated by the methods of the inventions have at least one minimal essential binding specificity determinant from a heavy chain or light chain CDR3 from the reference antibody. The method can be used, e.g., in humanization procedures. The invention also provides libraries and antibodies made in accordance with the methods.

Antibody specificity transfer using minimal essential binding determinants

Interaction-activated circularly permutated proteins are disclosed that depend for their functional reassembly into the parent protein on the interaction of heterologous polypeptides or other molecules which have been genetically or chemically conjugated to the break-point termini of engineered enzymes. In addition, methods are provided for identifying circularly permutated marker proteins that will optimally reassemble into a functional parent protein, and which are dependent on the association of heterologous interactor domains. The invention is exemplified by circular permutations of a Class A &#x3b2;-lactamase (TEM-1 of). Circularly permutated marker proteins that comprise molecular interaction-dependent enzymes particularly find use in (1) cell-based sensors for activation or inhibition of metabolic or signal transduction pathways for high-efficiency, (2) high-throughput screening for agonists/antagonists of the target pathway and in high-throughput mapping of pair-wise protein-protein interactions within and between the proteomes of cells, tissues, and pathogenic organisms, and in (3) cell-based screens for high-throughput selection of inhibitors of any protein-protein interaction.

Circularly permutated, interaction-activated proteins

This invention provides methods of obtaining a binding molecule, e.g., an antibody, that has enhanced affinity for a binding partner relative to a reference binding molecule.

Methods for affinity maturation

This invention provides methods and systems for linking the functional activation of a responder molecule to the interaction of two or more binding ensemble members of interest either in vitro or in vivo, and thereby producing a signal, phenotype, or other functional output in response to the interaction of the binding ensemble members. The systems comprise a responder, an inhibitor of the responder, and an inhibitor of the inhibitor, or 'reactivator' of the responder, and interacting components. Two binding ensemble members are complexed to the other components of the system in such a way that interaction of the binding ensemble members, either directly or via additional ensemble members, causes a shift in the equilibrium of inhibitor binding from the responder to the reactivator, thereby functionally activating the responder.

Reactivation-based molecular interaction sensors

ADMA is determined in a physiological sample by first removing interfering components: proteins by precipitation; and amines and citrulline, by means of a cation exchange column, followed by enzymatic hydrolysis of the ADMA to citrulline with DDAH. The citrulline is then determined spectrophotometrically.

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Palo Alto, CA, United States of America

Robert F Balint