The presently disclosed subject matter relates to “Randomized Configuration Targeted Integration” (also referred to herein as “Randomized Chain Targeted Integration”) (RCTI) strategies for the generation and identification of host cells capable of expressing recombinant proteins, e.g., monoclonal antibodies, as well as compositions derived from the same, e.g., bispecific antibodies, and other complex format proteins, e.g., membrane protein complexes and other difficult to express molecules.

Provided herein are methods that enable parallel evaluation of multiple functional nucleic acids in individual cells or subpopulations of cells, in the context of incubation with other types of single cells. The key insight is concurrent measurement of polynucleic acids derived from small populations of at least two different cell types, such that function in one cell type is linked to the clonal identity of another cell. These methods simultaneously process thousands, millions, or more single cells or small populations of cells. The method integrates molecular, algorithmic, and engineering approaches. This invention has broad and useful application in a number of biological and medical fields, including immunology and drug discovery.

Provided are methods and compositions for the production of novel antibodies that bind specifically to a target antigen. These methods and compositions are particularly useful for producing antibodies having the antigen binding specificity of a reference antibody but with improved properties (e.g., binding affinity, immunogenicity, and thermodynamic stability) relative to the reference antibody.

A method of broadening epitopic coverage of an antigen of interest, wherein a first sample of the antigen of interest is contacted with a first plurality of host cells collectively expressing a first library of antibodies. Host cells expressing antibodies that bind to the antigen are then collected from among the first plurality of host cells, and a composition is prepared comprising a polyclonal mixture of antibodies expressed by these host cells. A second sample of the antigen of interest is then contacted with an aliquot of the prepared composition and a second plurality of host cells collectively expressing a second library of antibodies. Host cells expressing antibodies that bind to the second sample of the antigen are then collected from among the second plurality of host cells.

Isolated or recombinant EphA5 or GRP78 targeting antibodies are provided. In some cases, antibodies of the embodiments can be used for the detection, diagnosis and/or therapeutic treatment of human diseases, such as cancer. A method of rapidly identifying antibodies or antibody fragments for the treatment of cancer using a combination of in vitro and in vivo methodologies is also provided.

The present invention relates to a method for engineering an immunoglobulin comprising a variable domain and at least one modification in at least two structural loops of said immunoglobulin and determining the binding of said immunoglobulin to an epitope of an antigen, wherein the unmodified immunoglobulin does not significantly bind to said epitope, comprising the steps of: providing a nucleic acid encoding an immunoglobulin comprising at least two structural loops, modifying at least one nucleotide residue of each of said structural loops, transferring said modified nucleic acid in an expression System, expressing said modified immunoglobulin, contacting the expressed modified immunoglobulin with an epitope, and determining whether said modified immunoglobulin binds to said epitope, immunoglobulins produced by such a method and libraries of immunoglobulins.

The present invention relates to a method for producing a population of 20 or more nucleic acids, each encoding at least one protein comprising at least one immunoglobulin variable domain having a rabbit-derived CDR3 amino acid sequence embedded in essentially human framework sequences, as well as to a population of nucleic acids and a population of proteins relates thereto and uses thereof.

The invention provides singleplex and multiplex assays for screening of antigen-binding molecules for their affinity to antigens by normalizing for the concentration of the antigen-binding molecule.

The present invention relates to a method for preparing a novel antibody library and a library prepared thereby. The antibody library prepared according to the present invention contains antibodies having excellent physical properties against a plurality of antigens, thereby having functional diversity and containing a plurality of unique sequences, and thus can be favorably used as an antibody library.

Provided herein are antibodies with conditional affinity for an antigen. The affinity of an antibody with conditional affinity for an antigen may be altered by a small molecule agent. Antibodies with conditional affinity may be used therapeutically alone or in combination with other therapeutics to treat cancer and other diseases.