2 - Project Summary Antibody Humanization.docx

Project Summary Antibody Humanization Antibody:

For each antibody:

Project_1: Parental Cyno Fv / Human Fc IgG1 - Minimum of 12 humanized IgG variants that aim to optimize expression in mammalian cells, stability and solubility, and minimize aggregation. An LS mutation will be inserted in the Fc region of the IgG1 to increase the half-life of the molecule.

Project_2: Parental Human patient Fv / Human Fc IgG1 - Minimum of 12 humanized IgG variants that aim to optimize expression in mammalian cells, stability and solubility, and minimize aggregation. An LS mutation will be inserted in the Fc region of the IgG1 to increase the half-life of the molecule.

Scale: Initially 0.5-1.0mg for each antibody Use: for In Vitro Purification: Protein A and buffer exchange Final concentration: 1mg/ml Final buffer: PBS Analytics: Sequence liabilities (in silico), Titre (Protein A HPLC), Purity (SDS-PAGE), Aggregate content (SEC-HPLC) Shipment: Priority overnight shipment on ice packs Sample Submission: Directions on how and where samples should be submitted/shipped for processing.

Please include an estimated timeline for each phase:

Phase One:

Phase Two:

Phase Three:

Phase Four:

Deliverables:

Deliverables of Phase One: Sequencing report containing DNA and protein sequences of VH and VL domains.

Deliverables of Phase Two: In silico humanization report including sequence liability analysis Protein VH and VL sequences for 12-16 humanized variants Deliverables of Phase Three: Approximately 1mg of each antibody. Report containing expression and aggregation data for each antibody. Report including DNA and protein sequences for the VH and VL domains of the antibodies produced.

Phases of Project:

Phase One: Hybridoma sequencing The antibody heavy and light variable domains of the hybridoma clone will be sequenced by whole transcriptome shotgun sequencing. If the client already has the sequences for their antibody this stage will be omitted.

Phase Two: In silico humanization A model of the parental VH and VL domains will be produced and used to guide the humanization process. The original mouse sequences will be aligned to all human germline sequences. The original mouse and closest matching germline sequences will be analyzed for sequence liabilities and the most appropriate germline frameworks selected. Complementarity determining regions (CDRs) from the parent antibody will be grafted onto an appropriate number of human frameworks and back mutations introduced as necessary. A minimum of 12 and maximum of 16 variants will be designed.

Phase Three: Antibody production Variable domains for the mouse and humanized sequences will be designed and optimized for expression in mammalian cells (HEK293) prior to being synthesized. The sequences will then be subcloned into an Absolute Antibody cloning and expression vector for the appropriate isotype and subtype of immunoglobulin heavy and light chains. HEK293 cells will be passaged to the optimum stage for transient transfection. Cells will be transiently transfected with heavy and light chain expression vectors and cultured for a further 6-14 days. An appropriate volume of cells will be transfected to obtain approximately 0.5-1.0 mg of antibody. Cultures will be harvested, and a one-step purification performed using affinity chromatography. Antibody concentration will be determined by UV spectroscopy. Antibody yield will be determined for all antibodies and aggregation levels will be measured by SEC-HPLC. A full report will be issued that includes a sequencing report containing DNA and protein sequences for the VH and VL domains of the antibodies produced.