Antibody Generation: challenges and solutions Glen Marszalowicz, PHD May 10, 2015 9AM GenScript – the most cited biology CRO Gene Services Peptide Services CRISPR Services Protein Services Antibody Services Discovery Biology Catalog Products Services Make Research Easy 2 Antibody Generation: challenges and solutions 1 Antibody applications 2 Traditional antibody generation 3 3 Common challenges and solutions 4 Related antibody generation services Make Research Easy 3 Antibody applications Research • Track biomarker location, expression -Enzyme linked immunosorbent assay(ELISA) -Fluorescent activated cell sorting (FACS) -Western Blot -Immunohistochemistry (IHC), Immunocytochemistry (ICC) -Neutralize receptors/ligands in lieu of knock-out systems Industry • Traditional assays, but also - Therapeutic Antibody Drugs - Antibody Drug Conjugates (ADCs) - Recombinant antibodies (rAbs) result in many formats Make Research Easy 4 Antibody Generation: challenges and solutions 1 Antibody applications 2 Traditional antibody generation 3 3 Common challenges and solutions 4 Related antibody generation services Make Research Easy 5 How are antibodies generated Antibodies are created in response to foreign (non-self) antigen via the adaptive immune system • B-cells recognize foreign antigens and, with further activation by Helper T cells, release antibodies to that antigen. B-cell recognizes Cognate helper T-cell Transformation to plasma cell exogenous antigen activates through cytokine signaling and antibody secretion Make Research Easy 6 Creating an immune response Primary immunization (prime) • Inject antigen, with carrier protein or adjuvant if required Measure response via ELISA Provided additional exposures (boost) with antigen in same or in various format (stable cell expressing, alternative carrier protein, etc.) Measure response via ELISA If satisfied with serum titer, proceed to antibody recovery Make Research Easy 7 How to recover antibodies Polyclonal antibodies: • Collect serum • (optional) purify by protein A/G or antigen affinity Monoclonal antibodies • Create hybridomas • Reach clonality through limiting dilutions • Select for positive clones • Expand, produce, purify, and preserve Make Research Easy 8 Creating hybridomas Lymphocyte fusion with myelomas via electrofusion. Selection of only lymphocyte/myeloma fusion products through HAT medium Make Research Easy 9 Achieving monoclonality Repetitive limiting dilutions statistically insures a single cell/well Selection of positive clones via ELISA Make Research Easy 10 Antibody Generation: challenges and solutions 1 Antibody applications 2 Traditional antibody generation 3 3 Common challenges and solutions 4 Related antibody generation services Make Research Easy 11 Challenges with Antibody Generation Antibody generation requires many steps, therefore many chances for difficulty including: Antigen Production • Difficult to produce antigens Immune response • Self-tolerance Final product • Low affinity antibodies Make Research Easy 12 Challenge: Difficult Antigens Possible antigen production difficulties • Low yield- not enough antigen produced • Low stability- antigen does not survive long enough for durable exposure • Low solubility- achieving concentrations necessary for immunization are difficult • Complexity- ex; GPCRs, ion channels Additional antigen issues • Toxicity- exposure harms or kills host Make Research Easy 13 Solution to difficult antigens: DNA Immunization Monoclonal Antibody Development Fusion & Screening Immune Response Immunization Cloning Target cDNA In vivo expression overcomes antigen generation issues. Make Research Easy 14 Case Study: GLP1R mAb by DNA immunization FACS ELISA Glucagon-like peptide receptor-1 (GLP1R): • 3 Disulfide bonds • N-terminal glycosylation Western Blot ICC Make Research Easy 15 Challenge: self-tolerance (to high homology antigens) Make Research Easy 16 Solution to self-tolerance Modify immunization such that signaling mimics foreign antigen exposure • GenScript proprietary ImmunoPlus™ technology Overcomes self tolerance through antigen modification Make Research Easy 17 Challenge: low affinity antibodies Despite high serum and hybridoma supernatant titers during initial screening, ultimate affinity is unknown until sufficient antibodies quantities can be generated Only low affinity antibodies may be recovered at which time the opportunity to generate high affinity antibodies in vivo is lost. Make Research Easy 18 Solution to low affinity antibodies Germinal Center Associated Nuclear Protein (GANP) transgenic mice B-cells experience somatic hypermutation (SHM) which create a wider variety of variable domains. Antibodies have greater chances of subtle changes in the complemetarity determining region (CDR), increasing the opportunity to recover high affinity antibodies. Make Research Easy 19 Conclusions Antibody generation has may steps for potential difficulties, but also many opportunities for optimization Novel techniques such as DNA immunization, immunomodulation, and GANP mice offer elegant solutions to previously insurmountable problems Immunization approach must be tailored to fit the characteristics of the target Ultimately, collaborating or outsourcing to experience hands can expedite the process Make Research Easy 20 Antibody Generation: challenges and solutions 1 Antibody applications 2 Traditional antibody generation 3 3 Common challenges and solutions 4 Related antibody generation services Make Research Easy 21 GenScript’s antibody generation solutions •antibodies in 45 days with just your target Polyclonal sequence starting at $649. •high quality, purified monoclonal antibodies Monoclonal guaranteed to work in your application Phospho- •monoclonal or polyclonal with <10% non- specific phospho peptide cross-reactivity guaranteed •Ideal for studying PK/PD of therapeutic Anti-idiotype antibodies DNA Immunization Applicable to ImmunoPlus™ any antibody GANP® Mice project! Make Research Easy 22 Q&A Thank you for attending! Type your questions into the “Questions” field on your Go to Webinar toolbar Please complete the survey you receive by email Check out upcoming and archived webinars at www.genscript.com/webinars.html Email me at [email protected] Make Research Easy 23 .