Antibody Generation: challenges and solutions. Glen Marszalowicz, PHD May 10, AM

Transcription

1 Antibody Generation: challenges and solutions Glen Marszalowicz, PHD May 10, AM

2 GenScript the most cited biology CRO CRISPR Services Gene Services Peptide Services Protein Services Antibody Services Discovery Biology Services Catalog Products 2

3 Antibody Generation: challenges and solutions Antibody applications Traditional antibody generation 3 Common challenges and solutions Related antibody generation services 3

4 Antibody applications Research Track biomarker location, expression Industry -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 Traditional assays, but also - Therapeutic Antibody Drugs - Antibody Drug Conjugates (ADCs) - Recombinant antibodies (rabs) result in many formats 4

5 Antibody Generation: challenges and solutions Antibody applications Traditional antibody generation 3 Common challenges and solutions Related antibody generation services 5

6 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 exogenous antigen Cognate helper T-cell activates through cytokine signaling Transformation to plasma cell and antibody secretion 6

7 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 7

8 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 8

9 Creating hybridomas Lymphocyte fusion with myelomas via electrofusion. Selection of only lymphocyte/myeloma fusion products through HAT medium 9

10 Achieving monoclonality Repetitive limiting dilutions statistically insures a single cell/well Selection of positive clones via ELISA 10

11 Antibody Generation: challenges and solutions Antibody applications Traditional antibody generation 3 Common challenges and solutions Related antibody generation services 11

12 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 12

13 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 13

14 Solution to difficult antigens: DNA Immunization Monoclonal Antibody Development Immunization Immune Response Fusion & Screening Cloning Target cdna In vivo expression overcomes antigen generation issues. 14

15 Case Study: GLP1R mab by DNA immunization Glucagon-like peptide receptor-1 (GLP1R): 3 Disulfide bonds N-terminal glycosylation FACS ELISA Western Blot ICC 15

16 Challenge: self-tolerance (to high homology antigens) 16

17 Solution to self-tolerance Modify immunization such that signaling mimics foreign antigen exposure GenScript proprietary ImmunoPlus technology Overcomes self tolerance through antigen modification 17

18 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. 18

19 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. 19

20 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 20

21 Antibody Generation: challenges and solutions Antibody applications Traditional antibody generation 3 Common challenges and solutions Related antibody generation services 21

22 GenScript s antibody generation solutions Polyclonal antibodies in 45 days with just your target sequence starting at $649. Monoclonal Phosphospecific high quality, purified monoclonal antibodies guaranteed to work in your application monoclonal or polyclonal with <10% nonphospho peptide cross-reactivity guaranteed Anti-idiotype Ideal for studying PK/PD of therapeutic antibodies DNA Immunization ImmunoPlus GANP Mice Applicable to any antibody project! 22

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