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Response: Experimental Protocol to Identify Antibodies Targeting IDH-Wildtype Glioblastoma Using Phage Display and Ginkgo Bioworks' Platforms

Objective: To discover and characterize monoclonal antibodies that specifically bind to isocitrate dehydrogenase (IDH)-wildtype glioblastoma cells using phage display technology, leveraging Ginkgo Bioworks' synthetic biology and high-throughput screening capabilities.

Background:

Glioblastoma is the most aggressive form of primary brain tumor, with IDH-wildtype being the most prevalent subtype. Developing antibodies that specifically target IDH-wildtype glioblastoma cells can aid in diagnostics and therapeutics. Phage display is a powerful method for selecting peptides or proteins with high affinity and specificity for a particular target. Ginkgo Bioworks specializes in synthetic biology and can facilitate library construction, high-throughput screening, and optimization processes.

Materials:

Cell Lines:
IDH-wildtype glioblastoma cell lines (e.g., U87MG)
IDH-mutant glioblastoma cell lines (as negative control)
Normal human astrocyte cells (as additional negative control)
Phage Display Components:
Phage display antibody library (e.g., human scFv or Fab library)
M13 filamentous bacteriophage vectors
Escherichia coli host strains (e.g., TG1, SS320, or ER2738)
Buffers and Reagents:
Phosphate-buffered saline (PBS)
Blocking buffers (e.g., 5% BSA in PBS)
Washing buffers (PBS with 0.1% Tween-20)
Elution buffer (e.g., 0.2 M glycine-HCl, pH 2.2)
Neutralization buffer (1 M Tris-HCl, pH 9.1)
Equipment:
Cell culture facilities
Magnetic beads (e.g., Dynabeads™ M-280 Streptavidin)
Biotinylation kit
ELISA plates and plate reader
Flow cytometer
PCR and sequencing equipment
Ginkgo Bioworks' high-throughput screening platforms

Methods:

1. Preparation of Target Antigens

1.1. Cell Culture:

Cultivate IDH-wildtype glioblastoma cells under appropriate conditions.
Similarly, culture IDH-mutant glioblastoma cells and normal astrocyte cells as controls.

1.2. Antigen Preparation:

Harvest cells and prepare a cell suspension.
Option A: Use whole, fixed cells as the antigen for phage panning.
Option B: Isolate and purify membrane proteins from IDH-wildtype glioblastoma cells.

1.3. Biotinylation (if using purified proteins):

Biotinylate the purified membrane proteins using a biotinylation kit, following the manufacturer's instructions.
Remove excess biotin through dialysis or desalting columns.

2. Negative Selection (Depletion of Non-Specific Binders)

Purpose: To remove phage that bind to common antigens present on non-target cells.

2.1. Incubation with Control Cells:

Incubate the phage display library with IDH-mutant glioblastoma cells and normal astrocyte cells.
Allow non-specific binders to attach to the control cells.

2.2. Separation:

Remove the cells with bound phage by centrifugation or using magnetic beads if cells are labeled.
Collect the supernatant containing unbound phage, which is enriched for phage not binding to control cells.

3. Positive Selection (Biopanning) with Target Cells

3.1. Incubation with Target Cells:

Incubate the pre-cleared phage library with IDH-wildtype glioblastoma cells.
Perform this step under gentle agitation to promote interactions.

3.2. Washing:

Wash the cells extensively with washing buffer to remove non-specifically bound phage.
Increase washing stringency in subsequent rounds by increasing the number of washes or detergent concentration.

3.3. Phage Elution:

Elute bound phage from the cells using an elution buffer (e.g., low pH glycine-HCl buffer).
Immediately neutralize the eluate with neutralization buffer.

3.4. Amplification:

Infect E. coli host cells with the eluted phage.
Grow the bacteria to amplify the phage particles.
Isolate phage from the bacterial culture supernatant.

3.5. Repeat Rounds:

Perform 3–5 rounds of biopanning, repeating steps 2 and 3.
Each round enriches the phage pool for binders with higher affinity and specificity.

4. Screening of Phage Clones

4.1. Phage ELISA:

Clone individual phage clones into E. coli and culture them to produce phage particles.
Coat ELISA plates with IDH-wildtype glioblastoma antigens.
Incubate phage clones with the coated plates.
Detect bound phage using anti-M13 HRP-conjugated antibodies.
Include wells coated with control antigens to assess specificity.

4.2. Flow Cytometry Analysis:

Incubate phage clones with IDH-wildtype glioblastoma cells.
Use anti-phage antibodies conjugated with fluorophores to detect binding.
Analyze samples using a flow cytometer.
Compare binding profiles with control cells.

5. Identification of Antibody Sequences

Extract phagemid DNA from positive phage clones.
Use PCR to amplify the antibody variable region genes.
Send PCR products for Sanger sequencing or use next-generation sequencing platforms.
Ginkgo Bioworks' Contribution:
Utilize their high-throughput sequencing and bioinformatics platforms to analyze large numbers of clones efficiently.
Perform sequence alignment and clustering to identify unique antibody sequences.

6. Expression and Purification of Antibodies

6.1. Subcloning:

Clone the variable region genes into appropriate expression vectors for full-length IgG, scFv-Fc, or other antibody formats.

6.2. Expression:

Transfect expression constructs into mammalian cell lines (e.g., HEK293, CHO cells).
Culture cells and collect supernatant containing secreted antibodies.

6.3. Purification:

Purify antibodies from the supernatant using Protein A/G affinity chromatography.
Assess purity by SDS-PAGE and quantify protein concentration.

7. Characterization of Antibodies

7.1. Binding Affinity Measurement:

Use surface plasmon resonance (SPR) or bio-layer interferometry (BLI) to measure the kinetic binding parameters (association/dissociation rates, affinity constants).

7.2. Specificity Testing:

Perform ELISA and flow cytometry assays against a panel of cell lines, including:
IDH-wildtype glioblastoma cells
IDH-mutant glioblastoma cells
Normal astrocytes
Other relevant brain cells
Confirm that antibodies bind specifically to IDH-wildtype glioblastoma cells.

7.3. Immunohistochemistry (IHC):

Use antibodies to stain tissue sections from glioblastoma patients.
Assess staining patterns to confirm specificity and potential diagnostic utility.

8. Functional Assays

8.1. Cell Proliferation Assays:

Treat IDH-wildtype glioblastoma cells with antibodies.
Measure effects on cell proliferation using assays like MTT or CellTiter-Glo.

8.2. Apoptosis Assays:

Analyze induction of apoptosis via flow cytometry using Annexin V/PI staining or caspase activation assays.

8.3. Antibody-Dependent Cellular Cytotoxicity (ADCC):

Evaluate if antibodies can mediate immune cell-mediated killing of target cells.

9. Antibody Optimization (Optional)

9.1. Affinity Maturation:

Generate libraries introducing mutations in complementarity-determining regions (CDRs).
Perform additional rounds of phage display selection to isolate higher-affinity variants.
Ginkgo Bioworks' Contribution:
Employ their automated DNA synthesis and high-throughput mutation platforms.
Utilize machine learning models to predict beneficial mutations.

9.2. Humanization:

If initial antibodies are from non-human sources, humanize them to reduce immunogenicity.
Use structural modeling and sequence alignment tools.

Safety and Compliance:

Biosafety:
Handle all biological materials in accordance with institutional biosafety guidelines.
Use appropriate containment for cell lines and recombinant DNA work.
Ethical Considerations:
Obtain necessary approvals for the use of human-derived cell lines.
Ensure compliance with relevant regulations, such as the Declaration of Helsinki.
Waste Disposal:
Dispose of all hazardous waste according to institutional policies and environmental regulations.

Ginkgo Bioworks' Role:

Synthetic Biology and Engineering:
Provide custom synthesis of antibody libraries with enhanced diversity.
Utilize their foundry for automated cloning and expression.
High-Throughput Screening:
Employ robotics and automation to screen large numbers of antibody variants rapidly.
Data Analysis and Machine Learning:
Apply bioinformatics tools to analyze sequencing data.
Use machine learning algorithms to predict antibody behaviors and optimize candidates.
Scalable Production:
Facilitate transition from discovery to production-scale antibody manufacturing processes.

Conclusion:

By integrating phage display technology with Ginkgo Bioworks' synthetic biology expertise, this protocol aims to efficiently identify and optimize antibodies that specifically target IDH-wildtype glioblastoma cells. The resulting antibodies have the potential to serve as valuable tools for diagnosis or as therapeutic agents against this aggressive brain tumor subtype.

References:

Smith, G. P., & Petrenko, V. A. (1997). Phage Display. Chemical Reviews, 97(2), 391–410.
Hoogenboom, H. R. (2005). Selecting and screening recombinant antibody libraries. Nature Biotechnology, 23(9), 1105–1116.
Paria, B. C., & Rahman, M. T. (2017). Synthetic Biology and its Application in Biotechnology. Journal of Molecular and Genetic Medicine, 11(1).

Note: The above protocol is a general guideline. Specific experimental conditions may need to be optimized based on laboratory resources and specific objectives.