out6

Response: Developing an Experimental Protocol to Identify Antibodies Against IDH-Wildtype Glioblastoma Using Phage Display and Engineering a BiTE Therapy Similar to Tebentafusp

Introduction

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor in adults, with poor prognosis and limited treatment options. The IDH-wildtype GBM represents the majority of GBM cases and is characterized by rapid progression and resistance to conventional therapies. Targeted immunotherapies offer a promising avenue for treating this malignancy.

Phage display technology is a powerful method for identifying peptides, proteins, or antibodies that bind specifically to a target antigen. By combining phage display with advanced synthetic biology platforms like Ginkgo Bioworks and high-throughput DNA synthesis technologies from companies like Twist Bioscience, we can accelerate the discovery of novel antibodies against GBM-specific antigens.

Once a specific antibody is identified, it can be engineered into a bispecific T-cell engager (BiTE) therapy, similar to tebentafusp. Tebentafusp is a BiTE that has demonstrated efficacy in targeting cancer cells by redirecting T-cells to kill tumor cells expressing a specific antigen.

Objective

To develop an experimental protocol to:

Identify antibodies that bind specifically to IDH-wildtype glioblastoma cells using phage display technology.
Engineer the identified antibody into a BiTE therapy that can engage T-cells to target and kill GBM cells, following the model of tebentafusp.

Materials and Methods

1. Antigen Selection and Preparation

a. Target Identification

Antigen Selection: Identify a cell surface antigen or peptide that is highly expressed on IDH-wildtype GBM cells but minimally expressed on normal tissues to reduce off-target effects. Potential targets include:
Epidermal growth factor receptor variant III (EGFRvIII)
Interleukin-13 receptor alpha 2 (IL13Rα2)
Ephrin type-A receptor 2 (EphA2)
Other GBM-specific markers identified through proteomic analysis

b. Antigen Synthesis

Gene Synthesis: Use Twist Bioscience's chip technology to synthesize the DNA sequence encoding the extracellular domain of the selected antigen.
Expression Vector Construction: Clone the synthesized gene into an appropriate expression vector with a suitable tag (e.g., His-tag) for purification.

c. Protein Expression and Purification

Expression System: Transfect the expression vector into a suitable host cell line (e.g., HEK293 or CHO cells).
Protein Purification:
Harvest the supernatant or cell lysate containing the expressed antigen.
Purify the antigen using affinity chromatography targeting the purification tag.
Verify purity and integrity using SDS-PAGE and Western blot analysis.

2. Phage Display Library Preparation

a. Library Selection

Obtain a human scFv (single-chain variable fragment) or Fab (fragment antigen-binding) phage display library to ensure minimal immunogenicity in eventual therapeutic applications.

b. Amplification and Titration

Amplify the phage library in an appropriate E. coli host strain.
Titrate the amplified library to determine phage concentration.

3. Biopanning (Selection of Specific Binders)

a. Immobilization of Antigen

Coat Plates or Beads: Immobilize the purified antigen onto microtiter plates or magnetic beads.
Blocking: Block non-specific binding sites with a blocking buffer containing BSA or skim milk.

b. Phage Incubation

Positive Selection:
Incubate the phage library with the immobilized antigen to allow specific binders to interact.
Optimize incubation time and conditions to improve binding specificity.
Negative Selection:
Perform a pre-incubation step where the phage library is exposed to irrelevant proteins or normal human cell lysates to remove non-specific binders.

c. Washing and Elution

Washing: Perform stringent washes to remove unbound and weakly bound phages.
Elution: Elute bound phages using an acidic buffer, competitive elution with soluble antigen, or enzymatic cleavage if applicable.

d. Phage Amplification

Infect E. coli with the eluted phages to amplify the selected phage pool.
Repeat biopanning for 3-5 rounds to enrich for high-affinity binders.

4. Screening and Characterization of Phage Clones

a. Individual Clone Isolation

Plate dilutions of the amplified phage onto agar plates to obtain individual colonies.
Pick single colonies and culture them for phage production.

b. Phage ELISA

Binding Assay:
Coat ELISA plates with the target antigen and control proteins.
Incubate with culture supernatants containing phage or expressed scFv/Fab fragments.
Detect bound phages using anti-M13 HRP-conjugated antibodies or anti-human Fc antibodies if applicable.
Analysis:
Identify clones that show strong binding to the target antigen but not to control proteins.
Select top candidates based on binding strength and specificity.

c. Sequence Analysis

Extract phagemid DNA from positive clones.
Sequence the variable regions to identify unique clones and assess diversity.

5. Expression and Purification of Selected Antibodies

a. Subcloning

Subclone the selected scFv or Fab fragments into an appropriate expression vector for soluble expression, possibly adding a human Fc region to create full-length antibodies if desired.

b. Expression

Transfect into an expression system (e.g., mammalian cells or yeast) for high-yield antibody production.

c. Purification

Purify antibodies using Protein A/G affinity chromatography.
Confirm purity and correct folding through SDS-PAGE and size-exclusion chromatography.

6. In Vitro Characterization of Antibodies

a. Binding Affinity Measurement

Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):
Assess the kinetic parameters (KD, kon, k_off) of antibody-antigen interactions.

b. Specificity Testing

Test binding against a panel of related proteins and normal human tissue antigens to confirm specificity.

c. Cellular Binding Assays

Flow Cytometry:
Incubate antibodies with IDH-wildtype GBM cell lines and control cells.
Use fluorescently labeled secondary antibodies to detect binding.
Analyze using flow cytometry to assess surface binding.
Immunofluorescence Microscopy:
Examine binding to GBM cells using fluorescence microscopy to visualize localization.

7. Engineering the BiTE Molecule

a. BiTE Design

Structure:
Fuse the variable domains of the anti-GBM antibody with the variable domains of an anti-CD3 antibody in a single-chain format.
Maintain a flexible linker between the two scFvs to allow proper folding and simultaneous binding.
Sequence Optimization:
Use molecular modeling to optimize the linker length and orientation.
Ensure the construct maintains stability and solubility.

b. Gene Synthesis and Cloning

Synthesis:
Use Twist Bioscience's DNA synthesis platform to synthesize the gene encoding the BiTE construct.
Cloning:
Clone the synthesized gene into an appropriate expression vector with signal peptides for secretion and necessary purification tags.

c. Expression and Purification

Expression System:
Utilize mammalian cell lines (e.g., HEK293, CHO cells) for proper folding and post-translational modifications.
Purification:
Purify the BiTE using affinity chromatography based on the attached tags.
Perform buffer exchange into a suitable formulation buffer.

8. In Vitro Functional Assays of the BiTE

a. T-cell Activation Assays

Co-culture Setup:
Incubate human peripheral blood mononuclear cells (PBMCs) or isolated T-cells with GBM cells in the presence of the BiTE.
Assessment:
Measure T-cell activation markers (CD69, CD25) by flow cytometry.
Quantify cytokine release (IFN-γ, IL-2, TNF-α) using ELISA or multiplex assays.

b. Cytotoxicity Assays

Real-Time Cell Analysis:
Use impedance-based systems to monitor cell viability over time.
LDH Release or Calcein-AM Assays:
Quantify cell death by measuring lactate dehydrogenase release or calcein-AM retention.
Flow Cytometry-Based Killing Assay:
Label GBM cells with a fluorescent dye and measure the reduction of viable target cells after co-culture with T-cells and BiTE.

c. Specificity Controls

Include control antibodies (irrelevant BiTE, anti-CD3 alone) to confirm that cytotoxicity is antigen-specific and dependent on BiTE-mediated T-cell engagement.

9. In Vivo Efficacy Studies

a. Animal Models

Xenograft Models:
Establish human GBM tumors in immunodeficient mice (e.g., NSG mice).
Humanize the immune system in mice by engrafting human T-cells or using humanized mice.

b. Treatment Groups

Administer the BiTE therapy at varying doses.
Include control groups receiving vehicle, non-specific BiTE, or standard treatments.

c. Monitoring

Tumor Growth:
Measure tumor size regularly using calipers or imaging techniques (MRI, bioluminescence).
Survival Analysis:
Record survival times and perform statistical analysis to assess the efficacy.
Immune Response:
Analyze T-cell infiltration into tumors via immunohistochemistry or flow cytometry.

d. Toxicology Studies

Assess potential off-target effects and cytokine release syndrome.
Monitor body weight, behavior, and clinical chemistry parameters.

10. Data Analysis and Optimization

a. Data Interpretation

Analyze binding affinities, specificity, T-cell activation levels, and cytotoxicity data.
Compare in vitro and in vivo efficacy between experimental groups.

b. Optimization

Based on results, optimize the BiTE molecule for:
Improved binding affinity and stability.
Reduced immunogenicity by modifying amino acid sequences or glycosylation patterns.
Consider Fc fusion to extend half-life if necessary.

11. Regulatory and Manufacturing Considerations

a. Scalability

Develop a scalable manufacturing process for GMP production.

b. Regulatory Compliance

Ensure all studies are conducted following regulatory guidelines for preclinical development.
Prepare documentation for Investigational New Drug (IND) application if proceeding to clinical trials.

Conclusion

By following this protocol, antibodies specifically targeting IDH-wildtype glioblastoma can be identified using phage display technology enhanced by platforms like Ginkgo Bioworks and Twist Bioscience. Engineering these antibodies into BiTE constructs enables the recruitment of T-cells to GBM cells, potentially providing an effective immunotherapeutic strategy similar to tebentafusp. Further optimization and preclinical testing will pave the way for clinical development and offer hope for patients with this challenging disease.

References

Sidhu, S. S., & Koide, A. (2015). Phage display methodologies and applications. Methods in Enzymology, 523, 283–309.
Chames, P., Van Regenmortel, M., Weiss, E., & Baty, D. (2009). Therapeutic antibodies: successes, limitations and hopes for the future. British Journal of Pharmacology, 157(2), 220–233.
Dreier, T., Baeuerle, P. A., & Fichter, M. (2015). Preclinical activity of MT110, a novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors. Molecular Cancer Therapeutics, 11(3), 637–644.
John, L. B., Devaud, C., Duong, C. P. M., Yong, C. S. M., Beavis, P. A., Haynes, N. M., … & Darcy, P. K. (2013). Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells. Clinical Cancer Research, 19(20), 5636–5646.

Note: This protocol is a generalized guideline and may need to be adapted based on specific laboratory resources, regulations, and recent scientific advancements. Always consult current literature and regulatory guidelines before proceeding with experimental work.