Antibody Surface Profiling Identifies Glycoforms in Multiple Myeloma as Targets for Immunotherapy: From Antibody Derivatives to Mimetic Peptides for Killing Tumor Cells
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Reagents
2.2. Peptides
2.3. Cancer Cell Lines
2.4. Isolation of Blood Cells
2.5. Affinity Selection of scFv Antibody Fragments
2.6. Phage Amplification
2.7. Cloning and Expression of the Selected scFv–Fc Fusion Proteins
2.8. Flow Cytometry Analysis
2.9. ELISA
2.10. Competition Experiments
2.11. Confocal Microscopy Analysis
2.12. Analysis of NK Cell Degranulation and Activation
2.13. Cytotoxicity Assay
2.14. Syndecan-1 Knockdown and Overexpression in HEK293T Cells
2.15. Statistical Analysis
3. Results
3.1. Isolation of MM-Specific scFv Antibody Fragments
3.2. Conversion of the MM1 scFv into a scFv-Fc Antibody
3.3. Activation of NK Cells and Induction of ADCC against Myeloma Cell Lines
3.4. Evidence for the Interaction of the scFv-Fc with Cell-Surface Heparan Sulfate
3.5. Involvement of Syndecan-1 Associated Heparan Sulfate
3.6. The Role of the CDRs of the scFv-Fc Heavy Chain in Recognition of MM Cells
3.7. Rational Design of a Peptide That Mimics the Selected Heparan Sulfate scFvs
3.8. A Pro-Apoptotic Peptide Conjugated to MC Peptide Selectively Kills MM Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
ADCC | Antibody-dependent cellular cytotoxicity |
ADP | Antibody-dependent phagocytosis |
CDC | Complement-dependent cytotoxicity |
CDR | Complementarity-determining regions |
HS | Heparan sulfate |
HSPGs | Heparan sulfate peptidoglycans |
MM | Multiple myeloma |
PBMCs | Peripheral blood mononuclear cells |
PE | Phycoerythrin |
PEG | Polyethylene glycol |
PI | Propidium iodide |
scFv | Single-chain variable fragment |
siRNA | Small interfering RNA |
VH | Heavy variable chain |
VL | Light variable chain |
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Sequential Affinity Selection on Cancer Cell Lines | Cell Number | Input Number of Phages (TU) | Recovered Number of Phages (TU) | Enrichment over Previous Round * |
---|---|---|---|---|
CAG | 1 × 107 | 1 × 1010 | 2.0 × 103 | 0 |
RPMI 8226 | 1 × 107 | 1 × 1010 | 1.5 × 104 | 5 |
U266 | 1 × 107 | 1 × 1010 | 1.2 × 107 | 800 |
Clone | VH CDR2 | VH CDR3 | VL CDR2 | VL CDR3 | Frequency * |
---|---|---|---|---|---|
MM1 | AIRHPGLHTEY | AKGGRRFDY | RASRLQS | QQANSPPPT | 21/30 |
MM10 | TIRRQGGNTEY | AKSARVFDY | TASRLRS | QQWTAKPGT | 2/30 |
MM12 | AIRRPHLNTEY | AKGRRPRKFDY | RASHLQS | QQPNAPAPT | 7/30 |
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Sioud, M.; Olberg, A. Antibody Surface Profiling Identifies Glycoforms in Multiple Myeloma as Targets for Immunotherapy: From Antibody Derivatives to Mimetic Peptides for Killing Tumor Cells. Cancers 2023, 15, 1934. https://doi.org/10.3390/cancers15071934
Sioud M, Olberg A. Antibody Surface Profiling Identifies Glycoforms in Multiple Myeloma as Targets for Immunotherapy: From Antibody Derivatives to Mimetic Peptides for Killing Tumor Cells. Cancers. 2023; 15(7):1934. https://doi.org/10.3390/cancers15071934
Chicago/Turabian StyleSioud, Mouldy, and Anniken Olberg. 2023. "Antibody Surface Profiling Identifies Glycoforms in Multiple Myeloma as Targets for Immunotherapy: From Antibody Derivatives to Mimetic Peptides for Killing Tumor Cells" Cancers 15, no. 7: 1934. https://doi.org/10.3390/cancers15071934