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Published on: February 3, 2015
Engineering PD-1-targeted small protein variants for in vitro diagnostics and in vivo PET imaging
Joanna Maria Mierzwicka1, Hana Petroková1, Leona Rašková Kafková2,3
1Laboratory of Ligand Engineering, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV Research Center, Průmyslová 595, 252 50, Vestec, Czech Republic.
Background:
Programmed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging.
Methods:
We designed a 13 kDa β-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1.
Results:
Binding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7-99.3% and in vitro stability in human serum after 120 min was in the range 94.6-98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors.
Conclusions:
Using directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.
Insights
Researchers developed novel small binding proteins to improve diagnostics for Programmed cell death 1 (PD-1) positive cells. These Myomedin scaffold anti-PD-1 variants show high affinity and potential for in vivo imaging in non-small cell lung cancer.
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- Programmed cell death 1 (PD-1) is a key immune checkpoint protein regulating immune responses.
- In non-small cell lung cancer (NSCLC), PD-1 expression on tumor-infiltrating lymphocytes correlates with therapeutic sensitivity and efficacy.
- Tumor heterogeneity necessitates improved methods for profiling PD-1+ populations for reliable diagnostics.
Purpose of the Study:
- To develop novel, high-affinity, low molecular weight diagnostic probes for PD-1.
- To enhance the reliability of PD-1 expression profiling in tumor biopsies.
- To improve in vivo mapping efficiency of PD-1+ tumor-infiltrating lymphocytes (TILs) using immune-PET imaging.
Main Methods:
- A 13 kDa β-sheet Myomedin scaffold combinatorial library was engineered with randomized mutable residues.
- Ribosome display technology was employed to identify anti-PD-1 Myomedin variants (MBA ligands).
- Binding affinity was assessed using LigandTracer, and in vivo imaging potential was evaluated using 68Gallium-labeled MBA proteins with PET/CT.
Main Results:
- Identified anti-PD-1 MBA ligands with high affinity for human and murine PD-1 (e.g., MBA066, MBA197, MBA414 with KD values in nanomolar range).
- Demonstrated successful in vivo imaging of PD-1+ populations in mice using 68Ga-labeled MBA proteins via PET/CT.
- Confirmed specificity of MBA proteins through co-staining of human tonsil and NSCLC tissue biopsies, showing potential for solid tumor mapping.
Conclusions:
- Directed evolution yielded a unique set of small binding proteins targeting PD-1.
- These novel MBA proteins offer improved diagnostic capabilities for PD-1+ cells both in vitro and in vivo.
- The developed probes show promise for enhancing PD-1-based diagnostics and imaging in cancer, particularly NSCLC.
