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.

• Klíčová slova
• Cancer diagnostic, Combinatorial library, Immune checkpoint, Non-small cell lung cancer, Programmed cell death 1, Protein engineering,
• MeSH
• antigeny CD279 * metabolismus   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory plic diagnostické zobrazování   patologie   metabolismus   genetika   MeSH
• nemalobuněčný karcinom plic diagnostické zobrazování   patologie   metabolismus   MeSH
• pozitronová emisní tomografie * metody   MeSH
• proteinové inženýrství * MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD279 * MeSH
• PDCD1 protein, human MeSH Prohlížeč

Compared to solid tumors, the role of PD-L1 in hematological malignancies is less explored, and the knowledge in this area is mostly limited to lymphomas. However, several studies indicated that PD-L1 is also overexpressed in myeloid malignancies. Successful treatment of the acute myeloid leukemia (AML) is likely associated with elimination of the residual disease by the immune system, and possible involvement of PD-L1 in this process remains to be elucidated. We analyzed PD-L1 expression on AML primary cells by flow cytometry and, in parallel, transcript levels were determined for the transcription variants v1 and v2. The ratio of v1/v2 cDNA correlated with the surface protein amount, and high v1/v2 levels were associated with worse overall survival (p = 0.0045). The prognostic impact of PD-L1 was limited to AML with mutated nucleophosmin and concomitant internal tandem duplications in the FLT3 gene (p less than 0.0001 for this particular AML subgroup).

• Klíčová slova
• AML, CD34, FLT3-ITD, NPM1, PD-1, PD-L1 transcript, leukemia,
• MeSH
• akutní myeloidní leukemie krev   genetika   MeSH
• antigeny CD274 krev   genetika   metabolismus   MeSH
• jaderné proteiny genetika   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• mutace MeSH
• nádorové biomarkery krev   genetika   metabolismus   MeSH
• nukleofosmin MeSH
• tyrosinkinasa 3 podobná fms genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD274 MeSH
• CD274 protein, human MeSH Prohlížeč
• FLT3 protein, human MeSH Prohlížeč
• jaderné proteiny MeSH
• messenger RNA MeSH
• nádorové biomarkery MeSH
• NPM1 protein, human MeSH Prohlížeč
• nukleofosmin MeSH
• tyrosinkinasa 3 podobná fms MeSH