1. Targeting CD47 in Sézary syndrome with SIRPαFc
by Lisa D. S. Johnson, Swati Banerjee, Oleg Kruglov, Natasja Nielsen Viller, Steven M. Horwitz, Alexander Lesokhin, Jasmine Zain, Christiane Querfeld, Robert Chen, Craig Okada, Ahmed Sawas, Owen A. O’Connor, Eric L. Sievers, Yaping Shou, Robert A. Uger, Mark Wong, and Oleg E. Akilov
BloodAdv
Volume 3(7):
April 9, 2019
© 2019 by The American Society of Hematology

2. Lisa D. S. Johnson et al. Blood Adv 2019;3:1145-1153
© 2019 by The American Society of Hematology

3. CD47 is overexpressed on Sézary cells and correlates with worse OS
CD47 is overexpressed on Sézary cells and correlates with worse OS. (A) Representative fluorescence-activated cell sorter (FACS) plots of gating strategy identifying CD47 expression on Sézary cells (with a dominant Vβ13.2+ T-cell population) in comparison with nonmalignant T cells (CD4+Vβ13.2−) in a patient with SS. CD47 expression was determined on Sézary cells (red histogram) compared with nonmalignant CD4+ cells (blue histogram) and isotype control (green histogram).
CD47 is overexpressed on Sézary cells and correlates with worse OS. (A) Representative fluorescence-activated cell sorter (FACS) plots of gating strategy identifying CD47 expression on Sézary cells (with a dominant Vβ13.2+ T-cell population) in comparison with nonmalignant T cells (CD4+Vβ13.2−) in a patient with SS. CD47 expression was determined on Sézary cells (red histogram) compared with nonmalignant CD4+ cells (blue histogram) and isotype control (green histogram). (B) The MFI of CD47 on CD4+Vβ+ (Sézary cells, red) and matched CD4+Vβ− (nonmalignant lymphocytes, blue) cells in patients with SS (n = 25). Two-tailed unpaired Student t test (****P < .0001). (C) Representative immunohistochemistry (IHC) samples of the skin biopsy from a patient with SS with low CD47 expression (left panel) and 1 with high CD47 expression (right panel) (anti-CD47 with red chromagen stain; original magnification ×40). The level of expression of CD47 on Sézary cells in the peripheral blood determined by flow cytometry provided on the right of the corresponding skin biopsy. (D) Kaplan-Meier curves of OS (months) of patients with SS having a low CD47 expression on Sézary cells (<6537 MFI) vs high CD47 expression (>6537 MFI) on Sézary cells (n = 25). Log-rank (Mantel-Cox) test (P < .001). FITC-A, fluorescein isothiocyanate area; FSC-A, forward scatter area; PE-A, phycoerythrin area; SSC-A, side scatter area.
Lisa D. S. Johnson et al. Blood Adv 2019;3:
© 2019 by The American Society of Hematology

4. CD47 expression on Sézary cells is under influence of Th2 cytokines.
CD47 expression on Sézary cells is under influence of Th2 cytokines. (A) Cytokine concentration in peripheral blood of Sézary patients (n = 11) vs age-matched healthy volunteers (n = 15). Mean ± SD is depicted. Two-tailed unpaired Student t test (*P < .05; **P < .01). (B) Percentage of CD47 on the surface of Sézary cells (CD4+Vβ+, red) vs nonmalignant T cells from the same patients (CD4+Vβ−, blue) in relationship to nonstimulated cells following 48-hour incubation with IL-2 (200 IU/mL), IL-4 (20 ng/mL), IL-6 (20 ng/mL), IL-7 (20 ng/mL), IL-10 (20 ng/mL), IL-12 (50 ng/mL), IL-13 (20 ng/mL), IL-17 (20 ng/mL), IL-23 (20 ng/mL), IFN-γ (20 ng/mL), or TNF-α (20 ng/mL). Mean ± SD is depicted. Two-tailed unpaired Student t test (*P < .05). ns, not significant.
Lisa D. S. Johnson et al. Blood Adv 2019;3:
© 2019 by The American Society of Hematology

5. Blockade of CD47 with SIRPαFc (TTI-621) promotes phagocytosis of Sézary cells but not nonmalignant CD4+ cells.
Blockade of CD47 with SIRPαFc (TTI-621) promotes phagocytosis of Sézary cells but not nonmalignant CD4+cells. (A) Cytospin preparation of representative samples 2 hours after phagocytosis in the presence of an IgG1 isotype control molecule (TTI-402) or TTI-621. Red arrows identify Sézary cells that are external to macrophages in the presence of isotype control but phagocytosed in the presence of TTI-621 (Romanowsky stain; original magnification ×100). (B) Macrophage-mediated phagocytosis of matched nonmalignant (blue) or Sézary cells (red) from 8 patients with SS in media (no treatment), 1 µM TTI-403 (isotype control), and 1 µM TTI-621. One-way analysis of variance with Tukey posttest (*P < .05; **P < .01).
Lisa D. S. Johnson et al. Blood Adv 2019;3:
© 2019 by The American Society of Hematology

6. TTI-621 (arrow) can be observed at the interface between the Sézary cell and macrophage.
TTI-621 (arrow) can be observed at the interface between the Sézary cell and macrophage. Gold-labeled TTI-621 was incubated with Sézary cells and macrophages for 5 minutes before scanning electron microscopy. (A) Sézary cell (SzC) attached to Mϕ (original magnification ×15 000). (B) Gold-labeled TTI-621 at the interface between the Sézary cell and the Mϕ (original magnification ×50 000).
Lisa D. S. Johnson et al. Blood Adv 2019;3:
© 2019 by The American Society of Hematology

7. Clinical benefit of TTI-621 in Sézary syndrome is accompanied by a decrease in circulating clone of Sézary cells.
Clinical benefit of TTI-621 in Sézary syndrome is accompanied by a decrease in circulating clone of Sézary cells. (A) Percentage change in clonal (Vβ+) CD4 cells in patients with Sézary syndrome (n = 5) who received a single dose of TTI-621. Comparison between day 1 pretreatment and 8 days after infusion. (B) Decrease in lactate dehydrogenase (LDH; right y-axis, blue) and absolute lymphocyte count (ALC; left y-axis, red) during treatment with TTI-621 (a representative patient). (C) A 52-year-old man with stage IVA1 (T4N1M0B2) SS enrolled in phase 1a of clinical trial NCT Images taken at screening and week 4 on study.
Lisa D. S. Johnson et al. Blood Adv 2019;3:
© 2019 by The American Society of Hematology