Fig 1: XmAb808 binds avidly to B7-H3 on target cells to costimulate T cells. A, Binding of XmAb808 or its analogue containing a single B7-H3–binding domain (mono–B7-H3×CD28) to target cells with varying B7-H3 densities (left axis) and binding of XmAb808 to human T cells (right axis) are plotted as mean fluorescence intensity (MFI). HEK293 cells express approximately 200,000 B7-H3 antigens; A431, 196,000; 22Rv1, 86,000; LOX-IMVI cells do not express B7-H3. Because mono–B7-H3×CD28 has a lower molecular weight than XmAb808 (145 vs. 192 kDa), the mono–B7-H3×CD28 concentrations in this figure were corrected by a factor of 1.3-fold to adjust for molarity. B, Human PBMCs from 20 different donors were incubated on plates coated with air-dried (immobilized) XmAb808, anti-CD28 (TGN1412), or anti-CD3 (OKT3) antibodies. IFNγ and IL2 were measured after 24 hours. Statistical significance is denoted by **, P < 0.01; ns, not significant. C, IL2 release from PBMCs cocultured with HEK293-αCD3 or HEK293-αCD3-B7-H3 KO cells at an E:T ratio of 25:1 was measured after 24 hours. Data are represented as means ± SEM of cocultures with PBMCs from eight different human donors.
Fig 2: CD28 costimulation drives activation and survival of T cells, leading to IL2-dependent killing of cancer cells. Target cells were cocultured with human T cells (E:T = 1:1) and treated with XmAb808. A, After 24 hours, IL2 (closed symbols) and IFNγ (open symbols) were measured in culture supernatants. B, The percentages of CD4+ or CD8+ T cells that were CD25+ or Bcl-xL+ from cocultures with 22Rv1-αCD3 cells following 24 hours of treatment are plotted. C, Real-time killing of 22Rv1-αCD3 cells in cocultures treated with (closed symbols) or without (open symbols) 1 µg/mL of XmAb808 plus an IL2-neutralizing antibody, an IFNγ-neutralizing antibody, or an isotype control antibody was monitored over time using an xCELLigence Real-Time Cell Analysis System. The arrow indicates the addition of T cells and antibodies at 24 hours after cancer cells were plated. Data are plotted as single points in A and B and as mean ± SEM of three replicates in C.
Fig 3: An XmAb808 analogue enhances antitumor activity of an EpCAM×CD3 TCE in human PBMC-engrafted mice. NSG-MHC I/II DKO mice were intradermally inoculated with HPAF-II cells. After 2 weeks, palpable tumors formed, and mice were then engrafted with human PBMCs together with 5 mg/kg of EpCAM×CD3 alone or in combination with 1 mg/kg of B7-H3×Null or 1 mg/kg of XmAb808s weekly. A, Tumor volumes are shown over time as mean ± SEM with n = 8–10 mice/group. B, Tumor volumes of individual mice at the end of the study. Each horizontal line represents mean values. C and D, Human T-cell counts in peripheral blood after 21 days of treatment. Each horizontal line represents the geometric mean. E, hCD45+ cells counted in the peripheral blood are shown over time as mean ± SEM with n = 8–10 mice/group. For B–D, asterisks denote statistical significance:*, P < 0.05; **, P < 0.01. XmAb808s is a surrogate B7-H3×CD28 bispecific identical to XmAb808 except it lacks the half-life–extending mutations in its Fc domain.
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