PMC

The 2.43 Mb retains Lyt2.2 antigen specificity. Primary cells isolated from the peripheral blood, thymus, spleen, and lymph nodes of B/6 and C3H mice were stained with phycoerythrin (PE)–anti-CD4 and either a commercial FITC–anti-CD8 antibody (A) or the Lyt2.2-specific FITC-2.43 Mb (B). The lack of CD8-FITC staining in Lyt2.1⁺ C3H mice shows the 2.43-Mb specificity for Lyt2.2⁺ B/6 mice.

Immuno-PET imaging of ⁶⁴Cu-NOTA-2.43 Mb in antigen-blocked and antigen-depleted B/6 mice is shown. Immuno-PET images were acquired 4 h p.i. of ⁶⁴Cu-NOTA-2.43 Mb into: WT B/6 (A), B/6 mice blocked with 4 mg/kg of cold Mb (B), and B/6 mice treated with an anti–CD8-depleting antibody (C). Solid white arrows (Upper, 20-mm coronal MIPs) indicate where the transverse images (Lower, 2-mm MIPs) are acquired. (Lower) Hollow white arrows indicate the location of the spleen.

Immuno-PET imaging of ⁶⁴Cu-NOTA-2.43 Mb and ⁶⁴Cu-NOTA-YTS169 Mb shows in vivo specificity of the 2.43 Mb to Lyt2.2⁺ mice. (A) ⁶⁴Cu-NOTA-2.43 Mb was injected into B/6, C3H, and NOD SCID gamma mice for immuno-PET imaging 4 h p.i. (B) ⁶⁴Cu-NOTA-YTS169 Mb was injected into B/6 and C3H mice for immuno-PET imaging 4 h p.i. Solid white arrows (Upper; 20-mm coronal MIPs) indicate where the transverse images (Lower; 2-mm MIPs) are acquired. Hollow white arrows indicate the location of the spleen.

Anti-CD8 Mb does not deplete CD8-expressing cells in vivo. B/6 mice were treated for three consecutive days with either 330 μg of anti-CD8 depleting antibody (clone 53-6.7) injected i.p. (A) or 250 μg of 2.43 Mb injected i.v. (B). Cells were then isolated from the peripheral blood, thymus, spleen, and lymph nodes for staining with anti–CD4-PE and FITC-conjugated 2.43 Mb.

Mb construction and epitope specificity are shown. (A) Anti-CD8 2.43 and YTS169 Mbs contain the rat V_H-V_L separated by an 18-aa linker, followed by the murine IgG2a hinge (h), murine C_H3, and a C-terminal hexahistidine (HisTag). (B) Murine CD8α (Lyt2) is expressed as two isoforms, Lyt2.1 and Lyt2.2, that differ in a single amino acid, and it is restricted to specific mouse strains. The 2.43 Mb binds CD8α only in Lyt2.2⁺ mouse strains, whereas the YTS169 Mb binds CD8α in all mouse strains.

Immuno-PET imaging of ⁶⁴Cu-NOTA-2.43 Mb 4 h p.i. is shown. Immuno-PET/CT images were acquired 4 h after i.v. injection in B/6 mice. The white arrows (2-mm transverse MIPs) are used to highlight uptake in various lymph nodes (Right) and the spleen seen in the whole-body 20-mm coronal MIPs (Left). A.LN, axillary lymph nodes; B, bone; C.LN, cervical lymph nodes; I.LN, inguinal lymph nodes; Li, liver; MIPs, maximum intensity projections; P.LN, popliteal lymph nodes; Sp, Spleen.