Table 1.

Design, binding avidities, and cytotoxic activities of CD33/CD3 TandAbs

TandAbaCD3 domainCD3 KD (nmol/L) Human T cellsCD33 domainCD33 KD (nmol/L) HL-60 cellsVH VL linker sequence (L2)CD25 induction EC50 (pmol/L)bCD69 Induction EC50 (pmol/L)bT-cell proliferation in PBMCs EC50 (pmol/L)cCytotoxicity HL-60 cells (% ± SEM)dCytotoxicity KG-1a cells (% ± SEM)d
T563641.3A50.4GGSG67782.9 ± 3.780.2 ± 1.9
T550641.5A40.3GGSG63284.7 ± 2.385.6 ± 1.6
T547061.9A50.5GGSG106648.0 ± 2.478.6 ± 2.3
T562642.1A30.3GGSG107686.0 ± 0.469.8 ± 5.7
T597642.1A89.7GGSGND22550012.4 ± 1.00.0 ± 0.2
T613642.3A105.6GGSGND5726424.5 ± 1.91.1 ± 0.2
T546062.4A30.5GGSG117943.2 ± 15.874.6 ± 3.2
T548062.6A60.3GGSG115652.7 ± 8.184.7 ± 1.4
T581643.3A75.0GGSG30114304.2 ± 0.20.7 ± 0.4
T605644.1A90.7GGSG104774.2 ± 7.444.4 ± 5.3
T564645.1A60.3GGSG12386.0 ± 1.481.3 ± 1.5
T589646.3A22.8GGSG95679.4 ± 3.583.8 ± 2.9
T5226449.7A113.7GGSGGS13465506.3 ± 3.32.1 ± 0.7
T5538955.7A40.2GGSG30222370.4 ± 2.51.3 ± 0.4
T4971169.5A51.0GGSGGS116747423.8 ± 6.90.3 ± 0.3
T4791169.8A40.2GGSG4227480.9 ± 3.64.6 ± 2.1
T4811179.3A60.5GGSG94624424.1 ± 4.00.7 ± 0.8
T4801181.9A51.1GGSG117876313.1 ± 3.60.0 ± 0.5
T4981186.3A60.4GGSGG39214845.7 ± 6.41.4 ± 0.2
T4781194.2A30.6GGSG9291898.0 ± 1.60.4 ± 0.4
T6098997.2A90.4GGSG41173773.7 ± 2.61.5 ± 0.3
T59389143.8A24.1GGSG98753831.2 ± 3.91.1 ± 0.3
  • Abbreviation: ND, no CD25 activation detectable.

  • aTandAbs are listed in order of increasing CD3 avidity.

  • bCD25 and CD69 induction was measured after 24 hours in unfractionated PBMC cultures.

  • cT-cell proliferation induced by CD33/CD3 TandAbs in unfractionated PBMCs with CD33+ cells present.

  • dCytotoxicity (%) after 48 hours of DAPI+ cells at a TandAb concentration of 25 pmol/L in the presence of healthy donor T cells at an E:T cell ratio of 5:1 from three independent experiments performed in duplicate wells.