Evaluation of antihuman T lymphocyte saporin immunotoxins potentially useful in human transplantation.
We have synthesized 3 immunotoxins (ITs) by covalently coupling the saporin-6 hemitoxin (SAP) to OKT11, SOT3, and SOT1a murine monoclonal antibodies that recognize human T lymphocyte CD2, CD3, and CD5 surface antigens, respectively. The resulting ITs, referred to as OKT11-SAP, SOT3-SAP, and SOT1a-SAP, are equally effective in inhibiting eukaryotic protein synthesis in a cell-free system, and all 3 ITs bind to human T lymphocytes in an almost comparable manner. However, these reagents differ markedly in their ability to kill target T lymphocytes as assessed by measuring the inhibition of DNA synthesis and growth of clonable T lymphocytes in response to mitogenic and allogeneic stimuli. Whereas the anti-CD2 IT, OKT11-SAP, shows moderate cytotoxicity against T lymphocytes, the anti-CD3 IT, SOT3-SAP, and the anti-CD5 IT, SOT1a-SAP, are highly effective in eliminating the same target cells. The concentrations inhibiting 50% (IC50) of T lymphocyte DNA synthesis are 60 nM, 4.5 nM, and 1.4 nM for OKT11-SAP, SOT3-SAP, and SOT1a-SAP, respectively. Among 3 tested lysosomotropic amines, i.e., ammonium chloride, chloroquine, and amantadine, the latter only moderately potentiates the cytotoxicity of SOT1a-SAP (IC50 0.36 nM). We show that the conditions under which T lymphocyte killing is accomplished require less than 10 min exposure of T lymphocytes to the ITs, in the absence of adjuvant molecules artificially added to the incubation medium and at physiologic culture pH. These experimental characteristics of unprecedented closeness to a physiologic in-vivo model are likely to reflect the biophysical properties of the SAP moiety of the ITs. We conclude that clinical studies are warranted to define the advantage of using SAP ITs over previously described immunoconjugates.
Siena
S
S
Cristina Gandini Bone Marrow Transplantation Unit, Division of Medical Oncology, Istituto Nazionale Tumori, Milan, Italy.
Bregni
M
M
Formosa
A
A
Martineau
D
D
Lappi
D A
DA
Bonadonna
G
G
Gianni
A M
AM
D000588:Amines / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D004357:Drug Synergism; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D008212:Lymphocyte Depletion; D008247:Lysosomes / Q000187:drug effects; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011817:Rabbits; D012156:Reticulocytes; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins; D013601:T-Lymphocytes / Q000187:drug effects*
D000588:Amines; D000922:Immunotoxins; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; D000076985:Saporins