Activity of a single-chain immunotoxin that selectively kills lymphoma and other B-lineage cells expressing the CD40 antigen.
We have constructed anti-CD40 immunotoxins consisting of the single chain Fv (sFv) region of the anti-human CD40 mAb G28-5 fused to a truncated form of Pseudomonas exotoxin, PE40. CD40 is an integral membrane glycoprotein found on the surface of B-lineage cells, including lymphomas and leukemias, as well as certain carcinomas. Two forms of the immunotoxin were constructed, one with the light chain variable (VL) region of the sFv preceding the heavy chain variable region (VH) [G28-5 sFv(VL-VH)-PE40] and the second with the sFv in the opposite orientation [G28-5 sFv(VH-VL)-PE40]. Although both forms of G28-5 sFv-PE40 specifically bound to CD40 in ELISAs, the binding of G28-5 sFv(VL-VH)-PE40 was > 10-fold higher. A number of malignant B- and T-cell lines were screened for CD40 expression and susceptibility to G28-5 sFv(VL-VH)-PE40. All of the B-lineage cells tested were CD40 positive and sensitive to the anti-CD40 immunotoxin, with EC50s ranging from 2.5-70 ng/ml, whereas none of the T cell leukemias or lymphomas were antigen positive or were affected by the immunotoxins. Consistent with the antigen-binding results, the VL-VH immunotoxin was > 10-fold more cytotoxic than the VH-VL immunotoxin. The anti-CD40 single-chain immunotoxin fusion protein G28-5 sFv(VL-VH)-PE40 represents a potent and specific cytotoxic agent for the elimination of normal and transformed B-lineage cells expressing CD40.
Francisco
J A
JA
Department of Molecular Immunology, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.
Gilliland
L K
LK
Stebbins
M R
MR
Norris
N A
NA
Ledbetter
J A
JA
Siegall
C B
CB
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D015703:Antigens, CD / Q000276:immunology* / Q000378:metabolism; D000944:Antigens, Differentiation, B-Lymphocyte / Q000276:immunology* / Q000378:metabolism; D001402:B-Lymphocytes / Q000187:drug effects* / Q000276:immunology*; D001427:Bacterial Toxins; D001483:Base Sequence; D019013:CD40 Antigens; D016923:Cell Death / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005098:Exotoxins / Q000633:toxicity*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D000922:Immunotoxins / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity*; D015448:Leukemia, B-Cell / Q000188:drug therapy* / Q000276:immunology* / Q000378:metabolism; D015458:Leukemia, T-Cell / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D016393:Lymphoma, B-Cell / Q000188:drug therapy / Q000276:immunology* / Q000473:pathology; D016399:Lymphoma, T-Cell / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D008969:Molecular Sequence Data; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; D001427:Bacterial Toxins; D019013:CD40 Antigens; D000939:Epitopes; D005098:Exotoxins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D000922:Immunotoxins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A