It has been observed that cAC10 , a monoclonal antibody that is chimeric, has the capacity to cause the in vitro arrest of the growing of the CD30+ cell lines in severe combined immunodeficiency (SCID)in models of mice having Hodgkin disease (Chiarle et al. , 1999; Weiner and Adams, 2000 ). Thus, it can `be said that these antibodies have antitumor activity.
The potency of CD30 +-targeted mAb activity was increased by generating an drug and antibody conjugate, “cAC10-vcMMAE”. MMAE is a synthetic analog of dolastatin 10. “cAC10-vcMMAE” has potent cytotoxic activity against CD30 expressing cell lines and has exhibited stability in physiological conditions.
The aim of the following experiment was to test the potency of “cAC10-vcMMAE” for causing tumor growth suppression in subcutaneous models in mice having tumor.
Materials and Methods
Cells and Reagents
Fresh human plasma, Anti-CD30 mAb Ki-1, cBR96, a human chimerical IgG1κ, CD30+ HD lines L540, KM-H2, HDLM-2, and L428 and the ALCL line Karpas 299 were the materials obtained for the experiment.
Fluorescence-Activated Cell Sorter Analysis
Relative levels of CD30 exhibiting their respective potencies as antitumor agents on different tumor cells were compared using flow cytometry.
Satrutauton binding of mAb and ADC was also studied similarly.
These assays were performed with Alamar Blue dye reduction assay.
Xenograft Hodgkin Disease Models of Humans
For ALCL- and HD-localized, subcutanoeus diseases models, Karpas 299 (5 × 106) or L540cy (2 × 107) cells were injected into the right flanks of C.B.-17/IcrHsd-SCID mice. Therapeutic interventions with “cAC10-vcMMAE” or controls were initiated when the size of tumor in each group of five animals was an average 100mm3.
Preparation of Antibody-Drug Conjugates
Binding of “cAC10-vcMMAE” and cAC10 to CD30+ Cells
It was found that the affinity of “cAC10-vcMMAE” to Karpas 299 cells was similar to that with parental mAb cAC10. Both the monoclonal antibody and ADC exhibited the very same dose-dependent activity and saturated at around 1μg/ml
In Vitro Selectivity and Effectiveness of “cAC10-vcMMAE”
The Alamar Blue assay revealed that “cAC10-vcMMAE” showed potent cytotoxic activity when Karpas 299 cells were exposed it. The IC50 value of mAb was 2.5 ng/ml, while the IC50 was 850 ng/ml for the control ADC.
Cell Cycle Effects of “cAC10-vcMMAE”
Cells treated with the same amount of “cAC10-vcMMAE” exhibited a pronounced increase in G2 cells, as shown by analysis of DNA content. There was also decrease of G1 cells occurring in 12 hours after being targeted. Along with this diminish, the number of G2 cells grew from 10% in cells subjected to no treatment to 47% following “cAC10-vcMMAE” exposure at 12 hours. Being targeted at 24 hours showed the G2 population to turn extremely diffuse, with sub-G2 and sub-G1 DNA content turning obvious. At 48 hours, around 53% of the cells displayed sub-G1 DNA content, seen with DNA fragmentation that can be described as decaying [Wahl et al., 2002].
Antitumor Activity of “cAC10-vcMMAE” In Vivo
The anti-tumor ability of “cAC10-vcMMAE” was later computed in subcutaneous models of HD and ALCL tumors in mice having SCID. In the Karpas 299 ALCL model, a dose of “cAC10-vcMMAE” of 1 mg/kg produced complete waning of tumor in all the animals.
Considered along with data on lethality, cAC10–vcMMAE generated enough tumor regression in subcutaneous tumor models in mice. The dose was less than 1/30th of the highest dose tolerated.
- Chiarle R., Podda A., Prolla G., Gong J., Thorbecke G.J., Inghirami G., 1999 CD30 in normal and neoplastic cells. Clinical Immunology, 90, pp. 57–164.
- Wahl A.F., Klussman K., Thompson J.D., et al., 2002, The anti-CD30 mAb SGN-30 promotes growth arrest and DNA fragmentation in vitro and affects antitumor activity in models of Hodgkin’s disease. Cancer Research, 62, pp. 3736–3742.
- Weiner L.M. and Adams G.P. 2000, New approaches to antibody therapy. Oncogene., 19, pp. 6144–6151.