Purging of contaminating breast cancer cells from hematopoietic stem cell grafts by adenoviral GAL-TEK gene therapy and magnetic antibody cell separation

The presence of contaminating tumor cells in autologous bone marrow or peri pheral blood stem cell (PB-SC) preparations increase the likelihood of rela pse in women receiving transplants for metastatic breast cancer. We describ e a new technique for purging breast cancer cells (BCCs) that combines two independent strategies: (a) the specific enrichment of CD34(+) progenitor s tem cells by magnetic antibody cell separation (MACS), and then (b) infecti on of the contaminating BCCs with a recombinant adGAL-TEK marker/suicide ge ne adenovirus (ad-v), followed by the addition of ganciclovir (GCV), Infect ion with this ad-v results in three to four times greater expression of ad- v-delivered reporter gene in BCCs than in CD34(+) cells, In addition -2 h, -low multiplicity of infection (50:1) adGAL-TEK infections of BCC lines (MC F-7 and BT474) eradicated >99% of BCCs after 72 h of exposure to 20 mu M GC V, However, exposure to both adenovirus and GCV at the MOIs and doses used had little effect on hematopoietic stem cells to form colonies in colony-fo rming unit assays, adGAL-TEK infection in our model system (10(3)-10(5) BCC s added into 10(7) HSCs) also resulted in the 3 to 5 log eradication of clo nogenic BCCs after the addition of GCV, MACS enrichment/purification of CD3 4(+) cells from PB-SC contaminated with 2 x 10(6) to 5 x 10(7) BCCs followe d by adGAL-TEK infection and GCV addition resulted in 5-7-log depletion of clonogenic BCCs as well as enrichment of CD34(+) progenitor cells to >98%, with the recovery of >70% of hematopoietic stem cells. This adenoviral purg ing system is so robust that poor MACS purification, resulting in 1.5-log d epletion of BCCs, still permits excellent ad-v infection and BCC killing.