Suicide genes: Monitoring cells in patients with a safety switch

Clinical trials increasingly incorporate suicide genes either as direct lytic agents for tumors or as safety switches in therapies based on genetically modified cells. Suicide genes can also be used as non-invasive reporters to monitor the biological consequences of administering genetically modified cells to patients and gather information relevant to patient safety. These genes can monitor therapeutic outcomes addressable by early clinical intervention. As an example, our recent clinical trial used 18F-9-(4-fluoro-3-hydroxymethylbutyl)guanine (18FHBG) and PET/CT scans to follow T cells transduced with herpes simplex virus thymidine kinase (TK) after administration to patients. Guided by preclinical data we ultimately hope to discern whether a particular pattern of transduced T cell migration within patients reflects early development of Graft vs. Host Disease (GvHD). Current difficulties in terms of choice of suicide gene, biodistribution of radiolabeled tracers in humans versus animal models, and threshold levels of genetically modified cells needed for detection by PET/CT are discussed. As alternative suicide genes are developed, additional radiolabel probes suitable for imaging in patients should be considered

Control of acute myeloid leukemia and generation of immune memory in vivo using AMV564, a bivalent bispecific CD33 x CD3 T cell engager

Off-the-shelf immunotherapeutics that suppress tumor growth and provide durable protection against relapse could enhance cancer treatment. We report preclinical studies on a CD33 x CD3 bivalent bispecific diabody, AMV564, that not only suppresses tumor growth, but also facilitates memory responses in a mouse model of acute myelogenous leukemia (AML). Mechanistically, a single 5-day treatment with AMV564 seems to reduce tumor burden by redirection of T cells, providing a time window for allogeneic or other T cells that innately recognize tumor antigens to become activated and proliferate. When the concentration of bispecific becomes negligible, the effector: target ratio has also shifted, and these activated T cells mediate long-term tumor control. To test the efficacy of AMV564 in vivo, we generated a CD33+ MOLM13CG bioluminescent human cell line and optimized conditions needed to control these cells for 62 days in vivo in NSG mice. Of note, not only did MOLM13CG become undetectable by bioluminescence imaging in response to infusion of human T cells plus AMV564, but also NSG mice that had cleared the tumor also resisted rechallenge with MOLM13CG in spite of no additional AMV564 treatment. In these mice, we identified effector and effector memory human CD4+ and CD8+ T cells in the peripheral blood immediately prior to rechallenge that expanded significantly during the subsequent 18 days. In addition to the anti-tumor effects of AMV564 on the clearance of MOLM13CG cells in vivo, similar effects were seen when primary CD33+ human AML cells were engrafted in NSG mice even when the human T cells made up only 2% of the peripheral blood cells and AML cells made up 98%. These studies suggest that AMV564 is a novel and effective bispecific diabody for the targeting of CD33+ AML that may provide long-term survival advantages in the clinic

Serial BLI in individual mice whose group mean BLI is shown in Fig 4C.

In the early treatment group, one mouse had no BLI signal, but required sacrifice on day 48 due to GvHD-like symptoms. For mice left untreated, treated only with T cells or only with AMV564, N = 5 mice/ group. For mice treated with both T cells and AMV564, N = 10 mice/group. (TIFF)</p

Morbundity assessment of animals.

To assess moribundity, points are assigned to each of the 5 criteria below. A total score of 6 or higher or 30% weight loss, regardless of score, requires euthanasia of the animal. (DOCX)</p

Effect of T cell and AMV564 dose on clearance of MOLM13^CG.

(A) Schema. On day 7 post i.v. injection of 3.3 x 103 MOLM13CG cells, mice were injected with a single dose of 8 x 106 T cells, i.v., and a single 5-day cycle of AMV564 (5 or 25 mcg/day, i.v.). (B) Mouse survival. *Untreated vs. T cells + either 5 or 25 mcg AMV564/day, p<0.0001. ns = no significant difference. (C) Tumor expansion measured by serial BLI, mean group signal. *Untreated vs. T cells + either dose on day 21, p<0.0001. **Difference in slope between T cells + 5 mcg vs. T cells +25 mcg, p = 0.0136, however difference in values between doses on day 21 was not significant (ns). (D) Serial BLI signal in individual mice treated with T cells and either 5 or 25 mcg of AMV564. (E) Number of T cells/ul in the blood of all mice on day 21 or total number in the (F) bone marrow and (G) spleen of surviving mice on day 51. N = 1 experiment. N = 7 animals in untreated group. N = 8 animals in 25 mcg AMV group and in group treated only with T cells. N = 10 in groups treated with T cells in combination with either 5 or 25 mcg AMV564.</p

Kinetics of tumor expansion as measured by BLI in mice injected with T cells only, or T cells plus one or two cycles of AMV564.

Same data as in Fig 2C, but rather than displaying data as group mean, the BLI signal in individual mice is shown. N = 5 mice/group for controls and n = 10 mice/group for mice treated with both T cells and AMV564. (TIFF)</p

On day 26 the number of T cells in the blood was greater and the number of tumor cells in the blood was lower in both the early and late AMV564 + T cells groups relative to only T cells.

In A, *p = 0.0039, ** = 0.0005. In B, *p = 0.0001, **p (TIFF)</p