BLOCKING NEGATIVE SIGNALS TO NK CELLS TO TREAT LEUKEMIA

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DESCRIPTION: (Adapted from the investigator's abstract) NK cells are cytolytic for tumor cells but clinical use of autologous NK cell treatment has been relatively unsuccessful. A major potential cause for this is that NK cells have receptors for 'self' class I transplantation antigens, and the majority of these receptors respond by sending negative signals that prevent NK cell lysis. This explains why NK cells preferentially lyse HLA or H2 allogeneic, or class I deficient target cells. During the first 3 years of this project, the investigators have obtained evidence that blocking negative signals for two inhibitory receptors, Ly-49I and C, with F(ab')2 5E6 MAbs enhanced survival of B6 mice infused with syngeneic C1498 myeloid leukemia cells. Use of T and B cell deficient mice indicated that NK cells were the effectors. The same treatment did not inhibit growth of syngeneic BMC in irradiated B6 mice (a safety concern) but did enhance the ability of mice to reject allogeneic BMC grafts. A new 8H7 anti-Ly-49I MAb at low doses blocks negative signals without depleting NK cells and can be used as a reagent with a longer half-life than MAb fragment. The F(ab')2 reagent is limited in function due to short half-life in serum (<18h). This renewal application has 5 specific aims: Aim 1. Generate more effective MAb reagents to block negative signals to NK cells without depleting them - the investigators have mutated the Fc region of 5E6 MAb to remove a critical N-carbohydrate attachment site that is required for the MAbs to deplete cells in vivo. Generate similar reagents against Ly-49G2, an inhibitory receptor expressed on a large fraction of murine NK cells.Aim 2. Develop rapid assays for growth assessment of leukemia cells in vivo to quicken the pace of developing new reagents, e.g., infusion of leukemic cells i.v. into irradiated hosts, and assessing proliferation 5 days later by measuring DNA synthesis, an assay for proliferating cells. Use 123I-iododeoxyuridine to label proliferating cells that can be used for imaging of growing tumors and for labeling leukemia cells that are infused so that survival can be determined by whole body counting. Aim 3. Test the reagents for the ability to 'purge' leukemia cells from syngeneic marrow cells 'spiked' with different numbers of leukemia cells.Aim 4. Expand the clinical treatment protocol to include supplemental treatment of mice with IL-2 after the infusion of syngeneic or allogeneic IL-2 activated NK cells coated with anti-5E5 and/or anti-Ly49G2 F(ab')2 MAbs. Aim 5. Extend the studies to the use of human myeloid leukemia cells, human NK cells, and immunodeficient SCID mice pretreated with asialo GM1 or SCID-NOD mice, which accept grafts of human leukemia cells. Non-depleting MAbs or fragments to human negative signaling receptors for class I antigens expressed on the leukemia cells will be tested for anti-leukemia effects. Success with these studies will hopefully determine if this approach has potential for clinical application.