BLOCKING NEGATIVE SIGNALS TO NK CELLS TO TREAT LEUKEMIA

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Natural killer (NK) cells are cytolytic for tumor cells, and stimulation of NK cells with low doses of interleukin-2 (IL2) have produced dramatic treatment results, especially in patients receiving T cell depleted allogeneic marrow transplants. Less success has been obtained by infusing autologous NK cells and giving high doses of IL2. NK cells are also involved in the rejection of incompatible bone marrow cell (BMC) grafts in rodents. The immunogenetics of BMC transplantation is unusual in that parental strain BMC are often rejected by F1 hybrid mice (hybrid resistance). Apparently NK cells will lyse target cells that fail to send negative signals to NK cells through certain receptors. These receptors are coded for by genes in the Ly49 NK gene family on mouse chromosome 6. One of these receptors, 5E6(Ly49C), receives negative signals from H2+ class I Ags. This explains why the 5E6 subset of Nk cells is responsible for the rejection of parental or allogeneic H2d but not H2b BMC grafts. A new in vitro assay mimics BMC grafts in vivo, which involves the lysis of lymphoblasts by NK cells. The inability of 5E6+ NK cells to lyse H2b target cells can be reversed by adding F(ab')2 anti-5E6 mAb, blocking the negative signals. In this assay syngeneic target cells are not lysed; even 5E6+ H2d NK cells fail to lyse syngeneic blasts unless F(ab'2 anti-5E6 is added. The knowledge that presence or absence of negative signals from class I Ags to NK cell receptors regulates lysis of target cells provides a new approach to utilize NK cells to treat syngeneic or autologous leukemia/lymphoma and other neoplasias. We have developed two specific aims for this proposal. In Aim 1, we will generate F(ab')2 anti5E6 by enzymatic digestion of whole mAbs and a recombinant anti-E6 mAb in which the murine Fc portion is replaced by human IgGI (Hgamma1)Fc fragment. To create this, the VDJ fragment of the anti-5E6 hybridoma IgG2a heavy chain gene will be spliced to the Hgamma1 gene in one vector, and the anti-5E6 kappa light chain gene and a neomycin resistance gene will be created in a second vector. These two vectors will be cotransfected into SP2/O hybridoma cells. The anti-5E6-Hgamma1 should not fix complement or cause antibody dependent cellular cytotoxicity (ADCC). We will also generate a recombinant 5E6 (extracellular domain)-Hgamma1 construct 5E6-Hgamma1 to be transfected into SP2/O cells. In Aim 2, we will first determine if the administration of F(ab')2 anti5E6, anti-5E6-Hgamma1 or 5E6-Hgamma1 to irradiated mice infused with syngeneic BMC can cause resistance to engraftment, presumably by blocking negative signals in a large fraction of 5E6+NK cells. We will then proceed to treat EL4 lymphomas and B16.F10 melanomas [B6 mice], L1210 lymphoma cells [DBA/2 and C.B-17 severe combined immune deficient (SCID) mice], and Friend virus leukemia [BALB/c mice]. Some mice will receive iL2 or interferon alpha/beta to boost NK cell activity. The methods will be to initially inject the reagents intraperitoneally (i.p.), and later the hybridomas secreting the anti-5E6- Hgamma1 or 5E6-Hgamma1 will be put in diffusion chambers i.p. to create a continuous supply of the reagents. Thus, this is a model of cancer therapy.