Southwestern In Vivo Cellular and Molecular Imaging Program

Project 5
Imaging Mouse Tumors to Evaluate Treatment by Blocking Negative Signals to NK Cells

Investigators and Areas of Expertise:

Michael Bennett, MD, Department of Pathology, is an immunologist and will be responsible for all tumor immunology studies except imaging. Padmakar Kulkarni, Ph.D., Department of Radiology, is a radiochemist, who will be responsible for designing radiochemical labeling. Dr. Anca Constantinescu will perform labeling and quality control of the labeled products and administer to animals. Dr. Oz will oversee all nuclear medicine imaging experiments and data will be transferred to the Informatics core (Dr. Roddy McColl) for analysis. MR studies will be coordinated by Dr. Mason.

Hypotheses and Specific Aims:

Hypotheses:

1) Blocking of inhibitory receptors for class I antigens on leukemia cells will result in decreased rate and amount of bone marrow involvement due to enhanced NK cell function.

2) The assessment of early stages in tumor progression will lead to more rapid and accurate results of experimental tumor therapies.

3) NK cells kill tumor cells both by causing necrosis and/or apoptosis, regulated in part by expression of 'death receptors' on particular tumor cells.

Specific Aims:

1) Develop magnetic resonance imaging (MRI) to detect involvement of bone marrow with leukemia as an assay for disease prior to overt leukemia and/or death. The early detection of anti-tumor activity by NK cells treated with blocking antibodies will accelerate research efforts in obtaining better reagents, and will avoid using long-term survival studies as the standard assay for efficacy.

2) Develop single photon and positron emitter (SPECT and PET) methods to image rapid destruction of infused tumor cells by NK cells treated with non-depleting mAbs to block negative signals. PET and SPECT imaging assays with radiolabeled reagents such as ^11IIn-oxine will be performed to develop methods to detect tumor lysis by NK cells in the lung after infusion.

3) Determine the mechanisms of tumor cell killing by NK cells, by imaging with agents that localize in areas of necrosis or apoptosis. SPECT or PET emitting isotopes conjugated with annexin can detect tumor apoptosis, while compounds like ^99mTc labeled glucarate or pyrophosphate can detect areas of necrosis similar to myocardial infarctions. In this system unlabeled cells are infused and the isotope is administered later to image 'hot spots' where apoptosis and/or necrosis are occurring.

Background and Significance:

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, but not cells sharing all of the ‘self’ class I antigens with the NK cells. This and other labs have been studying this peculiar property of NK cell immunogenetics since 1963 following the discovery of 'hybrid resistance', or the rejection of parental strain H2 homozygous marrow cells by F1 hybrid mice heterozygous for H2. The field has ‘exploded’ with the discovery of the receptors, which recognize class I antigens and are inhibitory. During the project (R01 CA70134) we have obtained evidence that blocking negative signals to two inhibitory receptors that recognize H2-K^b, Ly49I and C, with F(ab’)₂ 5E6 mAbs enhanced survival in 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 (bone marrow cells) in irradiated B6 mice (a safety concern), but did enhance the ability of mice to reject allogeneic BMC grafts. A new 8H7 anti-Ly49I mAb, used in low amounts, blocks negative signals without depleting NK cells, and can be used as a reagent with a longer half-life than mAb fragments. The F(ab’)₂ reagent is limited in function due to a short half-life in serum (<18 h). Specific aims in the R0170134 grant include 1) Generate more effective mAb reagents to block negative signals to NK cells without depleting them, e.g., we 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 Ly49G2, an inhibitory receptor expressed on a large fraction of murine NK cells that recognize primarily H2-D^d but also recognizes H2^b class I antigens in H2^b mice. 2) Test the reagents for the ability to ‘purge’ leukemia cells from syngeneic marrow cells ‘spiked’ with different numbers of leukemia cells. 3) Expand the clinical treatment protocol to include the infusion of syngeneic or allogeneic IL-2 activated NK cells coated with anti-5E6 and/or anti-Ly49G2 F(ab’)₂ mAbs, with treatment of mice with IL-2 to augment the NK cells treatment effect. 4) Extend the studies to the use of human myeloid leukemia cells, human NK cells, and immunodeficient SCID or SCID-NOD mice pretreated with asialo GM1 to deplete NK cells, because these mice accept grafts of human leukemia cells. Non-depleting mAbs or fragments to human negative signaling receptors of co-transferred NK cells for class I antigens expressed on the leukemia cells will be tested for anti-leukemia effects. Success with these studies will determine if this approach has potential for clinical utility. 5) To develop rapid assays for leukemia progression in mice to accelerate the pace of research, because survival studies require at least 60 days. The first ideal assay would detect leukemia development before it is clinically detectable. The second type of assay would be the detection of NK cell activity against the leukemia in vivo as soon as possible, an assay that correlates with survival and the clinically undetectable status mentioned above. 

In order to apply the use of NK cells as anti-tumor effector cells for BMT (bone marrow transplantation), several models can be used with clinical relevance. Autologous BMT has been hampered as a therapy for cancer due to its relatively high relapse rate, particularly when compared with allogeneic BMT. This relapse may be due to two sources, minimal residual disease (MRD) in the host after conditioning and contaminating tumor cells in the BMC graft itself. The use of NK cells, and importantly, blockade of inhibitory receptor interactions with the tumor cell, to purge the tumor in the BMC graft ex vivo may allow for significant reductions in relapse. In addition, these same methodologies may be applied post-BMT as an adoptive immunotherapy. In order to ascertain whether this concept can be applied to allogeneic BMT, one must determine whether such a blockade may result in autoreactivity. Autoreactivity itself may be a desirable result in cancer therapy as long as the tumor is destroyed and vital organs are not irreparably harmed. In addition, the recent success of delayed lymphocyte infusions for chronic myeloid leukemia (CML) suggest that at least one mechanism is the attack of host hematopoietic cells. Therefore, blockade of inhibitory receptors, even if NK cells attack host hematopoietic cells following allogeneic BMT, may yield significant anti-tumor responses as well as greater donor chimerism. Little is known with regard to the physiological role of these inhibitory receptors, both with NK cell development and function. Thus, these studies will not only provide the evaluation of inhibitory receptor blockade as a novel means for promoting anti-tumor responses but may provide insights into their role in NK development and role in diseases such as autoimmunity.

Images of ¹¹¹In-labeled (220 mCi) YAC-1 lymphoma cells (Figure)

References:

1. Koh, CY, George T, Bennett M, Blazar BR, Murphy WJ. Adoptive transfer of donor ALAK cells with blockade of Ly49 receptors using 5E6 mAb F(ab')2 fragments augments NK-mediated anti-tumor effects. FASEB J. 1999., 13,, A308.

2. Stoneman, E, Bennett M, An J, Chestnut KA, Wakeland EK, Scheerer M, Siciliano MJ, Kumar V, Mathew PA. Cloning and characterization of 5E6 (y49-C), a receptor expressed on a subset of murine natural killer cells. J. Exp. Med. 1995, 182, 305-313.

3. Bennett, M. Rejection of marrow allografts: importance of H2 homozygosity of donor cells. Transplantation 1972, 14, 289-298.

For Further Information Contact: RALPH  P. MASON, Ph.D.
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