ANTI-ICAM-R ANTIBODY-INDUCED APOPTOSIS

Abstract: Methods for inducing apoptosis in cell populations are disclosed which comprise the step of contacting the target cell population with an antibody immunoreactive with ICAM-R.

Description: ANTMCAM-R ANTIBODY-INDUCED APOPTOSIS FIELD OF THE INVENTION The present invention provides methods to induce apoptosis in a cell population by contacting the cell population with an antibody immunospecific for ICAM- R. BACKGROUND ICAM-R (see U.S. Patent 5, 525,487 issued June 11, 1996) is a member of the immunoglobulin superfamily of adhesion receptors. In normal subjects, protein expression is restricted to immature and mature hematopoietic cells of the lymphoid, myeloid, and monocytic lineages. The surface molecule is also expressed on acute and chronic myeloid leukemia cells, acute and chronic lymphocytic leukemia cells, hairy cell leukemia cells, multiple myeloma cells, and lymphoma cells. Ligands identified for ICAM- R include LFA- 1 (CD 1 1 a/CD 18) and α/CD 18 which bind to the IC AM-R extracellular region composed of five immunoglobulin-like domains. Presumably, ligand binding permits transmembrane signal transduction into the cell through the ICAM-R cytoplasmic domain. On T cells, ICAM-R acts as a co-stimulatory molecule [Hayflick, et al., J. Biol. Chem. :22207-14 (1997)] and plays a key role in the initiation of the immune response. ICAM-R also mediates T cell aggregation and spreading [Hayflick, et al., supra]. While the exact mechanisms by which these activities are carried out are not fully understood, it is believed that phosphoryiation of specific cytoplasmic serine residues of ICAM-R is required [Hayflick, et al., supra]. Other intracellular events have been observed in response to ICAM-R engagement, such as an activation of intracellular calcium flux, phosphoryiation of tyrosine residues of the tyrosine kinases p561ck and p59fyn, and phosphoryiation of cyclin-dependent kinase cdc2 [Chirathaworn, et al, J. Immunol. 755:5479-5482 (1995); Juan, J. Exp. Med 179:1747-1756 (1994)]. Both p561ck and p59fyn are involved in cell death signaling [Reichert, J. Immunol. 27:295-306 (1998); Schlottman, J. Leukoc. Biol. 60:546-554 (1996)] and phosphoryiation of cdc2 occurs on tyrosine 15, an event known to down-regulate cdc2 function and initiate cell cycle arrest. Programmed cell death (PCD), as distinguished from cell necrosis, is a normal process that occurs during development and homeostasis of multicellular organisms. See review by Schwartz et al, Immunology Today 74:582-590 (1993). PCD can be divided into two categories, apoptosis and non-apoptotic death. Apoptosis is defined as cell death that includes morphological changes including membrane bleb, or "apoptotic body" formation, and condensation/margination of nuclear chromatin. Non- apoptotic PCD occurs in the absence of the noted morphological changes. As part of the apoptotic process, cellular signaling pathways have been found to play important roles. Several cell surface receptors (TNFR I and Fas) have been shown to trigger apoptosis when engaged by specific ligands or antibodies. Work over the past decade has begun to define the intracellular protein components and their functions during the process of apoptosis, as well as methods to modulate the process. Thus there exists a need in the art to identify the molecular components of the normal hematopoietic cell's machinery involved in the subtle balance between blood cell production and destruction. Identification of modulators of hematopoietic cell growth and survival will lead to the development of new therapeutical strategies for the treatment of leukemias, myeloma and lymphomas. SUMMARY OF THE INVENTION The disclosures of all references cited herein are incorporated by reference. In general, the present invention provides methods to induce apoptosis in a cell population comprising the step of contacting the cell population with an antibody immunospecific for ICAM-R, a cellular adhesion molecule as defined in U.S. Patent 5, 525,487. Antibodies of the methods include monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, and CDR-grafted antibodies (including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) and other binding proteins specific for ICAM-R. The term "specific for" indicates that the variable regions of the antibodies recognize and bind ICAM-R polypeptides exclusively (i.e., able to distinguish ICAM-R polypeptides from the family of cell adhesion molecule polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), but may also interact with other proteins through contact in peptide domain other than the CDRs. Specific ICAM-R antibodies are described co-pending U.S. Patent Application Serial No. 08/863,790, incorporated herein by reference. In one embodiment, the present invention provides methods for inducing apoptosis in a cell population comprising the step of contacting the cell population with an antibody immunoreactive with domain 1 of ICAM-R. In another aspect, the invention provides methods for inducing apoptosis in a cell population comprising the step of contacting the cell population with an antibody that permits cross-linking of ICAM-R through domain 2. The invention further provides methods for inducing apoptosis in a cell population comprising the step of contacting the cell population with a fusion molecule comprising an antibody domain immunoreactive with domain 1 of ICAM-R. The invention also provides methods for inducing apoptosis in a cell population comprising the step of contacting a cell population with a fusion protein comprising an antibody domain that cross-link domain 2 of ICAM-R. In one aspect, the antibody is a fusion protein provided as a surface molecule expressed in a cell line modified ex vivo. Fusion proteins of the invention include those which comprise immunospecific domains of anti-ICAM-R antibodies in combination with additional components that improve biological activity of the immunospecific domains. For example, fusion proteins comprising additional sequences that improve solubility, increase circulating half-life, and/or reduce antigenicity of the antibody domain are contemplated. Fusion proteins comprising antibody regions fused to cytotoxic components that act on specific cell types to complement the apoptotic effect of antibody binding are also contemplated. In a preferred embodiment, the methods of the invention induce apoptosis in a cell population which comprises hematopoietic cells. In a most preferred embodiment, the cell population comprises tumor cells. In one aspect of the invention, the antibody is a humanized antibody, preferably, the antibody is a human antibody. Humanized ICAM-R antibodies contemplated include those as disclosed in U.S. Patent 5,837,822, issued November 17, 1998 and incorporated herein by reference. In a preferred embodiment, the antibody is immunoreactive with domain 1 of ICAM-R and inhibits ICAM-R binding to an ICAM-R binding partner. In a more preferred embodiment, the antibody is secreted by the hybridoma designated ICR1.1 (ATCC Accession No. HB 1 1053), the antibody secreted by the hybridoma designated ICR8.1 (ATCC Accession No: 11232) or humanized. The invention further contemplates use of antibody fragments comprising one or more variable immunoreactive domains. The methods of the invention embrace various modes of antibody or fusion protein administration to an animal, preferably to a mammal, and more preferably to a human. The invention further contemplates administration of antibodies or fusion proteins to other animals, for example, companion animals, including dogs and cats; farm animals, including cattle, horses, sheep, pigs, and goats; laboratory animals, including rats, mice, rabbits, and guinea pigs; birds; fish; and reptiles. Most preferred is parenteral administration, e.g., by a route selected from the group consisting of intravenous injection, intra-arterial injection, and intra-tumoral injection. Other routes of administration are also contemplated, including for example, oral, nasal, ocular, rectal, vaginal, urethral, intramuscular, and topical pathways. The invention further contemplates compositions comprising antibodies, fusion proteins, antibodies, or biologically active fragments thereof , in combination with one or more pharmaceutically acceptable carriers. The methods of the invention are preferably carried out as independent treatments. Alternatively, methods of the invention further comprise the step of subjecting the target cell population to one or more adjunctive treatments selected from the group consisting of chemotherapy and radiation therapy. The utility of the methods of the invention is manifest. Induction of apoptosis is particularly relevant in bone marrow transplant patients who have become refractory to traditional chemotherapy and radiotherapy. Induction of apoptosis through methods of the invention can also complement ongoing chemotherapy and radiotherapy to the extent that lower doses or shorter regimens of chemotherapy or radiotherapy can be employed, thereby reducing the side affects associated with those treatments.. DETAILED DESCRIPTION OF THE INVENTION The present invention is illustrated by the following examples which relate o materials and methods to induce apoptosis. Example 1 describes induction of apoptosis in normal marrow cells. Example 2 two relates to induction of apoptosis in leukemic cells from donors and leukemic cells in culture. Example 3 addresses modes of action for anti- ICAM-R antibody induce apoptosis. Example 4 relates to assessment of the role of CD18/CD1 la in ICAM-R antibody-induced apoptosis. Example 5 describes the role of F c receptor engagement in ICAM-R antibody-induced apoptosis. Example 6 addresses anti-ICAM-R antibody-induced apoptosis in vivo. Example 1 ICAM-R Antibody Induction of Apoptosis in Normal Marrow Cells Previous observations have indicated that ligand engagement of ICAM-R results in cytoplasmic phosphoryiation events which are directly or indirectly associated with components in known apoptotic pathways. In view of these findings, a first series of experiments were undertaken to determine what effects antibody engagement of ICAM-R had on continued viability of normal cell types. Marrow samples were obtained from 18 healthy human donors . Protocols and consent forms were approved by the Institutional Review Board of the Fred Hutchinson Cancer Research Center. Marrow aspirates were performed according to standard procedures [Hematol. Oncol. Clin. North Am. 8:651-63 (1994)]. Buffy coat cells were collected by centrifugation (20 minutes at 2000 rpm) of heparinized diluted (1 :3 dilution in Iscove's medium) marrow samples. Red cell hemolysis was performed by a 10 min. incubation in hemolytic buffer (0.83% ammonium chloride, 0.1% sodium bicarbonate, EDTA-free) at 37°C, followed by one wash. Cells were plated at 2 x 10 6 cells/ml in 24 well plates (1 ml well) or T 25 flasks (5 ml) and cultured in a 5% CO 2 humidifier at 37°C in media containing Iscove's 10% heat inactivated, endotoxin-free fetal bovine serum (FBS), 10% horse serum (HS), 2% L-glutamine, and 1% penicillin-streptomycin. Cells were incubated with either monoclonal antibody ICR1.1 (which recognizes domain 1 of ICAM-R and blocks LFA-1 binding to ICAM-R), monoclonal antibody ICR9.2 (which recognizes domain 2 of ICAM-R and does not block LFA-1 binding), or no antibody. ICR 1.1 and ICR 9.2 have been previously described [Sadhu, et al, Cell Adhes. Commun. 2:651-663 (1994); Nazeux, et al, Nature 360:485-488 (1992)]. Anti-ICAM-R monoclonal antibodies were purified from mouse ascites preparation, tested to be endotoxin free, and stored in PBS. Each monoclonal antibody was used at the final concentration of 10 μg/ml, and non-adherent cells were collected at different time points between three and seven days. Changes in the relative proportions of lymphoid, myeloid, and monocytic populations were determined by Fluorescence Activated Cell Sorting (FACS) (discussed below) with electronic gates drawn on forward/side scatter dot plots to permit data collection based on cell size and complexity. The number of dead cells at each time point was evaluated using propidium iodide (PI) staining according to manufacturer's suggested protocol. Briefly, an aliquot of non-adherent cells was collected, washed in PBS, and resuspended in PBS containing a final concentration of 2.5 μg/ml PI. Positive staining was detected by flow cytometry (n=9). In two experiments, both non-adherent and adherent cells were detached and analyzed simultaneously to rule out compartmentalization. In order to assess the onset of apoptosis, Annexin N FITC staining (performed according to manufacturer's suggested protocol) was carried out in combination with PI staining. Annexin V binds to phosphatidylserine which becomes exposed on the outer leaflet of the cell membrane during early apoptosis. In dead cells, the inner leaflet of the cell membrane is available for binding Annexin N, since the plasma membrane is disrupted. Double staining with a DΝA dye, such as PI, and FITC-labeled Annexin V allows the electronic exclusion of dead cells (Annexin N7PT) and quantitation of apoptotic cells (Annexin N + /PT) in FACS analysis. The double staining procedure therefore allows for quantitation of viable cells (Annexin N ' /PT), apoptotic cells (Annexin V7PI " ), and dead cells (Annexin N + /PT). At each time point, 0.3 to 1.0 x 10 6 normal cells per condition were collected, washed once in PBS, and resuspended in 200 μl ice-cold buffer containing 2.0 μl ready-to-use Annexin V FITC and 0.8 μl PI. Samples were incubated on ice for ten ® minutes in the dark and run on a FACScan . Parameters and regions were set up using no monoclonal antibody condition as the control, and fluorescence was analyzed on a logarithmic scale. Cells staining with PI (necrotic or late apoptotic) were gated out and the percentage of early apoptotic cells (staining Annexin N7PI ' ) in ICR 1.1 -treated cultures was compared to ICR9.2-treated cultures. In order to assess cell type in each aliquot per time point, the following analysis was carried out. Cells were collected, resuspended in 15% FBS-PBS, aliquoted to 0.2 to 0.5 x 10 6 cells/tube in 100 μl and incubated on ice. FITC-conjugated monoclonal antibodies to surface receptors were added to the cells at a final concentration of 10 μg/ml. The labeled cells were incubated 20 minutes on ice, washed twice, and resuspended in 250 μl PBS containing 1% BSA. Monoclonal antibodies used included anti-CD3 (marker for T cells), anti-CD 14 (myelomonocytic cells), anti-CD 15 (neutrophils, eosinophils, monocytes), anti-CD34 (hematopoietic precursor cells), anti-CD33 (myeloid and monocyte progenitor cells), anti-CD38 (activated T cells), anti-HLA-DR (MHC leukocyte antigen), and/or anti-CD50 (ICAM-R, all leukocytes) as indicated in this and following examples. Flow cytometry parameters and regions were set up using isotype- matched irrelevant monoclonal antibodies IgGl, IgG2a, IgG2b and IgM. Fluorescence was analyzed on a logarithmic scale and reported as percentage of cells expressing a specific marker above an type matched control. In nine experiments using nonadherent cells, the total cell population decreased following incubation with the ICR1.1 antibody as compared to cells incubated with the ICR9.2 antibody or to cells incubated with no antibody. The non-adherent cell population was found to express predominantly myeloid, not lymphoid or monocytic, surface markers. In comparison, the total adherent cell population was not affected by the presence of any antibody and the number of PI + cells in two experiments was the same following incubation with 1CR1.1 , ICR9.2, or no antibody. These observations suggested that the non-adherent cell population was being selectively reduced with ICR 1.1 incubation. In the non-adherent cell population, the number of cells staining AnnexinVPI " following incubation with ICR1.1 was significantly greater than when incubated with ICR9.2. The number of ICR9.2-incubated AnnexinTPT cells was essentially the same as the number detected following treatment with no antibody. In the non-adherent population, induction of the onset of apoptosis was detectable after eight to twelve hours of incubation with the antibody, and the difference between the ICR1.1 and ICR9.2 treated cell populations was noticeable for up to seven days. The depleted population was found to be cells that expressed myeloid markers (CD15, CD33) and lacked lymphoid and monocytic markers (CD3, CD38, HLA-DR, CD14). The ICR1.1 treated myeloid cell population decreased nearly 50% compared to no antibody or ICR 9.2 treatment. Example 2 ICAM-R Antibody Induction of Apoptosis in Leukemic Cells Leukemic Cells from Donors Having found that the antibody ICR1.1 significantly induced apoptosis in normal marrow cells, antibody incubation was carried out using marrow cells from eleven patients having newly diagnosed untreated leukemia and the number of apoptotic cells was quantitated. Cells were obtained as described above. Eight of the leukemic patients had been diagnosed with acute myeloid leukemia (AML) and three had been diagnosed with acute lymphoblastic leukemia (ALL). Diagnoses were established according to the French- American-British (FAB) classification. In all leukemic samples, greater than 50% of all cells were leukemic. Cells were cultured as described above except that cells from the leukemic donors were plated at 0.3 x 10 6 cells/ml in 24 well plates. The average number of apoptotic cells from all experiments as indicated by Annexin N7PI " staining was greatest following incubation with antibody ICRl .1 as compared to incubation with ICR9.2 or no antibody. In two patient samples wherein 100% and 98% of all cells were leukemic, incubation with ICR1.1 resulted in 75% and 78%, respectively, of the cells staining Annexin N7PI " . Since marrow aspirates from patients are comprised of a mixture of normal and leukemic cells, it could be argued that the antibody-triggered apoptotic response to ICR 1.1 occurred only in the normal cells and not the leukemic cells. Because induction of apoptosis was observed in marrow samples in which the majority of cells were leukemic, however, the results indicated that leukemic cells were indeed sensitive to the treatment. In cell samples from three of the eleven patients, however, ICR1.1 incubation did not increase apoptosis. It is possible that ICRl .1 -induced apoptosis requires some additional signal that was lacking in these cell samples. In vivo, acute leukemia cells can escape toxic injuries such as chemotherapy, leading to early or late treatment failure in the majority of acute leukemic patients. It is therefore not surprising that a subset of the samples examined were resistant to ICR 1.1- induced apoptosis. Cultured Cell Lines In view of these results, leukemic cell lines were similarly examined for induction of apoptosis following antibody incubation. U937 myelomonocytic leukemia cell line, Jurkat T cell leukemia cell line, and ICAM-3 -negative B cell lymphoblastoid cell line JY were maintained in Iscove's medium supplemented with 10% heat inactivated, endotoxin-free FBS, 2% L-glutamine, 1% penicillin-streptomycin prior to assay. Experiments were carried out as described above. In U937 and Jurkat cells, both of which express ICAM-R, the number of Annexin N7PI " cells was significantly greater following incubation with ICR1.1 as compared to incubation with either ICR9.2 or no antibody. In JY cells which do not express ICAM-R, the number of Annexin N + PI ' cells was essentially the same following ICRl .1, ICR9.2, or no antibody treatment. The result from the JY cells indicated that the ICR1.1 induced apoptosis of susceptible cells was not due to toxic effects of the monoclonal antibody or its preparation. In results obtained from experiments described in this example and Example 1, 1CR1.1 increased early apoptotic leukemic cells by 15%. This result must be put in perspective of the assay used and the biological nature of apoptosis. First, only early apoptotic events were considered in this study. Gating out PL cells allowed the exclusion of necrotic cells from the analysis, but also resulted in the exclusion of late apoptotic cells. As a result, the specificity of Annexin V staining increased but its sensitivity decreased. Second, as shown in the time-course experiments, ICR1.1 increased apoptosis continuously from 8 through 12 hours to 7 days. For these reasons, a 15% increase of early apoptotic events at one time point only represents a fraction of the total apoptotic response in the population over time. Example 3 Mode of Induction of Apoptosis by ICAM-R Antibodies As described in the first two examples the anti-ICAM-R antibody ICRl .1 was able to significantly induce apoptosis in marrow cells from normal and leukemic patients, as well as in certain leukemia cell lines compared to cells incubated with the ICAM-R antibody ICR9.2 or a cell population not treated with an antibody. In view of these results, experiments were designed to determine the mode of action by ICRl .1. Because Fas FasL and TNFα/TNFR interactions are known to be two main pathways involved in hematopoietic cell apoptosis, these two pathways were first examined as possible means by which ICRl .1 apoptotic activity is effected. In the first analysis, Jurkat T cells, total bone marrow cell samples, and ALL cells were separately incubated as described above with and without the monoclonal antibody 2B4 that neutralizes the Fas apoptosis pathway by blocking FasL Fas. binding. In eight experiments, ZB4 was added to Jurkat cells (n=5), or leukemic (n=2) or normal (n=l) donor cells at 100 ng/ml final concentration, alone or in the presence of ICR 1.1 or ICR 9.2. The positive control consisted of CHI 1 (agonistic anti-Fas monoclonal antibody) treated cells. In one experiment, both ZB4 and CH11 were added simultaneously as a control of the ability of ZB4 to block Fas (CH11) induced apoptosis in our system. Agonistic (CH11) and blocking (ZB4) monoclonal antibodies to Fas (CD95) were purchased from Immunotech (Westbrook, ME). In all cell types, the number of Annexin V7PI " cells increased following treatment with ICRl .1 irrespective of whether the neutralizing ZB4 monoclonal antibody was added. However, in control cells, the number of Annexin WPI " cells following incubation with the Fas agonist monoclonal antibody CH11 was 80% whereas the percentage was dramatically reduced following incubation with both ZB4 and CH11. These results indicated that ICRl .1 induction of apoptosis did not occur through the Fas pathway. In assessing the role of the TNFα pathway in ICRl.l activity, U937 (n=l) or Jurkat (n=3) cells were co-incubated with neutralizing anti-TNFα monoclonal antibody, either alone or in combination with ICR 1.1 or ICR 9.2 for 24 hours and apoptosis was assessed. As a control, neutralizing anti-TNFα monoclonal antibody was tested for its ability to block exogenous TNFα induced apoptosis of U937 cells (n=l). Recombinant human TNFα was purchased from R&D Systems (Minneapolis, MN). Rabbit anti-human TNFα polyclonal monoclonal antibody was purchased from Genzyme (Cambridge, MA). In Jurkat cells, the number of Annexin N7PT cells following incubation with TΝFα alone was greater than the number of Annexin AT/PI " cells following treatment with TΝFα and the neutralizing antibody. In cells treated with ICRl.l, however, the number of Annexin V + /PI " cells was similar regardless of inclusion of the TΝFα neutralizing antibody. The same results were observed using U937 cells, indicating that the ICR 1.1 -triggered apoptotic pathway is TNFα-independent. Example 4 Assessing the Role of CD18/CDlla in ICAM-R Antibody-Induced Apoptosis Since other ICAMs are ligands for LFA-1 binding, the role of CD18/CD1 la binding with ICAM-1 or ICAM-2 as a regulator for triggering apoptosis was evaluated using monoclonal antibodies to CD 18 or CD1 la. Cell samples from three AML patients were incubated with ICR 1.1, ICR 9.2, or without monoclonal antibody, with or without blocking monoclonal antibodies to CD1 la. In five experiments, blocking anti-CD 18 (TS1/18) or anti-CD 1 la (TS1/22) monoclonal antibodies were added to the cultures, alone or in the presence of ICR 1.1 or ICR 9.2, and the percentage of Annexin N7PI " cells were determined. In all experiments, ICR 1.1 strongly increased the percentage of Annexin N + /PT cells compared to the controls. By contrast, anti-CDl la itself had no effect on apoptosis, and did not block apoptosis when co-incubated with ICRl. l . Similarly, TS1/18 did not interfere with ICR 1.1 -induced U937 apoptosis (n=2). These results demonstrated that the ICAM-3 -triggered apoptotic signal did not require a co-stimulatory signal effected via ICAM- 1 or ICAM-2 binding with β 2 (CD 18) integrins. Example 5 Assessing the Role of F c Receptor Engagement in ICAM-R Antibody-Induced Apoptosis To investigate whether ICR 1.1 -triggered apoptosis involved F c receptor co-engagement, U937 or Jurkat cells were incubated with F(ab)' 2 fragments of ICR 1.1. Results indicated that in U937 cells, F(ab) ' 2 ICRl .1 induced a degree of apoptosis similar to that induced by the whole molecule. In Jurkat cells, F(ab)' 2 ICR 1.1 also induced apoptosis, but to a significantly higher extent than did the whole molecule. Similarly, the incubation of Jurkat cells with F(ab)' ICRl. l (n=3) increased the number of Annexin N + PI " cells relative to the number observed following incubation with F(ab)' ICR9.2. These results indicated that antibody-triggered induction of apoptosis was independent of Fc receptor binding and activation. In addition, cross-linking F(ab)' fragments of ICR 1.1 by adding anti-mouse kappa monoclonal antibody did not significantly increase apoptosis over that seen with F(ab)'. However, anti-mouse kappa monoclonal antibody added to F(ab)' ICR9.2 strongly induced apoptosis. Similarly, anti-mouse kappa monoclonal antibody added to intact ICRl.l only slightly increased apoptosis, compared to ICRl . l alone, whereas anti-mouse kappa monoclonal antibody co-incubated with ICR9.2 (n=3) strongly induced apoptosis. This finding demonstrated that the apoptotic signal can be transmitted through binding of either domain 1 or domain 2 of ICAM-3, and suggested that the valency with which ICAM-3 is engaged can regulate the resulting intensity of the apoptotic response. This result was not due to a toxic or non-specific effect of anti-kappa monoclonal antibody since anti-kappa monoclonal antibody alone did not induce Jurkat cell apoptosis. The result suggested that the apoptotic signal either required cell-cell interaction, or that ICAM-3 capping on the cell surface increased the apoptotic signal. F(ab)' 2 and F(ab)' fragments of ICRl . l induced comparable apoptosis, showing that bivalency of ICRl. l was not required for apoptosis. Therefore, the enhancing effect of antibody hypercross-linking on ICAM-3 -induced apoptosis seemed more likely due to a capping phenomenon than to cell-cell contact. Example 6 In Vivo Administration of ICAM-R Antibody The previous results demonstrated that anti-ICAM-R antibodies were able to induce apoptosis in cultured leukemia cell lines. In order to determine if an ICAM-R antibody behaved similarly in vivo, the following experiments were conducted. Male nude mice (BALB/c AnTac-nuf DF N9 homozygous), 6 to 8 weeks of age, were obtained from Tekonics. Animals were housed under controlled conventional conditions, which included sterile microisolator cages and autoclaved bedding. Sterile food and water were provided ad libitum. All procedures were performed under aseptic conditions in a laminar flow hood. Humanized ICR8.1 was supplied as a sterile 5 mg/ml solution formulated ® in 45.2 mM sodium acetate, 0.12 M NaCl, 0.02% Tween 20, pH 5.6. Placebo, Lot No. 98-0750, was identical to the humanized ICR8.1 formulation buffer. Solid tumors were induced in the mice as follows. Cells from the U937 human myeloid leukemia cell line (ATCC CRL-1593) were grown in Iscove's modified medium with 10% fetal bovine serum and penicillin/streptomycin, washed several times in Dulbecco's phosphate buffered saline (D-PBS), and resuspended in D-PBS at a concentration of 10 8 cells/ml. Fifteen mice were irradiated with 595 Rad emitted from a 137 Cs source (Mark I Irradiator, Shepard and Associates) and each animal was then injected subcutaneously with 1.5 x 10 7 U937 cells. Mice were monitored daily until the majority of mice developed observable tumors. Mice that developed observable tumors nine days after subcutaneous injection of U937 cells (15/15) were randomly divided into two groups. Animals received either 100 μl of humanized ICR8.1 (0.5 mg/mouse, approximately 25 mg/kg) or 100 μl of placebo by intravenous bolus injection via the tail vein on days 0, 3, and 5. Tumors were measured in each animal on days 0, 3, 4, 5, 6 and 7 using calipers. Approximate tumor volume was determined using the formula N=πLW 2 /6, where L is the longest diameter of the tumor and W is the diameter perpendicular to L (Clutterbuck, Arguello). Mice were sacrificed an Day 7 and tumors were excised, weighed and measured in three dimensions. Final tumor volume was determined using the formula V = πLVVD/6, where L, W and D are length, width and depth of the tumor. Excised tumors were divided in half, with one half frozen in OTC (Tissue Tek), a medium for sectioning, and the other imbedded in paraffin. Tumors sections were stained for the presence of humanized ICR8.1, ICAM-R, proliferation (Ki-67), and apoptosis, in addition to a general morphology assessment. Staining for proliferation was performed using rabbit polyclonal anti-sera Ki-67 (Νovacastra) that recognizes a nuclear antigen expressed in all proliferating human cells but not in cells in G 0 . All of the fifteen mice injected with human leukemic cells developed observable tumors at nine days post-injection. Mean tumor volume at the beginning of the treatment phase (Day 0) was calculated to be 38 ± 8 mm 3 for the placebo group, versus 41 ± 7 mm 3 for the humanized ICR8.1 group. Humanized ICR8.1 treatment resulted in significantly smaller tumor volumes on Days 4, 5, and 6, relative to placebo treatment While the differences between the humanized ICR8.1 and placebo treatments did not achieve statistical significance, there was a trend towards smaller tumors on Day 7 in the humanized ICR8.1 -treated animals. Numerous modifications and variations in the invention as set forth in the above illustrative examples are expected to occur to those skilled in the art. Consequently only such limitations as appear in the appended claims should be placed on the invention.

Claims: What is claimed is: 1. A method for inducing apoptosis in a cell population comprising the step of contacting said cell population with an antibody immunoreactive with domain 1 of ICAM-R in an amount sufficient to induce apoptosis. 2. A method for inducing apoptosis in a cell population comprising the step of contacting said cell population with an antibody that cross-links ICAM-R through binding domain 2. 3. The method according to claim 1 or 2 wherein the antibody is a fusion protein. 4. The method according to claim 1 or 2 wherein the cell population comprises a hematopoietic cell population. 5. The method according to claim 1 or 2 wherein the cell population comprises a tumor cell population 6. The method according to claim 1 or 2 wherein the antibody is a humanized antibody. 7. The method according to claim 1 or 2 wherein the antibody is a human antibody. 8. The method according to claim 1 wherein the antibody immunoreactive with domain 1 of ICAM-R inhibits ICAM-R binding to an ICAM-R binding partner. 9. The method according to claim 8 wherein the antibody is selected from the group consisting of the antibody secreted by hybridoma ICRl.l (ATCC Accession No. HB 1 1053), the antibody secreted by hybridoma designated ICR8.1 (ATCC Accession No: 11232), and humanized antibody ICR8.1. 10. The method according to claim 3 wherein the fusion molecule is a surface molecule expressed in a cell line modified ex vivo. 1 1. The method according to claim 1 or 2 wherein said contacting comprises parenterally administering the antibody to a mammal in need thereof. 12. The method according to claim 11 wherein the administering step is by a route selected from the group consisting of intravenous injection, intra-arterial injection, and intra-tumoral injection. 13. The method according to claim 1 or 2 further comprising the step of subjecting said cell population to an adjunct treatment selected from the group consisting of chemotherapy and radiation therapy.

Inventors: SANDMAIER BRENDA, STUCKI ANNE, SANDMAIER, BRENDA, STUCKI, ANNE
Applicants: HUTCHINSON FRED CANCER RES, FRED HUTCHINSON CANCER RESEARCH CENTER
Application year: 1999
Main classification: A61K39
IPC codes: A61K9, A61K38, A61K39, A61K45, A61N5, A61P35, A61P43, C07K16, C12N5, G01N33