JP2002529518A - Anti-ICAM-R antibody-induced apoptosis - Google Patents

Abstract

  (57) [Summary] Disclosed is a method for inducing apoptosis in a cell population, comprising contacting an antibody having immunoreactivity to ICAM-R with a target cell population.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention provides a method for inducing apoptosis in a cell population by contacting the cell population with an antibody having immunoreactivity to ICAM-R.

[0002]

2. Description of the Related Art

ICAM-R (see US Pat. No. 5,525,487, issued Jun. 11, 1996) is a member of the immunoglobulin superfamily of adhesive receptors. In healthy organisms, protein expression is restricted to immature and mature hematopoietic cells in lymphoid, myeloid and mononuclear cell lines. Surface molecules are 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 for identifying ICAM-R include LFA-1 (CD11a / CD18) and α _d / CD18, which bind to the ICAM-R extracellular region consisting of five immunoglobulin-like domains. Perhaps the ligand binding is ICA
Enables transmembrane signal transduction of cells via the cytoplasmic domain of MR.

[0003] ICAM-R functions as a costimulatory molecule on T cells [Hayflick, et al., J. Bio.
l. Chem .: 22207-14 (1997)], playing a major role in initiating the immune response.

[0004] ICAM-R is also mediates the diffusion and aggregation of T cells [Hayflick, et al., Supra].
The detailed mechanism leading to such activity is not completely understood, but is
It is believed to be necessary to phosphorylate specific cytoplasmic serine residue at AM-R [Hayflick, et al ., Supra]. Other intercellular phenomena, such as activation of calcium flux between cells, phosphorylation of tyrosine residues of tyrosine kinases p56lck and p59fyn, and phosphorylation of cyclin-dependent kinase cdc2, are likely to respond to ICAM-R involvement. [Chirathaworn, et al., J. Immunol. 155: 5479-5482
(1995); Juan, J. Exp. Med. 179: 1747-1756 (1994)]. p56lck and p59fyn
Are involved in cell death signals [Reichert, J. Immunol. 21: 295-30.
6 (1998); Schlottman, J. Leukoc. Biol. 60: 546-554 (1996)], phosphorylation of cdc2 occurs at tyrosine at position 15, which down-regulates cdc2 function and leads to cell cycle arrest. Known as the phenomenon to begin.

[0005] Programmed cell death (PCD), which is distinct from cell necrosis, is a normal process found in the growth processes and homeostasis of multicellular organs. Schwartz et al
., Immunology Today 14: 582-590 (1993). PCD can be divided into two categories: apoptotic and non-apoptotic death. Apoptosis is defined as cell death that involves the formation of membrane bubbles or "apoptotic bodies" and morphological changes including condensation / marginization of nuclear chromatin. It is known that cell signaling pathways play an important role as part of the apoptotic process.
Several cell surface receptors (TNFR I and Fas) have been found to cause apoptosis when certain ligands or antibodies are involved. Recent 10
Years of research began with defining the intercellular protein components and their function in the apoptotic process, and how to modulate this process.

[0006] Thus, there is a need in the art to identify molecular components in the normal hematopoietic cell machinery that affect the delicate balance between blood cell production and destruction.
Identifying modulators of hematopoietic cell growth and survival will establish new therapies for treating leukemia, myeloma and lymphoma.

[0007]

[Means for Solving the Problems]

 The disclosures of all references cited herein are hereby incorporated by reference.

[0008] In general, the invention provides a cell population comprising contacting an antibody immunoreactive with a cell adhesion molecule, ICAM-R, as defined in US Patent No. 5,525,487, to the cell population. To provide a method for inducing apoptosis. Antibodies used in these methods include monoclonal and polyclonal antibodies, single-chain antibodies, chimeric antibodies, and CDR-grafted antibodies (including compounds having CDR sequences that specifically recognize the polypeptides of the invention), and ICAM- There are other binding proteins specific for R. The term "specific" refers to the variable region of an antibody that recognizes and binds only to ICAM-R polypeptides (ie, where sequence identity, homology or similarity is recognized within a family of polypeptides). Nevertheless, ICAM-R polypeptides can be distinguished from the family of cell adhesion molecule polypeptides), but show that they can interact with other proteins by contacting peptide domains other than CDRs. is there. Specific ICAM-R antibodies are incorporated herein by reference.
It is described in pending US patent application Ser. No. 08 / 863,790.

In one embodiment, the present invention provides a method for inducing apoptosis in a cell population, comprising contacting the cell population with an antibody immunoreactive with domain I of ICAM-R. I do. According to another aspect, the invention relates to a method for administering an IC via domain 2.
Provided is a method for inducing apoptosis in a cell population, comprising a step of contacting the cell population with an antibody that allows crosslinking with AM-R.

[0010] The present invention further provides a method of inducing apoptosis in a cell population, comprising contacting the cell population with a fusion molecule comprising an antibody immunoreactive with ICAM-R domain I. The invention also provides a method of inducing apoptosis in a cell population, comprising contacting the cell population with a fusion protein comprising an antibody domain that crosslinks domain 2 of ICAM-R. According to one embodiment, the antibody is ex vivo
If it is a surface molecule expressed in a cell line modified with, it is a fusion protein.

[0011] Fusion proteins of the invention include those that include an immunospecific domain of an anti-ICAM-R antibody in combination with other components that improve the biological activity of the immunospecific domain. For example, fusion proteins comprising other sequences that improve solubility, increase circulating half-life, and / or reduce the antigenicity of the antibody domain are contemplated.
Also contemplated are fusion proteins comprising an antibody region fused to a cytotoxic component that functions for a specific cell type that complements the apoptotic effect of antibody binding.

[0012] In a preferred embodiment, the method of the present invention induces apoptosis in a cell population comprising hematopoietic cells. In a most preferred embodiment, the cell population is
Includes tumor cells.

[0013] In one aspect of the invention, the antibody is a humanized antibody, preferably the antibody is a human antibody. The contemplated humanized ICAM-R antibodies are elicited as described in US Pat. No. 5,837,822, issued Nov. 17, 1998, which is incorporated herein by reference. In a preferred embodiment, the antibody is immunoreactive with domain 1 of ICAM-R and inhibits binding of ICAM-R to an ICAM-R binding partner. In a more preferred embodiment, the antibody is produced by a hybridoma named ICR1.1 (ATCC Accession No.HB11053), an antibody produced by a hybridoma named ICR8.1 (ATCC Accession No.HB11232). It is a produced antibody or a humanized antibody. The present invention further contemplates the use of antibody fragments comprising one or more immunoreactive variable domains.

[0014] The methods of the invention encompass various forms of administration of the antibody or fusion protein to animals, preferably mammals, and more preferably humans. The invention relates to other animals,
For example, pet animals such as dogs and cats; domestic animals including cows, horses, sheep, pigs and goats; laboratory animals including rats, mice, rabbits and guinea pigs; antibodies or fusion proteins to birds; fish; Is further contemplated. Most preferred is parenteral administration, for example, by a route selected from the group consisting of intravenous, intraarterial and intratumoral injection. Other routes of administration are also contemplated, including, for example, oral, nasal, ocular, rectal, vaginal, urethral, intramuscular and local routes. The present invention further contemplates compositions comprising the antibody, fusion protein, antibody or biologically active fragment thereof in combination with one or more pharmaceutically acceptable carriers.

[0015] The method of the invention is preferably performed as a separate treatment. Alternatively, the method of the invention further comprises the step of administering to the cell population one or more adjuvant treatments selected from the group consisting of chemotherapy and radiation therapy.

The utility of the method of the present invention is clear. Induction of apoptosis is particularly relevant in bone marrow transplant patients who are resistant to conventional chemotherapy and radiation therapy. Induction of apoptosis by the methods of the present invention can also complement ongoing chemotherapy or radiation therapy, as long as low-dose or short-term chemotherapy or radiation therapy is available, thereby providing additional treatment for these. Related side effects are reduced.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION

The invention is illustrated according to the following examples of substances and methods for inducing apoptosis. Example 1 describes the induction of apoptosis in normal bone marrow cells. Example 2 relates to the induction of apoptosis in leukemia cells obtained from donors and in cultured leukemia cells. Example 3 describes the mode of action of anti-ICAM-R antibody-induced apoptosis. Example 4 demonstrates CD in ICAM-R antibody-induced apoptosis.
Regarding the role evaluation of 18 / CD11. Example 5 describes the role of _Fc receptors involved in ICAM-R antibody-induced apoptosis. Example 6 describes anti-ICAM-R antibody-induced apoptosis in vivo .

[0018]

【Example】

Example 1 Induction of Apoptosis by ICAM-R Antibodies in Normal Bone Marrow Cells From the previous studies, it has been shown that the involvement of ligands in ICAM-R makes it possible to directly or indirectly associate with known apoptotic pathway components of cytoplasmic phosphorus. It is known that it leads to an oxidation phenomenon. From these findings, the first series of experiments were performed to determine what ICAM-R antibody-related effects would continuously have on normal cell survival.

Bone marrow samples were obtained from 18 healthy individuals. The protocol and consent form were approved by the Fred Hutchison Cancer Research Center internal review committee. Bone marrow aspiration was performed according to standard procedures [Hematol. Oncol. Clin. North Am. 8: 651-63 (1994)]. Heparinized diluted bone marrow samples (diluted 1: 3 in Iscove's medium) were centrifuged (2000 rpm for 20 minutes) to collect buffy coat cells.
Hemolytic buffer (0.83% ammonium chloride, 0.1% sodium bicarbonate, no EDTA)
Incubate at 37 ° C for 10 minutes, then wash once,
Red blood cells were lysed. 2 × 10 ⁶ cells / ml were transferred to a 24-well plate (1 ml /
Wells) or T25 flasks (5 ml) and heat inactivated 10% Iscove, endotoxin-free fetal bovine serum (FBS), 10% horse serum (HS), 2% L-glutamine and 1% penicillin- The cells were cultured at 37 ° C. in a humidifier of 5% carbon dioxide using a medium containing streptomycin.

The cells were treated with the monoclonal antibody ICR1.1 (recognizing ICAM-R domain 1 and blocking the binding of LFA-1 to ICAM-R), (recognizing ICAM-R domain 2 and recognizing LCAM-1 Incubation was performed with either monoclonal antibody ICR9.2 (which does not block binding of one) or no antibody. ICR1.1 and ICR9.2 have already been described [Sadhu, et al., Cell Adhes. Commun. 2: 651-663 (1994); Vazeux, et al.
, Nature 360: 485-488 (1992)]. Anti-ICAM-R monoclonal antibodies were purified from mouse ascites preparations, verified to be endotoxin-free, and stored in PBS. Each monoclonal antibody was used at a final concentration of 10 μg / ml, and non-adherent cells were
Collected at different time points for 3-7 days. Fluorescence activation (described below) equipped with an electronic gate that draws forward / side scatter dot plots that allow data correction of changes in the relative proportions of lymphocyte, bone marrow and monocyte populations based on cell size and complexity Determined by cell sorter (FACS).

The number of dead cells at each time point was determined by propidium iodide (PI) staining according to the manufacturer's recommended protocol. That is, the fraction of non-adherent cells was collected, washed with PBS, and resuspended in PBS containing a final concentration of 2.5 μg / ml PI. Positive staining (n = 9) was detected by flow cytometry. In two experiments, both non-adherent and adherent cells were separated and analyzed simultaneously to separate without regard for correlation.

To study the onset of apoptosis, Annexin V FITC staining (performed according to the manufacturer's recommended protocol) was performed in combination with PI staining. Annexin V binds to phosphatidylserine, which is exposed to the outer lobe of the cell membrane early in apoptosis. In dead cells, the inner membrane of the cell membrane is available for binding to Annexin V because the plasma membrane is disrupted. Double staining with a DNA dye such as PI and FITC-labeled Annexin V resulted in dead cells (
Electron exclusion of Annexin V ⁺ / PI ⁺ ) and apoptotic cells (Annexin
V ⁺ / PI ^-) to enable the quantification of. Therefore, double staining allows the quantification of viable cells (Annexin V ⁻ / PI ⁻ ), apoptotic cells (Annexin V ⁺ / PI ⁻ ) and dead cells (Annexin V ⁺ / PI ⁺ ).

At each time point, 0.3-1.0 × 10 ⁶ normal cells were collected per condition, washed once with PBS, and 200 μl ice-cold buffer containing 2.0 μl prepared annexin V FITC and 0.8 μl PI And resuspended. Samples were incubated on ice for 10 minutes in the dark and applied to a FACScan®. Parameters and regions were set by using no monoclonal antibody as a control, and the fluorescence was analyzed on a log scale. Cells stained with PI (necrotic or late apoptosis) were gated out, and the percentage of early apoptotic cells (Annexin V ⁺ / PI ^- stained) in ICR1.1-treated cultures was determined by ICR9.2- Compared to that in the treated culture.

The following analysis was performed to examine the type of cells in each fraction at the time points. Cells were harvested, resuspended in 15% FBS-PBS, split into 0.2-0.5 × 10 ⁶ cells / 100 μl tube, and incubated on ice. FITC for surface receptors
-Conjugated monoclonal antibody was added to the cells at a final concentration of 10 μg / ml. The labeled cells are incubated on ice for 20 minutes, washed twice and 1% BSA
And resuspended in 250 μl of PBS. The monoclonal antibodies used in this example and the following examples include anti-CD3 (a marker for T cells), anti-CD14 (myeloid monocytes), anti-CD15 (neutrophils, eosinophil leukocytes, Spheres), anti-CD34 (hematopoietic progenitor cells), anti-CD33 (bone marrow and monocyte progenitor cells), anti-CD38 (activated T cells), anti-HLA
-DR (MHC leukocyte antigen) and / or anti-CD50 (ICAM-R, whole leukocyte).
Flow cytometry parameters and regions were set using monoclonal antibodies IgG1, IgG2a, IgG2b, and IgM, irrelevant for isotype compatibility. Fluorescence was analyzed on a logarithmic scale and the type matched to the control was reported as a percentage of cells expressing the particular marker described above.

In nine experiments with non-adherent cells, ICR1.1 was compared to cells incubated with the ICR9.2 antibody or cells incubated with no antibody.
The total number of cell populations incubated with the antibody was reduced. It was found that the majority of the non-adherent cell population was expressed on bone marrow surface markers and not on lymphocyte or monocyte surface markers. By comparison, the entire adherent cell population was unaffected by the presence of either antibody, and 2
The numbers of PI ⁺ cells after incubation with ICR1.1, ICR9.2 or antibody-free in the same experiment were identical. These observations suggested that non-adherent cell populations were selectively reduced by incubation with ICR1.1.

In a non-adherent cell population, annexin after incubation with ICR1.1
V ⁺ / PI ^- the number of cells staining was much often than when incubated at ICR9.2. The number of Annexin V ⁺ / PI ⁻ cells incubated with ICR9.2 was similar to the number detected after treatment with no antibody. In the non-adherent cell population, the induction of the onset of apoptosis became detectable 8-12 hours after incubation with the antibody, and the cell population treated with ICR1.1 and the cell population treated with ICR9.2. No difference between them could be observed over 7 days.

Cells and lymphocytes expressing bone marrow markers (CD15, CD33) and monocyte markers (
Population reduction was observed in cells lacking CD3, CD38, HLA-DR, CD14).
The bone marrow cell population treated with ICR1.1 was reduced by nearly 50% compared to those treated with no antibody or treated with ICR9.2.

Example 2 ICAM-R Antibody Induced Apoptosis in Leukemia Cells Leukemia cells derived from donors It has been confirmed that the induction of apoptosis by the antibody ICR1.1 in normal bone marrow cells was remarkable. Antibody incubations were performed using bone marrow cells from 11 diagnosed inexperienced patients and the number of apoptotic cells was quantified. Cells were obtained as follows. Eight leukemia patients were diagnosed with acute myeloid leukemia (AML) and three were diagnosed with acute lymphoblastic leukemia (ALL). Diagnosis was made according to the French-American-British (FAB) classification. Of all leukemia samples, more than 50% of all cells were from leukemia patients. Cells were cultured as described above, except that cells from leukemia donors were placed in 24-well plates at 0.3 × 10 ⁶ cells / ml.

The average number of apoptotic cells obtained from all tests by Annexin V ⁺ / PI ⁻ staining was lower for the antibody ICR1 compared to when incubated with ICR9.2 or no antibody.
Incubation at .1 was greatest. 100% and 98% of all cells
Incubation with antibody ICR1.1 on samples from two patients who were leukemic resulted in 75% and 78% annexin V ⁺ / PI ⁻ stained cells, respectively. Consider that antibody-induced apoptotic responses to ICR1.1 occur only in normal cells, not leukemia cells, because bone marrow aspirates from patients contain a mixture of normal and leukemic cells. Was completed.

However, these results are only indicative that leukemic cells are susceptible to treatment, as induction of apoptosis was observed in bone marrow samples where most cells are leukemic. However, in cell samples from three of the eleven patients, apoptosis was not increased by incubation with ICR1.1. Apoptosis induced by ICR1.1 can require other signals that are missing in these cell samples. In vivo,
Acute leukemia cells can avoid toxic damage, such as poor initial or late treatment chemotherapy in most acute leukemia patients. Therefore,
It is not surprising that some of the tested samples are resistant to apoptosis induced by ICR1.1.

Cultured cell lines Based on these results, leukemic cell lines were similarly validated using apoptosis induction followed by antibody incubation. U937 myelomonocytic leukemia cell line,
Jurkat T-cell leukemia cell line and ICAM-3-negative B-cell lymphoblastoid cell line JY
Prior to the assay, heat-inactivated endotoxin-free FBS, 2% L-glutamine and 1%
It was maintained in Iscove's medium supplemented with 5% penicillin-streptomycin.
The experiment was performed as described above.

In U937 and Jurkat cells, both of which express ICAM-R, the number of annexin V ⁺ / PI ⁻ cells was greater than that of incubation with ICR1.1 compared to incubation with ICR9.2 or no antibody. There was much more in the case of according to. In JY cells that do not express ICAM-R, the number of Annexin V ⁺ / PI ⁻ cells was about the same as when treated with ICR1.1, ICR9.2 or no antibody. The results obtained from JY cells indicated that ICR1.1-induced apoptosis of susceptible cells was not due to the toxic effects of the monoclonal antibody or its preparation.

According to the results obtained from the experiments described in this example and in Example 1, ICR 1.1
Early apoptotic leukemia cells had increased by 15%. This result must take into account the assay used and the biological characteristics of apoptosis. First, in this study, only early apoptosis was considered. Gated PI ⁺ cells exclude necrotic cells from the analysis but also result in the elimination of late apoptotic cells. As a result, the specificity of Annexin V staining improved, but its sensitivity decreased. Next, as shown in the time course experiments, ICR1.1 continuously enhanced apoptosis from 8-12 hours to 7 days. For these reasons, a 15% increase in the event of early apoptosis at a given time is only indicative of a fraction of all apoptotic responses in the population at all times.

Example 3 Forms of Inducing Apoptosis by ICAM-R Antibody As described in the previous two examples, the anti-ICAM-R antibody ICR1.1
Compared to cells incubated at 9.2 or cell populations not treated with antibody, it was possible to induce corresponding apoptosis in bone marrow cells from healthy and leukemic patients and in certain leukemic cell lines. Based on these results, ICR1
An experiment was designed to determine the mode of action according to .1. Fas / FasL and TNFα / TN
Since the interaction of FR is known to be the two main pathways involved in hematopoietic cell apoptosis, we first examined whether these two pathways are the means to make the ICR1.1 apoptotic activity effective.

In the first analysis, in the presence and absence of the monoclonal antibody ZB4, which neutralizes the Fas apoptotic pathway by blocking FasL / Fas binding, Jurkat T cells, whole bone marrow cell samples and ALL cells were incubated individually. In eight experiments, a final concentration of 100 ng / ml, alone or with ICR1
ZB4 was added to Jurkat cells (n = 5) or leukemia (n = 2) or normal (n = 1) donor cells in the presence of .1 or ICR9.2. Positive controls consisted of cells treated with CH11 (antagonistic anti-Fas monoclonal antibody). In one experiment, both ZB4 and CH11 were added simultaneously as a control for the ability of ZB4 to block Fas (CH11) -induced apoptosis in our system. Antagonist (CH11) and blocking (ZB4) monoclonal antibodies to Fas (CD95) were purchased from Immunotech (Westbrook, ME).

Subsequent treatment with ICR1.1, with or without the addition of ZB4 neutralizing monoclonal antibodies, increased the number of Annexin V ⁺ / PI ⁻ cells in all types of cells. However, in control cells, subsequent incubation with Fas antagonistic monoclonal antibody CH11 resulted in annexin V ⁺ / PI ⁻ cells of 80
%, But the percentage dropped sharply after incubation with both ZB4 and CH11. These results indicated that ICR1.1-induced apoptosis did not occur in the Fas pathway.

To examine the role of the TNFα pathway in ICR1.1 activity, U937 (n = 1) or Jurkat (n = 3)
Cells were co-incubated for 24 hours with neutralizing anti-TNFα monoclonal antibody alone or in combination with ICR1.1 or ICR9.2 and assessed for apoptosis. As a control, a neutralizing anti-TNFα monoclonal antibody was tested for its ability to block exogenous TNFα-induced apoptosis of U937 cells (n = 1). Recombinant human TNFα was purchased from R & D Systems (Minneapolis, MN). Rabbit anti-human TNFα polyclonal monoclonal antibody was purchased from Genzyme (Cambridge, Mass.).

In Jurkat cells, Annexin V subsequently incubated with TNFα alone⁺/ PI ^- Cell numbers were determined by Annexin V when subsequently treated with TNFα and neutralizing antibodies.⁺/ PI^-Cellular
There were more than numbers. However, in cells treated with ICR1.1, TNFα neutralizing anti-
Annexin V, despite including the body⁺/ PI^-The number of cells was similar. U937
The same results were obtained with cells, indicating that ICR1.1-induced apoptosis
This indicates that the cis pathway is TNFα-independent.

Example 4 Investigation of the role of CD18 / CD11a in ICAM-R antibody-induced apoptosis Since other ICAMs are ligands for binding LFA-1, ICAM-1 or ICAM-2 was used as a regulator to induce apoptosis. / CD11a binding role, CD18 or
Evaluation was performed using a monoclonal antibody against CD11a. Cell samples from three AML patients were incubated with or without ICR1.1 or ICR9.2 in the presence or absence of a blocking monoclonal antibody to CD11a. In five experiments, blocking anti-CD18 (TS1 / 18)
Alternatively, an anti-CD11a (TS1 / 22) monoclonal antibody alone or in the presence of ICR1.1 or ICR9.2 is added to the culture, and the percentage of Annexin V ⁺ / PI ^- cells is determined. Was.

In all experiments, ICR1.1 had a significantly higher percentage of Annexin V ⁺ / PI ⁻ cells compared to controls. In contrast, anti-CD11a itself had no effect on apoptosis and co-incubation with ICR1.1 did not block apoptosis. Similarly, TS1 / 18 is ICR1.1-induced U937
It did not interact with apoptosis (n = 2). These results, apoptotic signals ICAM-3 is caused _{is, β 2 (CD18) ICAM-} 1 or I using integrin
It demonstrated that co-stimulatory signals provided via CAM-2 binding were not required.

[0041] Apoptosis induced by F _c receptor study involved ICR1.1 in Example 5 ICAM-R antibodies induced apoptosis, in order to investigate the presence or absence of F _c receptors both involved, the U937 or Jurkat cells, ICR1 Incubation was performed with the F (ab) ' ₂ fragment of .1. In U937 cells, F (ab) ' ₂ ICR1.1 induced the same degree of apoptosis as was induced by all molecules. In Jurkat cells as well, F (ab) ' ₂ ICR1.1 induced apoptosis, but its levels were very high compared to the levels induced by all molecules. Similarly, F (ab) '
The number of Annexin V ⁺ / PI ⁻ cells in incubation of Jurkat cells (n = 3) with ICR1.1 was observed when subsequent incubation with F (ab) ′ ICR9.2 Was increasing with the number. These results indicated that the induction of apoptosis induced by the antibody was independent of _Fc receptor binding and activation.

In addition, the cross-linked F (ab) ′ fragment of ICR1.1 by addition of the anti-mouse κ monoclonal antibody did not significantly enhance apoptosis as was observed when using F (ab) ′. However, anti-mouse κ monoclonal antibody added to F (ab) ′ ICR9.2 strongly induced apoptosis. Similarly, the anti-mouse kappa monoclonal antibody added to intact ICR1.1 only slightly increased apoptosis compared to ICR1.1 alone, as opposed to using ICR9.2. Co-incubated anti-mouse kappa monoclonal antibody (n = 3) strongly induced apoptosis.

This finding suggests that the apoptotic signal may be associated with ICAM-3 domain 1 or domain 2.
, And suggested that the valency involving ICAM-3 could modulate the strength of the resulting apoptotic response. This result was not due to the toxic or non-specific effects of the anti-κ monoclonal antibody, as the anti-κ monoclonal antibody alone did not induce apoptosis in Jurkat cells. This result suggested that either the apoptotic signal required cell-cell interaction or that ICAM-3 capping at the cell surface enhanced the apoptotic signal.
F (ab) ' ₂ and F (ab)' fragments of ICR1.1 induced comparable apoptosis, indicating that ICR1.1 bivalent was not required for apoptosis It shows. Therefore, the enhancement of the supercross-linking effect of the antibody on ICAM-3-induced apoptosis seemed to be due to the capping phenomenon rather than the cell-cell contact.

Example 6 In Vivo Administration of ICAM-R Antibodies Previous results demonstrated that anti-ICAM-R antibodies can induce apoptosis in cultured leukemia cell lines. The following experiment was performed to determine whether the ICAM-R antibody exhibited the same behavior in vivo.

6-8 week old male nude mice (BALB / c AnTac-nufDF N9 homozygotes) were obtained from Tekonics. Animals were housed under conventional controlled conditions, including sterile microisolator cages and autoclaved flooring. Sterile food and water were provided ad libitum. All work was performed under sterile conditions under a laminar flow hood.

42.5 mM sodium acetate, 0.12 M sodium chloride, 0.02% Tween® 20,
Humanized ICR8.1 was obtained in the form of a 5 mg / ml sterile solution formulated at pH 5.6. lot
The placebo of No. 98-0750 was identical to the humanized ICR8.1 preparation buffer.

[0047] Solid tumors were induced in mice as follows. U937 human bone marrow leukemia cells
Cells from the strain (ATCC CRL-1593) were combined with 10% fetal bovine serum and penicillin / strept
Grow in Iscove's modified medium with mycin and add Dulbecco's phosphate buffered saline.
Wash several times with Immobilized Saline (D-PBS) and add 10⁸Cells in D-PBS at a concentration of cells / ml
Suspended.¹³⁷Released from Cs source (Mark I irradiator, Shepard and Associates)
595 rads were irradiated to 15 mice, and then 1.5 × 10 5 ⁷ U937 cells were injected. Until tumor formation is confirmed in most mice,
The mice were observed daily.

Mice that formed tumors 9 days after subcutaneous injection of U937 cells (15/15) were arbitrarily divided into two groups. On days 0, 3, and 5, 100 μl of humanized ICR8
.1 (0.5 mg / mouse, approximately 25 mg / kg) or 100 μl of placebo were injected into the tail vein of the animal in a bolus vein. On days 0, 3, 4, 5, 6, and 7, tumors of each animal were measured using calipers. A schematic tumor volume, the formula of V = πLW ^2/6, where
L was determined using the formula where L is the longest diameter of the tumor and W is the diameter perpendicular to L (Glutterbuck, Arguello).

On day 7, the mice were sacrificed, tumors were excised and weighed, and then three-dimensionally measured. The final volume of the tumor was determined using the formula of V = πLWD / 6, where L, W and D are the length, width and depth of the tumor.

The excised tumor was divided into two, and one was frozen in a fragmentation medium, OTC (Tissue Tek), and the other was embedded in paraffin. In addition to general morphological examination, tumor sections were stained for the presence of humanized ICR8.1, ICAM-R, proliferative (Ki-67) and apoptosis. Staining for proliferating, all recognize rabbit polyclonal nuclear antigen expressed by proliferating human cells anti except G ₀ cells - serum Ki-67 (Novacas
tra).

Nine days after injection, tumor formation was observed in all 15 mice injected with human leukemia cells. The mean tumor volume at the beginning of treatment (day 0) was calculated to be 38 ± 8 mm ³ in the placebo group, whereas in the humanized ICR 8.1 group
It was calculated to be 41 ± 7 mm ³ . Tumor volumes on days 4, 5 and 6 with treatment with humanized ICR8.1 were very small compared to placebo treatment. Although there was no statistical significance between humanized ICR8.1 treatment and placebo treatment, tumors in animals treated with humanized ICR8.1 tended to shrink on day 7. was there.

[0052] Numerous changes and modifications to the invention described in the above examples will occur to those skilled in the art. Accordingly, only the limitations set forth in the appended claims should be added to the invention.

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Claims (13)

[Claims] 1. A method for inducing apoptosis in a cell population, comprising contacting the cell population with an antibody immunoreactive with domain I of ICAM-R in an amount sufficient to induce apoptosis. A method that includes 2. A method of inducing apoptosis in a cell population, comprising the step of contacting an antibody that cross-links ICAM-R via domain II with said cell population. 3. The method according to claim 1, wherein the antibody is a fusion protein. 4. The method according to claim 1, wherein the cell population comprises a hematopoietic cell population. 5. The method according to claim 1, wherein the cell population comprises a tumor cell population. 6. The method according to claim 1, wherein the antibody is a humanized antibody. 7. The method according to claim 1, wherein the antibody is a human antibody. 8. The method of claim 1, wherein the antibody immunoreactive with ICAM-R domain I inhibits binding of ICAM-R to an ICAM-R binding partner. 9. The antibody according to claim 1, wherein the antibody is hybridoma ICR1.1 (ATCC Accession No. HB11053).
The method according to claim 8, wherein the antibody is selected from the group consisting of an antibody secreted by ICR8.1 (ATCC Accession No. HB11232) and an antibody secreted by the hybridoma ICR8.1. 10. The method of claim 3, wherein the fusion molecule is a surface molecule expressed on an ex vivo modified cell line. 11. The method of claim 1, wherein said contacting comprises parenterally administering said antibody to a mammal in need of said antibody. 12. The method of claim 11, wherein said administering is performed by a route selected from the group consisting of intravenous injection, intraarterial injection, and intratumoral injection. 13. The method of claim 1 or 2, wherein said method further comprises the step of administering to said cell population an adjuvant treatment selected from the group consisting of chemotherapy and radiation therapy.