COMPOSITIONS AND METHODS TO ENHANCE THE IMMUNE SYSTEM

Abstract

The invention relates to the field of molecular medicine. In particular, it relates to compositions and methods to enhance the clearance of aberrant cells, e.g. cancer cells or virus-infected cells, by the host's immune system. Provided is a composition comprising (i) a therapeutic compound that can trigger a host's immune effector cells against an aberrant cell, such as a therapeutic antibody, and (ii) at least one agent capable of reducing or preventing inhibitory signal transduction initiated via SIRPalpha.

Claims

1-18. (canceled)

19. A method of enhancing antibody-dependent cellular cytotoxicity (ADCC) comprising contacting a target cell with (i) an antibody comprising a human or non-human primate IgG Fc portion, wherein the antibody induces ADCC and (ii) an antagonist of the CD47-SIRPα interaction, wherein said contacting occurs in the presence of an effector cell, and wherein the target cell is a diseased cell.

20. The method of claim 19, wherein the diseased cell is a cancer cell.

21. The method of claim 20, wherein the cancer cell is a non-Hodgkin's lymphoma cell, a breast cancer cell, a chronic lymphocytic leukemia cell or a colorectal cancer cell.

22. The method of claim 19, wherein the antibody comprises a human IgG Fc portion.

23. The method of claim 22, wherein the human IgG Fc portion is a human IgG1 Fc portion.

24. The method of claim 22, wherein the human IgG Fc portion is a human IgG3 Fc portion.

25. The method of claim 19, wherein the antibody comprises a non-human primate IgG Fc portion.

26. The method of claim 19, wherein the antibody is rituximab, trastuzumab, alemtuzumab, bevacizumab, cetuximab or panitumumab.

27. The method of claim 19, wherein the contacting occurs in vitro.

28. The method of claim 20, wherein the contacting occurs in a human subject.

29. The method of claim 19, wherein the effector cell is a monocyte.

30. The method of claim 19, wherein the effector cell is a macrophage.

31. The method of claim 20, wherein the antibody is rituximab, trastuzumab, alemtuzumab, bevacizumab, cetuximab or panitumumab.

32. The method of claim 21, wherein the antibody is rituximab, trastuzumab, alemtuzumab, bevacizumab, cetuximab or panitumumab.

33. The method of claim 19, wherein the antagonist is a polypeptide capable of inhibiting the interaction between SIRPα and CD47.

34. The method of claim 19, wherein the antagonist comprises the N-terminal V-type immunoglobulin domain in SIRPα.

35. The method of claim 19, wherein the antagonist comprises the N-terminal V-type immunoglobulin domain in CD47.

36. The method of claim 22, wherein the antibody is rituximab, trastuzumab, alemtuzumab, bevacizumab, cetuximab or panitumumab.

37. The method of claim 23, wherein the antibody is rituximab, trastuzumab, alemtuzumab, bevacizumab, cetuximab or panitumumab.

38. The method of claim 24, wherein the antibody is rituximab, trastuzumab, alemtuzumab, bevacizumab, cetuximab or panitumumab.

Description

LEGENDS TO THE FIGURES

[0041] FIG. 1. Model for the role of CD47 and SIRPα in limiting the antibody-dependent killing of tumor cells by the immune system and potentiation of tumor cell destruction by blocking CD47-SIRPα interactions. Antibodies directed against the tumor cells are recognized by immune cell Fc-receptors and this induces tumor cell killing. Under normal conditions (left panel) this antibody-induced killing is limited by the interaction of CD47 on the tumor cells with SIRPα on the immune cell, which generates an intracellular signal that negatively regulates the killing response. By blocking the interaction between CD47 and SIRPα (right panel) the antibody-induced killing of tumor cells is enhanced because it is relieved from this limitation.

[0042] FIG. 2: Blocking CD47-SIRPα interactions with antagonistic monoclonal antibodies enhances CC52 antibody-dependent cellular phagocytosis of rat CC531 colon carcinoma cells by rat NR8383 macrophages. CC531 tumor cells and NR8383 macrophages were incubated in culture plates with medium in the absence or presence of CC52 antibody against CC531 cells and/or blocking monoclonal antibodies against either CD47 or SIRPα. After 1.5 hours the percentage of ADCP was determined. For details, see Example 1.

[0043] FIG. 3: SIRPα-derived signals limit the killing of B16 tumor cells in vivo. CD47-expressing B16 melanoma cells were injected into control (i.e. Wild type) mice or into mice lacking the SIRPα cytoplasmic tail that mediates intracellular signaling in immune cells (i.e. SIRPα−/−). Groups of mice were treated every other day for 2 weeks with a suboptimal dose of therapeutic antibody TA99 directed against the gp75 tumor antigen present on the B16 cells. One week afterwards the animals were sacrificed and lung tumor load was quantified. Representative pictures (panel A) from the lungs of these mice show that there is essentially no tumor formation in the antibody-treated SIRPα −/− mutant mice as compared to the antibody-treated wild type mice, identifying a negative role of SIRPα signaling in tumor cell elimination in vivo. Each point in the graph (panel B) represents evaluation of a single animal. *, p<0.005, students T-test.

[0044] FIG. 4: ADCC of human monocytes towards Jurkat acute T leukemia cells is enhanced by blocking CD47-SIRPα interactions. (A) Surface expression of CD3 (using CLB-T3/4.2a mAb) and CD47 (using B6H12 mAb) and on Jurkat cells as evaluated by flow cytometry. (B) ADCC of human monocytes towards Jurkat cells after pre-incubation with mouse IgG.sub.2a anti-CD3 (20 μg/ml) and/or B6H12 (50 μg/ml) anti-CD47 F(ab′).sub.2. Saturation of Jurkat cells with anti-CD47 F(ab′).sub.2 was confirmed by parallel flow cytometric staining (data not shown). Note that virtually no killing is induced by anti-CD3 in absence of CD47-SIRPα blocking, whereas substantial levels of killing are evoked in the presence of anti-CD47 F(ab′).sub.2. Values shown are means±SD (n=3) from a representative experiment out of three.

[0045] FIG. 5: Rituximab-mediated ADCC of human monocytes towards Raji Burkitt's B cell lymphoma cells is enhanced by blocking CD47-SIRPα interactions. (A) Surface expression of CD20 (using anti CD20 Rituximab) and CD47 (using anti-CD47 B6H12 mAb) and on Raji cells as evaluated by flow cytometry. Red histogram represents control and blue histogram represents the Rituximab (upper histogram) or CD47 staining (lower histogram) (B) ADCC of human monocytes towards Raji cells after pre-incubation with Rituximab (20 μg/ml) and/or B6H12 (10 μg/ml) anti-CD47. ADCC was performed at an effector:target ratio of 50:1. Note that the Rituximab-mediated killing of Raji cells is significantly enhanced by anti-CD47 mAb. Values shown are means±SD (n=3) from a representative experiment. * statistically significant difference, p<0.05.

THE INVENTION IS EXEMPLIFIED BY THE FOLLOWING EXAMPLES

EXAMPLE 1

In Vitro Evidence for a Role of CD47-SIRPα Interactions in ADCC

[0046] In order to investigate the contribution of the CD47-SIRPα interaction during ADCC of tumor cells by macrophages an assay was employed in which CC531 rat colon carcinoma cells were incubated with CC52 antibody and rat NR8383 effector cell macrophages.

Materials and Methods:

[0047] Rat CC531 colon carcinoma cells and NR8383 rat alveolar macrophages were routinely cultured in RPMI-1640 medium containing 10% fetal calf serum (FCS) (Gibco BRL) and antibiotics. CC531 were detached from the tissue flasks by scraping, washed in PBS, and labelled with 5 μM of DiI (Molecular Probes) for 15′ at RT. After washing 3.75×10.sup.5 CC531 cells, either preincubated or not for 15′ with 5 μg/ml anti-rat CD47 antibody OX101, were incubated, in a round-bottomed 96-well tissue culture plastic plate in 200μl of HEPES-buffered RPMI-1640 containing 0.5% BSA, with 1.25×10.sup.5 NR8383 cells, either preincubated or not for 15′ with 5 μg/ml anti-rat SIRPα antibody ED9 or its Fab′-fragments, in the presence or absence of CC531-reactive mAb CC52 (1 μg/ml). After incubation for 90′ at 37° C. the cells were washed and stained using the macrophage specific biotinylated antibody ED3 (directed against rat sialoadhesin) and FITC-labelled streptavidin. ADCP (expressed as the % of NR8383 having ingested DiI-labelled CC531 cells) was determined on a FACScan flow cytometer (Becton and Dickinson).

Results:

[0048] In the absence of blocking antibodies against CD47 (OX101) or SIRPα (ED9) only very little antibody-dependent cellular phagocytosis is observed, whereas in the presence of such antibodies CC531 are readily phagocytosed (FIG. 2). This shows that interactions between CD47-SIRPα on respectively tumor cells and macrophage effector cells can negatively regulate ADCC in vitro.

EXAMPLE 2

In Vivo Evidence for a Role of SIRPα Signalling in Antibody-Dependent Tumor Cell Killing

[0049] In order to demonstrate that SIRPα provides signals that inhibit tumor cell killing in vivo we compared antibody-dependent tumor cell killing in wild type and SIRPα-mutant mice (Yamao (2002) J Biol Chem. 277:39833-9) using an in vivo B16F10 mouse melanoma model (Bevaart L et al. (2006) Cancer Res. 66:1261-4). The SIRPα mutant mice lack the complete cytoplasmic tail, including the ITIM motifs that act as docking sites for SHP-1 and SHP-2.

Materials and Methods:

[0050] Young adult (7 weeks old) C57Bl/6 wild type or SIRPα-mutant mice (Yamao (2002) J Biol Chem. 277:39833-9) were injected i.v. 1.5×10.sup.5B16F10 melanoma cells (in 100 μL saline; obtained from the National Cancer Institute (Frederick, Md.), in the absence or presence of therapeutic antibody TA99 (10 μg/mouse at day 0, 2, 4, 7, 9, and 11 after tumor cell injection). After 21 days the animals were sacrificed and the number of metastases and tumor load in the lungs was determined as described (Bevaart L et al. (2006) Cancer Res. 66:1261-4).

Results:

[0051] As can be seen in FIG. 3 there was a significantly lower level of tumor development in the SIRPα-mutant mice as compared to wild type mice using suboptimal concentrations of therapeutic monoclonal antibody TA99. These results demonstrate that SIRPα is a negative regulator of tumor cell killing in vivo.

EXAMPLE 3

[0052] To provide further evidence for a negative role of CD47-SIRPα interactions in tumor cell killing by myeloid cells, we established an ADCC assay employing human CD47-expressing Jurkat T cell leukemic cells, opsonized with a murine IgG.sub.2a anti-CD3 antibody (Van Lier RA et al. Eur J Immunol. 1987; 17:1599-1604) as a target (FIG. 4A), and human SIRPα-expressing monocytes as effector cells, and used this to test the effect of antibodies that block CD47-SIRPα interactions.

ADCC Assay

[0053] Monocytes were isolated by magnetic cell sorting by using anti-CD14 coated beads according to the manufacturer's instructions (Miltenyi Biotec B.V., Utrecht, The Netherlands) from PBMC isolated by density centrifugation using isotonic Percoll (Pharmacia Uppsala, Sweden) from heparinized blood obtained from healthy volunteers. The cells were cultured for 16 h in complete RPMI supplemented with 5 ng/ml recombinant human GM-CSF (Pepro Tech Inc, USA), harvested by mild trypsin treatment, and washed. Jurkat cells (5-8×10.sup.6 cells) were collected and labeled with 100 μCi .sup.51Cr (Perkin-Elmer, USA) in 1 ml for 90 min at 37° C. Where indicated the cells were preincubated with anti-CD47 and/or anti-CD3, and washed again. Monocytes were harvested and seeded in 9-well U-bottom tissue culture plates in RPMI with 10% FCS medium. The target cells (5×10.sup.3/well) and effector cells were co-cultured in 96-well U-bottom tissue culture plates in complete medium at a ratio of E:T=50:1 for 4 hours at 37° C., 5% CO.sub.2. Aliquots of supernatant were harvested and analyzed for radioactivity in a gamma counter. The percent relative cytotoxicity was determined as [(experimental cpm−spontaneous cpm)/(Total cpm−spontaneous cpm)]×100%. All samples were tested in triplicate.

Results:

[0054] As can be seen in FIG. 4B. anti-CD3-mediated ADCC against Jurkat cells, which express high levels of surface CD47 (FIG. 4A), is potently and synergistically enhanced by saturating concentrations of F(ab)′.sub.2-fragments of the antibody B6H.sub.12 that blocks CD47 binding to SIRPα. Notably, in the absence of effector anti-CD3 antibody no effect of anti-CD47 F(ab)′.sub.2 was observed, suggesting that CD47-SIRPα interactions act selectively to restrict antibody- and Fc-receptor-mediated affects on tumor cell killing.

[0055] Collectively, these data demonstrate that CD47-SIRPα interactions, and the resultant intracellular signals generated via SIRPα in myeloid cells, form a barrier for antibody-mediated destruction of tumor cells. These results provide a rationale for employing antagonists of the CD47-SIRPα interaction in cancer patients, with the purpose of enhancing the clinical efficacy of cancer therapeutic antibodies.

EXAMPLE 4

Blocking of CD47-SIRPα Interactions Enhances the Effect of Anti-Cancer Therapeutic Antibodies.

[0056] In order to demonstrate that the blocking of CD47-SIRPα interactions indeed enhance the effect of established anti-cancer therapeutic antibodies, we developed an ADCC assay using human Raji Burkitt's B lymphoma cells as targets, human monocytes as effector cells, and an FDA-approved therapeutic antibody against CD20 (Rituximab). For experimental details see the legend to FIG. 5. As can be seen in FIG. 5, the blocking antibody against CD47, B6H12, was able to significantly enhance the Rituximab-mediated cytotoxicity towards Raji cells.