Immunoconjugates for the treatment of tumours

Abstract

A pharmaceutical composition comprising a conjugate of a cytokine and a tumor targeting moiety (TTM) and a pharmaceutically acceptable excipient, wherein the cytokine is present in an amount which does not induce a negative feedback mechanism.

Claims

1. A method of diagnosing cancer comprising the steps of: (a) administering to a patient in need of the same (i) a conjugate of TNFα and at least one tumor targeting moiety (TTM) containing the NGR motif, wherein the conjugate is administered in a dosage range of 5-15 ng/kg, and (ii) a tumor-imaging compound, wherein the conjugate increases the permeability of tumor cells and vessels to the tumor-imaging compound; and (b) detecting the tumor-imaging compound to determine the presence or absence of a tumor.

2. The method according to claim 1, wherein the TTM is a tumor vasculature targeting moiety (TVTM).

3. The method according to claim 2, wherein the TVTM is a binding partner of a tumor vasculature receptor, marker, or other extracellular component.

4. The method according to claim 1, wherein the TTM is a binding partner of a tumor receptor, marker, or other extracellular component.

5. The method according to claim 1, wherein the TTM is targeted to aminopeptidase N (CD13).

6. The method according to claim 1, wherein the TTM is CNGRCVSGCAGRC (SEQ ID NO: 1), NGRAHA (SEQ ID NO: 2), GNGRG (SEQ ID NO: 3), cycloCVLNGRMEC (SEQ ID NO: 16), or linear or cyclic CNGRC (SEQ ID NO: 5 or 17).

7. The method according to claim 1, wherein the TNFα is linked to the TTM through a spacer.

8. The method according to claim 1, wherein the TTM is CNGRC (SEQ ID NO: 5) and wherein the amino terminal of TNFα is linked to the CNGRC peptide (SEQ ID NO: 5) through the spacer G (glycine).

9. The method according to claim 1, wherein the conjugate is in the form of a fusion protein.

10. The method according to claim 1, wherein the tumor-imaging compound is a radiolabelled antibody.

Description

FIGURES AND EXAMPLES

(1) The present invention will further be described by reference to the following non-limiting Examples and Figures in which:

(2) FIG. 1 Effect of mTNF and NGR-mTNF on Tumor Growth and Body Weight of Animals Bearing RMA-T Lymphomas.

(3) Animals bearing RMA-T tumors (5 mice/group) were treated i.p. with NGR-mTNF or mTNF at day 12 after tumor implantation (A) or at day 10, 11 and 12 (B), in two separate experiments (Exp. 1 and Exp. 2). Tumor volumes in Exp. 1 (A) and Exp. 2 (B) and animal body weight in Exp. 1 (C) 1-4 days after treatment are shown. The arrowheads in panel C indicate the time of treatment.

(4) FIG. 2 Circulating Levels of sTNF-R2 and their Role in Regulating the Activity of NGR-mTNF and NGR-hTNF.

(5) Panel A: serum levels of sTNF-R1 and sTNF-R2 in B16F1 tumor bearing mice 1 hour after treatment with various doses of NGR-mTNF or mTNF. Animals (3 mice/group) were treated at day 6.

(6) Panel B: effect of the anti-sTNF-R2 mAb 6G1 on the anti-tumor activity of NGR-mTNF. MAb 6G1 (100 μg) was administered to animals bearing B16F1 tumors at day 5 and 8. One hour later each animal was treated with NGR-mTNF at the indicated doses, and 2 hours later with melphalan (90 μg, 5 mice/group).

(7) Panel C: effect NGR-hTNF and hTNF on the growth of RMA-T tumors. Mice were treated with various doses of each cytokine at day 11. N.S.: not significant (t-test).

(8) FIG. 3 Effect of Melphalan, Alone (A) or in Combination with NGR-mTNF (C) or mTNF (D), on the Tumor Growth (A-D) and Body Weight (E-F) of Mice Bearing B16F1 Melanoma.

(9) The animals were treated (i.p.) with the drugs and the doses indicated in each panel (5 animals/group) at day 4, 7 and 9 after tumor implantation (indicated by arrows).

(10) FIG. 4 Effect of Various Doses of Doxorubicin, Alone (White Bars) or in Combination with NGR-mTNF (Black Bars) on the Tumor Growth (A, B) Body Weight (C. D) and Survival (E) of Mice Bearing B16F1 Melanomas.

(11) The drugs were administered to the animals (5 mice/group i.p.) 5 days after tumor implantation.

(12) FIG. 5 Role of TNF Receptors in the Synergistic Activity of NGR-mTNF and Melphalan.

(13) Panel A: effect of mAb V1q (an anti-mTNF neutralizing antibody) on the anti-tumor activity of melphalan in combination with NGR-mTNF in the B16F1 model. The drugs were administered at day 5. Mab V1q and NGR-TNF were pre-mixed and incubated for 1 hour before injection into animals.

(14) Panel B: effect of melphalan in combination with NGR-hTNF at the indicated doses.

(15) FIG. 6 Effect of NGR-mTNF on the Penetration of Doxorubicin in B16F1 and RMA-T Tumors.

(16) Panel A: bright field (upper panels) and fluorescence (lower panels) microscopy of B16F1 cells incubated in vitro with 100 μg/ml doxorubicin (30 min, 37° C.). Inset: merge of bright field and fluorescence images.

(17) Panel B: stability of the B16F1 fluorescence signal after in vitro treatment with doxorubicin. B16F1 cells were incubated with various doses of doxorubicin in culture medium (30 min, 37° C.), washed with 0.9% sodium chloride, and fixed with 4% formaldehyde. The cells were then incubated for 0 hours or 24 hours in culture medium at 4° C., washed again and analyzed by FACS.

(18) Panel C, F: representative FACS analysis of cells recovered from B16F1 (C) or RMA-T (F) tumors 2 hours after in vitro administration of doxorubicin alone (320 μg) or in combination with NGR-mTNF (0.1 ng). Dashed lines indicate the fluorescence interval considered positive.

(19) Panel D, G: mean±SE fluorescence of B16F1 (D) or RMA-T (G) cells recovered from tumors.

(20) Panel E, H: mean±SE of positive cells recovered from B16F1 (E) RMA-T (H) tumors. Statistical analysis by two-tailed t-test, P<0.05 (*).

(21) FIG. 7 Schematic Representation of the Hypothetical Interations of Low (A), Moderate (B) and High (C) Doses of NGR-TNF with Soluble and Membrane Receptors in Normal Vessels (CD13-Negative) and in Tumor Associated Vessels (CD13-Positive).

(22) Black arrows indicate TNF receptor signalling or extracellular domain shedding.

(23) FIG. 8 Effect of RGD-TNF on the Tumor Growth (FIG. 8A) and Body Weight (FIG. 8B) of an Animal Bearing RMA-T.

(24) FIG. 9 Effect of a Single Treatment (Arrow) with IFNγ-NGR on the Growth of RMA Lymphoma Tumors in C57B6 Mice (FIG. 9A & FIG. 9C) and on Animal Weight (FIG. 9B).

(25) FIG. 10 Effect of NGR-TNF and Cisplatinum on RMA Tumors in C57/BL6 Mice.

EXAMPLE 1—TUMOR CELL LINES AND REAGENTS

(26) Mouse B16F1 melanoma and RMA-T lymphoma cells were cultured as described previously (14, 15). MAb 6G1 (rat anti-p75 mTNF receptor antagonist) was produced and characterized as described previously (16, 17). MAb V1q (rat anti-mTNF) (was kindly supplied by Dr. D. Mannel (University of Regensburg, Germany). Melphalan (Alkeran) was obtained from Glaxo-Wellcome (London, Great Britain). Doxorubicin (Adriblastina) was purchased from Pharmacia-Upjohn (Milan, Italy).

EXAMPLE 2—PREPARATION OF HUMAN AND MURINE TNF AND NGR-TNF

(27) Human and murine TNF and NGR-TNF (consisting of TNF fused with the C-terminus of CNGRCG (SEQ ID NO: 6)) were prepared by recombinant DNA technology and purified from E. coli cell extracts, as described (14). All solutions used in the chromatographic steps were prepared with sterile and endotoxin-free water (Salf, Bergamo, Italy). Protein concentration was measured with a commercial protein quantification assay kit (Pierce, Rockford, Ill.). The in vitro cytolytic activity of human TNF (hTNF), estimated from a standard cytolytic assay with L-M mouse fibroblasts (18), was 5.4×10.sup.7 Units/mg, whereas that of purified NGR-hTNF was 1.4×10.sup.8 Units/mg. The cytolytic activity of murine TNF (mTNF), was 7.6×10.sup.7 Units/mg, whereas that of NGR-mTNF was 9.1×10.sup.7 Units/mg. The hydrodynamic volumes of NGR-mTNF, NGR-hTNF and mTNF were similar to those of hTNF, a homotrimeric protein (19), by gel filtration chromatography on a Superdex 75 HR column (Pharmacia, Sweden). Electrospray mass spectrometry of each product determined the following molecular masses: NGR-hTNF, 17937.6±1.9 Da (expected for CNGRCG-hTNF.sub.1-157 monomers, 17939.4 Da) (SEQ ID NO: 21); hTNF, 17349±1.3 (expected for hTNF.sub.1-157, 17350.7); NGR-mTNF, 17841.16±2.5 (expected for CNGRCG-mTNF.sub.1-156, 17844.2) (SEQ ID NO: 22), mTNF, 17384.9±2 (expected for Met-mTNF.sub.1-156, 17386.7). The endotoxin content of each product, measured using the quantitative chromogenic limulus amoebocyte lysate (LAL) test (BioWhittaker), was: NGR-hTNF, 0.079 Units/μg; hTNF, 0.117 Units/μg; NGR-mTNF, 0.082 Units/μg; mTNF, 1.61 Units/μg.

EXAMPLE 3—IN VIVO STUDIES

(28) Studies on animal models were approved by the Ethical Committee of the San Raffaele H Scientific Institute and performed according to the prescribed guidelines. C57BL/6 mice (Charles River Laboratories, Calco, Italy) weighing 16-18 g were challenged with subcutaneous injection in the left flank of 5×10.sup.4 RMA-T or B16F1 living cells; 4-12 days later, the mice were treated with TNF or NGR-TNF solutions (100 μl) followed 2 hours later by administration of melphalan or doxorubicin solution (100 μl). Unless specified, all drugs were administered intraperitoneally (i.p.). All drugs were diluted with 0.9% sodium chloride, containing 100 μg/ml endotoxin-free human serum albumin (Farma-Biagini, Lucca, Italy), except for doxorubicin, which was diluted with 0.9% sodium chloride alone. Tumor growth was monitored daily by measuring the tumors with calipers as previously described (20). Animals were sacrificed before the tumors reached 1.0-1.5 cm in diameter. Tumor sizes are shown as mean±SE (5 animals/group).

EXAMPLE 4—SOLUBLE TNF RECEPTOR ASSAYS

(29) Soluble p55-TNF receptor (sTNF-R1) and soluble p75-TNF receptor (sTNF-R2) in animal sera were measured using the Quantikine M kit (R & D Systems, Minneapolis, Minn. 55413).

EXAMPLE 5—DETECTION OF DOXORUBICIN IN TUMORS

(30) C57/BL6 mice bearing B16F1 or RMA-T tumors (diameter, 0.5-1 cm) were treated with or without NGR-m TNF (0.1 ng, i.p.), followed 2 hours later by doxorubicin (320 μg, i.p.). After 2 hours the animals were sacrificed and the tumors were excised. Each tumor was weighed, disaggregated, resuspended in cold phosphate-buffered saline (PBS) and filtered through 70 μm filters. The cells were resuspended with cold PBS (50 ml), centrifuged (1500 rpm, 10 min, 4° C.), resuspended in cold PBS (2.5 ml/g of tumor tissue) and mixed with freshly prepared PBS containing 8% formaldehyde (2.5 ml/g of tissue). The cells were stored in the dark at 4° C. overnight, and then analyzed by FACS. The FACScan (Becton-Dickinson) was calibrated with cells recovered from untreated tumors. Each sample was then analyzed using the FL-3 filter and Cell Quest software.

EXAMPLE 6—DOSE-RESPONSE CURVES OF NGR-mTNF AND mTNF IN MURINE LYMPHOMA AND MELANOMA MODELS

(31) The anti-tumor activity of NGR-mTNF and mTNF was first characterized in the absence of chemotherapeutic drugs. To compare the dose-response curves of NGR-mTNF and mTNF we performed several experiments based on single or repeated administration (i.p.) of various doses of NGR-mTNF and mTNF (from 0.01 to 10000 ng) to RMA-T lymphoma- or B16F1 melanoma-bearing mice. Murine TNF delayed tumor growth when adminstered at high doses (10000 ng) (FIG. 1A); no effects were induced by doses lower than 100 ng, either with single (FIG. 1A) or repeated administrations (FIG. 1B). NGR-mTNF was markedly more potent. In this case we observed anti-tumor effects even with doses as low as 0.01 ng (FIG. 1A, B). However, the dose-response curve was more complex. For instance, the effect of 10 ng was surprisingly lower than that of 0.01-0.1 ng and 1000-10000 ng. A bell-shaped dose-response curve was observed in several other experiments conducted in the RMA-T model as well as in the B16F1 melanoma model (not shown). These results suggest that 1) the efficacy of low doses of NGR-mTNF is markedly higher than that of mTNF, and 2) doses of NGR-mTNF greater than 1-10 ng activate negative feed-back mechanisms that inhibit its potential anti-tumor activity.

EXAMPLE 7—NANOGRAM DOSES OF NGR-TNF, BUT NOT PICOGRAMS, INDUCE SOLUBLE TNF RECEPTOR SHEDDING

(32) The protective mechanisms responsible for the bell-shaped dose-response curve of NGR-mTNF were then investigated. Since exogenously administered TNF can induce soluble TNF receptor (sTNF-Rs) shedding in vivo (21), we hypothesized that the lower efficacy of 10 ng of NGR-TNF was related to induction of sTNF-R1 and/or sTNF-R2 and, consequently, to neutralization of its interaction with membrane receptors.

(33) To test this hypothesis, we measured the levels of sTNF-R1 and sTNF-R2 in the serum of tumor bearing mice collected 1 hour after administration of various doses of mTNF and NGR-mTNF. As expected, both products induced sTNF-R2 shedding, but not sTNF-R1 shedding, at doses greater than 4 ng (FIG. 2A).

(34) To assess whether sTNF-R2 shedding regulates the activity of NGR-mTNF, we coadministered this cytokine with mAb 6G1, an antagonist anti-sTNF-R2 antibody that prevents the binding of mTNF to soluble and membrane murine TNF-R2 (16). The anti-tumor activity of 10 ng of NGR-mTNF was potentiated by mAb 6G1 (FIG. 2B), in line with the hypothesis that sTNF-R2 plays a role in inhibiting the anti-tumor effects of NGR-mTNF.

(35) To further support this hypothesis we compared the in vivo dose-response curve of NGR-mTNF with that of NGR-hTNF, taking advantage of the fact that the human cytokine cannot bind murine sTNF-R2 (22). We found that the dose-response curve of NGR-hTNF was not bell-shaped and that 10 ng of NGR-hTNF is as active as 1 ng (FIG. 2C). It is also remarkable that 1 ng was sufficient to induce the maximum anti-tumor effect. This may suggest that receptor binding on vessels can be achieved with very low blood levels of NGR-hTNF.

(36) Taken together, the results of these experiments strongly suggest that NGR-mTNF and mTNF, at doses greater than 4 ng, induce shedding of sTNF-R2 in amounts sufficient to inhibit their anti-tumor activity.

EXAMPLE 8—PICOGRAM DOSES OF NGR-mTNF ARE SUFFICIENT TO ENHANCE THE THERAPEUTIC EFFECT OF MELPHALAN AND DOXORUBICIN

(37) We then investigated whether targeted delivery of low doses of NGR-mTNF to tumor vessles could enhance the anti-tumor activity of chemotherapeutic drugs. These experiments were conducted in the B16F1 model, a spontaneous mouse melanoma characterized by scarce immunogenicity and low sensitivity to melphalan. Melphalan (90 μg) was unable to effect the growth of tumors when injected alone (FIG. 3A). Similarly, mTNF (0.1 ng alone, i.p.) was virtually inactive, while the same dose of NGR-mTNF modestly delayed the tumor growth (FIG. 3B, upper panels). The combination of melphalan with 0.1 ng of NGR-mTNF induced stronger anti-tumor effects than the single agents, indicating a synergistic effect (FIG. 3C). Remarkably, the combination of melphalan with 0.1 ng of NGR-mTNF was more effective than the combination with 5000 ng of mTNF (FIG. 3C-D). We observed this synergisim even when NGR-mTNF (0.1 ng) was injected i.v. (not shown).

(38) Two similar experiments were conducted with doxorubicin in the B16F1 model. Animals were treated five days after tumor implantation, with or without NGR-mTNR and, 2 hours later, with various doses of doxorubicin (20-320 μg, i.p.). In both experiments, the effect of doxorubicin plus NGR-mTNF was stronger than that of doxorubicin alone (FIG. 4A, B, E), indicating that NGR-mTNF markedly improves the efficacy of this drug. For example, the effect of doxorubicin (40 μg) plus NGR-mTNF (0.1 ng) was stronger than that of 320 μg of doxorubicin alone (FIG. 4B), while, the effect of doxorubicin (20 μg) plus NGR-mTNF was lower (FIG. 4A). From these results we estimate that the activity of doxorubicin is potentiated 8-10 fold by NGR-mTNF.

(39) In conclusion, these results suggest that picogram doses of NGR-TNF are sufficient to improve the response of tumors to both melphalan and doxorubicin.

EXAMPLE 9—LOW DOSES OF NGR-mTNF ARE NOT TOXIC AND DO NOT INCREASE THE TOXICITY OF MELPHALAN

(40) To estimate the efficacy/toxicity ratio of each treatment, we monitored the animal body weight daily and animal survival after treatment. While therapeutic doses of mTNF (10000 ng) induced marked loss of body weight in RMA-T bearing animals (FIG. 1C, left), therapeutic doses of NGR-mTNF (0.01-1 ng) did not cause loss of body weight (FIG. 1C, right). Moreover, neither NGR-mTNF nor mTNF (1 ng each) increased the lethality of 200 μg of melphalan in mice bearing the RNA-T tumor (Table 1).

(41) TABLE-US-00004 TABLE 1 Effect of mTNF and NGR-mTNF on the toxicity of Melphalan (high dose) in tumor-bearing mice.sup.a No. of mice alive Treatment 3 days after treatment None  5/5 (100%) Melphalan 7/10 (70%) Melphalan + mTNF 8/10 (80%) Melphalan + NGR-mTNF 8/10 (80%) .sup.aC57BL6 mice, bearing 11-day old tumors, were treated (i.p.) with 1 ng of NGR-mTNF or mTNF and, 2 hours later, with 200 μg of melphalan.

(42) In the B16F1 model, therapeutic doses of NGR-mTNF (0.1 ng) did not cause loss of body weight, even when combined with melphalan (FIG. 3E). In contrast, melphalan combined with therapeutic doses of mTNF (5 μg) induced marked loss of body weight (FIG. 3F). In addition, NGR-mTNF (0.1 ng) did not increase the loss of body weight causes by high doses of doxorubicin (FIG. 4C-D).

(43) These results suggest that picogram doses of NGR-mTNF increase the response of tumors to melphalan and doxorubicin with no evidence of increased toxicity.

EXAMPLE 10—TNF-R1 ACTIVATION IS NECESSARY AND SUFFICIENT FOR THE SYNERGISM BETWEEN NGR-TNF AND CHEMOTHERAPEUTIC DRUGS

(44) The mechanisms of the synergism between low doses of NGR-mTNF and chemotherapy were then investigated.

(45) To assess whether these mechanisms rely on TNF-Rs activation we tested the effect of mAbV1q, a neutralizing anti-mTNF antibody, on the anti-tumor activity of NGR-mTNF (0.1 ng) in combination with melphalan (90 μg). MAb V1q inhibited, at least partially, the anti-tumor activity of these drugs in the B16F1 model (FIG. 5A). This suggests that the interaction between the TNF moiety and TNF-Rs is critical for the activity of the conjugate.

(46) The role of TNF-R1 and TNF-R2 was then studied. To this end, we evaluated the effect of melphalan in combination with 0.01 ng or 0.1 ng of NGR-hTNF, a TNF-R1 specific agonist (22). The effect of melphalan in the B16F1 model was potentiated by NGR-hTNF (FIG. 5B) suggesting that TNF-R1 activation is sufficient for the synergism.

EXAMPLE 11—THE SYNERGY BETWEEN NGR-TNF AND CHEMOTHERAPY IS NOT DEPENDENT ON TUMOR CELL CYTOTOXICITY

(47) To assess whether the synergism depends directly on cytotoxicity against tumor cells we measured the effect of each compound, alone or in combination, on cultured B16F1 cells. Neither melphalan or NGR-mTNF, alone or in combination, killed these cells in a 48 hour in vitro assay (not shown). Similarly, NGR-mTNF did not enhance the cytotoxic activity of doxorubicin in vitro (not shown). These results suggest that the synergism observed in vivo is not directly dependent on cytotoxic effects against tumor cells and points to an indirect role of a component of the tumor stroma, e.g. the endothelial lining of tumor vessels.

EXAMPLE 12—NGR-TNF INCREASES THE PENETRATION OF DOXORUBICIN IN MURINE MELANOMAS AND LYMPHOMAS

(48) We then investigated whether NGR-mTNF could increase the penetration of chemotherapeutic drugs in tumors. To this aim we measured the amount of doxorubicin that had penetrated B16F1 and RMA-T tumors, 2 hours after administration, taking advantage from the fluorescent properties of this drug (23). Preliminary experiments showed that the nuclei of B16F1 cells become fluorescent after these cells are exposed to doxorubicin in vitro (FIG. 6A). The fluorescence signal is dose-dependent and stable for at least 24 hours, when the cells are fixed with formaldehyde and kept at 4° C., as measured by FACS (FIG. 6B). Thus, the fluorescence intensity of tumor cells recovered from animals after treatment is an indication of the amount of doxorubicin that has penetrated tumors. We observed that 0.1 ng of NGR-mTNF, administered 2 hours before doxorubicin, increased the fluorescence intensity and the percentage of positive cells recovered from both B16F1 and RMA-T tumors, 2 hours after treatment (2-5-fold, FIG. 6C-H). This suggests that NGR-mTNF increased the number of cells that were reached by doxorubicin as well as the intracellular amount of drug.

EXAMPLE 13—EFFECT OF RGD-TNF ON TUMORS

(49) C57/BL6 mice bearing RMA-T tumors (5 mice/group) were treated intraperitoneally with melphalan alone, or in combination with RGD-TNF or NGR-TNF. The effect on tumor volume is shown in FIG. 8A and the effect on animal weight is shown in FIG. 8B. These results demonstrate that RGD-TNF is also active in the picogram range.

EXAMPLE 14—EFFECT OF IFNγ-NGR ON TUMORS

(50) C57/BL6 mice bearing RMA lymphoma tumors were treated with or without IFNγ (3 ng) or IFNγ-NGR (3 ng). After 21 days the animals were sacrificed. The results of tumor volume and of animal weight loss can be seen in FIGS. 9A and 9B. In addition, C57/BL6 mice bearing RMA lymphoma tumors were treated with or or without IFNγ (3 ng), IFNγ-NGR (3 ng), IFNγ-NGR (3 ng) plus anti-CD13 R3-63 mAb (25 μg) or IFNγ-NGR (3 ng) plus mAB 19E12 (50 μg). The mAB 19E12 is an irrelevant IgG used as a negative control in the experiment. The results demonstrate that IFNγ-NGR is active in the picogram range.

EXAMPLE 15—PICOGRAM DOSES OF NGR-TNF ARE SUFFICIENT TO ENHANCE THE THERAPEUTIC EFFECT OF CISPLATINUM

(51) We investigated whether targeted delivery of low doses of NGR-TNF to tumor vessels could enhance the anti-tumor activity of the anti-cancer drug, cisplatinum. These experiments were carried out in C57/BL6/N mice bearing RMA-T tumors of initial age 8 weeks. The mice were treated with or without cisplatinum or NGR-mTNF with treatment at day 10. The results are shown in FIG. 10. In this figure, Cys=Cisplatino Teva solution 0.5 mg/ml; NGR=NGR-mTNF. The diluent was HAS 100 mg/ml in NaCl 0.9%. The results suggest that pciogram doses of NGR-TNF are sufficient to enhance the therapeutic effect of cisplatinum.

(52) Summary of Advantages

(53) Alteration of vascular permeability and interstitial pressure, endothelial cell damage and fibrin deposition are important mechanisms for the anti-tumor activity of TNF, either alone or in combination with chemotherapeutic drugs. After, infusion in animals or patients TNF can also induce negative feedback mechanisms that neutralize most of these effects. For example, TNF, even at moderate doses, can induce the release of soluble p55 and p75 TNF receptors that may prevent its interaction with membrane receptors (21, 24). Although these soluble inhibitors may protect the body from the harmful effects of this cytokine, they may also prevent its anti-tumor activity and could explain, in part, the need of high doses of TNF for effective therapy. In this work we postulated that homing low doses of TNF to tumor vessels represents a new strategy to avoid toxic reactions as well as negative feedback mechanisms, while preserving its synergism with chemotherapy. To verify this hypothesis, we have investigated the anti-tumor activity of high and low doses of NGR-mTNF and mTNF, ranging from picogram to microgram quantities, in two murine models based on subcutaneous RMA-T lymphoma and B16F1 melanoma tumors. The study was carried out using these cytokines alone or in combination with melphalan or doxorubicin. While mTNF was virtually inactive in these models at lower doses than 100-1000 ng, we found that NGR-mTNF, even alone, could induce anti-tumor effects with doses as low as 0.01-0.1 ng. Since the LD.sub.50 values of mTNF and NGR-mTNF are similar and correspond to about 50,000 ng in RMA-T tumor-bearing mice (14), these results indicate that the efficacy/toxicity ratio of NGR-mTNF is 10.sup.4-10.sup.5 times greater than that of mTNF.

(54) Administration of minute amounts of NGR-mTNF (0.1 ng) to tumor-bearing animals potentiated the anti-tumor activity of melphalan and doxorubicin with no evidence of increased toxicity, as judged by tumor mass reduction, animal survival and weight loss after treatment. This suggests that NGR-mTNF improves the therapeutic index of these drugs. It is noteworthy that 5×10.sup.4-fold greater doses of mTNF were necessary to enhance the effect of melphalan to comparable levels, causing marked loss of body weight.

(55) The fact that both melphalan and doxorubicin at doses virtually inactive in the B16F1 model reduced tumor growth when combined with NGR-mTNF indicates that these drugs act synergistically. Studies on the mechanism of action showed that the synergism relies on the interaction of NGR-mTNF with TNF-R1 on stromal cells, most likely endothelial cells, and much less on tumor cells. In addition, we found that vascular targeting with NGR-mTNF improves cytotoxic drug penetration in tumors. Noteworthy, NGR-mTNF increased both the percentage of cancer cells that can be reached by doxorubicin in 2 hours as well as the intracellular amount of drug, suggesting that NGR-TNF can alter drug-penetration barriers. Previous studies showed that TNF can rapidly increase endothelial permeability (25, 26), and can decrease interstitial fluid pressure (8) that are believed to be important barriers for drug penetration in tumors (1). Possibly, these mechanisms increase convective transport of drugs through the tumor vessel wall and interstitium, finally resulting in increased drug uptake by tumor cells. The timing of administration is likely critical for these mechanisms, as TNF can also induce intravascular coagulation (27) leading to vessel occlusion and reduction of tumor perfusion. According to this view, we observed that the effect of melphalan was higher when this drug was administered 2 hours after NGR-TNF than after 6 hours (data not shown).

(56) The hypothesis that vascular targeting could avoid negative feedback mechanisms, usually associated with TNF therapy, is supported by the observation that picogram doses of NGR-mTNF do not induce soluble receptor shedding, while both NGR-mTNF and mTNF rapidly induce the release of sTNF-R2 into the circulation at doses greater than 4-10 ng. These levels of sTNF-R2 inhibited most of the anti-tumor activity of 10 ng of NGR-mTNF and may explain the paradoxical observation that 10 ng is less active than 0.1 ng. Likely, a large proportion of injected molecules were rapidly complexed by sTNF-Rs and their activity was blocked.

(57) The molecular mechanisms underlying the selective interaction of low doses of NGR-mTNF with tumor blood vessels has been partially elucidated. We have shown recently that different CD13 isoforms are expressed in tumor-associated vessels, in epithelia and in myeloid cells, and that the NGR domain of NGR-TNF selectively recognizes a CD13 isoform associated with tumor vessels (28). We hypothesize, therefore, that low blood levels of NGR-mTNF can rapidly interact with CD13-positive endothelial cells because of high-avidity multivalent binding involving both CD13 and TNF-Rs, and little or not at all with CD13-negative endothelial cells of normal vessels, because of lower avidity. A schematic representation of these concepts and of the hypothetical interactions of NGR-TNF with soluble and membrane receptors is shown in FIG. 7.

(58) We have also found that RGD-TNF and IFNγ-NGR are active in the picogram range. We have also shown that NGR-TNF increases the effect of cisplatinum.

(59) In conclusion, we have found that targeted delivery of picogram doses of cytokines to tumor vessels enhances the anti-tumor activity of chemotherapeutic drugs in mice without inducing soluble receptor shedding. Given that the CNGRC (SEQ ID NO: 5) motif is expected to target murine as well as human tumor associated vessels (29), our results suggest that the combination of low doses of targeted cytokines with anti-cancer drugs could increase their therapeutic index in human patients.

(60) All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

REFERENCES

(61) 1. Jain, R. K. 1994. Sci Am. 271:58-65. 2. Lienard, D., et al. 1992 J. Clin. Oncol. 10:52-60. 3. Eggermont, A. M., et al. 1996. J. Clin. Oncol. 14:2653-2665. 4. Lejeune, F. J. 1995. Eur. J. Cancer. 31A:1009-1016. 5. Fraker, D. L., et al. 1996. J. Clin. Oncol. 14:479-489. 6. Rossi, C. R., et al. 1999. Cancer. 86:1742-1749. 7. van der Veen, A. H., et al. 2000. Br. J. Cancer. 82:973-980. 8. Kristensen, C. A., et al. 1996. Br. J. Cancer. 74:533-536. 9. Suzuki, S., et al. 1990. Int. J. Cancer. 46:1095-1100. 10. de Wilt, J. H., et al. 2000. Br. J. Cancer. 82:1000-1003. 11. Fiers, W. 1995. Biologic therapy with TNF: preclinical studies. In Biologic therapy of cancer: principles and practice. V. De Vita, S. Hellman, and S. Rosenberg, editors. J. B. Lippincott Company, Philadelphia. 295-327. 12. Fraker, D. L., H. R. Alexander, and H. I. Pass. 1995. Biologic therapy with TNF: systemic administration and isolation-perfusion. In Biologic therapy of cancer: principles and practice. V. De Vita, S. Hellman and S. Rosenberg, editors. J.B. Lippincott Company, Philadelphia 329-345. 13. Corti, A., and F. Marcucci. 1998. J. Drug Targ. 5:403-413. 14. Curnis, F., A et al. 2000. Nat. Biotechnol. 18:1185-1190. 15. Moro, M., et al. 1997. Cancer Res. 57:1922-1928. 16. Corti, A., et al. 1999. Infect Immun. 67:5762-5767. 17. Pelagi, M., et al. 2000. Eur. Cytokine Net. 11:580-588. 18. Corti, A., et al. 1994. J. Immunol. Meth. 177:191-198. 19. Smith, R. A., and C. Baglioni. 1987. J. Biol. Chem. 262:6951-6954. 20. Gasparri, A., et al. 1999. Cancer Res. 59:2917-2923. 21. Aderka, D., et al. 1998. J. Clin. Invest. 101:650-659. 22. Tartaglia, L. A., et al. 1991. Proc. Natl. Acad. Sci. USA. 88:9292-9296. 23. Luk, C. K., and I. F. Tannock. 1989. J. Natl. Cancer Inst. 81:55-59. 24. Sella, A., et al. 1995. Cancer Biother. 10:225-235. 25. Brett, J., et al. 1989. J. Exp. Med. 169:1977-1991. 26. Goldblum, S. E., and W. L. Sun. 1990. Am. J. Physiol. 258:L57-67. 27. Clauss, M., et al. 1992. Modulation of endothelial cell hemostatic properties by TNF: insights into the role of endothelium in the host response to inflammatory stimuli. In Tumour necrosis factors: the molecules and their emerging roles in medicine. B. Beutler, editor, Raven Press, New York. 49-63. 28. Curnis, F., et al. 2002. Cancer Res.: 62:867-874. 29. Arap, W., et al. 1998. Science. 279:377-380.