EP1187633A1 - Use of anti-vegf antibody to enhance radiation in cancer therapy - Google Patents

Abstract

We have discovered that VEGF expression is induced following exposure tumors to ionizing radiation (IR) both in vitro and in vivo. We found that treatment of tumor-bearing mice with a neutralizing antibody to VEGF prior to irradiation is associated with greater than additive antitumor effects.

Description

USE OF ANTI-VEGF ANTIBODY TO ENHANCE RADIATION IN CNACER THERAPY

BACKGROUND

This work was supported by grants rom the National Cancer Institute, and the

government have certain rights in the invention.

Tumors influence the surrounding host strαma by inducing angiogenesis to supply their oxygen and nutrient needs, allowing them to grow. In normal tissues, angiogenesis is tightly

regulated by the balance between angiogenic nnd anti-ongiogenic fuc or$lι2. However, the induction of angiogenesis by tumor-derived pro-angiogcnic proteins is a discrete component of the malignant phcnotypc. Decreased production of angiogenesis inhibitors or increased

expression of ungiogenic peptides can shiA the balance towards u pro-angiogcnic state*, permitting tumor growth. As a tumor increases in size, it disrupts its surrounding stro a and recruits still more host blood vessels. This paracrine relationship between a tumor and its blood supply represents a potential point of attack for an itumor therapy.

Λ family of angiogenic peptides, isofor s of vascular cndothclial cell growth factor (VEGF), are expressed by many human tumors and normal cells^-S, VEGF is the only known angiogenic protein that is exclusively itogenic for endothelial cells in vύro and strongly

angiogenic in It is secreted by n wide variety of human tumors, and inhibition of VEGF- induced angiogenesis, either by neutralizing antibodies or a dominant negative soluble receptor,

blocks the growth of primary and metastatic experimental tumors""". Physiologic regulators of VEGF expression include hypoxia9ιl0 and cytokines^. In certain human tumors, oncogcnic mutations of ras and p53 are associated with increases in intratumoral VEGF levels and a poor

nrcαr.csis' ^ >' ^.

^{' "} ST τ\TM ARY OF THE INVENTION

In the present study, e examined the production of VEGF by L1X and human xcno graft tumors following exposure to ionizing radiation (IR), and found that VEGF expression is induced following IR- We next examined whether blocking the action of a positive regulator of angiogenesis could po^ten^tia^te ^the αnutumor effect of IR.Block.ng _this IR-mcdiatcd increase in VEGF using neutralizing an^tibodies against VF.GF resulted in increased cndotheϋal cell killing and produced grea^ter ^than addi^ti^ve anti-tumor effect, in aousc tumor model sys_tems, findings ^that suppor^t a model in which induction of VEGF by IR contribu_tes to the protec_tion of _tumor blood vessels from radiation-mediated cytotoxicity.

The inveπ.io„ provides a method of reducing tumor radio resistance or chemotherapy res.stance in a cancer patient being or to be treated with radiation or chemotherapy, by administering to the patient a substance that inhibits chemotherapy or rad^tation-induced VEGF expression or that blocks VEGF activity in the patient.

The substance can be an anti-VEGF antibody, and can be administered (preferably IV) shordy (_M hours) prior to chemotherapy or radiation treatment.

The dosages^, fⁱning, and duration of anti-VEGF antibody administration in

humans can be extrapolated from the antma! model results presented herem. Antibody

preferably is administered intravenously, either prior to, duπng, or following radiation or chemotherapy administration.

BRIEF DESCRIPTION OF THE PR AWTNrrς

FIGURE 1. VEGF levels in Lewis lung carcinoma in vivo and in vitro. A. VEGF mRNA levels in LLC tumors following IR exposure (40 Gy divided into two daily doses). Total RNΛ was isolated from representative tumors and probed with a cDNA encoding human VEGF- 165, after which they were stripped of probe and reprobed with a cDNA to GAPDH to demonstrate message integrity. By scanning densitomctry, normalized to GAPDH, VβGF mRNA is induced 3-fold following IR exposure. Blots from representative animals are displayed. B. VEGF protein levels in media conditioned by LLCs following ]R exposure. Cs were plated in six-well plates at low density (25% confluence), allowed to attach overnight, and then irradiated with 0, 5, ι vi, or 20 Gy. Conditioned media was collected every 24 hrs, and cells were detached with trypsin and counted. VEGF levels were normalized to the number of cells and reported as total pg VEGF/10⁶ cells. No VEGF was detectable in unconditioned media.

Figure 2. VEGF expression in human tumor cell line*. Subconflucnt cells from human tumor cell lines (Seg-1 esophageal adcnocarcinoma, SQ20B squamous cell carcinoma, Ul melanoma, and U87 and T98 glioblastoma) were exposed to 10 Gy of ioni ing radiation. Conditioned media from radiated and unirradiaicd cells was collected 24 hours later. VEGF levels in conditioned media were measured by EL1SA and normalized to cell number. An increase in VEGF secretion was observed in each cell line: Scg-1 (p-?), SQ20B (p-0.08), T98 (p=0.02), Ul (p=0.009), U87 (p=0.0009). No VEGF was detectable in unconditioned media.

FIGURE 3. Effect of VEGF blockade prior to treatment with ionizing radiation in mouse tumors and human xcnografts. LLC cells (1 x 10^δ) were injected subcutancously into the hindlimbs of female C57B1 6 mice. SQ20B squamous cell carcinoma cells (5 x 10⁶) and Scg-1 csophagca! adcnocarcinoma cells (3 xl O⁶) were injccicd into the hindlimbs of female a^lhymic nude mice. Tumors were allowed lo attain a mean size between 350-450 mm^-1 (LLC, 442 ± 14 mm³; SQ20B, 372 ± 16 mm³; Scg-1, 407 ± 20 mm⁵), after which treatment was begun. A. Effect of VEGF blockade prior to ionizing radiation in LLC tumors. Mice were treated as follows: IR, 40 Gy administered as two 20 Gy doses on days 0 and 1 ; IR (40 Gy) plus polyelonal goat anti-mouse VEGF-164 antibody. 10 μg were administered inirapcritonualry 16 and 3 hrs before the first IR treatment and 3 hours before the second IR treatment (3 doses total); goat anti- mouse VEGF-164 antibody alone administered as described. Untreated controls received nonimmune goal IgG. B. Effect of VEGF blockade prior to ionizing radiation in SQ20B xenografts. Mice were treated as follows: IR, 40 Gy administered as four 10 Gy doses on days 0, 1 ,2, and 3; IR (40 Gy) plus monoclonal anti-human VEGF-165 antibody, 10 μg administered intrapcritoncally two to three hours before each dose of IR; monoclonal anti-human VEGF-165 antibody alone administered identically to the combined treatment group. Untreated controls received nonimmune mouse IgG. C. Effect of VEGF blockade prior to ionizing radiation in Seg- 1. Mice were treated as follows: IR, 20 Gy administered as 4-5 Gy doses on days 0, 1,2, and 3; IR (20 Gy) plus monoclonal anti-human VEGF-165 antibody, 10 μg administered imraperitoneaily two to three hours before each dose of ]R; monoclonal ami-human VEGF-165 antibody alone administered identically lo the combined treatment group. Untreated controls received nonimmune mouse IgG. D. Mice bearing SQ20B xenografts from different treatment groups (day 22). Mice with tumor volumes closest to the mean for each group were chosen.

FIGURE 4. Effect of manipulating VEGF levels in vitro on rR_*rπcdiatcd vascular endothclial cell killing. For MTT assays, HUVECs were plated in 96-well plates al 1 x 103 cells/well and treated with either differing concentrations of rccombinant human VEGF-165 or monoclonal anti-human VEGF-165 antibody prior to treatment with IR, and abscrbancc readings measured at varying time points after IR (sec Methods). For clonogcnic survival assays,

HUVECs were treated with different concentrations of VEGF or a polyelonal goat anti-VEGF-

^] 65 antibody four hours prior to irradiation (sec Methods). A, MTT assay for HUVECs prctreaied with varying concentrations of recυmbinant human VEGF-165 four hours before IR treatment. Absorbance measurements were taken ai 96 hrs after IR and normalized to those obtained under standard conditions (no JR treatment and VEGF-10 ng/ml). B. Clυnogenic survival assay for HUVECs pretreated with 1, 10, or 50 ng/ml VEGF ± 200 and 900 cGy.

Surviving raction is normalized to plating efficiency for untrradiated cells. C. MTT assay for

HUVECs pretreated with monoclonal anti-VEGF-) 65 antibody four hours before IR treatment.

Absorbance measurements were taken at 96 hrs after IR and normalized to those obtained with no pretreatmenl with Ab. VEGF=10 ng/mlfor this experiment. D. Clonogcnic survival assay for

HUVECs pretreated with a monoclonal anti-VEGF- 165 antibody prior to irradiation.

DETAILED DESCRIPTION

Ionizing radiation induces tumor VEGF production In vivo and in vitro

We have examined the production of VEGF in Lewis lung carcinoma (LLC) tumors following exposure to ionizing radiation (IR). LLC cells (1 x 10*) were injected subcutancously in the hindlimbs of female C57BL/6 mice and allowed to grow to a volume of 510 ± 11 mm'

(approximately 2.5% body weight). Tumors were irradiated with 20 Gy on days 0 and 1 and then harvested at days 2, 5, or 14. VEGF levels were measured by ELISΛ and normalized to total tumor protein. VEGF levels in extracts from control tumors remained relatively constant (46 to

90 pg/mg total protein) for 14 days as the tumors grew to 6110 ± 582 mm³, or approximately

30% of body weight (Table I). By contrast, on day 2, the mean VEGF level in irradiated tumors was increased more than three-fold as compared to thai in uπirradiatcd tumors (234 ± 79 pg mg total extract protein, p=0,032). The mean VEGF level in irradiated tumors remained 2.2-foId higher than that in unirradiated tumors at day 14' (194 ± 47 pg/mg total extract protein, p-0.027).

Plasma VEGF levels remained low or undctcctablc in control and irradiated animals (data not shown). To confirm the effects of IR, VEGF mRNA levels were assessed in the same tumors by

Northern blol analysis. VEGF transcripts were induced 3-fold two days after exposure to I (Fi^βure 1 A). Moreover, VEGF mP: ., levels remained elevated for fourteen days. These fⁱndings demonstrate that IR induces VEGF expression in vivo.

To determine whether IR induces VEGF in tumor cells in vitro, subconfluent LLC ceils were exposed ^lo different doses of IR, nd conditioned media was harvested at various intervals for measurement of VEGF levels by ELISA. VEGF levels in LLC-condiiioned media exhibited an IR dose-dependent increase within 24 hours. At 72 hours, VEGF levels were nearly 6-fold higher in media from LLC irradiated with 20 Gy (Figure IB), as compared to that for control cells (p=0.009). VEGF expression was also studied in irradiated human tumor cell li_nes: Seg-1 (esophageal adenocarcinoma)13_; SQ20B (a radioresistant squamous cell carcinoma li„_e)14_; Ul (melanoma); and T98 and U87 (glioblastoma). Under basal conditions, these tumor ceil lines secreted widely differing levels of VEGF, with U87 cells producing the most VEGF and Ul meianoma cells the .east (Figure 2). All demonstrated an IR-dependem increase in VEGF production within 24 hours of treatment with 10 Gy (Figure 2). These findings demonstrate that IR induces VEGF expression in diverse tumor cell types.

Blocking VEGF action enhances the in vivo untitumor effect of ionizing radiation

To determine whether induction of VEGF secretion by tumors affects ant_i-tumor response, w_e treated LLC tumors with neutralizing antibodies to VEGF prior to IR exposure. Female C57B 6 mice bearing LLC tumors (559 ± 51 mm³) were treated with a polyelonal goat antibody directed against recombinant murine VEGF-164 (R & D Systems, 10 μg qd by intrapeπtoneal injection) or with nonimmune goat IgG. By day 7, tumors from control animals had attained a volume of 2713 * 346 mm³, while tumors in anti-VEGF-ireated mice were 101 1 _ 266 mm³ (p=0.02). Consistent with previous ob_Wrv.tio_M6.15-17 ^ _{Mngs indjωtc} ^ blocking VEGF activity inhibits tumor growth. To evaluate the ami umor effects of combining anti-VEGF an^libodics and IR, mice bearing LLC tumors were treated as follows: untreated control; IR _aiθne, 20 Gy on consecutive days (40 Gy total); anti-VEGF antibody; and IR plus anti-VEGF antibody (Figure 3Λ). Starting from a mean volume of 442 =_fc 14 mm³ at day 0, tumors in untreated controls reached a mean volume of 1389 ± 136 mm³ by day 6. Treatment with anti-VEGF antibody alone produced a 42.6% reduction in tumor volume (796 ± 41 mm³, P=0.004); IR alone, 43.0% reduction (792 ± 30 mm\ p-0.006); and the combination of IR and ^Bnti-VEGF antibody, 78.0% reduction (305 * 58 mm³, p=0.001 relative to IR alone), a greater than additive effect (Table II).

To extend these findings to other models for tumors, we examined ihe effect of combining ami-VEGF an^tibody with IR in human squamous cell carcinoma and esophageal adcnocarcinoma xenografts, both of which represent human tumors for which IR is a major therapeutic modality. First, athymic nude mice bearing radioresistant human head and neck squamous cell carcinoma xenografts (SQ20B)H were treated iih IR and a neutralizing monoclonal antibody against human VEGF-165 (R & D Systems, inc.). SQ20B cells (5 x J 0⁶) were implanted in the hindlimbs of female athymic nude mice and allowed to attain a volume of 372 ± 16 mm³ (Figure 3B), after which they were treated with IR alone (40 Gy given as four 10 Gy fractions), ami-VEGF antibody alone (10 μg inlraperitoneally each day for four doses), or combined IR and anti-VEGF antibody (10 μg antibody administered 3 hours prior lo treatment wilh IR). On day 19, tumors in untreated controls reached a mean volume of 3671 ± 790 mm³. Treatment with anti-VEGF antibody alone produced a 28.5% reduction in mean tumor volume (2624 ± 287 mm³); IR alone, a 48.8% reduction (1793 ± 279 mm³); and the combination of IR and anti-VKGF antibody, a 81.8% reduction (669 * 120 mm³, p=0.003 relative to IR alone). Next, we examined a xenogra t model for a human cancer that is seldom cured by IR alone, esophageal adcnocarcinoma. Seg-1 cells^ (3 x 10⁶) _wcrc implanted in the hindlimbs of athymic nude mice und allowed to attain α volume of 407 ± 20 mm³ (Figure 3C), after which ihey were treated with IR alone (20 Gy in daily 5 Gy fractions), anti-VEGF antibody as above, or combined therapy. Similar enhancement of the anutumor effect of IR by anli-VEGF antibody was observed. As was the case for LLC, in both human xenografts, the anti-tumor effects of combined therapy were greater than additive (Table 11). These findings suggest that blocking the effects of VEGF enhances the iimoricidal effects of IR.

Blocking VEGF increases cndothellal cell killing by ionizing radiation

To assess the potential effects of VEGF on IR-mediated killing of tumor cells and endothciia] cells, we measured in vitro survival of LLCs and human umbilical vein endothclial cells (HUVECs) following exposure to IR. By MTT assay, there was no delectable cytotoxicity of LLC or SQ20B cells following exposure to VEGF or anti-VEGF antibody (duta not shown). In addition, rccombinanl VEGF failed to protect LLC or SQ20B cells from IR-mediated killing (data not shown), and there was no interactive cytotoxicity of LLC when anti-VEGF antibody was combined with IR (data not shown). Next, the effect of exogenous VEGF protein on IR-

mediated cell killing of HUVECs was assessed by MTT^ and clonogenic assays*⁹ (Figure 4). As measured by the MTT assay 96 hours after IR, pretreatment with VEGF protected HUVECs against the cytotoxic effects of 10 Gy 111 in a dose-dependent fashion (Figure 4A-NKKD p VALUES). This effect was observed both in the presence and absence of serum (data not shown). Clonogenic survival was also increased in a dosc-dcpcndent fashion when VEGF was added to the HUVEC culture medium prior lo IR (Figure 4B). Next, we tested whether adding anti-VEGF increases endothclial cell sensitivity to I . Adding anti-VEGF anUbody to the culture medium prior to IR exposure decreased cell proliferation as measured by MTT assay in HUVECs but not SQ20B cells (Figure 4C) or LLC proliferation (data no_i shown). Similar decreases in endothelial cell survival after exposure lo IR were obse,-ved in clonogcnic assays when HUVECs were pretreated with anti-VEGF antibody (Figure 4D). To determine whether VEGF protects against IR-induced apoptosis, flow cytometry studies were performed after labeling cells with 7-A AD» _{Thcrc was no dif c} ^^ ^ ^^ ^^ ^

in HUVECs exposed to IR, concentrations of anti-VEGF monoclonal antibody as high as 100 ng/ml, or both IR and anti-VEGF antibody (DATA NEEDS TO BE FINALIZED). These results indicate that IR-induced VEGF production by tumors inhibits the lethal effects of IR on endothelial cells.

DISCUSSTON Our fⁱndings demonstrate that IR induces VEGF expression by tumors. Importantly, blocking the effect of VEGF in irradiated LLC and human tumors produces greater than additive antitumor effects /„ vivo. Also, blocking VEGF action produces increased clonogenic killing of vascular endothclial cells in vitro, whereas the addition of rccombinant VEGF blocks the killing of endothclial cells. Taken together, these data raise the possibility that blocking positive regulators of angiogenesis is effective in potentiating the antitumor effects of IR. The use of growth blockade for endothelial cells (anti-VEGF antibody) _and IR _msy disrupt the paracrine relationship between the tumor and its blood supply and emphasizes the potential importance of combining an angiogenesis inhibitor with a DNA damaging agent. IR is a major therapeutic modality that is effective in the treatment of relatively .mall tumors and of large tumors only with considerable toxicity to normal tissues. Depriving the tumor endothelium of VEGF using neutralizing antibodies prior lo IR exposure or pretreating tumor vessels with antiangiogcnic peptides represent strategics to increase the anti-tumor effects of IR with minimal toxicity to normal tissues.

METHODS

Cell culture

Lewis lung carcinoma cells (gift of J. Folkman) and SQ20B cells were grown _as previously described 19.21.22. _{Hurnan urnbilical vcin endothclial} ^ _{{HUWECs) were}

maintained in EGM-2 medium (Clonetics) ₊ 1% fetal bovine scrum (Clonetics). U87 and T98 human glioblastoma cells were maintained in RPMI-1640 (Life Technologies. Inc.) + 10% FBS (In^tergen); Ul melanoma cells, DMEM (75%) + F12 (25%) + _] 0% FBS.

Neutralizing antibodies against VEGF

For experimen^ts with LLC, neutralizing polyelonal goat antibody (IgG) against recombinant mouse VEGF-164 (R & D Systems, Inc.) was dissolved in PBS and administered via intrapcritoncal injection. Conlrol mice in these experiments received nonimmune goat IgG (Sigma). For experiments with human tumor xenografts, a neutralizing monoclonal antibody to recombinant human VEGF-165 (R & D Systems) was used. Control mice in these experiments received nonimmune mouse IgG (Sigma).

Tumor models

LLC cells were injected subcutaπeously into the right hind limb (1 X 10⁶ cells in PBS) of C57BL 6 female mice (Frederick Cancer Research Institute). SQ20B human squamous cell

carcinoma cells" (1 x ,0* cells) and Seg-1 esophageal adenocarcinoma cells" (3 x 10' cells) were injected subcutaneously into the hind limb of female athymic nude mice (Frederick Cancer Research Institute). Tumor volume was determined by direct measurement with calipers and calculated by the formula (length x width x depth 2) and reported as the mean volume ± s.e.m., as previously described 19,21. _{Tumors were allowed {Q} ^ _{tø Q} ^^ _{of m}^_QQ ^ ^

which time mice were divided into experimental groups and treatment begun. Tumors were irradiated using a GE Maxitron X-ray generator operating at 150 kV, 30 mA, using a 1 mm aluminum fil^ter at a dose ra^te of 188 cGy/min.. Mice were shielded with lead except for the ^tumor-bearing right hmdlimb. The care and treatment of animals was in accordance with institutional guidelines.

Me^asur^em^en^t of VEGF levels in tumor extracts and condi_tioned media

A^t various ^time points, mice were chosen from each LLC experimental group such _tha_t the overall group mean tumor volume was affected as little as possible and euthanized to ob_tain tumor ^tissue. Tumor extracts were prepared by homogenizing tumors in RTP A buffer (150 mM Nad, 10 mM Tris, 5 mM EDTΛ, Triton -100 0.S%, and dithi₀threitol 1 μM, _PH 7.5, PMSF 50 μM, lcupcptin 1 μg/ml, and apro inin 2 μg/mi). The homogenate was then subjected to three freeze-thaw cycles in liquid nitrogen to l_yse cells and then spun at 5000 G at 4« C _lo pellet debris. VEGF levels were measured in tumor extract supernatants by ELISA (R & D Systems), and protein assays were performed by Lowry assay. VEGF levels were normalized to total extract protein concentration and expressed as pg VEGF/mg total extract protein. VEGF levels in tumor cell conditioned media were also measured by ELISA and were normalized to cell number in each well. At least three wells per time point were measured. Λ - Ao

P =

A rmur/tt "» in where P- proliferation relative to control; A= absorbance at 515 nm (Λ_s, ,); A₀- A_31J at T=0 hr, and A^eon^^βA^jw for control cells (unirradiated, grown in 10 ng/ml VEGP-165).

Clonogenic assays

Clonogenic assays were performed as previously described^. Briefly, HUVECs and LCs were plated in EGM-2 media. Eighteen hours after plating, HUVEC media was replaced with media in which the VEGF supplied by the manufacturer was omitted, and a defined amount (0-50 ng/ml) of rccombinant VEGF-165 (R & D Systems, Inc.) had been added. Four hours later, cells were irradiated with doses of 0-900 cGy using , GE Maxitron X-ray generator operating at 250 kV, 26 mA, with a 0.5 mm copper filter at a dose rale r 1 18 cGy/min. Cultures were returned to ⁱhe incubator for 14-17 days, after which they were stained with crystal violet. Colonies were counted and surviving fractions were determined. Colonies containing >50 cells were scored as positive. For .ludies with antibodies, HUVECs were plated in serum-free EGM-2 containing 5 ng ml VEGF-165. Four hours before irradiation, polyelonal antibodies to human VEGF-165 (R & D Systems, Inc.) were added to the media. Media was replaced with serum- containing media 48 hours after IR and the cells incubated for colony counting.

Data analysis

Statistical significance was determined using one-way analysis of variance (ANOVA) or Student's t-test, as appropriate.

TΛflUSS AP TABLE LEGE DS TABLF_f 1 : VEGF Levels In Lewis lung carcinoma rumors after irradiation

UNTREATED CONTROLS IONIZING RADIATION (40 Gy)

VEGF (PB VEGF (pg

Mean tumor volume VEGF/mg Mean tumor volume VEGF/mg total

Day (mm*) ± s.e.m total protein) (mm³) * g.e.m. rotein)

2 947 ± 43 69 ± 21 641 ± 22 234 ± 79 ^•

5 1545 ± 93 46 ± 18 786 ± 52 135 ± 32 * 14 6110 ± 582 90 ± 23 2854 ± 338 194 ± 47 *

*p < 0.05 relative to VEGF levels untreated controls

TABLE II: Effect of combining anti-VEGF antibody and Ionizing radiation

Tumor volume (% untreated control volume for untreated controls)

Expected for Observed

Ionizing Anti-VEGF combined If volume for Observed;

Tumor ay radiation antibody additive combined CXDCCtcd

LLC 6 57.0% 57.4% 32.7% 22.0% 0.673

Seg-1 13 19.8% 77.8% 15.4% 6.9% 0.448

SQ20B 19 51.2% 71.5% 36.6% 18.2% 0.497

Northern blots

Total RNA was isolated from cultured cells and tumor tissue using the _βuanidinc thiocyanate method23 _{utilizing Trizol Ls (Lifc} ^^ ^ _{25 μg} ^ ^ ^ ^.^

on 1.2% agarose gels containing formaldehyde and blotted onto nylon membranes, then hybridized with a cDNA probe labeled by random priming and consisting of a cDNA encoding

human VEGF** Hybridizations were carried out at 60* C in 0.5 M sodium phosphate (pH 7.0),

7% sodium dodecyl sulfate, 1 mM EDTA, and 1 % bovine serum albumin^, and blots were washed as previously escribed. _{After autoradiθβraph>}, _h]ot£ ^ ^.^ ^^ ^

rehybridized to a labeled cDN A encoding rat glyceraldchyde 3-pho.ph.te dchydrogcnasc (GΛPDH) to demonstrate message integrity.

MTTΛssays

HUVECs were plated (1 x 10¹ cells/well in 96 well plates) in EGM-2 media and allowed to attach overnight. Media was replaced with EGM-2 media containing different concentrations of recombinant human vmV- 165 (R & D Systems, Inc.). In odier experiments, the concentration of VEGF-165 was kept constant and varying concentrations of either a neutralizing polyelonal or monoclonal anti-human VEGF-165 antibody (R & D Systems, Inc.) were added prior to treatment with IR. 72 or 96 hours after IR, cells were pulsed with 3-[4. 5-

dimethylthiazol-2yl]-2, 5-dipheny!tetrazolium bromide (Sigma Chemical Company)18 _{at 0}.5 mg^/ml culture volume for four hours, after which the media was removed and the dye solubilized in dimethyl sulfoxide. Absorbance was measured at 515 nm and normalized to untreated control cells by the following equation: KEFKRENΓIΓK

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Claims

What is claimed:

1. Use of a substance that inhibits VEGF expression of blockers VEGF activity in vivo in the preparation of a medicament for the mediation of radio resistance or chemotherapy resistance in a human cancer patient.

2. The use of claim 1, wherein said substance is an anti-VEGF antibody.