Synergy of ARCA and Conventional Therapy

Chemo Secrets From a Breast Cancer Survivor

Breast Cancer Survivors

Get Instant Access

Although conventional cancer therapies (surgery, chemotherapy, and radiation) are effective at curing early-stage disease, few human cancers are curable with a single modality. The utilization of a replication-competent cytolytic adenovirus as a therapeutic modality shows much promise. In our laboratory and clinical trials, CV706 and CV787 have been shown to be effective against human prostate tumors [21-23]. However, single-dose total eradication of human LNCaP xenografts in our animal model required as much as one-third of the lethal dose of virus alone [22, 75]. To set the bar for efficacy high, namely single-dose efficacy, is deliberate. While multiple doses always allowed total eradication of tumors with lower doses, in the clinic the patient immune response will tend to limit the number of doses that can be effectively delivered despite the SIAPA technology described below. Certainly, strategies to improve the efficacy of these viruses are desirable. However, in preclinical experimental animal models, it can be readily appreciated that genetic engineering allows creation of many different viruses with an associated rationale for why the new mutant may be an improvement with greater efficacy over its predecessor. Experimentally it is impossible to compare different viruses for their efficacy in animal models unless a standardized treatment regimen is used. Thus, we use only single-dose treatment regimens in our animal studies. Small changes in efficacy do not warrant replacing current clinical candidates; large changes in efficacy are most interesting. The largest changes in efficacy we have seen came with the addition of the E3 region and the synergy afforded by radiation and chemotherapy.

We studied neoadjuvant therapy consisting of combining ARCA with conventional therapy for additional reasons as well: (1) combinations of agents with different toxicological profiles can result in increased efficacy without increasing overall toxicity to the host; (2) overlapping resistance between ARCA and conventional therapeutics has not been described previously and a combination of agents may thwart the development of drug resistance; and (3) tumor cell populations have different drug sensitivity profiles, which allow the physician to take advantage of possible synergies between drugs, resulting in increased anticancer efficacy in patients.

A. Synergy of CV706 and Irradiation

Radiation therapy, either administered through external beam or seed implantation (brachytherapy), is one of the most widely used therapeutic modalities for treatment of clinically localized prostate cancer [76, 77]. However, nearly 30% of patients treated with potentially curative radiation doses suffer relapse as defined as three serial rises in serum PSA levels (biochemical failure). Specifically, the eradication of locally advanced or high-risk prostate cancer with radiation has proven more difficult than believed previously. The gains with high radiation dose have been modest and fraught with significant side-effects [78]. It is apparent that there is a need for novel methods of radiosensitization. We have examined the effects of a combination treatment involving CV706 and irradiation for localized prostate cancer. We have demonstrated a very high synergy when irradiation treatment is complemented with CV706.

Initial in vitro experiments demonstrated a synergistic cytotoxicity to LNCaP prostate cancer cells when CV706 was combined with radiation. LNCaP cells treated with CV706 and radiation had significantly decreased viability compared to cells treated with either agent alone. LNCaP cells treated with radiation exhibited a significantly greater burst size of CV706 (m.o.i. = 0.1) with more than 100 times more virus produced per cell. In addition, the combination treatment of CV706 with radiation did not alter the specificity of replication-mediated cytotoxicity.

A similar synergistic anti-tumor efficacy of combination therapy with CV706 and radiation was also observed in vivo in prostate LNCaP xenografts. Previous in vivo studies demonstrated that prostate tumors were completely eliminated within 6 weeks by a single intratumoral administration of CV706 at a dose 5 x 108 particles per mm3. Combination treatment of CV706 at a dose of 1 x 107 particles per mm3 with radiation 10 Gy eliminated tumors within 6 weeks. Under similar conditions CV706 or radiation alone could only slow down or inhibit further tumor growth (Fig. 2). Thus, a 50-fold lower dose of virus (1 x 107 particles) can be used in the combination treatment modality to achieve the same curative effect. Statistical analysis of the in vivo studies indicated that CV706 and the radiation combination group showed a significant anti-tumor synergy with a 1.5- to 6.7-fold higher inhibition of tumor

0 1 2 3 4 5 6

Weeks

  • Vehicle CV706 (dO) and EBRT (d 1 ) j
  • CV7Q6 (1x107p/mm3) (dO)

Figure 2 In vivo efficacy of intratumorally administered CV706 and radiation against nude mouse LNCaP xenografts. Tumor volume of LNCaP xenografts treated with either vehicle (diamond), CV706 alone (1 x 107 particles per mm3 of tumor, circle), radiation alone ( 10 Gy, triangle), CV706 ¡1 x 107 particles per mm3 of tumor) at day 0 plus radiation (10 Gy) at day 1 (close square), and radiation at day 0 plus CV706 at day 1 (open square).

growth over the additive effect during the entire treatment period. Subsequent studies have shown that a synergistic antitumor efficacy could be achieved when 1 x 107 particle CV706 was combined with only 5 Gy radiation (unpublished data, Chen et al.). Further in vivo studies are in progress to determine the effective minimum dose of CV706 in combination with radiation required for complete regression of tumors.

A series of experiments was then designed to examine the effects of the sequencing of the agents, the timing of radiation following virus administration, and radiation fractionation. Efficacy was highly dependent on the sequencing of the agents; treatment with CV706 24 h prior to radiation was significantly superior to radiation followed by CV706. The antitumor activity was decreased when the tumors were treated with radiation 7 days after CV706 administration. However, there was no significant difference in antitumor efficacy when the tumors were treated with CV706 followed by a single dose of radiation or four sequential daily fractional doses of radiation as long as the total does of radiation was the same.

A preliminary assessment of synergistic activity in the CV706 and radiation combination treatment reveals several mechanistic possibilities. First, radiation at the synergistic dose significantly increases virus replication. One-step growth curve study shows that although synergistic doses of irradiation did not alter virus replication kinetics, the irradiation significantly increases the burst size of CV706 in LNCaP cells. For example, burst size of CV706 in LNCaP cells treated with CV706 for 24 h followed by irradiation is 500-fold higher than that in cells treated with CV706 alone (0.01 m.o.i.). Irradiation kills mammalian cells in the reproductive (also known as clonogenic) death pathway, and breaks down DNA strands. Most radiation induced DNA double-stranded breaks are rapidly repaired by constituitively expressed DNA repair mechanisms [79]. DNA repair machinery becomes more active in irradiated cells, thus allowing for greater replication/multiplication of the episomal adenoviral DNA. Because of its small target size, the adenoviral genome (36 kb) is far less likely to sustain radiation-induced damage as it is 105-fold smaller than that of human cells (3 x 106 kb). Therefore, the more active cellular DNA synthesis machinery in the irradiated cells may facilitate viral DNA synthesis and virus replication. Second, CV706 may be augmenting the anti-tumor activity of radiation. The adenovirus El A gene is the only viral gene expressed during the first 2.5 h of infection and encodes a multifunctional transcriptional factor also known to induce apoptosis [32, 80]. It is believed that the adenovirus E1A gene is a potent inducer of radiosensitivity through p53-dependent and -independent mechanisms. Malignant tumors, when expressing adenovirus E1A, are very sensitive to treatment with DNA-damaging agents in vivo, including irradiation [81, 82]. In the tumors treated with CV706 and radiation, the histological features included intravascular thrombosis and massive necrosis positioned more centrally within tumors. This supports a growing concept for the involvement of an effect on the destruction of the vasculature leading to tumor reduction and elimination. This seems to be in agreement with a recent finding that inhibition of angiogenesis led to increased tumor radiosensitivity [83]. Indeed, CV706 in combination with radiation exerted a significant reduction of CD31 positive blood vessels, indicating an anti-angiogenesis effect of the combination treatment [84, 85].

As human tumors are composed of a mixture of cells having different genetic makeup, this inherent intratumoral heterogeneity may need treatment with multiple therapeutic modalities. As demonstrated here, radiation can be successfully combined with cytolytic adenoviral therapy. The advantages are as follows: the combination of CV706 with irradiation still limits the damaging effects of CV706 to the irradiated cells; the combination of CV706 with irradiation leads to significantly increased necrosis, marked decrease of blood vessel number, and inhibition of angiogenesis; The combination of CV706 with irradiation allows at least a 50-fold reduction in the amount of virus to achieve the same curative effect; animals receiving combination treatment appear healthier, characterized by a significant weight gain compared to the groups treated with either agent alone. Thus, combining cytolytic adenoviral therapy with radiation may augment the efficacy of standard cancer modalities.

B. Synergy of CV787 and Chemotherapy

Once prostate cancer enters a metastatic stage, the current treatment is androgen ablation therapy, which in 70% of men provides relief from otherwise uncontrollable bone pain and increases life expectancy by 6-18 months [86, 87], For many years, chemotherapy was felt not to play any role in the treatment of advanced prostate cancer. However, this negative impression is apparently changing, as significant activity is being seen with new drugs and drug combinations [87], Interim analysis of an ongoing Phase III trial of docetaxel in end-stage hormone refractory prostate cancer patients shows an improvement in life span to 30 months (Dan Petrylak, pers. commun.).

A synergistic antiproliferative effect in LNCaP prostate cancer cells was observed when CV787 was combined with various chemotherapeutic agents including cisplatin (Platinol), docetaxel (Taxotere), doxorubicin (Adri-amycin), estramustine (Emcyt capsules), etoposide (VePesid), gemcitabine HC1 (Gemzar), mitoxantrone (Novantrone), and paclitaxel (Taxol). In this chapter, we will discuss the synergistic antitumor efficacy of CV787 only when combined with the taxanes.

In vitro experiments demonstrated that LNCaP cells cultured with paclitaxel or docetaxel for 24 h before or after infection with CV787 had significantly lower rates of proliferation than cells treated with either agent alone [88]. Also, LNCaP cells exhibited a greater burst size of CV787, whereas no significant effect on viral growth kinetics was seen. No significant difference in the effectiveness of the combined therapy of taxane and CV787 infection was observed by varying the time of taxane administration in vitro, whereas varying the administration schedule of paclitaxel with Ad-p53 gene therapy can modulate the synergistic activity between these two agents in ovarian cancer [89]. Furthermore, in vitro combination treatment of CV787 with taxane did not alter the specificity of replication-mediated cytotoxicity. CV787 has been shown previously to replicate preferentially in PSA-producing human prostate cancer cells 10,000 times more efficiently than in non-PSA-producing cells [22]. In the presence of paclitaxel or docetaxel in the culture medium, CV787 replicates to the same degree of specificity in the non-PSA-producing human cell lines versus PSA+ cells. Cell viability assays with MTT further indicate that CV787 in combination with taxane remains fully selective.

The antitumor effects of combination therapy with CV787 and taxane were also evaluated in vivo. Previous studies have demonstrated that tumors were eliminated within 6 weeks by a single intravenous administration of CV787 at a dose of 1 x 10n particles [22], Clinically, docetaxel is administered intravenously at 12.5 mg/kg once per week for 6 weeks. A single cycle of docetaxel is not toxic, whereas the full course of six cycles is highly toxic. Combination treatment of CV787 and taxane (three courses of 5 mg/kg) eliminated tumors within 4 weeks, whereas CV787 alone at the same dose of 1 x 1010 particles per animal could only slow down tumor growth (Fig. 3A). The experiments described below are with a single cycle of docetaxel. In combination with the higher clinical dose of 12.5 mg/kg docetaxel, 1 x 109 particles and of 1 x 108 particles of CV787 led to a complete elimination of tumors within 4 weeks. Thus, the dose of CV787 required for complete remission has been reduced by 1000 times from 1 x 10n particles to 1 x 108. The LDo and LDioo single bolus dose of CV787 for Balb/c, nu/nu is 1.0 x 1011 particles and 2.5 x 1011 particles, respectively. Thus, the single-dose combination of docetaxel with CV787 has increased the potential curative therapeutic window, from 1 to 1000. Furthermore, healthier animals, characterized by body weight, were observed in the combination treatment group as compared to groups treated with either agent alone (data not shown).

The mechanism(s) of synergistic activity in the combination of taxane with CV787 is unknown at this time; however, our experiments suggest a few hypotheses. First, taxane at the synergistic dose may be augmenting viral replication. It has been previously shown that a low concentration of paclitaxel (1-14 nM) increased the number of cells transduced by recombinant adenovirus 3-35% in a dose-dependent manner [89]. Recently, it was found that taxane increases intracellular receptor trafficking. For example, more Coxsackie and adenovirus receptor (CAR) moved to outer membrane in the paclitaxel-treated cells (Nielsen Loretta, pers. commun.). It is suggested that adenovirus transduction efficiency may increase after the cells are treated with taxane. Indeed, our data show that although the synergistic dose of paclitaxel

Weeks After Treatment

-♦-Control -A- CV787 (1x1010p) CV787 (1x1011p) -*- Docetaxel {5 mg/kg) -«- CV787/Docetaxel

B Docetaxel CV787 CV787/Docetaxel

Figure 3 In vivo efficacy of intravenously administered CV787 and faxane against nude mouse LNCaP xenografts. (A) LNCaP tumor volume in mice treated with either vehicle, CV787 (1 x 1010 1 x 1011 particles per animal, iv), docetaxel (5 mg/kg at day 2, 5 and 8, iv), or CV787 (1 x 1010 particles per animal) and docetaxel (5 mg/kg at days 2, 5, and 8, iv), (n = 6). Tumor volumes measured weekly. Error bars represent the standard error of the mean. (B) Histological features (H&E staining) of LNCaP tumors treated with either docetaxel (12.5 mg/kg) (left), CV787(1 x 10'° particles per animal) (middle), and CV787 (1 x 1010 particles per animal) plus docetaxel (12.5 mg/kg) (right) at x 400 original magnification. LNCaP xenografts were harvested 14 days after treatment. The open arrow indicates cells with abnormal mitosis, the filled arrow indicates necrotic cells.

Figure 3 In vivo efficacy of intravenously administered CV787 and faxane against nude mouse LNCaP xenografts. (A) LNCaP tumor volume in mice treated with either vehicle, CV787 (1 x 1010 1 x 1011 particles per animal, iv), docetaxel (5 mg/kg at day 2, 5 and 8, iv), or CV787 (1 x 1010 particles per animal) and docetaxel (5 mg/kg at days 2, 5, and 8, iv), (n = 6). Tumor volumes measured weekly. Error bars represent the standard error of the mean. (B) Histological features (H&E staining) of LNCaP tumors treated with either docetaxel (12.5 mg/kg) (left), CV787(1 x 10'° particles per animal) (middle), and CV787 (1 x 1010 particles per animal) plus docetaxel (12.5 mg/kg) (right) at x 400 original magnification. LNCaP xenografts were harvested 14 days after treatment. The open arrow indicates cells with abnormal mitosis, the filled arrow indicates necrotic cells.

or docetaxel did not alter virus replication kinetics, the chemotherapy drugs slightly increased the burst size of CV787 in LNCaP cells. Second, CV787 may be augmenting the anti-tumor activity of taxane. E1A gene expression has been shown to increase cellular sensitivity to chemotherapeutic agents [90] and this enhanced sensitivity is partially caused by the induction of p53-dependent apoptosis by the ElA-induced sensitization of the cells [91]. Recently, Ueno et al. found that human ovarian cancer cells that were originally resistant to paclitaxel, became paclitaxel sensitive in E1A downregulated HER-2/neu cells [66]. In CV787, the E1A gene is intact and may be overexpressed in PSA-producing LNCaP cells, which may enable significant interaction between El A and taxane. In addition, E1A is a potent inducer of p53 protein expression in infected cells [92], p53 levels may increase following infection, thereby increasing cell sensitivity to chemotherapy-induced apoptosis. This is consistent with the observation that more apoptotic cells were seen in the LNCaP tumors that received combination treatment than in tumors that received either agent alone (Fig. 3B). Interestingly, in the combination-treated tumors, most of the cells were empty and virtually devoid of cellular content. Finally, the actions of the two agents may be occurring at two distinct points in the same apoptotic pathway, analogous to the activity of cyclosporine A and rapamycin on two distinct points in the T-cell activation signal transcription pathway [93]. Further investigation of the possible mechanism(s) of synergistic activity in the combination of taxane with CV787 is under way.

Was this article helpful?

0 0
10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook


Post a comment