Toxin-Based Cancer Gene Therapy

C H A P T E R

8 Toxin-Based Cancer Gene Therapy Under the Control of Oncofetal H19 Regulatory Sequences Patricia Ohana1, Imad Matouk, Doron Amit, Michal Gilon and Abraham Hochberg Hebrew University of Jerusalem, Jerusalem, Israel

K EY W O R D S DTA-H19 plasmid; H19; oncofetal; cancer; transcriptional targeting; diphtheria toxin; tumorspecific promoter; hypoxia

Abstract Specific targeting of cancer cells while sparing normal cells with therapeutic mediators is a critical advantage over the current conventional drugs. Although various strategies can potentially be followed to achieve this vital goal, this review focuses on the strategy of transcriptional targeting of tumor cells by employing a toxin gene. Tumor-specific regulatory sequences can be used for this purpose to drive the expression of a toxin specifically in tumor cells. It is of immense importance to choose regulatory sequences of a gene that is playing critical roles in various aspects of tumorigenesis to increase the therapeutic window. It would be beneficial to identify a strong cancer-specific promoter for treating a wide range of cancers while having minimal toxicity. In light of these prerequisites, we present our experience with the H19 gene and the successful use of its regulatory sequence to drive the expression of diphtheria toxin A in both preclinical and clinical studies.

INTRODUCTION Reviewing brochures on the conventional cancer drugs in use today is undoubtedly a horrible and terrifying event. Seldom is any vital system in the body unaffected by a cancer drug, and sometimes they are seriously affected. Lack of tumor specificity is mostly responsible for these adverse events, which cause the

therapeutic dose to be greatly limited. With few exceptions, these conventional therapeutic regimens also have narrow therapeutic windows; this is especially the case for locally advanced malignancies and for those that metastasize. Chemotherapy has resulted in a very modest survival gain and has not demonstrated a major impact on the downhill course of most common malignancies. Residual tumor cells that bypass even radical combinations of surgery, chemotherapy, and radiotherapy can lead to relapse, with an even more aggressive behavior. Moreover, the issue of the multidrug-resistant phenotype that some cancer cells acquire is a major obstacle [1]. This frustrating state-of the-art behooves us to double our efforts in the search of innovative treatment avenues. During the past decade, cancer gene therapy has been one of the most exiting areas of therapeutic research, and some research has led to advanced stages of clinical trials with promising outcomes. The clearest advantage for this targeted therapy approach is that normal tissue toxicity may be avoided if suitable targeted delivery and tumor-specific expression strategies are employed. The issue of very efficient targeted delivery, which is ultimately needed for successful cancer therapy, still poses a major challenge, and enormous effort has been made to overcome this challenge. Major advancements have been achieved with regard to the tumor-specific expression strategy, also called transcriptional targeting, as a result of great efforts to identify genes that are expressed specifically in tumors and marginally in normal cells. Different strategies to improve and design tumor-specific

1

The first and second authors contributed to this work equally.

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promoters have been reported [2]. In this approach, the accumulated understanding of gene regulation has been exploited to target the diseased cells successfully with the therapeutic gene expression. Other approaches for targeting tumor cells specifically have also been developed. The transductional targeting approach involves the genetic modification of viral vectors to enhance its tropism to achieve selective delivery of transgenes to target cells, and it may also result in cancer cell lyses when using oncolytic viruses. To achieve maximal specificity, transcriptional targeting may be combined with transductional targeting. Targeted therapy also involves small-molecule drugs and monoclonal antibodies. In the latter case, toxic molecules can be delivered specifically to tumor cells through monoclonal antibodies by exploiting our knowledge of the unique antigens that are expressed on tumor cells [3]. Moreover, cancer gene therapy approaches can be administered in combination with chemotherapy and radiotherapy. This chapter focuses on the successful use of H19 gene regulatory sequences for transcriptional targeting of cancer cells. The heterologous H19 promoter is transactivated by the same transcription factors and complexes that drive its transcriptional upregulation in the course of malignancy. The next step after determining high expression levels of H19 in numerous types of cancer was to characterize H19’s regulatory sequences. In our group, cloning of different fragments of the human H19 promoter was done and enabled the examination of their transcriptional activity in a variety of cancer cell lines. The region located between 2229 and 2134 bp contains a sequence element(s) with a positive transcriptional activity. The region between 85 and 261 bp is rich in CpGs, and it plays a significant role in the regulation of H19 transcription. This region contains transcription factors binding sites, including the CCATT box, which binds transcription factors from the C/EBP family. The sequence region between 285 and 2134 bp acts as a binding site for negative acting transcription factor(s) in carcinoma cells [4]. Therefore, expression of the therapeutic toxin—in our case, diphtheria toxin A driven by H19 promoter— is predicted to occur in a manner specific for tumor expressing or overexpressing H19 gene product. This approach has been applied in clinical trials in patients of bladder, ovarian, colon, liver metastases and pancreatic carcinomas with promising outcomes [5]. Preclinical validation of this approach is under development for other types of human carcinomas. Therapies that target the H19 RNA or that exploit its transcriptional regulatory element to drive the expression of a cytotoxic gene specifically to tumor cells would therefore be very valuable in treating

tumors that have a high level of H19 RNA. However, the potential of this transcriptional targeting approach might be even greater if two different regulatory sequences, selected from the cancer-specific promoters H19, IGF2-P3, and IGF2-P4, are utilized in the same therapeutic construct because many tumors that lack H19 gene expression have been found to express IGF2. In a second approach, a single promoter may be used to drive two cytotoxic genes having synergistic effect on a single construct. Both approaches have shown superior tumor growth inhibition activity in preclinical studies of bladder cancer [6 8].

H19 AND TUMORIGENICITY Understanding the function of the H19 gene is crucial if we hope to uncover its roles in cancer and harness this knowledge for therapeutic benefit. The H19 gene was the first imprinted noncoding RNA gene to be discovered [9]. It is highly expressed in most fetal organs [10] but repressed immediately after birth [11]. Regarding its role in tumorigenicity, accumulating data from our group and others support the oncogenic function of H19 RNA, although several groups have reported H19 as having a tumor suppressor activity [12]. The H19 locus harbors the potential to produce various products that may account for this discrepancy. From H19 exon 1, miR675 is produced [13]. However, this microRNA is predicted to have a tumor-promoting role by targeting retinoblastoma tumor suppressor [14]. Moreover, a short-lived, approximately 120-kb-long antisense transcript called 91H is produced at the imprinted H19/IGF2 locus, overlapping the H19 gene. 91H transcript is stabilized in breast cancer cells and overexpressed in human breast tumors [15]. Recently, an antisense transcript encoded by H19 has been identified and called HOTS (H19 opposite tumor suppressor); it is a protein coding transcript and has a probable tumor suppressor function [16]. H19 RNA is emerging as one of the key players in cancer biology. Although H19 was long ago reported to show aberrant expression in various types of human cancer, where the majority of human tumors frequently express the H19 gene, clear, causal evidence of its role in cancer has only recently come to light. H19 RNA contributes significantly to several aspects of the malignant phenotype, including proliferation, hypoxic stress response, angiogenesis, metastasis, and multidrug resistance [17,18]. Cancer cells devoid of H19 expression encounter a very significant retardation of tumor growth in vivo [19]. To shed light on H19’s mechanism of action, which remained obscure until recently, we and others utilized

I. VIRAL AND NONVIRAL VECTOR METHODOLOGIES: TARGETS AND APPROACHES

TARGETING TOXINS AND SUICIDE GENES IN GENE THERAPY

a strategy of identifying upstream effectors and also downstream targets by applying the global gene expression profiling to identify genes modulated by both H19 overexpression and knockdown. Consequently, our group and others identified numerous modulators of H19 gene expression, including chemical carcinogens (e.g., BBN and DEN); retinoic acid; steroid and peptide hormones; growth factors such as hepatocyte growth factor; transcription factors such as E2F, HIF1α, p53, and c-myc; and environmental factors such as hypoxia and serum starvation. Because a complete report is beyond the scope of this concise review, for in-depth discussion, readers are referred to references [17,18,20]. Here, we discuss principal findings. The identification of molecular mechanisms used to mediate the hypoxic cancer cellular response is of great interest to identify targets that might compromise the survival of hypoxic cells. Hypoxia is a major trigger for tumor angiogenesis, invasiveness, metastasis, chemoresistance, radioresistance, and loss of genomic stability. It is also associated with poor prognosis in some types of human cancers. Notably, most of these conditions are associated with an increase in the level of H19 RNA. Recently, we uncovered a molecular mechanism that integrates H19, p53, and HIF1-α to hypoxic stress response [21]. We demonstrated a tight correlation between H19 RNA elevation by hypoxia and the status of the p53 tumor suppressor. p53 suppresses H19 elevation in hypoxic cancer cells. Furthermore, we identified HIF1-α as the factor responsible for H19 elevation under hypoxic stress in the tumor cells in which p53 is mutated [21]. Consequently, H19 functions as a stress modulator and a survival factor, and it is involved in several fundamental processes, including epithelial mesenchymal transition (EMT), malignant transformation, cell cycle transition, metastasis, and neo-angiogenesis [19,22]. EMT is an important process on the way to the malignant phenotype; notably, H19 upregulation occurs in the stroma as well as in the epithelium [23]. In the metastatic tumor stage, which bears a striking similarity to the embryonic stage, H19 involvement appears to be essential: Adherent and cohesive cells lose their anchorage, migrate under stressful conditions to remote sites, and replicate with neovascular support. Thus, H19 seems to occupy a central role in the cancer embryonic shift [18]. Because hypoxia readily occurs in the majority of solid tumors driving critical steps in tumor development and metastasis and resistance to therapeutic modalities, placing the H19 gene product in this deadly circuit undoubtedly will have major impacts in its utility as a high-priority target for cancer gene therapy.

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TARGETING TOXINS AND SUICIDE GENES IN GENE THERAPY By definition, a suicide gene has a product that will cause a cell to kill itself through apoptosis. This product can be transcribed by a variety of promoters (e.g., constitutive promoters and tissue-specific promoters), but tumor selectivity can be approached through the utility of tumor-specific promoters. The specificity of promoter-targeted therapy can also derive from cancer-specific conditions such as hypoxia [24]. Inducible promoters affected, for example, by radiation, heat, and drugs have also been reported [25 28]. Finding a promoter that uniquely directs expression in cancer cells such as in the case of H19 is of great importance. Another example is the human telomerase reverse transcriptase (hTERT), the catalytic subunit of the telomerase—a critical factor for cell immortalization and tumorigenesis. As demonstrated by our group and others, the use of the hTERT promoter has provided targeted preclinical therapeutic results in bladder and hepatocellular carcinoma cells [29,30]. Moreover, our group used promoters of IGF2 gene for this purpose [7,31]. The main safety advantage of specific promoters therapy is sometimes accompanied by the disadvantage of weak activity, consequently resulting in a decrease in therapeutic efficacy. To improve specific but weak promoters, enhancers can be added and negative regulatory elements can be removed. For example, by insertion of four tandem copies of the synthetic androgen responsive element, a nearly 20-fold enhancement of activity over the native PSA promoter and enhancer (PSE) was achieved [32]. In the case of H19, this promoter has the great advantage of its specificity to cancerous cells and at the same time demonstrates strong promoter activity similar to SV40. Targeted therapy also involves small-molecule drugs and monoclonal antibodies. Monoclonal antibodies that recognize, for example, the CD20 molecule (tositumomab and 131I-tositumomab (Bexxar), and ibritumomab tiuxetan (Zevalin)) and CD30 molecules (brentuximab vedotin (Adcetris)) linked to toxic molecules have been approved by the U.S. Food and Drug Administration (FDA).

Toxin Gene Therapy Toxins have the ability to kill cells efficiently; thus, many toxins have been examined as potential anticancer agents. Targeted fusion toxins consist of a targeted protein such as a growth factor fused to a bacterial toxin such as diphtheria toxin. The fused toxin is directed to the tumor cells via the targeting molecule,

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directed into the cells through receptor endocytosis, and then the toxin is released, resulting in tumor cell death. Diphtheria Toxin A Chain Diphtheria toxin (DT) is one of the most studied molecules, demonstrating compelling activity as a suicide gene therapeutic reagent. It efficiently ADPribosylates elongation factor-2 (EF-2) and thus blocks the translational machinery of target cells. It is estimated that a single molecule of diphtheria toxin can kill target cells, and many studies have successfully used its toxicity to eradicate target cancer cells. Diphtheria toxin is secreted from Corynebacterium diphtheriae as a single polypeptide chain containing two major domains: DT-A, which carries the active site for ADP ribosylation of EF-2, and DT-B, which promotes binding of toxin to cells and the entry of the A chain into the cytosolic compartment. Although a very low level of DT-A expression suffices for cell killing, DT-A released from the lysed cells is not able to enter the neighboring cells in the absence of the DT-B chain The advantages of DT-A in gene therapy are described in the literature and include (1) high potency, with one molecule able to kill a cell; (2) independence of cell cycle and p53 status; (3) toxic effect localized to transfected cells because the DT-B chain, which is responsible for cell penetration, is absent; (4) bypassing of anti-DT immunity because DT-A protein is being produced endogenously via an expression cassette within the tumor cells (to escape neutralization by anti-DT antibodies that are ubiquitous in most people during systemic administration); and (5) the absence of cellular resistance to the toxin. All of these characteristics make the DT-A chain a very effective and thus frequently used component of targeted cancer therapeutic approaches including immunotoxins (protein conjugates of DT-A combined with either an antibody or a cytokine to specifically target delivery to cancer cells) and in gene therapy studies [33] (Table 8.1).

Other Toxins Plant-derived ricin and pseudomonas exotoxin use a similar mechanism as that of diphtheria toxin to kill target cells, and they have been examined as effective anticancer reagents [34,35]. As a selective pancreatic cancer suicide gene strategy, the use of suicide gene Escherichia coli purine nucleoside phosphorylase (ePNP) under the control of either CEA or MUC1 promoter sequences showed a preferential killing of CEA- and MUC1-producing pancreatic tumor cells [36].

In a comparative survival study, Rodriguez et al. showed the relative potencies of the following eight recombinant cellular toxins for irreparable prostate cancer cell death: Pseudomonas exotoxin A, ricin, tumor necrosis factor-α (TNF-α), diphtheria toxin (DT), Crotalus durissus terrificus toxin, Crotalus adamenteus toxin, Naja naja toxin, and Naja mocambique. Dosedependent cytotoxic activity against all human prostate cancer cell lines tested was only identified as highly potent for ricin and DT. TNF-α had modest cytostatic activity in the screen; however, the combination of TNF-α and DT resulted in marked acceleration of the time to prostate cancer cell death [37]. Our group demonstrated the cytotoxic effect of TNF-α cytokine, together with the diphtheria toxin, in the therapy of ovarian cancer. Intratumoral injection of the toxin vector into ectopically developed tumors caused 40% inhibition of tumor growth [38].

Suicide Gene Therapy Enzyme Prodrug Systems Chemotherapeutic suicide gene therapy approaches are known as gene-directed enzyme prodrug therapy. Suicide gene therapy approaches using deactivated drugs are known as gene-directed enzyme prodrug therapy (GDEPT) or gene-prodrug activation therapy (GPAT). GDEPT utilizes a gene encoding a foreign enzyme delivered to the tumor, after which a prodrug is administered and activates a cytotoxic drug that has been expressed in the tumor. Three of the most promising suicide gene/prodrug combinations are (1) herpes simplex virus thymidine kinase (HSV1-TK) with ganciclovir (GCV), (2) cytosine deaminase (CD) with 5-fluorocytodine (5-FC), and (3) bacterial nitroreductase (NTR) with 5-(azaridin-1-yl)-2,4-dinitrobenzamide (CB1954) [39]. Enzyme prodrug systems kill the targeted cancer cells by interfering with the DNA replication or transcription processes. Moreover, the toxic substances produced by the combinations mentioned previously can spread to the neighboring cancer cells and induce consecutive cell death (the bystander effect). The two possible drawbacks of these enzyme prodrug systems are that there is a prominent bystander effect and these systems tend to be less effective against cancer cells that are not actively dividing [40]. Another example of GDEPT is the CPG2 CMDA system. Kirn et al. developed a suicide gene therapy based on the bacterial enzyme carboxypeptidase G2 (CPG2). CPG2 has the advantage over the well-studied suicide genes HSV-TK and CD in that it activates

I. VIRAL AND NONVIRAL VECTOR METHODOLOGIES: TARGETS AND APPROACHES

TABLE 8.1 The Use of DT-A in Gene Therapy Full Name

Promoter/Immunotoxin/ Protein

BC-819

Target Tumor Type

Summary

References

H19

Bladder, ovarian, pancreas, colon liver metastases

Drives the expression of the therapeutic protein, the DTA chain. It has been used in both animals and humans.

[5,8,42,43,47,49]

hTERT-DTA

hTERT promoter

Bladder and hepatocellular carcinoma cells

Expression vectors containing the DTA gene were linked to hTER and hTERT transcriptional regulatory sequences, and inhibition of protein synthesis occurred in bladder and hepatocellular carcinoma cells.

[29]

Denileukin diftitox (Ontak)

Interleukin-2 (IL-2)

Cutaneous T cell lymphoma

The drug binds to cell surface IL-2 receptors, which are found on certain immune cells and some cancer cells (expressing the CD25 component of the IL-2 receptor), directing the cytotoxic action of the diphtheria toxin to these cells. (FDA approved for use in humans).

[50]

Rad51-DTA

RAD51

The majority of human tumor cells, including those of the prostate, pancreas, breast, lung, and cervix

RAD51 is a recombinase protein essential in repairing DNA double-strand breaks and stalled replication forks by homologous recombination. Rad51-DTA/jetPEI injections treat tumors in mice with HeLa xenografts (subcutaneous and i.p. tumor).

[51]

pMSLN/DTA, pHE-4/ DTA

MSLN and HE4 promoters

Ovarian

The promoter sequences of two genes that are highly active in ovarian tumor cells, MSLN and HE4, were used to target DTA expression to tumor cells. Administration of DTA nanoparticles directly to s.c. xenograft tumors and to the peritoneal cavity of mice bearing primary and metastatic ovarian tumors resulted in a significant reduction in tumor mass and a prolonged life span compared to control mice.

[52]

PGL3-DF3-DTA

DF3 promoter

Breast cancer

Recombinant expression vector containing human breast cancer DF3 promoter and diphtheria toxin A fragment was highly expressed in human breast cancer cell line of DF3 positive, and it could kill the human breast cancer cells not only in vitro but also in vivo. Thus, it could produce a specific killing effect on human breast cancer cell line of DF3 positive.

[53]

EGF-DTA

EGF

Glioma cells

Recombinant toxin consisting of EGF fused to diphtheria toxin (DAB389EGF) effectively kills glioma cells, and this molecule is viewed as a promising agent for treating malignant gliomas primary brain cancers.

[54]

pAF-DTA

Human α-fetoprotein (AFP) promoter/enhancer

Hepatocellular carcinoma cells

DTA under the control of human AFP promoter/enhancer directed to AFP-producing hepatocellular carcinoma cells (using cationic liposomes (DMRIE-C)). After pAF-DTA transfection, the growth of AFP-positive HuH-7 cells was inhibited. Also, the growth of HuH-7 transplanted on BALB/c nu/nu mice was inhibited by i.t. injection.

[55]

(Continued)

TABLE 8.1 (Continued) Full Name

Promoter/Immunotoxin/ Protein

Target Tumor Type

Summary

References

E-selectin-DTA

E-selectin promoter

Human endothelial cells (HUVEC)

HUVEC were killed via apoptosis. Maxwell et al. proposed that delivery of transcriptionally regulated expression plasmids for DTA in vivo, using cationic lipids that show preferential accumulation in activated or proliferating endothelium, may offer a novel means of inhibiting undesired angiogenesis.

[56]

hCG-DTA

Human chorionic gonadotropin promoter

Malignant ovarian cell lines

DTA-chain gene regulated by the hCG promoter directed to malignant ovarian cell lines demonstrated the preferential expression of the DTA and provides an avenue for targeting such cells for suicide, toxin, or cytokine genes.

[57]

Mesothelin-DTA

Mesothelin

Pancreatic cancer cell lines

Mesothelin is specifically overexpressed in pancreatic cancers and not in the adjacent normal tissue. Showalter et al. sought to target mesothelin-expressing pancreatic cancer cells with a potent suicide gene, DT-A. This work achieved dramatic inhibition of protein translation ( . 95%) in mesothelin-expressing pancreatic cancer cell lines.

[58]

pPSA-DTA

Degradable, poly(β-amino ester) polymer, poly(butane diol diacrylate co-amino pentanol) (C32), DTA driven by a prostate-specific promoter

Benign prostatic hyperplasia (BPH) and prostate cancer

Using a degradable, poly(β-amino ester) polymer, C32, DTA driven by a prostate-specific promoter was delivered to cells and the same C32/DTA nanoparticles were directly injected to the normal prostate and to prostate tumors in mice. Nearly 50% of normal prostates showed a significant reduction in size, attributable to cellular apoptosis. These results suggest that local delivery of poly(β-amino ester) polymer/DTA nanoparticles may have application in the treatment of BPH and prostate cancer.

[59]

i.p., intraperitoneally; i.t., intratumorally.

NONCLINICAL PHARMACOLOGY

prodrugs that are able to kill quiescent as well as proliferating cells. CPG2 cleaves the prodrug CMDA such that its cytotoxic drug is directly released and has the advantage that no further enzymatic processing is required for drug activation [41]. As mentioned previously, an important aspect of toxins/suicide gene therapy is the bystander effect, defined as the secondary effects on adjacent cells and tissues triggered by treatment of a primary target with a therapeutic agent. Such negative effect can be seen in this prodrug plasmid and virotherapy, which can affect the immune system. Thus, studies have been conducted using a polymer/biomaterial that mimics a virus but is safer as a delivery agent. However, it is important to note that whereas these plasmid prodrugs have significant bystander effect, DT-A—due to its properties as a toxin—is unable to re-enter a neighboring cell, has no bystander effect, and hence its role in gene therapy is significant.

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NONCLINICAL PHARMACOLOGY Nonclinical studies (in vitro/in vivo) have been performed to demonstrate the potential efficacy of BC-819 in the different targeted cancers (bladder cancer, pancreatic cancer, ovarian cancer, and hepatic metastases) having upregulated levels of H19 expression. In vitro pharmacology studies started by analyzing the expression of the H19 gene in patients’ tumor tissues and in human tumor cell lines. Then, the selective expression of a reporter gene (luciferase) and/or the DT-A chain controlled by the H19 gene promoter was assessed in vitro at different human cancer cell lines expressing H19. Finally, the efficacy of BC-819 in terms of protein synthesis inhibition was assessed in tumor cell lines of various origins. Once a proof of concept of efficacy was obtained in vitro, in vivo nonclinical studies were conducted in several animal models with BC-819 administered as a naked DNA (for ovarian, hepatic metastases and pancreatic cancer) and in a complex suspension with PEI (for bladder cancer).

EXPERIMENTAL STUDIES OF DTA-H19 The investigational agent BC-819, also referred to throughout this chapter as DTA-H19, is a doublestranded DNA plasmid of 4560 bp in length that carries the gene for the DT-A chain under the regulation of the H19 promoter sequence. This is a targeted cancer therapy because DT-A chain expression is triggered by the presence of transcription factors that upregulate H19 RNA expression in tumor cells. The selective initiation of toxin expression results in selective tumor cell destruction via inhibition of protein synthesis in the tumor cell, enabling highly targeted cancer treatment. DTA-H19 is being developed for the treatment of cancers that have upregulated levels of H19 expression. The first four indications under development are transitional cell carcinoma (TCC) of the bladder, pancreatic cancer, ovarian cancer, and hepatic cancer (either primary hepatic cell carcinoma or hepatic metastases of other primary cancers). DTAH19 is administered intravesically into the bladder of TCC patients after mixing it just prior to intravesical instillation with the cationic transfection agent polyethyleneimine (PEI) (in vivo jetPEI) to enhance in vivo transfection efficiency. For other indications cited previously, DTA-H19 is given by local/regional routes of delivery (intratumorally, intraperitoneally, or by hepatic artery infusion (HAI)), and in these cases it is not previously mixed with PEI but injected as naked DNA. In all clinical trials, patients’ tumors must be tested and be positive for H19 RNA in order to be eligible for treatment.

In Vitro Nonclinical Pharmacology Bladder Cell Lines The cell-specific activity of the DT-A chain driven by the H19 promoter region was demonstrated in human bladder carcinoma cells (T24P) and human fibroblast cells (IMR-90). There was dose-dependent inhibition of luciferase expression (the reporter gene) in T24P cells starting at 0.1 μg of co-transfected BC-819 and complete inhibition with 1.0 μg. This demonstrated that luciferase production was inhibited in cells by the expression of the DT-A chain under the regulation of the H19 promoter only in cells that express H19 RNA (i.e., T24P bladder cancer cells) and not in cells that do not express H19 RNA (i.e., the IMR-90 fibroblast cell line). It was further demonstrated that the inhibition of luciferase expression is indeed attributable to the toxic effects of the DT-A chain because this effect was not observed in 293-DT-Aresistant cells [42]. Pancreatic Carcinoma Cells Two human pancreatic carcinoma cell lines expressing high levels of H19 (CRL 2547 and CRL 2119) were co transfected with 2 μg of LucSV40 and different concentrations of BC-819 vector. This experiment demonstrated that DT-A expression resulted in successful inhibition of protein synthesis as reflected by the decrease of luciferase activity in pancreatic tumor cells cell lines expressing H19 [43].

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Bladder weight (mg)

750

500

250

0 Normal rate

Control group

Treatment group

FIGURE 8.1 In situ hybridization analysis of pancreatic adenocarcinoma using Dig-LNA H19 DNA probe. No expression was seen in adjacent normal tissue.

H19 EXPRESSION IN PANCREATIC TUMORS

FIGURE 8.2 Effect of intravesical administration of DTA-H19/PEI

Pancreatic tumors from 36 patients and normal distal pancreatic tissue of 7 patients with tumors (obtained from the pathology department at Hadassah Medical Center, Israel) were examined for H19 expression by in situ hybridization (ISH) and/or polymerase chain reaction (PCR). Of the 36 tumors tested, 70% were positive for H19 expression. According to the results obtained from the biopsies of the 16 patients with unresectable pancreatic cancer screened in the phase I/IIa study, 90% of the patients showed H19 expression in the tumor. A representative example of ISH of a pancreatic tumor is shown in Figure 8.1. No H19 expression was found in normal pancreatic tissue distal to tumor regions [43].

on the orthotopic bladder carcinoma in rats. DTA-H19 or Luc-H19 (control group) complexed with PEI were administrated intravesically into rat bladders, which were previously implanted with syngeneic bladder carcinoma cells. Normal healthy rats received the same treatments separately. (Top) The mean bladder weights of the DTA-H19- and Luc-H19-treated bladders measured at the end of the treatment, after the animals were sacrificed. (Bottom) Hematoxylin/ eosin-stained sections (403 magnification) and macroscopic appearance of bladders of the normal, control, and DTA-H19-treated rats.

Ovarian Carcinoma Cell Lines The effect on protein synthesis inhibition of BC-819 was tested on ovarian carcinoma cells (OV-CAR3, SK OV3, TOV-122D, and ES-2). The effect was dependent on the presence of H19 RNA in the cells and was BC819 dose-dependent [44]. Hepatic Metastases The presence of H19 RNA in hepatic metastases of representative paraffin wax blocks of metastatic carcinoma that were selected from the archives of several pathology departments in Israel was investigated. H19 expression was observed in 64 of the 80 metastases [45]. Overall, these above presented results indicate that BC-819 is active only in cell lines expressing H19 RNA, and that the majority of the cancer cells tested are H19 positive—a finding that enabled the in vivo studies presented next.

In Vivo Nonclinical Pharmacology Animal Models of Bladder Cancer A syngeneic orthotopic rat model was developed by implantation of rat bladder cancer cells into the rat bladder, providing a relevant model for the investigation of the DTA-H19 biology and therapy of bladder cancer. After intravesical instillation of NBT-II rat bladder carcinoma cells onto the wall of the rat bladder in vivo, inhibition of tumor growth was achieved with two instillations of 50 μg of DTA-H19/PEI. One week after NBT II cells were inoculated, the gene transfer efficiency of the vectors following intravesical instillation of 50 μg of DTA-H19/PEI demonstrated that the tumors were efficiently transfected by the plasmid and that the H19 promoter was active, which led to DT-A chain expression inside the transfected cells (Figure 8.2) [8]. The in vivo efficacy of DTA-H19/PEI was also studied in a carcinogen-induced bladder tumor model in Wistar rats. After several weeks of carcinogen exposure, significant increases in the expression of H19 genes, as determined by reverse transcriptase polymerase chain reaction (RT-PCR), were observed. Animals were then treated intravesically for 5 weeks with DTA-H19/PEI or control vector. DTA-H19/PEI-treated animals demonstrated a significant decrease in the mean tumor

I. VIRAL AND NONVIRAL VECTOR METHODOLOGIES: TARGETS AND APPROACHES

Tumor growth progression (%)

NONCLINICAL PHARMACOLOGY

300 250 200 150 100 50 0

DTA-H19

Luc-H19

FIGURE 8.3 Tumor progression: Growth ratios of pancreatic tumors in hamsters following two Intratumoral injections of BC819. Hamsters received 50 μg of DTA-H19 by intratumoral injection on Days 7 and 9. The average tumor growth progression of the DTAH19 (n 5 9) and Luc-H19 (n 5 7) treated groups was calculated as the ratio of the tumor size (measured in situ) at the end of the experiment (Day 12) compared to the size before treatment (Day 7).

volume and mean tumor area compared to control animals. Similar in vivo efficacy was also observed in a syngeneic and in a nude mouse bladder carcinoma heterotopic model in which animals were treated intratumorally with DTA-H19/PEI [8]. RT-112 human bladder carcinoma cells were injected subcutaneously into the backs of 6- to 8-weekold CD-1 female nude mice. Ten days after inoculation of the cells, the developed tumors were measured in two dimensions and subjected to treatments. Intratumoral injections of 25 μg of BC-819 and 25 μg of Luc-H19 (control group) were performed with PEI on Days 10 and 12 after cell inoculation. Two injections of BC-819 were able to inhibit further tumor growth compared to the control Luc-H19 treatment. Animal Models of Pancreatic Cancer A syngeneic orthotopic model in hamsters was developed by injection of hamster pancreatic carcinoma cells (PC1-0) into the pancreas of Syrian golden hamsters for the investigation of DTA-H19. After 7 days, all of the animals presented a single tumor in the pancreas. DTA-H19 was tested for in vivo tumor growth inhibition after intratumoral administration of DTA-H19 into the hamster tumors. After the first treatment with DTA-H19 (7 days post cell inoculation), tumors showed a significantly lower tumor growth progression (TGP) (80% lower; n 5 9) than that of the Luc-H19 control group. After two treatments (9 days post cell inoculation), the tumors treated with DTAH19 had a TGP 68% smaller than that of the controls (p 5 0.029). Ex vivo tumor sizes of the DTA-H19-treated group were 48.6% lower (p 5 0.007) than those of the control group, and tumor weights were 49.3% lower than those of the control group (p 5 0.012). This study demonstrated that the intratumoral administration of BC-819 in hamsters with pancreatic tumors translated

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into DT-A expression in the tumor and that this expression was associated with a lower TGP (50% lower) (Figure 8.3). It also showed that fewer animals treated with BC-819 had metastases (3/9 (33%)) at the end of the experiment compared to control animals (7/7 (100%)) [43]. CRL-1469 cells (human pancreatic) were injected subcutaneously into athymic nude mice. After tumor development (30 days), three intratumoral injections of 25 μg BC-819 (with PEI) or control vector (Luc-H19) were given. There was a 3-day interval between each injection. Animals were sacrificed 3 days after the third injection; tumors were excised for ex vivo measurements and for pathology and molecular studies. The tumor growth of the BC-819-treated group was significantly lower (p 5 0.036). This observation was confirmed by the final ex vivo tumor volume, which was significantly smaller (75%) for the BC-819 group compared to the control group (p 5 0.038) [43]. Another model of efficacy involved the injection of rat colon carcinoma cells (CC531) into the pancreas of Wag/Rij rats. The rats were injected intratumorally (two times every 3 days) with either DTA-H19 or LucH19 control. Three days after the last treatment, the rats were sacrificed and the ex vivo tumor volume was measured. There was a statistically significant difference (58% reduction) in tumor volume measurements ex vivo in rats treated with BC-819. Animal Models of Hepatic Cancer DTA-H19 was also evaluated for its potential to control the growth of hepatic tumors using intratumoral injection, hepatic vein injection, and hepatic intraarterial injection (HAI) into a syngeneic rat liver metastatic model. In this model, rat colon adenocarcinoma cells (CC531) were implanted under the liver capsule in the left and in the right side of the median liver lobe of Wag/Rij male rats. A series of studies were performed using this model to characterize the tumor response to DTA-H19 with and without added PEI. The conclusions from these studies are presented next. Two or three injections of 50 μg of DTA-H19 at 4- or 7-day intervals either injected as naked DNA or complexed with PEI were able to inhibit the growth of CC531 rat colon adenocarcinoma cells implanted into the subcapsular space of rat livers. The addition of PEI did not significantly improve the therapeutic effect of DTA-H19. Thus, further evaluations were performed with DTA-H19 alone (no complexing with PEI) [46]. When intratumoral injection of DTA-H19 was combined with intraperitoneal (i.p.) injection of DTA-H19, the combined administration approach was more effective (hepatic tumor volume reduction) than intratumoral injection alone. However, in this case, the

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Number of surviving mice

7

pH19-DTA pH19-Luc

6 5 4 3

The experiments summarized here clearly demonstrate that BC-819 has the potential to be effective in the targeted indications—that is, bladder, pancreatic, ovarian, and metastatic to the liver cancers. These nonclinical proof-of-concept studies support a possible clinical efficacy of BC-819 in patients whose tumor cells express H19 RNA.

2 1

Nonclinical Toxicology Data

0 0

1

2

3

4

5 6 7 8 9 10 11 12 13 14 15 Days from the first treatment

FIGURE 8.4 Effect of intraperitoneal injections of BC-819 on human ovarian carcinoma cells injected intraperitoneally in nude mice. Twelve nude mice were injected i.p. with ES-2 cells. Fifteen days later, 6 mice received three injections of 100 μg of DTA-H19, and the other 6 received three injections of 100 μg of Luc-H19 plasmid complexed with PEI at 1-day intervals (the days of treatments are marked by black arrows). Day 0 indicates the day of the first treatment. Survival was monitored daily.

addition of PEI to the i.p. dose was more effective than when PEI was not included. DTA-H19 administered by HAI in four divided doses spaced 3 days apart, such that a total dose of 2.5 mg was administered, resulted in significant shrinkage of 37.5% of the tumor metastases (all tumors treated with control plasmid increased in size) [47]. Animal Models of Ovarian Cancer The human ovarian cancer cell line (ES-2) was injected subcutaneously into the dorsa of nude mice. After developing measurable tumors, the mice were injected intratumorally every 2 days with either DTAH19 or Luc-H19 control plasmid. Both plasmids were complexed with PEI. DTA-H19 led to a significant inhibition of tumor growth in this model. Four injections of BC-819/PEI at 2-day intervals were able to inhibit tumor growth by 40% compared to four LucH19/PEI treatments (p , 0.05) [48]. An ascites model was developed by injecting nude mice intraperitoneally with ES-2 cells. After the mice developed ascites 15 days later, they were injected intraperitoneally with either DTA-H19 or Luc-H19 control plasmid for a total of three injections 1 day apart. The overall survival was monitored daily. By Day 6, five of the six control mice (83%) had died, but only two of the six (33%) mice treated with DTA-H19 had died. Although all of the mice died by Day 15, there was evidence of slowing of tumor cell growth in vivo in this aggressive model of ascites ovarian cancer (Figure 8.4).

Four repeat-dose toxicology studies were performed in accordance with good laboratory practices regulations in mice and rats. In support of the bladder cancer indication, two of these studies included (1) i.p. administration of DTA-H19/PEI in BALB/c mice and (2) intravesical administration of DTA-H19/PEI into the urinary bladder in Sprague Dawley rats. The studies supporting i.p. administration of DTA-H19 (not complexed with PEI) in ovarian cancer, intratumoral administration in pancreatic cancer and liver cancer, and hepatic artery infusion in liver cancer consisted of (1) repeat intravenous (i.v.) dose study in BALB/c mice and (2) repeat i.p. dose study in Sprague Dawley rats of DTA-H19 (not complexed with PEI). Studies Supporting Bladder Cancer Trials The toxicity of repeat i.p. doses of BC-819/PEI was tested in BALB/c mice (three dosing sessions/week for a total of 3 weeks): 5% glucose (control) or BC-819 was administered at doses of 25, 85, and 175 μg/animal/day. No mortality occurred in any of the test article- or vehicle control-treated animals prior to the scheduled termination time points. No obvious treatment-related adverse reactions were observed among the test article- or vehicle control-treated animals during the treatment period and throughout the entire observation period until the scheduled termination time points, even though some mild to moderate microscopic inflammation in the spleen, liver, pancreas, ovaries, bladder, uterus, and colon was reported in the high dose, which resolved at study termination. To test the effect of BC-819/PEI via the intravesical route, rats were subjected to once-weekly repeated intravesical instillations under general anesthesia by isoflurane inhalation, for a total duration of 7 weeks, at three doses of BC-819/PEI of 50, 150, and 300 μg/ animal/week or 5% glucose (controls). Half of the animals were terminated 1 day and the other half 14 days following the last intravesical dosing session. No mortality occurred in any of the animals prior to the scheduled study terminations. No noticeable toxicological effects were evident in any of the animals throughout the entire treatment period and the

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subsequent 14-day observation period. Pharmacokinetic studies showed that intravesical administration of 300 μg DTA-H19 complexed with PEI resulted in uptake of plasmid primarily in the urinary bladder. Sixteen-fold lower levels were also observed in most animals’ kidneys, with very low to undetectable levels in other organs and blood. This was the expected distribution of plasmid injected intravesically because the majority is excreted from the urinary bladder in humans after intravesical treatment with undetectable amounts in the blood when tested a few hours to days after treatment. The presence of plasmid in the kidneys is not unexpected because reflux from the bladder to the kidneys frequently occurs during this type of experimental manipulation in rats. Studies Supporting Pancreatic, Ovarian, and Hepatic Tumor Trials (Local/Regional Administration of DTA-H19) The potential toxicity and pharmacokinetics (PK) of BC-819 were tested by administering the drug either intravenously three times per week for 2 weeks to BABL/c mice or intraperitoneally to Sprague Dawley rats and by assessing the reversibility of any potential toxicity after a 14-day recovery. Four groups of animals were injected with either sterile saline or BC-819 (0.2, 2.0, and 10.0 mg/kg/administration). Safety endpoints included daily observations, clinical observations/physical exams, body weights, feed consumption, serum chemistry and hematology, gross pathology, organ weights, and histopathology. Satellite groups were used to assess the PK of BC-819 administered at the highest study dose after the first administration and again after the sixth administration of BC-819. There were no clinically significant findings regarding clinical observations, hematology, clinical chemistry, and gross and microscopic pathology. Absolute organ weight data showed possible treatment- and dose-related decreases in brain, heart, kidney, thymus, testes, liver, and salivary glands for males only. The toxicological significance of these findings is unclear because of the lack of a histopathological correlation, the small magnitude of the changes, and the absence of any response in the females. The no-observedadverse-effect level in this study was considered to be greater than 10 mg/kg. Rats dosed intraperitoneally with 10 mg/kg BC-819 had a mean maximum serum concentration 60 min after injection. After the sixth injection of BC-819, the maximum concentration was achieved at a later time point (240 min) compared to the 60-min time point observed after the first injection. By 24 hr, the serum concentration was reduced by more than 1000-fold from the peak concentration.

CLINICAL DATA Data to support four initial indications have been generated in both animal tumor models and human clinical trials. These initial indications are TCC of the bladder, advanced stage pancreatic cancer, ovarian cancer, and metastic disease in the liver. The product BC-819 for bladder cancer is the farthest along in development because a phase I/IIa dose escalation safety and preliminary efficacy study has been completed and a phase IIb clinical trial is underway. The hepatic cancer indication has been shown to have safety and efficacy in animal tumor models and preliminary evidence of tumor response in two compassionate use patients with hepatic metastases. A patient with ovarian cancer treated under a compassionate protocol showed a clinical response. In addition, a phase I/IIa dose escalation safety and preliminary efficacy study for the treatment of patients with advanced stage ovarian cancer is underway. A patient with pancreatic cancer was treated under compassionate protocol, and a phase I/IIa dose escalation safety and preliminary efficacy study for the treatment of patients with unresectable pancreatic cancer has been completed showing a good safety profile and clinical response. These four indications were selected because tumor specimens showed high levels of overexpression of H19 in the majority of tissues tested from each type of tumor, animal models supported efficacy in each of these tumors, and each of these malignancies is accessible for local/regional administration of plasmid.

Bladder Cancer In the bladder cancer indication, BC-819 in combination with PEI given by intravesical administration into the bladder has been investigated in a phase I/IIa dose escalation clinical trial in order to assess the safety and preliminary efficacy in 18 patients [5]. The study was designed to assess the safety and preliminary efficacy of five different doses of BC-819 given as six intravesical infusions into the bladder of 18 patients with superficial bladder cancer who failed standard treatments. No serious adverse events related to the plasmid were detected, and 56% of the patients did not experience recurrence of the tumor. A phase IIb study is currently ongoing to assess the treatment efficacy in terms of tumor response, as well as its safety. This study was designed to assess the efficacy and safety of DTA-H19/PEI given as six intravesical instillations of 20 mg of plasmid DNA complexed with PEI into the bladder of patients with intermediate risk superficial bladder cancer (recurrent stages Ta (low and high) and T1 (low grade) TCC who have

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DT-A 468 bp

2

24

48

72

FIGURE 8.5 Treatment of a compassionate patient with BC819 (DTA-H19). (A) ISH detection of H19 transcript in tissue section of bladder carcinoma from the patient, demonstrating diffuse and high H19 expression (arrowheads). (B and C) Video cystoscopy performed before starting the treatment (dotted tumor area) (B) and 3 weeks after the third treatment with the toxin vector (C). (D) Detection of the DTA-H19 plasmid in urine and blood samples by PCR analysis. Urine was collected during the first and second week of treatment before and 2 hr after plasmid instillation.

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Hours after instillation

failed prior intravesical therapies, including either bacille Calmette Gue´rin (BCG) or chemotherapy. The complex of BC819/PEI has also been administered to four patients in named patient compassionate use programs. Two patients had recurrent superficial TCC of the bladder and had failed multiple courses of BCG and chemotherapy. For a patient who underwent nephrourectomy due to a diagnosis of recurrent superficial TCC that showed BCG intolerance and now has only one remaining kidney, BC-819/PEI has prevented this patient from becoming anephric and having to undergo dialysis. For one of the compassionate use patients who was a candidate for cystectomy at the beginning of treatment, BC-819/PEI has allowed to defer cystectomy for a period of at least 6 years without evidence of progressive disease (Figure 8.5).

Ovarian Cancer Sixteen i.p. infusions of BC-819 at doses ranging from 80 to 140 mg of plasmid DNA per treatment for a total cumulative dose of 1.7 g were well tolerated by a patient enrolled in a named patient compassionate use study. The first 10 infusions were performed weekly. After that, weekly treatments alternated between chemotherapy (paclitaxel, docetaxel, and carboplatin) and BC-819. At the time when she began the compassionate protocol with DTA-H19, this ovarian cancer patient was suffering from accumulation of peritoneal ascites

fluid containing malignant cells, which did not respond to chemotherapy, including carboplatin and paclitaxel, topotecan, gemcitabine, doxil, and VP16 [44]. Ultrasound imaging was performed before each treatment and showed a reduction in the ascites volume particularly in the lower abdomen. From the time of the second treatment, the patient reported a significant improvement in abdominal pressure and pain; however, CA-125 levels remained stable throughout the 10 weeks of treatment with DTA-H19 at a concentration of approximately 250 U/ml. Although no ascites fluid was found in the peritoneum cavity by treatment 10, the CA-125 level did not decrease from 300 U/ml for the last 3 weeks. The protocol was modified to use a sequential treatment that included the biweekly intravenous combination of chemotherapy and DTA-H19, which resulted in a significant decrease in CA-125 level to 159 U/ml. Ascites fluid was found to be undetectable after the 15th treatment (Figure 8.6). The patient received 17 infusions of DTA-H19 during a 10-month period. Infusion of the DTA-H19 plasmid into the peritoneum of the patient resulted in complete resolution of the ascites with minimal adverse events. After this period of plasmid therapy, the patient was still alive, had no ascites, and resumed regular daily activities with an improved quality of life. Computed tomography (CT) and positron emission tomography (PET)/CT scans were performed 6 weeks and 3.5 months after the first treatment, respectively. Both scans showed that although there were still multiple

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FIGURE 8.6 Ultrasound images of peritoneal cavity from compassionate patient with ovarian cancer treated with BC-819 intraperitoneally. Ultrasound imaging was performed before each treatment to evaluate the amount of ascites fluid in the patient’s peritoneum. Arrows indicate the limits around the area of ascites. CT and PET/CT scans were performed 6 weeks and 3.5 months after the first treatment, respectively.

i.p. areas of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) uptake indicating the presence of cellular activity of the tumor, an arrest in disease progression and disappearance of ascites were noted [44]. A complete summary of the data obtained so far in the phase I/IIa study is not presented here because this study is still being completed, but in terms of safety, no dose-limiting toxicity was observed among the evaluable patients for safety or among the total number of patients who received at least one dose of BC-819. The purpose of this study is to assess the safety, tolerability, PK, and preliminary efficacy of DTA-H19 administered i.p. in patients with advanced stage ovarian cancer or primary peritoneal carcinoma.

Hepatic Metastases In the liver cancer compassionate use studies, palliative treatment of the patient with a single large hepatic metastasis that had extended into subcutaneous tissue with involvement of the right chest ribs was performed by intratumoral injection of DTA-H19 (two doses for a total of 12 mg of plasmid DNA). A PET/CT study performed 4 weeks after the second treatment demonstrated destruction of the tumor mass at the injection site (Figure 8.7). There were no local or systemic signs of toxicity and no deterioration of liver function during treatment. A low level of plasmid was detected in the serum only at 2 hr after each injection, and no plasmid was detectable in the serum or urine during the next 72 hr following both injections. Thus, intratumoral injection of

FIGURE 8.7 PET/CT scan of liver of compassionate patient treated intratumorally with BC-819 showing area of destruction of tumor mass (arrows).

DTA-H19 (two doses for a total of 12 mg of plasmid DNA) into a hepatic metastasis was tolerated, provided temporary palliative relief, and showed radiographic evidence of a large area of tumor shrinkage. The second case of hepatic metastatic disease was a patient with inoperable liver metastasis in both lobes

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of the liver. During a 4-year period, the patient underwent multiple courses of a variety of different chemotherapy regimens. Having failed these, the patient was given two courses of treatment consisting of three infusions of DTA-H19 via the common hepatic artery given once per week for 3 weeks, with the second course starting 1 month after the first infusion. The first course consisted of escalating doses of 16, 24, and 36 mg of DTA-H19 plasmid DNA. The second course consisted of escalating doses of 48, 64, and 82 mg of DTA-H19 plasmid. Side effects included a fever resulting from an infection likely due to the catheter placement for infusion. The infection resolved and did not recur during subsequent treatments. Liver function tests were similar to baseline levels for all analytes. Serum creatine was within normal limits at all assessments. The detection of the plasmid DNA in the urine and in the blood after treatment varied from one administration to another. On average, BC-819 was detected in the urine more or less approximately 1 hr and up to 6 hr after treatment. It was no longer detected at the 48-hr time point. In the blood, the plasmid was detected more or less approximately 1 hr after treatment and, interestingly, prior to the start of the second course of treatment, suggesting persistence in the blood of at least 1 week following a hepatic artery administration. Hepatic artery infusion of doses up to 48 mg of BC-819 per infusion resulted in stable disease during the 2-month period of treatment and assessments.

Pancreatic Cancer A total of 10 patients with pancreatic cancer have been treated with intratumoral BC-819—9 during the phase I/IIa trial and 1 during a named patient compassionate use program. The purpose of this study was to assess the safety, tolerability, PK, and preliminary efficacy of BC-819 administered intratumorally in patients with unresectable, locally advanced pancreatic cancer. Patients were enrolled into one of two cohorts with escalating doses of BC-819 from the first to second cohort. Each cohort received 2 weeks of twice-weekly intratumoral BC-819. Intratumoral injections of BC-819 were performed using a CT-guided PTA or endoscopic ultrasound (EUS). The first cohort received 4 mg of BC-819 per injection, and the second cohort received 8 mg of BC819 per injection. Patients were assessed by CT or PET/CT 4 weeks after the start of the treatment. The results of the study showed that BC-819 can be safely administered intratumorally via EUS or PTA at a dose of at least 8 mg in 2 ml per injection twice weekly. Although this study was primarily a safety study, it was of interest to evaluate the overall survival

of subjects for up to 1 year after the start of study treatment. Two of the subjects in the cohort that received four intratumoral injections of 8 mg of BC-819 were still alive at 1 year. This short course of treatment, which was safe, did not appear to have any impact on the subjects’ ability to receive other cancer treatments, and for these two subjects it may have contributed to their overall survival at 1 year post-treatment. BC-819 given locally in combination with systemic chemotherapy may provide an alternative to chemotherapy alone for the treatment of pancreatic cancer. Future studies may include higher doses, longer periods of treatment, and combination with other systemically administered drugs. The pancreatic cancer patient started the compassionate protocol after he participated in a phase I/IIa DTA-H19 safety trial and received four injections of 4 mg DTA-H19 directly to the tumor in the pancreas via EUS. BC-819 was injected into the pancreatic tumor under EUS guidance, once a week for 4 weeks, using a dose of 8 mg of BC-819 in a volume of 2 ml for the first two injections and then a dose of 12 mg of BC-819 in a volume of 3 ml for the last two injections. No discomfort was reported during these injections, and no adverse events occurred. Two months later during the assessment, the patient had maintained his weight and the CT showed that the pancreatic tumor remained stable since the last CT performed 3 months earlier. These results indicated that a dose up to 12 mg and a cumulative dose of 40 mg in 4 weeks can be administered into pancreatic tumors with no detectable product-related toxicity.

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