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Bacterial Vaccines

  • Paulina Chorobik
  • Joanna BeretaEmail author
Living reference work entry

Abstract

A contemporary approach to bacterial cancer immunotherapy takes advantage of substantial progress in the understanding of the tumor-immune system interplay, as well as the recent advances in genetic engineering. Safe, targeted therapies are being developed, in which genetically-modified pathogens are designed to trigger effective anti-tumor immune response. Here we describe the bacterial strains intended for cancer immunotherapies, the genetic modifications that attenuate their pathogenicity but strengthen anti-tumor potential and the desirable mechanisms of actions. The only FDA approved Bacillus Calmette-Guerin-based cancer therapy as well as numerous examples of ongoing clinical trials involving different bacterial strains are presented.

Keywords

Bacterial attenuation Cancer immunotherapy Intracellular pathogens Vaccine vectors 

Target

Over a century ago, Dr. William Coley for the first time intentionally administered bacteria to patients to treat inoperable tumors. Thanks to the dedication of a lifetime to perfect this therapy, which in many cases led to complete tumor eradication, Dr. Coley was acknowledged as the Father of Cancer Immunotherapy (Richardson et al. 1999). Since that time, the major principle of bacterial anticancer therapy has not changed much and is based on systemic application of pathogenic bacteria to alert the immune system and induce a potent, specific, antitumor immune response, resulting in the inhibition of tumor growth.

Despite its initial success, bacterial antitumor therapy became underexploited and neglected throughout the years of the domination of chemo- and radiotherapy, which were easily manageable due to the simplicity of the effector nature (radiation or chemotherapeutics) and relatively rapid assessment of the effectiveness of treatment. However, acute toxicity and other adverse effects of conventional tumor therapies call for a continuous search for less devastating alternatives.

Among biological therapies, the application of live bacteria may meet these expectations, especially as some bacterial species spontaneously colonize solid tumors. In consequence, this natural tumor targeting may reduce systemic adverse effects compared to other therapies.

Bacteria are applied in cancer treatment to address at least one of the following: (i) specifically target tumor tissue, (ii) preferentially deliver therapeutic agents to tumor tissue to limit systemic toxicity, and (iii) break the immune suppression, disarm mediators which promote tumor progression, and boost antitumor immunity.

Since the first attempts of bacterial cancer therapy, the advances in genetic engineering have enabled the attenuation of pathogen virulence and thus the application of live instead of killed microorganisms. The viability of therapeutic bacteria extends the potential benefits of treatment as it guarantees the features that are unique for bacteria-based therapy, described in the next section. Weakening of the virulence allows for a repeated dose scheme as long as the immunogenicity of the attenuated strain is balanced to guarantee its proliferative and stimulatory capacities, along with the safety of treatment.

With the aid of genetic engineering, bacteria may also acquire novel therapeutic features. The term “bacterial vaccine vector” was coined to underline the use of bacteria as a vehicle for the delivery of therapeutic molecules.

Biology of the Target

The live-attenuated bacteria-based approach represents a versatile therapeutic option for cancer therapy, as it provides the complex and complete array of immunostimulatory effects that address the drawbacks of many single-agent therapies. Its additional advantage over small molecule therapeutics, as well as over antibody- or cell-based immunotherapies, is that most of the bacteria under consideration are self-propulsive and actively penetrate tumor tissue, including necrotic and hypoxic regions. Live bacteria represent a therapeutic unit that can be robustly multiplied in vitro in relatively simple growth media. Moreover, they retain the ability to replicate and efficiently produce proteins after being applied to patients. Intracellular pathogens trigger a cellular type of immune response with a Th1 cytokine profile, essential for effective antitumor effects. Therefore, predominantly intracellular microorganisms, examples presented in Table 1, are applied in preclinical studies or clinical trials.
Table 1

Examples of bacteria candidates for cancer therapy

Species

Gram staining

Motile

Oxygen tolerance

Intracellular phase of infection

Clostridium sp.

Positive

Vegetative forms are motile

Anaerobe

Not applicable

Listeria monocytogenes

Positive

Yes

Facultative anaerobe

Facultatively intracellular, actively escape from phagosome to the cytosol of the infected cell

Mycobacterium bovis BCG

Positive

No

Obligate aerobe

Facultatively intracellular; survive and replicate in vacuoles of phagocytes

Salmonella enterica, sv. typhimurium and typhi

Negative

Yes

Facultative anaerobe

Facultatively intracellular, survive and replicate in vacuoles of phagocytes and also in the cytoplasm of epithelial cells

Shigella flexneri

Negative

No

Facultative anaerobe

Facultatively intracellular

The natural adjuvant-like properties of bacteria may further be augmented by genetic modifications that arm bacteria with heterologous molecules. Virtually any protein of bacterial, viral, or eukaryotic origin whose activity does not depend on posttranslational modifications can be produced by a bacterial vector. Depending on the adopted strategy, this additional effector molecule is synthesized outside the host cells or is delivered into host cells during the intracellular phase of infection. For example, when bacteria capable of expressing a tumor antigen invade and survive inside the antigen presenting cells, such as dendritic cells (DCs) and macrophages, they can produce and deliver a tumor protein to the MHC-I antigen presentation pathway. This approach resulted in enhanced CTL responses to tumor antigens after the application of attenuated bacterial vaccine vectors to tumor-bearing animals (in the case of S. typhimurium) or to either animals or humans (in the case of L. monocytogenes).

The suppressed pathogenicity is a prerequisite for a modern bacterial cancer therapy. It can be achieved through: (i) direct modification of virulence factors or (ii) indirectly by modifying bacterial metabolic pathways.

The first approach involves mitigating virulence factors that directly damage host cells or activate robust and devastating immune responses during the natural course of infection. Virulence factors that are often targets for attenuation belong to ubiquitous pathogen-associated molecular pattern (PAMP) molecules, e.g., LPS, or are genus specific, e.g., Listeria monocytogenes ActA, which mediates actin polymerization and intracellular motility or Clostridium novyi α-toxin, which destroys host cell cytoskeleton through the modification of small GTP-binding proteins. Another approach of attenuation involves modifications of metabolic capabilities to impair bacterial growth and limit or redirect bacterial spread in the infected organism through the addiction of bacteria to external sources of missing metabolites. The concomitant effect of the disruption of a bacterial metabolic pathway is an accumulation of bacteria in tumor tissue which is a rich source of metabolites. Indeed, the metabolic attenuation of bacteria is indicated as a possible cause of their preferential tumor colonization.

Historically, attenuation was achieved by a nondirected method based on the exposure of a pathogen to mutagenic factors and/or selection of less virulent strains in vitro under special conditions or in vivo in nonhost species. For example, Bacillus Calmette-Guérin (BCG), the vaccine strain of Mycobacterium bovis, was developed after in vitro selection in a special culture medium. At present, most often, although not exclusively, a site-directed mutagenesis that disrupts a chosen vital gene is used for the development of therapeutic bacterial strains.

Attenuated S. typhimurium strains were obtained by the disruption of genes coding for: (i) enzymes involved in amino acid synthesis (leucine and arginine auxotrophic strain, aromatic amino acid synthesis defective aroA and aroD mutant strains), (ii) enzymes involved in purine synthesis (purI or purD mutant strains), and (iii) global transcriptional regulatory factors essential for the synthesis of numerous enzymes of catabolic pathways (cya and crp mutant strains). Genetically modified therapeutic strain Clostridium novyi-NT (NT stands for nontoxin) was obtained by the disruption of the genes coding for the lethal α-toxin. Moreover, Clostridium sp. is strictly anaerobic; therefore, the spores which are administered to the animal or human organism do not multiply effectively unless they reach hypoxic areas of the tumor. The attenuation of a modified Listeria monocytogenes strain, which is currently in Phase 2 clinical trial, limits bacterial survival in vivo due to the inactivation of actA gene encoding a major virulence factor (Wallecha et al. 2009). Shigella flexneri aroA mutant has also been used in preclinical studies (Galmbacher et al. 2010).

Target Assessment

A couple of dozen treatment regimens are currently under evaluation in Phase 1 safety studies. The bacterial inocula are administered based on the number of colonies formed on solid media (CFU, colony forming units). Maximum tolerated doses (MTD) are determined for each therapeutic strain or its novel derivative. The complete blood cell count (CBC) and blood biochemical assays are performed to monitor the patient status after bacteria administration. To evaluate the level of tissue colonization and biodistribution as well as to monitor bacteremia, the level of bacteria in the tissues, blood, urine, and stool is measured by cultivating bacteria from specimens on proper selective media. Serum levels of cytokines and chemokines such as IL-1β, IL-2 , IL-6, IL-8, IL-10, IL-12, TNF , IFNγ, MCP-1, MIP-1α, and GM-CSF can be measured to assess the biological activity of the treatment or indicate the risk of cytokine-release syndrome (CRS). If relevant, the presence of antigen-specific T cells in peripheral blood is examined by ELISpot assays to assess the efficacy of bacterial vectors that express tumor antigen. Due to the differences in immune responses and severity of toxic effects, which are specific to different bacterial species tested, the dose-limiting adverse events are defined for each study, according to common toxicity criteria.

Role of the Target in Cancer: 10

Apart from a single example of Bacillus Calmette-Guérin (BCG) already used for over three decades for bladder cancer treatment (described below), applications where bacteria are used as antitumor agents are in most cases still in the preclinical phase of development and are thus categorized as experimental immunotherapies.

The primary concept of immunotherapy is to overcome the tumor immunosuppressive microenvironment and induce efficient tumor-specific immunity, including memory responses that would protect patients from tumor recurrence and metastatic disease. Bacteria are well suited for this purpose as they are the most potent activators of the immune system due to the engagement of numerous alarm-sensing pattern recognition receptors (PRR) including the majority of Toll-like receptors (TLRs) by bacterial pathogen-associated molecular patterns (PAMPs) such as cell wall components (LPS, lipopeptides), unmethylated CpG, flagellin, outer membrane proteins, and others. Additionally, an administration of intracellular bacteria is supposed to skew the immune response toward the desirable Th1 type. Therapeutic bacteria are often genetically modified and serve as vectors delivering gene coding for proteins of choice.

There are two major approaches to bacteria-based therapy. In the first one, bacteria engineered to express a given tumor antigen (TA) serve as its additional rich source delivered simultaneously with the strong bacterial alarm signals. In this approach, bacterial invasion of the tumor is not necessary. The second approach intends the accumulation of bacteria in the tumor. By infecting and disrupting cancer cells, bacteria may increase the cross-presentation of TAs by dendritic cells via increasing the availability of TAs as well as providing or inducing multiple signals for the maturation and activation of DCs. These comprise: (i) ligands for all TLRs as well as other danger signal receptors, including tumor cell-derived damage-associated molecular patterns (DAMPs) such as heat shock proteins, nucleotides, nuclear proteins, and bacterial PAMPs, and (ii) proinflammatory cytokines and, in case of using intracellular microorganisms, also type I interferon. By infecting tumor-associated immune cells which are partially responsible for immunosuppressive microenvironment, bacteria may help in breaking anergy or tolerance toward tumor antigens. The natural effects of bacterial infection are often strengthened by their genetic modifications, leading to the expression of: (i) proapoptotic proteins that increase death rate (e.g., apoptin, TRAIL ), (ii) proteins that modulate immune response (e.g., IL-2 , IL-18, TNF , LIGHT ), (iii) molecules that modulate the tumor microenvironment (e.g., IDO shRNA), (iv) molecules that modulate intracellular signaling (e.g., STAT3 shRNA), or (v) a combination of the above.

In contrast to mice, where preferential tumor localization of bacteria has been proven, insufficient tumor colonization in humans has been identified as the major limitation in developing effective therapies. Antibody-fragment-based targeting of bacteria toward cancer cells is proposed to overcome this drawback (Bereta et al. 2007; Massa et al. 2013).

High-Level Overview

Diagnostic, Prognostic, and Predictive

An increased number of suppressive immune cells in peripheral blood and in the tumor are negative prognostic factors for numerous malignancies. High frequency of immature MDSCs is correlated with poor prognosis in melanoma and gastrointestinal, lung, and breast cancers (Gabrilovich et al. 2012; Solito et al. 2014; Weide et al. 2014), and increased tumor infiltration with FOXP3+ regulatory T cells predicts reduced survival of breast, cervix, gastric, kidney, ovary, and pancreatic cancer patients (Martin et al. 2010).

Two main therapeutic objectives of bacterial anticancer therapies are (i) the delivery of multiple immunostimulatory signals to resolve chronic inflammation and (ii) the augmentation of effective tumor-specific cellular immune responses. Effective intervention should inhibit the accumulation of suppressive cells in the tumor and overcome immunosuppressive tumor microenvironment consisting of soluble factors promoting tumor progression, released by both tumor cells and immunosuppressive cells. The activation of the innate immune Toll-like receptors with bacteria-derived ligands stimulates the proliferation of hematopoietic stem cells as well as the synthesis of proinflammatory cytokines. Upon acute exposure to the cytokines, cells of monocytic lineage undergo differentiation and activation (Goldszmid et al. 2014) and may result in repopulation of tumors with myeloid cells without a suppressive phenotype.

The specific predictive biomarkers for bacterial anticancer therapies are not yet defined as most of them are only in Phase 1 safety studies. The main candidates for predictive factors are the serum levels of proinflammatory Th1 cytokines and additionally, if the therapeutic bacteria express a tumor antigen, the antigen-specific T-cell responses. The only approved bacterial anticancer agent is Bacillus Calmette-Guérin vaccine for the treatment of superficial bladder cancer. Recently, candidate predictive factors of intravesical BCG therapy for non-muscle-invasive bladder cancer have been evaluated (Lima et al. 2012). The results indicate that a higher number of tumor-associated macrophages (TAMs) in the tumor and surrounding tissue are associated with shorter relapse-free survival (RFS), whereas higher IL-2 , IL-6, and TNF levels in urine are associated with longer RFS. The predictive value of urine levels of other cytokines, such as IFNγ or GM-CSF , is inconsistent and requires further research (Lima et al. 2012).

Therapeutics

Over three decades ago, intravesical BCG therapy for the management of bladder cancer superseded cystectomy. It is currently the standard therapy applied after transurethral lesion resection of intermediate- and high-risk non-muscle-invasive bladder tumors of Ta and T1 stages and for the management of carcinoma in situ (Kawai et al. 2013). The exact immunological mechanism is not clear; however, the therapeutic effect was ascribed to the infection of urothelial and bladder cancer cells which trigger an immune response, evidenced by the presence of cytokines in the urine. Moreover, the antitumor effect of BCG was shown to be dependent on T cells, NK cells, and neutrophils (Kawai et al. 2013).

BCG is administered as an induction and maintenance therapy and effectively reduces cancer recurrence and progression. During the induction therapy, one dose is given weekly for 6 weeks, but evidence shows that for an improved response, the therapy should be prolonged (Kamat and Porten 2014). The optimal frequency and duration of the maintenance therapy are still under investigation, although the protocol described by Lamm and coworkers has been suggested to be optimal (Kamat and Porten 2014; Lamm et al. 2000). The regimen consists of a 6-week induction therapy with intravesical and percutaneous administrations, followed by three weekly intravesical and percutaneous treatments at 3 and 6 month and then every 6 months, for a total of 3 years from the start of induction therapy (Lamm et al. 2000). The clinical efficacy is high and reaches 83 % overall 5-year survival, with median 76.8 months of recurrence-free survival (Lamm et al. 2000). Despite the high efficacy, some patients do not respond to BCG. Moreover, local and systemic adverse events tend to escalate during the maintenance therapy, in severe cases forcing therapy cessation. These drawbacks prompted further development of BCG anticancer therapy toward genetically modified strains which produce recombinant cytokines or application of BCG-derived particles instead of whole live organisms.

Preclinical Summary

The idea of bacteria-based immunotherapy is gaining more and more interest. In addition to bacteria strains, listed in Table 1, especially well suited for tumor therapy due to their intracellular lifestyle, the usefulness of extracellular bacteria such as Escherichia coli (St Jean et al. 2008) and certain lactic acid bacteria from the Lactococcus, Lactobacillus, and Bifidobacterium genera (Tangney 2010) has also been tested in preclinical studies, although to a lesser extent.

The preclinical research addresses two major issues: (i) Does the expression of a given tumor antigen (TA) in the context of bacteria induce a more effective antitumor immune response than the antigen administered solely? (ii) How does the expression of additional modifiers at the tumor site affect antitumor immune response? In both cases a gene of interest may be placed under a bacterial promoter, and the bacterial transcriptional, translational, and secretory machineries are used to express and deliver a heterogeneous protein. Alternatively, a gene coding for a protein or regulatory RNA is placed under a strong eukaryotic promoter such as CMV, and bacteria are used simply as vehicles that deliver the gene to the mammalian cell expression machinery.

The response to the following bacteria-coding TAs has been studied in mouse models: protein E7 of human papillomavirus type 16 (HPV-16 E7), tyrosinase-related protein 2 (TRP2), prostate-specific antigen (PSA) , cancer/testis antigen 1 (NY-ESO-1 ), mesothelin, and survivin .

The applicability of the following modifiers has been studied in mouse models: (i) effector proteins enhancing presentation of TAs through induction of tumor cell death and TAs processing (TRAIL , apoptin, FasL, Noxa), (ii) effector molecules modifying immune response (TNF , IL-2 , IL-18, CCL21 , LIGHT , IDO shRNA, STAT3 shRNA), and (iii) molecules triggering antiangiogenic response (VEGFR-2) (Chorobik et al. 2013; Wood and Paterson 2014).

Salmonella and Listeria are the genera most commonly used in the preclinical studies. Bacteria of genus Listeria are predominantly exploited as vectors producing TAs inside antigen presenting cells . Hence, TAs are delivered in the context of bacterial immunostimulatory molecules to boost the immune response. As selected attenuated Salmonella strains accumulate preferentially in solid tumors over internal organs at a ratio of 1000:1, they are well suited for the delivery of therapeutic molecules locally to tumor tissue. This approach offers the advantage of the delivery of the molecules that are too toxic for systemic administration or the factors that for any reason would bring benefit when accumulated mostly in the tumor. Both Listeria and Salmonella in various experimental settings have been shown to induce strong tumor-specific immune responses leading to delay in tumor growth or even tumor eradication (Brockstedt et al. 2004; Chorobik et al. 2013; Wood and Paterson 2014). Tumor growth inhibition after systemic or intratumoral administration of attenuated S. typhimurium to tumor-bearing mice has been associated with the shift in the phenotype and activity of intratumoral MDSCs indicated by the upregulation of maturation and activation markers (MHC class II and co-stimulatory molecules), their reduced suppressive capacity, and increased TNF secretion (Hong et al. 2013; Kaimala et al. 2014). Moreover, the frequency of regulatory T cells in tumors has also been reduced (Hong et al. 2013). Unfortunately, the very promising preclinical studies do not always translate into encouraging clinical trials. For example, the colonization of tumors in humans by attenuated S. typhimurium proved to be infrequent and insufficient for the therapeutic effect, which is in contrast to a high level of tumor colonization in mice susceptible to systemic S. typhimurium infection. This may result from significant differences in immune systems between humans and model mouse strains. NRAMP1 (natural resistance-associated macrophage protein-1) is important for the fate of intracellular bacteria such as Mycobacterium and Salmonella, which may survive and replicate inside phagocytes. NRAMP1 is absent in two major model mouse strains used for tumor therapy studies, namely, Balb/c and C57Bl/6, and this may explain the differences in the extent of bacterial tumor colonization between mice and humans. Hopefully, research on the mechanisms of bacterial infections, as well as on differences in the immune response between different species and strains, will help to bring the promising therapies to the clinic.

Clinical Summary

Since its approval in 1990, the BCG repeated intravesical instillations for treatment of non-muscle-invasive bladder cancer and inoperable bladder carcinoma in situ appear to have been one of the most effective cancer immunotherapies. The administration of BCG as a single agent against melanoma or colorectal or lung cancer has not been proven to be more effective than conventional therapies, but its use as a potential adjuvant for various cancer vaccines has been proposed.

The safety and therapeutic applicability of novel genetically modified bacteria species are being evaluated in Phase 1 or 2 clinical trials. L. monocytogenes therapeutic strains of series CRS are attenuated derivatives of wild-type L. monocytogenes 10403S obtained by the deletion of two genes: actA coding for a major virulence protein, actin assembly inducing protein, which mediates intracellular Listeria motility, and internalin B, which mediates the invasion of nonphagocytic cells. Clinical trials of the CRS-100 strain (ANZ-100, ClinicalTrial.gov identifier NCT00327652) have proven its safety. Further genetic modifications leading to the production of TAs by Listeria have been introduced, and the potentially improved efficacy of new strains is under clinical evaluation. The examples of ongoing clinical trials involving different bacterial species and strains are listed in Table 2.
Table 2

Examples of ongoing clinical trials on anticancer immunotherapy with live-attenuated bacteria

Therapeutic strain

Title/statusa/identifierb

Tumor type/study objectives and outline/result

Clostridium novyi-NT

Safety study of intratumoral injection of Clostridium novyi-NT spores to treat patients with solid tumors that have not responded to standard therapies

O NCT01924689

 Advanced solid tumor malignancies

 Safety and tolerability of intratumoral administration of C. novyi-NT spore; additional outcome measures: antitumor activity, bacterial load in the blood, and host immune response

Listeria monocytogenes

ADXS11-001

A phase II evaluation of ADXS11-001 in the treatment of persistent or recurrent squamous or non-squamous cell carcinoma of the cervix

O NCT01266460

 Cervix carcinoma

 Three intravenous administrations (every 28 days) of bacteria expressing HPV-16-E7 tumor-associated antigen fused to fragment of listeriolysin O (LLO); adverse effects, progression-free and overall survival, objective tumor response, and serum cytokine assessment

Listeria monocytogenes

ADXS11-001

BrUOG 276: a phase I/II evaluation of ADXS11-001, mitomycin, 5-fluorouracil (5-FU), and IMRT for anal cancer

O NCT01671488

 Invasive primary squamous, basaloid, or cloacogenic carcinoma of the anal canal

 Safety study of four intravenous doses given once every 28 days in combination with standard chemoradiation (mitomycin, 5-FU, and IMRT); evaluation of 6-month clinical complete response rate, progression-free and overall survival, peripheral, and histologic markers of immune response (T-cell infiltration)

Listeria monocytogenes

ADXS11-001

A phase I, dose-escalation trial of recombinant Listeria monocytogenes (Lm)-based vaccine encoding human papillomavirus genotype 16 target antigens (ADXS11-001) in patients with HPV-16 +ve oropharyngeal carcinoma

O NCT01598792

 Oropharyngeal carcinoma (HPV-16 positive)

 Safety study combined with the assessment of vaccine-induced T-cell responses

Listeria monocytogenes

ADXS11-001

Window of opportunity trial of neoadjuvant ADXS 11-001 vaccination prior to robot-assisted resection of HPV-positive oropharyngeal squamous cell carcinoma

O NCT02002182

 Newly diagnosed squamous cell carcinoma of stage I–IV (T1-3, N0-2b) of the oropharynx

 Safety and efficacy study of two intravenous ADXS11-001 infusions prior to tumor resection; induction of HPV E6/E7 antigen-specific CTLs in peripheral blood will be assessed

Listeria monocytogenes

CRS-100 (currently ANZ-100)

Phase 1 dose-escalation study of safety and tolerability of intravenous CRS-100 in adults with carcinoma and liver metastases

O NCT00327652

 Carcinoma and hepatic metastases

 Safety study to assess maximum tolerated dose of single intravenous administration of L. monocytogenes CRS-100 strain, a derivative of wild-type 10403S strain, attenuated due to deletion of two virulence factors: ActA and internalin B (ΔactAinlB)

 Six patients with colorectal cancer (CRC), two with pancreatic ductal adenocarcinoma (PDA), and one melanoma patient, all with liver metastases, received a single intravenous dose (one of three escalating doses) to determine maximum tolerated dose; all doses were well tolerated; reported adverse events associated with cytokine release were transient

 Decrease of lymphocytes and NK cells numbers in peripheral blood was observed (Le et al. 2012)

Listeria monocytogenes

CRS-207

A phase 1B study to evaluate the safety and induction of immune response of CRS-207 in combination with pemetrexed and cisplatin as front-line therapy in adults with malignant pleural mesothelioma

O NCT01675765

 Malignant pleural mesothelioma

 CRS-207 is derived of CRS-100 modified to produce tumor-associated antigen – human mesothelin under the control of bacterial actA promoter; bacteria will be administered twice in two cycles, in combination with six cycles of chemotherapy; adverse events, induction of antigen-specific immune response, objective tumor response, time to progression, and serum mesothelin will be assessed

Listeria monocytogenes

CRS-207

A phase 2, randomized, multicenter, open-label study of the efficacy and immune response of the sequential administration of GVAX pancreas vaccine alone or followed by CRS-207 in adults with metastatic pancreatic adenocarcinoma

O NCT01417000

 Pancreatic cancer (malignant adenocarcinoma of the pancreas)

 Assessment of safety and immune response to combined treatment with cyclophosphamide, GVAX Pancreas vaccine, and CRS-207, L. monocytogenes bacteria expressing tumor-associated antigen, mesothelin

Listeria monocytogenes

CRS-207

A phase 2B, randomized, controlled, multicenter, open-label study of the efficacy and immune response of GVAX pancreas vaccine (with cyclophosphamide) and CRS 207 compared to chemotherapy or to CRS-207 alone in adults with previously treated metastatic pancreatic adenocarcinoma

O NCT02004262

 Metastatic pancreatic cancer

 Overall survival and adverse events of CRS-207 therapy alone or combined therapy: cyclophosphamide + GVAX Pancreas vaccine + CRS-207 will be assessed

Listeria monocytogenes

CRS-207

A phase 1, open-label, dose-escalation, multiple-dose study of the safety, tolerability, and immune response of CRS-207 in adult subjects with selected advanced solid tumors who have failed or who are not candidates for standard treatment

C NCT00585845

 Treatment-refractory mesothelioma, pancreatic ductal adenocarcinoma (PDA), non-small cell lung cancer (NSCLC), or ovarian cancer

 Safety study to determine dose-limiting toxicities; 17 patients (five mesothelioma, seven PDA, three NSCLC, and two ovarian cancer patients) received up to four doses; all applied doses were well tolerated; mesothelin -specific CD8+ T-cell responses were induced in six out of ten evaluated patients, but the response did not correlate with survival; 37 % of patients lived for at least 15 months (Le et al. 2012)

Listeria monocytogenes

ADU-623

Phase I study of safety and immunogenicity of ADU-623, a live-attenuated Listeria monocytogenes strain (ΔactA/ΔinlB) expressing the EGFRvIII-NY-ESO-1 vaccine, in patients with treated and recurrent WHO grade III/IV astrocytomas

O NCT01967758

 Grade III or grade IV astrocytic tumors

 Safety, tolerability, and immunogenicity of four intravenous doses (every 21 days) of L. monocytogenes strain ADU-623 producing EGFRvIII and NY-ESO-1 tumor antigens; maximum tolerated dose, tumor burden, and humoral and cellular immune responses will be determined

Salmonella typhimurium

VNP20009

A phase I trial of a live, genetically modified Salmonella typhimurium (VNP20009) for the treatment of cancer by intravenous administration

C NCT00004988

 Advanced and/or metastatic solid tumors

 Safety study of a single intravenous injection of attenuated S. typhimurium VNP20009 strain, modified to preferentially colonize tumors; evaluation of dose-related toxicities, selective replication in tumors, and antitumor effects

 Twenty-four patients with metastatic melanoma and one patient with metastatic renal cell carcinoma received a single intravenous dose, in either of three escalating doses to determine the maximum tolerated dose. Dose-limiting toxicities were observed for the highest dose. Tumor colonization was observed only in three patients; none of the patients experienced objective tumor regression

Salmonella typhimurium

VNP20009

A phase I trial of a live, genetically modified Salmonella typhimurium (VNP20009) for the treatment of cancer by intratumoral injection

C NCT00004216

 Advanced or metastatic solid tumors

 Determination of safety, efficacy, and maximum tolerated dose of intratumoral VNP20009 injection

 Maximum tolerated dose has not been reached

Salmonella typhi Ty21a

VXM01 phase I dose-escalation study in patients with locally advanced, inoperable, and stage IV pancreatic cancer to examine safety, tolerability, and immune response to the investigational VEGFR-2 DNA vaccine VXM01

O NCT01486329

 Stage IV pancreatic cancer

 Determination of dose-limiting toxicities and the maximum tolerated dose, immune response, tumor staging according to RECIST criteria, after oral administration of VXM01 – S. Typhi Ty21a strain carrying VEGFR2 coding sequence under the control of eukaryotic promoter in order to deliver cDNA to monocytes and dendritic cells for the antigen presentation

Mycobacterium bovis – Bacillus Calmette-Guérin (Tice strain, Chicago Research Laboratories)

The study conducted in the Surgery Branch of the National Cancer Institute (Bethesda, Maryland) from 1967 to 1970 or in the Division of Oncology, Department of Surgery, UCLA School of Medicine (Los Angeles, California) from 1971 to 1974 (Morton et al. 1974)

C

 Patients with recurrent melanoma, known residual disease, or a high risk of developing recurrence

 36 patients with intracutaneous lesions, treated with intratumoral injections, were the most likely responders to BCG when compared to the patients treated by any other investigated route or to the patients with subcutaneous or visceral metastatic lesions. In the group of 36 patients, 91 % of injected lesions underwent complete regression, 17 % of patients had regression of not injected melanoma nodules, and 31 % of patients remained free of disease from 6 to 74 months following the therapy (Morton et al. 1974)

Mycobacterium bovis – Bacillus Calmette-Guérin in combination with ipilimumab

A phase I study of intralesional Bacillus Calmette-Guérin (BCG) and followed by ipilimumab therapy in patients with advanced metastatic melanoma

O NCT01838200

 Metastatic melanoma

 Safety study of one intralesional BCG injection followed by four intravenous ipilimumab injections every 3 weeks, starting on day 36 after BCG; clinical efficacy and tumor-specific immune responses will be assessed; ipilimumab is an anti-CTLA-4 monoclonal antibody approved for melanoma treatment

Mycobacterium bovis – Bacillus Calmette-Guérin

The SILVA study: Survival in an International Phase III Prospective Randomized LD Small Cell Lung Cancer Vaccination Study with Adjuvant BEC2 and BCG

C NCT00006352

 Limited stage small-cell lung cancer (SCLC); patients must have achieved clinical response to first-line chemotherapy with no evidence of progression or relapse

 Determination of safety, progression-free survival and the patient’s quality of life; patients received five intradermal doses of BCG and monoclonal antibody BEC2 every 2 weeks; BEC2 is an anti-idiotypic antibody that mimics GD3 (ganglioside expressed on tumor cells); vaccination had no impact on outcome of patients nor quality of life (Giaccone et al. 2005)

Mycobacterium bovis – Bacillus Calmette-Guérin in combination with Melaxin (autologous dendritoma vaccine)

Phase II, open-label trial in patients with stage IV malignant melanoma using Melaxin as a cancer vaccine in conjunction with BCG

C NCT00671554

 Melanoma

 Safety and response to treatment were assessed; all three enrolled patients had progression of the disease within 18 months post study completion

aStatus: O, ongoing; C, closed (completed or terminated)

bIdentifier according to www.clinicaltrials.gov database (2014-07-31)

Anticipated High-Impact Results

The perspectives for the development of effective bacterial cancer therapies are within the scope of four vital areas.
  1. 1.

    Modification of bacterial metabolism or application of targeting molecules in order to promote preferential tumor localization and colonization

    The evidence from preclinical studies on Salmonella shows that co-localization of bacteria and tumor antigen is important for therapeutic efficacy, and the intratumoral injection of bacteria more efficiently inhibits tumor growth than systemic administration. The increased bacterial ability to selectively target tumors and infect predominantly cancer cells rather than normal cells was demonstrated to improve therapeutic efficacy (Bereta et al. 2007; Massa et al. 2013). Hence, a lack of tumor regression after S. typhimurium VNP20009 intravenous infusion to patients was initially thought to result from inefficient tumor colonization. However, it was only slightly improved when the bacteria were injected directly into the tumor (Nemunaitis et al. 2003), indicating that bacterial tumor targeting in humans is an issue more complex than expected. It is possible that the increased survival of bacteria in tumor or the increased retaining of bacteria at the tumor site or higher rate of infection of tumor cells or enhanced damage in the tumor tissue due to bacterial invasion of cancer cells, achieved by genetic modifications of bacteria, could improve the clinical outcome.

     
  2. 2.

    Delivery of new tumor antigens or novel molecules with immunostimulatory potential or the combinations of the above

     
  3. 3.

    Personalization of treatment according to clinical status and genetic profile including the genes with predictive value

    Genetic polymorphism may have the prognostic value for bacterial anticancer therapies, as it was already proposed for BCG treatment of bladder cancer. The allele variabilities of inflammation-associated genes (IL-8, TNF , IL-6, TGFβ , COX-2, NF-κB ) and factors that influence innate defense to intracellular parasites, such as NRAMP1 (SLC11A1), were associated with differences in the risk of recurrence (Lima et al. 2012).

     
  4. 4.

    Standardization of novel treatment regimens favoring the effectiveness of immunotherapy

    The superior preclinical efficacy of early intervention, i.e., the administration of bacteria soon after tumor implantation, rather than to animals with large tumors, as well as the efficacy of BCG in recurrence of superficial bladder cancers, supports the notion of bacterial immunotherapy as the therapeutic option for patients with minimal tumor burden.

    Repeated administrations of bacterial therapeutics may help to overcome tumor tolerance, as they would constantly deliver a danger signal consisting of PAMPs and DAMPs that stimulate the maturation of antigen presenting cells and effective T-cell responses. The maintenance therapy could bypass the drawback of single tumor antigen-specific approaches, as they inevitably drive the selection of a novel antigen repertoire. Sustained treatment with nonspecific agents should not accelerate the selection of novel tumor cell variants but rather promote effective tumor recognition and killing. The concept of repeated vaccination aimed at the delivery of a danger signal co-localized with antigen has been proposed by Polly Matzinger as the remedy for low effectiveness of tumor vaccines (Matzinger 2012). The clinical experience with BCG treatment for bladder cancer proved the efficacy of repeated vaccination localized to the tumor lesion.

     

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Cell BiochemistryFaculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian UniversityKrakówPoland

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