Gene Therapy in Treating Patients With Advanced Head and Neck

280 Novel Gene Therapy Approaches

FUS1-nanoparticles and Erlotinib in Stage IV Lung Cancer

Study to Determine the Maximum Tolerated Dose of LErafAON in Patients With Advanced Solid

EphA2 Gene Targeting Using Neutral Liposomal Small Interfering RNA Delivery

C-VISA BikDD: Liposome in Advanced Pancreatic Cancer

permission from reference [4].

Cancer

Tumors

**7.4. Polymeric gene delivery nanosystems**

Self-assembled micellar nanoformulations are another type of NPs that are widely used as gene delivery nanosystems, in which the positively charged polymers can interact with the negatively charged nucleic acids forming nanomicelles under sonication. Cationic polymers (e.g., linear and branched polyethyleneimine (l-PEI and b-PEI), poly(L-lysine), and polyami‐ doamine (PAMAM) dendrimers) can condense the nucleic acids forming polyplexes as either uni-molecular or multi-molecular complexes. Unfortunately, similar to CLs, cationic polymers can also induce intrinsic toxicogenomics [86] and cytotoxicity [16]. To achieve efficient targetspecific gene transfer, these polymers can covalently be modified by conjugating targeting ligands. The ligand-armed systems can then target the cells that present the specific cellular receptors. Because of their nano-scaled size (100-200 nm in diameter), they can be taken up by cells through receptor-mediated endocytosis. For example, a Trf-modified cyclodextrin polymer-based gene delivery nanosystem has been developed. These Trf-armed PEGylated cyclodextrin show increased stability in biological fluids and active targeting potential via transferrin, retaining high binding affinity toward Trf receptor and profound transfection of the target leukemia cells (K562 cells) through both passive and active targeting [105]. Recently, Hatefi's group engineered a novel multi-domain biopolymer, consisting of: 1) repeating units of arginine and histidine to condense pDNA and lyse endosome membranes, 2) a HER2 affibody as targeting moety, 3) a pH responsive fusogenic peptide to destabilize endosome membranes and enhance endosomolytic activity of histidine residues, and 4) a nuclear localization signal to enhance translocation of pDNA towards the cell nucleus [106]. They showed that pDNA was condensed into biopolymeric Nps and protected from serum endo‐ nucleases, while targeting HER2 positive cancer cells, and metabolized by endogenous furin enzymes to reduce potential toxicity. Later on, synthesis of a targeted PEI polymer was reported, in which the PEI polymer was conjugated to angiogenic vessel-homing peptide Ala-Pro-Arg-Pro-Gly (APRPG) through PEG spacer [107]. The PEI-PEG-APRPG was shown to effectively condense siRNA into 20-50 nm NPs that can substantially impose inhibitory effects in vitro with profound EPR in vivo targeting tumor vasculature through VEGF.

normal cells in brain using a rat model. It was found that the tumoricidal bystander effect in the HSV-TK gene therapy using MSCs and GCV does not injure normal brain tissues [114]. In another study, umbilical cord blood MSCs were used to deliver the transgenic LIGHT (TNFSF14) to the target tumor cells in vivo. The transfected MSCs with lentiviral vectors carrying LIGHT genes demonstrated a strong suppressive effect on tumor growth, in which pathological sections of the tumor tissues showed significant induction of apoptosis and occurrence of tumor necrosis in tumor cells [115]. The potential of genetically modified MSCs expressing IFN- β was assessed in an immuno-competent mouse model of prostate cancer lung metastasis. Significant reduction in tumor volume in lungs was seen following IFN- β ex‐ pressing MSC therapy, perhaps through induction of apoptosis and increase in the natural kill cell activity [116]. The MSC-based gene therapy is still in its infancy era and need much more investigation prior to its clinical applications even though the MSCs themselves are under

Cancer Gene Therapy: Targeted Genomedicines

http://dx.doi.org/10.5772/54739

283

Tissue-specific promoters (TSPs), a powerful tool for decreasing the toxicity of cancer gene therapy to normal tissues, have been used as targeted gene therapy approach. TSPs have been utilized for specific mutation compensation or delivery of prodrug-converting enzymes and also for controlling crucial viral replication regulators and consequent restriction of replication to tumor cells [118]. The safety and contingency of this approach has been shown in some initial clinical trials [119]. Of these, the cytomegalovirus (CMV) immediate-early promoter is often harnessed in gene therapy since it can express target genes at high levels in tumor cells. Lin et al. (2001) examined the effects of the involucrin (INV), keratin 14 (K14) and CMV promoters on the expression of the reporter gene beta-galactosidase. They introduce the plasmid DNA to BALB/c mice using a gene gun, and examined the skin biopsies. They found that the K14 and INV promoter constructs could induce the beta-galactosidase gene expression only in the epidermis, while the CMV promoter was able to elicit gene expression in both the dermis and epidermis [120]. To increase promoter strength while maintaining tissue specific‐ ity, Qiao et al. (2002) constructed a recombinant adenovirus encompassing a binary promoter system with a tumor-specific promoter carcinoembryonic antigen (CEA) driving a transcrip‐ tion transactivator with capability to express a HSV-TK. After successful application in vitro, they employed noninvasive nuclear imaging using a radioiodinated nucleoside (fraluridine (FIAU)) serving as a substrate for HSV-TK in BALB/C mice model. The results indicated the accumulated radioactivity only in the area of CEA-positive tumors after intratumoral injection, in which significantly less spread was observed to the adjacent liver tissue [121]. In another study, a vector with the human minimal tyrosinase promoter and two human enhancer elements (2hE-hTyrP) was compared with different hybrid promoter constructs containing tyrosinase regulatory sequences and the viral simian virus 40 (SV40) promoters. The hybrid SV40-based promoters were effective in vitro, and the in vivo tissue specificity of the 2hEhTyrP vector was demonstrated in subcutaneous xenografted tumors model [122]. Another plausible approach to specifically target tumor cells for gene expression is to harness promoter elements that become activated in chemotherapy-resistant tumor cells [123]. In addition to TSPs, inducible promoters (IPs) can be exploited to minimize target gene expression in normal

clinical trials [117].

**8.3. Tissue–specific promoters and inducible promoters**

## **8. Tissue specific gene therapy**

In addition to targeted nanomedicine gene therapies, tissue specific gene therapy provides a robust targeted approach. We describe some of the cell/tissue specific applications in the following sections.

#### **8.1. Cancer Stem Cells (CSCs)**

Some of the transit-amplifying cells in the cancer population appear to remain immature within the differentiating cells. These classes of cells that act as cancer cells progenitor are called cancer stem cells, which are deemed to be one of the reasons for cancer relapse that are hardly responsive for treatment. The poor prognosis and responsiveness of patients with relapsed aggressive metastatic tumors necessitate the development of more effective tumor-selective therapies towards cells that cause such relapse, while the conventional therapy target the differentiated cancer cells. Therefore, to be maximally effective, gene therapy of cancer should target both the resting stem cells and the proliferating cells of the cancer [108]. To this end, several translational approaches have been undertaken to target CSCs, including use of oncolytic viruses that may offer an effective way to specifically target and eradicate CSCs. Of these, conditionally replicative adenoviruses (CRAd) are considered as promising virotherapy systems [109]. Considering the plasticity of CSCs, apoptosis-inducing strategy can be used to eliminate these cells by harnessing genes such as TRAIL, BCL-2 family and XIAP as targeted therapies [110].

#### **8.2. Mesenchymal Stem Cells (MSCs) as a gene therapy carrier**

To date, as a personalized medicine, cell-based therapy of cancer has been considered as a promising modality. Of these, MSCs seem to hold great potential as targeted-delivery vehicle in cancer gene therapy [111]. Their propagation in culture is simple, also shows contingency toward genetic modification in order to express therapeutic proteins. Above all, MSCs possess inherent tumor-tropic and migratory properties that allow them to serve as robust cell based carrier as targeted drug delivery systems for isolated tumors and metastatic diseases [112]. In a study, the migration ability of MSCs toward prostate cancer cells (in vitro and in vivo) and incorporating into the tumor mass was investigated. The infected cells with HSV-TK gene were shown to maintain their tumor tropism capabilities and significantly inhibited the growth of subcutaneous PC3 prostate cancer xenografts in nude mice in the presence of GCV [113]. Similar strategy was applied to evaluate the impact of the suicide gene therapy by MSCs in normal cells in brain using a rat model. It was found that the tumoricidal bystander effect in the HSV-TK gene therapy using MSCs and GCV does not injure normal brain tissues [114]. In another study, umbilical cord blood MSCs were used to deliver the transgenic LIGHT (TNFSF14) to the target tumor cells in vivo. The transfected MSCs with lentiviral vectors carrying LIGHT genes demonstrated a strong suppressive effect on tumor growth, in which pathological sections of the tumor tissues showed significant induction of apoptosis and occurrence of tumor necrosis in tumor cells [115]. The potential of genetically modified MSCs expressing IFN- β was assessed in an immuno-competent mouse model of prostate cancer lung metastasis. Significant reduction in tumor volume in lungs was seen following IFN- β ex‐ pressing MSC therapy, perhaps through induction of apoptosis and increase in the natural kill cell activity [116]. The MSC-based gene therapy is still in its infancy era and need much more investigation prior to its clinical applications even though the MSCs themselves are under clinical trials [117].

#### **8.3. Tissue–specific promoters and inducible promoters**

nucleases, while targeting HER2 positive cancer cells, and metabolized by endogenous furin enzymes to reduce potential toxicity. Later on, synthesis of a targeted PEI polymer was reported, in which the PEI polymer was conjugated to angiogenic vessel-homing peptide Ala-Pro-Arg-Pro-Gly (APRPG) through PEG spacer [107]. The PEI-PEG-APRPG was shown to effectively condense siRNA into 20-50 nm NPs that can substantially impose inhibitory effects

In addition to targeted nanomedicine gene therapies, tissue specific gene therapy provides a robust targeted approach. We describe some of the cell/tissue specific applications in the

Some of the transit-amplifying cells in the cancer population appear to remain immature within the differentiating cells. These classes of cells that act as cancer cells progenitor are called cancer stem cells, which are deemed to be one of the reasons for cancer relapse that are hardly responsive for treatment. The poor prognosis and responsiveness of patients with relapsed aggressive metastatic tumors necessitate the development of more effective tumor-selective therapies towards cells that cause such relapse, while the conventional therapy target the differentiated cancer cells. Therefore, to be maximally effective, gene therapy of cancer should target both the resting stem cells and the proliferating cells of the cancer [108]. To this end, several translational approaches have been undertaken to target CSCs, including use of oncolytic viruses that may offer an effective way to specifically target and eradicate CSCs. Of these, conditionally replicative adenoviruses (CRAd) are considered as promising virotherapy systems [109]. Considering the plasticity of CSCs, apoptosis-inducing strategy can be used to eliminate these cells by harnessing genes such as TRAIL, BCL-2 family and XIAP as targeted

To date, as a personalized medicine, cell-based therapy of cancer has been considered as a promising modality. Of these, MSCs seem to hold great potential as targeted-delivery vehicle in cancer gene therapy [111]. Their propagation in culture is simple, also shows contingency toward genetic modification in order to express therapeutic proteins. Above all, MSCs possess inherent tumor-tropic and migratory properties that allow them to serve as robust cell based carrier as targeted drug delivery systems for isolated tumors and metastatic diseases [112]. In a study, the migration ability of MSCs toward prostate cancer cells (in vitro and in vivo) and incorporating into the tumor mass was investigated. The infected cells with HSV-TK gene were shown to maintain their tumor tropism capabilities and significantly inhibited the growth of subcutaneous PC3 prostate cancer xenografts in nude mice in the presence of GCV [113]. Similar strategy was applied to evaluate the impact of the suicide gene therapy by MSCs in

**8.2. Mesenchymal Stem Cells (MSCs) as a gene therapy carrier**

in vitro with profound EPR in vivo targeting tumor vasculature through VEGF.

**8. Tissue specific gene therapy**

following sections.

282 Novel Gene Therapy Approaches

therapies [110].

**8.1. Cancer Stem Cells (CSCs)**

Tissue-specific promoters (TSPs), a powerful tool for decreasing the toxicity of cancer gene therapy to normal tissues, have been used as targeted gene therapy approach. TSPs have been utilized for specific mutation compensation or delivery of prodrug-converting enzymes and also for controlling crucial viral replication regulators and consequent restriction of replication to tumor cells [118]. The safety and contingency of this approach has been shown in some initial clinical trials [119]. Of these, the cytomegalovirus (CMV) immediate-early promoter is often harnessed in gene therapy since it can express target genes at high levels in tumor cells. Lin et al. (2001) examined the effects of the involucrin (INV), keratin 14 (K14) and CMV promoters on the expression of the reporter gene beta-galactosidase. They introduce the plasmid DNA to BALB/c mice using a gene gun, and examined the skin biopsies. They found that the K14 and INV promoter constructs could induce the beta-galactosidase gene expression only in the epidermis, while the CMV promoter was able to elicit gene expression in both the dermis and epidermis [120]. To increase promoter strength while maintaining tissue specific‐ ity, Qiao et al. (2002) constructed a recombinant adenovirus encompassing a binary promoter system with a tumor-specific promoter carcinoembryonic antigen (CEA) driving a transcrip‐ tion transactivator with capability to express a HSV-TK. After successful application in vitro, they employed noninvasive nuclear imaging using a radioiodinated nucleoside (fraluridine (FIAU)) serving as a substrate for HSV-TK in BALB/C mice model. The results indicated the accumulated radioactivity only in the area of CEA-positive tumors after intratumoral injection, in which significantly less spread was observed to the adjacent liver tissue [121]. In another study, a vector with the human minimal tyrosinase promoter and two human enhancer elements (2hE-hTyrP) was compared with different hybrid promoter constructs containing tyrosinase regulatory sequences and the viral simian virus 40 (SV40) promoters. The hybrid SV40-based promoters were effective in vitro, and the in vivo tissue specificity of the 2hEhTyrP vector was demonstrated in subcutaneous xenografted tumors model [122]. Another plausible approach to specifically target tumor cells for gene expression is to harness promoter elements that become activated in chemotherapy-resistant tumor cells [123]. In addition to TSPs, inducible promoters (IPs) can be exploited to minimize target gene expression in normal cells. Harnessing the IPs, the timing of the gene expression can be modulated and controlled. Of a large number of inducible systems developed, only a few were translated into clinical gene therapy trials, including radiation-inducible genes [124]. Using this cancer gene therapy modality, promoters of radiation-inducible genes are exploited to drive transcription of transgenes in response to radiation, resulting in increased responsiveness of cancer cells to radiotherapy. These constructs, delivered by adenoviral vectors, can activate a transgene encoding a cytotoxic protein in tumor cells, in which the tumoricidal effects can be then localized temporally and spatially by X-rays. Perhaps, TNFerade (GenVec, Inc) is the best paradigm, which is an adenoviral vector containing radiation-inducible elements of the early growth response-1 promoter upstream of a cDNA encoding human TNF-α [125]. It has been translated into several clinical applications, e.g., as the first-line treatment of locally advanced pancreatic cancer in combination with 5-FU and radiotherapy [126]. However, it has not been approved.

## **9. Translational hurdles**

Over the last couple of decades, various gene-based medicines have been developed in vitro with great potential to be translated for in vivo uses in clinic. It has been evinced that the gene therapy approach by virtue carries a certain degree of risk, thus the design and development of such modality need to meet the entire scientific and regulatory requirements. Some of the risks are procedural hazards (e.g. for parenteral medicaments), while some others happen to be specific to the genomedicine per se (e.g., immunologic reaction of viral vectors or nonspe‐ cific impacts of the delivered genes). All scientific, ethical, legal and social implications of this novel modality to genetic disease are involved for its successful translation. Fig. 7 schematically epitomizes the complexity of the steps for development and translation of gene-medicine for immunogene therapy of ovarian cancer.

**10. Final remarks**

cine.

associated cells, stromal cells and CSCs.

Cancer gene therapy continues to grow even though clinical applications of this approach demand further investigations. Trajectory of gene therapy shows great impacts of genomedi‐ cines (i.e., As-ODNs, siRNA, Ribozymes, DNAzyme) both cell based and animal models, while tumor antigen-specific vaccines and DNA vaccines appear to be the most promising modali‐ ties. While suicide gene therapy, immunogene therapy and angiogenic gene therapy continue to become a mature modality, integration of nanotechnology toward development of multi‐ functional nanoparticles appear to provide a resilient, yet versatile, platform for targeted cancer gene therapy as "nano-genoceuticals". Rise of MSCs based cancer gene therapies may also open a new chapter as "cyto-genoceuticals". More than 65% of the gene therapy trials have been devoted to the cancer diseases; however, less than 3% of these trials have been progressed to the phase II/III stages and only few to the phase IV stage. The first approved cancer vaccination (Sipuleucel-T) has resulted in great clinical corollaries. Still many tumor suppressor and apoptosis-inducing genes can be evaluated for clinical applications. Attribut‐ able to intricate nature of malignant diseases, to achieve more effective gene therapy against cancer, genomedicines need to be advanced to be able to holistically target the most cancer causing genes. It is also essential to target both the tumor cells and other cancer associated players of the tumor microenvironment including: tumor microvasculature and tumor

**Figure 7.** Schematic representation of translational approach for immunogene therapy of ovarian cancer. A) Design of the immunogene therapy based on a) different strategies of gene therapy and b) different gene delivery systems. B) Anatomical and cellular complexity of the ovarian cancer cells that need to be specifically targeted using advanced gene delivery systems and cancer marker molecules (CMMs). C) Various stages for translation of immunogenetic medi‐

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285

In immunogene therapy, for example, stimulation of the cellular immune system to recognize and obliterate the cancer cells with genes encoding a variety of cytokines (e.g., interleukin-2 (IL-2), granulocyte - macrophage colony-stimulating factor (GM-CSF), co-stimulatory mole‐ cules such as CD80 and CD86) can 1) increase the immunogenicity of the transfected autolo‐ gous tumour cells, 2) increase the likelihood of generating a tumor-specific cytotoxic Tlymphocyte (CTL) response. In the ex vivo transfection of antigen-presenting cells (APCs), the cells are transfected with a gene encoding a tumor-specific antigen (e.g., carcinoembryonic antigen, CEA) that is presented by major histocompatibility complex (MHC) class I molecules to antigen-specific CTLs via the T-cell receptor (TCR). Stimulated CTLs can find and eradicate the residual CEA-expressing tumor cells, in which GM-CSF can increase the activation of APCs and their migration into the tumor microenvironment [1].

Further, some degree of knowledge of industrial drug development is critical for innovation in this new sector, while healthcare systems and industries need to undertake more translatable approaches to fasten the in terms of cancer gene therapy.

**Figure 7.** Schematic representation of translational approach for immunogene therapy of ovarian cancer. A) Design of the immunogene therapy based on a) different strategies of gene therapy and b) different gene delivery systems. B) Anatomical and cellular complexity of the ovarian cancer cells that need to be specifically targeted using advanced gene delivery systems and cancer marker molecules (CMMs). C) Various stages for translation of immunogenetic medi‐ cine.

## **10. Final remarks**

cells. Harnessing the IPs, the timing of the gene expression can be modulated and controlled. Of a large number of inducible systems developed, only a few were translated into clinical gene therapy trials, including radiation-inducible genes [124]. Using this cancer gene therapy modality, promoters of radiation-inducible genes are exploited to drive transcription of transgenes in response to radiation, resulting in increased responsiveness of cancer cells to radiotherapy. These constructs, delivered by adenoviral vectors, can activate a transgene encoding a cytotoxic protein in tumor cells, in which the tumoricidal effects can be then localized temporally and spatially by X-rays. Perhaps, TNFerade (GenVec, Inc) is the best paradigm, which is an adenoviral vector containing radiation-inducible elements of the early growth response-1 promoter upstream of a cDNA encoding human TNF-α [125]. It has been translated into several clinical applications, e.g., as the first-line treatment of locally advanced pancreatic cancer in combination with 5-FU and radiotherapy [126]. However, it has not been

Over the last couple of decades, various gene-based medicines have been developed in vitro with great potential to be translated for in vivo uses in clinic. It has been evinced that the gene therapy approach by virtue carries a certain degree of risk, thus the design and development of such modality need to meet the entire scientific and regulatory requirements. Some of the risks are procedural hazards (e.g. for parenteral medicaments), while some others happen to be specific to the genomedicine per se (e.g., immunologic reaction of viral vectors or nonspe‐ cific impacts of the delivered genes). All scientific, ethical, legal and social implications of this novel modality to genetic disease are involved for its successful translation. Fig. 7 schematically epitomizes the complexity of the steps for development and translation of gene-medicine for

In immunogene therapy, for example, stimulation of the cellular immune system to recognize and obliterate the cancer cells with genes encoding a variety of cytokines (e.g., interleukin-2 (IL-2), granulocyte - macrophage colony-stimulating factor (GM-CSF), co-stimulatory mole‐ cules such as CD80 and CD86) can 1) increase the immunogenicity of the transfected autolo‐ gous tumour cells, 2) increase the likelihood of generating a tumor-specific cytotoxic Tlymphocyte (CTL) response. In the ex vivo transfection of antigen-presenting cells (APCs), the cells are transfected with a gene encoding a tumor-specific antigen (e.g., carcinoembryonic antigen, CEA) that is presented by major histocompatibility complex (MHC) class I molecules to antigen-specific CTLs via the T-cell receptor (TCR). Stimulated CTLs can find and eradicate the residual CEA-expressing tumor cells, in which GM-CSF can increase the activation of APCs

Further, some degree of knowledge of industrial drug development is critical for innovation in this new sector, while healthcare systems and industries need to undertake more translatable

approved.

284 Novel Gene Therapy Approaches

**9. Translational hurdles**

immunogene therapy of ovarian cancer.

and their migration into the tumor microenvironment [1].

approaches to fasten the in terms of cancer gene therapy.

Cancer gene therapy continues to grow even though clinical applications of this approach demand further investigations. Trajectory of gene therapy shows great impacts of genomedi‐ cines (i.e., As-ODNs, siRNA, Ribozymes, DNAzyme) both cell based and animal models, while tumor antigen-specific vaccines and DNA vaccines appear to be the most promising modali‐ ties. While suicide gene therapy, immunogene therapy and angiogenic gene therapy continue to become a mature modality, integration of nanotechnology toward development of multi‐ functional nanoparticles appear to provide a resilient, yet versatile, platform for targeted cancer gene therapy as "nano-genoceuticals". Rise of MSCs based cancer gene therapies may also open a new chapter as "cyto-genoceuticals". More than 65% of the gene therapy trials have been devoted to the cancer diseases; however, less than 3% of these trials have been progressed to the phase II/III stages and only few to the phase IV stage. The first approved cancer vaccination (Sipuleucel-T) has resulted in great clinical corollaries. Still many tumor suppressor and apoptosis-inducing genes can be evaluated for clinical applications. Attribut‐ able to intricate nature of malignant diseases, to achieve more effective gene therapy against cancer, genomedicines need to be advanced to be able to holistically target the most cancer causing genes. It is also essential to target both the tumor cells and other cancer associated players of the tumor microenvironment including: tumor microvasculature and tumor associated cells, stromal cells and CSCs.

## **Author details**

Yadollah Omidi1,2, Jaleh Barar1,2 and George Coukos1,3

1 Ovarian Cancer Research Center, Perelman School of Medicine, University of Pennsylva‐ nia, Philadelphia, PA, USA

feron-beta gene therapy and 5-fluorouracil significantly reduces growth of metastatic human breast cancer in SCID mouse lungs. Cancer Invest. (2008). , 26(7), 662-70.

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http://dx.doi.org/10.5772/54739

287

[11] Stevenson, F. K, Ottensmeier, C. H, & Rice, J. DNA vaccines against cancer come of

[12] Vassaux, G, & Martin-duque, P. Use of suicide genes for cancer gene therapy: study

[13] Wacheck, V, & Zangemeister-wittke, U. Antisense molecules for targeted cancer ther‐

[14] Devi, G. R. siRNA-based approaches in cancer therapy. Cancer Gene Ther. (2006). ,

[15] Dougherty, G. J, & Dougherty, S. T. Exploiting the tumor microenvironment in the development of targeted cancer gene therapy. Cancer Gene Ther. (2009). , 16(3),

[16] Kafil, V, & Omidi, Y. Cytotoxic impacts of linear and branched polyethylenimine

[17] Omidi, Y, Hollins, A. J, Benboubetra, M, Drayton, R, Benter, I. F, & Akhtar, S. Toxico‐ genomics of non-viral vectors for gene therapy: a microarray study of lipofectin- and oligofectamine-induced gene expression changes in human epithelial cells. J Drug

[18] Omidi, Y, Hollins, A. J, Drayton, R. M, & Akhtar, S. Polypropylenimine dendrimerinduced gene expression changes: the effect of complexation with DNA, dendrimer

[19] Hollins, A. J, Omidi, Y, Benter, I. F, & Akhtar, S. Toxicogenomics of drug delivery systems: Exploiting delivery system-induced changes in target gene expression to en‐

[20] Omidi, Y, Barar, J, Heidari, H. R, Ahmadian, S, Yazdi, H. A, & Akhtar, S. Microarray analysis of the toxicogenomics and the genotoxic potential of a cationic lipid-based gene delivery nanosystem in human alveolar epithelial a549 cells. Toxicol Mech

[21] Barar, J, Hamzeiy, H, Mortazavi-tabatabaei, S. A, Hashemi-aghdam, S. E, & Omidi, Y. Genomic signature and toxicogenomics comparison of polycationic gene delivery nanosystems in human alveolar epithelial A549 cells. Daru. (2009). , 17(3), 139-47.

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2 Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz Univer‐ sity of Medical Sciences, Tabriz, Iran

3 Ludwig Center for Cancer Research, University of Lausanne, Lausanne, Switzerland

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**Author details**

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Yadollah Omidi1,2, Jaleh Barar1,2 and George Coukos1,3

1 Ovarian Cancer Research Center, Perelman School of Medicine, University of Pennsylva‐

2 Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz Univer‐

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**Chapter 12**

**Identification and Validation of Targets for Cancer**

The link between immune responses and cancer is evident from findings such as, a com‐ promised immune system resulting in an increased tumour incidence [1] and cancer pa‐ tient sera evidencing recognition of autologous cancer antigens [2]. The identification of tumour associated antigens (TAAs) plays a central role in our understanding of how can‐ cer cells can inhibit the immune system and how we can overcome this tumour immune suppression to break tolerance and achieve cancer destruction [3]. Antibodies reacting with TAAs on the surface of cancer cells provoke an extremely effective immune re‐ sponse [4] which can be exquisitely specific to the tumour cells present in the body. However not many surface proteins are present on tumour cells and limited otherwise in

TAAs are most often proteins which have acquired mutations or have elevated expression levels which are expressed at the sub-cellular level. The ideal immunotherapy targets should also play a role in tumour progression [5]. For example p53 [6] is one of the most desirable targets for immunotherapy – targeting p53 can kill both the evolving tumour cell population and any cancer "stem" cell which harbours this as an early tumourigenesis aberration and supports further tumour growth. In addition, a number of tumour antigens have been shown

In this chapter, we will examine how tumour antigens are identified and characterised to demonstrate their potential as immunotherapy targets and examine their role as biomarkers

and reproduction in any medium, provided the original work is properly cited.

© 2013 Khan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

for treatment response and patient survival, and targets for personalised therapies.

**Immunotherapy: From the Bench-to-Bedside**

Ghazala Khan, Suzanne E. Brooks, Frances Denniss,

Dagmar Sigurdardottir and Barbara-ann Guinn

Additional information is available at the end of the chapter

expression to healthy non-essential tissues.

to be useful biomarkers for cancer diagnosis [7] and survival [8].

http://dx.doi.org/10.5772/54698

**1. Introduction**

## **Identification and Validation of Targets for Cancer Immunotherapy: From the Bench-to-Bedside**

Ghazala Khan, Suzanne E. Brooks, Frances Denniss, Dagmar Sigurdardottir and Barbara-ann Guinn

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54698

## **1. Introduction**

The link between immune responses and cancer is evident from findings such as, a com‐ promised immune system resulting in an increased tumour incidence [1] and cancer pa‐ tient sera evidencing recognition of autologous cancer antigens [2]. The identification of tumour associated antigens (TAAs) plays a central role in our understanding of how can‐ cer cells can inhibit the immune system and how we can overcome this tumour immune suppression to break tolerance and achieve cancer destruction [3]. Antibodies reacting with TAAs on the surface of cancer cells provoke an extremely effective immune re‐ sponse [4] which can be exquisitely specific to the tumour cells present in the body. However not many surface proteins are present on tumour cells and limited otherwise in expression to healthy non-essential tissues.

TAAs are most often proteins which have acquired mutations or have elevated expression levels which are expressed at the sub-cellular level. The ideal immunotherapy targets should also play a role in tumour progression [5]. For example p53 [6] is one of the most desirable targets for immunotherapy – targeting p53 can kill both the evolving tumour cell population and any cancer "stem" cell which harbours this as an early tumourigenesis aberration and supports further tumour growth. In addition, a number of tumour antigens have been shown to be useful biomarkers for cancer diagnosis [7] and survival [8].

In this chapter, we will examine how tumour antigens are identified and characterised to demonstrate their potential as immunotherapy targets and examine their role as biomarkers for treatment response and patient survival, and targets for personalised therapies.

© 2013 Khan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. Which tumour antigens?**

Many of the tumour antigens identified by serological identification of antigens by recombi‐ nant expression cloning (SEREX) can be classified into one or more categories which are: cancer-testis, mutational, differentiation, amplified/overexpressed, splice variant and viral antigens [9]. Cancer-testis (CT) antigens [10] have been found to be highly expressed in tu‐ mours but not in normal tissues with the exception of immunologically protected sites (those tissues which lack major histocompatibility complex (MHC) class I and therefore do not present self-antigens). CT antigens make attractive targets as due to their limited expres‐ sion, they should therefore induce specific anti-tumour immune responses and less toxicity to healthy tissue [11]. However some debate remains as to the definition of a CT antigen [12] with some suggestion of differential levels of expression and others suggesting no expres‐ sion in normal tissues expect those in immunologically protected sites (such as ovary, pla‐ centa and testis). TAAs that are frequently found in tumours and provide excellent targets for immunotherapy include Wilms tumour 1 [13] and PRAME [14].

**3.3. Serological identification of antigens by recombinant expression cloning (SEREX)**

ry screening, prior to cDNA sequencing of phagemid inserts [31].

preparative gel and indentified using mass spectrometry [33, 34].

**3.4. Serological proteome analysis (SERPA)**

**3.5. CDNA microarrays**

**3.6. Mass Spectroscopy (MS)**

Serological identification of antigens by recombinant expression cloning (SEREX) provided a much needed boost to the area of antigen identification at a time when few cancer antigen identification options existed [2]. SEREX was not limited to immunogenic cancers such as melanoma and has now been used to identify more than 2,000 antigens [22-23] in a large range of different solid [24-26] and haematological malignancies [27-30]. cDNA libraries are created from tumour samples, cell lines or healthy normal donor cells (such as testes). RNA from these cells were reverse transcribed and inserted as cDNA into phage vectors and ex‐ pressed as recombinant proteins on the capsid surface of phage which survived on permis‐ sive E.coli. Expressed proteins were transferred to nitrocellulose membranes and following the removal of excess E.coli waste, phage plaques were immunoscreened using pre-cleared patient sera. Any positive plaques were isolated, eluted and used for secondary confirmato‐

Identification of Targets for Immunotherapy http://dx.doi.org/10.5772/54698 299

Serological proteome analysis (SERPA) was first described by Klade *et al* in 2001 [32]. Proteins were extracted from primary tumours or cell lines, separated concurrently on two 2D gels and transferred to nitrocellulose membranes. A third gel is stained with Coomassie Blue as a prepa‐ rative gel. The membranes are incubated with cancer patient's sera and normal control. The two gels are directly compared and any bright spots on the cancer sera membrane were cut from the

The differential expression of tumour antigens and/or protein biomarkers between cell and dis‐ ease subtypes have been directly compared on cDNA microarrays and has allowed our im‐ proved understanding of lymphomas [35] and aided our development of personalised therapies [36]. Microarray technology is able to distinguish between different subtypes of a par‐ ticular cancer as well as identify the expression of novel antigens [37]. Minimal residual disease is a very important tool in the detection of impending relapse in patients who have had some form of treatment. Markers for minimal residual disease in acute lymphocytic leukaemia were identified by gene profiling [38]. cDNA microarray has been used to identify the frequency of elevated tumour antigen expression in acute myeloid leukaemia [28] and also associations be‐ tween specific cytogenetic abnormalities and relative levels of tumour antigen expression [39]. Micorarray has also been used to elucidate the possible function of tumour antigens such as Synovial Sarcoma X breakpoint 2 Interacting Protein (SSX2IP) in the subversion of cells har‐ bouring cytogenetic abnormalities (t(8;21) associated with mitotic spindle failure and the asso‐ ciation between the elevated expression of some tumour antigens (SSX2IP, RHAMM and SURVIVIN) at disease presentation and patient survival [8] in acute myeloid leukaemia.

Mass Spectroscopy (MS) involves the analysis of peptides eluted from the MHC of antigen presenting cells [40-42] or proteins in serum [43]. This area is reviewed more completely in the

## **3. Identification of tumour antigens**

Strategies are required to help identify potential targets which can be used for cancer immuno‐ therapy. Some of the most commonly used and successful techniques are described as follows:-

#### **3.1. Reverse-Transcription-Polymerase Chain Reaction (RT-PCR) and real-time PCR (RQ-PCR)**

Reverse-transcription-polymerase chain reaction (RT-PCR) and real-time PCR (RQ-PCR) has been used to examine known TAA expression in a range of solid and haematological malig‐ nancies [15-19]. Although this has provided important expression information and a good starting point to identify potential antigenic targets in a range of cancers, these studies are entirely limited to tumour antigens which had already been discovered.

#### **3.2. Representational difference analysis**

Representational difference analysis was developed by Thierry Boon's group and used to discover a number of CT antigens [20, 21] including the MAGE family of antigens, typically from melanoma with one exception, RAGE, from renal cancer. Briefly, total RNA was ex‐ tracted from normal tissue (driver) and a tumour sample (tester) and used to construct dou‐ ble-stranded cDNA. Both cDNA samples were digested with the restriction enzymes DpnII and ligated to adapters which contained primer binding sites. The fragments were amplified by PCR, the adapters removed and new adapters for unrelated primers ligated to the tester. The tester and driver were then mixed and hybridized leading to three combinations of product: driver-driver (no amplification), tester-driver (linear amplification) and tester-test‐ er (exponential amplification). A further two hybridization and amplification steps generate greater variation in the products which are subsequently cloned and sequenced.

#### **3.3. Serological identification of antigens by recombinant expression cloning (SEREX)**

Serological identification of antigens by recombinant expression cloning (SEREX) provided a much needed boost to the area of antigen identification at a time when few cancer antigen identification options existed [2]. SEREX was not limited to immunogenic cancers such as melanoma and has now been used to identify more than 2,000 antigens [22-23] in a large range of different solid [24-26] and haematological malignancies [27-30]. cDNA libraries are created from tumour samples, cell lines or healthy normal donor cells (such as testes). RNA from these cells were reverse transcribed and inserted as cDNA into phage vectors and ex‐ pressed as recombinant proteins on the capsid surface of phage which survived on permis‐ sive E.coli. Expressed proteins were transferred to nitrocellulose membranes and following the removal of excess E.coli waste, phage plaques were immunoscreened using pre-cleared patient sera. Any positive plaques were isolated, eluted and used for secondary confirmato‐ ry screening, prior to cDNA sequencing of phagemid inserts [31].

#### **3.4. Serological proteome analysis (SERPA)**

Serological proteome analysis (SERPA) was first described by Klade *et al* in 2001 [32]. Proteins were extracted from primary tumours or cell lines, separated concurrently on two 2D gels and transferred to nitrocellulose membranes. A third gel is stained with Coomassie Blue as a prepa‐ rative gel. The membranes are incubated with cancer patient's sera and normal control. The two gels are directly compared and any bright spots on the cancer sera membrane were cut from the preparative gel and indentified using mass spectrometry [33, 34].

#### **3.5. CDNA microarrays**

**2. Which tumour antigens?**

298 Novel Gene Therapy Approaches

Many of the tumour antigens identified by serological identification of antigens by recombi‐ nant expression cloning (SEREX) can be classified into one or more categories which are: cancer-testis, mutational, differentiation, amplified/overexpressed, splice variant and viral antigens [9]. Cancer-testis (CT) antigens [10] have been found to be highly expressed in tu‐ mours but not in normal tissues with the exception of immunologically protected sites (those tissues which lack major histocompatibility complex (MHC) class I and therefore do not present self-antigens). CT antigens make attractive targets as due to their limited expres‐ sion, they should therefore induce specific anti-tumour immune responses and less toxicity to healthy tissue [11]. However some debate remains as to the definition of a CT antigen [12] with some suggestion of differential levels of expression and others suggesting no expres‐ sion in normal tissues expect those in immunologically protected sites (such as ovary, pla‐ centa and testis). TAAs that are frequently found in tumours and provide excellent targets

Strategies are required to help identify potential targets which can be used for cancer immuno‐ therapy. Some of the most commonly used and successful techniques are described as follows:-

**3.1. Reverse-Transcription-Polymerase Chain Reaction (RT-PCR) and real-time PCR (RQ-**

Reverse-transcription-polymerase chain reaction (RT-PCR) and real-time PCR (RQ-PCR) has been used to examine known TAA expression in a range of solid and haematological malig‐ nancies [15-19]. Although this has provided important expression information and a good starting point to identify potential antigenic targets in a range of cancers, these studies are

Representational difference analysis was developed by Thierry Boon's group and used to discover a number of CT antigens [20, 21] including the MAGE family of antigens, typically from melanoma with one exception, RAGE, from renal cancer. Briefly, total RNA was ex‐ tracted from normal tissue (driver) and a tumour sample (tester) and used to construct dou‐ ble-stranded cDNA. Both cDNA samples were digested with the restriction enzymes DpnII and ligated to adapters which contained primer binding sites. The fragments were amplified by PCR, the adapters removed and new adapters for unrelated primers ligated to the tester. The tester and driver were then mixed and hybridized leading to three combinations of product: driver-driver (no amplification), tester-driver (linear amplification) and tester-test‐ er (exponential amplification). A further two hybridization and amplification steps generate

greater variation in the products which are subsequently cloned and sequenced.

for immunotherapy include Wilms tumour 1 [13] and PRAME [14].

entirely limited to tumour antigens which had already been discovered.

**3. Identification of tumour antigens**

**3.2. Representational difference analysis**

**PCR)**

The differential expression of tumour antigens and/or protein biomarkers between cell and dis‐ ease subtypes have been directly compared on cDNA microarrays and has allowed our im‐ proved understanding of lymphomas [35] and aided our development of personalised therapies [36]. Microarray technology is able to distinguish between different subtypes of a par‐ ticular cancer as well as identify the expression of novel antigens [37]. Minimal residual disease is a very important tool in the detection of impending relapse in patients who have had some form of treatment. Markers for minimal residual disease in acute lymphocytic leukaemia were identified by gene profiling [38]. cDNA microarray has been used to identify the frequency of elevated tumour antigen expression in acute myeloid leukaemia [28] and also associations be‐ tween specific cytogenetic abnormalities and relative levels of tumour antigen expression [39]. Micorarray has also been used to elucidate the possible function of tumour antigens such as Synovial Sarcoma X breakpoint 2 Interacting Protein (SSX2IP) in the subversion of cells har‐ bouring cytogenetic abnormalities (t(8;21) associated with mitotic spindle failure and the asso‐ ciation between the elevated expression of some tumour antigens (SSX2IP, RHAMM and SURVIVIN) at disease presentation and patient survival [8] in acute myeloid leukaemia.

#### **3.6. Mass Spectroscopy (MS)**

Mass Spectroscopy (MS) involves the analysis of peptides eluted from the MHC of antigen presenting cells [40-42] or proteins in serum [43]. This area is reviewed more completely in the following reviews [44,45]. By using mass spectrometry, it has been demonstrated that as many as 10,000 different peptide species are presented by individual class I MHC alleles [46]. The technique, its strengths and limitations are extensively reviewed [47].

**4.4. Immunoprecipitation**

4,6'-diamino-2-phenylindole (DAPI).

sub-cellular localisation are not possible.

**4.5. Immunocytochemistry/histochemistry**

The protein of interest can be purified by incubating lysed cell extracts with its specific antibody in solution. Once the antibody has bonded with the protein, the resulting complex can be precipitated using agarose or G Sepharose beads which remove the required protein. The complex can be separated using sodium dodecyl sulphate-polyacrylamide gel electro‐ phoresis [55]. The sample can then be used to determine how much protein is present relative to other cells or other treatment conditions but denaturation is often required and details about

Identification of Targets for Immunotherapy http://dx.doi.org/10.5772/54698 301

The antigen of interest can be detected in cells (cytochemistry) or in tissues (histochemistry). The cells or tissue sections are fixed onto slides using a fixative such as paraformaldehyde to immobilise them. They are incubated with the primary antibody and then the secondary which is labelled with a detection molecule. The technique is qualitative informing the user about the sub-cellular localisation of the antigen in tissue and which cell types express it (Figure 1). However quantitation is often lacking and like most methods this requires optimised reagents.

**Figure 1.** Demonstration of the sub-cellular localisation of the tumour antigen SSX2IP in K562 cells using immuno‐ fluoresence microscopy. Cells were air dried for 4-18hours onto glass microscope slides and stored at -20oC wrapped in saranwrap. Cells were defrosted and stained with antigen specific primary and fluorescently labelled secondary an‐ tibodies. Using confocal microscopy we detected SSX2IP expression (observed as a green colour by virtue of anti-SSX2IP-fluorescein isothiocyanate) was detectable on the surface of the K562 cells. Cell nuclei were stained blue using

The advent of multiple tissue arrays from collaborators or commercial sources provides a

screening opportunity once the cancer(s) of interest for the antigen has been defined.

#### **3.7. Protein microarrays**

Protein microarrays involve the immunoscreening of protein arrays (approximately 9,000 full length proteins and functional domains) which may be purchased from companies such as Invitrogen, Functional Genomics or Cambridge Protein Arrays. Antibodies in sera from patients [33,48,49] can be detected using generic secondary antibodies (fluorescently conju‐ gated anti-human IgG) and visualised on microarray scanners.

## **4. Validation of the expression of tumour antigens in tumour cells**

Once TAAs have been identified their expression in tumour cells needs to be confirmed. There are a number of assays which can be used to validate the expression of antigens in tumour cells. Many of the most frequently used rely on an available antibody which has been validated [50,51]. Techniques frequently used include:-

#### **4.1. Reverse Transcription (RT-PCR)/Real-time PCR**

Total RNA is extracted from cells and used to make cDNA using reverse transcriptase. The cDNA product is amplified by PCR and run on an agarose gel to identify the presence of the transcribed gene in the cell [52]. This technique is sensitive and real-time PCR can provide relative quantitation, however both techniques only indicate the presence/level of gene ex‐ pression and not protein translation, which can vary greatly between antigens.

#### **4.2. Enzyme-Linked Immunosorbent Assay (ELISA)**

Enzyme-Linked Immunosorbent Assay (ELISA) is a straightforward procedure which can be used to detect an antigen using an antibody [53]. The antigen is attached to the bottom of a 96 well plate, or bound by a capture antibody on the bottom of a plate (in the case of a sandwich ELISA). The protein of interest is then incubated with a chemically labelled detection anti‐ body. In most experiments the chemical label is an enzyme and a substrate is added which will produce a colour change detectable by a microplate reader. The technique is sensitive and quan‐ titative when used in conjunction with appropriate protein concentration controls but is better fitted to the analysis of protein in urine and blood, rather than in tissues.

#### **4.3. Immunoblotting**

Other systems which use antigen-antibody interactions are techniques such as Western blot. Extracted proteins from tumours or cells are separated by 2-dimensional electrophoretic gels and then blotted onto nitrocellulose membranes. The membranes are incubated with primary and then secondary antibody. The secondary antibody is covalently labelled with an enzyme which reacts with a substrate solution generating colour, which then can be measured [54].

#### **4.4. Immunoprecipitation**

following reviews [44,45]. By using mass spectrometry, it has been demonstrated that as many as 10,000 different peptide species are presented by individual class I MHC alleles [46]. The

Protein microarrays involve the immunoscreening of protein arrays (approximately 9,000 full length proteins and functional domains) which may be purchased from companies such as Invitrogen, Functional Genomics or Cambridge Protein Arrays. Antibodies in sera from patients [33,48,49] can be detected using generic secondary antibodies (fluorescently conju‐

Once TAAs have been identified their expression in tumour cells needs to be confirmed. There are a number of assays which can be used to validate the expression of antigens in tumour cells. Many of the most frequently used rely on an available antibody which has

Total RNA is extracted from cells and used to make cDNA using reverse transcriptase. The cDNA product is amplified by PCR and run on an agarose gel to identify the presence of the transcribed gene in the cell [52]. This technique is sensitive and real-time PCR can provide relative quantitation, however both techniques only indicate the presence/level of gene ex‐

Enzyme-Linked Immunosorbent Assay (ELISA) is a straightforward procedure which can be used to detect an antigen using an antibody [53]. The antigen is attached to the bottom of a 96 well plate, or bound by a capture antibody on the bottom of a plate (in the case of a sandwich ELISA). The protein of interest is then incubated with a chemically labelled detection anti‐ body. In most experiments the chemical label is an enzyme and a substrate is added which will produce a colour change detectable by a microplate reader. The technique is sensitive and quan‐ titative when used in conjunction with appropriate protein concentration controls but is better

Other systems which use antigen-antibody interactions are techniques such as Western blot. Extracted proteins from tumours or cells are separated by 2-dimensional electrophoretic gels and then blotted onto nitrocellulose membranes. The membranes are incubated with primary and then secondary antibody. The secondary antibody is covalently labelled with an enzyme which reacts with a substrate solution generating colour, which then can be measured [54].

pression and not protein translation, which can vary greatly between antigens.

fitted to the analysis of protein in urine and blood, rather than in tissues.

**4. Validation of the expression of tumour antigens in tumour cells**

technique, its strengths and limitations are extensively reviewed [47].

gated anti-human IgG) and visualised on microarray scanners.

been validated [50,51]. Techniques frequently used include:-

**4.1. Reverse Transcription (RT-PCR)/Real-time PCR**

**4.2. Enzyme-Linked Immunosorbent Assay (ELISA)**

**3.7. Protein microarrays**

300 Novel Gene Therapy Approaches

**4.3. Immunoblotting**

The protein of interest can be purified by incubating lysed cell extracts with its specific antibody in solution. Once the antibody has bonded with the protein, the resulting complex can be precipitated using agarose or G Sepharose beads which remove the required protein. The complex can be separated using sodium dodecyl sulphate-polyacrylamide gel electro‐ phoresis [55]. The sample can then be used to determine how much protein is present relative to other cells or other treatment conditions but denaturation is often required and details about sub-cellular localisation are not possible.

#### **4.5. Immunocytochemistry/histochemistry**

The antigen of interest can be detected in cells (cytochemistry) or in tissues (histochemistry). The cells or tissue sections are fixed onto slides using a fixative such as paraformaldehyde to immobilise them. They are incubated with the primary antibody and then the secondary which is labelled with a detection molecule. The technique is qualitative informing the user about the sub-cellular localisation of the antigen in tissue and which cell types express it (Figure 1). However quantitation is often lacking and like most methods this requires optimised reagents.

**Figure 1.** Demonstration of the sub-cellular localisation of the tumour antigen SSX2IP in K562 cells using immuno‐ fluoresence microscopy. Cells were air dried for 4-18hours onto glass microscope slides and stored at -20oC wrapped in saranwrap. Cells were defrosted and stained with antigen specific primary and fluorescently labelled secondary an‐ tibodies. Using confocal microscopy we detected SSX2IP expression (observed as a green colour by virtue of anti-SSX2IP-fluorescein isothiocyanate) was detectable on the surface of the K562 cells. Cell nuclei were stained blue using 4,6'-diamino-2-phenylindole (DAPI).

The advent of multiple tissue arrays from collaborators or commercial sources provides a screening opportunity once the cancer(s) of interest for the antigen has been defined.

#### **4.6. Flow cytometry**

This technique allows the analyses of cells with a variety of parameters such as extracellular or intracellular markers, granularity, size and shape. Cancer cells are labelled with fluorescent antibodies for the required antigen. The cells are passed in a stream and intersected with a laser beam. The intensity of the fluorescence is measured and plotted in the form of dot plots and histograms. This technique is sensitive and informative to allow specific cell types to be "gated" by virtue of size, granularity and detectable protein expression. Machines can measure up to 19 parameters in the most sophisticated machines allowing multiple proteins and cell types to be analysed simultaneously [56]. However the technique requires validated antibodies that have been shown to be appropriate for fluorescence activated cell sorting analysis and enough tumour cells in suspension for analysis.

A2, indicating how long T cells will have to interact with peptide bound HLA-A2 before the

Identification of Targets for Immunotherapy http://dx.doi.org/10.5772/54698 303

There are a number of databases which can be mined to find epitopes which have already been shown to bind to HLA molecules. These have been used to identify established epitopes that

The SYFPEITHI database allows the prediction of MHC class I and II binding ligands for different mammalian species. When a search is carried out using a protein sequence, a prediction is made based on the amino acids in the anchor and auxiliary anchor posi‐ tions and other frequent amino acids which can bind to MHC molecules. A score is then calculated which follows certain rules which are: a numerical value of 10 is given to ami‐ no acids that regularly arise in anchor positions, the value 8 is set for amino acids occur‐ ring in a significant number of ligands, six is for unusual anchors such as auxiliary anchors and less frequent residues of the same set have a value of four. Preferred amino acids have coefficients between 1–4 depending on the signal strength in pool sequencing or the occurrence of individual sequences. Amino acids that are considered as having an adverse effect on binding have a coefficient of –1 to –3 [60]. SYFPEITHI database gets updated regularly and has been used to identify various ligands; p28 peptide as an epit‐ ope for the CT antigen PLAC1 in breast cancer [61], p101-111 is the first CTA-derived peptide which induces CD4(+), CD8(+), and B-cell responses *in vitro* [62], p43-57 epitope stimulates T cells in HCA587-derived tumours [63] and PASD1(1) – PASD1(5) [51].

Bioinformatics and Molecular Analysis Section (BIMAS) develops computational processes to analyse data generated from molecular biology and genetics research; and provides bioinfor‐ matics guidance, support and resources for the collection, management, and display of biological sequence and genomic information for scientists involved in genomics and genetic analysis [64]. Other online software which can be used to identify epitopes includes EpiJen,

There are a number of assays which can be used to determine if T cells are activated in response

**7. Cell based assays —** *In vitro* **demonstration of T cell reactivity**

complex falls apart.

**6.1. The SYFPEITHI**

**6.** *In silico* **identification of epitopes**

may be used in immunotherapy strategies.

**6.2. Bioinformatics and Molecular Analysis Section (BIMAS)**

Rankpep, nHLApred, NetCTL and Multipred [65].

to antigen.

## **5. Identification of HLA-binding epitopes —** *in vitro* **assays**

Immune responses in the body can ensure that any foreign matter is eliminated effectively. Class I and II major histocompatability complexes (MHC) are present on the surface of nucleated cells and present processed peptides from proteins inside the cell to T cells. T cells can destroy infected cells if peptides in the context of "danger" are detected [57]. MHC in humans is known as the human leukocyte antigen (HLA) system. MHC class I HLA molecules are highly polymorphic and generally the best defence against infections.

#### **5.1. MHC Peptide binding assay**

Peptide antigens are stripped from the HLA class I molecules by mild acid treatment, cells are then incubated with a fluorescent reference peptide together with different concentrations of the peptide of interest. The efficiency with which the required peptide competes for binding to the HLA class I molecules is examined by measuring the amount of HLA-bound reference peptide with fluorescence activated cell sorting analysis [58].

#### **5.2. T2** *in vitro* **HLA-A2 binding assay**

T2 *in vitro* HLA-A2 binding assay is more frequently used to determine the strength of peptide binding to the most common HLA molecule in Caucasian populations. The HLA-A2 express‐ ing, TAP-1 deficient human T-cell line T2 is used as an assay of HLA-A2 peptide binding efficiency. T2 cells are washed and resuspended in serum-free RPMI media and plated in 96 well microtitre plates. Human β2-microglobulin and often nonamers (nine amino acids long peptides) are added and the cells are incubated overnight at 37°C/5% CO2. The cells are washed and probed with a HLA-A2-specific monoclonal antibody and appropriate secondary anti‐ body prior to flow cytometry. Only HLA-A2 molecules bound to peptide are stabilised and detectable on the cell surface. Results are reported as a relative mean fluorescence index (MFI), calculated as the MFI of peptide-pulsed T2 cells compared with the MFI of unpulsed T2 cells [59]. Time course assays may be used to indicate how long the peptide remains on the HLA- A2, indicating how long T cells will have to interact with peptide bound HLA-A2 before the complex falls apart.

## **6.** *In silico* **identification of epitopes**

There are a number of databases which can be mined to find epitopes which have already been shown to bind to HLA molecules. These have been used to identify established epitopes that may be used in immunotherapy strategies.

#### **6.1. The SYFPEITHI**

**4.6. Flow cytometry**

302 Novel Gene Therapy Approaches

enough tumour cells in suspension for analysis.

**5.1. MHC Peptide binding assay**

**5.2. T2** *in vitro* **HLA-A2 binding assay**

**5. Identification of HLA-binding epitopes —** *in vitro* **assays**

are highly polymorphic and generally the best defence against infections.

peptide with fluorescence activated cell sorting analysis [58].

This technique allows the analyses of cells with a variety of parameters such as extracellular or intracellular markers, granularity, size and shape. Cancer cells are labelled with fluorescent antibodies for the required antigen. The cells are passed in a stream and intersected with a laser beam. The intensity of the fluorescence is measured and plotted in the form of dot plots and histograms. This technique is sensitive and informative to allow specific cell types to be "gated" by virtue of size, granularity and detectable protein expression. Machines can measure up to 19 parameters in the most sophisticated machines allowing multiple proteins and cell types to be analysed simultaneously [56]. However the technique requires validated antibodies that have been shown to be appropriate for fluorescence activated cell sorting analysis and

Immune responses in the body can ensure that any foreign matter is eliminated effectively. Class I and II major histocompatability complexes (MHC) are present on the surface of nucleated cells and present processed peptides from proteins inside the cell to T cells. T cells can destroy infected cells if peptides in the context of "danger" are detected [57]. MHC in humans is known as the human leukocyte antigen (HLA) system. MHC class I HLA molecules

Peptide antigens are stripped from the HLA class I molecules by mild acid treatment, cells are then incubated with a fluorescent reference peptide together with different concentrations of the peptide of interest. The efficiency with which the required peptide competes for binding to the HLA class I molecules is examined by measuring the amount of HLA-bound reference

T2 *in vitro* HLA-A2 binding assay is more frequently used to determine the strength of peptide binding to the most common HLA molecule in Caucasian populations. The HLA-A2 express‐ ing, TAP-1 deficient human T-cell line T2 is used as an assay of HLA-A2 peptide binding efficiency. T2 cells are washed and resuspended in serum-free RPMI media and plated in 96 well microtitre plates. Human β2-microglobulin and often nonamers (nine amino acids long peptides) are added and the cells are incubated overnight at 37°C/5% CO2. The cells are washed and probed with a HLA-A2-specific monoclonal antibody and appropriate secondary anti‐ body prior to flow cytometry. Only HLA-A2 molecules bound to peptide are stabilised and detectable on the cell surface. Results are reported as a relative mean fluorescence index (MFI), calculated as the MFI of peptide-pulsed T2 cells compared with the MFI of unpulsed T2 cells [59]. Time course assays may be used to indicate how long the peptide remains on the HLA-

The SYFPEITHI database allows the prediction of MHC class I and II binding ligands for different mammalian species. When a search is carried out using a protein sequence, a prediction is made based on the amino acids in the anchor and auxiliary anchor posi‐ tions and other frequent amino acids which can bind to MHC molecules. A score is then calculated which follows certain rules which are: a numerical value of 10 is given to ami‐ no acids that regularly arise in anchor positions, the value 8 is set for amino acids occur‐ ring in a significant number of ligands, six is for unusual anchors such as auxiliary anchors and less frequent residues of the same set have a value of four. Preferred amino acids have coefficients between 1–4 depending on the signal strength in pool sequencing or the occurrence of individual sequences. Amino acids that are considered as having an adverse effect on binding have a coefficient of –1 to –3 [60]. SYFPEITHI database gets updated regularly and has been used to identify various ligands; p28 peptide as an epit‐ ope for the CT antigen PLAC1 in breast cancer [61], p101-111 is the first CTA-derived peptide which induces CD4(+), CD8(+), and B-cell responses *in vitro* [62], p43-57 epitope stimulates T cells in HCA587-derived tumours [63] and PASD1(1) – PASD1(5) [51].

#### **6.2. Bioinformatics and Molecular Analysis Section (BIMAS)**

Bioinformatics and Molecular Analysis Section (BIMAS) develops computational processes to analyse data generated from molecular biology and genetics research; and provides bioinfor‐ matics guidance, support and resources for the collection, management, and display of biological sequence and genomic information for scientists involved in genomics and genetic analysis [64]. Other online software which can be used to identify epitopes includes EpiJen, Rankpep, nHLApred, NetCTL and Multipred [65].

## **7. Cell based assays —** *In vitro* **demonstration of T cell reactivity**

There are a number of assays which can be used to determine if T cells are activated in response to antigen.

### **7.1. Carboxyfluorescein diacetate Succinimidyl Ester (CFSE)**

Carboxyfluorescein diacetate succinimidyl ester (CFSE) is a cytoplasmic dye which is absorbed by all cell types. Once the CFSE labelled cells divide, the dye is shared amongst the daughter cells equally therefore the fluorescence is halved after each round of the cell cycle. This difference in fluorescence can be measured. The more cells proliferate, the greater the decrease in the fluorescent signal. The fluorescence peaks can be measured by flow cytometry [66]. CFSE labelling is increasingly used to measure target tumour cell killing [67], superseding radiation based assays, as well as T cell proliferation in response to tumour cells *in vivo* [68]. CFSE labelling can also be performed *in vivo* where the dye is injected into the host animal's spleen or lymph nodes, however the labelling is not uniform and it is sometimes difficult to obtain individual peaks once lymphocyte cell division has occurred [66].

been generated but won't determine which T cells, if they are indeed T cells, have been activated. pMHCs, often referred to as tetramers, can be used to identify antigen specific T cells. They are produced through the refolding of β2-microglobulin and heavy chains in MHC molecules with the appropriate epitope of interest. The pMHC is then labelled with biotin using BirA enzyme. A streptavidin molecule conjugated to a fluorescent detector binds to four (tetramers) pMHCs or can used to create multimers (for example dimers, pentamers, dex‐ tramers) of these constructed MHC molecules courtesy of the biotin-avidin interaction [73]. T cell populations are added to this mixture and T cells with the specific receptor for the epitope of interest will bind and be measurable by flow cytometry [74]. Shen *et al* [74] have found that cross-reactive T cells i.e. T cells which recognise two different antigens can be identified providing an extra tool in vaccine development. In some cases antigen specific T cells may not bind tetramers due to being undifferentiated and unable to accumulate T cell receptor (TCR) molecules close to the antigen. Another reason could be low affinity between TCR and MHC [75]. Other techniques based on the use of pMHCs include pMHC arrays [76] (Section 7.5), NACS [77] and the combinatorial approach [78,79]. These techniques all provide high through‐ put analysis of multiple T cell populations with a variety of pros and cons to each technique including issues with background, specificity/binding capacity of individual pMHC com‐ plexes, activated induced cell death of pMHC bound T cells, internalisation of pMHCs following T cell binding [80], cost and labour intensity. Sequencing of TCRs (2-3 million every 2-3 days) by companies such as TRON gGmbH (Johannes Gutenberg University Mainz, Germany) and Adaptive Biotechnologies (Seattle, USA) will provide a new way of analysing T cell populations which will be informative with regards to which TCRs are present but not necessarily whether they are present on mature, anergic, activated or functional T cells nor which sub-group of T cells are harbouring them (helper T cells, cytotoxic T cells, Th17 cells or indeed regulatory T cells (Tregs)). This technology allows the first opportunity to examine an extremely large number of TCRs in a very short time and will revolutionise how we examine

Identification of Targets for Immunotherapy http://dx.doi.org/10.5772/54698 305

pMHC or tetramer arrays [76,81] (Figure 2) provides a strategy to determine which spe‐ cific CD8+ T cell populations are present in the peripheral blood of patients. Antigens identified by the techniques described already can be used to help expand the pMHC ar‐ ray for future studies. In addition, the pMHC array provides a means to investigate epit‐ ope spreading and changes in T cell specificities with disease progression. The technique benefits from the low number of purified CD8+ T cells required for each array (0.5-2 x

), which can be purified from 20ml of patient peripheral blood using StemCell CD8+ negative isolation beads providing "untouched" T cells (Bonney, Guinn *et al*, in prepara‐ tion). The purified CD8+ T cells are then lipophyllically dyed with DiD (Molecular Probes), washed and incubated with the pMHC array. The pMHC array has a detection limit of 0.02% matching the sensitivity we can reproducibly achieve with flow cytometry when analysing patient samples. Where sample availability permits, pMHC array data should be validated by flow cytometry [82] using the same pMHC tetramers as in the pMHC array. The pMHC array has the added advantage that it can be used for the ini‐

T cell responses in patients in the future.

**7.5. pMHC arrays**

106

### **7.2. Lymphocyte proliferation assays**

Lymphocyte proliferation assays can be used to determine activation of T cells. Peripheral blood mononuclear cells are isolated and cultured in microtitre plates. The specific antigen is incubated with the cells, which causes the T cells to divide and grow. The MTT colorimetric assay is based upon (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazolium salt which gets cleaved by enzymes in the mitochondria to produce blue formazan. Viable, dividing cells will create more formazan which can be quantified using a plate reader. MTT assay is very convenient, however some considerations need to be assessed to avoid false positives such as cell densities, correct culture medium, filtration of media to remove precip‐ itate, optimisation of MTT concentrations and incubation times [69].

#### **7.3. [3 H]-Thymidine incorporation assay**

[ 3 H]-Thymidine incorporation assay is based on the use of [3 H]-thymidine a radioactive molecule which can be incorporated into DNA during the S-phase of cell division. As new DNA is synthesised, occasional thymidine bases are replaced by [3 H]-thymidine and subsequently the incorporated radioactivity is measured, following washes to remove un‐ incorporated radioactivity, using a Scintillation Counter [70]. As [3 H]-thymidine is radio‐ active an analogue called bromodeoxyuridine (BrdU) was developed to replace it in assays. BrdU integrates into DNA strands and can be measured using immunohisto‐ chemistry and flow cytometry protocols using fluorescent conjugates and can be ob‐ served over a longer duration. BrdU is also used to look at the number of cells in each part of the cell cycle by flow cytometry [71]. However BrdU has been found to be more toxic than [3 H]-thymidine, possibly because it is structurally very different to the original DNA nucleotides. It also adversely affects cell division, the pattern of cell migration, fi‐ nal position of migrating cells and the fate of labelled cells [72].

#### **7.4. Peptide-MHC (pMHCs)**

Peptide-MHC (pMHCs) based assays circumvent issues caused by measuring T cell prolifer‐ ation. T cell proliferation assays can provide information on whether an immune response has been generated but won't determine which T cells, if they are indeed T cells, have been activated. pMHCs, often referred to as tetramers, can be used to identify antigen specific T cells. They are produced through the refolding of β2-microglobulin and heavy chains in MHC molecules with the appropriate epitope of interest. The pMHC is then labelled with biotin using BirA enzyme. A streptavidin molecule conjugated to a fluorescent detector binds to four (tetramers) pMHCs or can used to create multimers (for example dimers, pentamers, dex‐ tramers) of these constructed MHC molecules courtesy of the biotin-avidin interaction [73]. T cell populations are added to this mixture and T cells with the specific receptor for the epitope of interest will bind and be measurable by flow cytometry [74]. Shen *et al* [74] have found that cross-reactive T cells i.e. T cells which recognise two different antigens can be identified providing an extra tool in vaccine development. In some cases antigen specific T cells may not bind tetramers due to being undifferentiated and unable to accumulate T cell receptor (TCR) molecules close to the antigen. Another reason could be low affinity between TCR and MHC [75]. Other techniques based on the use of pMHCs include pMHC arrays [76] (Section 7.5), NACS [77] and the combinatorial approach [78,79]. These techniques all provide high through‐ put analysis of multiple T cell populations with a variety of pros and cons to each technique including issues with background, specificity/binding capacity of individual pMHC com‐ plexes, activated induced cell death of pMHC bound T cells, internalisation of pMHCs following T cell binding [80], cost and labour intensity. Sequencing of TCRs (2-3 million every 2-3 days) by companies such as TRON gGmbH (Johannes Gutenberg University Mainz, Germany) and Adaptive Biotechnologies (Seattle, USA) will provide a new way of analysing T cell populations which will be informative with regards to which TCRs are present but not necessarily whether they are present on mature, anergic, activated or functional T cells nor which sub-group of T cells are harbouring them (helper T cells, cytotoxic T cells, Th17 cells or indeed regulatory T cells (Tregs)). This technology allows the first opportunity to examine an extremely large number of TCRs in a very short time and will revolutionise how we examine T cell responses in patients in the future.

#### **7.5. pMHC arrays**

**7.1. Carboxyfluorescein diacetate Succinimidyl Ester (CFSE)**

individual peaks once lymphocyte cell division has occurred [66].

itate, optimisation of MTT concentrations and incubation times [69].

H]-Thymidine incorporation assay is based on the use of [3

new DNA is synthesised, occasional thymidine bases are replaced by [3

incorporated radioactivity, using a Scintillation Counter [70]. As [3

nal position of migrating cells and the fate of labelled cells [72].

**7.2. Lymphocyte proliferation assays**

304 Novel Gene Therapy Approaches

**H]-Thymidine incorporation assay**

**7.3. [3**

toxic than [3

**7.4. Peptide-MHC (pMHCs)**

[ 3

Carboxyfluorescein diacetate succinimidyl ester (CFSE) is a cytoplasmic dye which is absorbed by all cell types. Once the CFSE labelled cells divide, the dye is shared amongst the daughter cells equally therefore the fluorescence is halved after each round of the cell cycle. This difference in fluorescence can be measured. The more cells proliferate, the greater the decrease in the fluorescent signal. The fluorescence peaks can be measured by flow cytometry [66]. CFSE labelling is increasingly used to measure target tumour cell killing [67], superseding radiation based assays, as well as T cell proliferation in response to tumour cells *in vivo* [68]. CFSE labelling can also be performed *in vivo* where the dye is injected into the host animal's spleen or lymph nodes, however the labelling is not uniform and it is sometimes difficult to obtain

Lymphocyte proliferation assays can be used to determine activation of T cells. Peripheral blood mononuclear cells are isolated and cultured in microtitre plates. The specific antigen is incubated with the cells, which causes the T cells to divide and grow. The MTT colorimetric assay is based upon (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazolium salt which gets cleaved by enzymes in the mitochondria to produce blue formazan. Viable, dividing cells will create more formazan which can be quantified using a plate reader. MTT assay is very convenient, however some considerations need to be assessed to avoid false positives such as cell densities, correct culture medium, filtration of media to remove precip‐

molecule which can be incorporated into DNA during the S-phase of cell division. As

subsequently the incorporated radioactivity is measured, following washes to remove un‐

active an analogue called bromodeoxyuridine (BrdU) was developed to replace it in assays. BrdU integrates into DNA strands and can be measured using immunohisto‐ chemistry and flow cytometry protocols using fluorescent conjugates and can be ob‐ served over a longer duration. BrdU is also used to look at the number of cells in each part of the cell cycle by flow cytometry [71]. However BrdU has been found to be more

DNA nucleotides. It also adversely affects cell division, the pattern of cell migration, fi‐

Peptide-MHC (pMHCs) based assays circumvent issues caused by measuring T cell prolifer‐ ation. T cell proliferation assays can provide information on whether an immune response has

H]-thymidine, possibly because it is structurally very different to the original

H]-thymidine a radioactive

H]-thymidine and

H]-thymidine is radio‐

pMHC or tetramer arrays [76,81] (Figure 2) provides a strategy to determine which spe‐ cific CD8+ T cell populations are present in the peripheral blood of patients. Antigens identified by the techniques described already can be used to help expand the pMHC ar‐ ray for future studies. In addition, the pMHC array provides a means to investigate epit‐ ope spreading and changes in T cell specificities with disease progression. The technique benefits from the low number of purified CD8+ T cells required for each array (0.5-2 x 106 ), which can be purified from 20ml of patient peripheral blood using StemCell CD8+ negative isolation beads providing "untouched" T cells (Bonney, Guinn *et al*, in prepara‐ tion). The purified CD8+ T cells are then lipophyllically dyed with DiD (Molecular Probes), washed and incubated with the pMHC array. The pMHC array has a detection limit of 0.02% matching the sensitivity we can reproducibly achieve with flow cytometry when analysing patient samples. Where sample availability permits, pMHC array data should be validated by flow cytometry [82] using the same pMHC tetramers as in the pMHC array. The pMHC array has the added advantage that it can be used for the ini‐ tial screening of a relatively small number of CD8+ T cells against a large number of pMHCs on the array, and a short-list of T cell populations which are shown to exist on the pMHC array can then be quantitated by flow cytometry (limiting the amount of sam‐ ple required in subsequent studies). The pMHC array can be used to analyse patient samples at a number of disease time-points (presentation, post-treatment (surgery and/or radiotherapy) and with disease progression) to examine how T cell responses to tumour antigens change with treatment, to examine epitope spreading and to correlate changing immune responses with clinical responses.

printed multiplexed AF532-conjugated pMHCs at a concentration of 0.5mg/ml so that 1ng was placed in each spot (shown as coloured ovals). Each spot is 400uM wide with a 700uM inter-spot distance. (B) At least six spots per pMHC are applied in each of two independent sites. Each hydrogel slide can hold up to 1,000 spots of tetramer in total. (C) We applied 0.8- 1.5 x 10<sup>6</sup> lipophillically-dyed CD8<sup>+</sup> T cells/slide (seen in red). pMHCs included a range of HLA restrictions and had already been shown to work in flow cytometry studies,(i) pMHCs are spotted across the gel, (seen as green spots) (ii) even some HLA-A2 positive patients show no reactivity with any pMHCs (iii) while others show the presence of multiple T cell populations which recognise tumour antigens. Based on figure in [144]. **Figure 2.** pMHC arrays for the simultaneous detection of T cell populations in patient peripheral blood. (A) Using a QArray2 printer and HPLF 0.3mm solid tip pins (Genetix) we printed multiplexed AF532-conjugated pMHCs at a con‐ centration of 0.5mg/ml so that 1ng was placed in each spot (shown as coloured ovals). Each spot is 400uM wide with a 700uM inter-spot distance. (B) At least six spots per pMHC are applied in each of two independent sites. Each hydro‐ gel slide can hold up to 1,000 spots of tetramer in total. (C) We applied 0.8-1.5 x 106 lipophillically-dyed CD8+ T cells/ slide (seen in red). pMHCs included a range of HLA restrictions and had already been shown to work in flow cytometry studies,(i) pMHCs are spotted across the gel, (seen as green spots) (ii) even some HLA-A2 positive patients show no reactivity with any pMHCs (iii) while others show the presence of multiple T cell populations which recognise tumour antigens. Based on figure in [144].

**Figure 2.** pMHC arrays for the simultaneous detection of T cell populations in patient peripheral blood. (A) Using a QArray2 printer and HPLF 0.3mm solid tip pins (Genetix) we

39

**7.6. Intracellular cytokine staining assay**

ing 6-16 hours of culture only IFN-γ cytokine was found [84].

cytotoxic T cell response in the presence of longer CD4<sup>+</sup>

sometimes longer proteins can inhibit CD8+

**8. Cell based assays —** *In vivo* **assays**

molecules as described in this section.

**8.1. Immunodeficient mice**

on the constituents of individual protein sequences.

Intracellular cytokine staining assay detects particular cytokines released by immune cells which can provide a useful insight into the responding T cell populations. Such cy‐ tokines could include interferon-gamma (IFNγ), interleukin-2 (IL-2), IL-4 and tumour ne‐ crosis factor-*α*. Cells are plated and incubated with the antigen to stimulate cytokine production. To prevent the cytokine from exiting the cell a transport inhibitor is added e.g. brefeldin A. The cells are then fixed in paraformaldehyde and permeabilized to al‐ low the anti-cytokine antibody to bind. The results are analysed by flow cytometry. The use of intracellular cytokine staining assay to detect the cytokine IFNγ shows high repro‐ ducibility and linearity with little background [83]. Duration of culture prior to antigen stimulation, as well as the cytokine accumulation period, are critical parameters of these methods. In both murine and cattle models, following 2-6 hours in culture, T cells pro‐ duced a mixture of cytokines IFNγ, IL-2 and tumour necrosis factor-α, however follow‐

Using multiple peptides from distinct TAAs to stimulate immune cells may prove the most effective for peptide vaccines. A cocktail of four multiple myeloma antigen peptides were used to stimulate T lymphocytes from HLA-A2 positive people to induce IFNγ production, cell proliferation and cytotoxicity against HLA-A2 positive multiple myeloma patients' cells [85]. Indeed long peptides may offer the advantage of allowing the immune system to choose the epitope(s) it can best process and present from a peptide sequence and induce an effective

There are a number of approaches that can be taken when using mouse models to detect T cell immune responses. Animals can be used in transplantable tumour (xenografts) models or genetically engineered tumour models. In xenograft models human tumour cells are taken and injected into immunodeficient mice so that complex immune respons‐ es involving multiple cell types can be investigated. In contrast, in genetically engineered models, genes known to cause cancer are activated or tumour suppressor genes are "switched off" to allow their effects on tumour growth to be examined. In addition transgenic mice can be used to examine T cell responses to epitopes presented on MHC

Immunodeficient mice such as athymic nude mice, severely compromised immunodefi‐ cient (SCID) mice and non-obese diabetic severe combined immunodeficiency mice will accept xenografts of human cells [88]. Depending upon the number of cells injected, or

helper motifs [86]. Conversely

Identification of Targets for Immunotherapy http://dx.doi.org/10.5772/54698 307

T cells responses [87] but this may vary depending

#### **7.6. Intracellular cytokine staining assay**

tial screening of a relatively small number of CD8+ T cells against a large number of pMHCs on the array, and a short-list of T cell populations which are shown to exist on the pMHC array can then be quantitated by flow cytometry (limiting the amount of sam‐ ple required in subsequent studies). The pMHC array can be used to analyse patient samples at a number of disease time-points (presentation, post-treatment (surgery and/or radiotherapy) and with disease progression) to examine how T cell responses to tumour antigens change with treatment, to examine epitope spreading and to correlate changing

**Figure 2.** pMHC arrays for the simultaneous detection of T cell populations in patient peripheral blood. (A) Using a QArray2 printer and HPLF 0.3mm solid tip pins (Genetix) we printed multiplexed AF532-conjugated pMHCs at a concentration of 0.5mg/ml so that 1ng was placed in each spot (shown as coloured ovals). Each spot is 400uM wide with a 700uM inter-spot distance. (B) At least six spots per pMHC are applied in each of two independent sites. Each hydrogel slide can hold up to 1,000 spots of tetramer in total. (C) We applied 0.8- 1.5 x 10<sup>6</sup> lipophillically-dyed CD8<sup>+</sup> T cells/slide (seen in red). pMHCs included a range of HLA restrictions and had already been shown to work in flow cytometry studies,(i) pMHCs are spotted across the gel, (seen as green spots) (ii) even some HLA-A2 positive patients show no reactivity with any pMHCs (iii) while others show the presence of multiple T cell

**Figure 2.** pMHC arrays for the simultaneous detection of T cell populations in patient peripheral blood. (A) Using a QArray2 printer and HPLF 0.3mm solid tip pins (Genetix) we printed multiplexed AF532-conjugated pMHCs at a con‐ centration of 0.5mg/ml so that 1ng was placed in each spot (shown as coloured ovals). Each spot is 400uM wide with a 700uM inter-spot distance. (B) At least six spots per pMHC are applied in each of two independent sites. Each hydro‐ gel slide can hold up to 1,000 spots of tetramer in total. (C) We applied 0.8-1.5 x 106 lipophillically-dyed CD8+ T cells/ slide (seen in red). pMHCs included a range of HLA restrictions and had already been shown to work in flow cytometry studies,(i) pMHCs are spotted across the gel, (seen as green spots) (ii) even some HLA-A2 positive patients show no reactivity with any pMHCs (iii) while others show the presence of multiple T cell populations which recognise tumour

populations which recognise tumour antigens. Based on figure in [144].

antigens. Based on figure in [144].

immune responses with clinical responses.

306 Novel Gene Therapy Approaches

Intracellular cytokine staining assay detects particular cytokines released by immune cells which can provide a useful insight into the responding T cell populations. Such cy‐ tokines could include interferon-gamma (IFNγ), interleukin-2 (IL-2), IL-4 and tumour ne‐ crosis factor-*α*. Cells are plated and incubated with the antigen to stimulate cytokine production. To prevent the cytokine from exiting the cell a transport inhibitor is added e.g. brefeldin A. The cells are then fixed in paraformaldehyde and permeabilized to al‐ low the anti-cytokine antibody to bind. The results are analysed by flow cytometry. The use of intracellular cytokine staining assay to detect the cytokine IFNγ shows high repro‐ ducibility and linearity with little background [83]. Duration of culture prior to antigen stimulation, as well as the cytokine accumulation period, are critical parameters of these methods. In both murine and cattle models, following 2-6 hours in culture, T cells pro‐ duced a mixture of cytokines IFNγ, IL-2 and tumour necrosis factor-α, however follow‐ ing 6-16 hours of culture only IFN-γ cytokine was found [84].

Using multiple peptides from distinct TAAs to stimulate immune cells may prove the most effective for peptide vaccines. A cocktail of four multiple myeloma antigen peptides were used to stimulate T lymphocytes from HLA-A2 positive people to induce IFNγ production, cell proliferation and cytotoxicity against HLA-A2 positive multiple myeloma patients' cells [85]. Indeed long peptides may offer the advantage of allowing the immune system to choose the epitope(s) it can best process and present from a peptide sequence and induce an effective cytotoxic T cell response in the presence of longer CD4<sup>+</sup> helper motifs [86]. Conversely sometimes longer proteins can inhibit CD8+ T cells responses [87] but this may vary depending on the constituents of individual protein sequences.

### **8. Cell based assays —** *In vivo* **assays**

There are a number of approaches that can be taken when using mouse models to detect T cell immune responses. Animals can be used in transplantable tumour (xenografts) models or genetically engineered tumour models. In xenograft models human tumour cells are taken and injected into immunodeficient mice so that complex immune respons‐ es involving multiple cell types can be investigated. In contrast, in genetically engineered models, genes known to cause cancer are activated or tumour suppressor genes are "switched off" to allow their effects on tumour growth to be examined. In addition transgenic mice can be used to examine T cell responses to epitopes presented on MHC molecules as described in this section.

#### **8.1. Immunodeficient mice**

39

Immunodeficient mice such as athymic nude mice, severely compromised immunodefi‐ cient (SCID) mice and non-obese diabetic severe combined immunodeficiency mice will accept xenografts of human cells [88]. Depending upon the number of cells injected, or the size of the tumour transplanted, the tumour can develop over weeks to months and the response to appropriate therapeutic agents studied *in vivo* [89]. Indeed such models have been used to examine the effectiveness of various immunotherapeutic strategies in‐ cluding whole cell vaccines [90], dendritic cell (DC) vaccines [91], peptide vaccines [92] and DNA vaccines [67,87,93].

monoclonal antibodies (mAb) have become standard treatments for cancer. Ultimately if there is an antibody and it recognises a surface antigen solely on cancer and non-essential cells then this will likely be the most effective way to cause tumour destruction. mAbs are derived by vaccinating an animal with the target antigen and testing to see if the B cells are producing antibodies against it. Then the B cells are extracted from its spleen and infused with myeloma cells to produce hybridomas. Hybridomas divide perpetually and produce the mAb to the

Rosenberg *et al* [107] showed that only 2.6% of immunotherapy clinical trials had worked and therefore an overhaul was needed in the practice of immunotherapy. Subse‐ quently the same group showed that adoptive T cell therapy could be very promising with cell numbers being returned to the patient [108] and their status – activated but not mature [109] and cell numbers being the main issues. It is also likely that the best strat‐ egy may include a combination of conventional and immunotherapy techniques [110] or even a combination of immunotherapy techniques as demonstrated in increasing num‐

DCs are antigen presenting cells therefore they have received some attention for possible use in cancer immunotherapy. DCs pulsed by peptide and injected into the skin showed a response rate of 28%. This percentage increased to 35.7% when immature DCs are injected straight into

Tumour-infiltrating T cells (TIL) therapy has been used in stage IV melanoma patients. TILs are obtained from the blood, lymph nodes or from a tumour tissue biopsy. TILs are isolated, activated and expanded using IL-2 *in vitro*. The patient undergoes lympho-depleting chemo‐

When a tumour antigen is secreted into the circulation in high levels immune tolerance

antigens in the blood, which can induce tolerance through Tregs or negative selection. Tregs are cells which are part of the tolerance system which prevents autoimmunity [115, 116]. Simultaneous Treg depletions (using anti-CD25 antibodies for instance) may aid the effectiveness of immunotherapy in some cancer types where Treg infiltration into the tu‐

There are a number of reviews in this area of research which aim to look into effective immunotherapy strategies for the future. These include cellular immunotherapy [119], whole call vaccines [120], multidrug resistance [121] and DCs [122]. Targeted therapeutic strategies along with ever improving designs in clinical trials pave the way for further

Clinical trials are undertaken after a large amount of data has been obtained on the antigen of interest in the lab. This data is required to ensure treatment safety and efficacy as far as is

, a subset of DCs, are able to capture tumour

Identification of Targets for Immunotherapy http://dx.doi.org/10.5772/54698 309

Sirpα<sup>+</sup>

the tumour and even higher to 40% for advanced pancreatic cancer [113].

therapy prior to the T cells being injected back in to the blood [114].

antigen in large amounts [106].

bers of mouse models [111] and clinical trials [112].

can be induced in the thymus. CD8α−

mour is rife [117,118].

success [123].

**10. Clinical trials**

#### **8.2. Genetically modified mice**

Genetically modified mice may have genes which are overexpressed or deleted and the effects of these genes on tumour development can be studied. Examples include p53 null and heterogenous mice [94,95] demonstrated that these genes can act as oncogenes and lead to tumour development. Possible therapies for these oncogenes/tumour suppressor genes are tested for their response in an *in vivo*, full organism context [89] and examples include Ad-p53, AAV-HGFK1 [96].

HLA-A2 transgenic mice have genes inserted into the DNA so they will express the HLA molecules known in mice as H-2. In order to prevent the presentation of murine H-2-restricted cytotoxic T lymphocyte (CTL) epitopes in HLA-A2 (AAD) transgenic mice, HLA-A2.1 transgenic/H-2 class I knockout mice (HHD mice) were created [97]. In HHD mice, the H-2 class I gene is knocked out, and a chimeric HLA-A2.1 monochain (HHD) is produced by linking the C terminal of the human β2-microglobulin (unit of the class 1 MHC) covalently to the Nterminus of the chimeric HLA-A2 heavy chain (which contain the α1&2 domains of HLA-A2.1 and the α3 domain of H-2Db) by means of a peptide bond. This guarantees the sole expression of the HHD molecule on the cell surface, making sure that any identified CTL epitopes are HLA-A2 restricted [98]. HHD mice allow epitopes which are presented on human HLA-A2 to be examined for their ability to induce T cell responses in a variety of studies; for example STEAP, a prostate tumour antigen has been shown to be targeted by anti-tumour T cells [99] and DNA vaccines encoding Wilms tumour antigen 1 induce cytotoxic responses in mice [100] using this model system.

## **9. Modes of immunotherapy**

One of the biggest debates in cancer immunotherapy remains which mode will be the most effective. The National Cancer Institute have suggested that immunotherapy stud‐ ies should focus on a limited number of antigenic targets to maximise the chances of success [101]. However for some cancers effective immunotherapy targets have yet to be discovered (i.e. ovarian cancer, adult acute lymphocytic leukaemia) and better targets may yet be determined.

When T cells were found to be able to identify cancer cells [102] it was thought that T cell therapies would be the most effective with the aim being to stimulate CD8+ /CTL cells to kill tumour cells. This can be achieved by a number of ways such as through the use of DCs [103], peptide vaccines [85], DNA vaccines [104] and natural killer cells [105]. In recent years monoclonal antibodies (mAb) have become standard treatments for cancer. Ultimately if there is an antibody and it recognises a surface antigen solely on cancer and non-essential cells then this will likely be the most effective way to cause tumour destruction. mAbs are derived by vaccinating an animal with the target antigen and testing to see if the B cells are producing antibodies against it. Then the B cells are extracted from its spleen and infused with myeloma cells to produce hybridomas. Hybridomas divide perpetually and produce the mAb to the antigen in large amounts [106].