**3.2 Genetic manipulations of T cells**

Sometimes in certain cases, tumor infiltrated T cells do not recognize tumor cells and hence they neither get activated nor proliferated *in-vivo*. In such cases, T cells' usefulness becomes redundant. To improve the functional properties of these cells including recognition of antigen on cancer cells, an alternative approach is adopted

#### **Figure 3.**

*Leading adoptive cellular therapy. Patients' T cells utilized in TILs, TCR T cells and CAR T cell therapy. Major steps of these therapies discussed in the boxes.*

where patient's T cells are genetically manipulated using gene editing technology. This also overcomes the problem of isolating pre-existing tumor-reacting T cells from patients with tumors of other types. Here the cells are made to express tumor antigen-specific TCRs, thus are effective in anti-tumor cytotoxic function [38].

There are two strategies to genetically modify the specificity of T cells. The patient's cells can be genetically modified by integrating genes encoding either conventional alpha-beta TCRs, or Chimeric Antigen Receptors (CARs), specific for tumor antigen(s). To develop ACT by these mechanisms, TCR T cells or CAR T cells are manufactured by autologous T cells which are amended *ex-vivo,* expanded and re-injected in patient to fight against cancer cells (**Figure 3b** and **c**). The only difference remains in the mode of recognition of tumor antigen by these T cells (**Figure 4**).

### *3.2.1 TCR T cell therapy*

The TCR is a specific receptor as well as characteristic marker on T cell surface. TCR complex is a di-sulphide linked membrane anchored heterodimer protein, consisting of two different peptide chains, TCR ɑ and TCR ß encircled by four CD3 chains [39]. These TCR ɑ and ß chains recognize the polypeptide fragment presented by MHC molecule on cancer cells. The principal objective behind TCR T cell technology is to modify TCR binding to tumor antigen which as such shows poor affinity for antigens making them incompetent to recognize and kill tumor cells effectively [40]. Thus, making of high affinity TCR T cell requires identification of specific targets on cancer cells. This way the genetically engineered TCR shows augmented recognition specificity and affinity for tumor cells.

In a study done by Rapoport AP et al., an autologous T cell was engineered to express a high affinity TCR specific to identify naturally processed peptide shared by cancer-testis antigen New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1) and L antigen family member 1 (LAGE-1) to be used for multiple myeloma patients showing encouraging clinical response in 16 of 20 (80%) patients with advanced disease [41].

#### **Figure 4.**

*Molecular insights of cellular therapy. The boxes illustrate the salient features to determine the choice of ACT. In TILs and TCR T cells, the TCR* ɑ *and ß chains recognize the antigen presented with MHC molecule on cancer cells whereas CAR T cells recognize the tumor antigen independent of MHC. In TCR T cell, genetically engineered high affinity TCR recognizes tumor cells. In CAR T cell, a CAR, a scFv derived from variable regions of heavy and light chains of a monoclonal antibody against tumor antigen recognize tumor cells. CAR also consists of a trans-membrane domain; a hinge; one or more than one intracellular co-stimulatory molecules and a CD3z signaling domain. TCR T cell and CAR T cell therapy highly depend upon identification of unique antigen on cancer cells.*

**125**

*Advances in Adoptive Cellular Therapy (ACT) DOI: http://dx.doi.org/10.5772/intechopen.95854*

dent of MHC [2, 42, 43].

*3.2.2 CAR T cell therapy*

and functional T cell [2, 44].

function into a single receptor (**Figure 4**).

**4. Opportunities and challenges**

In above mentioned TIL and TCR T cell-based therapies, there may be yet another problem which may be encountered when these expanded/modified cells still do not so efficiently recognize cancer cells. This happens when cancer cells smartly down regulate expression of specific MHC molecules and tumor antigen is presented without MHC complex and thus these T cells fail to recognize the target cancer cells. Fortunately, emergence of recombinant DNA technology and novel cell isolation techniques from blood have paved newer ways to cancer immunotherapy. Hence, to overcome this evading mechanism by cancer cells, T cells are genetically modified in such a way that they recognize cancer cells by a mechanism indepen-

This newer modality of modifying T cells is yet another type of ACT, where T cell are armed with a CAR, which can now make the T cells recognize cancer antigen without MHC molecules. Use of such modified T cells bearing CAR is called as CAR T cell for therapy. The concept of CAR T cell is based on the ability of genetically engineered patient's own T cells to express a CAR, which is specific for a tumor antigen and therefore better in fighting cancer cells. A CAR consists of a scFv derived from variable region of heavy and light chains of a monoclonal antibody against tumor antigen to recognize tumor cells. Apart from this, a trans-membrane domain; a hinge; one or more than one intracellular co-stimulatory molecules and a CD3 zeta signaling domain are the part of CAR construct to make fully activated

Four generations of CARs have been developed, with subsequent generations being better than the previous ones with respect to cytotoxicity and shelf-life. First generation CARs had only single chain variable fragment (scFv) linked to CD3ζ or Fc receptor gamma signaling domain. With the subsequent additions of co-stimulatory domains like CD28, CD 137, or OX40, second and third generation CARs have been created. Fourth generation CARs also called TRUCK (T cell redirected for universal cytokine killing) are armed with immune stimulatory cytokines that ameliorate the performance of CAR T cell with respect to its expansion, persistence, and resistance even in immunosuppressive tumor microenvironment [45, 46]. This therapy harnesses the power of immune system to fight the cancer but in contrast to the regular T cell receptor (TCRs), that recognize Ag, only when presented with MHC, CARs have the aptitude to redirect the effector function of T cell toward any tumor associated antigen (TAA) expressed on the tumor surface even without MHC. Once CAR of the T cell binds to its specific TAA, T cell gets activated through phosphorylation of immune receptor tyrosine-based activation motifs leading to cytokine secretion, T cell proliferation and cytotoxicity [47]. The word chimeric here signifies both, antigen binding, independent of MHC and T cell activation

Advantage of TCR T cell therapy over CAR T cell therapy is that these can recognize even deep-seated antigens fragment presented on MHC molecule in contrast to CAR T cell that recognize only cell surface proteins. So, TCR T cell therapy offer wider range of application but it is also MHC restricted and recognizes only those antigens presented on MHC molecule, a major drawback of TCR T cell therapy.

Like any other therapy, ACT also has success and failures. The therapy deals with T cells in two different ways, firstly, the natural T cell with antitumor activity *Advances in Adoptive Cellular Therapy (ACT) DOI: http://dx.doi.org/10.5772/intechopen.95854*

In above mentioned TIL and TCR T cell-based therapies, there may be yet another problem which may be encountered when these expanded/modified cells still do not so efficiently recognize cancer cells. This happens when cancer cells smartly down regulate expression of specific MHC molecules and tumor antigen is presented without MHC complex and thus these T cells fail to recognize the target cancer cells. Fortunately, emergence of recombinant DNA technology and novel cell isolation techniques from blood have paved newer ways to cancer immunotherapy. Hence, to overcome this evading mechanism by cancer cells, T cells are genetically modified in such a way that they recognize cancer cells by a mechanism independent of MHC [2, 42, 43].

#### *3.2.2 CAR T cell therapy*

*Advances in Precision Medicine Oncology*

*3.2.1 TCR T cell therapy*

with advanced disease [41].

where patient's T cells are genetically manipulated using gene editing technology. This also overcomes the problem of isolating pre-existing tumor-reacting T cells from patients with tumors of other types. Here the cells are made to express tumor antigen-specific TCRs, thus are effective in anti-tumor cytotoxic function [38]. There are two strategies to genetically modify the specificity of T cells. The patient's cells can be genetically modified by integrating genes encoding either conventional alpha-beta TCRs, or Chimeric Antigen Receptors (CARs), specific for tumor antigen(s). To develop ACT by these mechanisms, TCR T cells or CAR T cells are manufactured by autologous T cells which are amended *ex-vivo,* expanded and re-injected in patient to fight against cancer cells (**Figure 3b** and **c**). The only difference remains in the mode of recognition of tumor antigen by these T cells (**Figure 4**).

The TCR is a specific receptor as well as characteristic marker on T cell surface. TCR complex is a di-sulphide linked membrane anchored heterodimer protein, consisting of two different peptide chains, TCR ɑ and TCR ß encircled by four CD3 chains [39]. These TCR ɑ and ß chains recognize the polypeptide fragment presented by MHC molecule on cancer cells. The principal objective behind TCR T cell technology is to modify TCR binding to tumor antigen which as such shows poor affinity for antigens making them incompetent to recognize and kill tumor cells effectively [40]. Thus, making of high affinity TCR T cell requires identification of specific targets on cancer cells. This way the genetically engineered TCR shows

In a study done by Rapoport AP et al., an autologous T cell was engineered to express a high affinity TCR specific to identify naturally processed peptide shared by cancer-testis antigen New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1) and L antigen family member 1 (LAGE-1) to be used for multiple myeloma patients showing encouraging clinical response in 16 of 20 (80%) patients

*Molecular insights of cellular therapy. The boxes illustrate the salient features to determine the choice of ACT. In TILs and TCR T cells, the TCR* ɑ *and ß chains recognize the antigen presented with MHC molecule on cancer cells whereas CAR T cells recognize the tumor antigen independent of MHC. In TCR T cell, genetically engineered high affinity TCR recognizes tumor cells. In CAR T cell, a CAR, a scFv derived from variable regions of heavy and light chains of a monoclonal antibody against tumor antigen recognize tumor cells. CAR also consists of a trans-membrane domain; a hinge; one or more than one intracellular co-stimulatory molecules and a CD3z signaling domain. TCR T cell and CAR T cell therapy highly depend upon identification* 

augmented recognition specificity and affinity for tumor cells.

**124**

**Figure 4.**

*of unique antigen on cancer cells.*

This newer modality of modifying T cells is yet another type of ACT, where T cell are armed with a CAR, which can now make the T cells recognize cancer antigen without MHC molecules. Use of such modified T cells bearing CAR is called as CAR T cell for therapy. The concept of CAR T cell is based on the ability of genetically engineered patient's own T cells to express a CAR, which is specific for a tumor antigen and therefore better in fighting cancer cells. A CAR consists of a scFv derived from variable region of heavy and light chains of a monoclonal antibody against tumor antigen to recognize tumor cells. Apart from this, a trans-membrane domain; a hinge; one or more than one intracellular co-stimulatory molecules and a CD3 zeta signaling domain are the part of CAR construct to make fully activated and functional T cell [2, 44].

Four generations of CARs have been developed, with subsequent generations being better than the previous ones with respect to cytotoxicity and shelf-life. First generation CARs had only single chain variable fragment (scFv) linked to CD3ζ or Fc receptor gamma signaling domain. With the subsequent additions of co-stimulatory domains like CD28, CD 137, or OX40, second and third generation CARs have been created. Fourth generation CARs also called TRUCK (T cell redirected for universal cytokine killing) are armed with immune stimulatory cytokines that ameliorate the performance of CAR T cell with respect to its expansion, persistence, and resistance even in immunosuppressive tumor microenvironment [45, 46]. This therapy harnesses the power of immune system to fight the cancer but in contrast to the regular T cell receptor (TCRs), that recognize Ag, only when presented with MHC, CARs have the aptitude to redirect the effector function of T cell toward any tumor associated antigen (TAA) expressed on the tumor surface even without MHC. Once CAR of the T cell binds to its specific TAA, T cell gets activated through phosphorylation of immune receptor tyrosine-based activation motifs leading to cytokine secretion, T cell proliferation and cytotoxicity [47]. The word chimeric here signifies both, antigen binding, independent of MHC and T cell activation function into a single receptor (**Figure 4**).

Advantage of TCR T cell therapy over CAR T cell therapy is that these can recognize even deep-seated antigens fragment presented on MHC molecule in contrast to CAR T cell that recognize only cell surface proteins. So, TCR T cell therapy offer wider range of application but it is also MHC restricted and recognizes only those antigens presented on MHC molecule, a major drawback of TCR T cell therapy.

#### **4. Opportunities and challenges**

Like any other therapy, ACT also has success and failures. The therapy deals with T cells in two different ways, firstly, the natural T cell with antitumor activity and secondly, the genetically manipulated T cells, either the TCRs engineered or the receptor is chimeric. Thus, T cells' immune responsiveness for tumor targeting functions discretely. Also, during the therapy, responsiveness and un-responsiveness of host is guided by many factors. Similarly, many criteria in preparation of the cells for therapy are the deciding factor for the effectiveness of the therapy, which in turn is decided by extent of tumor regression.

#### **4.1 Tumor regression**

Dramatic regression of variety of cancers, like melanoma, cervical cancer, lymphoma, leukemia, bile duct cancer, and neuroblastoma has been reported by the use ACT based approaches [2]. A number of cancer patients showed success using TILs, however, TIL therapy requires surgery and to obtain enough TILs is always a huge challenge technically. TILs expansion approach has been best used for the treatment of metastatic melanoma by Rosenberg et al. [48].

Applications of genetic manipulations in ACT have greatly contributed in improving the remission rate of treatment of various type of cancers [49]. For relapsed and refractory B-cell precursor Acute Lymphoblastic Leukemia in children and young adults, CAR T cell therapy was successful in 52 of 63 patients and three of every four patients did not show relapse in six months [50]. In another trial to treat refractory large B-cell lymphoma, CAR T cell therapy has shown promising results as it completely cured 54% patients, slowed tumor growth in 82% patients and there was no relapse in 40% patient even after 15.4 months [51]. CAR T cell therapy using B cell maturation antigen was tried on multiple myeloma patients and showed remission in 74% of patients [52].

#### **4.2 Challenges**

The failures of the therapy in any form have been associated with many factors. The extent to which the tumor cells can evade immune recognition and successfully employ immune suppression mechanisms leads to failure of ACT.

#### *4.2.1 Cell selection*

One of the factors which guards the successful use of ACT in humans is the identification of cells that can target antigens selectively expressed on the cancer and not on essential normal tissues. This criterion is the basis of the success of ACT. Also, sometimes immune cells lose their natural tendency to recognize and kill the tumor cells, leads to failure of therapy. Therefore, even though the cell selection is appropriate, sometimes success is not achieved.

#### *4.2.2 Tumor microenvironment*

Activities of tumor cells also play a key role in suppressing the effector function of immune cells used in ACT. The tumor cells along with their neighborhood constitute a unique environment which is called as tumor microenvironment (TME). This has an ability to suppress host's immune system by various mechanisms such as a) T cell exhaustion due to continuously changing antigen signatures on them; b) affects the cytotoxic function of T cells at the site of tumor and c) also the T cell trafficking [53]. To counter these immune suppressive mechanisms of TME, there are possible technologies available that locally deliver T cells to the TME and increase their proliferation, thus, could provide a means to treat inoperable solid tumors [54].

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*Advances in Adoptive Cellular Therapy (ACT) DOI: http://dx.doi.org/10.5772/intechopen.95854*

precautions to avoid technological pitfalls.

ACT involves different stages to generate clinical grade therapeutic cells and trained personnel to execute the technology. Therefore, it needs great care and

The technical issues are related to the manufacturing process of adoptive cellular material and the delivery platforms which also account for the success or failure of the ACT. During the *ex-vivo* expansion and genetic modification process, factors such as cell culture time, use of cytokines and use of vectors for gene transfer are the major concerns deciding the efficacy and survival of T cells being used in the therapy. Besides sometimes cancer specific T cells may not grow that well and not sufficient for infusion. At the same time efficacy of T cell may also change in *ex-vivo*

Major hurdle with regard to TCR T cell technology is related to its expansion which includes identifying of a good target, along with specific TCRs, screening for desirable TCR affinity. Also, TCR T cell therapy is MHC dependent and there is grave peril of hybridization between exogenous and endogenous chain causing recognition of auto-antigens thereby leading to graft-versus host disease [43].

Failures of the therapy with adverse outcomes have been reported due to some chromosomal DNA translocations and rearrangements during the preparation of

A major limitation of adoptive T cell therapies is the delivery technology used in the patient. It is noted that the viability and function of the transplanted cells rapidly decline after administration [58]. Hence, different delivery technologies (nanoparticles or scaffolds) have been explored to improve success of ACT. Adjuvant-loaded nanoparticles, chemically conjugated to the surface of T cells to stimulate transplanted cells and minimize the systemic side effects have been designed [59]. Advantages of having a delivery system of T cells which has some immune stimulating mechanisms linked, may help the T cells to enter the tumor site and perform better results of the therapy. Apart from systemic administration route, biomaterials-based strategies have also been explored to locally deliver adoptive T cells to solid tumors [60, 61] as successful targeting of T cells to most solid cancers remain challenging [62]. To overcome the barrier of secluded location of solid tumors, local injection of T cell in brain tumors in mouse model has been tried

Overall, biomaterial-mediated local T cell delivery approaches could improve the efficiency of adoptive T cell therapies for treating inoperable solid tumors by overcoming local immunosuppressive barriers. The usefulness of these therapies depends on how quickly T cells can be generated in tumors *in-vivo* using this

Regulatory guidelines are other part of the story that limit the use of any therapy where biological samples are used for therapeutic. There are certain considerations to be followed for minimal manipulation and homologous use of human cells, tissues, and cellular and tissue-based products. Cellular & Gene Therapies are complex

and has shown better outcome than systemic administration [54].

approach relative to the time it takes to expand T cells *ex-vivo*.

*4.2.4 Regulatory guidelines and cost*

*4.2.3 Technical glitches*

*4.2.3.1 Manufacturing*

growth conditions [55].

the cells [56, 57].

*4.2.3.2 Delivery system*

### *4.2.3 Technical glitches*

*Advances in Precision Medicine Oncology*

**4.1 Tumor regression**

**4.2 Challenges**

*4.2.1 Cell selection*

turn is decided by extent of tumor regression.

of metastatic melanoma by Rosenberg et al. [48].

showed remission in 74% of patients [52].

appropriate, sometimes success is not achieved.

*4.2.2 Tumor microenvironment*

and secondly, the genetically manipulated T cells, either the TCRs engineered or the receptor is chimeric. Thus, T cells' immune responsiveness for tumor targeting functions discretely. Also, during the therapy, responsiveness and un-responsiveness of host is guided by many factors. Similarly, many criteria in preparation of the cells for therapy are the deciding factor for the effectiveness of the therapy, which in

Dramatic regression of variety of cancers, like melanoma, cervical cancer, lymphoma, leukemia, bile duct cancer, and neuroblastoma has been reported by the use ACT based approaches [2]. A number of cancer patients showed success using TILs, however, TIL therapy requires surgery and to obtain enough TILs is always a huge challenge technically. TILs expansion approach has been best used for the treatment

Applications of genetic manipulations in ACT have greatly contributed in improving the remission rate of treatment of various type of cancers [49]. For relapsed and refractory B-cell precursor Acute Lymphoblastic Leukemia in children and young adults, CAR T cell therapy was successful in 52 of 63 patients and three of every four patients did not show relapse in six months [50]. In another trial to treat refractory large B-cell lymphoma, CAR T cell therapy has shown promising results as it completely cured 54% patients, slowed tumor growth in 82% patients and there was no relapse in 40% patient even after 15.4 months [51]. CAR T cell therapy using B cell maturation antigen was tried on multiple myeloma patients and

The failures of the therapy in any form have been associated with many factors. The extent to which the tumor cells can evade immune recognition and successfully

One of the factors which guards the successful use of ACT in humans is the identification of cells that can target antigens selectively expressed on the cancer and not on essential normal tissues. This criterion is the basis of the success of ACT. Also, sometimes immune cells lose their natural tendency to recognize and kill the tumor cells, leads to failure of therapy. Therefore, even though the cell selection is

Activities of tumor cells also play a key role in suppressing the effector function of immune cells used in ACT. The tumor cells along with their neighborhood constitute a unique environment which is called as tumor microenvironment (TME). This has an ability to suppress host's immune system by various mechanisms such as a) T cell exhaustion due to continuously changing antigen signatures on them; b) affects the cytotoxic function of T cells at the site of tumor and c) also the T cell trafficking [53]. To counter these immune suppressive mechanisms of TME, there are possible technologies available that locally deliver T cells to the TME and increase their proliferation, thus, could provide a means to treat inoperable solid

employ immune suppression mechanisms leads to failure of ACT.

**126**

tumors [54].

ACT involves different stages to generate clinical grade therapeutic cells and trained personnel to execute the technology. Therefore, it needs great care and precautions to avoid technological pitfalls.
