**5. Enhancing efficacy of ACT**

The journey of development of ACT for cancer treatment has faced success and failures of the therapy in different cancer types. This has led to newer researches and explorations in various domains of the treatment by ACT. Work on improving effectiveness of therapy has contributed enormously and made difference in its outcomes. Following are some areas, which have been mentioned in this section about the efforts made toward enhancing efficacy of ACT.

#### **5.1 Measuring effectiveness**

Measuring effectiveness of any therapy is an essential part of it. It is critical to measure of cellular therapy's effectiveness as variations may occur in various steps, starting from isolation of cells to its re-infusion and homing of effective cells to target the cancer cells (**Figure 5**). For this purpose, a therapeutic index (TI) has been developed [65].

TI of a drug is a quantitative assessment of the ratio of a drug dose that produces toxicity to the dose that yields a clinically effective response. ACT has complex biodistribution and also content dependent potency, so for this TI estimation depends on other factors as well. These include functional fitness of the product, vague pharmacokinetics due to trapping, sequestration and extravasations in nearby tissue and inconstant rate of expansion *in-vivo*. Addition factors also influence pharmacokinetics in case of solid tumors like difference in trafficking to benign and cancer tissue, immune suppression and cellular dysfunction due to unfavorable hostile metabolic state.

**129**

related) [65].

**Figure 5.**

**5.2 Host conditioning**

**5.3 Affinity of T cells**

*5.3.1 Selection of CARs with moderate affinity*

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

*the seesaw game. Increasing the scope will determine its future.*

Thus, TI of the ACT largely depends on generic factors (T cell potency and fitness, Dispersion, Dysfunction, combinatorial therapies, comorbidities and microbiome) and TME-specific factors (Antigen availability, tumor, immune response

*Scope to enhance therapeutic efficacy of ACT. The scope and limitations are two ends ACT. It may be viewed as* 

It is important to understand that anti-tumor efficacy of ACT greatly depends on the persistence of adoptively transferred T cells in the host. This is achieved by an optimal pre-conditioning of the host, an important part of pre-treatment protocol where lymphodepletion by chemotherapy and/or radiation therapy is done prior to therapy. The process of lymphodepletion is important to deplete T regulatory cells and lymphocytes, as these cells compete with the transferred cells for homeostatic cytokines, interleukin 7 and IL15 and this needs to be minimized [66]. This may also be important to avoid excessive cytokine release by lymphocytes which causes adverse effect during the therapy. Host conditioning by either nonmyeloablative chemotherapy or irradiation may induce high levels of IL-1β which increase the number and functionality of adoptively transferred T cells within the tumor and thus improves efficacy of ACT [67]. An FDA-approved reagent, fludarabine has predictable lymphodepleting kinetics and duration of action. Its use in a conditioning regimen, promotes homeostatic upregulation of cytokines and growth signals for T cell persistence [6]. The use of cytokine IL2 has also been recommended for

better proliferation of the cells being used in this cellular therapy [2, 6].

Affinity of the immune cells in ACT is a highly critical criterion while using them for therapy. Optimum affinity is ensured during making of the cells. In TILs based therapy, it may not be that critical as there are no manipulations involved as such except increasing their numbers. However, while doing genetic manipulations, *Advances in Adoptive Cellular Therapy (ACT) DOI: http://dx.doi.org/10.5772/intechopen.95854*

#### **Figure 5.**

*Advances in Precision Medicine Oncology*

majority of cancer patients [64].

**4.3 Advantages**

life span *in-vivo* [64]*.*

also not MHC restricted.

**5. Enhancing efficacy of ACT**

**5.1 Measuring effectiveness**

been developed [65].

metabolic state.

about the efforts made toward enhancing efficacy of ACT.

products which are regulated by the Food and Drug Administration (FDA) in the United States. The European Union (EU), governs the regulation of all medicinal products for human use, including advanced therapy medicinal products (ATMPs), i.e., medicinal products comprised of cells, genes, or tissues to ensure the quality, safety, and efficacy of medicines placed on the market in the EU. The aims of EU are

A very high treatment cost of ACT based immunotherapies has been another concern for its limited use [20, 63]. Cost of CAR-T cell has been curtailed by developing Universal CAR-T cell and CAR NK cell so that this therapy can be a hope for

There are many advantages with ACT that outweigh the conventional therapy. One major advantage of TCR-T cell therapy is that it can target many TAA, even when these lie intracellular and are deep seated. Site directed injection of T cells into tumor giving superior result than systemic administration, is yet another favorable approach in cell-based therapies. A successful example of such application is reported in brain tumors where T cells are injected into CSF directly [43, 54]. Similarly, next generation of CARs have enhanced the ability of T cells to destroy tumor by infiltrating into diseased tissue site and have potential to moderate tumor microenvironment by secreting pro-inflammatory cytokines and expand their own

CARs have another unique ability to recognize not only peptide but also carbohydrate and glycolipid antigens, thus increasing their target antigen number and are

The journey of development of ACT for cancer treatment has faced success and failures of the therapy in different cancer types. This has led to newer researches and explorations in various domains of the treatment by ACT. Work on improving effectiveness of therapy has contributed enormously and made difference in its outcomes. Following are some areas, which have been mentioned in this section

Measuring effectiveness of any therapy is an essential part of it. It is critical to measure of cellular therapy's effectiveness as variations may occur in various steps, starting from isolation of cells to its re-infusion and homing of effective cells to target the cancer cells (**Figure 5**). For this purpose, a therapeutic index (TI) has

TI of a drug is a quantitative assessment of the ratio of a drug dose that produces toxicity to the dose that yields a clinically effective response. ACT has complex biodistribution and also content dependent potency, so for this TI estimation depends on other factors as well. These include functional fitness of the product, vague pharmacokinetics due to trapping, sequestration and extravasations in nearby tissue and inconstant rate of expansion *in-vivo*. Addition factors also influence pharmacokinetics in case of solid tumors like difference in trafficking to benign and cancer tissue, immune suppression and cellular dysfunction due to unfavorable hostile

to ensure the quality, efficacy and most importantly safety of public health.

**128**

*Scope to enhance therapeutic efficacy of ACT. The scope and limitations are two ends ACT. It may be viewed as the seesaw game. Increasing the scope will determine its future.*

Thus, TI of the ACT largely depends on generic factors (T cell potency and fitness, Dispersion, Dysfunction, combinatorial therapies, comorbidities and microbiome) and TME-specific factors (Antigen availability, tumor, immune response related) [65].

#### **5.2 Host conditioning**

It is important to understand that anti-tumor efficacy of ACT greatly depends on the persistence of adoptively transferred T cells in the host. This is achieved by an optimal pre-conditioning of the host, an important part of pre-treatment protocol where lymphodepletion by chemotherapy and/or radiation therapy is done prior to therapy. The process of lymphodepletion is important to deplete T regulatory cells and lymphocytes, as these cells compete with the transferred cells for homeostatic cytokines, interleukin 7 and IL15 and this needs to be minimized [66]. This may also be important to avoid excessive cytokine release by lymphocytes which causes adverse effect during the therapy. Host conditioning by either nonmyeloablative chemotherapy or irradiation may induce high levels of IL-1β which increase the number and functionality of adoptively transferred T cells within the tumor and thus improves efficacy of ACT [67]. An FDA-approved reagent, fludarabine has predictable lymphodepleting kinetics and duration of action. Its use in a conditioning regimen, promotes homeostatic upregulation of cytokines and growth signals for T cell persistence [6]. The use of cytokine IL2 has also been recommended for better proliferation of the cells being used in this cellular therapy [2, 6].

#### **5.3 Affinity of T cells**

#### *5.3.1 Selection of CARs with moderate affinity*

Affinity of the immune cells in ACT is a highly critical criterion while using them for therapy. Optimum affinity is ensured during making of the cells. In TILs based therapy, it may not be that critical as there are no manipulations involved as such except increasing their numbers. However, while doing genetic manipulations, it needs care especially when a gene fragment is being incorporated into the cell to express a chimeric receptor as in the case of CAR T cell therapy. High affinity CAR bearing cells are *not* the choice of cells as these might recognize the TAA present on the normal cells too and may cause *on target/off tumor* effect, an effect occurs when CAR T cells attack non-tumor cells expressing the target antigen. Thus, CAR selection is very important and ensured to be of low affinity so that it recognizes the antigen when it is present in high number as in case of tumor cells. This helps in recognizing *only* tumor cells and sparing normal cells [68].

### *5.3.2 Countering loss of antigen on cancer cells*

Sometimes problem of therapy arises when the cancer cells start losing CARtargeted antigen on them and escape their detection by CAR T cells, thus avoid their killing. Such situation is countered by targeting multiple antigens with multiple CARs [69]. For this, anti-tag CARs (AT-CARs) have been developed by adding affinity-enhanced monomeric streptavidin2 (mSA2) biotin-binding domain in the CAR construct. Such novel mSA2CARs have an advantage that the T cells expressing such CARs can bind cancer cells coated with biotinylated antibodies [69]. Binding of such antibodies to cancer cells probably avoids the loss of antigen being targeted on them. Thus, recognition of cancer cells occurs followed by their killing by such CAR T cells without fail.

#### *5.3.3 Formation of synapse*

To further improve efficacy of CARs, small sized antibodies (variable heavy homodimers) or nanobodies are recommended to be used with CAR T cell preparation for infusion. These antibodies cause tight synapse formation between the target and effector cell, which is important for the initiation of immune signaling, thus effective T cell mediated killing [70].

### *5.3.4 CARs expressing Heparanase*

It has been discovered that heparanase enzyme expression needs to be upregulated in CAR T cells to penetrated tumor stroma which consists of heparin sulfate proteoglycane. In vitro expanded T cells show reduced heparanase expression as compared to activated immune cell, suggesting their compromised migration [71]. This drawback has been overcome by designing better CAR T cells which were engineered to express heparanase enzyme and therefore show greater capacity to infiltrate tumor stroma with enhanced anti-tumor activity in neuroblastoma xenograft model [72].

#### **5.4 Dose**

Next important part of therapy is the dose, i.e., number of the cells in the prepared fraction/dose. The dose frequency and the number of cells per dose to be used for infusion, both play a crucial role in outcome of the therapy. Proportion of immune cells responsible for tumor regression controls the success of ACT. Such as CD8+ enriched "young" tumor infiltrating lymphocytes show better response in the regression of metastatic melanoma compared to the crude fraction containing both CD8+ and CD4+ both proportions [73].

It has also been reported that the number of transfused CAR T cell needed for single transfusion is much less than that needed for TCR T cell therapy to produce equivocal response [43].

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spared.

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

ACT involves manipulation of immune system to improve its efficacy for specific killing of cancer cells. Such alterations may lead to exaggerated immune response and cause toxicities which are different from other cancer therapies. Thus, depending upon the type of mechanisms involved in these toxicities, discrete approaches are needed to minimize them. The possible toxicities observed and their

a.On target/off tumor recognition develops a toxicity due to shared expression of target antigens by normal tissue leading to varying severity of adverse event from B cell aplasia to death. Hypogammaglobulinemia in B cell aplasia can be

b.Anaphylaxis is seen in patients receiving genetically modified T cell as their antigen recognition domain in derived from murine mAb. Efforts are being

c.Graft versus host disease is commonly observed phenomena in immunothera-

d.Cytokine Release Syndrome (CRS) is associated with overt activation of T cell, which leads to immune activation process with markedly elevated cytokines. It is seen in CAR T cell therapy and called as CAR T cell toxicity. CRS can be minimized by controlling the activity of CAR T cells. For this a bispecific adaptor has been designed which is a cancer specific ligand conjugated with fluorescein. This specifically binds with cancer cells and tag them with fluorescein. CAR is so devised that it recognizes fluorescein and not tumor antigen. Thus, the bispecific adaptor bridges CAR T cell and its tumor target. Thus, availability of bispecific adaptor regulates the killing of tumor cells and can control the CRS. Also, to subdue any CRS, rupture of bridge between CAR T cell to cancer cell can be lifesaving [74]. Other successful approaches to control CRS are immuno-suppression by systemic corticosteroids, IL-6 receptor blockade with

treated with intravenous immunoglobulin replacement therapy.

pies. Infusion of isolated autologous TILs is the way to curtail it.

e.Immune effector cell associated neurotoxicity syndrome has also been reported, plausible explanation being elevated cytokine level.

f. Toxicity of T cell activation is also managed by inclusion of an "on switch" in CAR design which can make a hold on functional intensity and T cell activation. This requires selection of two target antigens that are co-expressed on malignant tissue making dual antigen binding a must for complete T cell activation. So normal tissue expressing one target antigen cannot provide complete activation and so limits this toxicity. Conversely, if dual antigens presentation is exclusive to normal tissue, inhibitory signaling in CAR design allow for selective targeting of malignant tissue expressing one antigen while normal tissue is

g.Severe neurological side effects leading to coma and death are reported in patients treated with ACT using autologous T cell when T cells were modified with Melanoma-associated antigen 3 (MAGE-A3) antigen specific TCRs (MAGE-A3 is cancer testis antigen never expressed in normal tissue). These TCRs recognize the different but similar epitopes of especially MAGE-A12 and

made to humanize the expressed protein [74].

mAb or lymphodepleting chemotherapy.

**5.5 Minimizing toxicities**

management are as follow:
