**5.5 Minimizing toxicities**

*Advances in Precision Medicine Oncology*

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

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

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

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

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

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

recognizing *only* tumor cells and sparing normal cells [68].

*5.3.2 Countering loss of antigen on cancer cells*

CAR T cells without fail.

*5.3.3 Formation of synapse*

effective T cell mediated killing [70].

CD8+ and CD4+ both proportions [73].

*5.3.4 CARs expressing Heparanase*

xenograft model [72].

equivocal response [43].

**5.4 Dose**

**130**

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 management are as follow:


possibly MAGE-A1, A8, A9 expressed in human brain. So unfortunately, strategies to enhance TCR affinity to neoantigens on tumor may lead to unanticipated toxicities thus warrants need of improved preclinical testing methods to better enable prediction of TCRs specificity.
