External objective reasons
Different co-stimulatory molecules
The antitumor effects of different co-stimulatory molecules in CART19 cells are different as well. CD19 CAR-T cells in ZUMA-1, JULIET and TRANSCEND studies have the similar structure containing same single-chain variable fragment (FMC63) and use CD3 for intracellular signaling but different combinations of transmembrane and costimulatory domains, leading to disparity on efficacy (Table 1).
Long et al. selected PD-1, LAG-3 and TIM-3 suppressor receptors as the detection markers for T-cell senescence. Through animal experimentation, it was found that CD28-CART19 cells had a strong tumor killing effect, whereas 4-1BB-CART19 cells was proven to be less potent but increased antitumor persistence.
Distinct manufacture methods
As lentivirus transfection is prone to give rise to insert mutations, CRISPR/Cas9 gene editing technology has become a prospective method in the manufacturing of CART19 cells . However, recent research  found that CRISPR/Cas9 system causes genomic damage and complex rearrangements, which may lead to pathogenic consequences. The CRISPR/Cas9 was not as precise and accurate as we expected. Recent study indicates that CART19 cells exhibits better differentiated ability and effector function when harvested from cultures at day 3 or 5 rather than at the routine period of 9–14 days in vitro.
Various categories and dosage of CART19 cells
Even in conventional CART19 cells, different preparation methods are taken in different centers, leading to distinct T-cell amplification. Furthermore, combinatorial antigen sensing developed to enhance tumor specificity [8▪], dual-CAR, tandem CAR and bi-epitopic CART cells [9▪], that targeted two tumor-specific antigens or epitopes, which can reduce tumor antigen escape rate and tumor relapse rate was also applied to clinical use [10–12]. There are also novel CAR-modified cell varieties in treating other hematological malignancies, such as multiple myeloma and acute leukemia with armored CART [13▪,14], CD44v6-targeted T cells , CAR-NK [16▪], CS1 CAR-Redirected T cells , anti-BCMA CART cell and anti-CD138-Kappa-light-chain CART cell, and so forth, making it hard to fully understand the relapse mechanism of post-CART cell treatment on B-cell malignancies.
Internal immunological and genetic reasons
Different B-cell malignancies have distinct tumor cells and are treated by different chemotherapy regimens. Even in homologous tumors, nontumor cells impact factors, such as somatic cells, transcriptional alterations, epigenetic modifications and molecular interactions can cause diverse disease attributes and lead to distinct prognosis. In addition, given the heterogeneous nature of the patients’ baseline conditions including age, disease and risk stratification, prior chemotherapy regimens and lines, whether using targeted drugs or not, curative efficacy evaluation pre-CART therapy are factors that need to be taken into consideration.
Medication history of targeted drugs and immunomodulatory drugs
As immune checkpoints were proved to have a critical role in immunotherapies and tumor microenvironment, antiprogrammed death-1 (PD-1) and programmed death ligands 1 (PD-L1) are currently widely used in relapsed/refractory B-NHL exhibiting high PD1 expression by T cells. Studies  reported increased expression of co-inhibitory molecular PD-1 in CART cells after infusion, and the obvious increasing of PD-1-expressed CAR19 T cells occurred between the time of infusion and the time of reaching peak CAR19 blood levels. As well, PD-1 expression is weaker in the CD19-negative CART cells than in CD19-positive CART cells. Zhang et al. [19▪] demonstrated that the combination of CD19 CART cells with a dose-adjusted PD-1 inhibitor shows synergistic antitumor capacity in a mouse trial, so the PD-1 inhibitor treatment before CART cell therapy might affect the efficacy positively.
IKZF1 and IKZF3 are transcription factors that are critical to the differentiation of B cells, lenalidomide can increase serum IL-2 level in vitro by down-regulating the expression of IKZF1/3 , thereby promoting the proliferation of natural killer (NK) cells, NK/T cells and CD4+ T cells. In-vitro studies showed that lenalidomide can decrease the amount of IL-6 that was secreted by monocytes and recede the immunosuppression on CART19 cell through the mechanism of reducing the quantity of CD8+CD28− Treg cells .
Bruton Tyrosine Kinase inhibitor ibrutinib
Due to the significant sequence and functional homology between BTK (Bruton Tyrosine Kinase) and ITK (IL-2-inducible kinase) , ibrutinib can inhibit the ITK signal pathway that is expressed on the surface of NK cells, NK-T cells and especially T cells including CART cells. There is another hypothesis about the interaction between ibrutinib and CD19 CART cell therapy as ibrutinib could cause depletion of targeted B cells in peripheral blood, the consequence of low-tumor burden might cause the loss of immunogenicity, thereby impact the CART cell expansion and proliferation. On the contrast, Ruella et al.  conducted experiments of combining CTL019 CART cells and ibrutinib to treat mantle cell lymphoma (MCL). Although ibrutinib changes the balance of Th1/Th2 cells in vitro, it has lower PD-1 expression and been proved to increase T-lymphocyte counts without changing T subsets by triggering T cells’ mobilization into peripheral blood in-vivo experiments. Ibrutinib—CART-cell interaction is complex and remains a controversial issue. We are looking forward to the results of ZUMA-2 study (NCT02601313) .
CART19 cell abnormality
Limited CART19 expansion and amplification
Application of humanized CART cells: as the immune response induced by murine-derived single chain fragment variable (scFv) region may limit the continuous expansion of CART cells in vivo and increase the risk of leukemia relapse, Maude et al. developed a humanized scFv, which was derived from mouse FMC63 antibody. Results showed that hCART19 cells therapy was effective for R/R ALL patients and those who relapsed after conventional CART-cell therapy.
CART19 cell exhaustion
T-cell exhaustion is the specific stage of T-cell differentiation caused by repeated antigen exposure, which weakens the function of effector T cells. CART cells, however, will inevitably be consumed because of the presence of co-stimulatory molecules, even without sustained antigen exposure. CART19 cells will be exhausted rapidly because of a high tumor burden, whereas a low-tumor burden reduces proliferation and differentiation of CART19 cells because of the lack of antigen stimulation. Many patients with a low tumor burden as well as low normal blood B-cell level were less likely to obtain remission after CART19 cell infusion . This phenomenon indicated that endogenous CD19+ cells could enhance proliferation of CART19 cells and speculated that CD19+ cellular vaccines might be another avenue to overcome immunological unresponsiveness in CART-cell therapy.
The continuous activation of T cells can cause T-cell senescence that is considered irreversible and is mainly related to age. Treg cells have been proven to be capable of enforcing CD8+ cytotoxic T cells, CD4+ helper T cells and effector T cells into senescence. The degradation of the effect function of senile T cells is accompanied by high expression of inhibitive receptors , such as PD-1, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4, or CD152), T-cell immunoglobulin and ITIM domain (TIGIT), lymphocyte activation gene-3 (LAG-3), CD244, CD160, T-cell immunoglobulin and mucin domain-containing-3 (TIM3), and so forth. At the same time, high expression of CD57 can damage the proliferation capacity of T cells, while increasing the ligand number of Killer Cell Lectin-like Receptor Subfamily G1 (KLRG-1) can increase the proliferation of T cells . If these two above-mentioned biomarkers have high expression, both can cause the CART cells lose the co-stimulatory signals, such as CD27 and CD28, whereas the down-regulation of CD28 expression is related to the loss of human telomerase RNA component (hTERC), which causes the loss of telomerase activity and leads to subsequent telomere damage, with a consequence of T cells duplicative senescence .
In summary, the mAb targeting the inhibitory receptor TIGIT can administrate immunotherapy by strengthening the antitumor function of NK cells, which is also of great significance to improve the efficacy of existing tumor immunotherapy. We envisage that CART-cell therapy, when combined with mAb to TIGIT, might further increase immune responses to cancer.
Immune escape mechanisms
Malignant tumors utilize various strategies to avoid the antitumor immunological effects of the adaptive immune system by establishing a microenvironment of immunosuppression. Immune escape mechanisms include regulating the expression of G1 regulatory proteins, producing immunosuppressive factors IL-10, TGF-β and IDO, and generating immunosuppressive receptors, such as the recruitment of PD-L1 and Treg cells.
It is hard to find a breakthrough in the complex immune system, so we turned our attention to the mechanism of CD19- relapse category. However, CD19− relapse is resistant to CART19-cell reinfusion  and cannot be prevented by extending the persistence of T cells. According to the recent estimates, the CD19− ALL relapsed after blinatumomab ranging between 10 and 30% in retrospective studies , another statistics showed that CD19− relapse accounts for 10–20% of post-CART19 therapy ALL patients [31,32]. Therefore, physicians should maintain a high level of suspicion for the evolution of post-CART malignancies.
The preexistence of CD19− clones
Preexistence of a minor CD19− population in the leukemia bulk has been proposed as a mechanism of resistance to blinatumomab and subsequent emergence of a CD19- relapse . Grupp et al. compared the samples of one pediatric case before CART19 therapy and CD19- relapse after CART19 therapy by flow cytometry, the results which was coincident with Fisher et al. and Ruella et al. demonstrated that rare CD19- blasts were existing in some samples before treatment in patients with CD19+ ALL. They hypothesized that these preexisting cells might be the trigger of CD19- relapse which developed as the dominant clone under the selective pressure of CART19 therapy and eventually resulted in CD19- relapse.
The loss or down-regulation of CD19 expression and the intervention
CD19 is not an essential condition of survival and proliferation of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells . It was found as a common phenomenon of CD19− relapse after CART19 therapy. As CD19 is located on chromosome 16p11.2, experiments conducted by Sotillo et al. found that entire chromosome 16 loss or alternative RNA splicing on exon 2, which was induced by serine and arginine-rich splicing factor 3 (SRSF3) occurred on CD19- xenograft tumor mice models. In their experiments, the CD19 gene was tested with the methods of whole exome sequencing (WES) and RNA-sequencing, finding de novo frameshift and missense mutations in exon 2 of CD19. The mutations did not result in the silencing of CD19 expression, but expressed the truncated protein with the presence of alternative exon 2 splicing of CD19, thus it could escape from the tumor killing effect as the CD19 epitope could not be recognized by CART19 cells. As the result, future CARs and other antibody based therapeutics should be designed to target essential exons, as a way to prevent escape .
Importantly, another mechanism of rapidly relapsing leukemia, especially in MLL gene rearranged pediatric leukemia, is lineage-switch from lymphoid to myeloid that results from reprogramming by down-regulating the B-cell transcript factors -- PAX5 and EBF1 [39,40]. CD19− relapse was not only found to have occurred through lineage switch of B-precursor cells from the lymphoid lineage to a CD14+ myeloid lineage in 4% of B-precursor ALL [39,41] but also reported that CD22 expression was maintained in the CD19- phenotype relapses , reminding us that dual/sequential CART cell infusion may play a role in preventing CD19− relapse.
CD22: Jacoby et al. suggested that simultaneous pressure on CD19 and CD22 might be an avenue to reduce the possibility of lineage switching, but anti-CD22 CART cells seemed to have only limited activity when B-cell malignancies was CD19− relapse.
CD123: CD123 is the IL-3 receptor expressing on hematopoietic progenitor cells. The studies [11,42] proved that combining CART19/123 cells could effectively prevent relapse caused by the loss of CD19 phenotype and the patients who developed CD19− relapse could be treated by CART123 cells.
FUTURE PERSPECTIVES: SPLIT, UNIVERSAL AND PROGRAMMABLE CHIMERIC ANTIGEN RECEPTOR
To alleviate the various limitations of CART cells, Cho et al.[43▪▪] presented a split, universal, and programmable (SUPRA) CAR system, which has the ability to switch targets without reinfusing other antigen-specific T cells, and can logically respond to multiple antigens by tuning T-cell activation precisely.
Conventional CART has a fixed structure of invariable antigen-specific scFv and intracellular signaling domains. This SUPRA CAR is composed of a universal receptor on T cell (zipCAR) and tumor targeting scFv adaptor molecule (zipFv). The zipCAR universal receptor is generated from the fusion of intracellular signaling domains and a leucine zipper as the extracellular domain. The zipFv adaptor molecule is generated from the fusion of a cognate leucine zipper and a scFv. The scFv of the zipFv binds to the tumor antigen and the leucine zipper binds and activates the zipCAR on the T cells [43▪▪].
When one of the controllable region is over activated and causes severe cytokine release syndrome (CRS), we can mitigate these toxicities by controlling other variable regions to regulate T-cell activation level. In order to reduce the extent of CRS, a competitive zipFv, which can prevent zipCAR from being activated by binding to the rest of zipFv has been developed (Table 2).
This SUPRA CAR system can also combat the antigen escape and achieve the antitumor effect equal to conventional Dual CART cell therapy. Of note, different antigens can easily be targeted without re-manipulation because of the SUPRA CAR platform.
In addition, SUPRA components have been proven to be effective in reducing immunogenicity while being humanized. Furthermore, the experiment also used orthogonal SUPRA CARs to regulate different T-cell signaling domains and T-cell subtypes independently to increase the range of the immune responses.
The SUPRA CAR system is a prospective product with inducible and logical control capabilities that can improve the safety and efficacy of current immunotherapy. However, further research is intensively needed to explore the toxicity and side-effects, the interaction of which with novel agents and the immune system affects the persistence and expansion of these SUPRA CART cells.
We would like to thank Xuefeng Gao, Shun Rao, Youhai Chen for their revision with the review.
Financial support and sponsorship
The relapse mechanism of genetic mutation of relapsed/refractory lymphoma after CART treatment has been approved by Shenzhen Science and Technology Innovation Committee, the amount RMB 500,000, Grant No. JCYJ20180228163509339. CART-38 cell therapy for disease progression/relapse multiple myeloma has been approved by Shenzhen municipal Health Commission Research Project, Project No. 201606021.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
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A prospective novel CART cell entity with logical operated system that simultaneously encompasses multiple critical ‘upgrades’, such as the ability to switch targets without re-engineering the T cells, finely tune T-cell activation strength, and sense and logically respond to multiple antigens.
B-cell hematological cancers; novel agent and novel chimeric antigen receptor T-cell system; the relapse mechanism after CART19 cells
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