Immunotherapies have revolutionized the cancer therapy landscape (1,2) and hold potential in other areas, including autoimmune diseases, allergies, (3) and organ transplant. (1,4,5) There are several types of immunotherapies, each of which aims to mount an immune response against diseased cells. This article will focus on adoptive T-cell therapies and the role of immunosequencing in understanding how these therapies affect the immune system.
Harnessing T cells to target tumors
Tumors develop several mechanisms to escape T-cell-mediated immune responses, including downregulating major histocompatibility complex (MHC) proteins, which are necessary for antigen presentation to T cells, (2,6,7) upregulating immune checkpoint receptors that inhibit T-cell activation, and reprogramming the tumor microenvironment to be immunosuppressive. (7,8) The goal of adoptive T-cell therapy (ACT) is to overcome this immunosuppression by introducing endogenous antigen-specific or modified T cells that recognize and eliminate tumor cells.
Type of T-cell therapies
There are three main types of ACT used in cancer therapy: tumor-infiltrating lymphocyte (TIL) therapy, engineered T-cell receptor (TCR) T-cell therapy, and chimeric antigen receptor (CAR) T-cell therapy (summarized in Figure 1 and Table 1). (2)
Figure 1. Overview of TIL, engineered TCR, and CAR T-cell therapies. In TIL, tumor-resident T cells are isolated, expanded ex vivo, and returned to the patient. In engineered TCR and CAR T-cell therapies, peripheral T cells are leukopheresed from the patient and transduced to express a TCR or CAR, respectively, that is designed to target tumor antigens. ACT: adoptive cell therapy; CAR: chimeric antigen receptor; TCR: T-cell receptor; TIL: tumor-infiltrating lymphocyte. Adapted from Rohaan et al. (2)
Table 1: Overview of adoptive T-cell therapies
|T-cell source||Ex vivo modification||Target||Examples|
|TIL therapy||Autologous TILs||None||MHC-restricted||LN-145*|
|Engineered TCR T-cell therapy||Autologous peripheral cells||Transduced with antigen-specific TCR||MHC-restricted||NY-ESO-1 TCR-transduced T cells*|
WT1 TCR-transduced T cells*
|CAR T-cell therapy||Autologous peripheral cells||Transduced with antigen-specific CAR||Non-MHC surface proteins and glycans||Tisagenlecleucel|
In both transgenic TCR T-cell therapy and CAR T-cell therapy, peripheral T cells are isolated from a patient and modified ex vivo to express either a TCR or CAR that is designed to target specific tumor antigens. The modified T cells are subsequently infused back into the individual to induce antitumor activity. Figure 2 shows how a modified CAR T cell interacts with an antigen on tumor cells.
Figure 2. A first-generation CAR T-cell binding to a cell surface antigen on a tumor cell. CAR-T: chimeric antigen receptor-modified T cell; CD3ζ: cluster of differentiation 3-ζ; TM: transmembrane domain; VH: variable heavy chain; VL: variable light chain. Adapted from Dees et al. (9)
Monitoring the effects of T-cell therapies with the immunoSEQ® Assay
CAR T-cell therapies have demonstrated impressive response rates in patients with hematologic malignancies. Response rates to approved CAR T-cell therapies can reach 90% in patients with B-cell acute lymphoblastic leukemia (B-ALL) and 60% in patients with non-Hodgkin’s lymphoma (NHL). However, many patients develop resistance and experience relapse. (10,11)
To investigate the characteristics of T-cell therapies associated with response or resistance, researchers are using immunosequencing to profile both the T-cell infusion product and the recipient’s T-cell repertoire response to the product.
Our immunoSEQ Assay is a next-generation sequencing technology that enables high-throughput and quantitative insights into the T-cell repertoire. It enables researchers to track the expansion and contraction of T-cell clones over time or in response to treatment. To learn more about this technology, view our webinar on Utilizing Immunosequencing In Immuno-Oncology And Immunotherapy Research.
In the next section, we’ll look at how the immunoSEQ Assay has been used to analyze changes in the T-cell repertoire before, during, and after T-cell therapy to:
- identify mechanisms of resistance;
- track immune reconstitution after infusion; and
- track the expansion and contraction of modified T cells post infusion.
Identifying mechanisms of resistance
Anti-CD19 CAR T-cell products, such as tisagenlecleucel (tisa-cel), target the CD19 antigen on malignant B cells and have been approved for several types of B-cell malignancies. While these therapies have a high rate of complete responses, most patients relapse, often due to loss of the target antigen. (10)
Ruella et al. (12) used the immunoSEQ Assay to understand the potential causes of relapse in an individual with B-ALL treated with tisa-cel who had initially achieved complete remission. Flow cytometry was used to characterize leukemic cells following relapse and showed that they were negative for CD19 protein. Antigen loss is a common mechanism for resistance to CAR T-cell therapy; however, what was unique in this case is that CD19 transcripts were detectable via other methods, and CD19 transcript levels correlated with disease dynamics. (12)
The authors showed that leukemia cells following relapse were CAR-transduced B (CARB) cells. To identify the origins of these CARB cells, they characterized the B-cell repertoire using the immunoSEQ IGH Assay in the CAR T-cell infusion product as well as in the subject’s periphery following infusion. They found that a single leukemic B cell was inadvertently transduced with the CAR lentivirus during the manufacturing of the CAR T-cell product. The expression of anti-CD19 CAR on leukemia cells thereby masked CD19 proteins on the cell surface so that they were not detectable by either CD19 CAR T cells or anti-CD19 antibodies (Figure 3), eventually driving aggressive lymphoma. While rare, understanding the mechanisms behind this event will be important for improving CAR T-cell therapy manufacturing methods. (12)
Figure 3. Proposed CAR epitope masking model. Resistance to CAR T-cell therapy induced by transduction of a single leukemic B cell with anti-CD19 CAR.
Profiling TCR clonotypes
While anti-CD19 CAR T-cell therapies have proven to be effective against a number of cancers, additional CAR T-cell therapies are still required. Recently, Shah et al. (13) conducted a Phase I clinical trial to assess the efficacy of an anti-CD22 CAR T-cell product in the treatment of B-ALL. (13) Following infusion in a 28-year-old subject, the authors observed a significant increase in the number of white blood cells and lymphocytes, with 97.8% of all T cells being CD22 CAR+. (13) Subsequently, using the immunoSEQ hsTCRB sequencing kit, the authors identified a dominant TCR clonotype (TCRBV20/TCRBD02-01*02/TCRBJ02-07*01 CSARDKASGRMYEQYF) at days 51 and 53 post-infusion. Frequency analyses revealed that this clonotype accounted for 16.7% and 44% of all circulating T cells, respectively. The subject was negative for Epstein–Barr virus and cytomegalovirus (CMV), and the identified TCR sequences did not match known TCR sequences to other antigens across various TCR databases.
Taken together, these data suggested that the identified clonotype likely expanded due to the anti-CD22 CAR T cells, and not because of an infection with a known pathogen. Importantly, this TCR sequence may serve as an important marker for assessing the effects of anti-CD22 CAR T-cell therapies in the future. (13)
Tracking immune reconstitution
The immunoSEQ Assay has also been used to track immune reconstitution over time after CAR T-cell therapy. Hegde et al. (14) carried out a Phase I clinical trial to assess the safety of multiple infusions of autologous HER2 CAR T-cell therapy in subjects with metastatic rhabdomyosarcoma (RMS). (14) The authors used our immunoSEQ Assay to monitor immune reconstitution of peripheral T cells in a 7-year-old boy with RMS in response to the HER2 CAR T-cell therapy.
These data revealed an increase in the number of unique productive TCRB rearrangements in the peripheral blood after each CAR T-cell infusion (Figure 4A), suggesting remodeling of the T-cell repertoire. Of the 20 immunodominant clones tested, eight were undetectable in the peripheral blood before CAR T-cell therapy (Figure 4B; highlighted in orange) or in the infused CAR T-cell product. The authors suggested that each CAR T-cell infusion triggered an endogenous adaptive immune response to metastatic disease. Interestingly, rather than having a scenario whereby targeted CAR T-cell therapy elicits an immune response against just one antigen, there may be a significant contribution from the endogenous adaptive immune system against additional antigens. These two factors may act together to achieve tumor control.
Figure 4. (A) Fate of TCRB CDR3 rearrangements that developed after HER2 CAR T-cell infusions, from top 250 rearrangements (n = 127). (B) Longitudinal tracking of the productive TCRB CDR3 rearrangements expanding in the peripheral blood. Eight T-cell clones not detected pre-infusion or in the infused T-cell product were detected in the peripheral blood following CAR T-cell infusions (highlighted in orange). Adapted from Hegde et al. (14)
Tracking CAR T cells over time
To understand the changes in CAR T-cell composition after infusion, Sheih et al. (15) used the immunoSEQ Assay to profile peripheral CD8+ CAR T cells from individuals undergoing CD19 CAR T-cell therapy. They showed that CAR T-cell clonal diversity peaks in the infusion product and decreases following infusion (Figure 5). They also tracked the dynamics of the top CAR T-cell clones and found that in some subjects the dominant T-cell clones in the infusion product persisted after infusion while in others they decreased. Additional studies are needed to determine how these dynamics may correlate with responses to therapy. (15)
Figure 5. Decreased clonal diversity in CD8+ CAR T cells after infusion. Statistical differences between samples were evaluated with paired t-tests. *p < 0.05, **p < 0.005, ***p < 0.001. Adapted from Sheih et al. (15)
In another study, Fraietta et al. (16) used the immunoSEQ Assay to track the peripheral TCRB repertoire of a study subject after CAR T-cell therapy. While the TCRB repertoire remained polyclonal 1 month after infusion (Figure 6), after 2 months there was a significant expansion of the TCRVB5.1 clonotype, with clonal dominance occurring in CD8+ CTL019 cells. Subsequent analysis showed that 94% of the CD8+ CAR T-cell repertoire consisted of a single clone that was not detected at the time of infusion or even 1 month after the second infusion. (16) This again highlights the utility of our immunoSEQ technology for monitoring T-cell dynamics over time.
Figure 6. Frequency of TCRVB gene segment usage 1 month (left) and 2 months (middle) after the second CAR T-cell infusion. TCRVB clonotype frequencies in sorted CD8+ CAR T cells at the peak of expansion are also shown (right). Adapted from Fraietta et al. (16)
Tracking transgenic TCR T cells over time
Preclinical data suggest that transgenic TCR T-cell therapy targeting New York esophageal squamous cell carcinoma 1 (NY-ESO-1) can improve dendritic cell (DC) vaccination and immune checkpoint blockade in some tumors. (17)
To study the safety and feasibility of this treatment regimen, Nowicki et al. (17) treated four individuals with transgenic TCR T cells and DC vaccination with or without an anti-CTLA-4 antibody. Immunosequencing of the TCR repertoire following therapy showed that peripheral reconstitution of NY-ESO-1-specific T cells peaked 2 weeks post T-cell infusion due to rapid in vivo expansion. CTLA-4 blockade did not impact the persistence of transgenic T cells or clinical outcomes. Cancer clinical trials are often costly and time-consuming, with it sometimes taking years to establish whether treatment improves patient survival. However, smaller pilot studies (such as the study described here) that characterize the additive effects (or lack thereof) of combination therapies on the immune repertoire may prevent the costly and unnecessary inclusion of additional therapeutic agents during treatment with T-cell therapy. (17)
Monitoring virus-specific T cells in SCID patients
Severe combined immunodeficiencies (SCIDs) are a group of disorders characterized by congenital defects in adaptive immunity that prevent the development or survival of T cells. (18) The primary treatment for SCID is hematopoietic stem cell transplantation (HSCT). (19) Viral infections are prevalent and can be fatal in individuals with SCID and other immunodeficiency disorders before and after HSCT. (18)
Adoptive T-cell therapy with virus-specific T cells (VSTs) has been used to treat many individuals following HSCT. Miller et al. (18) describe the use of VSTs prior to HSCT in an infant with SCID and treatment-refractory adenovirus infection. VST treatment from a matched unrelated donor successfully cleared the adenovirus infection and the subject was successfully transplanted using reduced conditioning. They tracked the TCRB repertoire following HSCT and showed a decrease in diversity (Figure 7A), as well as an expansion of adenovirus-specific clonotypes after the second VST infusion (Figure 7B). These data indicate that VSTs administered prior to HSCT persist after transplant and may contribute to continued protection against infection. (18)
Figure 7. (A) Adenovirus-specific TCRB clonotype persistence in peripheral blood. (B) TCRB repertoire diversity post HSCT. HSCT: hematopoietic stem cell transplant; TCR: T-cell receptor; VST: virus-specific T cells; Adapted from Miller et al. (18)
Identifying immune-related markers of treatment resistance
Human CMV is a ubiquitous viral infection that establishes lifelong latency following acute infection. (20) In most people, infection is harmless as viral reactivation can be controlled by the immune system. However, aberrant T-cell immunity in solid-organ transplant recipients is associated with infection-related morbidity and mortality. (20)
In a recent study, the impact of autologous CMV-specific ACT was investigated in solid-organ transplant recipients experiencing complications due to drug-resistant CMV. Smith et al. (20) used the immunoSEQ Assay to assess the effect of ACT on the T-cell repertoire. They showed that clinical responses occur alongside significant remodeling of the TCRB repertoire following ACT. This restructuring was also linked to an increase in the number of effector memory T cells in responding patients. In contrast, subjects who did not respond to treatment showed dramatic T-cell expansion before ACT with minimal change post ACT. These data shed light on underlying immune features in non-responders that can be used to inform strategies to improve response. (20)
One of the challenges in cancer therapy is overcoming tumor-mediated immune evasion. Immunotherapies are key anticancer modalities because they reprogram the adaptive immune system to overcome this immunosuppression and detect and eliminate tumor cells.
The immunoSEQ Assay is a powerful tool that can comprehensively profile T- and B-cell repertoires before, during, and after immunotherapy. The studies highlighted here demonstrate the utility of our technology to reveal important insights into disease dynamics, immune responses following treatment, and modes of disease relapse.
Once generated, your immunoSEQ Data can be efficiently analyzed using the immunoSEQ Analyzer, our easy-to-use analytics platform. Moreover, we can offer you end-to-end support in your immunosequencing projects. To get started, get in touch with our product team.
For Research Use Only. Not for use in diagnostic procedures.
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- Shah NN, Qin H, Yates B, Su L, Shalabi H, Raffeld M, et al. Clonal expansion of CAR T cells harboring lentivector integration in the CBL gene following anti-CD22 CAR T-cell therapy. Blood Adv. 2019;3(15):2317–22.
- Hegde M, Joseph SK, Pashankar F, DeRenzo C, Sanber K, Navai S, et al. Tumor response and endogenous immune reactivity after administration of HER2 CAR T cells in a child with metastatic rhabdomyosarcoma. Nat Commun. 2020;11(1):3549.
- Sheih A, Voillet V, Hanafi L-A, DeBerg HA, Yajima M, Hawkins R, et al. Clonal kinetics and single-cell transcriptional profiling of CAR-T cells in patients undergoing CD19 CAR-T immunotherapy. Nat Commun. 2020;11(1):219.
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