T cells are immune cells crucial for human immune system surveillance, including rejecting oncogenic tumors. To identify foreign cells or cancer cells, T cells use their T-cell receptors (TCRs) to recognize antigenic peptides on the surface of antigen-presenting cells. However, early malignant cells evolve to evade this immune attack (Baumeister et al., 2016). Therefore, much research and development have been devoted to circumventing this immune avoidance through adoptive cell transfer (ACT) immunotherapy (Cohen et al., 2017).
In ACT, T cells taken from a patient are grown in large numbers in a laboratory and then returned to the patient. Conceptually, this process allows patient-derived T cells that have already been primed against the patient’s cancer to expand in vitro before being returned to the patient to help the immune system fight the cancer.
While in the laboratory, the patient-derived T cells are also modified to improve their cancer-targeting abilities. Among the types of ACT, chimeric antigen receptor (CAR) T-cell therapy has received increasing attention due to its early success in treating childhood leukemia patients.
CAR T-cell modifications for cancer immunotherapy initially employed genetic transformation methods with plasmid or viral vectors. This process is now more safely achieved by IVT-mediated transient expression of mRNA. Examples of recent advances in cancer immunotherapy using this newer mRNA-based approach are provided herein and feature products obtained from TriLink BioTechnologies. A comprehensive review of IVT-made mRNA CAR T-cells for hematologic and solid tumor management is available elsewhere (Rajan et al., 2020).
Autologous CAR T Cells
As reviewed by Hughes-Parry et al., the original CAR T-cell design (Figure 1) was comprised of three parts:
- an extracellular antibody-derived antigen-binding domain (single-chain fragment variable, scFv);
- a spacer linked to the transmembrane domain;
- an intracellular co-stimulation domain consisting of a CD3 ζ signaling tail.
Critically, antigen-binding induced signaling via CD3 ζ facilitates exocytosis of cytotoxic granules and apoptotic killing of the antigen-expressing cancer cell.
FIGURE 1. Chimeric antigen receptor (CAR) T-cell design. The single-chain variable fragment (scFv) of the CAR is derived from the heavy and light chains of the antibody variable region, whereas the CAR CD3ζ domain is derived from the T-cell receptor’s intracellular signaling domains. Taken from Hughes-Parry et al. (2020) and free to use under the Creative Commons Attribution License.
Following this initial “first generation” CAR design, a series of increasingly more sophisticated “next generation” CAR design strategies were developed for engineering a cancer patient’s T cells for autologous reinfusion. This progress led to two FDA approvals in 2017: Kymriah for acute B-cell lymphoblastic leukemia and Yescarta for diffuse large B-cell lymphoma. However, these autologous biological products require individual patient-specific manufacturing, which is complex and expensive. Unfortunately, CAR T-cell products were also often adversely affected by chemotherapeutic pre-treatment of patients, causing CAR T-cell production failure. Fortunately, these problems are addressed with further advancements.
Allogeneic CAR T Cells
Umbilical cord blood stem cells or T cells from healthy donors can provide cells for generating allogeneic CAR T cells. Once donated, these T cells are transduced and expanded for administration to the genetically unrelated patient following lymphodepleting chemotherapy (Pampusch et al., 2019; Bechman and Maher, 2021) (Figure 2). Allogeneic CAR T cells were first referred to as “universal” or “off-the-shelf” by Eshhar and collaborators. They published a proof-of-concept study for ACT using tumor-specific allogeneic CAR T cells (Marcus et al., 2011).
FIGURE 2. Allogeneic (“off-the-shelf”) CAR T cells generation and sources of T cells. Taken from Bedoya et al. (2021) and free to use under Budapest Open Access.
To avoid these problems, TCR-deficient T cells are engineered using site-specific gene-editing tools such as TALENs and CRISPR-Cas9. Subsequent gene-editing methods improved approaches to universal allogeneic T cells that also addressed allogeneic T cell-induced graft-versus-host disease (GVHD) and allorejection (Ingulli, 2010). Specific examples of these approaches are detailed in the following sections.
Allogeneic CAR T Cells for Treatment of Renal Cell Carcinoma
In 2022 in the United States, the American Cancer Society predicts ~79,000 new cases and ~14,000 deaths from renal cell carcinoma (RCC, a.k.a kidney cancer). Chemotherapy is minimally effective against advanced kidney cancer, so developing targeted immunotherapy for this disease is a research priority.
One target that has recently been investigated and is in the early phases of clinical trials for RCC is CD70. CD70 is highly expressed in RCC, has limited expression in normal tissue, and is an attractive target for CAR T-cell immunotherapy (Panowski et al., 2022). Recently, these researchers generated and characterized a panel of anti-CD70 scFv-based CAR T cells. TALEN-based gene editing (Figure 3) of allogeneic anti-CD70 CAR T cells was used to edit the cells and reduce the chance of GVHD in this setting. TriLink TALEN mRNA was used to successfully edit these CAR T cells in the laboratory (Figure 3).
FIGURE 3. Schematic representation of transcription activator-like effector nucleases (TALENs), which are comprised of TALE protein domains for DNA-specific binding connected to the FokI endonuclease (green) for double-strand breaks (arrows) to enable gene disruption, correction, or insertion. Taken from Rahimmanesh et al. (2022) and free to use under CC BY 4.0 license.
The resultant allogeneic anti-CD70-CAR T cells were produced at a large scale to enable advancement as a candidate immunotherapeutic (ALLO-316) in Phase 1 clinical trial sponsored by Allogene Therapeutics. This is a dose-escalation study of the safety, efficacy, and cell kinetics of ALLO-316 in adult patients with advanced or metastatic RCC after lymphodepleting chemotherapy. The trial is actively recruiting patients and expects the readout on primary outcomes by the end of 2022.
Allogeneic CAR T Cells for Treatment of T-Cell Malignancies
T-cell malignancies encompass a heterogeneous group of diseases, each resulting from a clonal evolution of dysfunctional T cells from various stages of development. T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% and 25% of childhood and adult ALL cases, respectively, and is the most common form of T-cell cancer seen in children (Fleischer et al. 2019).
While most T-ALLs highly express the transmembrane glycoprotein CD7, Cooper et al. (2018) observed that 86% of normal T cells, including those used to engineer CAR T, also express CD7. This shared T-cell antigen expression results in what immunologists often refer to as “fratricide,” or self-killing of the CAR T cells. As expected, this process interferes with anti-tumor activity (Figure 4).
FIGURE 4. Two potential outcomes of CAR T-cell therapy in a patient with T-cell disease. Upon re-infusion into a patient, CAR T cells recognize their cognate antigen, expanding upon this recognition and initiating an attack. Due to shared antigen expression on CAR T cells and tumor cells, fratricide and anti-tumor activity are possible. Taken from Fleischer et al. (2019) and free to use under Budapest Open Access.
To prevent fratricide yet preserve anti-tumor activity, Cooper et al. envisaged CRISPR-Cas9 deletion of endogenous CD7 and disruption of T-cell antigen binding while also transducing these same T cells with a CD7 targeting CAR. Briefly, CD7-CAR T cells (termed CART7) were generated using commercial gene synthesis of a known anti-CD7 scFv sequence. Deletion of endogenous CD7 and TRAC and in CART7 was then achieved by electroporation with the following gene-editing reagents from TriLink: Streptococcus pyogenes Cas9 (spCas9) mRNA (5meC, Ψ) and sgRNAs for TRAC and CD7 incorporating 2’-O-methyl ribonucleoside and phosphorothioate linkages at the three terminal bases of the 5’ and 3’ ends of the gRNAs to protect against nuclease activity.
Cooper et al. referred to these doubly deleted cells as universal CART7 (UCART7). In vitro assays demonstrated that UCART7 efficiently killed (95%) primary T-ALL blasts using samples from three patients. Next, they tested the capacity of UCART7 to kill primary T-ALL in vivo using a patient-derived xenograft model in groups of mice. Kaplan–Meier survival data showed a median survival of 60 days for UCART7 treated mice vs. 30 days death/dying for all untreated control mice. There was no evidence of UCART7-induced xenogeneic GVHD.
More recently, Georgiadis et al. (2021) used base editing to implement an analogous strategy to target T-cell malignancies with fratricide-resistant CAR T cells. In this instance, T-ALL cells with shared antigens CD7 and CD3 were targeted by combinatorial treatment with separately engineered CD7 CAR T cells and CD3 CAR T cells. The expression of CD7 and CD3 in these CAR T cells was blocked by CRISPR-guided base editing that caused deamination to create novel stop codons or disrupt splice donor/acceptor sites. They used BE3 (Komor et al. 2016) comprised of a deactivated Cas9 nickase fused to rat APOBEC1 deaminase and a single uracyl glycosylase inhibitor; all encoded in TriLink custom-synthesized CleanCap® (Cap 1) mRNA. Co-culturing these base-edited CAR T cells exhibited the highest cytotoxicity against CD3+/CD7 + T-ALL targets in vitro and in vivo human/murine chimeric models.
Challenges and Opportunities
For all of their wonderful successes in leukemias, CAR T-cell efficacy in solid tumors has significant room for improvement (Marofi et al., 2021). The pivotal challenges for CAR T cells include recognition, trafficking, and survival in the tumor microenvironment. Solid tumors have different levels of antigen expression as well as more antigen diversity at various tumor sites, which complicates CAR T-cell recognition. Compared to blood cancers, CAR T-cell therapy is also more limited in solid tumors due to the T cells’ inability to penetrate tumor tissue and infiltrate the immunosuppressive tumor microenvironment.
Marofi et al. emphasize that each challenge represents an opportunity for novel approaches. New topics being investigated include multiplexed CARs to improve recognition, local infusion into solid tumors to improve trafficking, and inclusion of tumor microenvironment modulators to improve survival in the tumor microenvironment. Importantly, IVT-mRNA facilitates production of multiple antigen receptors to potentially achieve improved recognition of heterogeneous cancer cells having different surface antigens. IVT-mRNA can also be employed to code for transient expression of tumor microenvironment modulators, such as heparinase, which promotes tumor infiltration and antitumor activity of CAR T-cells (Rafiq et al., 2020).
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