Specific Infections Require Specific Terminators: CAR T-cells

Seshadri Dutta, NIIT University

Introduction

Chimeric antigen receptor (CAR) T-cells are a type of genetically engineered T- cells, modified in order to produce an artificial T-cell receptor that can be further used for immunotherapy. These receptor proteins are engineered in a way that it gives the T-cells the ability to target a specific protein. Following its approval in 2017 for the treatment of B-cell malignancies, chimeric antigen receptor (CAR) T-cell therapy has been a huge success. Currently, four CAR T-cell therapies – Kymriah® (B-cell precursor acute lymphoblastic leukemia), Yescarta® (non-Hodgkin lymphoma), Tecartus® (relapsed or refractory mantle cell lymphoma), and Actemra® (CAR T-cell induced severe or life-threatening cytokine release syndrome) have been approved by the FDA for treatments of respective disorders.

While CAR T-cells are being used mostly for anti-tumor therapy, its original intention was to target membrane antigens, which could lead to a wide range of applications. Since then, various researches have looked into how CAR T-cells might be used to treat non-malignant conditions such as autoimmune disorders, viral disorders, and, more recently, cardiac fibrosis and cellular senescence. Viral and opportunistic fungal infections are a major threat to immunodeficient patients. Despite several antifungal and antiviral drugs, the mortality rate of these patients is significantly high. A few studies on CAR T-cell therapy have achieved success in targeting viral infections, such as Human Immunodeficiency Virus (HIV), Hepatitis B, and also opportunistic fungal infections.

Mechanism

• Car T-cell in treating HIV:-

In the early 1980s, HIV and the associated acute immunodeficiency syndrome were first diagnosed, and the disease had quickly spread over the world. Despite the fact that the modified antiretroviral therapy (CAR-T) is highly efficient in limiting viral replication, it fails to destroy the virus, and viral presence from the latent reservoir following treatment interruption as per reports. Initially, during the 1990s, the first attempts were made to find a cure for HIV using CAR T-cell therapy but clinical studies revealed that, while the technique was viable and safe, it frequently failed to reduce HIV replication. With the evolution of the CAR T-cell design and a few key factors, in-vitro activity against HIV and in-vivo persistence showed better clinical results along with a few upcoming challenges such as long-term persistence, optimal extracellular target, and ability to target latent viral reservoir.

• CD4 based CAR T-cell:-

The identification of CD4, mediated by the viral envelope protein gp120 binding, is a crucial phase in cellular invasion for HIV, which is a highly mutating virus. CD4 is an appealing extracellular CAR domain because this domain is an important target, and the gp120 binding site is well conserved. CD4 expression, on the other hand, makes CAR T-cells more vulnerable to lytic infection and destruction. In reality, clinical experiments using CD4 and the zeta chain indicated that these first-generation CARs were unsuccessful. Hence, in order to improve the CAR T-cell activity and persistence, CD4-based CAR was re-engineered into second and third-generation CARs. While CAR T-cells with the CD28 costimulatory domain produced more cytokines and had better control over HIV replication in vitro, CARs with the 4-1BB costimulatory domain were more effective in suppressing HIV infection in vivo. It was found that second-generation CAR T-cells were more effective at reducing HIV replication in vitro when compared to first-generation CAR T-cells. However, it has been demonstrated that CD4-based CARs make CAR T-cells susceptible to HIV infection. To overcome this limitation, CD4-based CAR was outfitted with either a viral fusion inhibitor (C46 peptide) or small hairpin RNAs capable of knocking out the HIV-1 co-receptor CC-chemokine receptor (CCR5) and degrading viral RNA. Both strategies were successful in rendering CD4-based CAR-T immune to HIV infection and conferring long persistence and HIV infection control in vivo.

Furthermore, numerous genome editing techniques were employed to knock-out CCR5 in T-cells, conferring them with long-term HIV resistance. These methods include – ZFNs (Zinc finger nucleases), which are currently under clinical investigation; TALEN (Transcription activator-like nucleases) and CRISPR-CAS9 in pre-clinical studies. Because these endonucleases were initially employed to break down T-cell receptors for the development of universal CAR T-cells, it is expected that using them to knock out CCR5 in HIV-CAR T-cells may be beneficial.

• Scfv based CARs:-

CARs targeting conserved regions in the envelope protein with scFv (single-chain variable fragment) generated from broadly neutralizing antibodies (bNAbs) have also been produced in order to avoid using CD4 as a targeting element.

The first-generation anti-gp120 CARs promoted effective activation and cytokine secretion in gene-modified T-cells, as well as facilitated lysis of envelope-expressing cells and HIV-1-infected CD4+ T-lymphocytes in vitro. CAR T-cells using second-generation CARs for diverse targets were able to kill HIV-1-infected cells, however, their antiviral efficacy varied depending on the virus strain. The third-generation scfv CARs showed more efficacy than CD4-based CARs in knocking down anti-gp120 expressing cells in vitro. VRC01, a third-generation CAR containing a scFv generated from a Nab directed against gp120, is now undergoing clinical trials.

However, the HIV viral escape mutation mechanism, which can abrogate the antibody-binding site and render CAR T-cell therapy ineffective, is one key obstacle to developing scFvs-based CAR T-cell treatment.

• Bi and tri-specific CARs:-

To bypass the HIV mutation escape mechanism, bi- and tri-specific CAR-expressing T-cells targeting up to three HIV antigens were developed.

T-cells have recently been modified to have up to three functionally different HIV envelope-binding domains, resulting in bispecific and trispecific anti-HIV CAR T-cells. Two different CARs were expressed on one T-cell, or one CAR with two targeting elements was joined together in these cells.

Bi- and tri-specific CAR T-cells demonstrated powerful anti-HIV activity in vitro and in vivo in a humanized mouse model, successfully destroying HIV-infected cells while protecting CAR T-cells from infection.

Despite the challenges, anti-HIV CAR T-cell therapy has made tremendous progress in terms of antiviral effectiveness, CAR T-cell protection from HIV infection, and HIV escape mechanisms. At least two clinical trials targeting the eradication of latent reservoirs are now underway, one using modified bNAb-based CAR T-cell therapy and the other using CD4-based CAR T-cell therapy with CCR5 ablation.

• CAR T-cells in other infectious diseases:-

In addition to HIV, in a CAR, specifically with hepatitis C virus (HCV), which is based on the scFv (single-chain variable fragment), targeting the E2 glycoprotein epitope that is found in the hepatitis C virus capsid, have been developed recently. Sautto et al. examined the performance of the activities of the anti-HCV positive CAR T-cells in vitro, which can induce lysis of the target cells transfected with the HCV-E2. However, this anti-HCV CAR-T has not yet been tested in vivo. Since the structural features of the E2 glycoprotein, are not yet fully understood, research is going on to expand on this line of thinking.

After HBV infection and subsequent anti-HBV CAR-T treatment, a reduction in plasmatic HBV antigens and DNA was reported in vivo, although total eradication of the virus could not be achieved. However, using anti-HBV CAR-T-cells is still more promising than using anti-HBV treatments that are already recognized and in use, such as nucleoside analogs.

The use of CARs has also been evaluated in models of CMV (Human Cytomegalovirus) infection and also in recognizing EBV (Epstein Barr Virus) proteins. Proff et al. reported in 2016 that CMV-infected cells were resistant to anti-CMV CAR T-cell killing. Following that, it was feasible to confirm that various anti-apoptotic viral proteins, which can prevent infected host cells from dying, contribute to reducing T-cell cytotoxicity. A second-generation CAR specific for the EBV latent membrane protein 1 (LMP1) was described to target EBV-associated cancers. As a result, anti-EBV lymphocytes kill neural progenitor cells overexpressing LMP1. Tang et al. also found that anti-EBV lymphocytes kill neural progenitor cells overexpressing LMP1. Nonetheless, the current study demonstrates that this strategy is promising and deserves to be further developed for EBV-related malignancies.

Another recent attempt to apply CAR-T technology to infectious disorders was the creation of a CAR T-cell to fight opportunistic Aspergillus fungal infections, which are a serious threat to immunocompromised people. D-CAR is a second-generation CAR that uses the extracellular domain of dectin-1 as a targeting element to target Aspergillus fumigatus. Few pieces of research have shown that CAR T-cells could be used to treat acute fungal infections in addition to cancer and chronic viral infections, while promising results of the research have yet to be received and are being investigated.

• Emerging applications of CAR T-cells in fibrotic diseases:-

Cardiovascular disease is still one of the world’s major causes of death. Most myocardial illnesses result in cardiac fibrosis, which reduces organ function and accelerates heart failure. The number of fibrosis reversing medicines is highly restricted, necessitating the development of novel therapeutic approaches. The idea of employing CAR T=cells to target cellular senescence and reverse senescence-associated diseases, as demonstrated by Amor et al., has recently attracted a lot of attention. An anti-mouse uPAR single-chain variable fragment coupled to the CD28 costimulatory protein and the CD3zeta signaling domain was used to create a second-generation uPAR-specific CAR. Using mouse models of lung adenocarcinoma treated with senescence-inducing medications and two models of liver fibrosis, in which senescent hepatic stellate cells contribute to disease progression, those CAR T-cells were efficient in ablating senescent cells in vitro and in vivo. Although it’s too early for clinical use, and more safety research is needed, this model suggests that senolytic CAR T-cells could be a viable treatment for a variety of senescence-related diseases.

Conclusion and Future Prospects:-

In the last decade, CAR T-cell therapy has achieved enormous advances since its clinical applications were approved for notably fighting against hematological malignancies. Great efforts have been made to develop similar treatments for patients suffering from chronic viral and acute invasive fungal infections, which are based on the acquisition of knowledge through T-cell technology in cancer research. Pathogen escape mechanisms and reservoirs remain key challenges, even though targets are more exact and unique to the pathogen, making it easier to avoid off-targets.

Significant progress has been made in the anti-HIV T-cell therapy, which is currently in clinical trials. Apart from HIV, CART-cells targeting HBV, HCV, CMV, EBV, and Aspergillus have been developed, although they are still in the early stages of development with minimal efficiency. To date, large-scale results have been reported, which demonstrates the feasibility of the use of CAR T-cells. However, there is still a lot of work to do in order to improve the safety and efficacy of this concept. More pathogen targets should be examined for relevant animal models in vitro and in vivo.

Also read: Nanoparticles to treat a common type of cancer- neuroblastoma

References:-

1. Liu, D., Tian, S., Zhang, K., Xiong, W., Lubaki, N. M., Chen, Z., & Han, W. (2018). Erratum to: Chimeric antigen receptor (CAR)-modified natural killer cell-based immunotherapy and immunological synapse formation in cancer and HIV. Protein Cell, 9(10), 902. DOI: 10.1007/s13238-017-0427-1
2. Sterner, R. C., & Sterner, R. M. (2021). CAR-T cell therapy: current limitations and potential strategies. Blood Cancer Journal, 11, 69. https://www.nature.com/articles/s41408-021-00459-7
3. (picture credits) Mazzi, M. T., Hajdu, K. L., Ribeiro, P. R., & Bonamino, M. H. (2021). CAR-T cells leave the comfort zone: current and future applications beyond cancer. Immunotherapy Advances, 1(1), ltaa006. https://doi.org/10.1093/immadv/ltaa006
4. Seif, M., Einsele, H., & Löffler, J. (2019). CAR T Cells Beyond Cancer: Hope for Immunomodulatory Therapy of Infectious Diseases. Frontiers in Immunology. 10, 2711. DOI: 10.3389/fimmu.2019.02711

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