Next generations of CAR-T cells - new therapeutic opportunities in hematology?

Abstract

In recent years, the introduction of chimeric antigen receptor (CAR) T-cell therapies into clinics has been a breakthrough in treating relapsed or refractory malignancies in hematology and oncology. To date, Food and Drug Administration (FDA) has approved six CAR-T therapies for specific non-Hodgkin lymphomas, B-cell acute lymphoblastic leukemia, and multiple myeloma. All registered treatments and most clinical trials are based on so-called 2nd generation CARs, which consist of an extracellular antigen-binding region, one costimulatory domain, and a CD3z signaling domain. Unfortunately, despite remarkable overall treatment outcomes, a relatively high percentage of patients do not benefit from CAR-T therapy (overall response rate varies between 50 and 100%, with following relapse rates as high as 66% due to limited durability of the response). Moreover, it is associated with adverse effects such as cytokine release syndrome and neurotoxicity. Advances in immunology and molecular engineering have facilitated the construction of the next generation of CAR-T cells equipped with various molecular mechanisms. These include additional costimulatory domains (3rd generation), safety switches, immune-checkpoint modulation, cytokine expression, or knockout of therapy-interfering molecules, to name just a few. Implementation of next-generation CAR T-cells may allow overcoming current limitations of CAR-T therapies, decreasing unwanted side effects, and targeting other hematological malignancies. Accordingly, some clinical trials are currently evaluating the safety and efficacy of novel CAR-T therapies. This review describes the CAR-T cell constructs concerning the clinical application, summarizes completed and ongoing clinical trials of next-generation CAR-T therapies, and presents future perspectives.

Keywords: CAR-T cells; CRISPR; CRS; acute lymphoblastic leukemia; allogeneic; cytokine release syndrome; immunotherapy; lymphocyte.

Figure 1

Generations of CAR-T cells. (A) First-generation CAR-T cells – equipped with an extracellular antigen-recognizing domain combined with intracellular CD3z accounting for signal transduction. (B) Second-generation CAR-T cells - equipped with an extracellular antigen-recognizing domain combined with two intracellular domains: CD3z and an additional costimulatory domain (e.g., CD28 or 4-1BB). (C) Third-generation CAR-T cells - equipped with an extracellular antigen-recognizing domain combined with three intracellular domains: CD3z and two additional costimulatory domains. (D) Fourth/Next-generation CAR-T cells – a diversified group of CAR-T constructs embracing armored CAR-T cells, cytokine-expressing CAR-T cells (illustrated above), switchable CAR-T cells, and universal CAR-T cells. Created with BioRender.com.

Figure 2

Armored CAR-T cells – immune checkpoint modulation. (A) Transforming the inhibitory signal into stimulation. This construct embodies the extracellular PD-1 domain fused to the intracellular CD28. Interaction between PD-1 and PD-L1 (expressed in the tumor microenvironment) is transformed into activating signal and leads to the enhanced CAR-T response. (B) Secretion of PD-1 Fc to block PD-L1 inhibitory signaling. The CAR-T cell is programmed to express and secrete a protein that combines the PD-1 domain and fragment crystallizable region (Fc) of an antibody. The secreted protein blocks PD-L1 molecules of malignant cells and makes them susceptible to innate immune cells. (C) Downregulation of PD-1 expression. The CAR-T cells are transfected with short hairpin RNA (shRNA) and subsequently, PD-1 expression is silenced via RNA interference. Created with BioRender.com.

Figure 3

TRUCKs – cytokine-expressing CAR-T cells. TRUCKs are next-generation CAR-T cells engineered to express certain cytokines to augment CAR-T cells’ antitumor efficacy, improve their persistence, and alte tumor microenvironment characteristics. Following antigen recognition, in addition to cytotoxic activity, the engineered cells release selected cytokines. Depending on the type, cytokines may promote the proliferation and survival of CAR-T cells and act as chemoattractants and enhancers of antitumor activity. Created with BioRender.com.

Figure 4

Switchable CAR-T cells – safety-switch strategies. (A) Surface antigen safety-switch strategy. The CAR-T cell is programmed to express a surface protein that can be targeted by a specific antibody. The binding of the antibody enables the removal of the CAR-T cells via antibody-dependent cytotoxicity or complement-dependent cytotoxicity. (B) Induction of apoptosis. Administration of AP1903 elicits dimerization of inducible caspase 9 (iCasp9) which in turn leads to activation of proapoptotic molecules and subsequent apoptosis of the iCasp9-transduced CAR-T cell. (C) HSV-TK safety-switch strategy. The HSV-TK gene codes an enzyme that transforms an inactive prodrug (GCV) into a competitive inhibitor of DNA polymerase. As a result, DNA replication is disrupted, and CAR-T cell undergoes apoptosis. Created with BioRender.com.

Figure 5

Universal CAR-T cells – genome editing strategies. (A) TALEN genome editing. TALEN is a genome-editing system in which a targeted DNA sequence is recognized by a pair of individually designed DNA-binding domains. Then, a pair of nuclease domains cause a double-stranded DNA break and subsequent knockout of the targeted gene. (B) CRISPR/Cas9 genome editing. CRISPR/Cas9 is a genome-editing system in which a targeted DNA sequence is recognized by guide RNA associated with Cas9 endonuclease that cleaves DNA strand, thus causing the knockout of a selected gene. Created with BioRender.com.