Terapia cu celulele CAR-T

Chimeric antigen receptor (CAR) T-cell therapy is a developing form of cancer therapy that involves collecting immune system cells from the patient, genetically programming them to recognize the tumor target, in vitro culture, and reinfusing, aimed at responding to and preventing the progression of various types of malignancies. Currently, there are several forms of adoption cell transfer in the study, but the first to enter clinical practice is with CAR T cells.

1. T CAR lymphocyte technology

CRISPR/Cas9 technology (clustered regularly interspaced short palindromic repeats associated protein-9 nuclease) has its origins in CRISPR/Cas9 type II systems, which involve the acquisition of specific immunity by bacteria to viruses or plasmids. These systems incorporate sequences from the “invader” DNA between the repeated CRISPR sequences encoded in the genome of the host (in this case, the bacterium). Transcripts from CRISPR repeat matrices are processed into CRISPR RNA (cRNA), each of which has a variable sequence transcribed from “invading” DNA, called a protospacer, and a fragment from the CRISPR repeat. Each cRNA sequence hybridizes to a second RNA sequence which is known as transactivating RNA (transcr RNA) forming a complex with DNA-Cas9 endonuclease. The protospacer-encoded portion of the cRNA leads Cas9 to the complementary target DNA sequences and cleaves them if they are adjacent to the PAM sequences (protospacer adjacent motifs)⁽¹⁾.

Other studies have shown that, in order to cleave a DNA sequence, the presence of the RNA complex is not mandatory and this process can be performed with RNA synthesized in the laboratory (chimeric single guide RNAsg)⁽²⁾.

The CRISPR/Cas9 system can be applied to human cells by transferring genetical material via a plasmid encoding Cas9 and gsRNA. Lentiviruses, retroviruses, adenoviruses and ribonucleoproteins have also been used as a vector for the transmission of genetic material⁽³⁾.

2. Chimeric antigen receptor

CAR is a synthetic protein consisting of two components: the extracellular domain of antigen recognition represented by a variable domain consisting of a single chain (single-chain variable fragment – scVf) specific to the target antigen which recognizes a specific protein on the surface of malignant cells (e.g., CD19 on the surface of the B lymphocyte) and the intracellular activation domain which provides the T cell receptor (TCR) signal required to activate CAR T cell effector functions (Figure 1).

If the first generation of CAR T cells showed an intracellular domain structurally identical to the z chain of the CD3 molecule of TCR, a co-stimulating intracellular domain was added to the second and third generations (e.g., CD28, 41BB, CD3z-CD28-41BB, CD3z-CD28-OX40). This improvement has led to the ability of CAR T cells to proliferate, expand and persist in vivo⁽⁴⁾.

The modified T lymphocytes are reinfused to the patient 2-14 days after the induction of bone marrow aplasia by the administration of chemotherapy. Once infused, the cells continue to proliferate and bind the malignant cells through the added receptor, resulting in cell death. The presence of CAR T cells was reported three years after infusion⁽⁴⁾.

3. Clinical experience

The steps of T CAR lymphocyte therapy are the identification of the target antigen and the development of T CAR lymphocytes directed against them. An ideal antigen would be one that has high expression in the target tissues and is absent on the surface of normal ones. These features would increase the effectiveness of therapy and minimize the adverse side effects in healthy tissue. These concepts are the basis of successful anti-CD19 therapy.

The anti-CD19 agent – tisagenlecleucel – is the first CAR T-cell therapy to enter clinical practice. In August 2017, FDA (US Food and Drug Administration) approved the use of tisagenlecleucel in the treatment of B-cell ALL (acute lymphoblastic leukemia) in patients up to 25 years old in case of recurrence or refractory disease to first-line therapy. Its approval was based on the results of a study performed on a group of 88 patients (Study B2202). A number of 68 patients of these were treated and 63 were evaluated for efficacy, of which 52 responded to the treatment: in 40 patients, complete remission was noted three months after the infusion, and in the other 12, complete remission was observed with incomplete recovery of the number of blood cells⁽⁶⁾.

The second anti-CD19 CAR T-cell therapy to enter clinical practice – axicabtagene ciloleucel – was approved by FDA in October 2017. It is indicated in adult patients with diffuse large B-cell lymphoma who have not responded to first-line chemotherapy or whose disease has recurred after at least two different treatment regimens. Its indications are represented by diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma and follicular origin diffuse large B-cell lymphoma. This therapy is not indicated in central nervous system lymphoma⁽⁷⁾.

Several clinical trials based on the T-cell therapy use in solid tumors have shown that patients may experience severe toxicity, although the target antigen level has minimal expression in healthy tissues. CAR T cells specific for carbonic anhydrase IX used in renal cell carcinoma have led to hepatotoxicity due to its expression on the bile duct epithelium⁽⁸⁾. CAR T cells specific for HER2 used in metastatic colorectal cancer have caused pulmonary toxicity accompanied by multiple organ failure syndrome⁽⁹⁾.

Although other tumor antigens have been identified and studies have shown a higher safety profile, their research has been discontinued due to limited clinical efficacy⁽¹⁰⁾.

CAR T cells specific for HER2 were also tested in a group of 17 patients with progressive glioblastoma and the data provided suggest a satisfactory level of safety and feasibility, obtaining partial remission in one of the patients for more than nine months and stable disease for seven of these between 8 weeks and 29 months⁽¹¹⁾.

CAR T cells specific for EGFRvIII were used in a phase I trial of ten patients with recurrent glioblastoma. Posttherapy, seven of them underwent surgery. Tissue analysis performed on the resection pieces showed a large lymphocytic infiltrate, the number of intratumorally CAR T cells being much higher compared to that of peripheral blood. This is important, demonstrating the affinity of CAR T cells for the target antigen and the developmental potential of therapy⁽¹²⁾.

In a case of glioblastoma, CAR T cells specific for IL13Ra2 were infused locally (intracranially) to a patient with recurrent glioblastoma with multiple intracranial and spinal solid tumors, a therapy followed by their regression with a favorable response at seven and a half months after infusion⁽¹³⁾.

The Melanoma Antigen Gene (MAGE) protein family is a group of antigens identified in several neoplastic pathologies. MAGE-A4 is a member of this family and it has been described in ovarian cancer, melanoma, non-small-cell lung carcinoma and squamous-cell carcinoma⁽¹⁴⁾.

A patient diagnosed with uterine leiomyosarcoma was treated surgically in 2014. A few months later she was diagnosed with progressive disease through secondary dissemination and she received chemotherapy and biologic therapy (bevacizumab). At the beginning of 2016 she received CAR T-cell therapy specific for MAGE-A4, and the complete remission of the disease was observed. Three years after the administration of the therapy, no major side effects were described, the patient being in remission⁽¹⁴⁾.

Conclusions

Given the efficacy of already approved CAR T-cell therapies in hematologic malignancies and the potential of other identified antigens in solid tumors to become feasible targets, CAR T-cell immunotherapy may become a future standard in cancer therapy.  

Conflict of interests: The author declares no con­flict of interests.