An extra gene

Fighting the depletion of CAR-T lymphocytes has increased the effectiveness of cancer immunotherapy

Oleg Lischuk, N+1

American researchers have discovered a gene that plays a key role in the depletion of T-lymphocytes. In an experiment on laboratory mice, the removal of this gene increased the effectiveness of the most promising cancer immunotherapy using lymphocytes with chimeric antigen receptors. The results of the work are published in the Journal for Immunotherapy of Cancer (Kumar et al., Deletion of Cbl-b inhibits CD8+ T-cell exhaustion and promotes CAR T-cell function).

The principle of the technology of chimeric antigen receptors (chimeric antigen receptor, CAR) is that in human own cytotoxic T-lymphocytes, the main receptor complex (TCR + CD3), necessary for the recognition of foreign cells, is replaced by a CAR specific to a specific type of cancer. Such artificial receptors are called chimeric because they consist of modules of different origin: a fragment of a monoclonal antibody (scFv) for recognizing a tumor antigen, a hinge region, an activating domain (cd3ζ), as well as various coactivators, expression regulators and other transgenes. To obtain CAR-T lymphocytes from the patient's blood, his own cells are taken, a composite chimeric receptor gene is injected into them with the help of a viral vector, cultured on a nutrient medium and injected back into the body. There, transgenic lymphocytes recognize the tumor and are activated by attacking its cells and releasing cytokines – interferon γ (IF-γ), tumor necrosis factor α (TNF-α) and others that attract various immune cells. You can read more about CAR technology in the material "Chimera against Cancer".

T-lymphocytes with conventional (left) and chimeric (right) receptors and their interaction with target cells.Shivani Srivastava, Stanley R. Riddell / Trends in Immunology, 2015

In clinical trials, CAR-T lymphocytes made it possible to achieve a complete cure of cancer and prevent its recurrence in a number of patients. However, such successes were achieved mainly with blood neoplasms. It turned out to be more difficult to defeat cancer of other body tissues; one of the main reasons for this was the rapid depletion of lymphocytes in the tumor microenvironment. Such depleted cells gradually lose effector functions, such as cytokine synthesis (IF-γ, TNF-α) and destruction of cancer cells, and also express inhibitory receptors PD1, Tim3 and LAG3. Previous studies have shown that the deficiency of the regulatory protein Cbl-b from the E3 family of ubiquitin ligases is associated with tumor rejection, mainly due to the activity of cytotoxic T-lymphocytes, but its exact role in this process remained unspecified.

To understand this issue and to find out the therapeutic potential of exposure to Cbl-b, a group of scientists from several research centers led by Venuprasad Poojary from the Southwest Medical Center of the University of Texas conducted a series of laboratory experiments.

First, the researchers isolated T-lymphocytes from foci of artificially vaccinated colon cancer in mice and analyzed their RNA using real-time polymerase chain reaction and immunoblotting. He showed that depleted cells expressing PD1 and Tim3 receptors (PD1+Tim3+) produce significantly more Cbl-b (p=0.00005) than non-depleted (PD1-Tim3-).

Then the experimenters "turned off" the CBLB gene encoding Cbl-b in isolated antitumor PD1+Tim3+ T-lymphocytes using CRISPR-Cas9 technology. This restored the production of cytokines IF-γ, TNF-α, interleukin-2 (IL-2) and cytotoxic enzyme granzyme B (GrB) in depleted cells.

To study the effects of Cbl-b in vivo, mice with the presence (CBLB+/+) and absence (CBLB-/-) of the corresponding gene were inoculated with colon cancer cells expressing cancer-embryonic antigen (CEA, CEA) – biomarker of many human malignancies. It turned out that in CBLB-/-mice, the tumor grows much slower (p=0.001) and is more strongly infiltrated by cytotoxic T-lymphocytes. Also, these animals had much fewer depleted PD1+Tim3+T-lymphocytes (18.4 vs. 45.6 percent), and they retained the ability to synthesize cytokines and GrB, unlike CBLB+/+ cells of mice.

Restriction of tumor growth in mice without Cbl-b.Kumar et al.
/ Journal for Immunotherapy of Cancer, 2021

To assess the therapeutic value of Cbl-b, the researchers provided CBLB+/+ and CBLB-/-T lymphocytes of mice with a chimeric antigen receptor hCEAscFv-CD28-cd3ζ.GFP recognizing human CEA (hCEA). The resulting CAR-T cells were injected into mice with colon cancer expressing this antigen. Animals treated with CBLB-/-CAR-T had much better survival and a smaller tumor size compared to CBLB+/+ cell therapy and lack of treatment.

Among the CAR-T lymphocytes isolated from tumors, CBLB-/- showed significantly lower "depletion" compared to CBLB+/+ (4-6 vs. 30-35 percent). In addition, unlike the latter, they retained the ability to destroy cancer cells in culture and synthesize IF-γ, TNF-α and GrB. Thus, the removal of CBLB potentiates the antitumor activity of CAR-T cells, the researchers concluded.

Since Cbl-b deficiency in T-lymphocytes is associated with the development of autoimmune diseases, the authors conducted additional morphological and histological examination of the spleen, colon, lungs and liver of mice receiving therapy. No signs of damage were observed when using both CBLB+/+ and CBLB-/- cells.

In all experiments, the statistical significance of the results was determined as p<0.05.

Improving the efficiency of CAR-T lymphocytes by removing the Cbl-b gene.Kumar et al.
/ Journal for Immunotherapy of Cancer, 2021

"Our study represents an important step towards the development of CAR-T cells to fight tumors of various organs. [This approach] can overcome the limitations of some modern cancer immunotherapy strategies," Pujari said.

Currently, various research teams are searching for new applications of CAR-T lymphocytes and improving their effectiveness. Certain successes have been achieved not only in oncology, but also in the treatment of viral infections, and even in the fight against cellular aging.

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