CAR-T cell therapy brings new hope to cancer patients with its unique targeted therapy. The 10 CAR-T drugs currently on the market are mainly autologous CAR-T cell transplantation treatments targeting CD19 or BCMA. According to statistics, only more than 35,000 patients have been treated so far, which is a drop in the bucket compared to the total number of patients worldwide. The most important reason is that personalized customization leads to unfriendly prices. In addition, immune cell therapies are currently all autologous transplantation treatments, so clinical treatment also faces a long preparation time. After bridging treatment, patients need to clear lymph and recover to a physical state suitable for reinfusion of CAR-T. More importantly, due to the influence of autoimmunity and chemotherapy, some patients' autologous T cells will be exhausted, aged, and functionally defective, which is not enough to prepare enough CAR-T cells in vitro, thus affecting the final treatment effect. The cell therapy industry is rapidly realizing technological iteration and moving towards commercialization to cope with huge market demand. In particular, there are technical barriers to the underlying technology viral vectors and efficient gene editing, which face pain points such as long R&D cycles, high production costs, and low conversion rates, resulting in limited production capacity and high prices, making its commercialization path extremely challenging. Therefore, it is particularly important to develop universal cell products.

In order to allow more patients to benefit from immune cell therapy, researchers are now developing safe and effective allogeneic transplant immune cells and preparing universal CAR-immune cells. The successful development of such products will greatly reduce production costs, shorten production time, and better ensure product efficacy, so that more patients can benefit. The most important challenges of immune cell allogeneic transplantation are graft-versus-host disease (GvHD) and host-versus-graft response (HvGR). To address this challenge, researchers mainly address it from the following aspects:

1. T cells can destroy GvHD by knocking out TRAC, and additional gene editing can resist host rejection, such as modification of MHC-I-related sites in the graft (B2M-HLA I).

2. Selecting γδT cells and NK cells from healthy donors can avoid or reduce GvHD.

Universal CAR-T and TCR-T cells

The researchers used CRISPR-Cas9 to integrate CD19-CAR into the TRAC site, with the goal of knocking out the TCR of the donor cells and introducing specific CAR molecules targeting CD19 at the same time, so that the transplantation of universal CAR-T cells avoided GvHD, and CD19-CAR was expressed under the natural TCR promoter. This genetic engineering modification enhanced the function of CAR-T cells and better controlled the disease progression of pre-B-ALL. However, there are also related problems. For example, among the 21 patients treated with “UCART19”, 14 patients achieved complete remission or complete remission with incomplete blood recovery 28 days after infusion. Therefore, host rejection of allogeneic CAR-T is still a major factor. The main way to avoid rejection of allogeneic cells by the host is to prevent autologous immune cells from identifying transplanted cells as foreign cells and killing them. Some researchers have also constructed an allogeneic immune defense receptor (ADR) through genetic engineering, which can selectively recognize 4-1BB, a cell surface receptor that is transiently upregulated on the surface of activated lymphocytes. CAR-T cells expressing ADR can effectively resist allogeneic reactive T cells both in vivo and in vitro. In hematological tumor and solid tumor mouse models, allogeneic CD19-CAR-T therapy expressing ADR showed persistent tumor elimination. This method may have a good prospect for the development of universal CAR-T in the future.

Figure 1. Manufacturing of off-the-shelf CAR-T cells. (Sadeqi Nezhad M, et al., 2021)

Universal NK and CAR-NK cells

Studies have found that compared with peripheral blood stem cell transplantation, patients who received allogeneic umbilical cord blood transplantation had lower levels of proinflammatory cytokine release and significantly reduced GvHD. Other sources, such as immune effector cells obtained from induced pluripotent stem cells, are also being explored.

NK cells are a type of cytotoxic cell that plays an important role in the innate immune response against virally or bacterially infected or damaged cells. Since they do not play a killing function by recognizing allogeneic HLA and do not cause GvHD, NK cells have the potential to be used as allogeneic cell immunotherapy. NK cell activation is regulated by a variety of transmembrane receptors, including activating receptors, inhibitory receptors, cytokine receptors, and chemokine receptors. MHC downregulation is a common feature of tumor cells, which provides an activation and killing mechanism for NK cells. When the inhibitory killer immunoglobulin receptor KIR on the surface of NK cells binds to tumor cells with MHC-I downregulation, the NK inhibitory signal is weakened, thereby inducing the killing function of NK cells. The inhibitory receptor CD94 (NKG2A or NKG2C heterodimer) on the surface of NK cells can recognize non-classical HLA-E molecules. Malignant tumors or virus-infected cells can escape the killing of the immune system through this molecular pathway. The activation signal of NKG2C can activate NK cells to exert their killing function, so NKG2C-positive NK cells can better enable NK cells to resist the escape of HLA-E-positive tumors. In addition, in addition to the natural killing ability of NK cells, CAR-NK can also specifically recognize tumor antigens through CAR molecules to exert anti-tumor effects. However, allogeneic NK cells are still susceptible to allogeneic rejection by the host immune system. The most commonly used approach is to knock out HLA-I class molecules (B2M gene) in NK cells and express single-chain HLA-E molecules to escape the killing of host T cells, NK and macrophages, and also prevent transplanted NK cells from killing each other.

Figure 2. Strategies for stealth NK cell products. (Berrien-Elliott M M, et al., 2023)

Universal γδT and CAR-γδT cells

T cells are divided into two categories, αβT cells and γδT cells, according to different TCRs. Human peripheral blood lymphocytes are mainly αβT cells, and γδT cells generally account for only 1%-5%. Although γδT cells account for a small proportion, they can directly kill tumor cells through NK cell receptors on their cell surface, ADCC effects, and cytokines (IFN-γ, TNF-α) secreted by them. In addition, γδT cells can also act as antigen-presenting cells to activate αβT cells, or induce anti-tumor cytotoxicity of NK cells through the 4-1BB co-stimulation pathway, thereby achieving indirect killing of tumors. In addition, γδT cells do not have the risk of GvHD during allogeneic cell transplantation, so they are safer. In addition to its natural anti-tumor effect, CAR-γδT can further specifically identify tumor-associated antigens through CAR molecules and play a direct role in killing tumors.

Figure 3. The anti-tumor and pro-tumor functions of γδT cells mediated by cytokines and receptor-ligand interactions.

Creative Biogene has achieved breakthroughs in the preparation processes of CAR-T, NK, CAR-NK, γδT and CAR-γδT, especially in CAR-T. Please feel free to contact us if you have a need, and our experienced specialists will be happy to assist you.