Editor's Note: The breakthrough achievements of CAR-T therapy in the field of hematologic malignancies are remarkable. However, challenges such as target limitations, limited cell sources, and durability of efficacy continue to necessitate further exploration. Dr. Jianxiang Wang's team at the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences , stands as a pioneer in the field of immunotherapy, advancing both basic and clinical research to push the boundaries of hematologic cancer immunotherapy. In this issue of Hematology Frontier, Dr. Jianxiang Wang shares the research outcomes and forward-looking insights of his team in the realm of immunotherapy. The aim is to inspire new clinical thinking and collaboratively open a new chapter in hematologic cancer immunotherapy, bringing more hope and possibilities to patients.

Exploration of New Targets and Therapies in Immunotherapy

In the journey of immunotherapy for hematologic malignancies, including antibody and cell-based immunotherapy, Chinese scholars have conducted extensive exploratory research, placing China’s CAR-T cell therapy research at the forefront of the world. Our research group has independently developed a CD19 CAR-T therapeutic product, which has already been approved for the market, completing the entire process from research and development to productization. Additionally, we have expanded our focus to other known targets, including CD33 and CD123 for myeloid tumors; CD20 and CD22 for B-lymphoid tumors; CD5 and CD7 for T-lymphoid tumors; and BCMA and CD38 for plasma cell tumors.

However, despite the outstanding work by Chinese scholars in the field of immunotherapy for hematologic malignancies, many unresolved issues remain, such as insufficient target diversity, concentrated cell sources, short-lived efficacy, and significant toxicity. Below, I will share insights and findings from our team’s research in exploring new immunotherapy targets and therapies, as well as expanding and optimizing effector cells.

Exploration of New Immunotherapy Targets and Therapies

According to the “multiple hit” hypothesis of leukemia development, leukemia arises from different “hits” on hematopoietic cells, which can lead to the development of acute myeloid leukemia (AML), acute T-lymphoblastic leukemia (T-ALL), and acute B-lymphoblastic leukemia (B-ALL). Besides targeting hematopoietic cells, the tumor microenvironment also plays a crucial role in the development and progression of leukemia, including supporting hematopoietic cells, inhibiting the immune microenvironment, and possibly directly stimulating leukemia cells. We conducted a study to explore immune evasion and its direct impact on leukemia development.

01. Is the impact of Treg cells on mouse survival solely due to immune evasion?

In the leukemia microenvironment, we clearly observed a significant increase in regulatory T cells (Treg cells). It is well-known that an increase in Treg cells, along with enhanced suppressive activity, can lead to abnormal polarization of Th1 and Th2 cells, making immune suppression and evasion more likely. Therefore, Treg cells are related to disease survival and progression in mice. But is the impact of Treg cells on mouse survival entirely due to immune evasion? Our research found that Treg cells enhance the stemness of leukemia cells in mice, including an increase in the proportion of side population (SP) cells, a higher proportion of G0 phase cells, and enhanced primitive colony formation ability. We further validated these findings in primary cells.

02. Through what mechanism do Treg cells exert their effects?

By measuring cytokine secretion from Treg cells, we discovered a significant increase in interleukin-10 (IL-10) secretion. To verify the relationship between IL-10, leukemia relapse, and survival, we conducted further functional experiments using both cell lines and primary cells. The results showed that IL-10 can directly incubate leukemia cells, increasing their stemness, including the proportion of SP cells, G0 phase cells, and leukemia cell colonies.

This led us to hypothesize that Treg cells exert their effects through IL-10. How can this hypothesis be proven? We employed two methods: First, by using an antibody-blocking approach, we found that after adding an antibody against the IL-10 receptor, the number of SP cells, G0 phase cells, and leukemia cell colonies decreased, confirming that the IL-10 receptor antibody effectively blocked this pathway. Second, by knocking out the IL-10 receptor in endogenous leukemia cells, we observed that the effect of IL-10 on enhancing leukemia stemness was eliminated with the receptor knockout, further confirming that Treg cells exert their effects by binding IL-10 to its receptor, thus stimulating leukemia stemness.

Having established this mechanism, we delved into the IL-10 signaling pathway, hoping to identify a therapeutic target within this pathway. However, after a series of phosphorylation studies, we found that this pathway is not unique and mainly operates through the PI3K/AKT signaling pathway. The PI3K/AKT pathway is well-known as an anti-apoptotic pathway, and numerous inhibitor studies have made PI3K a promising therapeutic target. PI3K has three subunits: α, β, and γ, but not all are effective in treating hematologic malignancies, necessitating the identification of specific PI3K inhibitors for particular tumors. In summary, IL-10 promotes leukemia stemness through PI3K/AKT phosphorylation.

03. Are there any deeper mechanisms to explore regarding leukemia cell stemness?

Our research showed that Treg cells significantly increase the expression of OCT4 and Nanog through the PI3K/AKT signaling pathway. In other words, during the reprogramming process of cells, when stem cells are “rejuvenated,” the transcription factors OCT4 and NANOG play critical roles. Additionally, by adding inhibitors targeting the PI3K/AKT pathway, we confirmed that downregulating the PI3K/AKT signaling pathway could reverse stemness in both leukemia cell lines and primary cells.

In conclusion, through this study, we demonstrated that Treg cells enhance leukemia cell proliferation by suppressing the immune response and stimulate leukemia stemness through the IL-10 receptor on leukemia cells. Consequently, we designed a new CAR-T targeting the IL-10 receptor.

04. Construction of IL-10 CAR-T

Since our laboratory currently lacks an antibody against the IL-10 receptor, we used a ligand instead of a single-chain antibody to construct IL-10 CAR-T cells. We screened target cells expressing the IL-10 receptor in vitro. In leukemia cell incubation experiments, we observed that IL-10 receptor-positive target cells specifically activated IL-10 CAR-T cells, killing leukemia cells effectively both in vitro and in primary cells. In vivo experiments also showed that IL-10 CAR-T could prolong the survival of AML-bearing mice.

05. Exploration of New Targets in Leukemia Membrane Proteomics

It has become increasingly difficult to identify new targets for leukemia cells solely based on differential gene expression analysis. Another critical issue is the significant disparity between gene expression profiles and protein expression profiles. Large differences in gene expression may not correspond to differences in protein expression, and vice versa. Therefore, ultimate target identification should focus on the expression of membrane proteins on leukemia cells to discover new targets. However, this is challenging because it is difficult to isolate sufficient amounts of highly pure membrane proteins for mass spectrometry analysis. Nevertheless, through technological research, we have essentially overcome these two challenges.

Equally important is the choice of reference standards for cell analysis. We need to prepare membrane proteins from normal hematopoietic stem cells. However, normal hematopoietic cells expressing CD34 (i.e., normal hematopoietic stem cells) are very scarce. We collected over 100 samples of normal hematopoietic stem cells in the apheresis unit, from which we isolated normal CD34 membrane proteins. Currently, no database contains a standard membrane protein library for CD34 from normal individuals, as such resources are scarce, and it is difficult to obtain pure membrane proteins. Now that we have isolated them, we have a solid reference for cell analysis.

Through cell analysis, we aim to identify membrane proteins that are expressed at low levels in normal hematopoietic cells, including normal CD34 cells and progenitor cells, but highly expressed in leukemia cells, representing relatively specific membrane proteins and potential targets. Additionally, we need to prepare antibodies using two approaches: One is to prepare humanized antibodies through phage display, and the other is to use traditional hybridoma technology. We have screened five antibody clones and quickly produced CAR-T cells. These CAR-T cells targeting potential new targets have demonstrated significant cytotoxicity in both in vitro and in vivo experiments.

Currently, we are transducing viruses for this new target CAR-T and preparing to apply for clinical trials. At the same time, we have also validated the reported CD70 target using this new CAR-T approach. The membrane protein database confirms that CD70 is highly expressed on leukemia cells. Accordingly, we have produced CAR-T cells targeting CD70, and the virus has been prepared, ready for clinical trials. This demonstrates the feasibility of discovering new targets through membrane protein exploration. In addition to CD70 CAR-T, we have also developed universal autologous CAR-T cells and produced humanized antibodies. The methods for producing humanized antibodies include: (1) preparing fully humanized antibodies through phage display, and (2) producing humanized antibodies from mouse antibodies, ensuring reduced immune-inflammatory responses in vivo and achieving the desired effects.

Expanding Cell Sources

In expanding cell sources, the focus is on NK cells and addressing key scientific issues, including NK cell expansion and transduction. For expansion, trophoblast cells serve as a universal and efficient method for activating primary NK cells. In terms of transduction, we optimized solutions to the challenges of difficult transduction and expression in mature autologous NK cells. We screened promoter elements for NK cells and found that the CMV promoter could efficiently initiate gene expression, enabling stable expression of target genes in NK cells. The combination of RetroNectin and Polybrene significantly increased lentiviral transduction efficiency in NK cells.

Moreover, NK cells have innate antiviral properties, and to further improve transduction efficiency, we explored inhibiting specific pathways in NK cells. The results showed that adding relevant inhibitors partially increased transduction efficiency, but fundamental improvement was not achieved. Meanwhile, in enhancing NK cell activity, we used the NK-92 cell line as a model for high-throughput CRISPR gene screening, identifying potential targets. The next step involves validating these findings to identify precise targets for boosting NK cell activity.

In conclusion, beyond exploring existing cell therapies in clinical research, we have also investigated leukemia pathogenesis, developed new targets and therapies, and optimized and expanded effector cells. Our goal is to improve the efficacy, safety, and accessibility of cell therapy. In fact, throughout the translational medicine process, clinicians play an indispensable role, participating in various stages, including proposing significant scientific questions, discovering new mechanisms and technologies through experiments, conducting clinical trials after companies translate technologies into products, and applying research-validated drugs in clinical practice, ultimately contributing to society.

References:

1. Xu Y, et al., Leukemia, 2022, 36:403-415.
2. Nat. Rev. Clin. Oncol., 2020, 17:147-167.

Expert Profile:

Dr. Jianxiang Wang

• Chief Clinical Expert, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences
• Director, National Center for Clinical Medicine Research on Hematologic Diseases
• Professor, Chief Physician, Doctoral Supervisor
• One of the First Long-term Professors at Peking Union Medical College, Chinese Academy of Medical Sciences
• National-Level Talent in the National Hundred, Thousand, and Ten Thousand Talent Project
• Chair, 10th Hematology Committee, Chinese Medical Association
• Vice President, Internal Medicine Physicians Branch and Hematology Physicians Branch, Chinese Medical Doctor Association
• Tianjin Expert, Haihe Medical Scholar, First Tianjin Famous Doctor, Tianjin’s “Top Ten” Medical Workers, “China’s Good Doctor” of the Month
• With extensive clinical experience in diagnosing and treating various hematologic diseases, currently focused on clinical and basic research in leukemia and hematologic malignancies.