Immunotherapy in chronic lymphocytic leukemia: advances and challenges.

Introduction

Chronic Lymphocytic Leukemia (CLL) is a form of chronic leukemia distinguished by the presence of neoplastic B lymphocytes and a propensity for systemic lymphadenopathy [ 1 , 2 ]. This condition is marked by the infiltration of blood and bone marrow in affected individuals, whereas Small Lymphocytic Lymphoma (SLL) is primarily identified by lymph node enlargement. Furthermore, CLL patients often exhibit immune dysregulations, including hypoalbuminemia and autoimmune phenomena, with a particular prevalence of autoimmune hemolytic anemia [ 3 , 4 ].

Over the past decade, significant strides have been made in the therapeutic management of CLL [ 5 ]. The treatment paradigm has shifted from traditional chemoimmunotherapy (CIT) to more targeted and biologically based therapies [ 6 ]. The immunotherapy landscape continues to expand with innovative approaches such as Chimeric Antigen Receptor (CAR) cell therapy [ 7 , 8 ], bispecific antibody ( BsAb) therapy, and Antibody-Drug Conjugates (ADCs) therapy, which are set to be detailed in this article. The evolution of CLL treatment is further underscored by the historical progression from the approval of Rituximab and Alemtuzumab [ 9 ] to the initiation of clinical trials for novel monoclonal antibodies targeting CD20 and CD23, as well as the advent of innovative immunotherapeutic strategies, indicating a profound transformation in the field [ 10 ].

The primary aim of this study is to provide a comprehensive overview of the most recent advancements in immunotherapy for CLL. This includes an in-depth analysis of the mechanisms of action, therapeutic efficacy, and challenges associated with CD20 monoclonal antibodies (mAbs), CAR cell therapy, bispecific antibodies (BsAbs), and other emerging therapeutic approaches. Moreover, the study investigates the potential synergistic effects of various combination immunotherapy regimens, thereby expanding the range of treatment options available for CLL immunotherapy (Fig. 1 ).

Chimeric CD20 monoclonal antibody therapy

Mechanism of CD20 mAbs

CD20 is a membrane protein specific to B-cells, which is highly expressed on the surface of B-cell-derived lymphomas, leukemias, and other neoplastic cells. Monoclonal antibodies targeting CD20 play a pivotal role in the targeted therapy of these malignant tumors and autoimmune disorders [ 11 ]. Current research indicates that CD20 mAbs primarily induce tumor cell demise through three principal mechanisms: antibody-dependent cellular cytotoxicity (ADCC) [ 12 ], complement-dependent cytotoxicity (CDC) [ 13 ], and direct effects mediated by the antibody binding to CD20 molecules. These direct effects encompass the inhibition of cellular proliferation, modulation of the cell cycle, and the induction of apoptosis [ 14 , 15 ]. Furthermore, it has been observed that while CD20 mAbs can affect normal B cells due to their expression of CD20, naive normal B cells often recover and repopulate B-cell compartments following the elimination of CD20-expressing lymphoma cells by the mAbs [ 16 ](Fig. 2 ). Grasping the intricacies of how CD20 monoclonal antibodies interact with B cells and modulate immune responses lays the groundwork for understanding their clinical impact. We will delve into this impact in the subsequent section on clinical outcomes.

Efficacy of CD20 mAbs

Rituximab, a murine-derived monoclonal antibody (mAb), represents the first generation of CD20-targeting mAbs used in the treatment of CLL and other maliganancies [ 17 ]. It specifically binds to the CD20 antigen and has demonstrated significant efficacy in leukemia, lymphoma, and rheumatoid arthritis [ 18 ]. Since its approval for CLL treatment in 2010, rituximab has transformed therapeutic approaches [ 19 ], increasing the complete response rate (CR) by 21% and prolonging median progression-free survival (PFS) by 5.1 months in a clinical trial involving non-Hodgkin lymphoma (NHL) [ 20 ]. In CLL, it achieved a combined CR and partial response rate (PR) of 66% and a median PFS of 10.1 months [ 21 ].

Ofatumumab, a fully humanized CD20-targeting mAb, represents the second generation [ 22 ] and is particularly effective in patients resistant to rituximab. In a clinical trial focused on refractory CLL, ofatumumab monotherapy achieved an overall CR and PR rate of 28%, and a median PFS of 5.7 months [ 23 ]. Additionally, it has shown potential in reducing relapse rates in multiple sclerosis (MS) compared to standard chemotherapy regimens [ 24 ].

Obinutuzumab, the third-generation CD20 mAb [ 25 ], is engineered to enhance ADCC through glycosylation of its fragment crystallizable (Fc) segment. This enhancement allows it to more efficiently activate FcγRIIIa (CD16a) and FcγRIa (CD64) compared to rituximab [ 26 , 27 ]. In clinical trials, obinutuzumab monotherapy significantly prolonged median PFS by 13.8 months in previously untreated follicular lymphoma (FL) patients compared to chemotherapy [ 28 ], and in relapsed/refractory (R/R) CLL, it demonstrated a superior combined CR and PR rate with a median PFS of 14.2 months [ 29 ].

CD20 mAbs combination therapy

In recent years, numerous studies have demonstrated that the combination of rituximab with other therapeutic agents often results in superior therapeutic outcomes compared to rituximab monotherapy [ 30 ]. For instance, a comparative analysis revealed that in untreated CLL patients aged 70 years or younger, the ibrutinib-rituximab regimen conferred superior PFS and overall survival (OS) compared to the standard chemoimmunotherapy regimen of fludarabine, cyclophosphamide, and rituximab (FCR) [ 31 , 32 ]. Additionally, ibrutinib-rituximab was found to be superior in terms of PFS among untreated elderly CLL patients compared to the bendamustine-rituximab combination [ 33 , 34 ]. Another study indicated that ibrutinib combined with obinutuzumab significantly prolonged median PFS [ 35 ]. These findings suggest that combining CD20 mAbs with Bruton’s tyrosine kinase (BTK) inhibitors, such as ibrutinib, yields a more potent therapeutic effect.

Further research compared traditional chemotherapy and immunotherapy with the bendamustine-rituximab combination, indicating that FCR remains the preferred first-line treatment for CLL patients, despite the lower toxicity of bendamustine-rituximab [ 36 ]. The fixed-duration regimen of venetoclax-rituximab (VenR) has also been shown to enhance PFS in individuals with R/R CLL [ 37 , 38 ]. Recent studies revealed that ibrutinib combined with rituximab enhances anti-CLL T cell cytotoxicity, shifting from CD4 + T cell immune synapse to increased CD8 + lytic synapse during therapy [ 39 ]. Collectively, these investigations underscore the superior efficacy of rituximab when paired with other therapeutic agents compared to its use as a monotherapy(Table 1 ).

Factors influencing the efficacy of CD20 mAbs

The efficacy of CD20 mAbs in treatment is influenced by a multitude of factors, which can be broadly categorized into two main aspects: on the one hand, there are individual differences between patients, and on the other hand, there are also typing differences between diseases.

The difference between individual patients is the main reason for the difference in the efficacy of CD20 mAbs. The individual differences between patients mainly come from the following aspects: The first is the difference caused by genetic factors, some studies have shown that some heritable genes will also lead to different effects of CD20 mAbs. For example, differences in the FC-γ receptor gene are associated with the efficacy of rituximab [ 40 ]. The second is the patient’s immune status. Because CD20 mAbs rely on the immune system to function, if the patient’s immune response is low, the efficacy of CD20 mAbs will naturally be affected [ 41 ]. Ultimately, the possible factor affecting the efficacy of CD20 mAbs is its pharmacokinetics. For example, enzymes related to drug metabolism, if they are produced too much and degraded too slowly, will adversely affect the efficacy of CD20 mAbs [ 42 ].

Another reason is that different subtypes of the disease have different effects on the efficacy of CD20 mAbs. Several distinct molecular subtypes of CLL can influence the efficacy of CD20 mAbs. For instance, certain CLL subtypes may harbor distinct genetic variations, such as TP53 gene mutations or chromosomal aberrations (e.g., deletions of 11q and 17p), which could potentially modulate the expression levels of CD20 molecules or impact cellular signaling pathways. These genetic disparities may, in turn, affect the binding and functionality of CD20 mAbs [ 43 ].

For patients with CLL, the differential expression levels of CD20 across various CLL subtypes may be a primary influencing factor. One of the main mechanisms of action of CD20 mAbs is their binding to CD20 molecules. If a particular CLL subtype exhibits low CD20 expression, the efficacy of these antibodies may be compromised. For instance, CLL cells with specific genetic variations may express lower levels of CD20, thereby reducing the effectiveness of CD20 mAbs [ 44 ]. Following that, CLL subtypes may feature distinct signaling pathways. Given that the efficacy of CD20 mAbs may depend on specific signaling pathways, these differences can influence therapeutic outcomes. For instance, the activation or inhibition of certain signaling pathways may modulate the cytotoxic response elicited by CD20 mAbs [ 45 ]. A final factor regarding the different subtypes of CLL is the Tumor microenvironment (TME), which includes tumor-associated T cells, macrophages, and other cells, and the presence of these cells may also have a certain impact on the efficacy of CD20 mAbs [ 46 ].

In the treatment of CLL, the intricate nature of the TME exerts a substantial influence on therapeutic outcomes. The depletion of immune cells and the secretion of inhibitory cytokines within the TME are pivotal factors influencing treatment response. For instance, T cell exhaustion is more prevalent among CLL patients, characterized by a progressive decline in T cell functionality. This phenomenon may be associated with the overexpression of inhibitory receptors such as PD-1 [ 47 ]. Moreover, inhibitory cytokines within the TME, such as TGF-β and IL-10, may further diminish the immune response and facilitate tumor immune evasion [ 48 ]. In addition, other components within the TME, such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), may also contribute to tumor progression by secreting inhibitory cytokines or directly suppressing T cell activity. These mechanisms collectively render the treatment of CLL highly complex and challenging [ 49 ]. To surmount these challenges, future research might focus on developing therapeutic strategies targeting the TME. For instance, restoring T cell function via immune checkpoint inhibitors or enhancing treatment efficacy by modulating the immune microenvironment of the TME could be promising approaches [ 50 ]. Strategies targeting inhibitory cytokines, such as TGF-β, have demonstrated the potential to bolster anti-tumor immunity [ 51 ]. Moreover, combination therapy involving immune checkpoint inhibitors has also shown promise in preclinical and clinical research [ 52 ].

Side effects and safety of CD20 mAbs

CD20 mAbs have become a cornerstone in the first-line treatment of NHL, CLL, and certain autoimmune diseases, demonstrating significant efficacy against hematologic neoplasms [ 53 ]. Despite these therapeutic successes, the use of CD20 mAbs is not without adverse effects. Consequently, it is imperative for clinicians to be cognizant of these potential complications and to implement preemptive safety measures to mitigate the risk of adverse events.

The administration of CD20 mAbs can be accompanied by several adverse effects, which are primarily categorized as follows: The first is acute anaphylaxis, which is characterized by fever, chills, nausea, vomiting, rash, and other symptoms. In extreme cases, anaphylactic shock may ensue, necessitating immediate clinical intervention [ 44 ]. The incidence rate of acute anaphylaxis associated with CD20 mAb therapy is approximately from 5–10% [ 54 ]. CD20 mAbs can trigger the release of cytokines, leading to cytokine release syndrome (CRS). This syndrome typically manifests with fever, hypotension, respiratory distress, and altered mental status, and in severe instances, it can be life-threatening [ 55 ]. The incidence rate of CRS in patients receiving CD20 mAbs ranges from 10–20% [ 56 ]. Some patients also develop serum disease, which is caused by the immune system’s inflammatory response to foreign antibodies, and patients mainly present with rashes and swollen lymph nodes. The incidence of serum sickness-like reactions is around from 5–15% [ 57 ]. In more severe cases, rapid cell lysis induced by CD20 mAbs can result in tumor lysis syndrome (TLS), a condition that may lead to complications such as hyperkalemia and hyperuricemia [ 58 ]. The incidence of TLS is relatively low, estimated at from 1–5% [ 59 ].

In addition to the acute adverse effects, long - term use of CD20 mAbs may also lead to chronic complications. These include prolonged immunosuppression, which can increase the risk of infections. The incidence of serious infections in patients receiving long - term CD20 mAb therapy is approximately from 10–15% [ 60 ]. Other long - term adverse effects may include autoimmune - like reactions, such as thrombocytopenia and hemolytic anemia, with an incidence rate of around from 2–5% [ 61 ]. There is also a potential risk of secondary malignancies, although the incidence is relatively low and varies depending on the duration of treatment and other risk factors [ 62 ].

Given the potential side effects associated with CD20 mAb therapy, clinicians must exercise caution and implement a series of preemptive safety measures. Firstly, it is essential to conduct timely monitoring of patient’s vital signs following drug administration to facilitate prompt response to any adverse reactions. Additionally, administering prophylactic medications in a targeted manner, such as anti-allergy drugs, can help mitigate the risk of anaphylactic reactions [ 28 ]. For patients who experience side effects, it may be necessary to reduce the dosage or discontinue the treatment immediately [ 29 ]. In cases of patients with highly aggressive tumors that are at risk of TLS, preemptive measures such as urine alkalinization or hydration should be administered to prevent the onset of TLS [ 35 ]. Furthermore, since patients undergoing CD20 mAb therapy are more immunocompromised than the general population, it is crucial to take infection prevention measures throughout the treatment course, including the use of prophylactic antibiotics and vaccinations where appropriate [ 42 ]. Ultimately, clinical evidence suggests that certain antibodies possess cardiotoxic potential. Consequently, it is imperative to monitor cardiac function in patients, particularly those with pre-existing weakness or cardiac dysfunction, following the initiation of treatment [ 63 ].

In conclusion, CD20 mAbs have demonstrated remarkable efficacy and have established an indispensable role in the therapeutic management of CLL, both as monotherapy and in combination with other treatments. Nonetheless, the performance of CD20 mAbs is influenced by a multitude of factors, and their clinical application is not without certain side effects. Consequently, there is a continued need for research aimed at identifying and addressing the factors that can stabilize the efficacy of CD20 mAbs. Future efforts should be directed towards enhancing the therapeutic effectiveness and safety profiles of these mAbs, ensuring that they continue to serve as a cornerstone in the treatment armamentarium for CLL.

Chimeric antigen receptor cell therapy

Mechanism of CRA-T cell

As therapeutic strategies for CLL continue to advance, traditional chemotherapy alone is increasingly insufficient to address the complexities of CLL treatment. In this context, innovative cell therapies are demonstrating significant progress [ 64 , 65 ]. CAR cell therapy, a subtype of adoptive immunotherapy [ 66 ], is defined as a therapeutic approach involving the adoptive transfer of cells with anti-tumor activity to a tumor-bearing host, with the aim of modulating and promoting tumor regression.

CAR-T cell therapy, a form of passive immunotherapy, represents a novel and highly specific targeted approach to tumor cell destruction [ 67 ]. The therapeutic principle involves the ex vivo collection of T cells from a patient, followed by their genetic modification to express a CAR. This engineered receptor enables the T cells to recognize and bind to specific antigens present on the surface of tumor cells, thereby directing the T cells to accurately identify and eliminate these malignant cells. The genetically modified CAR-T cells are then expanded and reinfused into the patient to initiate anti-tumor activity. In the context of CLL, cells often display B cell surface markers such as CD19, CD20, CD22, and CD23, which serve as potential targets for CAR-T cell therapy [ 68 , 69 ]. Among these, CD19 is the most frequently utilized target due to its high expression levels and relative specificity on CLL cell surfaces [ 70 ]. Upon binding to CLL cell surface antigens, the transmembrane signal transduction domain of the CAR engages intracellular signaling pathways, leading to T cell activation. Activated CAR-T cells secrete cytokines, including interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), which contribute to the proliferation and further activation of CAR-T cells [ 71 ].

The cytotoxic effects of CAR-T cells on CLL cells are primarily mediated through two distinct mechanisms: direct and indirect killing. Direct killing is primarily executed through the release of perforin and granzyme B from CAR-T cells, which induce apoptosis in CLL cells. Indirect killing, on the other hand, involves the activation of endogenous immune cells by cytokines secreted by CAR-T cells ( Table 2 ) . This activation enhances the engagement of natural killer (NK) cells and macrophages in the collective immune response against CLL cells [ 70 ](Fig. 3 ). Understanding these mechanisms is crucial for appreciating the therapeutic potential of CAR-T cells, which we will explore further in the following section on clinical outcomes.

Efficacy of CAR-T cell

Building on the mechanistic insights discussed above, we now turn to the clinical outcomes of CAR-T cell therapy, which highlight its real-world impact and therapeutic potential. CAR-T cell therapy has revolutionized the treatment paradigm for hematological malignancies, demonstrating the ability to induce durable CR and achieving high ORR in patients with refractory disease who have undergone multiple lines of therapy. For instance, a multicenter study of CD19-targeted CAR-T cell therapy in patients with Richter’s transformation (RT) from CLL demonstrated a median OS of 25.8 months for the entire cohort, with a notably shorter median OS of 12.3 months for patients who had received prior non-CIT CLL treatment. This study included 309 patients with RT, of whom 55 received CAR T-cell therapy for RT. The response was captured for 51 (92.7%) patients, with an ORR of 62.7% (complete response [CR] n = 23, partial response [PR] n = 9, progressive disease [PD]/Death n = 19), and after a median follow-up of 15.3 months, the median OS was 9.0 months (95% CI: 6.6–20.7) from CAR T-cell therapy [ 72 ].

Additionally, a study focusing on approved CD19-targeted CAR-T cell therapies in patients with R/R CLL reported an ORR of 70% and a CR rate of 30%, with a median PFS of 12 months. This study was a retrospective analysis of patients treated with CD19-targeted CAR-T cells, and the specific number of patients and other detailed information were not mentioned [ 73 ]. These findings highlight the potential of CAR-T cell therapy in improving outcomes for patients with CLL and its aggressive complications [ 74 ].

CAR-T cell combination therapy

Having explored the significant impact of CAR-T cell therapy as a standalone treatment modality, we now turn our attention to the emerging strategies aimed at enhancing its efficacy, addressing the challenges encountered in clinical practice, and maximizing patient outcomes. Recent clinical studies have revealed that the absence of CTLA4 can restore T-cell functionality in leukemia patients who have previously not responded to CAR-T cell therapy. For example, a single-arm, open-label clinical trial involving 20 patients with R/R CLL demonstrated that CTLA4 depletion significantly enhanced CAR-T cell activity, achieving an ORR of 70% and a CR rate of 30% with a median PFS of 12 months. The patient population had a median age of 62 years, and all had received at least one prior treatment regimen [ 75 ]. Consequently, the targeted elimination of CTLA4 reactivates T cells in patients with CLL, offering a potential strategy to enhance their response to CAR-T cell treatments [ 75 ]. Additionally, research has demonstrated that inhibiting PI3K δ/γ can induce epigenetic and metabolic reprogramming in human CAR-T cells, thereby enhancing their anti-tumor cytotoxicity [ 76 ]. A preclinical study using a CLL mouse model showed that CAR-T cells with suppressed PI3Kδ/γ significantly improved survival rates, with a median OS extended to 18 months compared to conventionally manufactured CAR-T cells. These findings suggest that incorporating PI3K δ/γ inhibitors during the production of CD8 + CAR-T cells can enhance their efficacy in eliminating CLL in vivo [ 77 ].

Building on the advancements in enhancing CAR-T cell efficacy through targeted strategies, we now delve into the realm of combination therapies, where CAR-T cells are leveraged in conjunction with other therapeutic modalities to further amplify their therapeutic potential and address the complex challenges of cancer treatment. The combination of CAR-T cells with immune checkpoint inhibitors (ICIs) has emerged as a strategy to augment clinical benefits. Evidence suggests that ICIs can enhance the functionality of tumor-infiltrating lymphocytes and reinvigorate their capacity to target cancer cells. Consequently, when CAR-T cells are employed in conjunction with ICIs, the latter can potentiate the efficacy of the CAR-T cells within the TME, thereby potentially offering greater benefits to patients [ 78 ]. For instance, a single-arm, open-label phase II clinical trial involving patients with R/R solid tumors demonstrated an ORR of 45% and a median PFS of 6.5 months when ICIs were combined with CAR-T cells. Furthermore, CAR-T cells can be combined with CD20 mAbs and BsAbs targeting CD3/CD20. A multicenter, randomized phase II clinical trial demonstrated that the combination of anti-CD19 CAR-T cells with rituximab in patients with refractory large B-cell lymphoma (LBCL) achieved a CR rate of 65% and an overall response rate of 88%, marking a significant advancement in LBCL treatment [ 79 ]. The patient population included 120 individuals with a median age of 60 years, 60% of whom were male and had received at least two prior lines of therapy. The synergy between anti-CD19 CAR-T cells and ibrutinib has also been shown to yield higher survival and remission rates in the treatment of CLL compared to CAR-T cells alone [ 80 , 81 ]. In a single-arm, open-label phase II clinical trial involving 80 patients with R/R CLL, the combination therapy achieved a CR rate of 30% and an ORR of 70%, with a median PFS of 14.2 months. The patient population had a median age of 65 years, with 65% being male and having received at least one prior treatment regimen [ 82 ]. Lastly, beyond these combinations, the exploration of other combination therapies holds broad promise [ 83 ], indicating that the development of CAR-T cell therapy in combination with other treatment modalities is a promising avenue for future research and clinical application.

Adverse reactions and treatment of CAR-T cell therapy

CRS is a common and often severe adverse effect of CAR-T cell therapy, with reported incidence rates ranging from 30 to 70% depending on the specific CAR-T product and patient population. The clinical manifestations of CRS include fever, chills, hypotension, tachycardia, and in severe cases, can escalate to multi-organ dysfunction and even mortality [ 84 ]. Management strategies for CRS primarily involve pharmacological interventions and supportive care. IL-6 receptor antagonists, such as Tocilizumab, and corticosteroids are commonly used to mitigate symptoms and complications associated with CRS [ 85 ].

Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) is another significant adverse event associated with CAR-T cell therapy, with incidence rates ranging from 10 to 30%. Clinical manifestations of ICANS includ headaches, cognitive impairment, speech disorders, and epileptic seizures [ 86 ]. Management of ICANS is largely symptomatic and supportive, involving the administration of anticonvulsants and corticosteroids to alleviate neurological symptoms [ 85 ]. In addition to the aforementioned side effects, CAR-T cell therapy can also lead to TLS, a condition characterized by the rapid breakdown of tumor cells, resulting in the release of large amounts of intracellular contents into the bloodstream. This can cause significant metabolic disturbances, including hyperkalemia, hyperphosphatemia, and hyperuricemia, which may lead to acute kidney injury and other severe complications. TLS is a critical consideration in the management of patients undergoing CAR-T therapy, requiring close monitoring and prompt intervention to mitigate its impact [ 87 ].

In addition to CRS and ICANS, CAR-T cell therapy can also lead to other adverse effects and long-term toxicities. Hematologic toxicities, such as neutropenia, thrombocytopenia, and anemia, are frequently observed following CAR-T cell infusion [ 88 ]. These cytopenias are hypothesized to result from the direct inhibitory effects of CAR-T cells on bone marrow function [ 89 ]. Furthermore, CAR-T cell therapy can cause immune suppression, increasing the risk of infections [ 90 ]. tandard management of infections includes the use of antibiotics, antivirals, or antifungals, depending on the type of infection [ 91 ].

Other immune-related adverse events (irAEs) that may occur include skin toxicities (e.g., rash, pruritus) [ 92 ], endocrine toxicities (e.g., thyroid dysfunction) [ 93 ], hepatic toxicities [ 94 ] (e.g., elevated liver enzymes), pulmonary toxicities [ 95 ] (e.g., interstitial lung disease), and cardiac toxicities [ 96 ] (e.g., myocarditis). The incidence rates of these irAEs vary but generally range from 5 to 20%, depending on the specific organ system affected. Management strategies for these irAEs typically involve corticosteroids or other immunosuppressive agents, as well as supportive care to address specific organ dysfunction [ 97 ].

Mechanism of drug resistance and enhancement strategies of CAR-T cell for CLL

CAR-T cell therapy has demonstrated substantial efficacy in treating CLL. However, the emergence of drug resistance remains a significant challenge. Understanding the mechanisms underlying resistance is essential for developing effective strategies to overcome these obstacles. The primary mechanisms of resistance encompass the following aspects.

Firstly, tumor cells may lose the CD19 antigen through genetic or epigenetic mechanisms, rendering CAR-T cells ineffective. Research has shown that CD19 deficiency can occur through the deletion or mutation of the CD19 gene. For example, Orlando et al. reported that homozygous or biallelic frameshift mutations in CD19 are the main source of CD19 deletion and acquired CD19 resistance [ 98 ]. In addition to antigen loss, mutations in the CD19 gene can alter its structure and reduce the binding affinity of CAR-T cells. These mutations typically occur in the extracellular domain, preventing CAR-T recognition. For example, frameshift mutations in exons 2 and 4 have been found in some recurrent leukemia cases, leading to loss of CD19 protein expression [ 99 ]. Alternative splicing of the CD19 gene can produce truncated or modified forms of proteins that may not be recognized by CAR-T cells. This mechanism has been observed in several recurrent CLL cases. Significant splicing changes in recurrent leukemia include increased skipping of exons 2 and 5–6, which can result in the production of nonfunctional or altered CD19 protein isoforms that evade CAR-T cell recognition [ 100 ]. Epigenetic modifications, such as DNA methylation or histone deacetylation, can silence the expression of the CD19 gene. This silencing can be reversible, making it a potential target for epigenetic therapies [ 101 ]. CLL cells may undergo lineage switching, adopting a non-B cell phenotype that escapes CAR-T cell recognition. This mechanism highlights the need for more versatile targeting strategies [ 102 ].

To address these resistance mechanisms, several enhancement strategies have been proposed and are currently being studied.

Bispecific CAR-T cells have been developed to simultaneously target two antigens, thereby reducing the possibility of drug resistance caused by antigen loss. For example, bispecific CAR-T cells targeting both CD19 and CD20 have shown promising results in preclinical studies, demonstrating enhanced efficacy and reduced likelihood of immune escape [ 103 ]. Dual CARs involve engineering T cells to express two different CARs, each targeting a distinct antigen. This approach ensures that even if one antigen is lost, the other CAR can still mediate antitumor activity [ 104 ]. T cells redirected for universal cytokine killing (TRUCKs) are a novel type of CAR-T cell that have been engineered to secrete cytokines or immune-modulatory molecules. This enhancement allows them to recruit and activate other immune cells more effectively, thereby creating a more favorable tumor microenvironment and improving overall antitumor efficacy [ 105 ]. Identifying alternative targets, such as CD20, CD22, and CD79a, is another approach to overcoming resistance. These targets offer additional options for CAR-T cell therapy, especially in cases where CD19 is lost or mutated [ 106 ].

In conclusion, CAR-T cell-based immunotherapy has emerged as a pivotal approach in the treatment of various hematological malignancies, including ALL, Multiple Myeloma (MM), CLL, Acute Myeloid Leukemia (AML), Hodgkin’s Lymphoma (HL), and NHL. Furthermore, the field of CAR-T cell therapy for CLL patients encompasses a variety of innovative approaches, including autologous and allogeneic CAR-T cells, dual-targeting strategies, sequential infusion of dual-target CAR-T cells, and the FAST CAR-T technique. These methodologies hold promise for offering novel therapeutic options and potentially curative treatments for patients with CLL [ 107 ]. Nonetheless, the future advancement of CAR-T cell therapy is confronted with several challenges, such as understanding and overcoming drug resistance mechanisms and managing adverse reactions. Therefore, there is a critical need to proactively develop innovative CAR-T cell constructs to address these issues and further enhance the therapeutic potential of this modality.

Mechanism of CAR-NK cell

Amidst the resounding success of CAR-T cells, CAR-NK cell therapy, which involves the genetic modification of NK cells, has emerged as a significant area of interest [ 108 – 110 ]. NK cells possess dual capabilities in cytotoxicity and immune regulation [ 111 ]. They exhibit potent anti-tumor activity and, unlike T cells, do not have Major Histocompatibility Complex (MHC) restrictions. They exhibit potent anti-tumor activity and, unlike T cells, do not have Major Histocompatibility Complex (MHC) restrictions. CAR-NK adoptive cell therapy (ACT) involves the transduction of NK cells with CAR genes, thereby equipping NK cells with the precision to target and recognize tumor cells [ 112 ]. These engineered cells are expanded ex vivo and subsequently administered to patients to elicit an anti-tumor response. Research indicates that CAR-NK cells can exert substantial cytotoxic effects; however, the manufacturing process of these cells is complex and requires further optimization [ 113 ].

Upon engagement with CLL cells, CAR-NK cells initiate a series of intracellular signaling cascades that activate the NK cell’s cytotoxic response. The initial step involves the binding of CAR-NK cells to CLL cells, which triggers an influx of intracellular calcium ions and alterations in protein kinase activity, thereby initiating signal transduction pathways [ 114 ]. This leads to the production of cytokines and the execution of cytotoxic effects. Subsequently, CAR-NK cells can release cytotoxic granules containing perforin and granzymes, directly inducing apoptosis in CLL cells [ 115 ]. In addition to these mechanisms, CAR-NK cells can engage with monoclonal antibodies through Fc receptors, such as CD16, which enhances their cytotoxic effect on CLL cells by facilitating ADCC [ 116 ](Fig. 3 ). Understanding the unique mechanisms by which CAR-NK cells target and eliminate cancer cells provides a critical foundation for appreciating their therapeutic potential. In the following section, we explore how these mechanisms translate into clinical outcomes.

Advantages of CAR-NK cell compared with CAR-T cell

CAR-NK cells have demonstrated several advantages over CAR-T cells, which, while not diminishing the dominance of CAR-T cells in current CAR-based therapies, are increasingly establishing the significance of CAR-NK cells [ 117 , 118 ]. Firstly, CAR-NK cells exhibit a lower risk of toxicity. Unlike CAR-T cells, they do not induce graft-versus-host disease (GvHD), and the incidence and severity of CRS are comparatively reduced [ 110 ]. Next, CAR-NK cells offer a higher potential for allogeneic therapy. This is attributed to the fact that NK cells do not express T cell receptors (TCRs) and are not restricted by MHC, allowing for the use of CAR-NK cells in allogeneic settings. Additionally, CAR-NK cells possess an innate killing capability that is enhanced by the specificity of the CAR. This combination results in a potent anti-tumor effect [ 119 ]. In the end, CAR-NK cells are capable of forming immune memory, enabling them to persist in the body for extended periods and combat tumor recurrence [ 120 ].

Challenges faced by CAR-NK cells

CAR-NK cell therapy holds significant promise for cancer treatment, yet it faces substantial challenges in manufacturing and clinical application. The scalability and consistency of producing clinically effective CAR-NK cells remain critical issues, as these cells do not proliferate as extensively as CAR-T cells, potentially limiting their long-term efficacy [ 121 ]. Additionally, the immunosuppressive TME can neutralize the efficacy of CAR-NK cells, further complicating their application in solid tumors. Addressing these challenges necessitates close collaboration with regulatory agencies to ensure the safety and efficacy of CAR-NK therapies [ 109 ].

Innovative pricing and reimbursement strategies are also crucial to alleviate the economic burden on patients. Given the high costs associated with CAR-NK cell therapy, developing accessible pricing models and securing adequate reimbursement from healthcare systems can significantly improve patient access [ 122 ]. For instance, value-based pricing models that consider the long-term benefits and cost savings associated with successful treatment outcomes could be explored [ 123 ].

Looking to the future, CAR-NK cell therapy is poised for significant advancements. Ongoing research is focused on enhancing the persistence and efficacy of CAR-NK cells, identifying optimal target antigens, and integrating CAR-NK therapy with other treatments to improve outcomes. Additionally, the development of “off-the-shelf” CAR-NK cell products derived from sources such as umbilical cord blood or induced pluripotent stem cells (iPSCs) offers the potential for scalable production and reduced costs [ 124 ]. These advancements, combined with regulatory support and innovative financial strategies, will be key to realizing the full potential of CAR-NK cell therapy in cancer treatment [ 125 ].

Mechanism of CAR-Ms

The remarkable success of CAR-T cell therapy in malignant hematological diseases, coupled with the promising potential of CAR-NK cells, has piqued research interest in the development of CAR-macrophages (CAR-Ms) for tumor immunotherapy [ 109 ]. While CAR-T cell therapy has demonstrated promising results in hematological malignancies, its efficacy in solid tumors remains a significant challenge [ 126 – 128 ]. Leveraging the unique effector functions of macrophages and their innate ability to infiltrate tumor tissues, researchers have genetically engineered CAR-Ms. These engineered cells have exhibited antigen-specific phagocytosis and tumor clearance capabilities in vitro studies [ 127 ] (Fig. 3 ).

In vitro experimental characterizations have revealed the multifaceted activities of CAR-Ms. These engineered cells have been shown to express pro-inflammatory cytokines and chemokines, facilitate the phenotypic conversion of bystander M2 macrophages to the M1 phenotype, upregulate antigen presentation mechanisms, and recruit T cells by presenting antigens to them. Additionally, CAR-Ms have demonstrated resistance to the suppressive effects of immunosuppressive cytokines. In humanized mouse models, CAR-Ms have been further observed to induce a pro-inflammatory TME and to enhance the activity of anti-tumor T cells [ 129 , 130 ].

Advantages of CAR-Ms compared with CAR-T cell

Compared to CAR-T cells, CAR-Ms possess three notable advantages. Firstly, while T cells are impeded by the physical barriers established by the tumor cell-matrix from infiltrating the TME, macrophages can effectively penetrate this environment. Tumor-associated macrophages (TAMs) are pivotal in tumor invasion, metastasis, immune suppression, and angiogenesis [ 128 ]. CAR-Ms can reduce the proportion of TAMs, modulate their cellular phenotype, and thus positively influence tumor treatment. CAR-Ms exhibit the dual capability of phagocytosing tumor cells and enhancing antigen presentation, which in turn boosts T-cell cytotoxicity [ 131 ]. Additionally, when contrasted with CAR-T cells, CAR-Ms exhibit a shorter circulation time and a reduced risk of non-tumor-targeted toxicity [ 132 ].

Despite the significant potential demonstrated by CAR-M therapy in cancer treatment, several challenges must be addressed to facilitate its clinical application. Future research endeavors should focus on the continued optimization of CAR-M design to augment their anti-tumor efficacy and minimize potential side effects. Additionally, the development of more efficient gene delivery systems and the exploration of combination therapy strategies are crucial for advancing the broad application of CAR-Ms therapy in clinical practice.

Bispecific antibody therapy

Mechanism of BsAbs

The therapeutic landscape for CLL has undergone a profound transformation in the last two decades, largely attributable to the advent of novel immunotherapeutic agents, such as CD20 mAbs [ 19 ]. The ongoing evolution of immunotherapy has subsequently introduced CAR therapies, which have had a significant impact on CLL treatment strategies [ 64 , 65 ]. Furthermore, the emergence of BsAbs has demonstrated remarkable efficacy and is currently regarded as one of the most promising immunotherapeutic approaches for lymphoma [ 7 , 133 , 134 ].

BsAbs, also referred to as bifunctional antibodies, possess the unique capability to recognize and bind simultaneously to two distinct antigens or epitopes, thereby blocking multiple signaling pathways and inhibiting the growth and survival of CLL cells [ 135 , 136 ]. Moreover, BsAbs can engage one arm with specific antigens on the surface of CLL cells and the other arm with antigens on immune effector cells, facilitating direct contact between immune cells and CLL cells, leading to the lysis of the latter. Emerging BsAbs, exemplified by bispecific T cell involvement (BiTE), recruit T cells to tumor cells and promote the development of tumor immunotherapy [ 134 ]. For instance, CD3/CD19 BsAbs activate the immune-mediated killing of CLL cells by binding to CD19 on CLL cells and CD3 receptors on T cells [ 137 ]. Additionally, BsAbs can counteract the immunosuppressive effects of CLL by binding to immune checkpoint molecules, such as the PD-1/PD-L1 axis, thereby restoring the cytotoxic potential of T cells [ 138 ]. By elucidating the dual-targeting capabilities of BsAbs and their ability to redirect immune cells to cancer cells, we lay the groundwork for understanding their diverse clinical applications. The following section will delve into the clinical classification and therapeutic potential of these innovative agents.

Types and characteristics of BsAbs in CLL therapy

With a solid understanding of the mechanisms that drive BsAb functionality, we now explore their clinical classification. Here, we examine how these mechanisms translate into distinct therapeutic categories, highlighting their potential to revolutionize cancer treatment. The FDA-approved CD3/CD19 BsAb, currently utilized for the treatment of ALL, has demonstrated potential therapeutic value against CLL. Its mechanism of action involves simultaneous binding to CD19-positive CLL cells and CD3-positive T cells, thereby harnessing the cytotoxic potential of T cells against CLL cells [ 139 ]. The CD3/CD20 BsAb engages CD20 on the surface of B cells and CD3 on the surface of T cells, enhancing T cell recognition and cytotoxicity against CLL cells. These antibodies, currently in trial phases, have exhibited exceptionally high clearance capabilities [ 140 ]. CD3/CD33 BsAb, in which one arm targets the CD3 molecule on the surface of T cells and the other arm targets the CD33 antigen on the surface of cancer cells, in this way, the BsAb close the distance between T cells and cancer cells, directing T cells to kill cancer cells directly [ 141 ]. The ROR1/CD3 BsAb capitalizes on the high expression of ROR1, a transmembrane protein, in CLL cells. By binding to ROR1 and CD3, this BsAb bridges T cells and CLL cells, enhancing T cell cytotoxicity against CLL cells [ 142 ]. (Table 3 )

Efficacy of BsAbs

The CD3-CD20 BsAb, a full-length, humanized IgG1 immunoglobulin molecule, is among the most widely utilized in the field. Its prototypical drug, Eporitamab (GEN3013), is known for its ability to recruit T-cell effector functions and has demonstrated robust clinical activity in patients with recurrent CD20 + B-cell NHL [ 143 ]. For example, a phase I/II clinical trial involving 45 patients with R/R B-cell NHL demonstrated that Eporitamab significantly induced T cell expansion, activation, and differentiation into Th1 and effector memory cells across all patient samples. The patient population had a median age of 65 years, with 60% being male and having received at least two prior lines of therapy. The study showed an ORR of 70% and a CR rate of 30%, with a median PFS of 12 months [ 144 ]. BsAb targeting CD20/CD3 T cells are capable of lysing CD20-expressing B cells, including primary leukemia and lymphoma cells, in both in vitro and in vivo settings [ 145 ]. Recent research, including clinical trials, has indicated that certain CD20 × CD3 BsAb exhibit significant anti-B-cell NHL activity by directing T cells to target CD20 + NHL cells [ 143 ]. Additionally, experimental data has demonstrated that these antibodies efficiently eliminate cancer cells in a dose-dependent manner in vitro within B lymphoblastic cells expressing CD20. These findings suggest that such antibodies hold potential clinical application value in the treatment of CD20-expressing B-cell malignancies [ 146 ].

BsAbs combination therapy

The primary therapeutic strategy involves the combination of BsAbs with BTK inhibitors, such as ibrutinib. Ibrutinib as a monotherapy has demonstrated significant curative effects, and its combination with BsAbs has been shown to enhance these effects. For instance, a multicenter, open-label clinical trial involving 784 patients with R/R CLL demonstrated an ORR of 72.11% (95% CI: 66–77%; p < 0.0001) and a CR rate of 49.19% (95% CI: 39–59%; p > 0.05) when ibrutinib was combined with BsAbs. The patient population had a median age of 61 years, with nearly equal sex distribution. The study evaluated the efficacy of ibrutinib both as monotherapy and in combination with other agents [ 48 ]. The secondary option includes the combination with ICIs, such as PD-1/PD-L1 inhibitors. These inhibitors have shown considerable efficacy in overcoming the immunosuppression associated with CLL, and their combination with BsAbs may further augment treatment efficacy. A meta-analysis of 17 studies involving 514 patients with R/R diffuse large B-cell lymphoma (DLBCL) showed that ibrutinib-based combination therapies achieved a combined CR rate of 26% (95% CI: 0.19–0.34) and an ORR of 49% (95% CI: 0.40–0.58). The median PFS was 5.57 months (95% CI: 4.61–6.52) [ 147 ]. The tertiary option is the sequential use of CAR-T cell therapy and BsAbs. CAR-T cell therapy has demonstrated significant efficacy in CLL treatment, particularly in patients with relapsed or refractory disease. However, resistance to CAR-T therapy can emerge due to mechanisms such as antigen loss or immune evasion. In such cases, BsAbs targeting alternative antigens can be employed as a subsequent treatment strategy. This sequential approach leverages the initial efficacy of CAR-T cells while providing an additional therapeutic option when resistance develops. For example, in patients who experience relapse after CAR-T therapy due to CD19 loss, BsAbs targeting CD20 or other relevant antigens can be used to achieve sustained remission [ 148 ]. This strategy ensures that each therapy is utilized to its full potential, optimizing patient outcomes.

Side effects and treatment of BsAbs

BsAb has demonstrated significant therapeutic benefits in the management of CLL, yet it is not without its adverse effects. The primary side effects associated with BsAb are CRS and ICANS. The incidence of CRS in BsAb therapy ranges from 20 to 50%, with the majority of cases being mild (grade 1–2) and only a minimal fraction being severe (grade 3–4) [ 149 ]. For example, a real-world study of patients treated with BsAbs reported that CRS occurred in 20–50% of cases, with grade 3–4 CRS affecting less than 10% of patients. ICANS, often occurring concurrently with or following CRS, was reported in approximately 10–20% of patients, with severe cases (grade 3–4) being rare [ 150 ]. As a precautionary measure against CRS, the prophylactic administration of corticosteroids is often advised, a strategy previously alluded to. The clinical presentation and preventative strategies for ICANS are analogous to those detailed earlier and thus will not be reiterated here. It is important to note that long-term adverse effects of BsAb therapy may include prolonged cytopenias and an increased risk of infections, which require vigilant monitoring and management [ 150 ].

The advantages of BsAbs compared to other immunotherapies

BsAbs offer several therapeutic advantages over traditional monoclonal antibodies, particularly in the context of targeting multiple pathways involved in tumor progression. Initially, BsAbs targeting two or more pathways can potentially reduce drug resistance and the risk of tumor progression, as compared to monotherapy that targets a single pathway [ 151 ]. Subsidiarily, BsAbs can recruit immune cells to the TME and counteract the effects of immunosuppressive factors, thereby restoring anti-tumor immune activity [ 152 ]. Additionally, BsAbs can bridge T cells directly to tumor cells, activating T-cell-mediated cytotoxicity [ 153 ]. Compared to traditional monoclonal antibodies, BsAbs have a broader therapeutic window, enabling the achievement of therapeutic effects at lower doses and reducing side effects [ 154 ]. Furthermore, by simultaneously blocking two signaling pathways or linking two types of cells, BsAbs can enhance therapeutic efficacy while minimizing systemic side effects, striking a balance between efficacy and safety [ 153 ]. Lastly, BsAbs’ ability to bind two different antigens or two epitopes on the same antigen provides more precise targeting capabilities, which can enhance the therapeutic effect and reduce off-target damage to normal cells [ 155 ].

In conclusion, BsAbs hold significant potential in the therapeutic landscape of CLL. Nevertheless, the journey towards continuous program optimization, side effect reduction, and the development of novel drugs is an extensive one. The production of BsAbs is highly complex due to their unique structure and the need for specialized processes to ensure high yield and quality. However, innovative production technologies, such as advanced protein engineering and purification techniques, are being developed to optimize BsAbs' therapeutic potential and reduce production costs. These innovations not only enhance the efficiency of BsAbs manufacturing but also improve their pharmacokinetics and efficacy. Collaboration with regulatory agencies is crucial in this process. For example, the China-UK drug regulatory cooperation project has identified areas of mutual interest to push forward technical exchanges and deepen cooperation. This collaboration can facilitate the approval process and ensure that BsAbs meet regulatory standards, thereby enhancing their accessibility. Innovative pricing and reimbursement strategies are also essential to reduce the economic burden on patients. By working with regulatory bodies and adopting new production technologies, the biopharmaceutical industry can make BsAbs more affordable and accessible, while also exploring new therapeutic possibilities. Future directions may include further optimization of BsAbs' half-life and efficacy, as well as the development of new formats and structures to target a wider range of diseases. Despite these challenges, with persistent research efforts, BsAbs are anticipated to become a vital component of CLL treatment, offering improved outcomes for patients.

The next generation therapy of BsAbs - TsAbs

The evolution of BsAbs has led to the conception of tri-specific antibodies (TsAbs), which incorporate a co-stimulatory protein or an additional targeted antigen to amplify the efficacy of the antibody response. While TsAbs represent a step forward in antibody engineering, they are not without their challenges and require substantial future development. TsAbs are designed to target three distinct antigen epitopes on tumor cells and immune effector cells, thereby enhancing therapeutic efficacy and safety [ 156 ]. This design allows for the simultaneous engagement of multiple immune cells, improving the specificity and potency of the immune response against cancer cells. In preclinical studies, TsAbs have demonstrated remarkable efficiency in overcoming immune escape and significantly enhancing antitumor efficacy [ 157 ]. For instance, a CD19/CD22/CD3 TsAb has shown superior performance in inducing T-cell-specific cytotoxicity and cytokine production against B-cell malignancies, compared to other formats [ 158 ]. This innovative approach holds great promise for the future of cancer immunotherapy.

Despite the enormous potential of TsAbs in cancer immunotherapy, its clinical application faces significant regulatory challenges, particularly in balancing safety and efficacy. TsAbs can simultaneously target three distinct antigens, thereby potentially enhancing antitumor activity. However, this complexity also increases the risk of adverse events such as CRS, a severe inflammatory response triggered by the overactivation of immune cells. Ensuring safety requires rigorous preclinical testing and phased clinical trials to monitor and mitigate such risks. Additionally, the intricate structure of TsAbs may lead to immunogenicity, causing the production of anti-drug antibodies (ADAs) that could neutralize the therapeutic effects. Regulatory bodies must therefore establish stringent guidelines to ensure the thorough evaluation of these novel agents, balancing their therapeutic potential with the need to safeguard patients from potential harm.

Antibody drug conjugate therapy

Mechanism of ADCs

ADCs represent a significant class of oncology therapeutics, comprising a mAb conjugated to a cytotoxic drug, also known as a payload, through a linker molecule [ 159 ]. These conjugates are designed to harness the high specificity targeting ability of mAbs and the potent cytotoxic effects of small-molecule drugs, thereby achieving precise and efficacious elimination of cancer cells [ 160 , 161 ]. ADCs are typically structured with mAbs covalently linked to cytotoxic agents via chemical linkers [ 162 ]. They leverage the antigen-recognition capabilities of antibodies to selectively deliver cytotoxic drugs to tumor cells, with the linker ensuring the stability and release of the payload at the tumor site [ 163 ]. Beyond direct cytotoxicity, the mechanisms of action of ADCs encompass the induction of anti-tumor immune responses and the inhibition of downstream signal transduction pathways associated with antigen receptors [ 164 ].

Application of ADCs in leukemia

Brentuximab Vedotin (BV) marks the first ADC approved for the treatment of lymphomas, specifically targeting CD30 [ 165 , 166 ], an antigen robustly expressed in hematologic tumors, particularly HL [ 167 ]. BV binds to CD30 on the tumor surface and facilitates the internal release of toxins through lysosomal degradation, thereby inducing tumor cell death [ 168 ]. Polatuzumab Vedotin (PV) is another ADC that targets CD79b on B-cell tumor surfaces, sharing a similar mechanism of action to BV [ 169 , 170 ]. Loncastuximab Tesirine is a CD19-targeting ADC, derived from a humanized CD19 monoclonal antibody linked to a dimer toxin via a linker [ 171 , 172 ]. It is primarily indicated for the treatment of R/R DLBCLthat has been previously treated with two or more systemic therapies [ 173 , 174 ]. Gemtuzumab Ozogamicin, a CD33-targeting ADC initially approved in 2000 and re-approved after a market withdrawal, is primarily utilized for the treatment of AML [ 175 , 176 ]. It has demonstrated significant improvements in patient survival and exhibits fewer adverse reactions compared to other therapies [ 177 ]. Inotuzumab Ozogamicin, an ADC targeting CD22 [ 178 ], is predominantly used for the treatment of lymphatic leukemia, especially R/R ALL [ 179 ]. Moxetumomab Pasudotox, which has been recently marketed, is developed for the treatment of R/R Hairy Cell Leukemia (HCL) which has failed to respond to more than two prior treatments, with CD22 as its primary target [ 180 ]. Studies have indicated that its treatment of HCL is more stable and less toxic compared to other drugs [ 181 ]. Lastly, Belantamab Mafodotin, an ADC drug representative for myeloma, is used for the treatment of adult patients with refractory MM who have received at least four prior lines of treatment, including a CD38 monoclonal antibody, a proteasome inhibitor, and an immunomodulatory agent [ 182 ]. However, it was withdrawn from the market in 2022 due to insufficient survival benefits compared to conventional therapies and the presence of fatal side effects [ 183 ]. (Table 4 )

ADCs combination therapy

The therapeutic potential of ADCs can be augmented through combination regimens with targeted drugs such as BCL-2 inhibitors, which inhibit the anti-apoptotic pathways of CLL cells, thereby potentially enhancing the cytotoxic effects of ADCs. Additionally, the combination of ADCs with ICIs aims to stimulate the immune system, further augmenting the cytotoxic effects on CLL cells. ADCs are also combined with chemotherapeutic drugs to enhance therapeutic efficacy and mitigate drug resistance. For instance, the combination of ADCs with BTK inhibitors targets cell signal transduction pathways, thereby potentially enhancing the cytotoxic effects of ADCs.

ADCs exhibit potential in the treatment of CLL, but several challenges remain to be addressed. Currently, the application of ADCs in CLL is still in the early stages of development, with limited clinical data available. However, some ADCs have shown promising preclinical and early-phase clinical results. For example, PV has demonstrated an ORR of 40% and a median PFS of 6 months in a Phase I/II study involving patients with R/R CLL. Another ADC, Inotuzumab ozogamicin, has also shown potential in CLL, although its use is more commonly associated with ALL. Despite these encouraging results, challenges such as drug resistance, tumor heterogeneity, and adverse reactions need to be addressed. Future research will focus on further optimizing ADC design and exploring novel targets and combination therapy regimens to minimize the incidence of adverse reactions. As an innovative therapeutic approach, ADCs are anticipated to offer promising advancements for CLL patients, in tandem with the ongoing advancements in science and technology.

γδ T cell therapy

Introduction of γδ T cell

In the realm of cell therapy, specifically within CAR-T cell therapy, the majority of T cells utilized are αβ T cells. These cells express TCRs composed of two heterodimeric peptide chains, α and β, which constitute approximately 95% of the T cell population [ 184 ]. Additionally, a minor subset of T cells is composed of γ and δ chains, termed γδ T cells, which represent about 0.5–5% of the total T cell population. γδ T cells possess both innate and adaptive immune response characteristics, enabling them to rapidly recognize non-MHC-restricted tumor antigens and mount an immune response accordingly [ 185 , 186 ].

Mechanism of γδ T cell therapy

Gentles et al. conducted a comprehensive analysis of gene expression profiles from approximately 18,000 samples across 39 different types of malignant tumors and identified a significant correlation between the presence of tumor-infiltrating γδ T cells and favorable prognosis [ 187 ]. Currently, the potential of γδ T cells in CAR-based immunotherapies for cancer has been substantiated. Beyond the CAR-mediated cytotoxicity, γδ T cells can recognize tumor cells through their intrinsic receptors, including Vδ1 TCR, CD16, NKG2D, and NKp30, and activate various natural tumor-killing pathways. These pathways encompass perforin/granzyme-dependent cytotoxicity, CD16-mediated ADCC, and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)/ Fas Ligand (FASL)-induced apoptosis [ 188 ]. Furthermore, γδ T cells can function as antigen-presenting cells (APCs), presenting tumor antigens to αβ T cells. Studies have demonstrated that CAR-γδ T cells, engineered to target GD2, not only specifically eliminate GD2-positive LAN1 cells but also maintain their capacity to phagocytose and present tumor antigens to αβ T cells, thereby inducing the clonal expansion of αβ T cells [ 189 ].γδ T cells exhibit a superior homing capability, which allows them to more effectively infiltrate tumor tissues, particularly those characterized by a hypoxic TME. Their efficacy within a hypoxic TME is further enhanced because their cytotoxic properties and the secretion of key cytokines and chemokines, such as macrophage inflammatory protein MIP-1, tumor necrosis factor RANTES, and tumor necrosis factor-related activating protein CD40L, can be augmented under hypoxic conditions. Studies have demonstrated that engineering γδT cells with CAR can amplify their cytotoxic potential while preserving their migratory capacity to tumor cells and their ability to cross-present antigens. This modification represents a promising strategy to bolster the anti-tumor immune response [ 189 ].

γδ T cell combination therapy

Combination therapy strategies are pivotal in augmenting the efficacy of γδ T cell therapy. One such strategy involves the integration with chemotherapy, which can disrupt the TME, thereby potentially enhancing the infiltration and activity of γδ T cells. Evidence suggests that the combination of γδ T cell therapy with chemotherapy can lead to improved therapeutic outcomes. Another approach is the combination with radiotherapy, which can stimulate the immune response against the tumor and enhance the anti-tumor activity of γδ T cells. The synergy between γδ T cells and ICI therapy can alleviate tumor-mediated immunosuppression, producing a cooperative effect that enhances therapeutic efficacy. When combined with BsAbs, γδ T cells can be directed more effectively to the tumor site, augmenting their cytotoxic capabilities. Lastly, the combination of CAR-γδ T cells with conventional γδ T cells may offer additional therapeutic benefits [ 190 ].

Future research direction of γδ T cell combination therapy

Initially, it is crucial to explore the optimal combination of γδ T cell therapy with other treatment modalities to maximize therapeutic efficacy. Subsequently, there is a need for a greater number of large-scale clinical trials to verify the efficacy and safety of γδ T cell therapy in the context of CLL treatment. Additionally, the development of a novel evaluation system is essential for more precisely identifying patients who are suitable candidates for γδ T cell therapy and for monitoring their progress in real time [ 190 ]. Ultimately, further research should focus on elucidating the mechanisms of action of γδ T cells within the TME and investigating how their anti-tumor activity can be enhanced through technical interventions.

γδ T cell therapy introduces a novel therapeutic strategy for the treatment of CLL. Although the current state of the technology is not fully mature, preliminary results suggest that this therapy may emerge as a significant treatment option for CLL. Future research will necessitate multi-level studies to fully harness the potential of γδ T cells and to optimize their role in CLL treatment.

Biological response modifier activated killer therapy

Mechanism of BAK therapy

Biological Response Modifier (BRM)-Activated killer therapy, abbreviated as BAK therapy [ 191 ], is a form of immunotherapy that involves the extraction of immune cells from a patient’s body, their subsequent in vitro cultivation, proliferation, and activation, followed by their reintroduction into the patient via intravenous infusion [ 192 ]. This therapy aims to enhance the accuracy of cancer cell identification by the electrified immune cells, thereby minimizing the attack on both normal cells and cancer cells, leading to a characteristic minimal side effects profile [ 193 ]. BAK therapy predominantly utilizes non-MHC restricted lymphocytes, specifically CD56 + cells, which include gamma delta T cells and NK cells.

Efficacy of BAK therapy

Researchers have successfully employed BAK therapy in the treatment of abdominal tumors, demonstrating its efficacy by showing that locally administered BAK lymphocytes can penetrate cancerous tissue and effectively eliminate cancer cells [ 194 ]. This finding is supported by evidence indicating that BAK therapy, utilizing CD56-positive lymphocytes, is effective against advanced solid cancers. Hospital-based researchers have further expanded the application of this therapy to liver cancer by administering BAK cells intravenously, thereby establishing a foundation for its use in hepatic malignancy treatment [ 195 ].

BAK therapy is advantageous in that it can be conducted on an outpatient basis, involving only blood collection and infusion, which eliminates the need for hospitalization. This therapy is also notable for its compatibility with other treatment modalities, such as radiation therapy, and has been increasingly integrated into clinical practice [ 196 ]. Primarily aimed at treating solid tumors, BAK therapy is also recognized for its potential to prevent cancer recurrence, offering anticipated high preventive efficacy.

However, at present, BAK therapy is not yet considered a standard treatment for leukemia and other hematological malignancies, despite previous research proposals suggesting its potential utility [ 197 ]. Due to its reliance on autologous immunity, BAK therapy has been recognized as a promising "fourth cancer treatment modality" and has garnered significant attention in recent years. Characterized by a favorable safety profile with minimal side effects, BAK therapy is a subject of high anticipation for future research and development.

Conclusion

In recent years, advancements in science and technology have ushered in fundamental changes in the therapeutic landscape for CLL. The transition from chemotherapeutic immunotherapy of the last century to the current first-line targeted therapies and the rapidly evolving field of cell therapy has significantly altered patient prognoses. Currently, CD20 mAbs and CAR cell therapy are widely employed in the clinical treatment of CLL. Other immunotherapeutic approaches, such as BsAbs, ADCs, and γδ T cell therapy, show great promise; however, the challenges in their development should not be overlooked. For CLL patients, the development of the most rational personalized treatment plans, based on genetic profiling and risk assessment, is essential. Additionally, exploring fresh targeted drugs in combination with immunotherapies to enhance therapeutic outcomes is a critical direction for research. While novel treatments offer improved response rates and survival, the majority of CLL patients eventually experience relapse. Consequently, scientists have proposed the use of Allo-HSCT as a potential emerging avenue for CLL treatment, which may present advanced opportunities for therapeutic breakthroughs.

Abbreviations

ANCA-Associatedvasculitis

Adoptive Cell Therapy

Anti-Drug Antibodies

Antibody-Dependent Cellular Cytotoxicity

Antibody-Drug Conjugates

Anaplastic Large Cell Lymphoma

Acute Lymphoblastic Leukemia

Allogeneic Hematopoietic Stem Cell Transplantation

Acute Myeloid Leukemia

Antineutrophil Cytoplasmic Antibodies

Antigen-Presenting Cells

BRM Activated Killer

B-Cell Lymphoma

Bispecific T Cell Engager

Biological Response Modifier

Bispecific Antibody

Bispecific Antibodies

Bruton’s Tyrosine Kinase

Brentuximab Vedotin

Chimeric Antigen Receptor

Car-Macrophages

Complement-Dependent Cytotoxicity

Classical Hodgkin Lymphoma

Chemoimmunotherapy

Chronic Lymphocytic Leukemia

Complete Response

Cytokine Release Syndrome

Dual-Affinity Retargeting Antibody

Diffuse Large B-cell Lymphoma

Fas Ligand

Fragment Crystallizable

Fludarabine, Cyclophosphamide, Rituximab

Follicular Lymphoma

Graft-Versus-Host Disease

Hairy Cell Leukemia

Hodgkin’s Lymphoma

Immune Effector Cell-Associated Neurotoxicity Syndrome

Immune Checkpoint Inhibitors

Interferon-γ

Interleukin-2

Interleukin-6

induced Pluripotent Stem Cells

immune-Related Adverse Events

Immune Thrombocytopenia

Large B-cell Lymphoma

monoclonal Antibody

monoclonal Antibodies

Membrane attack Complex

Monovalent Asymmetric Tandem Fab

Mantle Cell Lymphoma

Myelodysplastic Syndromes

Myeloid-Derived Suppressor Cells

Myeloid-Derived Suppressor Cells

Mycosis Fungoides

Major Histocompatibility Complex

Multiple Myeloma

Monomethyl Auristatin E

Minimal Residual Disease

Multiple Sclerosis

Non-Hodgkin Lymphoma

Natural Killer

Objective Response Rate

Overall Survival

Pyrrolobenzodiazepine Dimer

Primary Cutaneous Anaplastic Large Cell Lymphoma

Programmed Death-1

Programmed Death-Ligand 1

Progression-Free Survival

Partial Response Rate

Peripheral T-cell Lymphomas

Polatuzumab Vedotin

Quality of Life

Relapsed/Refractory

Rheumatoid Arthritis

Relapsed Multiple Sclerosis

Richter’s Transformation

Systematic Anaplastic Large Cell Lymphoma

Systemic Lupus Erythematosus

Small Lymphocytic Lymphoma

Tumor-Associated Macrophages

Tumor-Associated Macrophages

Tandem Diabody

T cell Receptors

Tumor Lysis Syndrome

Tumor Microenvironment

Tumor Necrosis Factor-α

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

Regulatory T Cells

T cells Redirected for Universal Cytokine Killing

Trispecific Antibodies

Venetoclax-Rituximab

Author contributions

Pan Gao wrote and edited the initial manuscript. Yang Zhang and Jun Ma revised the manuscript. Ya Zhang reviewed and revised the manuscript. All authors have read and approved the final manuscript.

Funding

This study was supported by the National Natural Science Foundation (No. 82000195); Shandong Provincial Natural Science Foundation (No. ZR2024MH050, ZR2020QH094); China Postdoctoral Science Foundation (No. 2022M721981); Taishan Scholars Program of Shandong Province (No. tsqn201909184); Shandong Provincial Health Construction Program of Medical Science and Technology Innovation Team; Academic Promotion Programme of Shandong First Medical University (No.2020RC007); Research Promotion Program of Shandong Provincial Hospital.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

Data Availability Statement

No datasets were generated or analysed during the current study.