Factors Affecting the Cancer Immunotherapeutic Efficacy of T Cell Bispecific Antibodies and Strategies for Improvement

ABSTRACT

T-cell bispecific antibodies (T-BsAbs) are a new class of cancer immunotherapy drugs that can simultaneously bind to tumor-associated antigens on target cells and to the CD3 subunit of the T-cell receptor (TCR) on T cells. In the last decade, numerous T-BsAbs have been developed for the treatment of both hematological malignancies and solid tumors. Among them, blinatumomab has been successfully used to treat CD19 positive malignancies and has been approved by the FDA as standard care for acute lymphoblastic leukemia (ALL). However, in many clinical scenarios, the efficacy of T-BsAbs remains unsatisfactory. To further improve T-BsAb therapy, it will be crucial to better understand the factors affecting treatment efficacy and the nature of the T-BsAb-induced immune response. Herein, we first review the studies on the potential mechanisms by which T-BsAbs activate T-cells and how they elicit efficient target killing despite suboptimal costimulatory support. We focus on analyzing reports from clinical trials and preclinical studies, and summarize the factors that have been identified to impact the efficacy of T-BsAbs. Lastly, we review current and propose new approaches to improve the clinical efficacy of T-BsAbs.

KEYWORDS:

• Cancer immunotherapy
• bispecific antibody
• T cell activation

Current status of T cell engaging bispecific antibodies in cancer immunotherapy

T cell engaging bispecific antibodies (T-BsAb) have become a promising immunotherapy strategy for treating cancer. T-BsAbs are off-the-shelf products that appear to work even in patients with advanced lymphoma that progresses after Chimeric antigen receptor T-cell therapy. Typical T-BsAbs contain two binding sites: a CD3-binding site binding to T cells and another site recognizing the target antigen expressed on tumor cells (Frankel and Baeuerle 2013). Many different structures and forms of T-BsAbs have been developed, which can be roughly categorized into IgG-like versus non-IgG like biochemically (Ma et al. 2021). The non-IgG likely BsAbs lack the Fc segment and are usually smaller in size. One classical non-IgG like platform is BiTE (bispecific T cell engager), represented by blinatumomab. It contains two scFvs (one for CD3 and one for CD19) linked through a glycine/serine (GGGGS) linker. In contrast, a classical IgG-like type of BsAb, such as catumaxomab, combines two Fab segments with an intact Fc region. Hence, in addition to binding CD3 and EpCAM through the heterodimerized Fab region, the intact Fc region provides a third binding site to recruit and activate immune effector cells. All T-BsAbs redirect T cells to target tumor cells via their CD3 and the target-antigen binding sites, serving as a bridge. Moreover, CD3 is part of TCR complex, and cross-linking of CD3 by T-BsAbs triggers TCR signaling in the presence of the target antigen. Numerous in vitro experiments have demonstrated that T-BsAbs induce activation of T cells, leading to immune synapse formation and ultimately the killing of tumor cells in a “target-antigen-specific” manner (Goebeler and Bargou 2020; Wang, et al. 2019; Wu and Cheung 2018).

Catumaxomab is an anti-EpCAM x anti-CD3 bispecific antibody that consists of full-length mouse IgG2a and rat IgG2b that allows it to simultaneously target CD3 and EpCAM. Catumaxomab engages EpCAM positive tumor cells and T cells, inducing T cell-mediated tumor cell killing. Moreover, the fully functional Fc region of Catumaxomab binds to Fc receptors (FcRs) on macrophages and NK cells, inducing antibody (Ab)-dependent cell cytotoxicity (ADCC) or Ab-dependent cell phagocytosis (ADCP) as alternative mechanisms to kill tumor cells. It was approved in the European Union in 2009 for the intraperitoneal treatment of malignant ascites in patients with EpCAM-positive carcinomas, but was withdrawn from the market in 2017 for commercial considerations (Eyvazi et al. 2018; Linke et al. 2010).

Blinatumomab, that targets CD19 and CD3, has been successfully used in patients with acute lymphoblastic leukemia (ALL), leading to its approval by the FDA for relapsed/refractory ALL, for which the rate for complete remission (CR) is approximately 40 to 50%. Blinatumomab has also demonstrated impressive single-agent efficacy in other CD19 expressing tumors. For relapsed/refractory lymphoma patients, at the target dose of 60 μg/m²/day, the CR rate was 37% and the overall response rate (ORR) was 69%. Impressively, some patients demonstrated durable remissions without further treatment (Goebeler et al. 2016; Smits and Sentman 2016). A subsequent phase II trial confirmed efficacy for blinatumomab in heavily pretreated relapsed/refractory diffuse large B cell lymphoma (DLBCL) with ORR of 43% and CR of 19% after once cycle of therapy (Viardot et al. 2016). Blinatumomab demonstrated much more impressive efficacy in eliminating residual diseases in MRD (minimal residual disease) positive patients. In a phase 2 study of patients who had persistent MRD or MRD relapse after intensive chemotherapy, blinatumomab cleared MRD in 16 out of 20 patients (Topp et al. 2012). The preliminary results of a large phase II study (BLAST) of 116 MRD+ patients also demonstrated a MRD clearance rate of 80%, with most responses (78%) occurred during the first cycle of treatment (Goekbuget et al. 2014; Gökbuget et al. 2017). Therefore, blinatumomab is highly effective when used to treat the persistent MRD. However, in the setting of high disease burdens (e.g. r/r ALL and lymphoma), the therapeutic efficacy of blinatumomab seems to be subpar comparing to CD19 chimeric antigen receptor (CAR) T cells (Abramson et al. 2016; Brentjens et al. 2013; Gardner et al. 2016; Grupp et al. 2015; Lee et al. 2015; Lee et al. 2016; Locke et al. 2017; Neelapu et al. 2017; Schuster et al. 2015; Turtle et al. 2016a; Turtle et al. 2016b). While increasing dosages usually improves therapeutic efficacy, severe neurotoxicity and cytokine release syndrome are common dose limiting toxicities, and hence limits their usage in elderly and frail patients (Jacobs et al. 2017; Jacobs et al. 2018; Westervelt et al. 2018).

Tebentafusp (formerly IMCgp100) is another bispecific T cell engager that has been approved by FDA for treatment of unresectable or metastatic uveal melanoma in HLA-A2 positive patients. It belongs a new class of T-cell – redirecting bispecific fusion proteins termed immune-mobilizing monoclonal T-cell receptors against cancer (ImmTAC). Like typical T-BsAbs, Tebentafusp has an anti-CD3 single-chain variable fragment for binding to T cells. However, the target binding arm is an affinity-enhanced T-cell receptor (TCR) that recognizes the glycoprotein 100 (gp100) YLEPGPVTA epitope presented by HLA-A *02:01, as an MHC-peptide complex, on target cells (Middleton et al. 2020). In a clinical trial of 378 advanced uveal melanoma patients, the group treated with tebentafusp demonstrated superior overall survival (OS) at 1 year comparing to the standard care group (73% versus 59%). The progression free survival (PFS) was also better with tebentafusp (31% at 6 months) than standard care (19%), and also showed a good safety profile, with low incidence of high-grade CRS (1% for Grade >3 CRS) and other severe toxicities (Nathan et al. 2021).

There are numerous T-BsAbs be tested in clinical trials for both hematological malignancies and solid tumors, and many have demonstrated promising efficacy. Multiple research groups and pharmaceuticals have developed T-BsAb against CD20. These CD20x CD3 BsAbs have demonstrated impressive efficacy on B cell Non-Hodgkin Lymphomas (NHL) (Bröske et al. 2022; Brouwer-Visser et al. 2021; Budde et al. 2022; Hutchings et al. 2021; Patel et al. 2021). Moreover, none of these CD20 BsAbs cause severe neurotoxicity, which is the dose limiting toxicity of CD19 targeting BsAb such as blinatumomab. Multiple myeloma is another disease that has surface expression of several tumor-associated antigens that can be targeted. T-BsAbs against BCMA (Cho et al. 2020; Lesokhin et al. 2022; Minnema et al. 2022; Topp et al. 2020; Usmani et al. 2021; Zonder et al. 2021), GPRC5D (Minnema et al. 2022) and FcRH5 (Nakamura et al. 2020) have all demonstrated promising clinical activity in myeloma patients. For acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), there are ongoing clinical trials with T-BsAb targeting CD33 and CD123, with clinical response seen in selected patient populations (Agarwal et al. 2020; Ravandi et al. 2020a; Ravandi et al. 2020b; Uckun et al. 2021; Uy et al. 2021; Vadakekolathu et al. 2020b; Westervelt et al. 2019). For solid tumors, T-BsAb targeting tumor associated-antigens such as EpCAM (Kebenko et al. 2018), CEA (Moek et al. 2018; Tabernero et al. 2017), DLL3 (Owonikoko et al. 2021), HER2 (Haense et al. 2016; Lum et al. 2021), and EGFR (Lum et al. 2020) are undergoing clinical trials. On-target off-tumor toxicities have been found to be dose limiting in some trials (e.g., the GI toxicity with EpCAM and CEA BsAb). T-BsAbs targeting PMSA in prostate cancer seem to be the most promising and have demonstrated meaningful clinical efficacy with acceptable safety profiles (Bono et al. 2021; Deegen et al. 2021; Hummel et al. 2020; Subudhi et al. 2021). There are also more immune-mobilizing monoclonal T-cell receptors (ImmTAC) under development (He et al. 2019; Zhao et al. 2021a), targeting intracellular tumor associated antigens such as WT1 (Augsberger et al. 2021), NY-ESO-1 (Lopez et al. 2019) and MAGE-A4 (Davar et al. 2021). Some of these have already entered early phase clinical trials and are showing promising efficacy.

Efficient target killing by T cells with suboptimal signal 2: lessons from blinatumomab

According to the classic two-signal theory of T cell activation, productive T-cell activation in vivo requires two signals: signal 1 is from TCR stimulation, which is the main signal that determines the antigen specificity of the T-cell response, and signal 2 from costimulatory molecules, which modify the quality and outcome of the response (Bretscher 1999; Jenkins et al. 2001). T-cell activation when there is a lack of or suboptimal costimulatory signals typically leads to the deletion or functional tolerance of T cells. Nevertheless, typical T-BsAbs crosslink and stimulate T-cell receptors without eliciting costimulatory signals. There are several potential mechanisms, discussed below, by which a T-BsAb can achieve therapeutic efficacy without optimal costimulatory signals.

Intensity of TCR stimulation

Strong TCR stimulation could be sufficient for the activation of naïve CD8+ T-cells with no or suboptimal costimulatory signals. While costimulatory molecules such as CD28 play important roles in CD4 T-cell activation, studies have shown that CD8 T-cells can be activated without costimulatory signals (Goldstein et al. 1998; Wang et al. 2000). Activated with a strong TCR signal alone, naïve CD8 T-cells were able to proliferate, make effector cytokines and express cytolytic functions (Wang et al. 2000). Several in vivo studies have also shown that without CD28 costimulation, T cells can be full activated and demonstrates effector and memory functions (Mittrücker et al. 2001; Shahinian et al. 1993; Suresh et al. 2001b). While a pure TCR signal 1 can be delivered using anti-CD3 antibodies or peptide/MHC tetramers in vitro, it is difficult to stringently avoid costimulatory signals in vivo studies using conventional methods such as genetic knockout and/or antibody blockade. Earlier studies demonstrated that CD28 is dispensable for in vivo CD8 T-cell response in certain circumstances. However, in most scenarios, T-cell activation was found to be suboptimal when costimulatory signals were lacking or suboptimal (Nurieva et al. 2006).

Cytolytic function is more resistant to tolerance induction

Under conditions that eventually lead to T-cell tolerance/anergy, the functional impairment of CD8 T-cells occurs in a hierarchical pattern. In particular, the cytolytic function of effector T cells is retained or less affected than other functions, such as proliferation and cytokine production (Hombach et al. 2001; Mittrücker et al. 2001; Suresh et al. 2001b). This type of “split dysfunctional status” has also been reported with activation-induced nonresponsiveness (AINR), where CD8 T-cells are stimulated with some costimulatory signals and acquire effector functions. However, for a period of time, the T-cells show signs of split anergy, with compromised capability for proliferation and producing IL-2, while cytolytic function remains intact (Deeths et al. 1999; Mescher et al. 2007; Tham et al. 2002). In the case of T-BsAb therapy, with all T-cells of broad antigen specificity being redirected toward target cells, maintaining T-cell cytolytic function is likely crucial for efficacy, whereas proliferation might be less important. Further, diminished cytokine production might actually be desirable to avoid cytokine release syndrome.

Effector/memory T cells

In humans, a sizable proportion of peripheral T-cells have a memory/effector phenotype. Particularly in leukemia/lymphoma patients receiving T-BsAb therapy, the percentage of memory/effector T-cells is even higher since most of these patients have already experienced repeated infections and multiple lines of chemotherapy. A key feature of memory/effector T-cells is that they have a lower threshold for activation and are less dependent on costimulatory signals. Although current evidence predominantly suggests that although for memory/effector T cells costimulatory signals help to boost the recall response and are required for optimal T-cell activation and effector functions, multiple studies have shown that memory T-cells can be activated to express effector functions (including cytolytic function) with no or suboptimal costimulatory signals (Croft et al. 1994; Fuse et al. 2008; London et al. 2000; Suresh et al. 2001a). In the case of T-BsAb therapy, in vitro studies have shown that effector memory T-cells (TEM) constitute the majority of proliferating T cells, while naïve T-cells largely remain unchanged (Wong et al. 2013). In patients receiving blinatumomab, it has also been found that T-cell subset distribution is skewed toward the CD45RA-/CCR7-effector memory phenotype. Additionally, blinatumomab causes B-cell depletion from peripheral blood within hours (Wong et al. 2013). In vitro studies using human T cells (which contain a high frequency of effector/memory T cells) also demonstrated that T-cell-mediated cytotoxicity occurs in hours at a low E:T ratio, while naïve T-cells need a much longer priming period to become functional (Rogala et al. 2015). Thus, these clinical and pre-clinical evidence support that memory T-cell subsets are the main effector cells accounting for most of the therapeutic efficacy by T-BsAb.

Duration of antigen exposure

Another critical factor affecting the outcome of T-cell fate post TCR engagement is the duration of antigen exposure. In vivo TCR stimulation with poor costimulatory signals (e.g., i.v. peptide injection or antigen expressed by autologous cells without inflammatory signals) is usually considered a typical tolerogenic condition where T-cells eventually become dysfunctional/anergic. However, if the duration of such antigen exposure is limited, T-cells can be primed to become functional effector cells, clear the antigen and generate long-term memory (Aichele, et al. 1995; Zinkernagel 2000). On the other hand, prolonged antigen exposure (e.g., with LCMV infection in mice), even with adequate costimulatory signals can lead to T-cell dysfunction/exhaustion (Zajac et al. 1998). With T-BsAb therapy, if the tumor burden is relatively low, antigen-bearing cells could be cleared or significantly reduced within days to weeks. The time of exposure may not be enough to induce tolerance, and most T-cells remain functional and avoid the fate of tolerance. This may also help to explain some clinical findings, such as patients with low tumor burden being more likely to respond to blinatumomab therapy. Additionally, in patients who do respond to the therapy, the response usually occurred within the first cycle of treatment in most cases.

Costimulation provided by target cells

As discussed above, a typical T-BsAb does not engage costimulatory molecules. However, T-BsAb treatment can deliver “signal 2” to T-cells by costimulatory molecules expressed by target cells. To date, T-BsAbs have achieved more success in hematological malignancies of B-cell lineage (ALL, NHL). Both normal and malignant B cells can express costimulatory molecules. Resting B cells are usually considered to be tolerogenic when acting as antigen-presenting cells. However, large-scale T-cell activation by T-BsAbs inevitably causes inflammatory signals, which in turn will activate B cells and upregulate the expression of costimulatory molecules. Normal B cells can express significant amounts of class I and II MHC as well as costimulatory molecules such as CD70 (ligand for CD27), CD86 and CD80 (ligand for CD28) in the resting state. 4-1BBL and OX-40 L are not expressed by resting B cells, but can be induced in activated B cells with inflammatory stimuli that can be invoked upon T-BsAb treatment. It has been reported that B cells, as antigen-presenting cells, can elicit a productive T-cell immune response under the right conditions (Hong et al. 2018). Even in the case of MRD, blinatumomab therapy usually causes significant elevation of inflammatory cytokines and causes cytokine release syndrome in some patients, which likely reflects the significant antigen load coming from normal B cells. Most malignant B cells from leukemia/lymphoma patients also express costimulatory molecules. However, the type and levels of expressed costimulatory molecules are dependent on the type of B-cell malignancy and vary between individual patients. Malignant B cells can also express immunosuppressive molecules such as CD200, PD-L1 and PD-L2.

Design features of blinatumomab that enable the formation of immune synapses that allow efficient target killing

The overall design and structural features of T-BsAbs also affect their capacity to active T-cells. For many years, blinatumomab has been the only FDA-approved T-BsAb. From the 1990s to early 2000s, there were many other T-BsAbs under development. However, most of these early T-BsAbs were much less potent and did not achieve meaningful efficacy in vitro and in vivo without supplementation with costimulatory signals (e.g., CD28 agonists) (Bohlen et al. 1993; Bohlen et al. 1997; Cochlovius et al. 2000a; Cochlovius et al. 2000b; Csóka et al. 1996; Daniel et al. 1998; Demanet et al. 1996; Kipriyanov et al. 2002). Thus, for some time, blinatumomab was the only T-BsAb that demonstrated meaningful efficacy in cancer patients.

Blinatumomab is a small single-chain bispecific antibody constructed based on the “BiTE” platform (Nagorsen et al. 2009). One unique feature is its small size and the level of flexibility between the two binding domains, thus allowing T-cells and target cells to be brought into close proximity. This expedites optimal crosslinking and efficient immune synapse formation, ultimately leading to strong TCR activation without intentionally engaging costimulatory signals (Löffler et al. 2003; Mølhøj, et al. 2007; Rogala et al. 2015). The short distance between T-cells and target cells also allows efficient delivery of perforin and granzyme B into the target cell, hence enhancing target killing efficiency. Another unique feature of blinatumomab is that its affinity for CD3 and CD19 is optimally balanced (Hoffmann et al. 2005). The affinity for the target antigen CD19 is higher, with a Kd of approximately 10^-9 M, which is approximately the average among typical antibodies. The affinity for CD3 on T-cells is lower, with a Kd of approximately 2.6 × 10^-7 M, only slightly higher than the affinity at which a typical TCR binds its MHC-presented cognate antigen. With this “skewed” affinity to target cells and T cells, blinatumomab preferentially distributes to the surface of target cells, to subsequentially enable the membrane bound anti-CD3 moiety to readily crosslink and activate the TCR complex on T cells. Moreover, the affinity to CD3 is at the appropriate range to sustain proper T-target cell interactions, and allow the formation of immune synapses and the delivery of cytotoxicity, while avoiding protracted interactions that delay T-cell scanning for the next target. This unique design of blinatumomab turns T-cells into serial killers capable of killing multiple target cells. This capability is more critical when the effector/target (E:T) ratio is low (which is almost always the case for B-cell antigen targeting T-BsAb, since even in a healthy person without tumor burden, B cells normally outnumber T-cells). Moreover, as mentioned before, prolonged antigen exposure has a tendency to induce functional tolerance in T cells. Thus, this relatively fast off rate (but not too fast to preclude target killing) expedites target clearance while reducing the duration of antigen exposure that might otherwise induce tolerance. Moreover, the low affinity for CD3 also reduces antigen-independent activation of T cells, thus reducing the potential for cytokine release syndrome.

Factors affecting T-BsAb efficacy

Checkpoint molecules and costimulatory molecules

Immune checkpoint blockade has demonstrated impressive clinical efficacy across multiple types of cancer and has been a major breakthrough of immunotherapy in the past decade. Checkpoint molecules, such as PD-1 and CTLA-4, can attenuate T-cell activation and suppress T-cell-mediated antitumor immunity via multiple mechanisms, and their effects on T-BsAb therapy have been intensely investigated.

Laszlo et al. reported that the expression of checkpoint molecules (PD-L1 and L2) by AML cell lines reduces the cytolytic activity of T-cells in the presence of AMG330 (CD33x CD3 BsAb) (Laszlo et al. 2015). For primary AML cell samples, there was generally low to absent expression of these checkpoint molecule, although PD-L1 expression can be induced on primary leukemia cells after treatment with T-cells and BsAb in vitro, and blocking PD-1/PDL-1 interaction increased the efficacy of AMG330-mediated lysis of primary leukemia blasts. This effect of checkpoint blockade was most prominent with low E:T ratios. At an E:T ratio of 1:5, T-BsAb (AMG 330)-mediated lysis was increased by 14% with PD-L1 blockade, whereas no or minor enhancement could be observed for an E:T ratio of 1:1 (Krupka et al. 2016). Thus, checkpoint blockade might be particularly helpful in patients with a high tumor burden and low T-cell numbers (low in vivo E:T ratio). Marcinek et al. also reported that PD-L1 blockade increases LFA-1 accumulation within the immune synapse formed, and thus promotes immune synapse formation between T-cells and target cells in the presence of AMG330 (Marcinek et al. 2021).

Meermeier et al. studied BCMA x CD3× CD3 BsAb in vivo using a syngeneic murine model of multiple myeloma, and found that checkpoint ligands such as PD-L1 were upregulated by IFN-γ and were expressed on nearly all tumor cells during the second week of anti-BCMA/CD3 treatment. Simultaneously, PD-1 on T-cells also increased, with a similar trend noted for other checkpoint molecules such as KLRG1 and LAG3. These results suggest that T-cells become functionally exhausted and less responsive to anti-BCMA/CD3 over time (Meermeier et al. 2021). Increased PD-L1 expression in tumor samples was also observed in human patients treated with T-BsAbs. In one study, a more than twofold increase in PD-L1 expression was observed in patients treated with tebentafusp (HLA-A2/gp100 × CD3) from serial tumor biopsy samples (Middleton et al. 2020).

Upregulation of PD-L1 and other checkpoint molecules has also been observed after treatment with other T-BsAbs. Checkpoint blockade has been found to enhance the therapeutic efficacy of T-BsAbs targeting different antigens expressed by both solid tumors and hematological malignancies (Bacac et al. 2016; Junttila et al. 2014; Mathur et al. 2020; Osada et al. 2015; Sam et al. 2020). Additionally, this effect was demonstrated in vivo using both PDX/CDX models and murine syngeneic tumor models. However, there are limited clinical correlation data regarding how baseline and/or on-treatment checkpoint molecules affect the outcome of T-BsAb therapy in human patients. As mentioned above, melanoma cell expression of PD-L1 was upregulated with treatment by tebentafusp; however, the relationship between PD-L1/PD-1 levels and treatment response could not be established due to the limited number of patients with serial tumor samples. At the 2020 ASH meeting, Ravandi et al. reported the results of a clinical trial using vibecotamab (CD3XCD123 T-BsAb) to treat patients with AML. It was found that responders had low T cell expression of PD-1. The variation was large due to the limited number of patients analyzed (seven responders); however, the result for PD-1 expression by CD4 T-cells reached statistical significance (Ravandi et al. 2020a). For lymphoma patients treated with the CD20 BsAb glofitamab, complete response was associated with a low PD-1 high T-cell signature on transcriptome analysis of baseline tumor samples (Bröske et al. 2022). Increased PD-L1 expression by leukemia cells has been found in an ALL patients resistant to blinatumomab therapy (Köhnke et al. 2015). Additionally, baseline PD-L1 expression by myeloma cells had no association with the clinical outcomes of myeloma patients treated with BCMA x CD3 BsAb (AMG420) (Topp et al. 2020). Many clinical trials are combining checkpoint blockade with T-BsAbs. However, most of these trials are still in the early phase, with preliminary outcome analyses still pending. Nevertheless, preliminary results of a phase 1b clinical trial combining blinatumomab with PD-1 and CTLA-4 inhibition have been reported. Despite the limited number of cases, the 80% CR rate among heavily pretreated relapsed/refractory ALL patients (compared to the 40–50% CR rate from historical results of single-agent blinatumomab therapy) appears highly promising (Webster et al. 2018).

T-BsAbs activates T-cells while bypassing professional antigen-presenting cells, and in many cases, the target tumor cells do not express costimulatory molecules (or do so at very low levels). Thus, a unique feature of the T-cell response elicited by T-BsAbs is the lack of, or suboptimal costimulation.

One costimulatory molecule of interest is 4-1BB. A preclinical study of APVO603 (CD123 BsAb) demonstrated that supplementation with 4-1BB and O×40 costimulation with agonist antibodies can markedly promote in vitro cytotoxicity against target tumor cells (Gottschalk et al. 2022). Chiu et al. also reported that in a murine syngeneic model of prostate cancer, a 4-1BB agonist improves the antitumor efficacy of a PMSA x CD3 BsAb, especially when the tumor burden is high. The 4-1BB agonist promotes sustained T-cell activation from several hours to up to several days. Sustained T-cell activation also leads to enhanced expansion of cytotoxic effector T-cells, but not regulatory T cells. Thus, effector T-cell number, functionality and CD8+ to Treg ratio all improved with 4-1BB stimulation, which translated into markedly enhanced antitumor efficacy (Chiu et al. 2020b). Belmontes et al. reported similar findings in multiple murine syngeneic tumor models. Their research also suggests that enhanced intra-tumoral T-cell expansion and an increased CD8 to Treg ratio are the two key mechanisms of this synergistic effect (Belmontes et al. 2021).

OX40 might be another key costimulatory molecule that significantly affects the outcome of T-BsAb treatment and has been found to acting synergistically with 4-1BB to enhance T cell immunity(Lee et al. 2007; Qui et al. 2011). By analyzing the publicly available large-cohort data, which included gene expression profiles from bone marrow samples from 576 B-ALL patients, Huo et al. found that the O × 40 ligand was expressed at high levels in nearly 34% of B-ALL patients (Huo et al. 2021). They further found that the O×40-L expression level in leukemia cell lines is positively associated with blinatumomab-redirected cytotoxicity by T cells. O×40-L/OX40 signaling has been found to enhance the proliferation, survival and functionality of conventional T-cell populations but has a negative impact on the immunosuppressive capability of regulatory T cells.

CD28 is the canonical costimulatory molecule. In vitro studies have found that the expression of CD80 and CD86 (ligands for CD28) on target tumor cells enhances the cytotoxic activity triggered by AMG 330 (CD33 BsAb) (Laszlo et al. 2015) and a BsAb against MLSN (Shen et al. 2022). CD28 agonists has also been shown to improve the efficacy of T-BsAbs. Several T-BsAbs developed in the 1990s and early 2000s showed no or little single reagent activity and can only work in vitro and in vivo when combined with CD28 agonists (Bohlen et al. 1993; Bohlen et al. 1997; Cochlovius et al. 2000a; Cochlovius et al. 2000b; Csóka et al. 1996; Daniel, et al. 1998; Demanet et al. 1996; Kipriyanov et al. 2002).

CD28 agonists are not safe for human use due to catastrophic cytokine release syndrome (Suntharalingam et al. 2006). More recent studies have focused on targeted delivery of CD28 agonist to the tumor site by constructing bispecific antibodies in which one end engages CD28 while the other binds to tumor-associated antigens. In fact, this strategy had been attempted decades ago. Brandl et al. developed a CD20 X CD28 bispecific in the 1990s, and found that the combination of target x CD3 with target x CD28 specificity induces vigorous autologous T-cell activation and killing of malignant cells in vitro (Brandl et al. 1999). In the last several years, more of these types of bispecific antibodies have been developed (e.g., CD19 X 28, EGFR xCD28, PD-L1 x CD28) and show promising pre-clinical efficacy with an acceptable toxicity/safety profile (Lakhani et al. 2021; Moore et al. 2021; Skokos et al. 2020; Waite et al. 2020).

T-cell trafficking and intratumoral T-cell infiltration

A key for successful T-BsAb-based immunotherapy is the presence of T-cells that have migrated into the tumor site. The trafficking of T-cells into tumors depends upon the compatibility between chemokines found in tumors and chemokine receptors expressed on T cells (Donnadieu et al. 2020). There is increasing evidence from clinical studies that tumors enriched in T-cells are more susceptible to immunotherapy with checkpoint blockade, while tumors with few infiltrated T-cells are likely refractory to such treatments (Herbst et al. 2014). Thus, the status of intratumoral infiltration of T-cells could also be a crucial factor in determining the outcome of T-BsAb therapy.

Belmontes et al. found that in multiple murine syngeneic tumor models (Belmontes et al. 2021), pretreatment intratumor T-cell infiltration is a key predicting factor of T-BsAb treatment efficacy. The “T-cell cold” responded poorly to T-BsAbs, and concomitant treatment with PD-1 blockade and/or 4-1BB agonists led to marked local T-cell expansion, turned the tumor into an immune hot spot and overcame resistance to T-BsAb treatment. Another group also reported that intratumoral infiltration of T-cells is crucial for the efficacy of T-BsAbs (Li et al. 2018). However, they found that this efficacy is less dependent on baseline pretreatment T-cell infiltration, and rather is mostly mediated by de novo-recruited T cells. They proposed that BsAb treatment activates T-cells to produce proinflammatory cytokines, for instance, IFNγ that can stimulate cells in the tumor microenvironment to secrete chemokines such as CXCL-9, −10 and −11. These chemokines subsequently recruit more T-cells, via their interaction with CXCR3, to the tumor site. The importance of chemokines such as CXCL10 in recruiting T-cells has been demonstrated in other tumor models (Mulligan et al. 2013; Yang et al. 2006), and higher intratumoral levels have been found to be associated with improved survival in human cancer patients (Bronger et al. 2016; Reschke et al. 2021; Suyama et al. 2005).

Correlative studies for clinical trials of tebentafusp also support the importance of de novo recruitment of T-cells via the CXCL10-CXCR3 axis. Tebentafusp is a bispecific fusion protein that redirects CD3+ T-cells to target gp100 (a melanoma-associated antigen) presented by HLA-A2 that has been approved for the treatment of advanced uveal melanoma. Middleton et al. investigated immunological biomarkers to help predict clinical outcomes (Middleton et al. 2020). They found that a greater increase in serum CXCL10 and CXCL11 levels was associated with both longer overall survival and greater tumor shrinkage. Transient reduction of circulating CXCR3+ CD8 T-cells was observed shortly after starting Tebentafusp infusion, which reflects T-cell infiltration/sequestration in the tumor site via CXCL10 and CXCL11. A greater transient decrease in CXCR3+ CD8 T-cells was also found to predict favorable clinical outcomes.

Intratumoral T-cell infiltration has also been found to predict favorable clinical outcomes in hematological malignancies when treated with T-BsAbs. Odronextamab (CD3 x 20 BsAb) has been studied in clinical trials treating relapsed/refractory B-cell non-Hodgkin lymphoma patients. It has been reported that higher levels of tumor-infiltrating T-cells (TILs) at baseline were associated with a better chance for complete or partial response (Brouwer-Visser et al. 2020). For glofitamab, another CD20 BsAb, clinical correlative analysis showed a trend toward a higher percentage of CD8+ TIL cells at baseline in patients who achieved complete remission. Additionally, transcriptome analysis showed a trend toward a higher effector-like CD8+ T-cell count in tumors from patients who achieved complete remission (Bröske et al. 2022). Clinical trials with Mosunetuzumab, also a CD20 BsAb, found that on-treatment CD8+ tumor infiltrating T-cell levels were significantly higher in responders and is associated with favorable clinical outcome (Hernandez et al. 2019).

BFCR4350A is a CD3 X FcRH5 bispecific antibody that has been developed to treat multiple myeloma. In clinical trials, it has been found that the level of T cell infiltration in bone marrow was higher in responding than in nonresponding patients (Nakamura et al. 2020). Additionally, it has been found that for AML patients treated with Flotetuzumab(CD3 x CD123 BsAb), clinical response is associated with immune-infiltrated, T-cell-rich bone marrow at baseline. Gene expression analysis revealed that the response also correlated with increased IFNʏ stimulated genes and T-cell recruiting factors, including CXCL-10, STAT1 and IRF1 (Uy et al. 2021; Vadakekolathu et al. 2020a). Thus, both basic and clinical studies support that enhanced T-cell recruitment into the tumor site by the IFNγ-CXCL10/CXCR3 axis is crucial for the therapeutic efficacy of T-BsAbs in both solid tumors and hematological malignancies.

Regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSC)

Regulatory T (Treg) cells play a key role in preventing autoimmune diseases and limiting damage by inflammatory processes. However, they also limit beneficial immune responses and are one of the main mechanisms by which tumor cells evade attacks by the immune system. T-BsAbs elicit global activation of all T-cell subsets regardless of antigen specificity. Thus, activation of Treg cells may profoundly affect the efficacy and outcome of T-BsAb therapy.

Treg cells also have killing potential under right circumstances. One major mechanism by which Treg cells suppress the immune response is by killing immune cells (e.g., antigen-presenting cells) via the granzyme-perforin pathway. Interestingly, Choi et al. reported that an EGFRvIII x CD3 BsAb can potently activate human Treg cells (freshly purified or in vitro expanded) to elevate the expression of granzymes and perforin specifically in the presence of tumor cells expressing EGFRvIII (Choi et al. 2013). The activated human Tregs are capable of killing EGFRvIII-expressing tumors in the presence of T-BsAb. Additionally, human glioblastoma samples also displayed diffuse infiltration of activated, granzyme-producing Treg cells, indicating that Treg cells with potent effector functions may already be present in tumors under typical clinical situations. Thus, the group proposed that T-BsAb therapy has the potential to bypass Treg-mediated suppression and turn those activated, tumor-infiltrating Treg cells into effector cells to kill tumor cells. However, another research group also found that T-BsAbs activate Treg cells against target-expressing tumor cells, albeit the activated Treg cells produced immunosuppressive cytokines such as IL-10 and almost no inflammatory cytokines (Koristka et al. 2012). Additionally, they found that T-BsAb-activated regulatory T-cells are able to suppress the effector functions of activated autologous T cells both in vitro and in vivo. The group further tested T-BsAb-induced killing against tumor cells using highly purified (>95%) fresh or expanded human Treg cells and found no measurable cytolytic effects mediated by Treg cells. They also found that while Treg cells were able to express granzyme, no or only marginal upregulation of CD107a (marker for degranulation) was detected upon activation by T-BsAbs. In vivo, using an immune compromised mouse model engrafted with the human tumor cell line PC3, they demonstrated that injection of Tregs in combination with T-BsAbs did not reduce tumor outgrowth in mice. In contrast, Treg administration suppressed the antitumor effect of co-injected conventional T-cells and significantly enhanced tumor growth in this scenario (Koristka et al. 2015; Koristka et al. 2014). Of note, these findings are in line with chimeric antigen receptor Treg (CAR-Treg) cells. Regulatory T-cells have been engineered with chimeric immune receptors, and it has been found by several groups that instead of contributing to target-cell killing, CAR-Tregs efficiently protect target cells from the cytotoxicity of effector T-cells in vivo (Arjomandnejad, et al. 2022; Dawson et al. 2020; Hombach et al. 2009). Regarding the discrepancy between the two groups, Koristka et al. argued that the Treg cell purity might be key. For the BsAb-mediated cytotoxicity assay, both groups used a high E:T ratio (20:1 for Choi et al.). Thus, contamination of conventional T cells, even at low levels, may account for all or majority of the cytotoxicity observed. Koristka et al. used a two-step purification method to ensure high purity (>95%) of CD4+CD25+CD127lo Tregs, while Choi et al. used one-step sorting strategy. Another difference is the E:T ratio used for the in vitro killing assay. Choi et al. used a very high E:T ratio of 20:1, while Koristka et al. used a more conventional ratio of 5:1 (up to 10:1 in some cases). Thus, a high E:T ratio could lead to a greater contribution of observed cytotoxicity by contaminating conventional T cells. However, it is also possible that the Treg-mediated cytotoxicity was very weak and could only be detected at very high E:T ratios.

In another in vitro study combining functional studies with single-cell transcriptome analysis, it was found that a higher frequency of activated CD8 T-cells and conventional CD4 T-cells compared to Treg cells is correlated with stronger blinatumomab-mediated cytotoxicity (Zhao et al. 2021b). Several other in vivo studies using syngeneic murine tumor models (both solid tumors and hematological malignancies) also suggest that a reduced ratio of Treg cells to conventional CD4 or CD8 T-cells is associated with superior therapeutic efficacy with T-BsAbs (Belmontes et al. 2021; Chiu et al. 2020b; Meermeier et al. 2021). The antitumor effect of T-BsAbs can be further enhanced by depleting Treg cells, either by depletion using a pan-CD4 antibody or more via more selective depletion of intratumoral Treg cells using a CTLA-4 antibody (Belmontes et al. 2021).

The frequency of regulatory T-cells has also been found to correlate with the clinical outcome of ALL patients treated with blinatumomab (CD19x CD3). Duell et al. performed a retrospective correlative study of 42 ALL patients enrolled in clinical trials (MT103–206, 211, 311) to identify factors that can help to predict the outcome of blinatumomab therapy (Duell et al. 2017). They found that ALL patients responding to blinatumomab have significantly fewer regulatory T-cells (Tregs) than those who did not respond, with a median baseline Treg frequency of 8.75% in the responder group compared to 14.25% in the nonresponders. Using the predicting model developed, with a primary split of baseline Treg frequency defined as 8.5%, they predicted nonresponders with an accuracy of 100% (14/0). The authors also found that blinatumomab activated Tregs to secrete high levels of IL-10 and demonstrated a strong inhibitory effect on the proliferation and cytotoxicity of conventional T-cells stimulated with blinatumomab. A more recent retrospective study used single-cell RNA sequencing to analyze samples from 44 ALL patients treated with blinatumomab (Zhao et al. 2021b). However, they found no apparent difference in the frequency of T-reg cells between responders and nonresponders (5% vs. 3.7%; P = 1.34 × 10^−4). This discrepancy could be mainly due to different techniques to identify Treg cells – flow cytometry versus single-cell RNA sequencing, with the latter remaining technically challenging (Andreatta et al. 2021). Further, conventional T-cells can also transiently express Foxp3 when activated by T-BsAb. Another major difference is the sample source. While Duell et al. focused on Treg frequency from baseline peripheral blood, Zhao et al. analyzed their cases using both blood and marrow biopsies depending on sample availability. The Treg frequency is usually 2-3-fold higher in peripheral blood than in bone marrow, which could also mask the difference between responders versus nonresponders in the works of Zhao et al.

Myeloid-derived suppressor cells (MDSCs) and/or tumor-associated macrophages (TAMs) are also major immunosuppressive cell types. They have been found to inhibit T-cell infiltration into tumors and induce T-cell dysfunction via the production of immunosuppressive factors or cytokines, inducing Tregs and the surface expression of checkpoint molecules such as PD-L1. Thus, these myeloid cell types can also play a key role in dampening the antitumor immune response induced by T-BsAbs. However, there are relatively few studies to date. It has been demonstrated that MDSCs can potently suppress T cell proliferation, cytotoxicity, and cytokine production induced by T-BsAbs (Thakur et al. 2012). A more recent study demonstrated that depletion of MDSC cells and/or tumor associated macrophages (TAM) enhances the antitumor efficacy of T-BsAb using xenograft murine models, with the depletion of TAMs seeming to be most effective (Park et al. 2021).

Tumor microenvironment (TME)

The tumor microenvironment (TME) is among the major factors affecting the outcome of T-cell immunotherapy (Fearon 2017; Höpken and Rehm 2019; Zhao et al. 2022). The TME is the complex ecosystem that surrounds tumor parenchymal cells that includes nonhematopoietic stromal cells, blood vessels, extracellular matrix and infiltrated immune cells such as lymphocytes and myeloid cell subpopulations. The TME can profoundly affect T-cell immunity via multiple mechanisms that involve immunosuppressive surface molecules such as CD200 and PD-L1 that are expressed on various cell types, or secreted immunosuppressive factors such as TGF-beta and IL-10. Excessive production of inflammatory signals can also have immunosuppressive effects. Immunosuppressive effects can also be imposed by small molecules produced by tumor cells or other cell types, such as adenosine and tryptophan metabolites catalyzed by Indoleamine 2, 3-Dioxygenase (IDO1).

The impact of several common TME-related immunosuppressive factors on T-BsAb-induced antitumor immunity has been evaluated in vitro using AMG110 (EpCAM x CD3) (Deisting et al. 2015). It has been found that tumor-derived serpin PI-9, Bcl-2, TGF-β and PD-L1 significantly compromise BsAb redirected cytotoxicity against tumor cells. The most substantial inhibition of T-cell proliferation was seen by IDO. Serpin PI-9 is a protease inhibitor that can directly block the enzymatic activity of granzyme B that is delivered into the cytosol of cancer cells through the cytolytic synapse formed with T cells. Bcl-2 is known for its role as an anti-apoptotic protein that can decrease the susceptibility of cancer cells to apoptosis induced by granzyme B. TGF-beta can broadly downmodulate effector T-cell functions by suppressing the expression of granzymes A and B, perforin and cytokines such as IFNγ. In another study, Cho et al. showed that various cell types in the bone marrow microenvironment, including stromal cells, osteoclasts/osteocytes and bone marrow-associated myeloid cells, can reduce T-BsAb (AMG701, a BCMA x CD3 BsAb)-mediated cytotoxicity (Cho et al. 2020). Galectin-1 (Gal-1) is a glycan-binding protein with broad anti-inflammatory and immunosuppressive effects (Sundblad et al. 2017). It is overexpressed by various cancer cells and can compromise antitumor immunity through multiple mechanisms. Zhang et al. studied an EPCAM x CD3 BsAb and evaluated its function in vitro and in vivo using a murine model of liver cancer (Zhang et al. 2014). They found that cancer cells expressing higher levels of Gal-1 are more resistant to T-BsAb treatment. Gal-1 expression was found to increase the effective concentration (EC₅₀) of BsAb 4-fold. It also negatively impacted the therapeutic efficacy of T-BsAbs in vivo, with much better control of tumors that express low Gal-1 levels.

On the other hand, the inflammatory process within TME seems to be positively correlated to T-BsAb efficacy. Uy et al. reported that an immune-infiltrated, inflammatory tumor (marrow) microenvironment predicts a favorable clinical response to flotetuzumab (CD3 X CD123 BsAb) in AML patients (Uy et al. 2021; Vadakekolathu et al. 2020a). Combining multiplexed digital spatial profiling (for protein expression) and immune transcriptomic analysis (RNA expression), they were able to classify the AML tumor microenvironment (TME) into immune-infiltrated and immune-depleted subgroups. The immune-infiltrated, inflammatory TME was found to predict a poor response to conventional chemotherapy but a much better likelihood of responding to T-BsAb immunotherapy. Complete response from flotetuzumab was observed in 21%, 44.4%, and 60% of patients with low, intermediate, and high immune infiltration, respectively. The immune-infiltrated profiles were associated with increased expression of IFN-γ stimulated genes and T-cell recruiting factors (STAT1, CXCL10, IRF1), T-cell markers and cytolytic effectors, immune checkpoints, and molecules involved in antigen processing and presentation. Immune checkpoints negatively impact immunotherapy. Antigen processing and presentation are also unlikely to contribute to T-BsAb’s efficacy, as previous studies have shown that MHC I expression is dispensable for the activity of T-BsAb (Brischwein et al. 2007; Offner et al. 2006). Thus, this gene expression profile likely reflects a preexisting IFN-γ-driven adaptive immune response, which creates inflammatory signals and helps to recruit T-cells into the TME. This immune response was nonproductive due to suppression by multiple checkpoint molecules. However, with T-BsAb therapy, this inflammatory microenvironment enhances BsAb-redirected cytotoxicity against tumor cells and promotes tumor cell clearance directly and/or indirectly. Thus, baseline immune infiltration and preexisting IFNγ-driven inflammatory processes are crucial in determining clinical outcome during flotetuzumab treatment.

Similar findings were also reported from clinical correlative studies of melanoma patients treated with tebentafusp (gp100 X CD3) (Middleton et al. 2020). Gene expression analysis of paired baseline and on-treatment biopsies revealed that in responders, there was enrichment of three categories of genes related to cytotoxicity, antigen processing and T-cell function. This gene expression pattern again reflects the inflammatory response initiated by IFNγ secretion, which in turn drives the local production of multiple inflammatory cytokines and chemokines. Among them, CXCR3 ligands (CXCL9, 10 and 11) play a key role in recruiting T-cells to the tumor site, which is crucial for T-BsAb efficacy.

In a retrospective study of 44 relapsed/refractory ALL patients, Zhao et al. reported that the baseline immune signature within the bone marrow microenvironment is correlated with clinical outcome (Zhao et al. 2021b). They also found that CRLF2 (cytokine receptor-like Factor 2) gene rearrangement, which is associated with poor clinical outcome with conventional chemotherapies, predicts a favorable response to blinatumomab. Thus, CRLF2 rearrangement leads to the activation of downstream JAK/STAT pathways, and gain-of-function JAK mutations are present in approximately half of CRLF2 rearranged cases. These findings again support the notion that the preexisting immune/inflammatory process within the TME leads to activation of the IFNγ-JAK-STAT signaling pathway, which fosters a favorable tumor microenvironment for immunotherapy with T-BsAb (Figure 1).

Figure 1. The IFNγ-JAK-STAT signaling pathway enhances T-BsAb efficacy by recruiting T cells to TME via the CXCL10/CXCR3 axis. IFN-γ produced by innate immune cells (e.g., NK cells) promotes local production of chemokines (CXCL9, 10 and 11) by immune and tumor cells, which in turn recruit more T cells to the TME via CXCR3. Newly recruited T cells are subsequently activated by T-BsAb in the presence of target antigen to produce more IFN-γ, hence supporting a positive feedback loop.

Intrinsic resistance to T-cell cytotoxicity by tumor cells

Another major mechanism by which tumor cells evade immune attack is intrinsic resistance to T-cell-mediated cytotoxicity. One mechanism identified is impaired IFNγ-R signaling in tumor cells. Recently, Larson et al. reported that tumor cells can develop resistance to killing by CAR-T-cells by loss-of-function mutations in the IFNγ receptor signaling pathway (Larson et al. 2022). In searching for mutations that confer tumor cell resistance to CAR T-cell cytotoxicity using a genome-wide CRISPR screen approach, they found that the loss of genes involved in the IFNγ-R signaling pathway, including IFNGR1, JAK1 and JAK2, rendered glioblastoma and other solid tumors more resistant to killing by CAR T-cells, and the resistance is in part contributed by inefficient formation of immune synapses from inadequate upregulation of adhesion molecules such as ICAM-1. They confirmed this finding using multiple different CAR-T constructs and solid tumor models in vitro and in vivo. Interestingly, deficiency in the IFNʏ-R pathway does not render hematological malignancies (such as leukemias and lymphomas) resistant to CAR T-cell-mediated killing. The IFNγ-IFNʏR-JAK signaling has also been found to affect tumor cell sensitivity to T-BsAb-mediated killing. Arenas et al. explored gene mutations in tumor cells that affect sensitivity to T-BsAb-mediated cytotoxicity using another screening strategy (Arenas et al. 2021). They cocultured tumor cells expressing HER2 with a HER2 x CD3 BsAb along with T cells, and then selected for tumor cells that survived multiple rounds of T-BsAb redirected killing, which had acquired an IC50 approximately tenfold higher than that of parental cells. Transcriptome analysis revealed that resistant tumor cells have acquired deficiency in the IFN-γ signaling pathway, with a marked reduction in JAK2 expression. They further confirmed that inhibiting the IFNʏ-R signaling pathway in tumor cells (IFN-γ-blocking antibodies or knockdown of IFNʏ receptor) caused resistance to HER2x CD3 in vitro and in vivo using both cell line- and patient-derived xenograft models. Finally, sensitivity to T-BsAb-redirected cytotoxicity could be restored by overexpression of JAK2 by tumor cells.

In another correlative study of lymphoma patients treated with golimumab (CD20 x CD3 BsAb), gene expression and mutational profile of pretreatment biopsy samples were analyzed to identify biomarkers that are associated with treatment response (Blagih et al. 2020). It was found that upregulated MYC targets and downregulated TP53 target signatures were enriched in patients who failed to achieve complete remission. TP53 mutation status also demonstrated a trend toward an association with poor outcomes (p = 0.07). These gene mutations in cancers affect the recruitment and dampen the activation of T cells, thus enabling resistance to T-BsAb treatment

CD58, the ligand of the CD2 T-cell costimulatory receptor, is another adhesion molecule that has been found to affect immune synapse formation and cytotoxicity by CAR-T cells (Majzner et al. 2020). CAR T-cells demonstrated significantly reduced cytolytic activity against CD58 knockout vs. wild-type tumor cells. In vivo, mice inoculated with wild type leukemia cells (Nalm-6) and treated with CAR-T-cells demonstrated complete responses and prolonged leukemia-free survival, while mice inoculated with CD58KO leukemia cells only had partial, temporary responses followed by tumor progression and death from leukemia. CD58 gene expression is frequently altered in cancer cells. Correlative analysis of lymphoma treated with CD19 CAR-T-cells (Axicabtagene ciloleucel) found that patients with CD58 alterations had inferior clinical outcomes; only 1 out of 12 patients achieved a durable, complete response, while the remaining 11 patients progressed, most commonly after a short period of initial response. Tumor expression of CD58 has also been shown to play an important role in T-BsAb-mediated cytotoxicity. Shen et al. explored the potential cancer cell-intrinsic factors affecting T-BsAb-mediated cytotoxicity by genome-wide CRISPR loss-of-function and gain-of-function screens (Shen et al. 2022). They found that loss of CD58 in tumor cells led to decreased T-BsAb-mediated cytotoxicity, T-cell activation and antitumor efficacy in vitro and in vivo. Moreover, the effects of CD58 loss were found to act synergistically with concurrent loss of CD80/CD86 (ligands for CD28) to reduce T-BsAb-mediated killing of tumor cells, indicating a nonredundant role of CD58/CD2 signaling for full-scale T-cell activation by BsAb.

Shen et al. also found that other cancer cell-intrinsic genes with functions in autophagy, T-cell costimulation, the apoptosis pathway, chromatin remodeling, and cytokine signaling could alter the responsiveness of tumor cells to BsAb-mediated killing (Shen et al. 2022). Particularly, tumor cell sensitivity to killing by BsAb is profoundly affected by changes in genes involved in cell death and apoptosis pathways, such as BID, BCL2L1 and CFLAR (Caspase-8 and FADD Like Apoptosis Regulator). Other groups have made similar findings that overexpression of the protease inhibitor serpin PI-9 (blocking the enzymatic activity of granzyme B) and/or the antiapoptotic gene Bcl-2 helps to protect target cells from T-BsAb-mediated cytotoxicity (Deisting et al. 2015).

Antigen escape

Loss of surface expression of target antigens is another major mechanism by which tumor cells escape immunotherapy by T-BsAbs. Among all the T-BsAbs approved or under clinical trials, blinatumomab has the most clinical experience. CD19-negative relapse is not as common with blinatumomab as with CD19 CAR-T therapy. Gore et al. analyzed 67 ALL patients whose disease failed or relapsed post blinatumomab therapy and found that only 4 patients (6%) had negative CD19 surface expression (Gore et al. 2018). As shown in Table 1 across different studies, it has been reported that loss of target antigen CD19 was observed in 10–30% of patients upon relapse or treatment failure post blinatumomab, and the incidence was similar between adult and pediatric patients. Zhao et al. found loss of CD19 expression in 7 out of 11 relapsed patients, which may be due to the high prevalence of patients of Hispanic ancestry, whose leukemia is more likely to have ph-like features and CRLF2 aberrations.

Table 1. Incidences of antigen loss with major T-BsAb clinical trials.

Type of cancer treated	T-BsAb used	Number of patients failed/relapsed after BsAb treatment	Percentage of CD19/CD20 negative cases on relapse.	reference
r/r ALL, pediatric	Blinatumomab	N=67 (blinatumomab failure)	6% (4 out of 67 patients).	(Gore et al. 2018)
r/r ALL, adult	Blinatumomab	N=68 (blinatumomab failure)	8% (5 out of 68 patients).	(Jabbour et al. 2018)
r/r ALL, pediatric	Blinatumomab	N=18 (blinatumomab failure)	22% (4/18)	(Mejstríková et al. 2017)
r/r ALL, pediatric	Blinatumomab	N=23	26% (6/23)	(Locatelli et al. 2020)
r/r ALL, adult	Blinatumomab	N=10	30% (3 out of 10 patients).	(Topp et al. 2014)
r/r ALL, pediatric	Blinatumomab	N=30	30% (9/30)	(Mikhailova et al. 2021)
r/r ALL	Blinatumomab	N=11, high percentage of patients with Hispanic ancestry with ALL of ph-like features	64% 7/11	(Zhao et al. 2021b)
R/R B-NHL	odronextamab	N=11	82% (9/11).	(Brouwer-Visser et al. 2021)

There are limited reports regarding antigen loss with other T-BsAbs. Brouwer et al. found that for lymphoma patients treated with odronextamab (CD20 X CD3 BsAb), there was a high incidence of loss of target antigen (CD20) in patients who failed the therapy (Brouwer-Visser et al. 2021). Among patients whose disease progressed, 11 had paired biopsy samples available, and loss of CD20 expression by IHC was found in 9 post-treatment samples. An interesting finding is that patients whose tumors harbored CD20-negative lymphoma cells still had a possibility of achieving durable clinical responses, suggesting that a bystander immune effect may be induced by T-BsAb therapy. Similarly, Middleton et al. studied serial tumor biopsy samples from melanoma patients before and after treatment with tebentafusp (gp100 x CD3); however, they did not find loss or reduction of gp100 expression in posttreatment samples (Middleton et al. 2020). Loss of BCMA expression has been reported to confer resistance to anti-BCMA CAR T-cell therapy for multiple myeloma patients (Samur et al. 2020). In a study of BCMA x CD3 BsAb (AMG420), it was found that there was no difference in BCMA expression levels in myeloma cells between responders and nonresponders. However, regarding BCMA expression after relapse, the tumor cell infiltration was too low in the few patients from whom the data were available to draw conclusions. Thus, the incidence of antigen loss and its contribution to treatment resistance by T-BsAbs is dependent on the type of tumor as well as the antigen targeted.

Tumor burden, T-cell number and E:T ratio

To date, most clinical correlative studies of T-BsAbs have been performed with blinatumomab. Tumor burden has been found by many blinatumomab clinical studies to be a key factor affecting clinical outcome and treatment efficacy. A lower tumor burden (marrow blast percentage <50%) has been found to be associated with a better CR rate and longer survival across all kinds of clinical situations for ALLs: pediatric versus adult cases, ph-positive versus ph-negative ALL, and morphologically proven leukemia versus minimal residual disease (Gökbuget et al. 2018; Locatelli et al. 2020; Martinelli et al. 2017; Stein et al. 2019; Topp et al. 2015; von Stackelberg et al. 2016; Wei et al. 2021; Zhao et al. 2021b). The same trend was found when blinatumomab was used to treat CD19-positive B-cell lymphomas (Nägele et al. 2021; Viardot et al. 2020).

Lower tumor burden, as defined by lower WBC and lower blast percentage in marrow and peripheral blood, was also found to predict better clinical outcomes in AML patients treated with CD33xCD3 (AMG330) and CD123xCD3 (Vibecotamab and APVO436) T-BsAbs (Agarwal et al. 2020; Ravandi et al. 2020a; Ravandi, et al. 2020b; Uckun, et al. 2021). For multiple myeloma patients, one study suggested that baseline tumor burden did not affect the treatment efficacy of a BCMA x CD3 BsAb (AMG420), as there was no difference between responders and nonresponders in the percentage of myeloma cells in the bone marrow at baseline (Topp et al. 2020). Interestingly, in a preclinical study using syngeneic murine myeloma models, the therapeutic efficacy of BCMA x CD3 BsAb was less efficacious at high tumor burden, which can be easily measured by blood M-protein spike and the gamma/albumin (G/A) ratio (Meermeier et al. 2021). In another preclinical study, similar findings were made with solid tumor murine models treated with PMSA x CD3 BsAb (Chiu et al. 2020b). One puzzling finding from both preclinical studies is that larger tumors did not cause reduced T-cell infiltration or activation at the tumor site, nor did larger tumors cause more severe systemic T-cell dysfunction.

Tumor load may be a marker of the aggressiveness of cancer cells, and as mentioned above, cytoreduction by prephase chemotherapy does not impair, and may even improve the efficacy of T-BsAbs. A high tumor load may reflect an unfavorable effector-to-target (E:T) ratio, which is crucial for the outcome of both in vitro and in vivo cytotoxicity assays. Thus, the other side of the E:T ratio equation, T-cell number and expansion, has also been found by many studies to be a predicting factor for T-BsAb efficacy. For blinatumomab therapy, it has been found that a higher frequency of T-cells with a memory/stem memory phenotype in the blood or bone marrow at baseline is associated with a favorable clinical response for ALL patients (Zhao et al. 2021b). Analysis of a phase 2 study of ALL patients treated with blinatumomab revealed that greater on-treatment expansion of T-cell numbers is associated with a better chance of long-term survival without additional treatment (Zugmaier et al. 2015). For lymphoma patients treated with blinatumomab, on-treatment T-cell expansion (both CD4 and CD8 T cells) was more pronounced in patients who achieved complete remission (Nägele et al. 2021). However, another phase 2 study of DLBCL patients did not find notable differences in T-cell expansion between responders versus nonresponders after blinatumomab therapy (Katz et al. 2022a). For EPCORITAMAB (CD20 x CD3 BsAb), it has been found that there is a trend toward greater expansion of activated and total T-cells in lymphoma patients who had a partial or complete response to epcoritamab (Chiu et al. 2020a). Similar observations were made for Glofitamab, another CD20 BsAb (Bröske et al. 2022). While baseline levels of peripheral blood T-cells did not show a significant association with clinical response, on-treatment expansion of T-cells with an effector/memory phenotype was associated with clinical benefit by lymphoma patients treated with this CD20x CD3 BsAb. Nakamura et al. also reported that for myeloma patients treated with FcRH5 x CD3 BsAb(BFCR4350A), there was a more pronounced T-cell expansion, irrespective of baseline T-cell levels, in responders (Nakamura et al. 2020).

Strategies to improve therapeutic efficacy

Modify signal 2 through checkpoint blockade and/or costimulatory molecule agonists

Checkpoint molecule blockade has achieved great success as a standalone immunotherapy for cancer patients. The expression of checkpoint molecules such as PD-1 can be induced upon T-cell activation by BsAb treatment and has been identified as one of the major resistance mechanisms. Webster et al. first reported that blinatumomab combined with PD-1 and/or CTLA-4 blockade is well tolerated in relapsed/refractory ALL patients and achieved an impressive complete remission rate of 80% in the 6 patients enrolled in the phase I part of the trial compared to a 40–50% CR rate with blinatumomab single therapy in this patient population from historical data (Webster et al. 2018). Since then, there have been many clinical trials incorporating checkpoint molecule blockade with T-BsAb therapy, mostly for the treatment of lymphoid malignancies (Zhou et al. 2021).

T-BsAbs do not provide costimulatory signals (“signal 2”) for T-cells on their own, and the target cells usually express no or low levels of costimulatory molecules. Thus, providing additional signal 2 via agonist antibodies for costimulatory molecules is another intriguing approach to enhance the efficacy of T-BsAbs. However, this is a concern due to side effects and toxicities, especially cytokine release syndrome. A CD28 agonist is well known to cause a dangerous cytokine storm as learned from the disastrous TGN1412 trial (Suntharalingam et al. 2006). High-affinity agonist antibody for 4-1BB has demonstrated single-agent clinical efficacy in multiple types of solid tumors. However, it also comes with the risk of fatal liver toxicity (Bartkowiak et al. 2018). To mitigate excessive toxicity, researchers have focused on bispecific strategies. Agonist antibodies to costimulatory molecules such as CD28 and 4-1BB have been constructed into a bispecific platform along with the target (e.g., CD20) binding arm for targeted delivery to tumor sites. These costimulatory BsAbs have been tested along with T-BsAbs in preclinical studies and have demonstrated enhanced efficacy as well as promising safety profiles (Claus et al. 2019; Moore et al. 2021 Skokos et al. 2020). Some costimulatory BsAbs have been put into clinical trials, although most of them are still in the early phase, and the data regarding efficacy and safety are still pending (Ku et al. 2020; Muik et al. 2022; Zhang et al. 2021).

Optimizing the design of T-BsAbs

Another approach to improve the efficacy and therapeutic window is to optimize the design of BsAbs. T-BsAbs activate T-cells by crosslinking the TCR complex and forming immune synapses between T-cells and target cells. Subsequently, perforin and granzyme are delivered through this synapse, which results in target cell killing. This process can be modulated by multiple factors, including binding arm length, linker design and flexibility between CD3 and target antigen binding sites, affinity/valency to CD3 and target antigen (Staufer et al. 2022).

One factor is the affinity of T-BsAbs toward CD3 and target antigens. With a natural immune response, T-cells recognize the antigen peptide-MHC complex with low affinities (Kd values of approximately 1–100 µM). The affinity of a typical antibody toward its cognate antigen is usually much higher, with a Kd in the nM range. While higher binding affinity to both CD3 and target antigen are positively correlated with in vitro potency of T-BsAb, this also comes with increased cytokine production by T-cells and the risk for cytokine release syndrome during clinical use. Ideally, a well-designed T-BsAb should be able to induce adequate cytotoxicity against target cells without eliciting excessive production of inflammatory cytokines. Fortunately, for T cells the activation threshold for cytolytic activity is much lower than that for cytokine production (Faroudi et al. 2003). The CD3 binding arm of blinatumomab was developed from the OKT3 backbone and had a reduced Kd of the uM range. Companies such as Xencor and Macrogenics have also developed CD3 binding arms based on another clone of anti-CD3 antibody (SP34). Teneobio also developed novel F2 family antibodies (Trinklein et al. 2019; Vafa and Trinklein 2020). All these novel CD3 binding arms have a relatively low affinity for CD3, with the hope of allowing T-BsAbs to retain strong cytotoxicity while causing less cytokine release.

The relative affinity toward CD3 versus target antigen also affects the in vivo distribution of T-BsAbs. T-BsAbs with higher affinity toward target antigens preferentially bind first to cancer cells, and this affinity gap minimizes free BsAb binding to T-cells and thus reduces antigen-independent activation and antibody-induced apoptosis of T-cells while retaining efficient killing of target tumor cells (Mandikian et al. 2018; Wang et al. 2021).

The specific binding site on the CD3 molecule also impacts T-cell activation. CD3 is part of the TCR/CD3 complex and is composed of γ, δ, ε and ζ subunits. Most CD3 antibodies predominantly bind to the CD3ε unit. The F2 family CD3 binders developed by Tenebrio preferentially bind to CD3εδ heterodimers with intermediate affinity. T-BsAbs with CD3 binding arms based on F2 family binders showed much lower levels of cytokine production while retaining significant cytotoxicity against target cells. Moreover, the developers claim that BsAbs with the F2 family CD3 binding arm preferentially bind to CD8 T-cells over regulatory T-cells, and cause much less upregulation of checkpoint molecules such as PD-1 and CTLA-4 (Vafa and Trinklein 2020).

Appropriate spacing between T-cells and target cell that have been linked by T-BsAbs has been found to be crucial for efficient T-cell activation and target killing. Blinatumomab demonstrated superior killing efficacy over many other CD19-targeting BsAb constructs developed during the same era, and one major advantage of its design has been postulated to be the ability to bring T-cells and target cells in close proximity, thus expediting immune synapse formation and transfer of cytotoxic molecules. Recently, Staufer et al. again demonstrated the importance of T-BsAbs’ capability to elicit close contacts between target cells and T-cells (<16 nM). A typical T-cell immune synapse formed between the TCR and peptide-MHC complex (pMHC) creates a space of 14 nm between target cells and T cells (Choudhuri et al. 2009; Velas et al. 2021). Thus, it has been speculated that T-BsAbs will be more potent if optimized to recapitulate this spacing between the T-cell and target. The spacing of T-BsAb-induced immune synapses was determined by adding up three factors: 1) the distance between the CD3 epitope and the T-cell membrane, 2) the distance between the two binding arms of the T-BsAb, and 3) the distance from the TAA epitope to the target cell membrane. For a BsAb targeting a certain antigen, usually 1) and 3) are fixed. The distance between the two arms of BsAb binding sites can be adjusted to some extent by modifying the structure of the T-BsAb. Different formats of BsAb also have their own typical distance between the two binding arms. For example, diabodies have a relatively short binding arm distance of 3–6 nm, while the distance between the two binding arms of IgG-based BsAbs is approximately 9–15 nm.

Recently, Chen et al. nicely demonstrated that the right spacing between T-cells and the target membrane critically impacts the potency of T-BsAbs (Chen et al. 2021). They chose BCMA as the target antigen, in which the BsAb binding epitope is close to the cell membrane (2.5 nm). By adding EGF-like domains (3 nm each) as rigid spacers, they created BCM-EGF hybrid molecules with BsAb binding epitopes at different distances from the cell membrane. They found that for the original BCMA target, IgG-based BsAb (9–15 nm between binding arms) has better efficacy than diabody-based BsAbs (3–6 nm between arms). However, when the distance from the BCMA epitope to the membrane was increased by adding more EGF spacer domains, the potency of IgG-based BsAb was reduced, while the potency of the diabody-based BsAb was improved. Most strikingly, they found that cytotoxicity and cytokine production are affected differently by this spacing factor. This finding is exciting in that it suggests by refining the BsAb design to allow for optimized spacing between target cells and T cells, it is possible to reduce cytokine secretion while retaining strong cytotoxicity against target cells.

Reduce tumor burden and improve the E:T ratio

As discussed before, tumor burden is another key factor predicting the clinical efficacy of T-BsAbs. T-cells can work as serial killers and destroy multiple target cells during T-BsAb therapy. However, there is likely a limitation of the number of target cells that one T-cell can target. In most cases, the T-BsAbs induce T-cell activation with suboptimal costimulatory support, and the T-cells eventually become deleted or become dysfunctional after prolonged encounter with target cells. Hence, cutting the tumor burden by prephase cytoreduction therapy should help to improve the ratio of T-cells (effector) to tumor cells (target), aka the E:T ratio, and thus improve the clinical response to T-BsAb therapy.

For most tumor types, “debulking” requires cytotoxic chemotherapy, which unfortunately could also negatively impact the E:T ratio by killing T cells. Nevertheless, as learned from murine models, as long as the tumor is more sensitive to chemotherapy, prephase cytoreduction can improve the efficacy of T-BsAbs. In addition, cytotoxic chemotherapy could boost immunotherapy by preferential depletion of immunosuppressive cell types such as Treg cells. T-BsAb treatment given after “debulking” by full-scale chemotherapy has been tested in clinical trials for patients with ALL (Brown et al. 2021; Fleming et al. 2019; Short et al. 2020) and lymphoma (Falchi et al. 2022; Ghosh et al. 2021; Katz et al. 2022b; Phillips et al. 2020), and the preliminary results have been promising.

For some malignancies, targeted therapy or immunotherapy could provide an alternative approach to achieve significant debulking and even remission with minimal negative impact on T-cell number and function. Inotuzumab ozogamicin is an antibody‒drug conjugate targeting CD22 that is expressed by most acute lymphoblastic leukemia (ALL). Sequential treatment with the single agent inotuzumab followed by blinatumomab has been demonstrated to have good efficacy and safety profiles in clinical trials with ALL patients (Ueda et al. 2022). Similarly, tyrosine kinase inhibitors have been used for prephase debulking for Ph-positive ALL before initiating treatment with blinatumomab in several clinical trials (Advani et al. 2021; Short et al. 2021). For multiple myeloma patients, there are more options for debulking without harming T-cell immunity. CD38 antibodies, proteasome inhibitors and immunomodulatory drugs (IMiDs) are all standard treatment options. Proteosome inhibitors and CD38 antibodies have a much lower impact on T-cell immunity than cytotoxic chemotherapies. IMiDs such as lenalidomide and pomalidomide have been found to enhance the potency of BCMA BsAbs in multiple myeloma preclinical models (Ghobadi et al. 2020). Thus, myeloma patients can achieve significant cytoreduction and even complete remission without significantly compromising T-cell immunity. Currently, there are multiple ongoing clinical trials combining IMiDs and/or proteasome inhibitors with BCMA BsAb.

Modify factors within tumor microenvironment

Different types of tumors can employ diverse mechanisms within TME to suppress T cell immunity. Thus, it is important to identify the key mediators to intervene. Gal-1 is one of the ideal targets for intervention within TME since it is overexpressed by many types of cancers and has been found to suppress T-BsAb mediated killing. Several human gal-1 neutralizing antibody have demonstrated efficacy in pre-clinical tumor models (Croci et al. 2014; Croci et al. 2012; Ouyang et al. 2011; Pérez Sáez et al. 2021; van Beijnum et al. 2016), although none have yet entered clinical trials. Alternative Gal-1 inhibitors, including glycan-based competitors, allosteric antagonists or peptidomimetics and Polymeric Nanoparticles have been developed and evaluated in pre-clinical tumor models (Gu et al. 2021; Rabinovich and Conejo-García 2016). Among these reagents, OTX008 demonstrated anti-cancer effects both in vitro and in vivo and had been evaluated in clinical trials for advanced solid tumors (Delord et al. 2013). GM-CT-01 is an inhibitor of both Gal-1 and Gal-3 that has progressed into phase I and II clinical trials and has demonstrated modest single agent efficacy in patients with metastatic colorectal cancer (Klyosov et al. 2012; Wdowiak et al. 2018). Another promising immunotherapy target within TME that has been found to compromise T-BsAb’s efficacy is IDO1. To date, many of IDO1 inhibitors have been developed and tested in clinical trials (Tang et al. 2021). Overall single agent efficacy of Gal-1 and IDO inhibitors is lackluster; however, these reagents can act synergistically with T-BsAb to overcome tolerogenic mechanisms within the TME and significantly enhance the therapeutic efficacy.

Regulatory T cells (Treg) is another key element of immunosuppressive TME that has been found to have significant impact on the clinical efficacy by T-BsAb. As reviewed elsewhere (Dees et al. 2021), many immunotherapy approaches targeting Tregs have developed, although none have been tested in combination with T-BsAb. Treg cells express high level of CD25, the high affinity receptor for interleukin-2 (IL-2). Denileukin diftitox, a fusion protein of IL-2 and diphtheria toxin can deplete Treg cells in animal models (Litzinger et al. 2007), and has been tested in several clinical trials. Depletion of Treg cells in human patients was not obvious in melanoma patients (Luke et al. 2016). For ovarian cancer patients, Denileukin diftitox did deplete the Treg cells, but clinical benefit was observed when used in combination with IFN-α (Curiel et al. 2014; Thibodeaux et al. 2021). CCR4 is another molecule expressed by activated/effector Treg cells and targeting CCR4 has been found to decrease the frequency of tumor infiltrating Treg cells and increase antitumor immunity. CCR4 antibodies (e.g. Mogamulizumab) and small molecule inhibitors (e.g. FLX475) have entered clinical trials and Treg depletion in human patients had been observed (Goyal et al. 2021; Ni et al. 2015; Zamarin et al. 2020). Foxp3 is the most specific marker of Treg cells. Although an intracellular protein and not accessible to antibodies, Foxp3 peptide epitopes presented by MHC molecules can be specifically recognized by T cells. A T cell receptor mimic antibody (Foxp3-#32) has been developed that recognizes a Foxp3-derived epitope in the context of HLA-A *02:01, and has demonstrated the capability to deplete Treg cells in preclinical studies (Dao et al. 2019). AZD8701, an antisense oligonucleotide for Foxp3, was found to relieve immunosuppression by Treg cells in tumor models (Revenko et al. 2022) and has entered clinical trials for advanced solid tumors (NCT04504669). Treg cell depletion can also be achieved by targeting TGF-β receptor and associated molecules (Moreau et al. 2022). Glycoprotein-A repetitions predominant (GARP), a cell surface docking receptor for TGF-β, is highly expressed on Treg cells. GARP antibody DS-1055a has been found to efficiently depleted Treg cells in animal models and is now in early phase clinical trial (NCT04419532) (Satoh et al. 2021).

Intra-tumoral inflammatory signals initiated by IFN-γ play a crucial role in recruiting cytotoxic T cells via the CXCL10/CXCR3 axis. Systemic therapy with IFN-γ is toxic and the side effects overlap with those observed with cytokine release syndrome, which makes it difficult to tolerate when used in combination with T-BsAb. Alternative delivery methods have been developed with the purpose of mitigating systemics toxicity. Intratumoral injection is a straightforward approach. Several methods have been developed and optimized to promote local delivery of IFN-γ, including liposomes, polymer gels, biodegradable microspheres, gene therapy, and magnetic or albumin nanoparticles (Castro et al. 2018). Moreover, proinflammatory stimulants such as Toll like receptor (TLR) agonist, which can induce local IFN-γ production by innate immune system, can be delivered in lieu of IFN-γ. Oncolytic virus (OV) can efficiently enhance inflammatory cytokines levels within the TME via activation of innate immune cells as well as directly in infected tumor cells (Bourgeois-Daigneault et al. 2016). As reviewed elsewhere, intra-tumoral/localized delivery can be successfully achieved for most inflammatory factors, including cytokines, TLR ligands, oncolytic virus and chemotherapy drugs (Humeau et al. 2021). Targeted delivery can also be achieved for systemically administrated therapeutic reagents. For example, systemically administrated immune stimulants can be directed to tumor site with single chain antibody fragment (Cauwels et al. 2018; Schau et al. 2019), or by other tumor targeting ligands such as GCNGRC (a CD13 ligand to target tumor vasculature) (Curnis et al. 2005). Tumor targeting peptides, such as poly-specific integrin-binding peptide (PIP), can be conjugated to cytokines or immunostimulants to trigger intratumoral production of interferon and enhances T cell infiltration (Miller, et al. 2022; Raab-Westphal et al. 2017; Shen, et al. 2015). Local production of T cell chemotaxis factors within the TME can also be enhanced by small molecule drugs targeting immune cell signaling pathways. Recently it has been reported that a MEK inhibitor synergizes with chemotherapeutic drugs to trigger CXCL10 secretion by cancer cells, thus improving T cell recruitment and sensitizing the tumor to immunotherapy (Limagne et al. 2022). RAR-related orphan receptor gamma (RORγt) is the master transcription factor for Th17 lineage. RORγt agonist (LYC -55,716), which is currently used in clinical trials for advanced solid tumors (Mahalingam et al. 2019), has been found to increase local production of CXCL10 by monocytes/dendritic cells to increase intratumoral T cell infiltration (Xia et al. 2022).

Conclusion remarks

T-BsAbs are one of the most promising immunotherapy strategies for treating cancer. Like CAR-T cells, T-BsAbs recognize surface expressed tumor-associated antigens via antibody scFvs fragments and engages T cells’ cytotoxicity machinery to achieve target cell killing. Compared to CAR T cells, T-BsAbs are off-the-shelf products that require minimal time for manufacturing and do not involve the biological and ethical risks associated with infusion of genetically engineered live human cells. T-BsAbs also have a better safety profile compared to CAR-T therapy, as treatment can be readily interrupted or terminated. However, to date, the clinical efficacies of almost all T-BsAbs are somewhat inferior to CAR-T cells targeting the same antigens. As discussed in this review, by studying the factors affecting T-BsAb’s efficacy, we can gain greater insight into the underlying mechanisms of treatment resistance, and therefore design novel combinational therapy strategies to improve efficacy, making it on par or even superior to corresponding CAR-T therapies.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the American Cancer Society, MRSG-19-033-01(ML) and American Society of Hematology, ASH Scholar Award (ML).