Cytotoxic T cell lymphocyte-associated protein 4 (CTLA4) is a transmembrane protein member of the CD28 family receptor expressed by T cells, constitutively by CD4+ CD25+ regulatory T cells (T_reg) and only following activation by cytotoxic CD8+ effector T cells (T_eff) (13). In general, CTLA4 counteracts the activity of CD28, a co-stimulatory molecule expressed by T cells. Following antigen recognition, CD28 binds to CD80 (also known as B7-1) and CD86 (also known as B7-2), expressed by activated antigen-presenting cells (APCs), and transmits a co-stimulatory signal for T cell activation and proliferation (14). CTLA4 is placed in intracellular vesicles and directed on the T cell surface only after T cell receptor (TR) activation (15), where it binds to CD80/CD86 with greater affinity than that of CD28, transmitting co-inhibitory signals to control T_eff cell activation (14, 16) and preventing the potential damage by an excessive inflammatory response (14–16).

IMGT/mAb-DB lists eight mAbs targeting CTLA4 for cancer immunotherapy. Ipilimumab (YERVOY®, IMGT/mAb-DB ID: mAbID 180) blocks the binding of CTLA4 with its ligands, then inhibits CTLA4-mediated downregulation of T cells and promotes the interaction of CD80/CD86 with CD28. This interaction stimulates the immune response by increasing T cell expansion and by enhancing the cytotoxic T lymphocyte (CTL)-mediated anti-tumor immune response (17). By its effector properties, the IgG1-Fc region of ipilimumab induces antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for enhanced anti-tumor efficacy by reducing T_reg cells (17, 18) ( Figure 2 ). Thus, ipilimumab’s MOA is ‘Blocking - Immunostimulant, Fc-effector function’ ( Table 2 ). In contrast, tremelimumab (mAbID 248) differs from ipilimumab in its Fc region and its ability to engage FcγRs. The IgG2-Fc region of tremelimumab shows reduced affinity to various FcγRs (19) and presents minimal ADCC activity against cancer cells (20). Therefore, tremelimumab’s MOA is ‘Blocking - Immunostimulant’ without Fc-effector function to deplete T_reg cells.

It is worth noting that antibodies with high affinity to FcγRs may increase the anti-tumor activity (18). Thus, enhancing FcγR binding by modifying the Fc region provided a generation of engineered anti-CTLA4 antibodies with increased anti-tumor activity by T_reg cells depletion (18). These engineered antibodies act in the same way as ipilimumab, blocking CTLA4 to stimulate CTL activity, and enhancing Fc-effector function against T_reg cells ( Table 2 ). Botensilimab (mAbID 1123), an anti-CTLA4 antibody with enhanced Fc-effector function, has been engineered to improve FcγRIIIa affinity while decreasing FcγRIIb binding and boosting effector functions such as ADCC (21). Table S1 and IMGT Biotechnology page (https://www.imgt.org/IMGTbiotechnology/ > Antibody glycosylation and effector properties > IMGT engineered variant nomenclature: IGHG variants) describe in detail and in a standardized format all engineered antibodies Fc variants (22) provided in this study.

Programmed death receptor 1 (PDCD1, PD-1) is a member of the CD28/CTLA4 family receptors that downregulates T cell activation, proliferation, and cytotoxic activity in peripheral tissues during inflammatory responses. PDCD1 is expressed by activated T cells, B cells as well as natural killer (NK) cells and upregulated on T cells after persistent antigen exposure, preventing autoimmunity (23). The expression of its ligands, programmed death ligand 1 (CD274, PD-L1) and programmed death ligand 2 (PDCD1LG2, PD-L2, CD273), is induced by inflammatory cytokines released after TR activation, on tumor cells (23, 24). Thus, the binding of PDCD1 to its ligands downregulates T_eff cell activity and promotes tumor escape (23, 25). PDCD1 and other co-inhibitory molecules, such as LAG3, could lead to T cell exhaustion. Exhausted T_eff cells lose several functions such as interleukin 2 (IL2) production, proliferative capacity, and cytotoxicity (26).

Monoclonal antibodies targeting the PDCD1 axis include 1) molecules directed to PDCD1, blocking receptor interaction with both ligands, as well as 2) antibodies against CD274, blocking ligand interaction with PDCD1. Both therapeutic approaches enhance immune mediated anti-tumor responses in several cancer types, including melanoma and bladder cancer (27, 28).

Currently, IMGT/mAb-DB includes 32 anti-PDCD1 mAbs for cancer immunotherapy. To date, four anti-PDCD1 mAbs have been approved by FDA and/or EMA for different cancer types, nivolumab (OPDIVO®, mAbID 424), pembrolizumab (KEYTRUDA®, mAbID 472), dostarlimab (JEMPERLI, mAbID 849) and cemiplimab (LIBTAYO®, mAbID 846). These antibodies block the PDCD1 receptor from binding to both ligands, CD274 and PDCD1LG2. This process reverses T cell inactivation and restores immune function through the activation of CTL against tumor cells (29, 30) ( Figure 3 ). Therefore, the antibodies’ MOA is ‘Blocking - Immunostimulant’ ( Table S2 ).

Most of the anti-PDCD1 mAbs are IgG4 subclass, which presents low affinity to FcγRs (31) and little ability to mediate cellular cytotoxic effector functions against T_eff cells. Therapeutic IgG4 mAbs are designed to stabilize and prevent half-IG molecules by a single mutation (IMGT variant G4v5 h P10) introduced in the hinge region (32, 33). Attempts have been made to introduce mutations in IgG4 antibodies to completely abolish FcγR binding and avoid any cytotoxicity, an example being tislelizumab (mAbID 757) (34, 35). The usage of IgG1 subclass with abolished FcγR binding was explored, as in the case of penpulimab (mAbID 1093) (36) and some other additional mAbs ( Table S2 ). For more details about IMGT engineered variants, see Table S1 .

As mentioned above, there exist antibodies which bind to CD274, blocking the ligand-receptor interaction. IMGT/mAb-DB includes 17 anti-CD274 mAbs, of which three have been approved by FDA and/or EMA, atezolizumab (TECENTRIQ®, mAbID 526), durvalumab (IMFINZI™, mAbID 528) and avelumab (BAVENCIO®, mAbID 512).

Blockade caused by anti-CD274 mAb binding restores T_eff cell activation and enhances cytotoxic immune response against tumor cells. The anti-CD274 antibody does not block the PDCD1/PDCD1LG2 pathway, allowing inhibitory signals to maintain immune tolerance (37) ( Figure 4 ). Several anti-CD274 antibodies have been developed to avoid Fc effector functions by mutations in the IgG1-Fc region or by using IgG4 subclass ( Table S1 ), to prevent T_eff cells depletion, such as atezolizumab (38) and durvalumab (39). Their main MOA is ‘Blocking - Immunostimulant’ ( Table 3 ).

Unlike other anti-CD274 mAbs which are designed to eliminate any ADCC/CDC activity as a precaution of off-tumor cytotoxicity, the IgG1-Fc region of avelumab binds to FcγRs on NK cells and directly mediates cellular cytotoxicity against tumor cells (40). According to its properties, its MOA is classified as ‘Blocking - Immunostimulant, Fc-effector function’.

Inducible T cell co-stimulator (ICOS) belongs to the CD28/CTLA4 family of receptors that stimulates immune response and homeostasis (41). ICOS is a homodimeric transmembrane protein expressed upon TR engagement and CD28 signaling on activated T cells (42, 43). The binding of ICOS with its ligand, ICOSLG (CD275) expressed by APCs (44), promotes proliferation and differentiation of T_eff and T_reg cells (45).

ICOS co-stimulation promotes, on the one hand, anti-tumor CTL activation which produces inflammatory cytokines (43, 45) and, on the other hand, T_reg cells proliferation which enhances tumor activity (46). This dual controversy effect of ICOS/ICOSLG interaction represents an attractive target to explore for mAbs engineering. It is worth noting that ICOS expression varies depending on T cell subtypes and on their localization; intratumoral T_reg cells exhibit higher ICOS expression than T_eff cells (43). This differential expression plays an important role in the MOA of the engineered mAbs as presented below.

mAbs targeting ICOS have been developed, however none of them have reached the clinic yet. IMGT/mAb-DB lists three mAbs targeting ICOS, with an assigned INN name, for cancer immunotherapy. A first approach was to develop agonist mAbs targeting ICOS that activate this signaling and exert anti-tumor activity by co-stimulating low ICOS+ T_eff cells, thereby promoting activation and expansion of T_eff cells, which in turn increase cytotoxic activity against tumor cells.

Feladilimab (mAbID 1010), a humanized IgG4-kappa mAb, acts as an agonist of ICOS, activating T_eff cells, which mediate a cytotoxic anti-tumor immune response (47). Thus, feladilimab’s main MOA is ‘Agonist - Immunostimulant’ ( Figure 5 and Table 4 ). Feladilimab has an engineered Fc region (IMGT variant G4v3 CH2 E1.2) which reduces C1q and FcγR binding ( Table S1 ) and avoids ICOS+ T_eff cells depletion by ADCC activity. However, its agonist activity is not sufficient to induce anti-tumor cytotoxicity (41), therefore feladilimab was discontinued in phase II trials due to cancer progression.

An alternative approach aims to deplete intratumoral high ICOS+ T_reg cells by Fc effector functions (48, 49). Equivalently to feladilimab, alomfilimab (mAbID 1120) and vopratelimab (mAbID 801) activate and induce the proliferation of low ICOS+ T_eff cells, enhancing CTL-mediated anti-tumor immune response. Nevertheless, in addition to feladilimab’s MOA, these antibodies have an effect of ‘Fc-effector function’ in cancer treatment and preferentially deplete high ICOS+ T_reg cells by Fc effector function (50, 51) ( Figure 5 and Table 4 ). The T_reg cells depletion ability is triggered when high levels of antigen are found on the surface of the target cell, the case of T_reg cells, promoting signaling through FcγR clusters in immune effector cells and strong ADCC against target cells (52).

Lymphocyte activation gene-3 protein (LAG3, also known as CD223) is a member of IG superfamily expressed by T cells upon antigen stimulation, B cells and NK cells (53, 54). The LAG3 signaling pathway plays a critical role inhibiting T cell activation and proliferation while stimulating differentiation into T_reg cells which leads to immunosuppression (55). The LAG3 inhibitory function is closely correlated with the LAG3 expression levels on the T cell surface (56). Thereby, a constant antigen stimulation of the T cells in the tumor microenvironment (TME) leads to a high expression of LAG3 and of other co-inhibitory receptors on T cells, such as PDCD1, promoting immune escape in tumors and exhaustion of T cells that lose their effector functions (57, 58).

LAG3 binds the major histocompatibility class II (MH2) and several other ligands, including galectin-3 (LGALS3, Gal-3), liver-secreted fibrinogen-like protein 1 (FGL1), and C-type lectin domain family 4 member G (CLEC4G, LSECtin). Binding to MH2, LAG3 inhibits CD4+ T cells activation (59). LGALS3 is secreted by many tumor cells and associated with neoplastic transformation. LAG3-LGALS3 interaction inhibits T cell responses promoting cancer progression (60). FGL1 plays a role in proliferation and metabolism and can be expressed by tumor cells. Its binding to LAG3 results in T cell depletion, mechanisms of immune evasion of the cancer and resistance to anti-PDCD1/CD274 therapy (61). Ultimately, CLEC4G is a type II transmembrane protein expressed by APCs and certain types of cancer. It interacts with LAG3 and prevents T_eff cell responses in melanoma cells (62).

There are several anti-LAG3 mAbs in different clinical trials; most of them are hinge-stabilized IgG4 antibodies (IMGT variant G4v5 h P10) to limit the FcγR binding. IMGT/mAb-DB includes seven anti-LAG3 antibodies, with an assigned INN name. Relatlimab (OPDUALAG™, mAbID 781), recently approved by FDA in 2022, binds to LAG3 overexpressed by T cells in the TME and blocks its binding to its ligands, activating exhausted T cells and enhancing the CTL-mediated immune response against tumor cells (63) ( Figure 6 ). In combination with nivolumab, relatlimab demonstrated a progression-free survival increased when compared to nivolumab alone (64, 65). The main MOA of relatlimab is ‘Blocking – Immunostimulant’ ( Table 5 ).

Assays have been made to introduce mutations in IgG4 antibodies in order to eliminate FcγR binding, such as fianlimab (mAbID 950) (66). The usage of IgG1 subclass with abolished FcγR binding was also explored, as in the case of tuparstobart (mAbID 1371) (67) ( Tables 5 , S1 ). This approach allows to design blocking mAbs totally silent to avoid cytotoxic activity against the target immune cells.

CD40 (also known as TNFRSF5) is a co-stimulatory immunoreceptor member of the TNFR superfamily which is expressed by APCs and B cells. CD40 is involved in T cell activation by dendritic cells (DCs) and in antibody class-switching in B cells (68, 69). Upon activation by its ligand, CD40LG (CD154) expressed by activated T cells, CD40 promotes APC activation and differentiation. It also promotes a bi-directional signaling between T cells and APCs, that amplifies a stimulatory immune response, increasing T cell expansion and enhancing CTL activity in the TME (70). In cancer immunotherapy, through the binding with agonist mAbs, CD40 can stimulate T cells and APCs to attack tumor cells (71, 72). In contrast, CD40 is highly expressed by B cell malignancies including non-Hodgkin’s lymphoma (NHL), chronic lymphocytic leukemia (CLL) and myeloma, and in that case, it promotes proliferation and inhibits the apoptosis of malignant B cells (73).

Monoclonal antibody therapy targeting CD40 acts via multiple mechanisms to stimulate anti-tumor immunity in a wide range of lymphoid and solid malignancies (74). Several mAbs targeting CD40 have been developed in the oncology domain. Twelve mAbs targeting CD40, with an assigned INN name, are integrated in IMGT/mAb-DB. However, none of them has reached the clinic yet. Agonist mAbs stimulate CD40 signaling of different pathways depending on the target cell type. However, in all cases, they promote cell activation and proliferation of several immune cells that contribute to anti-tumor activity. An ideal agonist mAb can lead to CD40 crosslinking to promote greatest agonist and anti-tumor activities with minimal adverse events (75).

The anti-CD40 mAb, selicrelumab (mAbID 723), has a strong agonist activity, since it does not block CD40LG binding site, and reduces CDC and ADCC activities against the target cell, thanks to its belonging to the IgG2 subclass (76, 77). In addition, the IgG2 subclass aids the agonist effect due to its lack of flexibility in the hinge region, which may trigger CD40 aggregation without FcγR engagement (78). The MOA of selicrelumab is ‘Agonist - Immunostimulant’.

Most agonist mAbs targeting CD40 are of the IgG1 subclass with different levels of affinity to CD40 and depend on crosslinking with FcγRs in order to facilitate CD40 aggregation for APCs activation (79, 80) as well as an ADCC activity against tumor cells (81, 82) ( Table 6 ). Engineered mAbs with enhanced binding to FcγRIIIa and ADCC activity have been developed to increase CD40 crosslinking ( Table S1 ). However, an increased affinity for FcγR could enhance not only anti-tumor activity but also adverse events, such as thrombocytopenia and transaminitis (80).

On the other hand, agonist IgG1 mAbs targeting CD40 with Fc engineered to enhance FcγRIIb binding may mediate antibody crosslinking and strong CD40 signaling while reducing the binding with FcγRIIIa in order to inhibit ADCC activity on APCs expressing CD40 (83, 84) ( Figure 7 ). As FcγRIIb is mainly expressed by B cells in tumor tissues, the antibodies are expected to be more active in the TME, depending on the FcγR crosslinking and with lower toxicity. The MOA of these antibodies is ‘Agonist - Immunostimulant, FcγR crosslinking’.

In contrast to the agonist mAbs, antagonist anti-CD40 mAbs block the CD40/CD40LG pathway to inhibit the proliferation of malignant B cells, which highly express CD40, such as in CLL and NHL. In IMGT/mAb-DB, we find one blocking mAb anti-CD40, lucatumumab (mAbID 176), that mediates ADCC and ADCP against tumor cells (85). Thus, lucatumumab’s MOA is ‘Blocking - Fc-effector function’. However, this antibody was discontinued in 2013 after being explored for the treatment of multiple myeloma and follicular lymphoma with modest efficacy as monotherapy (86).