Human CCR8 Stable Cell Line-CHO-K1

CCR8 is primarily found on tumor-infiltrating Treg cells. Due to the anti-tumor effects demonstrated by CCR8-antibody-mediated Treg depletion, CCR8 has become a highly sought-after target. Single-cell RNA sequencing analysis revealed that CCR8-positive Treg cells constitute a small subpopulation that co-expresses CCR8 and CTLA-4. Therefore, researchers have proposed a novel bispecific antibody targeting both CCR8 and CTLA-4, which has the potential to enhance T cell activation while selectively depleting tumor-infiltrating Treg cells. The candidate molecule, 2MW4691, was developed with a tetravalent symmetric structure, maintaining strong binding affinity for CCR8 while exhibiting relatively weak binding to CTLA-4. This selective binding ability enables 2MW4691 to target and deplete tumor-infiltrating Treg cells with enhanced specificity. In vitro studies demonstrated that the antibody exhibited antibody-dependent cellular cytotoxicity (ADCC) against Treg cells expressing high levels of CTLA-4, but had no effect on CD8 T cells with relatively low cell surface CTLA-4 levels. Furthermore, 2MW4691 inhibited the CTLA-4 pathway and enhanced T cell activation. The in vivo therapeutic efficacy of 2MW4691 was further demonstrated using humanized hCCR8 or hCTLA-4 mouse models, as well as an hCCR8/hCTLA-4 double knock-in mouse model. In cynomolgus monkeys, 2MW4691 was well tolerated, exhibited expected pharmacokinetic properties, and had minimal effects on peripheral T cell populations. These encouraging preclinical results support further evaluation of 2MW4691 as a next-generation Treg therapy in clinical trials.

The ADCC activity of 2MW4691 was assessed using a reporter gene bioassay, using FcγRIIIa-expressing Jurkat cells as effector cells and hCCR8 overexpressing CHOK1 cells or hCTLA-4 overexpressing CHOK1 cells as target cells. The results showed that 2MW4691 exhibited potent ADCC activity against hCCR8 overexpressing CHOK1 cells, similar to that of the parental antibody, B9B11, with an EC50 value of 0.062 nM for 2MW4691 and 0.027 nM for B9B11 (Figure 1A). The C-terminal structure of the CTLA-4 binding arm attenuates its ADCC activity. However, a non-fucosylated Fc fragment with enhanced hFcγRIIIa binding capacity restored its function. As shown, the ADCC activity of 2MW4691 against CHOK1-hCTLA-4 was significantly stronger than that of the parental ipilimumab antibody using a normal human IgG1 Fc (Figure 1B). This suggests that the structural design of 2MW4691 does not impair the interaction between non-fucosylated hIgG1Fc and hFcγRIIIa. Therefore, 2MW4691, through its dual binding to CCR8 and CTLA-4 and enhanced ADCC activity, can maximize the depletion of intratumoral Tregs.

Figure 1. (A) ADCC effect of 2MW4691 on CHOK1-hCCR8 was performed based on a reporter-based bioassay. (B) ADCC effects of 2MW4691 on CHOK1-hCTLA4. (Guo C, et al., 2024)