LALA Mutations Technology

A specific combination of amino acid mutations in the Fc region can alter the binding affinity of the antibody to FcγR, thereby altering the strength of the antibody's effector function. The most common LALA mutations (L234A/L235A) result in altered antibody affinity for FcγR (elimination of binding to low-affinity FcγR and decreased binding to FcγRI), resulting in significantly reduced ADCC and CDC activity.

Background

Glucocorticoid-induced TNF receptors (GITRs) are members of the TNF receptor superfamily. As a stimulatory molecule of CD4⁺ and CD8⁺ naïve T cells, GITR has the ability to induce T cell proliferation and enhance effectors. It is expressed in cytotoxic T cells and Treg cells. Tumor antigens can activate and increase the expression of GITR on T cells. Preclinical studies have shown that the activation of GITR could enhance immunity by activating T cells and inhibiting Treg cell activity. However, clinically, GITR agonists have not shown good therapeutic efficacy. This may be caused by the inability of agonists to effectively induce receptor-mediated multimerization.

Research Results

The aggregation of GITR is essential for T cell activation.
Fig.1 The aggregation of GITR is essential for T cell activation.1,2	In this study, different forms of GITR-L fusion proteins and different forms of GITR agonist antibodies were constructed (Fig.1 d), and these molecules with different structures could induce the aggregation of GITR to varying degrees. The results showed that the Fc-fusion GITR-L trimer (([(GITR-L)3]2)6-Fc-RGY) with RGY mutation (promoting the formation of six concrete) had the best effect and was much stronger than the RGY-mutated GITR agonist antibody (GITR-RGY mAb). This also proves that the activation of GITR-related pathways can be independent of the aggregation of GITR by the antibody FcγR. Therefore, in this study, a PD-1/GITR-L antibody fusion protein was constructed, hoping to use PD-1 to aggregate GITR and activate related signaling pathways through GITR-L trimers, thereby activating and promoting the proliferation of T cells. In CD3-activated NFκB-GITR+ reporter cells, the activation of the NFκB pathway by PD-1/GITR-L is dependent on the expression of PD-1 (Fig.1 h-i).
Co-expression of PD-1/GITR on T cells and construction of PD-1/GITR-L bispecific molecules.
Fig.2 Co-expression of PD-1/GITR on T cells and construction of PD-1/GITR-L bispecific molecules.1,2	The basis for the effect of the PD-1/GITR-L antibody fusion protein is the co-expression of PD-1 and GITR, which were co-expressed on TCR-activated T cells by T cell analysis of PBMCs, and also co-expressed on tumor-infiltrating lymphocytes (Fig.2 a-b). In addition, PD-1 and GITR were found to be co-expressed on T cells in head and neck squamous cell carcinoma (Fig.2 c-e). The PD-1/GITR-L antibody fusion protein is constructed with a 2+2 structure, in which PD-1 uses a 12A11 monoclonal antibody, while GITR-L is fused to the end of Fc in the form of a trimer. For Fc, LALA mutations are used to reduce the associated effector function of Fc (ADCC, CDC, etc.). In vitro binding, the PD-1/GITR-L antibody fusion protein binds well to both targets at both the protein and cellular levels (Fig.2 f-m).
In vivo efficacy of PD-1/GITR-L antibody fusion proteins.
Fig.3 In vivo efficacy of PD-1/GITR-L antibody fusion proteins.1,2	In this study, PD-1 or GITR single humanized mice, or double humanized mice of both, were constructed, and the corresponding hybrid antibody (anti-muPD-1-huGITR-L or anti-huPD-1-muGITR-L) was constructed. In a mouse model, these hybrid antibodies were only effective against their mono-humanized mice (Fig.3 a-b), suggesting that the efficacy of the antibody fusion protein requires simultaneous binding of two pairs of two targets. Similarly, PD-1/GITR-L also inhibited tumor growth in a dual-target humanized mouse model (Fig.3 i-j). The number of TIGIT+CD4+, Ki67+, TCM CD4+, and CD8+ T cells in the blood of mice increased after treatment with PD-1/GITR-L antibody fusion protein. In tumors, the number of Ki67+, CD226+, and KCNA3+ CD8T cells increased, while the number of SLAMF6–TIM3+ Treg and CD8+ TTE (tired T cells) decreased (Fig.3 c-h).

Creative Biolabs offers a range of Fc modification services to help clients achieve their desired treatment results. You can visit our Therapeutic Fc Engineering Technology unit for more information!

References

1. Chan, Sarah et al. "An anti-PD-1-GITR-L bispecific agonist induces GITR clustering-mediated T cell activation for cancer immunotherapy." Nature Cancer. 3,3 (2022): 337-354.
2. Distributed under Open Access license CC BY 4.0, without modification.