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Abstract
Background Multiple myeloma (MM) is a malignant plasma-cell disorder that accounts for accounts for 1% of all cancers and 10% to 15% of all hematologic cancers. Primary refractory to treatment and relapse resulted in a high death rate of MM patients. Bispecific antibodies (BsAbs) are promising therapeutic stratergies against MM by targeting CD3 on T cells and MM specific cell surface markers. GPRC5D is expressed on malignant bone marrow plasma cells, whereas normal tissue expression is limited to the hair follicle. Upregulated GPRC5D has been associated with unfavorable prognosis. Moreover, because GPRC5D expression is independent of BCMA, it could also be a promising target for patients who relapsed from BCMA-directed therapy. Here we report a novel 2+1 GPRC5D x CD3 TCE, ATG-021, which showed GPRC5D-dependent CD3 binding, and potent in vivo anti-tumor efficacy with reduced cytokine release.
Methods In the current study, we developed humanized antibodies targeting GPRC5D and CD3. The candidate parental antibodies were both derived from hybridoma of mouse and then antibody humanization was performed. The binding affinity of CD3 antibody was an important consideration impacting the distribution, efficacy and safety of the BsAbs. Therefore, CD3 antibodies with a wide affinity range were screened and compared. ATG-021 was constructed with two Fab arms targeting GPRC5D and one high affinity anti-CD3 scFv. It was evaluated through a series of preclinical in vitro and in vivo assays for efficacy and safety.
Results Compared to clinical benchmark TCE, Talquetamab and RG2634, ATG-021 has a higher affinity to GPRC5D positive cells. Although the parental CD3 antibody has high binding affinity with a single digit nM cellular binding EC50. ATG-021 exhibited extremely low binding capability to CD3+ Jurkat cells without GPRC5D-crosslinking. Whilst ATG-021 induced more potent T cell dependent cytotoxicity of MM cell lines expressing different level of GPRC5D compared with benchmarks, while demonstrating no effect on GPRC5D negative cells (figure 1). ATG-021 induced lower ex-vivo cytokine release by human PBMC compared with benchmarks. The in vivo efficacy of ATG-021 was evaluated in PBMC humanized MM.1S myeloma xenograft model. ATG-021 showed better in vivo efficacy compared with Talquetamab, resulted in complete tumor remission in all mice. Notably, the serum concentration of proinflammatory cytokines, such as IL-2, INF-γ, and TNF-α, were significantly lower in ATG-021 treated group, suggesting low risk of cytokine release syndrome (figure 2).
Conclusions The result demonstrates the optimum efficacy and safety of ATG-021 which warrants further clinical evaluation against MM.
Ethics Approval Crownbio: 001243
ATG-021 induced T cell-mediated cytotoxicity against MM cells in GPRC5D-expression-dependent manner. The cell surface copy numbers of GPRC5D on MM cell lines were determined by flow cytometry analysis. Daudi is a GPRC5D negative cell line. The result showed that ATG-021 exhibited specific and more potent in vitro T cell dependent cytotoxicity compared with BMKs. The cytotoxicity was positively correlated with surface copy numbers of GPRC5D.
ATG-021 demonstrates in vivo efficacy and good safety profile in human PBMC reestablished MM tumor model. (A) Schematic of the treatment design for the PBMC humanized MM1S CDX model (top panel) and average tumor growth curves (bottom) of MM1S. Each mouse was inoculated subcutaneously in the right front flank region with MM.1S tumor cells (1x107) in 0.1 ml of PBS for tumor development on day - 12; hPBMC (1 x 107/mouse, i.v., 0.1mL) was inoculated on day -5; The randomization was started when the mean tumor size reached approximately 75 mm3. Blood samples were collected at 2 hours after the first dosing. ATG-021 produced significant anti-tumor efficacy with TGI value of 102.46% compared with 57.82% of the BMK on D20. Meanwhile, All of ATG-021 treated mice were tumor free. (B) The concentration of cytokines in the serum collected 2 hours after the first dosing from mice shown in (A) Compared with BMK, ATG-021 induced reduced release of cytokines, such as IL-2, INF-γ and TNF-α, indicating lower risk of cytokine release syndrome.
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