HPN328, a new T cell–activating construct for treating solid tumors

‍‍

Molloy et al. HPN328, a Trispecific T Cell–Activating Protein Construct Targeting DLL3-Expressing Solid Tumors, Molecular Cancer Therapeutics, 2024

‍
Small-cell lung cancers (SCLC) are very aggressive and lethal types of cancer, with limited and not very effective treatment options¹. DLL3 (delta-like ligand 3) is known for being highly expressed on the surface of SCLC tumor cells and other malignancies, but only expressed at low levels in healthy tissue, making DLL3 a promising target for anti SCLC-therapies^2,3. In recent years, different types of immunotherapies have been developed to specifically target DLL3, and with the aim of improving the outcome of small-cell lung cancers⁴. Of relevance is the use of T-cell engagers, which are antibodies that direct T cells to a specific tumor antigen⁵.

‍

A new T-cell engager with extended half-life that selectively kills DLL3-expressing cancer cells

Molloy and colleagues described a new tri-specific T-cell activating construct (TriTAC), named HPN328⁶. This construct comprises three domains: 1) a single-domain antibody (sdAb) that recognizes DLL3, allowing the specific recognition of cancer cells, 2) a sdAb that binds to serum albumin, which prolongs the construct’s half-life, and 3) a single-chain fragment variable that is specific to the CD3ε subunit of the T-cell receptor and allows for T-cell activation.

The cytotoxicity potential and specificity of HPN328 was confirmed upon observation of T-cell-dependent cytotoxicity exclusively of cancer cells that expressed DLL3. Furthermore, analysis of the binding affinity and cross-reactivity of HPN328 showed that the construct had a high affinity to both human and cynomolgus DLL3, CD3ε and albumin. The authors tested if HPN328 induced T-cell activation in vitro and observed that HPN328 treatment of co-cultures of DLL3-expressing tumor cells and T cells from diverse donors led to a dose-dependent increase in CD25 and CD69 expression, and the secretion of IFNγ and TNFα, validating this construct as a T-cell activator.

‍

The anti-tumor activity of HPN328 was confirmed in a humanized CD3ε mouse model

In vivo, HPN328 treatment of DLL3-expressing SCLC xenografts in immunocompromised mice resulted in tumor growth inhibition, in addition to extensive T-cell recruitment to the tumors. To assess the activity of HPN328 in immunocompetent mice, a mouse model expressing the human CD3ε fragment that binds to TriTAC constructs was developed by genOway. A similar prevalence of the different subpopulations of lymphoid cells, dendritic cells and macrophages was observed between WT and hCD3ε mice. Additionally, T cells derived from hCD3ε mice activated ex vivo by CD3 antibodies secreted IFNγ and IL-2, confirming the functionality of the CD3-TCR complex in the model. Tumor inhibition by HPN328 was then confirmed in hCD3ε mice inoculated with MC38 cells expressing human DLL3 (MC38-hDLL3) (see figure below, panel A), as well as T-cell activation (panel B). The construct induced long-term anti-tumor activity, as hCD3ε mice inoculated with MC38-hDLL3 cells and treated with the highest dose of HPN328 were refractory to rechallenge with tumor cells (panels C, D, E). The development of immune memory was further confirmed by quantification of the presence of CD8⁺ memory T cells (panel F).

Figure 4 in the paper: A) MC38-hDLL3 cells were implanted into hCD3ε mice and treated with HPN328 or vehicle on days 9–18. B) Percentage of CD69 and CD25 of CD8-positive T cells in tumors at 2 days post treatment. C) Schematic for rechallenge study design. D) MC38-hDLL3 cells were implanted into hCD3ε mice and treated with HPN328 or vehicle on days 8–17. E) Mice previously cured from (D) were rechallenged with parental MC38 tumor cells on study day 30. F) On study day 60, the spleens from mice in (E) were processed and memory T cells were analyzed by flow cytometry. Adapted from Molloy et al., 2024⁶.

Given the cross-reactivity of HPN328 to cynomolgus CD3, DLL3 and albumin, PK/PD studies of HPN328 were performed in cynomolgus monkeys. HPN328 displayed an extended half-life in serum, was well tolerated, and induced a short-term boost in cytokine release.

Altogether, the data proves that DLL3 can be used as a valuable and specific target by TriTACs. T-cell engagers are a promising strategy for treatment of solid tumors, as shown by the recruitment of T cells to the tumor microenvironment, and the tumor-inhibition effect. Moreover, the development of the hCD3ε mouse model by genOway enables the study of immunotherapies such as T-cell engagers in immunocompetent in vivo models with high predictability, helping to improve the outcome of clinical studies. This model was also shown to be a valuable asset for the study of long-term immunological memory. Thanks to the preclinical efficacy and safety studies in humanized mice and cynomolgus monkeys, HPN328 is currently in clinical trials in patients with DLL3-expressing cancers.

A hCD3ε mouse model is available off the shelf at genOway, designer and provider of multiple preclinical models in several research areas, including immuno-oncology, metabolism, cardiovascular diseases and neuroscience.

‍

References

1. Blackhall, F. et al. Treatment patterns and outcomes among patients with small-cell lung cancer (SCLC) in Europe: a retrospective cohort study. BMJ Open 13, e052556 (2023).
2. Owen, D. H. et al. DLL3: an emerging target in small cell lung cancer. J. Hematol. Oncol.J Hematol Oncol 12, 61 (2019).
3. Sabari, J. K., Lok, B. H., Laird, J. H., Poirier, J. T. & Rudin, C. M. Unravelling the biology of SCLC: implications for therapy. Nat. Rev. Clin. Oncol. 14, 549–561 (2017).
4. Iams, W. T., Porter, J. & Horn, L. Immunotherapeutic approaches for small-cell lung cancer. Nat. Rev. Clin. Oncol. 17, 300–312 (2020).
5. Staerz, U. D., Kanagawa, O. & Bevan, M. J. Hybrid antibodies can target sites for attack by T cells. Nature 314, 628–631 (1985).
6. Molloy, M. E. et al. HPN328, a Trispecific T Cell–Activating Protein Construct Targeting DLL3-Expressing Solid Tumors. Mol. Cancer Ther. OF1–OF11 (2024) doi:10.1158/1535-7163.MCT-23-0524.