1061 CLSP-1025, a novel bispecific T-cell engager targeting a p53 R175H mutant peptide presented by HLA-A*02:01

Background

CLSP-1025 is a T-cell engager (TCE) designed to treat p53 R175H mutant tumors in patients who are positive for HLA-A*02:01. CLSP-1025 is an asymmetric bispecific single-chain diabody (scDb) Fc fusion protein with monovalent binding specific to the p53(R175H)_168–176 peptide bound to HLA-A*02:01 on cancer cells and monovalent binding to CD3ε on T cells. The anti-peptide HLA (pHLA) variable domain of CLSP-1025 was specifically developed to recognize cells presenting the mutant p53(R175H)_168–176 peptide bound to HLA-A*02:01 and ignore cells presenting the wild-type p53_168–176 peptide.¹

Methods

To demonstrate CLSP-1025 pHLA binding specificity, cocultures of PBMCs with various cell lines expressing HLA-A*02:01 and/or p53(R175H) were conducted. Presentation of potential cross-reactive peptides from the human proteome on HLA-A*02:01 was assessed using a positional scanning peptide array added to a coculture of T2 and Jurkat-NFAT reporter cells. To evaluate potential off-target binding of CLSP-1025 to other HLA alleles, T cell activation was assessed in cocultures with K562 cells individually expressing >250 HLA alleles, representing >95% of the population. The pharmacokinetics (PK) of CLSP-1025 were measured in animal models to evaluate the effect of fusing the scDb to an Fc domain. A structure-based model was generated with simultaneous binding to CD3 and the p53(R175H)_168–176 peptide bound to HLA-A*02:01.

Results

Only cell lines that expressed both HLA-A*02:01 and the mutant p53(R175H) could be recognized by CLSP-1025 and induce T cell activation. Based on the activity of CLSP-1025 in a cell-based assay with an array of positional scanning peptides, CLSP-1025 specifically recognized p53(R175H)_168–176 and did not recognize any naturally occurring peptides bound to HLA-A*02:01. In addition, the vast majority of the HLA alleles expressed on K562 cells showed no off-target activity with CLSP-1025. The HLA alleles that showed activity with CLSP-1025 will be used as exclusion criteria in our planned clinical trial. The PK profile of CLSP-1025 in animal models was similar to an IgG4 control antibody, with an increased half-life compared to the scDb control (without the Fc fusion).

Conclusions

The nonclinical data presented are in support of CLSP-1025 being evaluated in a clinical trial of HLA-A*02:01-positive patients with an advanced solid tumor expressing the R175H mutant p53. We expect that CLSP-1025 will be the first TCE targeting a common cancer neoantigen to be evaluated in the clinic. An IND application filing is planned for Q4 of 2024.

Acknowledgements

We would like to express our gratitude to Drew Pardoll, Bert Vogelstein, Nickolas Papadopoulos, Shibin Zhou, Ken Kinzler, and Kellie Smith for their invaluable scientific contributions. Their expertise, dedication, and insightful input were instrumental in shaping and refining our work. Thank you for your continued unwavering support and collaboration.

Reference

Hsiue EH, Wright KM, Douglass J, Hwang MS, Mog BJ, Pearlman AH, Paul S, DiNapoli SR, Konig MF, Wang Q, Schaefer A, Miller MS, Skora AD, Azurmendi PA, Murphy MB, Liu Q, Watson E, Li Y, Pardoll DM, Bettegowda C, Papadopoulos N, Kinzler KW, Vogelstein B, Gabelli SB, Zhou S. Targeting a neoantigen derive common TP53 mutation. Science. 2021;371(6533):eabc8697.

Ethics Approval

All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Charles River Accelerator and Development Labs.