A bispecific T cell engager targeting Glypican-1 redirects T cell cytolytic activity to kill prostate cancer cells

Abstract

Background Glypican-1 is a heparan sulfate proteoglycan that is overexpressed in prostate cancer (PCa), and a variety of solid tumors. Importantly, expression is restricted in normal tissue, making it an ideal tumor targeting antigen. Since there is clinical and preclinical evidence of the efficacy of Bispecific T cell Engager (BiTE) therapy in PCa, we sought to produce and test the efficacy of a GPC-1 targeted BiTE construct based on the Miltuximab® sequence. Miltuximab® is a clinical stage anti-GPC-1 antibody that has proven safe in first in human trials. Methods The single chain variable fragment (scFv) of Miltuximab® and the CD3 binding sequence of Blinatumomab were combined in a standard BiTE format. Binding of the construct to immobilised recombinant CD3 and GPC-1 antigens was assessed by ELISA and BiaCore, and binding to cell surface-expressed antigens was measured by flow cytometry. The ability of MIL-38-CD3 to activate T cells was assessed using in vitro co-culture assays with tumour cell lines of varying GPC-1 expression by measurement of CD69 and CD25 expression, before cytolytic activity was assessed in a similar co-culture. The release of inflammatory cytokines from T cells was measured by ELISA and expression of PD-1 on the T cell surface was measured by flow cytometry. Results Binding activity of MIL-38-CD3 to both cell surface-expressed and immobilised recombinant GPC-1 and CD3 was retained. MIL-38-CD3 was able to mediate the activation of peripheral blood T cells from healthy individuals, resulting in the release of inflammatory cytokines TNF and IFN-g. Activation was reliant on GPC-1 expression as MIL-38-CD3 mediated only low level T cell activation in the presence of C3 cells (constitutively low GPC-1 expression). Activated T cells were redirected to lyse PCa cell lines PC3 and DU-145 (GPC-1 moderate or high expression, respectively) but could not kill GPC-1 negative Raji cells. The expression of PD-1 was up-regulated on the surface of MIL-38-CD3 activated T cells, suggesting potential for synergy with checkpoint inhibition. Conclusions This study reports preclinical findings into the efficacy of targeting GPC-1 in PCa with BiTE construct MIL-38-CD3. We show the specificity and efficacy of the construct, supporting its further preclinical development.

Role of glypican-1 in regulating multiple cellular signaling pathways

“…In addition to its potential diagnostic value, GPC1 has been evaluated as a potential target for antibody-based strategies, including anti-GPC1 mAb (11,88), antibodydrug conjugates (ADC) (89,90), CAR-T cell therapy (88,91), radiotherapy (92), photoimmunotherapy (93), and bispecific T cell engager (BiTE) (94) in preclinical settings (Table 2).…”

“…Moreover, a BiTE (MIL-38-CD3) was generated by combining the single chain variable fragment (scFv) of Miltuximab ® and the CD3 binding sequence of Blinatumomab. MIL-38-CD3 caused the activation of peripheral blood T cells, induced the release of inflammatory cytokines TNF and IFN-g, and eventually redirected those activated T cells to lyse GPC1 positive prostate cancer cells without any killing to GPC1 negative Raji cells(94).…”

Immunotherapy in Advanced Prostate Cancer—Light at the End of the Tunnel?

“…Thus, a BiTE targeting Glypican-1, a heparan sulfate proteoglycan that is overexpressed in PCa with a correlation to the Gleason score, has been designed on the basis of the CD3 binding sequence of blinatumomab in a standard BiTE format. Promising preclinical results have been reported including T cell activation and cytokine release [ 161 ]. In addition, a BiTE targeting disintegrin and metalloproteinase 17 (ADAM17), a transmembrane protease, and an anti-ADAM17 BiTEs-mediated specific lysis of ADAM17-expressing cells including PCa cell lines have been analyzed [ 162 ].…”

T Cell Bispecific Antibodies: An Antibody-Based Delivery System for Inducing Antitumor Immunity

“…Glypicans are a family of six heparan sulfate proteoglycans in vertebrates, and some have found to be expressed specifically in cancer [121]. The tumor-specific glypicans that have been utilized in T-BsAbs are glypican 1 and glypican 3 [55,122]. Mucin 16 is an example of a membrane glycoprotein targeted by a T-BsAb [123].…”

“…[ 97,111,116,120] • Aimed at mitigating on-target toxicity to normal tissues, more tumor-specific antigens, such as mutant proteins and complexes of a glycan and a protein, have been exploited as a T-BsAb target. [110,122,123] Format Selection…”