Abstract
BackgroundImmunotherapeutic approaches designed to augment T and B cell mediated killing of tumor cells has met with clinical success in recent years suggesting tremendous potential for treatment in a broad spectrum of tumor types. After complex recognition of target cells by T and B cells, delivery of the serine protease granzyme B (GrB) to tumor cells comprises the cytotoxic insult resulting in a well-characterized, multimodal apoptotic cascade.MethodsWe designed a recombinant fusion construct, GrB-Fc-4D5, composed of a humanized anti-HER2 scFv fused to active GrB for recognition of tumor cells and internal delivery of GrB, simulating T and B cell therapy. We assessed the construct’s antigen-binding specificity and GrB enzymatic activity, as well as in vitro cytotoxicity and internalization into target and control cells. We also assessed pharmacokinetic and toxicology parameters in vivo.ResultsGrB-Fc-4D5 was highly cytotoxic to Her2 positive cells such as SKBR3, MCF7 and MDA-MB-231 with IC50 values of 56, 99 and 27 nM, respectively, and against a panel of HER2+ cell lines regardless of endogenous expression levels of the PI-9 inhibitor. Contemporaneous studies with Kadcyla demonstrated similar levels of in vitro activity against virtually all cells tested. GrB-Fc-4D5 internalized rapidly into target SKOV3 cells within 1 h of exposure rapidly delivering GrB to the cytoplasmic compartment. In keeping with its relatively high molecular weight (160 kDa), the construct demonstrated a terminal-phase serum half-life in mice of 39.2 h. Toxicity studies conducted on BALB/c mice demonstrated no statistically significant changes in SGPT, SGOT or serum LDH. Histopathologic analysis of tissues from treated mice demonstrated no drug-related changes in any tissues examined.ConclusionGrB-Fc-4D5 shows excellent, specific cytotoxicity and demonstrates no significant toxicity in normal, antigen-negative murine models. This construct constitutes a novel approach against HER2-expressing tumors and is an excellent candidate for further development.
Highlights
Immunotherapeutic approaches designed to augment T and B cell mediated killing of tumor cells has met with clinical success in recent years suggesting tremendous potential for treatment in a broad spectrum of tumor types
The current study extends our initial observations of impressive biological activity of granzyme B (GrB)-containing anti-HER2/ neu fusion constructs and provides a comparison to the FDA-approved Antibody drug conjugate (ADC) Kadcyla
Construction, expression, and purification of GrB-Fc-Humanized anti-HER2/neu scFv (4D5) fusion protein Following expression and protein purification through Nickel-IMAC, the fusion protein was incubated with recombinant enterokinase to remove the N-terminal purification tag
Summary
Immunotherapeutic approaches designed to augment T and B cell mediated killing of tumor cells has met with clinical success in recent years suggesting tremendous potential for treatment in a broad spectrum of tumor types. GrB has been well validated as a highly cytotoxic payload with a unique mechanism of action when compared to other payloads employed in targeted therapeutic constructs including antibody drug conjugates (ADCs) and fusion toxins [27,28,29,30,31,32]. The addition of a highly cytotoxic payload emtansine to Herceptin has resulted in an agent Kadcyla (T-DM1) with excellent clinical therapeutic properties [42,43,44,45], as well as a number of follow-on ADC products for a wide-range of therapeutic targets, including new designs and novel payloads with unique mechanisms of action [46,47,48,49,50]. The emergence of resistance mechanisms which limit treatment success with these agents is driving innovation to overcome these issues
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