Development of a human immuno-oncology therapeutic agent targeting HER2: targeted delivery of granzyme B

Abstract

Background: 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. 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.

Methods: We 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.

Results: GrB-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.

Conclusion: GrB-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.

Keywords: Granzyme B; HER2; Immunotherapy; Kadcyla; Pharmacokinetics.

Fig. 1

Design and expression of the GrB-Fc-4D5 construct. a cDNA of GrB, IgG-Fc region and an anti-HER2 scFv were fused together with a flexible GGGGS linker by overlapping PCR. The resulting plasmid was then cloned into the mammalian expression pSECTag-A vector for either transient HEK-293E or stable CHO expression of the fusion protein. b SDS-PAGE of GrB-Fc-4D5 and GrB(S195A)-Fc-4D5 confirmed > 95% purity of the fusion proteins and that both constructs are monomers under reducing conditions and homodimers under non-reducing conditions

Fig. 2

ELISA binding of GrB-Fc-4D5 to Her2/ECD. Purified HER2/neu ECD were coated on 96-well ELISA plates. After incubation with the target protein (1 h) and wash steps, human anti-GrB antibody was added and incubated, followed by the addition of horseradish peroxidase-conjugated goat anti-human immunoglobulin antibody. Absorbance at 405 nm was measured after 30 mins. a Various concentrations of either GrB-Fc-4D5 or GrB(S195A)-Fc-4D5 protein to determine Kd of the 4D5 scFv. b Various concentrations of GrB-Fc-4D5 and an unrelated GrB-Fc-scFv (as a specificity control) to confirm specificity of the 4D5 scFv for HER2 ECD

Fig. 3

Internalization and localization of GrB-Fc-4D5. a Internalization into SKOV3 target cells assessed by a polyclonal anti-GrB antibody demonstrated rapid internalization. SKOV3 cells were either untreated or treated with 50 nmol/L of GrB-Fc-4D5 for 1, 5.5 and 24 h. The cells were fixed, acid washed to remove surface bound fusion protein, and then permeabilized and immunostained for the presence of GrB by using a polyclonal rabbit anti-GrB antibody (green). Cells were counterstained with DAPI (blue) to identify nuclei and visualized using a fluorescent microscope. b Intracellular localization of GrB-Fc-4D5. A549 (top) and MDA-MB-231 (bottom) cells were treated with AF-594-GrB-Fc-4D5 (red) for two hours followed by addition of Lysotracker dye (blue). Cells were imaged live by confocal microscopy. Magnification, 20x

Fig. 4

Mechanistic studies of GrB-Fc-4D5. a Western blot analysis of MEF3.5^−/− (antigen negative) and MDA-MB-231 (targeted) cell extracts for pAkt, Caspase 9 and cleaved PARP after treatment with 20 nmol/L of GrB-Fc-4D5 for 0, 24 and 48 h. b Western blot analysis of cell extract for pHer2 (Y877) was assayed against SKBR3 after treatment with 100 nmol/L of either Herceptin or GrB-Fc-4D5 for 0, 24 and 48 h. β-actin was used as a control for protein loading

Fig. 5

Pharmacokinetic clearance of GrB-Fc-4D5 after IV administration in BALB/c mice. 200 μg of GrB-Fc-4D5 were injected IV into BALB/c mice. Groups of mice (3 mice/group) were sacrificed at various time point after injection. The concentration of fusion protein in plasma was assessed by ELISA according to the protocol in material and method, and the mean blood concentration to time profile of GrB-Fc-4D5 was generated using a least squares nonlinear regression

Fig. 6

Toxicity assessment of GrB-Fc-4D5 in BALB/c mice. a Body weight in control group (PBS) and Grb-Fc-4D5-treated (100 mg/kg) mice. The values represent the weights measured in BALB/c mice before treatment, one day after treatment (acute group) and 4 weeks after treatment (recovery group). b Liver enzymes detected in serum of BALB/c treated mice. The enzymatic activity of Alanine Transferase (SGPT), Aspartate transferase (SGOT) and Lactate Dehydrogenase (LDH) was measured in acute and recovery groups. No statistical difference was found between mice treated with control or GrB-Fc-4D5 in the acute or recovery groups