A Broad-Based Characterization of a Cell-Penetrating, Single Domain Camelid Bi-Specific Antibody Monomer That Targets STAT3 and KRAS Dependent Cancers.

STAT3 and KRAS regulate cell proliferation, survival, apoptosis, cell migration, and angiogenesis. Aberrant expression of STAT3 and mutant active forms of KRAS have been well-established in the induction and maintenance of multiple cancers. STAT3 and KRAS mutant proteins have been considered anti-cancer targets; however, they are also considered to be clinically "undruggable" intracellular molecules, except for KRAS(G12C). Here we report a first-in-class molecule, a novel, single domain camelid VHH antibody (15 kDa), SBT-100, that binds to both STAT3 and KRAS and can penetrate the tumor cell membrane, and significantly inhibit cancer cell growth. Additionally, SBT-100 inhibits KRAS GTPase activity and downstream phosphorylation of ERK in vitro. In addition, SBT-100 inhibits the growth of multiple human cancers in vitro and in vivo. These results demonstrate the feasibility of targeting hard-to-reach aberrant intracellular transcription factors and signaling proteins simultaneously with one VHH to improve cancer therapies.

TNF Apoptosis Protection Fraction (TAPF) prevents apoptosis induced by TNF, but not by Fas or TRAIL, via NF-κB-induced increase in cFLIP.

Tumor Necrosis Factor α (TNFα) induces both the apoptotic pathway and anti-apoptotic factors. Incubation of human dermal fibroblasts with TAPF (TNF Apoptosis Protection Fraction) protects them from apoptosis induced by the subsequent addition of TNF and cycloheximide (CHX). TAPF does not protect against apoptosis induced by CHX in combination with either TRAIL (TNF related apoptosis inducing ligand) or an agonistic Fas antibody, or against apoptosis induced by the chemotherapeutic agent doxorubicin. Incubation with TAPF does not affect the quantity of TNF that binds to the cell. TAPF prevents TNF-induced cleavage of caspases 8, 9, 3 and 7 and the apoptotic substrate PARP (poly-ADP ribose polymerase), but has no effect when these molecules are induced by an agonistic Fas antibody. TAPF induces rapid phosphorylation of the NF-κB/p65 (nuclear factor-κB) transcription factor at serine 536 which is indicative of its activation. TAPF increases the expression of cFLIP (cellular FLICE-inhibitory protein) which is a potent inhibitor of apoptosis that acts by preventing the cleavage of caspase 8. This increase in cFLIP is coincident with protection from TNF-induced apoptosis. Decreasing cFLIP levels using shRNA (short hairpin RNA) decreases protection by TAPF. TAPF also induced the anti-apoptotic A20 protein. These data indicate that TAPF protects human dermal fibroblasts from TNF-induced apoptosis by induction of cFLIP and subsequent inhibition of caspase 8 cleavage.

Doxorubicin plus interleukin-2 chemoimmunotherapy against breast cancer in mice.

As recently characterized, following s.c. implantation into syngeneic C57BL/6 mice, E0771 tumor invades locally into dermal layers and peritoneum, metastasizes to the lung, and induces a nonspecific immunosuppression in the host. Using this breast medullar adenocarcinoma model, a therapy consisting of a single moderate dose of doxorubicin followed by twice daily moderate doses of interleukin-2 for 30 days was examined for efficacy and mechanism of action when given to animals with established disease. This combination treatment, but not combinants alone, resulted in tumor-free long-term survival of 40% of the mice without significant toxicity and 83% of survivors had immune memory specific for E0771 cells. Treatment also decreased immune suppression induced by E0771 tumor. Full response to treatment required functional CD8(+) T cells, whereas depletion of natural killer cells caused only a reduction in response rate. A serum "biomarker" profile that correlated with, and seemed predictive of, response to treatment was identified by nuclear magnetic resonance-based metabonomic analysis. The efficacy of this nontoxic treatment and the potential to be able to predict which individual is responding to treatment are characteristics that make this chemoimmunotherapy attractive for clinical testing.

Doxorubicin induces specific immune functions and cytokine expression in peritoneal cells.

To examine the basis of the immune modulation induced by the anticancer agent doxorubicin (DOX), the immunophenotype, tumoricidal activity, cytokine protein and mRNA expression were determined using peritoneal exudate cells (PEC) from saline-treated (untreated) and DOX-treated mice. A greater percentage of PEC from DOX-treated mice than from untreated mice were adherent to plastic, had characteristics of granulocytes, and were positive for the NK1.1, CD11b/Mac-1, and CD3 markers. DOX decreased the percentage of CD45R/B220+ cells. PEC from DOX-treated mice had greater tumoricidal potential than those from untreated mice since IL2, LPS, or IFNgamma alone increased the cytolytic activity of PEC from DOX-treated mice, whereas PEC from untreated mice required both LPS and IFNgamma to become cytolytic. DOX treatment modulated the expression of specific cytokines. Following stimulation in culture, PEC from DOX-treated mice produced more TNF, IL1, and IFNgamma than PEC from untreated mice. DOX treatment increased the levels of TNF, but not IL1, mRNA and decreased the levels of IL6 mRNA and protein. These data demonstrate that a single DOX injection induces specific effects in PEC and, as a consequence, increases the tumoricidal potential of cells of the macrophage and natural killer types.