Clarke E., Kyriss B., Mergaert A., Society of Toxicology 53rd Annual Meeting & ToxExpo 2014, Poster Abstract No.1920. ReachBio Research Labs, Seattle, WA.

 INTRODUCTION

Combination therapies are being increasingly employed to treat diseases where single agents do not have the desired clinical benefit. Acute myeloid leukemia (AML) is a malignant disease characterized by rapid growth of myeloid cells in BM. This year, 14,500 new cases will be diagnosed in the US, while claiming the lives of approximately 10,000. Nearly 40 years have passed since the development and implementation of cytarabine (Ara-C) and daunorubicin (DNR) combination therapy as a standard of care. However, in clinical trials additional drugs have been introduced as novel treatment regimes, either alone or in combination with standard treatments. A relevent ex vivo  assay that reflects the potential of novel treatments may facilitate the discovery of more useful drug candidates. Flow cytometric and CFC assays provide ex vivo platforms in which the diversity of the disease parameters as well asthe utility of novel compounds can be tested. To this end we used eight color flow cytometric analysis to determine the cellular phenotypes of AML and NBM. Using the CFC assays, we evaluated the effects of drug combinations on NBM progenitors and AML CFU-L.

MATERIALS and METHODS

Eight color flow cytometric analysis was used to analyze mononuclear cells from nine AML BM and six NBM donors with Becton Dickinson LSR II and analyzed using Kaluza®. The list of flurochromes is provided in Table 1. Using CFC assays evaluated the toxicity of AML drugs, DNR, Ara-C, clofarabine and sorafenib on AML “cancer stem cells” and NBM. A panel of AML patient marrow samples were analyzed and compared to a panel of normal donor marrow in CFC assays (Table 2). Bone marrow cells from either AML or normal donors were mixed with the compounds at an extended concentration range in ColonyGel™ 1102 and plated in 35 mm dishes (3 replicates per concentration). The cultures were incubated on a humidified incubator at 37°C, 5% CO₂. CFU-GM or CFU-L (AML-Blast like CFC) were enumerated on day 14 and IC₅₀ values were determined. For CFU-L , IC₂₅ values were also determined. Based on these IC₂₅ values,standard combination treatment of Ara-C/DNR were compared to treatment regimens with additional concentrations of clofarabine or sorafenib in these ex vivo assays.

TABLE 1: Fluorochromes Used in Multi-parameter Analysis

TABLE 2: AML Patient and NMB Donor Characteristics

FIGURE 1A: NBM Flow Cytometric Profile                                                                              FIGURE 1B: AML Blasts Flow Cytometric Profile

These data represent one of the NBM and one of the AML patient BM profiles.  Marrow from normal donor had consistent profiles as 1a above shows, but AML patient profiles differed from each other and from the NBM profile.

TABLE 3: Prevalence of Antigens in NBM and AML Blasts 

TABLE 4: IC₅₀ and IC₂₅ in NBM and AML 

                FIGURE 2A: Sorafenib IC_{50                                                                                             FIGURE 2A: Clofarabine IC50}   

_{There was no appreciable difference in the IC50  in response to sorafenib. However there were significant differences (p=0.0008) in IC50 values between NBM CFU-GM and AML CFU-L. One AML Patient (colored in red above) had a very different response (>2 SD from the mean IC50 value from the group) to clofarabine. The same patient also demonstrated as unusual response to other drugs.}       

FIGURE 3: Combination Therapy Using ARA-C, DNR, CLOFARBINE AND SORAFENIB 

_{The combination therapy of Ara-C and DNR had a greater inhibitory effect on the AML CFU-L as compared to NMB CFU-GM. The addition of sorafenib significantly enhanced the killing of  both CFU-L and CFU-GM, whereas clofarabine had a selective inhibitory effect on CFU-L but not CFU-GM.}

_{TABLE 5: Percent Inhibition of CFU-GM and CFU-L with Single Agents and Combination Therapy}

RESULTS

• Eight color flow cytometry was used to elucidate the complex and varied phenotypic presentation of AML (Figure 1) and to assess if there
  was a correlation between phenotype and CFU-L clonal growth in the CFC assay. Only five of nine AML samples supported CFU-L proliferation and there was no correlation between phenotype and clonal growth in the CFC assay (Table 3).
• Selected compounds when tested alone in CFC assays were generally less toxic to NBM CFY-GM than AML CFU-L. There was a significant reduction (p< 0.05) for Ara-C, DNR, and clofarabine in AML CFU-L as compared to NBM CFU-GM (Table 4). However, some variation in responsiveness to the drugs was seen in the AML samples. Sorafenib demonstrated no preferential inhibition on CFU-L as compared to CFU-GM (Table 4 and Figure 2).
• Ara-C and DNR in combination at their IC₂₅ values on CFU-L were significantly (p< 0.01) more toxic to CFU-L than either drug alone.  This combination was not significantly more inhibitory to NBM CFU-GM (Table 5 and Figure 3).
• The addition of sorafenib, but not clofarabine, to the Ara-C/DNR combination was additionally and significantly (p=0.007) toxic to CFU-L (Figure 3). However, this combination was also significantly toxic to NBM CFU-GM (p<0.00001).
• Sorafenib does not selectively inhibit the growth of AML CFU-L as no significant difference between the inhibition of NBM CFU-GM and AML CFU-L was determined.

CONCLUSIONS

• Investigational therapeutics in their soluble (ie. not plastic-immobilized) form can be evaluated in vitro for their ability to trigger pro-inflammatory cytokine secretion by using a human primary cell HUVEC/PBMC co-culture system.
• Clinically, anti-CD28SA was associated with severe CS in 6 out of 6 patients. Using Method 2 and saturating anti-CD28SA, we observed high concentrations (in excess of 1000 pg/ml) of all pro-inflammatory cytokines tested.
• Using Method 2 and saturating anti-CD3, there was more donor variability of the four pro-inflammatory cytokines. Clinically, patient response to anti-CD3 therapy is also variable.
• The method my have potential uses both at the  development state, to identify potential problems with an investigational biotherapeutics, and at the clinical stage, to identify at-risk patients prior to first infusion.
• IL2 levels in excess of 1000 pg/ml may be a good benchmark for estimating risk of CRS or CS. Other pro-inflammation cytokines should also be measured.

REFERENCES

Römer,P et al. Preculture of PBMCsat high density increases sensitivity of T cell responses, revealing cytokine release by CD28 superagonist TGN1412. Blood 118 (2011): 6772-6782

Findlay, L et al. Endothelial cells co-stimulate peripheral blood mononuclear cell responses to monoclonal antibody TGN1412 in culture. Cytokine 55 (2011): 141-151

Eastwood, D et al. Severity of the TGN1412 trial disaster cytokine storm correlated with IL-2 release. Br J Clin Pharmacol 76 (2013): 299-315

ACKNOWLEDGEMENTS

NHP PBMCs were obtained from the Washington National Primate Research Center at the University of Washington which is supported by the Office of Research Infrastructure Programs (ORIP) of the National Institutes of Health through Grant Number P51 OD 010425