Trop-2-targeting tetrakis-ranpirnase has potent antitumor activity against triple-negative breast cancer

Background Ranpirnase (Rap) is an amphibian ribonuclease with reported antitumor activity, minimal toxicity, and negligible immunogenicity in clinical studies, but the unfavorable pharmacokinetics and suboptimal efficacy hampered its further clinical development. To improve the potential of Rap-based therapeutics, we have used the DOCK-AND-LOCK™ (DNL™) method to construct a class of novel IgG-Rap immunoRNases. In the present study, a pair of these constructs, (Rap)2-E1-(Rap)2 and (Rap)2-E1*-(Rap)2, comprising four copies of Rap linked to the CH3 and CK termini of hRS7 (humanized anti-Trop-2), respectively, were evaluated as potential therapeutics for triple-negative breast cancer (TNBC). Methods The DNL-based immunoRNases, (Rap)2-E1-(Rap)2 and (Rap)2-E1*-(Rap)2, were characterized and tested for biological activities in vitro on a panel of breast cancer cell lines and in vivo in a MDA-MB-468 xenograft model. Results (Rap)2-E1-(Rap)2 was highly purified (>95%), exhibited specific cell binding and rapid internalization in MDA-MB-468, a Trop-2-expressing TNBC line, and displayed potent in vitro cytotoxicity (EC50 ≤ 1 nM) against diverse breast cancer cell lines with moderate to high expression of Trop-2, including MDA-MB-468, BT-20, HCC1806, SKBR-3, and MCF-7. In comparison, structural counterparts of (Rap)2-E1-(Rap)2, generated by substituting hRS7 with selective non-Trop-2-binding antibodies, such as epratuzumab (anti-CD22), were at least 50-fold less potent than (Rap)2-E1-(Rap)2 in MDA-MB-468 and BT-20 cells, both lacking the expression of the cognate antigen. Moreover, (Rap)2-E1-(Rap)2 was less effective (EC50 > 50 nM) in MDA-MB-231 (low Trop-2) or HCC1395 (no Trop-2), and did not show any toxicity to human peripheral blood mononuclear cells. In a mouse TNBC model, a significant survival benefit was achieved with (Rap)2-E1*-(Rap)2 when given the maximal tolerated dose. Conclusions A new class of immunoRNases was generated with enhanced potency for targeted therapy of cancer. The promising results from (Rap)2-E1-(Rap)2 and (Rap)2-E1*-(Rap)2 support their further investigation as a potential treatment option for TNBC and other Trop-2-expressing cancers.


Background
Breast cancer is the leading cause of cancer deaths in women and the second most common cancer worldwide after lung cancer [1]. In the USA, about 232,340 new cases of this cancer will be diagnosed and about 39,620 women will die from this disease in 2013 [2]. During the past two decades, important advances have been made in the treatment of hormone receptor (HR)-positive [3,4] and human epidermal growth factor receptor type 2 (HER2)-overexpressing breast cancers [5,6]. However, effective therapies for patients with triple-negative breast cancer (TNBC), which lacks the expression of estrogen receptor (ER), progesterone receptor (PR) and HER2, are still urgently needed [7]. TNBC, afflicting approximately 12 to 17% of women with breast cancer [8], is a heterogeneous disease with varying prognoses according to molecular, pathological, and clinical factors [9,10]. Because of the absence of clear targets like HER2, ER, and PR, chemotherapy with doxorubicin plus cyclophosphamide followed by paclitaxel is recommended as the standard of care for systemic treatment of TNBC in the adjuvant setting [11], whereas for patients with metastatic disease, there is no standard first-line agent. To date, various approved agents, including platinum-based compounds, ixabepilone, erlotinib, bevacizumab, cetuximab, and several investigative drugs, in particular, inhibitors of PARP, tyrosine kinases, or mTOR, are being evaluated alone or in combination in different phases of clinical development for TNBC [11]. An arduous challenge in TNBC chemotherapy is primary or acquired drug resistance, resulting in incomplete response, relapse and poor survival.
Rap is a single-chain protein of 104 amino acids (MW 12 kDa) originally isolated from the oocytes of Rana pipiens, a Northern leopard frog [12]. Rap exhibits cytostatic and cytotoxic effects on a variety of tumor cell lines in vitro [13], as well as antitumor activity in vivo [14]. The amphibian ribonuclease enters cells via AP-2/ clathrin-mediated endocytosis [15] and once internalized into the cytosol, it selectively degrades tRNA [16], thereby inhibiting protein synthesis and inducing apoptosis [17]. In addition, Rap was shown to enhance both the in vitro and in vivo antitumor activity of vincristine against HT-29 human colorectal cancer cells that had been rendered multidrug-resistant by overexpressing the mdr1 gene [18], and induce caspase-independent cell death in both drug-sensitive and -resistant neuroblastoma cells and tumor xenografts [19]. Clinical studies of Rap in patients with unresectable malignant mesothelioma showed a significant impact on the survival of patients treated with doxorubicin plus Rap compared to doxorubicin alone [20], and a dose-limiting renal toxicity that was reversible upon discontinuation of treatment [21]. Notably, an earlier Phase I trial of Rap in patients with solid cancers reported a lack of untoward immune response upon repeated weekly injections [22].
Trop-2, also known as EGP-1 (epithelial glycoprotein-1), is a cell-surface glycoprotein overexpressed by a variety of epithelial carcinomas relative to corresponding normal tissues [23]. The expression of Trop-2 was shown to be necessary for tumorigenesis and invasiveness of colon cancer cells, which could be reduced effectively with a polyclonal antibody against the extracellular domain of Trop-2 [24]. More recently, the biological function of Trop-2 in promoting self-renewal and hyperplasia in the prostate was attributed to the accumulation in the nucleus of the intracellular domain of Trop-2 following its cleavage via regulated intramembrane proteolysis [25]. Because of the well-documented clinical evidence in breast cancer [26], colorectal cancer [27,28] and other cancers [29] that overexpressed Trop-2 is associated with increased tumor aggressiveness, metastasis, and decreased patient survival, there is a growing interest in Trop-2 as a therapeutic target for solid cancers [30]. For example, the humanized anti-Trop-2 monoclonal antibody (mAb), hRS7, is currently under clinical investigation as a drug delivery moiety for patients with advanced epithelial cancers (NCT01631552).
The DOCK-AND-LOCK™ (DNL™) method [31][32][33] is a platform technology for production of multivalent, multifunctional conjugates by utilizing the naturally occurring interaction between the dimerization and docking domain (DDD) of cAMP-dependent protein kinase A (PKA) and the anchoring domain (AD) of an A-kinase anchoring protein (AKAP). The established strategy involves the use of a specific pair of DDD and AD peptides, termed DDD2 and AD2, to generate two distinct modules, which upon mixing under redox conditions, self-assemble into a DNL conjugate with retained activity and defined composition. We previously reported the antitumor activity of a novel immunotoxin, designated Rap(Q)-hRS7, in Trop-2-expressing cancer cells [34]. The recombinantly-produced Rap(Q)-hRS7 comprises two molecules of Rap(p.N69Q) or N69Q-Rap, a variant of Rap with the putative N-glycosylation site removed by replacing the 69 th residue of asparagine (N69) with glutamine (Q), each fused to a light chain of hRS7 at the amino terminus [34].
To further explore the potential of Rap-based immunotoxins, we applied the DNL method to site-specifically tether a dimerized Rap, denoted as (Rap) 2 , where (Rap) represents Rap-DDD2, at each of the carboxyl termini of either the heavy chain (the C H 3-format) or the light chain (the C K -format) of a humanized IgG, resulting in a class of novel immunoconjugates (Table 1) with tetrakis Rap ( Figure 1). As exemplified herein by (Rap) 2 -E1-(Rap) 2 , comprising four copies of Rap linked to the AD2-fused C H 3 termini of hRS7 IgG (denoted as E1), and by (Rap) 2 -E1*-(Rap) 2 , comprising four copies of Rap linked to the AD2-fused C K termini of hRS7 IgG (denoted as E1*), these DNL-based immunoRNases could offer a distinct advantage over their recombinantlyproduced fusion counterparts, such as Rap(Q)-hRS7, in

MTD in nude mice
Among the four groups (20, 40, 60 or 80 μg) tested, only those mice that received the four injections of (Rap) 2 -22*-(Rap) 2 at 20 μg survived without acute toxicity or death. One of the three animals lost~12% of its body

In vivo activity
We selected (Rap) 2 -E1*-(Rap) 2 for in vivo studies because the C K -based conjugates exhibited superior Fc-effector functions in vitro, as well as improved pharmacokinetics, stability, and activity in vivo, as demonstrated for two types of DNL conjugates having a similar structure to IgG-Rap; namely, a bispecific hexavalent antibody comprising a pair of dimeric Fab linked to an IgG, and a multivalent immunocytokine comprising a pair of dimeric IFN-α linked to an IgG [35]. In the TNBC model of mice bearing MDA-MB-468 tumors, all mice tolerated the two cycles of treatment with no mouse losing more than 9% of starting body weight during dosing (Additional file 6: Figure S5). Based on area under the curve (AUC) determined on day 48, (Rap) 2 -E1*-(Rap) 2 significantly inhibited tumor growth compared to the saline control ( Figure 5A; P = 0.0254). In addition, a significant survival benefit ( Figure 5B To further demonstrate the targeting specificity of (Rap) 2 -E1*-(Rap) 2 vs. (Rap) 2 -22*-(Rap) 2 , we performed another study to compare their potency in a CD22expressing, but Trop-2-negative, tumor model. Mice bearing disseminated Burkitt-NHL (Daudi) were treated 7 days after the animals were administered the cells to ensure a large tumor-burden at the time of therapy initiation ( Figure 5C). All mice in the saline control group succumbed to disease progression by day 32 (median = 32 days). Likewise, mice treated with the MTD of (Rap) 2 -E1*-(Rap) 2 were all dead by day 48 (median = 36 days). Conversely, all mice that received the MTD of (Rap) 2 -22*-(Rap) 2 or half this dose were still alive and tumor-free at the time the experiment was ended on day 84. Overall, treatment with (Rap) 2 -22*-(Rap) 2 resulted in a significant survival benefit when compared to the saline or treatment control (P < 0.0034).

Discussion
Rap is a promising antitumor toxin with several attractive properties, including minimal toxicity, negligible immunogenicity, and the potential to overcome multiple drug resistance via unusual mechanisms of internalization and cytotoxicity. Over the years, we have made considerable progress to enhance the potency of Rap by targeting it to cancer cells expressing CD22, CD74 and Trop-2, as exemplified by LL2-onconase [36], Rap-hLL1 [37], and Rap(Q)-hRS7 [34], respectively, but have also encountered difficulty in scaled-up production of these prototypes due to relatively low productivity of Rap-hLL1 or Rap(Q)-hRS7 in mammalian cell cultures. We believe the DNL™ platform technology, which conveniently tethers four copies of Rap to the C-terminus of different IgG modules, has key production advantages for Rap-based immunotoxins. Moreover, these DNL-Rap conjugates ensure targeted delivery of Rap with significantly improved potency in cancer cells expressing the cognate antigens, as exemplified by (Rap) 2 -E1-(Rap) 2 in the current study with breast cancer cell lines. It is noteworthy that in MDA-MB-468 cells with a moderate level of Trop-2, the cytotoxicity of the DNL-generated (Rap) 2 -E1-(Rap) 2 (EC 50 = 0.03 nM) was 100-fold more potent than the recombinant Rap(Q)-hRS7 (EC 50 = 3.8 nM), and 3,000-fold more potent than unconjugated Rap (EC 50 > 100 nM). On the other hand, (Rap) 2 -E1-(Rap) 2 inhibited the proliferation of Trop-2-negative HCC1395 cells only at much higher concentrations (EC 50 > 50 nM) and yet was not toxic to PBMCs, indicating the likely existence of a relatively large therapeutic window.
We reported previously that in MDA-MB-468 cells, Rap(Q)-hRS7 co-localized with human transferrin in the endosomes after a 2-h incubation at 37°C [34], whereas in the current study under the same conditions, (Rap) 2 -E1-(Rap) 2 did not co-localize with human transferrin in the endosomes, suggesting a different internalization pathway, which may explain its greater potency in MDA-MB-468 than observed for Rap(Q)-hRS7.  5 and 25 nM). The CD20-targeting (Rap) 2 -20-(Rap) 2 and hA20 served as negative controls; B, following incubation, pH 2.5 treatment, and fixation as described in the Methods, cells were stained with GAH-Alexa Fluor 488 (green) to detect intracellular (Rap) 2 -E1-(Rap) 2 , and also with Hoechst 33258 (blue) and human transferrin-conjugated Alexa Fluor 568 (red) to reveal the nucleus and endosomes, respectively.
We noted that unlike the bispecific hexavalent antibodies, for which the C K -format was demonstrated to be superior to the C H 3-format [35], the potential advantage of a C K -format of DNL-Rap vs. its C H 3-counterpart is not readily apparent from the present data, and will require further studies.
The potency and specificity of DNL-Rap conjugates for targeted cancer therapy is also supported by the statistically significant antitumor activity of (Rap) 2 -E1*-(Rap) 2 in suppressing growth of MDA-MB-468 xenografts, and by the observed cure of all five mice with disseminated Daudi lymphoma following treatment with four 20-μg doses of (Rap) 2 -22*-(Rap) 2 given 4 days apart, both of which should reduce the potential concern that the larger size of DNL-Rap conjugates would negatively impact their in vivo efficacy, particularly in solid cancer, due to poor penetration.
Although hRS7 binds to selected human epithelial cells, recent studies with a drug conjugate of hRS7 in cynomolgus monkeys, whose tissues cross-react with hRS7, showed that tolerated doses occurred at clinically-relevant concentrations, and that dose-limiting toxicities to normal tissues were no different from those reported previously with the free drug [38]. These data suggest that having Rap attached to hRS7 may not add substantially its toxicity to normal tissues, and that (Rap) 2 -E1-(Rap) 2 or (Rap) 2 -E1*-(Rap) 2 may be tolerated at clinically-relevant concentrations. In addition, because the structural motif (x)D(y) identified to be responsible for the binding of ricin A-chain or interleukin-2 to endothelial cells is absent in the native sequence of Rap, and hRS7 is not cross-reactive with human endothelial cells, we consider the likelihood of (Rap) 2 -E1-(Rap) 2 or (Rap) 2 -E1*-(Rap) 2 causing vascular leak syndrome as remote. Regarding the immunogenicity of DNL-Rap conjugates, it needs to be addressed in a relevant species as well as in future human studies.

Conclusions
We have generated a new class of immunoRNases and demonstrated their potential for targeted therapy of cancer. These promising results warrant further studies to advance the development of (Rap) 2 -E1-(Rap) 2 or (Rap) 2 -E1*-(Rap) 2 , in particular, and other DNL-Rap conjugates, in general, as potential novel therapeutics for triplenegative breast cancer or other Trop-2-expressing cancers.

Expression and purification of DNL modules
To produce the Rap-DDD2 module used for DNL conjugation, a DNA fragment encoding Rap and a GGGGS linker sequence was amplified by PCR from rPRL2#26 plasmid [34] and inserted into the MscI and XhoI restriction sites of the pET-26b vector to generate rap-GS-pET26b. Subsequently, the DDD2 coding sequence was amplified by PCR from IFNα2b-DDD2-pdHL2 [33] and inserted into XhoI site of rap-pET26b to generate the expression vector rap-GS-DDD2-pET26b. Competent Rosetta-pLysS cells (EMD Millipore, Billerica, MA) were transformed with rap-GS-DDD2-pET26b and cultured in shaker flasks at 37°C in Difco 2xYT broth (Becton Dickinson, Franklin Lakes, NJ), supplemented with 100 μg/mL kanamycin sulfate and 34 μg/mL chloramphenicol. When the cell density reached OD 600 = 1.0, cultures were switched to 30°C and protein expression was induced with 0.4 mM IPTG for 4 h. Cell pellets were frozen, thawed and homogenized in a lysis buffer comprising 2% Triton X-100, 5 mM MgSO 4 , 10 units/mL benzonase (Novagen EMD Millipore), 100 μM 4-(2-aminoethyl) benzenesulfonyl fluoride (Sigma-Aldrich, St. Louis, MO), and 20 mM Tris-HCl, pH 8.0. The insoluble material, containing inclusion bodies, was pelleted by centrifugation, re-homogenized in 1% Triton X-100 in PBS, and re-pelleted. Inclusion bodies were solubilized in 6 M guanidine-HCl, 100 mM Na-phosphate, pH 8.0, and applied to a His-Select affinity column (GE Healthcare, Piscataway, NJ). The denatured protein was eluted in 4 M guanidine-HCl, 100 mM NaH 2 PO 4 , pH 4.5. The eluate was neutralized with 3 M Tris-HCl, pH 8.6, added dithioerythreitol (DTE) to 60 mM, and held at room temperature overnight, to which was added rapidly 10-fold volume of 0.5 M arginine, 20 mM oxidized glutathione, 2 mM EDTA,100 mM Tris, pH 8.0, followed by dialysis against 5 L of a renaturation buffer (0.5 M arginine, 2 mM oxidized glutathione, 0.6 mM DTE, 2 mM EDTA, 20 mM Tris, pH 8.0) for 72 h at 4°C, and subsequently against PBS buffer.
Expression vectors for the C H 3-AD2-IgG modules were engineered from cognate IgG-pdHL2 expression vectors, as described previously [39]. The C K -AD2-IgG modules were generated by fusing AD2 and a hinge linker sequence to the C-terminal end of the kappa light chain [35]. These modules were produced in myeloma cell culture of SpESFX-10 cells [40] and isolated from culture broths using MabSelect affinity chromatography (GE Healthcare).

DNL conjugation
The Rap-DDD2 module was reacted with a C H 3-AD2-IgG or a C K -AD2-IgG of choice to generate a panel of DNL-Rap conjugates, as listed in Table 1, with the structures of the C H 3-and C K -types shown schematically in Figure 1. Typically, an AD2-IgG was combined with approximately two mole-equivalents of Rap-DDD2, followed by the addition of reduced glutathione to a final concentration of 2 mM. After incubation at room temperature overnight, oxidized glutathione was added to a final concentration of 4 mM on the next day, the incubation continued for another 24 h, and the resulting DNL-Rap conjugate was purified by MabSelect affinity chromatography.

Flow cytometry
Cells were trypsinized briefly, washed, re-suspended in 1% BSA-PBS, incubated with hRS7 or hA20 IgG, and detected with FITC-GAH. All incubations were 45 min at 4°C with 1% BSA-PBS washes between incubations. Cell binding was measured by flow cytometry using a BD FACSCalibur (BD Biosciences, San Jose, CA).

In vitro proliferation
Cells were seeded in 96-well plates at 1,000-2,000 cells/ well and held at 37°C overnight prior to incubation with the indicated agents for 4 days. Viable cells were measured using MTS substrate Cell Titer96® AQueous One Solution (Promega, Madison, WI).

Toxicity in human PBMC
Buffy coats from healthy donors were purchased from the Blood Center of New Jersey, with approval by the New England Institutional Review Board. PBMCs were isolated from buffy coats by standard density-gradient centrifugation over Ficoll-Paque, and treated with (Rap) 2 -E1-(Rap) 2 at 37°C in 5% CO 2 for 16 h. After incubation, the cells were stained with Alexa Fluor® 488-labeled annexin V, then with 7-aminoactinomycin-D, and analyzed by flow cytometry.

Determination of maximal tolerated dose (MTD) in mice
Female athymic nude mice (8 weeks old) were divided into four groups of three each, with each group receiving (Rap) 2 -22*-(Rap) 2 at one of four different doses (20,40,60 or 80 μg). Animals were dosed i.v. every four days for a total of four injections (q4dx4). Toxicity was assessed by daily observations and weights taken twice weekly. Mice were deemed to have lethal toxicity if they lost more than 15% of starting body weight or were otherwise deemed moribund. The MTD was the highest dose administered in which all the mice in the group survived the injections.

In vivo therapeutic activity
All animal studies were approved by the University of Medicine and Dentistry of New Jersey's Institutional Animal Care and Use Committee (IACUC) and performed in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care, U.S. Department of Agriculture, and Department of Health and Human Services regulations. Female NCr athymic nude (nu/nu) mice, 5 weeks old, and SCID mice, 7 weeks old, were purchased from Taconic Farms (Hudson, NY). Two tumor models, solid and liquid cancers, were used to evaluate the efficacy and specificity of DNL-generated immunoRNases, (Rap) 2  approximately 0.2 cm 3 in size, the animals were divided into three groups of 8 to 9 each. The treatment group received i.v. administration of the MTD of (Rap) 2 -E1*-(Rap) 2 (20 μg i.v. q4dx4), followed by a second cycle 24 days after the last injection of the first cycle. The control group received the same dose/schedule of a CD22targeting (Rap) 2 -22*-(Rap) 2 , which served as an alternative non-specific counterpart of (Rap) 2 -E1*-(Rap) 2 . The third group remained untreated and only received saline (100 μl). After treatment commenced, mice were weighed and tumors measured weekly. For both models, tumor volume (TV) was determined by measurements in two dimensions using calipers, with volumes defined as: L × w 2 /2, where L is the longest dimension of the tumor and w the shortest. When a tumor in individual mice ulcerated or exceeded 1.0 cm 3 in measured volume, the animal was deemed to have succumbed to disease progression and was euthanized.
The model for disseminating human Burkitt lymphoma was established by injecting SCID mice i.v. with 1.5×10 7 Daudi cells. On the day cells were administered, the mice were randomized into groups of 5 each. Therapy began 7 days later. The treatment groups received (Rap) 2 -22*-(Rap) 2 at either the MTD (20 μg, q4dx4) or at half that dose (10 μg q4dx4). For the three controls, one group received the same amount of (Rap) 2 -E1*-(Rap) 2 , another group received the parental hLL2 IgG/ epratuzumab (25 μg, q4dx4), and the third group received only saline (100 μL). Mice were deemed to have succumbed to disease progression when hind-limb paralysis developed, or if they lost more than 15% of their initial body weight, or if they became otherwise moribund.
Statistical analysis for the tumor growth data was based on AUC as well as survival time. The profile of tumor growth in each mouse was obtained through linear curve modeling. As a consequence of incompleteness of some of the growth curves (due to deaths), statistical comparisons of AUC was only performed up to the time at which the first animal within a group was euthanized due to disease progression. An f-test was employed to determine equality of variance between groups prior to statistical analysis of growth curves. A two-tailed t-test was used to assess statistical significance (P ≤ 0.05) between treatment groups except for the saline control in which a one-tailed t-test was utilized. Survival studies were analyzed from Kaplan-Meier plots (log-rank analysis), using the Prism GraphPad Software (v4.03) software package (Advanced Graphics Software, Rancho Santa Fe, CA).