TumorSelect® Technology Enhancing the Safety and Ecacy of Cancer Chemotherapy

Veiled Therapeutics has developed an anticancer technology, TumorSelect ® Technology, which 2 combines proprietary anticancer prodrugs and nanotechnology, which takes advantage from 3 current knowledge of human physiology. Tumors have a voracious appetite for cholesterol 4 which facilitates tumor growth and fuels their proliferation. We have transformed this need into 5 a stealth delivery system to disguise and deliver anticancer drugs with the assistance of both the 6 human body and the tumor cell. Veiled ’ s designer prodrugs are assembled within pseudo-LDL 7 nanoparticulates which carry them to tumor tissues where they are taken up, internalized and 8 transformed into active drug and kill the cancer cells. This three-prong approach delivers the 9 anticancer drug selectively to the tumors and thereby avoids or reduces the severe side effect 10 toxicities associated with current chemotherapy. Reduction of side effect toxicity of cancer 11 therapy by our technology will improve patient quality of life, patient retention in treatment 12 regimes, more rapid patient recovery post treatment, and overall patient benefit. Tumor concentrations of paclitaxel are significantly higher in tumors of mice injected with 1 formulated TumorSelect ® paclitaxel compared with the mice injected with Cremophor ® 2 EL/EtOH paclitaxel (194%). Plasma and heart concentrations of paclitaxel are significantly lower in tumored vs. non- 4 tumored animals injected with formulated TumorSelect ® paclitaxel (<80%). 5 Selective cellular uptake of TumorSelect ® paclitaxel by tumors actively expressing LDL- 6 receptors has been demonstrated. 7 Tumor suppression observed was sustained for 63 days after Q1Dx5 dosing with 8 TumorSelect ® paclitaxel. 9 TumorSelect ® technology represents a potential major improvement in the clinical treatment 10 of cancer through enhanced efficacy due to tumor-facilitated targeted delivery and reduced 11 patient toxicity with its associated deleterious side effects. 12 2.


Abstract 1
Veiled Therapeutics has developed an anticancer technology, TumorSelect ® Technology, which 2 combines proprietary anticancer prodrugs and nanotechnology, which takes advantage from 3 current knowledge of human physiology. Tumors have a voracious appetite for cholesterol 4 which facilitates tumor growth and fuels their proliferation. We have transformed this need into 5 a stealth delivery system to disguise and deliver anticancer drugs with the assistance of both the 6 human body and the tumor cell. Veiled's designer prodrugs are assembled within pseudo- LDL 7 nanoparticulates which carry them to tumor tissues where they are taken up, internalized and 8 transformed into active drug and kill the cancer cells. This three-prong approach delivers the 9 anticancer drug selectively to the tumors and thereby avoids or reduces the severe side effect 10 toxicities associated with current chemotherapy. Reduction of side effect toxicity of cancer 11 therapy by our technology will improve patient quality of life, patient retention in treatment 12 regimes, more rapid patient recovery post treatment, and overall patient benefit. 13

A. Background 14
The costs of cancer, measured in terms of mortality, morbidity, direct costs of treatment, and 15 costs of lost productivity are high. TumorSelect ® is a delivery system designed for tumor-targeting delivery of drugs thereby 1 overcoming the low therapeutic index limitation. TumorSelect ® includes a prodrug of 2 sufficient lipophilicity to be entrapped in a lipid nanoparticle of composition similar to that 3 of the lipid core of human LDL particles. Previous work by Maranhão and coworkers has 4 demonstrated that after injection such a pseudo-LDL nanoparticle lipid nanoparticle adsorbs 5 a molecule of exchangeable apo-lipoprotein, the ligand which mediates internalization of the 6 particle by the LDL receptors (LDLR) that are overexpressed by rapidly dividing tumor 7 cells. 45,46,47 This biological phenomenon is the rationale for TumorSelect ® chemotherapeutic 8 delivery technology. 9 As a proof of concept of the technology, paclitaxel, a very widely clinically utilized 10 cytotoxic chemotherapeutic, was selected for evaluation for selective delivery via 11 TumorSelect ® delivery. 12 The work described in this manuscript characterizes the preclinical performance of 13 resistance on the part of the tumors by expression of paclitaxel efflux by ABC transporters 12 (P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), multi-drug resistance related 13 protein (MRP-1)), expression of mutated ß1 tubulin and aberrant tubulin isoforms, and 14 enhanced expression of cell survival mechanisms. 48 15 In order to incorporate the current clinically approved chemotherapeutic into the pseudo-16 LDL particle, the hydrophilic chemotherapeutic was converted to a lipophilic acid-labile 17 prodrug which is capable of being retained within the pseudo-LDL particle until the particle 18 is internalized into the tumor cells by uptake by the LDL receptor and processed by the 19 endosome/lysosome cascade, ultimately releasing the active chemotherapeutic drug moiety. 20 This strategy is directly applicable to the majority of clinically approved and widely utilized 21 chemotherapeutics. 22

Materials and methods 1
To address question onepreparation of a pseudo-LDL delivery formulation vehicle we 2 undertook efforts to prepare such a material via the technique of microfluidization. 3

A. Pseudo-LDL nanoparticle dispersion preparation 4
The TumorSelect ® lipid nanoparticle formulation was prepared through extensive 5 development efforts as previously described. 49 6 i. Homogenization 7 Quantities of the components which make up the nanoparticles (dispersed in 0.9% NaCl 8 in 10 mM Acetate buffer, pH 5.5) of TumorSelect ® paclitaxel are shown in Table 1  9 below. 10 The target amounts of formulation components (PC, DMPC, TG, FC, CE, U and Vit E) 3 were weighed into a suitably sized beaker to form a lipid premix (LPM). The target 4 amount of the acid labile, lipophilic prodrug (ART 207) was weighed into the beaker. 5 The LPM was homogenized by dissolution in dichloromethane (DCM), 10 mL per 100 6 mL formulation. The DCM was removed by gentle heating in a chemical fume hood in 7 a warm water bath with gentle agitation with nitrogen flow. Once the DCM had 8 evaporated, the LPM was held in a 45°C vacuum oven at 100 µm for at least 1 hour to 9 remove residual DCM. The LPM was stored at -5 °C prior to high-pressure 1 homogenization. 2 The LPM was removed from the freezer and allowed to warm to RT; 100 mL of 3 formulation buffer (0.9% NaCl in 10 mM Acetate buffer of pH 5.5 at 65 °C) was added 4 and the mixture was homogenized with a hand blender with short pulses for 1 to 2 5 minutes to form a coarse emulsion. The coarse emulsion was transferred to a 6 MicroFluidizer (MF) (MicroFluidics, Inc., Model 110P with temperature-controlled 7 recirculation bath) and processed at 30,000 psi and 60 °C, monitoring particle size of 8 processed material at 10 minutes intervals by dynamic light scattering until an intensity-9 weighted mean particle size of less than 65 nm was achieved. The nanoparticle 10 dispersion was sterile filtered through a 0.22-µm polyethersulfone polymer membrane 11 filter. 12

ii. Characterization of nanoparticle dispersions 13
The HPLC analytical method used to quantitate ART 207 in lipid emulsions was 14 performed on an Agilent 1100 quaternary pump and single wavelength system. The 15 column was a Phenomenex 4.6 x 50 mm Luna 5µm C18(2) 100Å, part number 00B-16 4252-E0. The method conditions were: flow rate: 1.5 mL/minute, detection: 230 nm, 17 column temperature: 55ºC, and injection volume: 3 µL. The gradient program is shown 18 in Table 2 below. 19 Discovery of a suitable cytotoxic derivative for incorporation (answering question 2 above) 6 into the pseudo-LDL nanoparticle delivery vehicle recognized the derivative must be 7 lipophilic, would readily release active cytotoxic moiety upon delivery to tumor tissue and 8 be readily accessible via chemical synthesis. 9 The lipophilic paclitaxel prodrug ART-207 was synthesized as previously described. 50 The 10 reaction sequence is shown in Figure 1 below. 11 1 Figure 1 Synthesis of ART-207 2 The reactions are described in Table 3 below. 3

C. Biological Evaluation 2
Accepted in vivo animal models were selected to demonstrate TumorSelect ® technology's 3 potential. 4

i. ATL-1 and 2 5
To assess the toxicity and, preliminarily, the efficacy of TumorSelect ® paclitaxel, two 6 small studies were commissioned at Southern Research Institute, Birmingham, AL 7

USA. 8
These studies were designed to address question 3 outlined above-does the proposed 9 TumorSelect construct of cytotoxic derivative and delivery vehicle improve TI of the 10 cytotoxic and still retain efficacy? 11 a. Experiment ATL- 1 12 This study was to determine the toxicity of the TumorSelect ® paclitaxel 13 formulation. 14 The study consisted of six groups of five non-tumored mice per group for a total of

b. Experiment ATL-2 16
This study was commissioned to assess preliminary efficacy of the TumorSelect ® 17 paclitaxel. 18 The study consisted of six groups of five mice per group for a total of 30 mice were treated with a drug-free formulation (injection volume of 0.21 mL/20 g of 8 body weight). Animals in Group 6 were treated with paclitaxel at a dose of 15 9 mg/kg/injection (injection volume of 0.1 mL/10 g of body weight). 10

c. ATL-3 Mouse breast-cancer xenograft safety and efficacy study 11
The purpose of the experiment ATL-3 (contracted for execution at Southern where: Tumor volume is expressed in mm 3 ; W is width (the shorter dimension) in mm; 18 and L is length (the longer dimension) in mm. Mouse weights were measured every 2 19 or 3 days. 20 Beginning on day 24 after implantation (when the tumors reached 200-250 mm 3 ), the 1 mice were divided into three groups and were injected daily with Abraxane (15 mg/kg 2 paclitaxel equivalent), TumorSelect vehicle, or TumorSelect paclitaxel (37.5 mg/kg 3 paclitaxel equivalent) for 5 consecutive days (total Abraxane paclitaxel dose of 75 4 mg/kg, total TumorSelect paclitaxel dose of . Total Abraxane dose of 75 mg/kg and 5 formulated ART-207 dose of 187.5 mg/kg for a paclitaxel equivalent dose of 132.5 6 mg/kg.) 7

A. Material characterization 9
The ART-207 content and mean particle-size data for TumorSelect ® paclitaxel nanoparticle 10 dispersions used in animal studies are shown in Table 4 below. 11

B. Biological evaluation 14
The results of the mouse safety, pharmacokinetics, and efficacy studies are shown below. In 15 all displays, doses are expressed in paclitaxel equivalents. 16

i. ATL-3 Breast-cancer mouse xenograft safety and efficacy study 1
The following graphs show animal survival, animal weights, and tumor weights in the 2 breast-cancer mouse xenograft study. In all figures the TumorSelect ® and Cremophor ® 3 administered doses are expressed in paclitaxel equivalents per injection which provides 4 a total of 5-times the daily injection for the experimental drug exposure. 5

a. Animal survival data 6
The survival of animals in Study ATL-3 is illustrated in Figure 2     Difference between free paclitaxel levels of tumored mice injected with Taxol 3 (paclitaxel) or TumorSelect ® paclitaxel was significant for all assessed tissues (p < 4 0.05). 5 As illustrated in Figure 6 below, in Study ATL-5, tumor concentrations of free 6 active paclitaxel are significantly higher from TumorSelect ® paclitaxel than those 7 from generic Cremophor ® paclitaxel: (ATL5).  Table 7 below shows the volumes of distribution of the TumorSelect ® prodrug 5 ART-207 and of paclitaxel delivered by by the Cremophor ® /EtOH formulation in 6 Studies ATL-4 and 5. 7  cholesterol, triglycerides and phospholipids. 55 Those essential nutrients are available to be 8 taken up from LDL particles in the plasma. As a result, tumors dramatically overexpress 9 LDL receptors (LDLR) by as much as eleven-fold relative to normal healthy systemic 10 tissues. 56,57 High LDLR expression is associated with rapid tumor growth 58,59 and a poor 11 prognosis for the patient. 60 Pseudo-LDL particles can increase the utility of clinically approved chemotherapeutic 3 agents by altering their PK/PD properties and dramatically raising their therapeutic indices. 4 In order to incorporate the current clinically approved chemotherapeutic into the pseudo-5 LDL particle, the hydrophilic chemotherapeutic (selected to be hydrophilic in order to be 6 administered by intravenous infusion in aqueous infusion fluids) is necessarily converted to 7 a lipophilic metabolically labile prodrug which will be retained within the pseudo-LDL 8 particle until the particle is internalized into the tumor cells by uptake by the LDL receptor 9 and processed by the endosome/lysosome cascade. This strategy is directly applicable to the 10 majority of clinically approved and widely utilized chemotherapeutics. 11 The proposed mechanism for the cleavage of the promoiety from ART-207, and liberation 12 of paclitaxel, in the acidic environment of the endosome/lysosome cascade is shown in 13 Figure 10 below. This hydrolysis which takes place intracellularly delivers the active 14 cytotoxic, paclitaxel, and the lipophilic promoiety is cleaved to the nontoxic products, 15 carbon dioxide, glycerol, and oleoyl aldehyde which is rapidly transformed to the fatty acid 16 oleic acid. 17 1 Figure 10 Cleavage of TumorSelect ® paclitaxel prodrug 2 Veiled Therapeutics has developed the strategy as TumorSelect ® technology and licensed 3 TumorSelect ® Paclitaxel to Cloaked Therapeutics Development, LLC to demonstrate its 4 utility of promise to improve both cytotoxic chemotherapy efficacy and tolerability when 5 compared with standard generic Cremophor ® as well as Abraxane ® formulations of 6 paclitaxel. Since many of paclitaxel's issues can be traced to its well-characterized PK/PD 7 limitations, investigators expect that improvements in paclitaxel's PK/PD profile should 8 translate to improved clinical outcomes. TumorSelect ® paclitaxel has overcome the 9 limitations of conventional paclitaxel delivery systems as demonstrated by its superior 10 efficacy and apparent lack of toxicity in established preclinical in vivo mouse models of 11 breast cancer and pancreatic cancer. These experimentally observed benefits support the 12 validity of the TumorSelect® technology and strategy. 13

Conclusions 14
The future of traditional cytotoxic chemotherapy : 15 Clinical practice for the foreseeable future will continue to rely on cytotoxic chemotherapy with 16 hundreds of thousands of patients treated annually. Also, it is clear from recently published 17 studies, such as the NEJM atezolizumab plus Abraxane ® in triple negative breast cancer study, 40  that traditional chemotherapy will remain a mainstay of cancer treatment for the foreseeable 1 future. In particular, there is evidence that taxanes can potentiate immunotherapy. 64 2 Abraxane ® upon administration allows for a higher fraction of free paclitaxel than even generic 3 Cremophor ® paclitaxel and thus drives wider tissue distribution than standard paclitaxel. 65 4 Albumin-bound drug may leave capillaries by active transport across vessel walls, but there is 5 little or no active uptake into tumors and thus little or no selectivity for tumor cells. In short, 6 with Abraxane ® both normal and tumor tissue are non-selectively exposed to free paclitaxel. 7 Abraxane ® 's pharmacokinetic "improvements" are the direct opposite of those that are 8 theoretically desired, and thus opposite to those seen with the superior TumorSelect® paclitaxel 9 formulation. TumorSelect ® dramatically decreases normal tissue exposure while selectively 10 increasing tumor exposure. 11 Abraxane ® and TumorSelect ® paclitaxel were compared in both a pancreatic and an ovarian 12 model with results similar to the Cremophor ® paclitaxel results. However, TumorSelect ® 13 paclitaxel was better tolerated than standard generic Cremophor ® paclitaxel or Abraxane ® and 14 can be dosed more intensively (2.5 equivalents of the MTD in mice of either Cremophor ® 15 paclitaxel or Abraxane ® ) than either the generic Cremophor ® paclitaxel or the Abraxane® 16 formulation. In a tumored mouse model with various dose levels of TumorSelect ® paclitaxel a 17 measure of weight loss post dosing, as an indication of toxicity, showed that TumorSelect ® 18 paclitaxel could be dosed at 2.5 equivalents of paclitaxel with minimal weight loss in the 19 animals, demonstrating that the TumorSelect ® paclitaxel formulation was less toxic than 20 Abraxane ® . 21 In addition to allowing more intense dosing due to lower systemic toxicity, the mechanism of 22 receptor mediated uptake will bypass the common mechanism of resistance of ABC transporter 1 activity (PGP, MDR, etc.) which is membrane located. Both these attributes should enhance the 2 efficacy and reduce the systemic side effect toxicity of the chemotherapeutic agents. 3 The demonstrated advantages of TumorSelect ® paclitaxel include: 4  Better efficacy-in the animals treated with Abraxane ® , the tumors continued their steady 5 growth, while in those treated with TumorSelect ® paclitaxel (at 2.5X the paclitaxel 6 equivalent of Abraxane ® ) the tumors had nearly disappeared by the day 56 of study. 7  Less toxicity-there was substantially less weight loss observed in the animals treated 8 with TumorSelect ® paclitaxel at 2.5X the paclitaxel-equivalent dose given to Abraxane ® 9 treated animals. 10 Unlike TumorSelect ® paclitaxel, Abraxane ® allows for a higher fraction of free paclitaxel and 11 thus drives wider tissue distribution than Cremophor ® -formulated paclitaxel. While tumor 12 response rates are improved, the debilitating side-effects of paclitaxel are also made substantially 13 worse. Nonetheless, Abraxane ® , which has never more than barely penetrated its total potential 14 market, has become a blockbuster commercial success with sales of nearly $1 billion. 15 TumorSelect ® paclitaxel has the ideal and directly opposite PK/PD profile of Abraxane ® and 16 would therefore be expected to improve tumor responses while substantially decreasing side-17 effects. 18

Additional TumorSelect ® Advantages 19
 It does not contain or require human proteins as part of its manufacturing process 20  It is not a liposome technology and is comprised of well-known, safe, and easily sourced 21

D. Availability of data and materials 8
The datasets used and/or analyzed during the current study are available from the 9 corresponding author on reasonable request. 10

E. Competing interests 11
The authors declare that they have no competing interests. 12

F. Funding 13
The work described in this paper was funded by Veiled Therapeutics, LLC (formerly Arbor 14 Therapeutics, LLC) and by Cloaked Therapeutics, LLC.