TeoffflScal Field;

There Is real need to advance Ghemoifrerapeuiie treatment of tripe egative breast cance te ^: reduce the non- specific side effect of chemotherapeuties:

Mitochondrial dysfunction is an important hail mark of cellular dysfunction associated with cancer. Cancer cells that fall to go into apoptosis in response to treatment provide a very real oerrier to successful combinatorial chemofhefapeutie therapy. Cancer driven mitochondrial override mechanisms include but are not limited to: decreased apoptosss, decreased oxidativ phosphorylation, and increased aerobic glycolysis. The wort? of fv!iiane et- af 2011) demonstrates thstiranslertt cellular hypoxia contributes to multi-drug resistance (: DR) in many re-oceurring triple negative breast cancer case (TN8C).

In tit past the liver toxicity associated: with mufti-drug therapies in treating (TNSG) occurrence out weights the positive benefits of using cocktail treatments:, in the current invention we disclose a targeted multi-drug therapy that has little or no systemic toxicity that eiectively targets the energ systems (mitochondria) in TNBC cancer,

Nanomedicln offers an exceptional opportunity in drug design for new cancer therapies; the opportunity to increase specificity through "active targeting" of molecular residues relevant: to a particular ip e otype, the opportunity to achieve combination therapy In one formulation, the opportunity to deliver drugs and ^■ biologies ^' that cannot fee delivered in free solution, and the opportunity to use a lower dose of antineoplastic agents and decrease residual to lPlty of treatment. There are oyer 40 nanomediclne formulations approved by the FOA or equivalent agencies, with almost 10 FDA nanomedicine therapies for cancer treatment and over 15 rsanomedicine therapies in clinical trials for cancer treatment £41- Based on the disclosed advance In t is application, a rniiochondrlotro lo nahornedlclhe therapy for reversing multi-drug resistant C PR} In TNBC is now a viable and effective treatment approach for managing breast cancer.

In t current invention we disclose the novel application for fv1Df¾ TNBC; the development of a dual targeted naoonwdicine therapy that targets the epidermal growth factor receptor oh the surface of TNBC cancers ceils - _. (this receptor is often overexpressed in TNBC} and subcellular argefihg of mitochondria through mitofusin 2 fMF!Si2) targeting (mitofusin mediates fcf~mito¾h©n fisi fusion and fusion of mitochondria wit the endoplasmic reticulum). The novel combination therap will deliver an MFN2-peptide- polymer construct for blocking F.N2 along with a low dose of BAM? (a BAX activator). Transien blocking of MFH2 reduces cellular energy capacity (through decreased mitochondrial fusion) ., decrease total protein production (by decreased mitochondrial coupling to the endoplasmic reticulum}, increases tie susceptibilit of the cell to paclitaxel or BAM? (increased efficacy of lower dose), with minimal: toxicity to normal cells (as MFN2 blocking inhibits mitochondria! fusion not mitochondrial function). Slocking the ability of m tochondria to fuse together and with other organelles through a nanomedicine therapy significantly Increase the efficacy of TNBC treatment and address global health concern.

Brief P jO ¾Qft Q wi ngs

Figure 1 Is a depletion .schema for a treatment schema for cancer treatment employing nanocarhers modified with both EGFR peptide and PN2 peptide.

Figure 2a Illustrates how MF 2 padicipates in fusion of mitochondrial and endoplasmic reticulum: Fl#.2(:h) depicts how mitochondrisi fusion may be blocked by FN2*pepiide Polymer in conjunction ifh a dual targeted EGFR MF 2 nanoparticle.

Figure 3 depicts a representative nanoparticle design for a dual targeted £0F& WF 2 having R¾ modification.

£ e¾orl t|or M 'Mm ^ Mi .

This new dasg/fr a ment scheme is depicted in. Figure 1. The surface of the nanocarrlers (1) was modified wit both an EGF peptide (2) and an MFN2 peptide (4), B6F targeting allowed active targeting of T BC eels. When the nsnocarder binds to the EGFR receptor {18} it s internalized via a flip-flop mechanism, once inside the ceil, the FN2 residues on the surface of the nanocar ier binds to MFN2 on mitochondria (8) and blocks mitochondria! fusion with each other and with the endoplasmic reticulum.. As the nanoparticle degrades, (10) the thefapeytics are released (12) arid the fvlFNS-pepiide- polymer construct a) blocks MFN2 (12) and render a ceil susceptible to the proapoptoiie agent (14), This treatment scheme improved therapeutic outcomes for M&R T BG b disabling the Ijioenergetic network {mitochondria! fusion) and maintaining fission (requirement for apoptosis}.

The scheme shown in Figure 1 can be summarized as;

1. Targeting and binding to MF 2 increases apoptosis - as mitochondria! fission is a necessary stage in the apoptotic process.

2. The result of this decreased energy capacity and decreased protein synthesis capacit renders a ΜύΗ T BC ceii more susceptible to a Sow dose of a proapopfofie agent delivered in the nanopartiele formulation _. (increased efficacy). Dose range 20-200 mg/kg.

3. Dual targeting of EGFR and MF 2 enables specific eel! and organelle delivery; through targeting EGFR: overexpression in TN8C cells and MFN2 targeting of mitochondria.

4. The dual targeting system does not caus overt toxicity upon systemic

administration as mitochondrial function is completely inhibited (onl

mitochondrial fusion will be inhibited); targeting and inhibiting M 2 with a

FN2- eptsde-polymer construct i less toxic than silencing PFN2 through siRNA as blocking £M2 with the peptide-polyrner construct Is a transient process-

A, SACS e^QUND AND SI 3NIFiCANCE

C.i < usio a««t f ission

Contrary lo misconc ptions, mitochondri ar not static organelles that are merely the "powerhouses" of the cell. Mitochondria are highly plastic organelles that undergo Intracellular fission and fusion, out of phase with cell division, Mitochondria are active and mobile, they use the mitochondria! GTP-ase MIR© and its effector MILTO to move bi-directionally along microtubules £5]. Mitochondria certainly function as Isolated organelles, but we now know they also function as complex networks to accomplish specific cellular tasks | |. lyBtochondriai copies per cell depend on the function and energy demands of the tissue, with red tissue such as heart having the highest copy number per cell Mitochondrial morphology is also a tissue variant with hepatoeytes having more spherical mitochondria while fibroblast mitochondria are elongated Perpetual mitochondrial fusio and fission is an important form of cellular quality control,, is used to correct for damaged -mitochondria, i essential to localizing and migrating mitochondria to specific subcellular regions such as the synapse of a neuron, and is a response to metabolic changes j¾l,?J. Not only are mitochondria capable of functioning in networks and in continual contact with each other, but recen biologica investigations have revealed that mitochondria are also In direct membrane contact with the endoplasmic reticulum, functioning in mitochondrial fission., in intracellular calcium regulation, and apoptosis [8-1 i], Mitochondria have also been reported to have direct membrane association with meianosorrses, iysosome related organelles Involved in the synthesis and transfer of melanin in pigment cells Mitochondrial association with th EF¾ and with melanosomes involves similar protein anchors including ltofusion 2 pj. Mtoc ondrial/nielanosome contacts have been correlated with melanogenesis p¾- This insight depicts scenario of mitochondria being recruited to and establishing direct membrane association with organelles undergoing active biogenesis (perpetual contact with ER and transient contact with other organelles as they are involved in biosynthesis), inhibiting mitochondrial fusion is the targeting therapy in this invention for treating cancer,

C J, it chond ial Dysfunction in Cancer

Cancer ceil mitochondria have long been established as dysfunctional; Increased mtDNA mutations, increased RGS production, decreased OXPHOS, and failure to induce apoptosis [12- ], Due to the central role of mitochondria in programmed cell death and th©: Inherent resistance to apoptosis of cancer cells, cancer is very much a mitochondrial disease. Most types of cancers are resistant to bot extrinsic and intrinsic apoptotio signaling [1 Sj. Apoptosome dysregulation has been linked to the carcinogenesis of ma different cancers |12|, Mutation in the tumor suppressor gene pS3 a e the most common mutations In human: cancers; p53 functions In apoptosis regulatio Bcl-2, an anfk apeptot!c protein, is over-expressed in many tumors conferring resistance of cancer cells to apoptosis [13J. Mitochondria are central to energy and apoptotio dysfunction in cancer. Th mechanism of action of MPU2 fusion and therapeutic blocking is demonstrated in R trn- 2, As shown in Panel A mitofusion 2 (20) a protei in humans encoded by the ΙΑ Η2 gene Is em edded m the outer membrane of the mitochondria (22), MFM2 mediates fusio of mitochondria or mitochondria and endoplasmic reticulum ^' {24% The therapeutic dual targeted E6F F 2 nanopartioSes (26) of the present invention {Panel B) blocks mitochondria! fusion in conjunction with MFM2~peptlde polymer (28), The WH2 peptide binds to the surface of the nanoparticle and the encapsulated F 2 peptide functions to block FM2 mediated mitochondria! fesion,

Preferred Em odiments':

A selection of FGF.R positive cells from ATCC ^' s triple negative breast cancer pane! is used -{MOA- B-231 , MDA-.M8- 68, and BT-20) along with SKGV3 ovarian cancer ceils and MQR cells (Included an established WM eel! line was used as positive control), and MOA- 8-43S cells (for an EGFR negative control). The MD&- B-231 ceils, MDA-M -M®, S OY3, and MDA- &-43S ^' cells are also part of the GI- 0 Human Cancer Ceil Line Screen fo developmental therapeutics. Hypoxic derivatives of the ceil lines were created using a modular incubation chamber were flushed with a O.S% Ch, 5% COsi nitrogen balanced gas for five minutes and Incubated at 3? ^* C. Hypoxic, normoxlc, and MQR ceils were incubated at 37 ^* C and maintained in P&$-1S40 media supplementesi with 10% fetal bovine serum and 1 penici!lin/strepiomyciniamphotericin B mixture.

To determine efficacy of MFN2 blocking in the panel of hypoxic, normoxic, and MDR cell lines, a dose response study was conducted with a range of time points and ooncehifatsons using MFH2 sIRNA (positive control), an MFN2 antibody, m FN2- peptide, an ¾IF 2-pept!de~polymer construct, a combination of the antibody and peptide (cem etit!ve binding study between antibody and peptide), and a combination of the antibody and peptide-cpnstruc (competitive binding study between antibody and peptide- construct). The MFN2-pepflde~poiymer construct was used in a second embodiment

At each tim point, the BCA assay is used to measure basal protein concentration of all samples; results are compared to untreated cells (and normalized to cell number) to assess the effect of MFN2 blocking on protein production {possible outcome of decreased mitochondrial binding to the endoplasmics .reticulum) ^' , To assess ATP concentration, fhe iteehondnal To Gio Assay (Proniega) was used; this assa measures ATP concentration as ell as mitochondrial membrane potential (combined provide data for mfetoxiclty), Western blots were performed on nucleic and cytoplasmic protein factions to evaluate the tviOR phenoty/pe of the celis; proteins to be examined include Epidermal Growth Factor Receptor (EOF R), P-giyoeprotein (F-gp; drug efflux pump), Multi-drug Resistance Protein 1 ( RP!; drug efflux pump). Hypoxia Inducible Factor ies (HiF~1a; transcription factor upreguiated in MDR Hypoxi Inducible Factor 2ct (HIF~2u; transcription factor upreguiat d in DR), Glucose transporter 1 {Giut-1}, Hexokinase Si (HX^2 _; first enzyme of glycolytic pathway), Complex V (CMPLX V; ATP producing unit in oxidative phosphorylation)* Cytochrome C (Qyt C; electron transport chain component, apopfosome component). KSitofusin 1 ( F _. Ni ; mitochondrial fusion protein); yitofusin 2 (MFU2: mitochondrial fusion protein also mediates mitochondrial fusion with the endoplasmic reticulum), and Optic Atrophy 1 (OPa-1; inner mitochondrial membrane fusion protein). Cell viability { TS assay) was conducted.

Evatetten of activity (Compound Efficacy) ti Dismvoty: af the appropriatep - eptide; develop and optimize a long c ain pofymer-peptido construct (far surface modification) and a short chmn po!ymor-pepMde construct (for encapsulation}.

The optimum chain to poiynwr -peptide construct is with & < 2500 kfWpoiymer and-® <2S ammo add peptide.

An existing anti-mitofusin 2 peptide w s used as a guide for initial peptide design and o timisatio (sigma-aldrietrs 6 44 corresponding to amino acid residues 55?-576 of human MFN2), Using previously established methods of peptide-polymer conjugation, the peptide was linked to a low molecular weight Polyiethyiene glycol) to create a short chain construct and linked to Poly(eihyien glycol)-poiy-Sactic-giycollc acid conjugate to create a long chain construct (21-23]. The peptide-polymer chemistry will be optimised to promote extended receptor occupancy (¾SFN2 binding); the constructs will be included In Aim 1 studies for ev luation, MMR will be used to characterize the constructs. $y tti&$j$ & ^Q ptimizat o, ansf eft8fmt&fiMtion- f-clm1 te*g&i&ti- {&t$Fftw& A FAf¾ ptiiyffi ric na pmticies modulating MFN2-pepM&"pQiym&r (shmi chain) m<j pac!itaxel

Polymeric nanopartio es were synthesized according to a previously established solvent displacement method [21-23], The previously employed synthesis schema (for combination t erapy EOFR-targeted nanopartioies) was modified to include two targeting e wtrycts and adapted to maximise d u encapsulation. Therefore this nvention represents a significant advance over the state-of-the-art developed by Milan© et at, (2Ϊ11). The nanopartscl design is depicted in Figure 3. PES modification {30} prevents aggregation of the dual targeted EGFR-peptide conjugate (32} and PN2 -peptide polymer conjugate 34) about a polymer .core (36) in this cure containing MFN2-peptsde- poiymer fragments and an adjuvant chemotherapeutic agent, combination therapy system.Nanoparticles were oliaracterlzed for size and zeta potential (using a ZetaPlus Particle Analyzer or similar instrumentation). Surface modification was determined using E QA analysis and nanoparticles will be imaged b SEW. The optimal dose combination of FN2-peptide~poiymer and paclitaxel are determined throug a dose response stud in the panel of cells, Nanoparticies are loaded with the optimum molar ratio of the agents and loading efficiency was measured by iyophiliaog the nanoparticles, rupturing th washed particles, and measuring drug load. Likewise, using iyophilized naneparficles, drug release kinetics was measured over the time course of 15 minutes to 10 days. The dried nanopartioies are suspended in two different puffers (one at pH 7.4 and one at pH J to mirror the ofte acidic microe vlmnment of a tumor). Samples were removed and quantified (a sorbance; plate reader), and buffer wa replaced to prevent sink conditions. Burst release phenomena or high drug retention has lead to formulation optimization. Active targeting of EGF ¾nd MFN2 was evaluated through competitive fnanopartleies verses antibodies) biding studies, visualized through microscopy.

Assessing he tt mp&utio effcaoy of m part ie mmbmation i mpy with MFN2- papM0 constructs arid pa itami m MDR _f hypoxic, et hormoxic wit lines.

Cell viability studies ( TS were conducted to determine the tCsa values of single agent treatment combination therapy, in solution, d as hanoparticies. Results are compared to non-targeted nanoparfiefes (no peptides on surface), unloaded (blank) nanoparticles, media (no treatment), and poly(ethyleneimine) (positive control). The combination index of F 2~p0 s de- olyfT¾er and paclla eS therapy ar determined y comparing cornoinaSon therapy to solitary treatment.

Calculated effective range of doses: 2—200 mg/kg

Safety Studies: Evaluated c&ttuM toxicity and safety gf te mnom&ciic tm therapy

To evaluate the cellular toxicity and safety of the nanomedlcine therapy, a panel of noncancerous cells were evaluated using the MTS assay and the Mitochondria! ToxGio Assay (to assess mitochondrial toxicity). Toxicity of single agent treatment a d combination therapy in solution forms and nanopartscle formulations were evaluated. Toxicity was compared to MFH2 silencing using FN2 sI NA,

Data Analysts

Staisfieal analysis was completed using GrapfiPad Prism Φ software and icrosoft Excel.

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