Pei Zhou

Shuang Wu

Mohammad Hegazy

Hong Li

Xueju Xu

He Lu

Xin Huang
email: xinhuang@hit.edu.cn

Engineered borate ester conjugated protein-polymer nanoconjugates for pH-responsive drug delivery

Keywords: protein polymer nanoconjugates, self-assembly, borate ester bond, pH-sensitive, anti-cancer

To improve the clinical efficiency of cytotoxic anticancer drugs e.g. doxorubicin (DOX), reduce the severe off-target side effects, and allow the more biocompatible and biodegradable drug penetration into tumor cells, our research efforts developed a new DOX-conjugated protein polymer nanoconjugates (PPNCs) prodrugs delivery system. Briefly, DOX was conjugated to bovine serum albumin (BSA) and the complex was treated with lactobionic acid (LA) as well as folic acid (FA) to enhance drug endocytosis and targeting selectivity. Such functionalized BSA could be conjugated with a designed phenylboronic acid functionalized poly(N-isopropylacrylamide) (PNIPAAm) via forming a pH-sensitive borate ester bond to give the functionalized PPNCs prodrugs. The potential of the PPNCs prodrugs on tumor cells therapy was systematically evaluated in dose/time-dependent effects. In vitro results showed a rapid accumulation of the prodrugs into the MDA-MB-231 tumor cell during the first 30 minutes and reached maximum at 24

hours. Moreover, the cell-killing effect was observed quickly after 4 hours incubation with an IC50 of 0.5 mg/mL (≈ 4 µM/L). In general, given the efficient pH-dependent DOX release of these constructed nanoconjugates, it is anticipated to contribute a potential delivery strategy for cancer therapy.

Introduction

Cancer is a dominant intimidation to human health [START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF]. Toxic drugs are widely used to treat cancer, however, their side effects constitute a major obstacle in clinic. To overcome this matter, one can use prodrugs instead of drugs as a promising strategy to improve delivery of toxic effects within tumor cells greatly [START_REF] Tang | Polymeric nanostructured materials for biomedical applications[END_REF][START_REF] Seidi | Designing smart polymer conjugates for controlled release of payloads[END_REF]. Up to now, despite the innovation of numerous conventional drug delivery systems, the efficacy and clinical use of prodrug is still restricted by metabolism clearing mechanism, nonspecific biodistribution and multidrug resistence [START_REF] Blanco | Principles of nanoparticle design for overcoming biological barriers to drug delivery[END_REF][START_REF] Prabhakar | Challenges and key considerations of the enhanced permeability and retention effect for nanomedicine drug delivery in oncology[END_REF][START_REF] Allen | Liposomal drug delivery systems: from concept to clinical applications[END_REF]. To address these restrictions, strategies toward drug delivery and delineated nanomedicine have been developed to improve the efficacy [START_REF] Maksimenko | A unique squalenoylated and nonpegylated doxorubicin nanomedicine with systemic long-circulating properties and anticancer activity[END_REF][START_REF] Zhang | Improving paclitaxel delivery: in vitro and in vivo characterization of PEGylated polyphosphoester-based nanocarriers[END_REF][START_REF] Pang | pH-responsive polymer-drug conjugates: design and progress[END_REF][START_REF] Zhang | Targeted chemo-photodynamic combination platform based on the DOX prodrug nanoparticles for enhanced cancer therapy[END_REF]. Such strategies widely use diverse drug delivery vehicles that are able to enhance drug penetration and detection, including liposomes [START_REF] Hansen | Positron emission tomography based elucidation of the enhanced permeability and retention effect in dogs with cancer using copper-64 liposomes[END_REF][START_REF] Liu | Irinotecan delivery by lipid-coated mesoporous silica nanoparticles shows improved efficacy and safety over liposomes for pancreatic cancer[END_REF][START_REF] Feng | Cisplatin-prodrug-constructed liposomes as a versatile theranostic nanoplatform for bimodal imaging guided combination cancer therapy[END_REF],

polymersomes [START_REF] Wong | Polymersomes prepared from thermoresponsive fluorescent protein-polymer bioconjugates: capture of and report on drug and protein payloads[END_REF][START_REF] Gaitzsch | Engineering functional polymer capsules toward smart nanoreactors[END_REF][START_REF] Iyisan | Multifunctional and dual-responsive polymersomes as robust nanocontainers: design, formation by sequential post-conjugations, and pH-controlled drug release[END_REF], dendrimerosomes [START_REF] Kainz | Polymer-and dendrimer-coated magnetic nanoparticles as versatile supports for catalysts, scavengers, and reagents[END_REF][START_REF] Wei | Anticancer drug nanomicelles formed by self-assembling amphiphilic dendrimer to combat cancer drug resistance[END_REF][START_REF] Hu | Bioinspired Design of Stereospecific d-Protein Nanomimics for High-Efficiency Autophagy Induction[END_REF], inorganic particles [START_REF] Soenen | Intra) cellular stability of inorganic nanoparticles: effects on cytotoxicity, particle functionality, and biomedical applications[END_REF][START_REF] Anselmo | Impact of particle elasticity on particle-based drug delivery systems[END_REF][START_REF] Hegazy | Construction of polymer coated core-shell magnetic mesoporous silica nanoparticles with triple responsive drug delivery[END_REF], coacervates [START_REF] Wang | Non-covalent microgel particles containing functional payloads: coacervation of PEG-based triblocks via microfluidics[END_REF],

proteinosome [START_REF] Zhou | Multifunctional and Programmable Modulated Interface Reactions on Proteinosomes[END_REF][START_REF] Liu | Hierarchical proteinosomes for programmed release of multiple components[END_REF][START_REF] Zhou | In situ gelation-induced death of cancer cells based on proteinosomes[END_REF], and molecularly targeted nanoparticles [START_REF] Li | Ratiometric fluorescence platform based on modified silicon quantum dots and its logic gate performance[END_REF][START_REF] Li | Modulation of assembly and disassembly of a new tetraphenylethene based nanosensor for highly selective detection of hyaluronidase[END_REF]. Besides, they could be used to construct drug carriers, targeted-drug delivery and nanodrugs [START_REF] Huang | Combination of small molecule prodrug and nanodrug delivery: amphiphilic drug-drug conjugate for cancer therapy[END_REF][START_REF] Chen | An imagable and photothermal "abraxane-like" nanodrug for combination cancer therapy to treat subcutaneous and metastatic breast tumors[END_REF][START_REF] Qiu | A cell-targeted, size-photocontrollable, nuclear-uptake nanodrug delivery system for drug-resistant cancer therapy[END_REF]. In spite of these great developments, the side-effects into tissues or organs in the process of transportation, communication, metabolism and excretion, was still due to the limited biodegradability and long chronic toxic effect. Therefore, the development of novel drug delivery systems are expected to provide new materials with improved biocompatibility and better drug payloads with selectivity, still needs our great efforts.

Recently, much attention has been paid to a new concept of directly conjugating hydrophobic drugs with good biocompatible macromolecules to form drug delivery systems. Bovine serum albumin (BSA) is a natural macromolecule endowed with many advantages for drug targeting and it is known to improve the pharmacokinetic profile of peptides, because of its good biocompatibility, facile modification, low immunogenicity, high safety and excellent biodegradability as a versatile carrier [START_REF] Thakkar | Albumin microspheres as carriers for the antiarthritic drug celecoxib[END_REF][START_REF] Zhou | In Situ Generation of Core-Shell Protein-Based Microcapsules with Regulated Ion Absorbance Capacity[END_REF][START_REF] Liu | Simple bioconjugate chemistry serves great clinical advances: albumin as a versatile platform for diagnosis and precision therapy[END_REF]. In comparison with other carriers, such as liposomes, polymeric micelles and inorganic particles, BSA offers the capacity of loading and delivering to the pathological sites.

Additionally, folic acid (FA) has been widely employed as an active targeting agent to improve the targeting ability [START_REF] Low | Folate-targeted therapeutic and imaging agents for cancer[END_REF][START_REF] Xia | Folate-targeted therapies for cancer[END_REF][START_REF] Chen | Structural basis for molecular recognition of folic acid by folate receptors[END_REF][START_REF] Porta | Folic acid-modified mesoporous silica nanoparticles for cellular and nuclear targeted drug delivery[END_REF]. For this reason, FA has been used

to deliver anticancer prodrugs because of its high binding affinity with tumor cells in liver, kidney, ovary, brain, uterus and heart [START_REF] Paulos | Folate receptor-mediated targeting of therapeutic and imaging agents to activated macrophages in rheumatoid arthritis[END_REF][START_REF] Kim | Doxorubicin loaded pH-sensitive micelle: antitumoral efficacy against ovarian A2780/DOX R tumor[END_REF]. This effect was most likely due to FA-prodrug could be accumulated at the tumor sites, resulting in augmentation of drug uptake through folate receptor-mediated endocytosis by cancer cells [START_REF] Chen | Poly (L-lysine)-graft-folic acid-coupled poly (2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA): A bioactive copolymer for specific targeting to folate receptor-positive cancer cells[END_REF][START_REF] Cal | Targeting cancer cells with folic acid-iminoboronate fluorescent conjugates[END_REF][START_REF] Wang | Folic acid-functionalized mesoporous silica nanospheres hybridized with AIE luminogens for targeted cancer cell imaging[END_REF]. Among these versatile nanocarriers, boronic ester conjugated with polymer was assessed as an ideal pH-responsive biomaterial to construct nanoconjugates by self-assembly, spontaneously formed a sensitive boronic ester bond in a moderate pH (≈ 9-10) [START_REF] Fu | Multifunctional lactobionic acid-modified dendrimers for targeted drug delivery to liver cancer cells: investigating the role played by PEG spacer[END_REF][START_REF] Zhao | Lactose-functionalized gold nanorods for sensitive and rapid serological diagnosis of cancer[END_REF][START_REF] Obayashi | Electronic structure of reaction intermediate of cytochrome P450nor in its nitric oxide reduction[END_REF] and rapidly disassembled in a weak acid environment [START_REF] Zhang | Synthesis and volume phase transitions of glucose-sensitive microgels[END_REF][START_REF] Roy | Sugar-responsive block copolymers by direct RAFT polymerization of unprotected boronic acid monomers[END_REF][START_REF] Su | Catechol polymers for pH-responsive, targeted drug delivery to cancer cells[END_REF][START_REF] Chen | Dual-responsive boronate crosslinked micelles for targeted drug delivery[END_REF][START_REF] Li | Well-defined, Reversible boronate crosslinked nanocarriers for targeted drug delivery in response to acidic pH values and cis-diols[END_REF][START_REF] Ellis | Boronate-mediated biologic delivery[END_REF][START_REF] Zhu | Efficient nuclear drug translocation and improved drug efficacy mediated by acidity-responsive boronate-linked dextran/cholesterol nanoassembly[END_REF]. Thus, borate ester was well explored in sugar sensing [START_REF] Wu | Selective sensing of saccharides using simple boronic acids and their aggregates[END_REF][START_REF] Jin | Carbohydrate recognition by boronolectins, small molecules, and lectins[END_REF], protein modification [START_REF] Ellis | Boronate-mediated biologic delivery[END_REF][START_REF] Hoeg-Jensen | Reversible insulin self-assembly under carbohydrate control[END_REF][START_REF] Brustad | A genetically encoded boronate-containing amino acid[END_REF][START_REF] Liu | Evolution of proteins with genetically encoded "chemical warheads[END_REF][START_REF] Cal | Boronate-mediated biologic delivery[END_REF], and reactive oxygen/nitrogen species detecting [START_REF] Lippert | Iminoboronates: a new strategy for reversible protein modification[END_REF][START_REF] Wang | Boronate oxidation as a bioorthogonal reaction approach for studying the chemistry of hydrogen peroxide in living systems[END_REF][START_REF] Z.-J. Chen | Genetically encoded fluorescent probe for the selective detection of peroxynitrite[END_REF] as well as surface immobilizing [START_REF] Liu | Dual-responsive surfaces modified with phenylboronic acid-containing polymer brush to reversibly capture and release cancer cells[END_REF][START_REF] Pan | Dynamic introduction of cell adhesive factor via reversible multicovalent phenylboronic acid/cis-diol polymeric complexes[END_REF][START_REF] Bull | Exploiting the reversible covalent bonding of boronic acids: recognition, sensing, and assembly[END_REF].

Importantly, borate ester derivatives have a binding affinity to monosaccharides through a dynamic reversible process. Shin and co-workers showed a binding affinity (Keq ≈ 17800 M -1 ) of a phenylboronic acid-salicylhydroxamic acid conjugation system [START_REF] Shin | Assembling ligands in situ using bioorthogonal boronate ester synthesis[END_REF]. As the coupling of borate ester derivatives with biological diols showed a considerable number of properties such as high stability, fast rates, potential reversibility and high pH sensitivity, it represents an additional asset when targets the cancer surface. These properties advocate considerably the merits of borate ester derivatives in modification of biocarriers in order to achieve surface immobilization along with reversible reparation.

In an attempt to develop a new drug delivery system with favorable biocompatibility, high drug payloads, cancer targeted properties, effective releasing as well as multiple functionalization, we explored a novel multifunctional pH-sensitive borate ester bridging protein polymer strategy based on biocompatible and biodegradable DOX-conjugated prodrug with FA modified surface (Scheme 1, left).

In the experiments, DOX was conjugated onto BSA and treated with lactobionic acid (LA) and folic acid, then reacted with phenylboronic acid (PBA) functionalized thermosensitive polymer (PNIPAAm). The resultant products were used to generate a stable pH-sensitive borate ester bridging protein polymer nanoconjugates via a covalent coupling reaction under weak alkaline conditions. Biological and pharmaceutical properties of the final product as an anticancer agent in vitro were discussed.

Scheme 1. Schematic design of PPNCs: (left) pH-sensitive borate ester bridged BSA and PNIPAAm nanoconjugated drug-delivery system, whereas (right) formation and mechanism of protein polymer nanoconjugates (PPNCs) prodrug.

Experimental section

Synthesis of end-capped mercaptothiazoline-activated PNIPAAm by RAFT polymerization

The modification of mercaptothiazoline-activated PNIPAAm was prepared according to the previously reported method [START_REF] Huang | Interfacial assembly of protein-polymer nano-conjugates into stimulus-responsive biomimetic protocells[END_REF][START_REF] Huang | Design and construction of higher-order structure and function in proteinosome-based protocells[END_REF][START_REF] Zhou | Programmable modulation of membrane permeability of proteinosome upon multiple sdtimuli responses[END_REF][START_REF] Su | Enzyme-modulated anaerobic encapsulation of chlorella cells allows switching from O2 to H2 production[END_REF]. Briefly, mercaptothiazoline-activated trithiol-RAFT agent (18.9 mg, 50 μmol), AIBN (1.7 mg, 10 μmol), NIPAAm (847 mg, 7.5 mmol) and acetonitrile (8 mL) were added to a 25 mL round-bottom flask. The flask was then sealed and the solution degassed via four freeze pump-thaw cycles. The polymerization was carried out at 60 °C for 8 h (conversion 45%), then purified by three times precipitation in diethyl ether/hexane (2:

1 volume ratio). The obtained polymer was characterized via 1 H NMR spectroscopy in CDCl3. The proton signal (δ, 4.58, 3.3 ppm) from mercaptothiazoline at the end of PNIPAAm chains was clearly visible in 1 H NMR spectrum. The molecular weight of the obtained PNIPAAm was determined through 1 H NMR by comparing the proton integral for CH signal at δ = 7-7.5 ppm in phenylboronic acid with that of the characteristic CH signal at δ = 4.01 ppm in the repeated NIPAAm units (Mn 10000 g/mol).

Synthesis of end-capped phenylboronic acid-activated PNIPAAm

The conjugation of 4-aminophenylboronic acid pinacol ester and end-capped mercaptothiazoline-activated PNIPAAm was carried out in an aqueous solution (pH 8.5, sodium carbonate buffer). The synthetic process was shown in scheme 1.

End-capped mercaptothiazoline-activated PNIPAAm (Mn 10000 g mol -1 , 40 mg in 10 mL of water) was added to a stirred solution of 4-aminophenylboronic acid pinacol ester (4 mg, in 10 mL of PBS buffer pH 8.5) to give polymer chains for 4-aminophenylboronic acid pinacol ester with molar ratio of 1 : 5. The mixed solution was stirred for 24 h, then the product was dialyzed to remove any unreacted 4-aminophenylboronic acid pinacol ester or salts against HEPES buffer (10 mM, pH 7) three times over 24 hours and against Milli-Q water three times over 2 days. Finally, the product phenylboronic acid end-capped PNIPAAm was lyophilized and stored at -20 o C.

Synthesis of BSA-LA complex

The conjugation of LA to BSA via an amide bond was performed by the N-hydroxysuccinimide activated carboxyl group of LA and the amino group of BSA.

Briefly, lactobionic acid (LA, 10.74 mg, 0.03 mmol), N-hydroxysuccinimide (NHS, 3.45 mg, 0.03 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 5.76 mg, 0.03 mmol) were dissolved together in 25 mL of PBS buffer (pH 5.0, 100 mM). The reaction was carried out in dark for 60 min to activate the carboxylic groups of LA. Then, a solution of bovine serum albumin (BSA, 100 mg in 25 mL of distilled water) was added dropwise to this stirred solution and stirred for another 24 h at room temperature in the dark, as well. After the reaction completion, the solution was purified by centrifuging to remove any precipitate, after that the supernatant was dialyzed (dialysistubing 12-14 kDa MWCO) extensively against Milli-Q water.

Synthesis of DOX-BSA-LA complex

The conjugation of DOX to BSA-LA was via an amide bond formation in aqueous solutions. In brief, BSA-LA (100 mg), N-hydroxysuccinimide (NHS, 1.725 mg, 0.015 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 2.88 mg, 0.015 mmol) were dissolved together in distilled water (50 mL) in the dark for 60 min to activate the carboxylic groups of BSA. The pH of the reaction mixture was immediately adjusted to 5.0 by carefully adding HCl (0.5 M). Then, a solution of doxorubicin (DOX, 8.7 mg, 0.015mmol in 25 mL of distilled water) was added slowly to the mixed solution and stirred for a further 24 h at room temperature in the dark.

After finishing the reaction, the mixed solution was purified and separated from the free DOX through centrifugation to remove any precipitate, thereafter the supernatant was dialyzed (dialysis tubing 12-14 kDa MWCO) extensively against Milli-Q water.

Synthesis of Folic acid-modified LA-BSA-DOX complex (BLDF)

Folic acid (FA) was conjugated to LA-BSA-DOX via an amide bond reaction that is similar to the reaction between the residual amino group of BSA and the abundant carboxyl group of FA. Briefly, FA (6.615 mg, 0.015 mmol), N-hydroxysuccinimide (NHS, 1.73 mg, 0.015 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 2.88 mg, 0.015 mmol) were dissolved together in 25 mL of PBS buffer (pH 5.0, 100 mM). The solution was stirred in the dark to activate the carboxylic groups of FA. After adding the solution of LA-BSA-DOX (100 mg in 25 mL of distilled water), the mixed solution was stirred in the dark for another 24 h at 25 °C. The precipitates of solution were removed by centrifuging, then the supernatant was dialyzed (dialysistubing 12-14 kDa MWCO) twice against Milli-Q water.

Construction of protein polymer nanoconjugates (PPNCs)

Phenylboronic acid-activated PNIPAAm was added dropwise to a stirred solution of BLDF (1 mg in 5 mL of PBS buffer at pH 9.3). The mixed solution was incubated for 12 h at room temperature in the dark, then purified by a millipore filter (Φ 13 mm, 0.45 μm) to remove any precipitate. After filtration, the concentrated protein polymer nanoconjugates were obtained.

Characterization of protein polymer nanoconjugates (PPNCs) prodrug

The absorption spectrum of the synthesized building blocks was measured on a PerkinElmer spectrophotometer (Lambda 750S, USA) to determine the components of LA, DOX and FA in the PPNCs prodrug. The average particle size and size distribution of the nanoconjugates (0.2 mg mL -1 , pH 7.4, 5.0 mM PBS buffer) were characterized by dynamic light scattering (DLS) with an ALV-5000/E DLS instrument (Malvern Instruments, UK) at a fixed scattering angle of 90°, after being filtered by 0.45 μm Milli-pore filters. Zeta potential studies of sample solutions (0.2 mg mL -1 , pH 6.8, 5.0 mM PBS buffer) were carried out at 25 °C using a ZETASIZER Nano series instrument (Malvern Instruments, UK). Transmission electron microscopy (TEM) analysis was undertaken on a JEM-1400 using a LaB6 filament at 120 kV in bright field mode. Samples were prepared by adding one drop of nanoconjugates solution (0.1 mg mL -1 ) onto a 300 mesh carbon film coated copper grid and the specimens were then dried in vaccum for one day. The samples for TEM observations were prepared according to the similar procedure used for SEM. SEM images were obtained on a HITACHI UHR FE-SEM SU8000 with samples sputter-coated with 10 nm platinum. Optical and fluorescence microscopy was performed on a Leica DMI8 manual inverted fluorescence microscope at 10x, 20x, 40x and 100x magnification.

CLSM images were acquired using a confocal laser scanning microscope (CLSM, Nikon-A1 system, Japan), whereas the imaging parameters were kept constant for different groups.

The pH sensitivity of the prodrug

To evaluate the pH sensitivity of the prodrug (PPNCs), in vitro release profiles of BLDF from the PPNCs with different ratios were determined at 25 o C in PBS buffer under different pH conditions (pH 5.5, 7.4 and 9.3). PPNCs and 1 mL of ARS solution were conducted in a shaking incubator at room temperature with PBS buffer under different pH conditions (pH 5.5, 7.4 and 9.3). At predetermined time intervals, the whole solution was removed and the amount of released prodrug BLDF was calculated by UV-vis spectrometry in comparison to a standard curve. A new absorption peak at 480 nm assigned to the generated ARS-PBA and released BLDF was measured to calculate the release efficiency as described above. All experiments were performed in triplicate.

Cell culture and treatment

The MDA-MB-231 human breast cancer cells (HtB-26, atCC®) were cultured at 37 o C with 5 % of CO2 in RPMI-1640 medium (Eurobio, Les Ulis, France) containing 10 % FBS, 1% L-glutamine and 0.1% antibiotics (penicillin/streptomycin). While HMEC-1 (human microvascular endothelial cells) were cultured in MCDB131 medium containing 15 % FBS, 1 % L-glutamine and 0.1 % antibiotics (penicillin/streptomycin). The cells were treated with the prodrug (PPNCs) alone or in combination with different building blocks (BSA, BSA-LA, LA-BSA-FA and PBA-Polymer) at indicated concentrations.

Fluorescence microscopy imaging

To investigate the targeting ability of the prodrug to tumor cells, MDA-MB-231 with folate receptor overexpressing were selected. The cancer cells were incubated at 37 o C in the presence of 0.5 mg/mL of prodrug. After incubation for 1 h, the cells were washed three times with PBS, then fixed by 1 % paraformaldehyde and finally the nuclei were stained with DAPI. Fluorescence images were examined and collected under a ZEISS Microscopy Imaging System (Germany).

Flow cytometry analysis

In flow cytometry studies, MDA-MB-231 cells were seeded in a six-well tissue culture plate (2 mL medium) at a density of 2.0 × 10 5 cells per well. After 24 h, the cells were cultured with a medium containing 0.5 mg/mL of Prodrug and after incubation for 0.5 h, 4 h, 16 h, 24 h, 48 h or 72 h, the cells were trypsinized, washed resuspended in PBS and at last subjected to flow cytometry analysis (BD FACS CATON II, USA).

Cellular uptake study

Cellular uptakes of BSA, BSA-LA, LA-BSA-FA, PBA-Polymer and prodrug were quantitatively estimated by flow cytometry. To investigate the uptake along with various concentrations, MDA-MB-231 cells were treated with different concentrations (0, 0.3125, 0.625, 1.25, 2.5 mg/mL) of diverse building blocks for 48 h.

While for the uptake investigation along with various times, the same mass concentration of diverse building blocks was incubated for 0.5 h, 4 h, 16 h, 24 h, 48 h and 72 h, respectively. After incubation, the cells were trypsinized, washed and resuspended in PBS. Then, the suspended cells were filtrated and examined by flow cytometry (BD FACS CATON II, USA). The instrument was calibrated with non-treated cells (negative control) to identify viable cells.

In vitro cytotoxicity

Following overnight seeding into 48-well culture plates, the cancer cells MDA-MB-231 and normal cells HMEC-1 were treated with different concentrations of prodrug (PPNCs) with or without modified DOX, thereafter incubated for 24 h, 72 h and 120 h, respectively. In addition, cells treated only with the medium were incubated at the same time as a control. At last, the cells were collected and counted with a Coulter counter (Beckman, Z2, USA) to evaluate the cytotoxicity of prodrug with or without modified DOX against cancer cells MDA and the normal cell HEMC-1.

Results and discussion

Self-assembly between BLDF and Polymer-PBA

In order to reduce the adverse effects of cytotoxic drug DOX, BSA was used as a main matrix to construct a prodrug involving favorable characteristics. Since albumin possesses large amounts of biodegradable free amino and carboxyl groups, non-toxic, low immunogenic and extremely stable in pH range of 4-9 at 60 o C, hence, these unique advantages make it suitable for prolonging activity of fast-clearance drugs when used as a drug carrier. In this study, we showed that different functional groups could be conjugated onto the surface of BSA. These groups included doxorubicin hydrochloride (DOX), lactobionic acid (which was used for conjugating to a polymeric chain by forming pH sensitive borate ester bond in the following step) and finally folic acid (which was used for promoting endocytosis along with targeting toward cancerous cells). The successful construction of these intermediate products, that involving BSA-lactobionic acid (BL), BSA-lactobionic acid-DOX (BLD) and BSA-lactobionic acid-DOX-folic acid (BLDF) were confirmed by dynamic light scattering (DLS) measurements which showed a gradual increase in the hydrodynamic diameter of the constructed intermediate building blocks from BSA (4.8 nm) to BL (5.6 nm) then to BLD (6.5 nm) and finally to BLDF (7.6 nm) (Figure 1a), as well as a corresponding change in zeta potential values from -16 mV to -21 mV then to 8.32 mV and lastly to 12.11 mV for BSA, BL, BLD and BLDF, respectively (Figure 1b). Also, the appropriate molecular weight differences arisen by conjugating with different functional groups were shown in MALDI-TOF mass spectrum profiles (Figure 1c). Moreover, from UV-vis absorption analysis, the specific absorption peaks for the different functional units were 490 nm for DOX and 350 nm for folic acid (Figure 1d). Interestingly, from primary amine titration measurements, the content of each group related to BSA molecule was about 14 of lactobionic acid (Figure S1), 4 of DOX (Figure S2) and 9 of folic acid (Figure S3).

These results were consistent with the study of MADI-TOF MS data. The end-capped phenylboronic acid-functionalized poly(N-isopropylacrylamide) (PBA-PNIPAAm) was synthesized according to a previous procedure using a well-known amide reaction between the end-capped mercaptothiazoline-activated PNIPAAm and aminophenylboronic acid. [68] As shown in Figure S4, the characteristic proton signals of phenyl group in PBA at 7.0~7.5 ppm could be discerned clearly, therefore by comparing the integration ratio between the specific signals of PBA at 7.5 ppm and those of CH group in the monomer units at 4.0 ppm, the molecular weight of PBA-PNIPAAm was estimated to be 10000 g/mol (Mn, monomer repeat units = 86). Given that the lactobionic acid involved in BLDF building blocks contains a unit of glucose and also it is a good bridging moiety, then it can allow the covalent conjugation of BLDF with boronic acid in PBA-PNIPAAm by forming a dynamic boronate ester bond. To monitor such conjugation procedure, alizarin Red S (ARS) was employed as an indicator (Figure S5), since it can bind with boronic acid to generate a blue shift on the characteristic UV-vis absorbance spectrum and a strong appeared fluorescence. As shown in Figure S6, by mixing ARS in a weak alkaline environment (pH 9.3) with PNIPAAm-PBA, there was a blue shift of 45.67 nm at wavelength of 520 nm (Figure S6a) and also a clear fluorescence emission peak at 600 nm (excitation wavelength 450 nm) (Figure S7a). By considering the strong complexation ability between lactose and PBA, thus when mixing the ARS containing PNIPAAm-PBA with BLDF, the displaced ARS resulted in a red shift of UV-vis absorbance peak besides an obvious fluorescence quenching, which well suggested the conjugation between both polymer and protein (Figure S6b,S7b). Moreover, the study of DLS at 20 o C showed that BLDF-PNIPAAm nanoconjugates gave a mean hydrodynamic diameter of 10.12 nm, so compared with the size of BLDF (7.56 nm), it yielded an increase of ~2.56 nm which possibly is due to the presence of conjugated PNIPAAm chains (Figure 1a). In contrast, the hydrodynamic diameter of the aqueous BLDF-PNIPAAm nanoconjugates was approximately 300 nm. We attributed this large increase in the size to the aggregation of protein-polymer nanoconjugates in water.

These aggregations are perhaps because of the increased hydrophobicity of hydrophobic DOX along with PBA esters, leading to their envelopment in the hydrophilic protein carrier as hydrophobic centers, thence, the self-assembly of such nanoconjugates can be occurred in the aqueous solution.

Differing from the widely reported regular amphiphilic copolymers which could self-assemble into micelle-like nanoparticles, our constructed star-like protein-polymer nanoconjugates were composed of hydrophobic DOX and PNIPAAm distributed on the surface of BSA, so they can aggregate easily into nanoparticles due to the random hydrophobic intertwining. 69 In this study, by using the feed molar ratio of protein to polymer (1:10) (Figure S9), and when the concentration of BLDF-PNIPAAm conjugates reached to be 0.1 mg/mL, they could aggregate into nanoparticles in the cell culture environment widely with a hydrodynamic size of 300 nm in average (DLS measurement, Figure 1e). Significantly, the formed protein-polymer nanoconjugates showed a good stability against diluting the concentration from 4 mg/mL to 0.5 mg/mL (Figure S8), as well as these nanoconjugates could keep well-dispersed in 10 % fetal bovine serum (FBS) solution without any aggregation or disassembly for over one week (Figure 1e). In general, the formed spherical structure is clearly observed from both scanning electron microscope (SEM) and transmission electron microscopy (TEM) images (Figure 1f,g). Also, the corresponding SEM boron elemental mapping image of a single nanoparticle, whereas boron element is widely distributed inside the formed nanoparticle, suggests a random distribution of PNIPAAm during the aggregation process (Figure 1h,i). Moreover, it is worth mentioning that by varying the feed ratio of protein carrier and polymer (from 1:3 to 1:5 then 1:7), the generated protein-polymer nanoconjugates could also aggregate into nanoparticles with a size ranging from 280 nm to 330 nm with a relatively coarse surface (Figure S9). Accordingly, such constructed protein-enriched nanoparticles that composed of covalently conjugated DOX and folic acid groups onto the surface of BSA, as a favorable type of prodrug models, are expected to show good biocompatibility, high biodegradability and low side effects, which may represent a difference over other types of comparable micelles carrier models.

pH-dependent stability of protein polymer nanoconjugates (PPNCs)

Given the pH sensitive boronate ester bond inside the formed nanoparticles, its dissociation under acidic environment would allow the disassembly of the protein polymer nanoconjugates. As summarized in the schematic illustration of pH-dependent dissociation for the constructed PPNCs prodrug, the dissociation in a weak acid environment and recombination in a moderate pH (≈9.0-9.5) could be circulated many times (Figure 2a). The corresponding dynamic light scattering experiments showed the stable conjugated nanoparticles with negligible size changes under a weak alkaline environment, while the size decreased seriously by transforming to a weak acid environment, suggesting the evident disassembly of nanoconjugates (Figure 2b). Such characters would allow the release of payloads from the constructed nanoparticles, especially in intracellular weak acidic environment, which is key role for antitumor efficacy. For a drug delivery system with long circulation and antitumor efficacy, it is necessary to have a high stability in the body circular system besides a fast release in the cell. To quantitatively determine the release of DOX-BSA from BSA-PNIPAAm nanoparticles, the nanoparticles were poured into a typically weak alkaline environment (pH 9.3), then in PBS buffer at pH 7.4 (corresponding to pH of blood) and finally in PBS buffer at pH 5.5 (corresponding to pH of endosome). The amount of released DOX-BSA at different predetermined time points was measured by fluorescence detector with excitation wavelength of 480 nm and emission wavelength of 580 nm. The results in Figure 2c showed that, the release efficiency of DOX-BSA from the aggregated nanoparticles was 4.95 % in the weak alkaline condition (pH 9.3) and 11.74 % in the neutral one (pH 7.4) after 48 hours. While when pH decreased to 5.5, there was an accelerated dissociation of nanoconjugates and about 50 % of DOX-BSA were released within the first 0.5 hour and 97 % were released within 4 hours. These studies well confirmed that, the constructed protein polymer nanocojugates as a novel synthesized prodrug could behave inactively during the long blood circulation, whilst they could efficiently release their targeted drug in environment of the relatively acidic cancer cells.

In vitro anticancer activity of protein polymer nanoconjugates prodrug

To evaluate the effect of PPNCs prodrug on tumor cells, two cell lines were used in Flow cytometric profiles against MDA-MB-231 cells incubated with PPNCs prodrug in different concentration (0, 0.3125 mg/mL, 0.625 mg/mL, 1.25 mg/mL and 2.5 mg/mL, respectively).

The dose-dependent effect of nanoconjugates prodrug on tumor cells

Subsequently, flow cytometry was used to quantify endocytosis of the prodrug by cancer cells at different concentrations of nanoconjugates (Figure 3c). By increasing the concentration of nanoconjugates from 0.3125 to 2.5 mg/mL, the fluorescence of MDA had increased in a dose dependent manner, indicating that the fluorescence was accompanied by the increasing concentration of nanoconjugates. However, the nanoconjugates without DOX were also endocytosed by MDA cells with the same manner. Flow cytometry analysis also showed that, treatment of nanoconjugates with DOX induced the cell swelling, whereas the nanoaggregates without DOX were not able to collapse MDA (Figure S10). This confirms that only DOX-containing nanoconjugates can kill these cancer cells and inhibit their growth.

The time-dependent effect of nanoconjugates prodrug on tumor cells

Confocal microscopy was performed to observe the cell-killing process of nanoconjugates during first 2 hours. Nanoconjugates were gently formed small patches in the endocytosis process and most likely these patches were created in cytoplasmic macrovesicles within the first 30 minutes as shown in Figure 4a. Then, along with the accumulation of nanoconjugates in MDA for one hour, the cytoplasmic macrovesicles fused and diffused within the MDA cells. The cytotoxic effect of nanoconjugates could be clearly observed at the end of 2 hours. Since the fluorescence was clearly dispersed in the cells, this suggesting that the disintegration of nanoconjugates was successful and started to act on the MDA cells. Actually, the accumulated nanoconjugates were between a period of 30 minutes to 2 hours within the MDA cancer cells, indicating that the uptake of nanoconjugates was time-dependent. Then, by prolonging the time of uptake, sequential analysis of the endocytosis process of MDA was carried out for 72 hours (Figure S11). In contrast to prodrug, nanoconjugates without DOX were added to MDA and the number was systematically measured by flow cytometry during 72 hours (Figure S12). The results showed a time dependent endocytosis via the endocytosis amount that rapidly reached a maximum during the first 24 hours and then stabilized during the following 48 hours.

Importantly, the control experiments of nanoconjugates without DOX showed no such effect and were not able to disintegrate the cancer cells. The cytotoxic effect of nanoconjugates and different building blocks was further assessed in vitro with cell counting method (Figure 4b). It was found that, after incubating the cells for 24 hours with different control building blocks at a concentration ≤ 2.5 mg/mL, over 80 % of the cancer cells survived. It indicated that the building blocks of nanoconjugates without DOX have no noticeable toxicity for MDA cells. By contrast, when the concentration of DOX-nanoconjugates reached 0.625 mg/mL, the survival rate of MDA dropped to 30 % with an IC50 of about 0.5 mg/mL. When the concentration reached 2.5 mg/mL, the survival rate of MDA dropped to below 10 %, showing that nanoconjugates have a strong cytotoxic effect for MDA cells. The cytotoxicity of tumor cells was visible after 24 hours incubation and the cells growth was completely suppressed (Figure 4c). It is well known that, human blood contains albumin at a concentration of 35 -50 g/L (3.5 -5.0 g/dL). As albumin is a very stable protein in blood circulation and its turnover is very long (more than 24 hours), nanoconjugates after injection would be expected to circulate and remain with a high enough concentration for achieving the therapeutic aim.

Therefore, these nanoconjugates (prodrug) were suitable for carrying chemotherapeutic drugs, such as DOX in cancer therapy. Considering that the cytotoxicity of doxorubicin alone has been well characterized clinically besides its severe side effects including inhibition of human hematopoiesis and lowering in heart pumping capability, the biologic vehicles carrying such drugs are expected to protect the healthy organs and increase the therapeutic effects. Herein, we showed the generation of nanoconjugates prodrug which were able to deliver the drugs inside the cancer cells effectively, therefore, we expect this study will promote the advance in pharmaceutical technology.

Conclusions

In summary, an efficient approach to generate pH-sensitive protein-based nanoconjugates with size of ~300 nm was developed successfully by using protein polymer as building blocks connected together via borate ester bond. Both DOX and folic acid were incorporated onto the surface of protein covalently, and as a prodrug model, it demonstrated a lot of merits e.g. satisfactory biocompatibility, low cytotoxicity, targeted-anticancer ability as well as pH-sensitive releasing. The very low toxicity of PPNCs drug model effectively avoided the high toxicity of DOX, which is expected to not only reduce toxic side effects, but also improve anticancer efficiency in vitro examinations. Hence, the development of this pH-sensitive borate ester bridging protein polymer nanoconjugates model may provide a promising strategy for practically cancer therapy and enrich the field of pharmaceutical science, especially on-demand drug-delivery systems.

Figure 1 .

 1 Figure 1. Characterization of protein polymer nanoconjugates (PPNCs) prodrug. (a,b)

Figure 2 .

 2 Figure 2. (a) Schematic illustration of pH-dependent dissociation for the constructed

  vitro system: folate receptor overexpressing MDA-MB-231 cells and a low expression human normal cell HEMC-1. In this study, imaging DOX autofluorescence was evaluated by observing the distribution of PPNCs in intracellular organelles in order to determine whether PPNCs is isolated by lysosomes or lysosome-based fluorescent tracers (Lyso-Green). Consistently, Lyso-Green can label lysosomes and use fluorescent double labeling to determine whether PPNCs is surrounded by lysosomes (by observing the overlap of PPNCs and fluorescently labeled cell vesicles) or not. The entrance of the prodrug into living cells was examined by microscopic examination as shown in Figure 3a. After 0.5 h incubation of MDA cells with PPNCs, the bright particles appeared within the cell lysosomes, indicating that nanoconjugates have been significantly endocytosed by MDA cells. The red fluorescent DOX combined nanoconjugates gave the similar result and demonstrated the quick endocytosis of the prodrug, which suggesting that the constructed nanoconjugates were easily endocytosed by MDA cells. Finally, DOX was released fromnanoconjugates and accompanied by migration to lysosomes of the acidic inner environment, then it entered the nucleus gradually, which was necessary for the drug to be efficient in cancerous cells killing. The corresponding enlarged region clearly showed that the endocytic graininess nanoconjugates were gradually dissociated near the nucleus (Figure3b).

Figure 3 .

 3 Figure 3. Cellular uptake of PPNCs prodrug in MDA-MB-231 cells. (a) CLSM

Figure 4 .

 4 Figure 4. Intracellular delivery of PPNCs prodrug in MDA-MB-231 cells. (a) CLSM

Acknowledgements

The authors acknowledge the financial support of NSFC (21474025, 21871069), the Fundamental Research Funds for the Central Universities (No.HIT.NSRIF.201830), Open Project of Key Laboratory of Microsystems and Microstructures Manufacturing (2016KM003) and the Thousand Young Talent Program for financial support.

Conflict of interest

The authors declare no competing financial interest.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡ These authors contributed equally.


Rémy Duléry

Juliana Bastos

Annalisa Paviglianiti

Florent Malard

Eolia Brissot

Giorgia Battipaglia

Clémence Médiavilla

Federica Giannotti

Anne Banet

Zoé Van De Wyngaert

Tounes Ledraa

Ramdane Belhocine

Simona Sestili

Rosa Adaeva

Simona Lapusan

Françoise Isnard

Ollivier Legrand

Anne Vekhoff

Marie-Thérèse Rubio

Annalisa Ruggeri

Mohamad Mohty
email: mohamad.mohty@inserm.fr

Prof Mohamad Mohty

Thiotepa, Busulfan, and Fludarabine Conditioning Regimen in T Cell-Replete HLA-Haploidentical Hematopoietic Stem Cell Transplantation

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

1

Financial disclosure statement:

M Mohty, R Dulery, F Malard and MT Rubio received honoraria for lectures from Keocyt whose drug was included in this study.

The other authors declare no competing financial interests.

INTRODUCTION

The widespread use of haploidentical (haplo) family donors allows the quick identification of an available donor for the majority of patients who need hematopoietic stem cell transplantation (HSCT).

T-cell replete haplo transplants have now become feasible with the use of post-transplant cyclophosphamide (PT-Cy) [START_REF] Luznik | HLA-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide[END_REF]. Recent retrospective studies suggest non-inferior survival outcomes when compared to matched-unrelated 10/10 (MUD), mismatched-unrelated 9/10 or umbilical cord blood donors [START_REF] Bashey | Comparison of Outcomes of Hematopoietic Cell Transplants from T-Replete Haploidentical Donors Using Post-Transplantation Cyclophosphamide with 10 of 10 HLA-A, -B, -C, -DRB1, and -DQB1 Allele-Matched Unrelated Donors and HLA-Identical Sibling Donors: A Multivariable Analysis Including Disease Risk Index[END_REF][START_REF] Raiola | Unmanipulated haploidentical transplants compared with other alternative donors and matched sibling grafts[END_REF][START_REF] Burroughs | Comparison of outcomes of HLA-matched related, unrelated, or HLA-haploidentical related hematopoietic cell transplantation following nonmyeloablative conditioning for relapsed or refractory Hodgkin lymphoma[END_REF][START_REF] Ringdén | Is there a stronger graft-versus-leukemia effect using HLA-haploidentical donors compared with HLA-identical siblings?[END_REF][START_REF] Ruggeri | Comparison of outcomes after unrelated cord blood and unmanipulated haploidentical stem cell transplantation in adults with acute leukemia[END_REF][START_REF] Martínez | Post-Transplantation Cyclophosphamide-Based Haploidentical Transplantation as Alternative to Matched Sibling or Unrelated Donor Transplantation for Hodgkin Lymphoma: A Registry Study of the Lymphoma Working Party of the European Society for Blood and Marrow Transplantation[END_REF][START_REF] Gauthier | Better outcome with haploidentical over HLA-matched related donors in patients with Hodgkin's lymphoma undergoing allogeneic haematopoietic cell transplantation-a study by the Francophone Society of Bone Marrow Transplantation and Cellular Therapy[END_REF][START_REF] Ciurea | Haploidentical transplant with posttransplant cyclophosphamide vs matched unrelated donor transplant for acute myeloid leukemia[END_REF][START_REF] Duléry | Sequential Conditioning with Thiotepa in T Cell-Replete Hematopoietic Stem Cell Transplantation for the Treatment of Refractory Hematologic Malignancies: Comparison with Matched Related, Haplo-Mismatched, and Unrelated Donors[END_REF]. However, the optimal conditioning regimen in the setting of haplo-HSCT remains yet to be defined.

Thiotepa, an alkylating agent, has been used in HSCT with increasing frequency. Its antineoplastic, myelosupressive and immunosuppressive activities, associated with its ability to penetrate the bloodbrain barrier and a good safety profile, make it an attractive agent in the setting of HSCT for hematological malignancies [START_REF] Bacigalupo | Thiotepabased reduced intensity conditioning regimen: a 10 year follow up[END_REF][START_REF] Eder | Thiotepa-based conditioning for allogeneic stem cell transplantation in acute lymphoblastic leukemia-A survey from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation[END_REF][START_REF] Heideman | Phase I and pharmacokinetic evaluation of thiotepa in the cerebrospinal fluid and plasma of pediatric patients: evidence for dose-dependent plasma clearance of thiotepa[END_REF]. The preparative regimen associating the two alkylating agents, thiotepa and busulfan, with fludarabine (TBF), was initially reported in umbilical cord blood transplantation by Sanz et al, as capable of improving engraftment and survival outcomes [START_REF] Sanz | Single-unit umbilical cord blood transplantation from unrelated donors in patients with hematological malignancy using busulfan, thiotepa, fludarabine and ATG as myeloablative conditioning regimen[END_REF]. TBF with myeloblative (MAC) busulfan dose as conditioning regimen has been compared to busulfanfludarabine (BF) MAC in acute myeloid leukemia (AML) patients in first remission who were transplanted with matched-sibling (MSD) or MUD donors. Patients who underwent TBF-MAC conditioning had lower relapse than BF-MAC. However, the non-relapse mortality (NRM) was increased with TBF-MAC, which counterbalanced the stronger anti-leukemic effect, thus resulting in similar survival [START_REF] Saraceni | Thiotepa-busulfanfludarabine compared to busulfan-fludarabine for sibling and unrelated donor transplant in acute myeloid leukemia in first remission[END_REF]. This regimen was also applied in the haploidentical setting by Bacigalupo et al. (thiotepa 10 mg/kg, busulfan 9.6 mg/kg, or 6.4 mg/kg for patients over 60 years or in poor clinical condition, and fludarabine 150 mg/m 2 ), using unmanipulated bone marrow graft and PT-Cy (50 mg/Kg/day) at day +3 and +5 in association with cyclosporine (from day 0) and mycophenolate mofetil (MMF) (from day +6) as graft-versus-host disease (GvHD) prophylaxis [START_REF] Bacigalupo | Unmanipulated haploidentical bone marrow transplantation and post-transplant cyclophosphamide for hematologic malignanices following a myeloablative conditioning: an update[END_REF][START_REF] Raiola | Unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning[END_REF]. They reported lower rates of graft rejection (0.7%) than with the reduced-intensity (RIC) non-myeloablative Baltimore conditioning regimen (13%) [START_REF] Luznik | HLA-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide[END_REF], acceptable NRM (18% at 1 year), low risk of acute (aGVHD) and chronic GVHD (cGVHD) and relapse rates comparable to MUD and MSD results.

In this study, peripheral blood was the preferred stem cell source in our center, and we used antithymocyte globulin (ATG) in the conditioning regimen. ATG can reduce the GVHD risk in conventional HSCT settings with MSD and MUD [START_REF] Baron | Anti-thymocyte globulin as graft-versus-host disease prevention in the setting of allogeneic peripheral blood stem cell transplantation: a review from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation[END_REF][START_REF] Devillier | Impact of antithymocyte globulin doses in reduced intensity conditioning before allogeneic transplantation from matched sibling donor for patients with acute myeloid leukemia: a report from the acute leukemia working party of European group of Bone Marrow Transplantation[END_REF][START_REF] Storek | Rabbit anti-T cell globulin in allogeneic hematopoietic cell transplantation[END_REF]. However, data on the use of ATG are scarce in a haplo setting with PT-Cy. Thus, we aimed to evaluate the clinical outcomes of the patients receiving an unmanipulated haplo-HSCT in our center with TBF conditioning.

PATIENTS AND METHODS

Patient selection

We report a single-center retrospective analysis of all consecutive patients receiving a haplo-HSCT after a TBF conditioning regimen from August 2013 to December 2017 at Saint Antoine Hospital (Paris, France). Fifty-one patients ≥ 16 years old were included. All patients who proceeded to transplantation provided written informed consent for the use of their data for clinical research, in accordance with the modified Declaration of Helsinki and the local Ethical Committee. Patients were stratified according to the disease risk index (DRI) [START_REF] Armand | A disease risk index for patients undergoing allogeneic stem cell transplantation[END_REF].

Conditioning regimens and GVHD prophylaxis

All patients received a TBF conditioning regimen (table 2). According to our center policy, all but 6 patients transplanted in 2013 and 2014 received a total dose of 10 mg/kg of thiotepa (5 mg/kg on days -7 and -6), that was reduced to 5 mg/kg (at day-6) in those transplanted between 2015 and 2017. A total dose of 150mg/m 2 of fludarabine was administered to all patients (30 mg/m 2 from day -5 to day -1) and the total dose of intravenous busulfan was 6.4 or 9.6 mg/kg (3.2 mg/kg/day on days -4, -3 or -4, -3, -2) according to the patient's age, comorbidities and clinical condition. The source of stem cells was peripheral blood stem cells (PBSC) or bone marrow (BM), with no ex-vivo T-cell depletion.

The dose of PT-Cy was adjusted according to the stem cell source. BM recipients were scheduled to receive 1 dose of PT-Cy (50 mg/kg/day) at day+3. PBSC recipients were given 2 doses of PT-Cy at days +3 and +5. GVHD prophylaxis consisted of cyclosporine A (starting from day-3 as a 3 mg/kg/day as continuous intravenous infusion and adjusting to blood levels) and MMF (starting from day +6 until day +35 at 15 mg/kg/12h intravenously) in all patients. In the absence of GVHD, cyclosporine A was tapered over 4 to 8 weeks from day +60 in high-risk diseases and day+90 in intermediate-risk. Patients could receive rabbit ATG (Thymoglobulin, Genzyme) at a total dose of 5 mg/kg (between 2013 and 2014) or 2.5 mg/kg (between 2015 and 2017). aGVHD and cGVHD were diagnosed and graded according to standard criteria [START_REF] Przepiorka | Consensus Conference on Acute GVHD Grading[END_REF][START_REF] Jagasia | National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: I. The 2014 Diagnosis and Staging Working Group report[END_REF].

Supportive care

Granulocyte colony-stimulating factor was given from day+6 until neutrophil engraftment. Mesna and hyperhydration were administered for prevention of the uroepithelial damage of Cy, and prophylactic clonazepam was given in association with busulfan. Prophylaxis of veno-occlusive hepatic disease (VOD) was performed with orally applied ursodeoxycholic acid and low-dose unfractioned heparin.

Patients at high-risk of VOD according to the EBMT criteria received defibrotide instead of heparin [START_REF] Mohty | Revised diagnosis and severity criteria for sinusoidal obstruction syndrome/veno-occlusive disease in adult patients: a new classification from the European Society for Blood and Marrow Transplantation[END_REF][START_REF] Picod | Defibrotide for Sinusoidal Obstruction Syndrome/Veno-Occlusive Disease Prophylaxis in High-Risk Adult Patients: A Single-Center Experience Study[END_REF].

Antimicrobial prophylaxis consisted of valacyclovir or acyclovir at the start of the conditioning regimen. After engraftment, patients received amoxicilline and atovaquone or trimethoprimsulfamethoxazole. Primary antifungal prophylaxis was not administered routinely. Patients were monitored weekly by real time polymerase chain reaction (PCR) for cytomegalovirus (CMV), human herpesvirus 6 (HHV6), Epstein Barr virus (EBV) and toxoplasma until day+100. Monitoring of BK virus in blood and urine was done according to clinical signs, as we previously reported [START_REF] Ruggeri | Incidence and risk factors for hemorrhagic cystitis in unmanipulated haploidentical transplant recipients[END_REF].

Statistical analysis

Continuous variables were recorded as median and range and were compared using the Mann-Whitney U test. Qualitative variables were recorded as frequency and percent, and were compared using the chi-square test. Overall survival (OS), defined as the time from transplant to death from any cause, disease-free survival (DFS), defined as the time from transplant to relapse or progression or death from any cause, whichever came first, and GVHD-free, relapse-free survival (GRFS) [START_REF] Ruggeri | Definition of GvHD-free, relapse-free survival for registry-based studies: an ALWP-EBMT analysis on patients with AML in remission[END_REF], defined as being alive with neither grade III-IV aGVHD, severe cGVHD nor disease relapse were calculated by the Kaplan-Meier method, and the differences between groups were compared using the log-rank test.

Neutrophil engraftment was defined as an absolute neutrophil count (ANC) ≥0.5 x 10 9 /L for 3 consecutive days, and platelet engraftment was defined as achieving platelet count ≥20 x 10 9 /L unsupported by platelet transfusions for 7 days. Full donor chimerism was measured by PCR and defined as ≥95% leucocytes of donor origin in the peripheral blood. Engraftment, GVHD, relapse, and NRM were calculated using the cumulative incidence (CI) method and analyzed in a time-dependent fashion. For acute and chronic GVHD or relapse, death was considered a competing risk of the event.

For NRM, the competing event was relapse. All variables having a significance level of p<0.10 in univariate analysis were introduced in a multivariable Cox regression model, with backward selection [START_REF] Fine | A Proportional Hazards Model for the Subdistribution of A Competing Risk[END_REF]. Interactions between main effect and adjusting were tested, and variables with significant interaction were excluded from the final model. Adjusted hazard ratios (HR) and 95% confidence intervals (95%CI) were computed and p<0.05 was considered statistically significant. Statistical analyses were performed with SPSS 22 (SPSS Inc./IBM, Armonk, NY) and R 3.2 (R Development Core Team, Vienna, Austria) software packages.

RESULTS

Patients, disease and transplant characteristics

Patients and disease characteristics are summarized in table 1. Median age at HSCT was 55 years (range, 16-72). Patients had AML (n=23), acute lymphoblastic leukemia (ALL; n=8), lymphoid neoplasm (n=12), myeloproliferative neoplasm (n=5) and myelodysplastic syndrome (n=3). Thirtyseven patients were in complete remission (CR) at transplant (27 in first CR, CR1), 6 in partial remission and 8 had progressive disease. DRI stratified patients in intermediate (n=35), high (n=14) and very-high (n=2). Nine patients had failed a previous allogeneic (n=3) or autologous (n=6) transplantation. Twenty-seven patients received a dose of 6.4 mg/kg of busulfan (table 2). Thirty-nine patients received ATG associated to the conditioning regimen, at a dose of 2.5 mg/kg (n=29) or 5mg/kg (n=10). BM recipients (n=9) received mainly (n=8) 1 dose of PT-Cy (50 mg/kg/day) at day+3. PBSC recipients (n=42) were given 2 doses of PT-Cy at days +3 and +5, except for 2 patients (one with pre-transplant left ventricular ejection fraction of 40% and another who received a graft with a low CD34+ cells count) (table 2). Nine patients received defibrotide prophylaxis for VOD.

Engraftment

Of the 51 evaluable patients, all achieved neutrophil engraftment, within a median time of 17 days (range, 12-34 days). Platelet counts > 20 x10 9 /L were reached in 48 patients within a median time of 18 days (range, 7-130), with 7 non-evaluable patients (due to transfusion support at superior platelet number cutoffs) (table 3). Forty-five patients (88%) had a platelet count recovery to >50 x10 9 /L within a median time of 28 days (range, 9-406). All patients had full donor chimerism at day+30, +60 and +90, except for one patient presenting a 94% donor chimerism at day+30, and a full donor chimerism at day+60 and +90. No graft rejection was observed. Four patients received a successfully CD34+cell boost from the same donor for poor graft function, after initial engraftment, and achieved neutrophil recovery with in a period of 4, 5, 7 and 11 months post-HSCT, respectively.

Transplant-related complications and infections

Transplant-related complications and infections are outlined in table 4. VOD developed in two patients (4%) and was fatal in one. Signs of transplant-associated microangiopathy were observed in 8 patients (16%), requiring plasma exchanges in one patient and responding to cyclosporine withdrawal alone in the other patients. Grade 3-4 mucositis occurred in 16 patients (31%) and hepatic, cardiac, or neurological toxicities of grade ≥3 after the conditioning chemotherapy were observed in 5, 4 and 1 patients, respectively. At last follow-up, hemorrhagic cystitis associated with BK virus reactivation occurred in 24 patients (47%) and required bladder irrigation in 13 (26%). Four patients received cidofovir and 9 polyvalent intravenous immunoglobulins. CMV reactivation occurred in 34 patients (67%). All patients received preemptive therapy with foscarnet or gancyclovir (according to hematopoietic recovery and renal function). One patient developed CMV retinitis 9 months after HSCT, which resolved with intraocular injections of ganciclovir. EBV reactivation (viral load > 5000 UI/mL) occurred in 30 patients (59%) and was preemptively treated by rituximab. Two patients (4%) had an EBV-related post-transplant lymphoproliferative disease (PTLD), treated successfully by rituximab alone in one case and chemotherapy in the second. HHV6 reactivation occurred in 31/46 evaluable patients (67%). Two patients developed symptomatic adenovirus infection and 5 had varicella-zoster virus reactivation. Bacteremia occurred in 33 patients (64%), including 16 (31%)

Staphyloccocus positive blood culture and 19 (37%) catheter-related bloodstream infections. Invasive fungal infection was documented in 5 patients (10%), including possible or probable invasive aspergillosis in 2 patients (4%).

The use of ATG and ATG doses were not significantly associated with an increased incidence of infection. In patients receiving ATG, CMV, EBV and HHV6 reactivations occurred in 67%, 59% and 62% cases, respectively. BK virus hemorrhagic cystitis developed in 38% patients and required irrigations in 23%. Bacteremia and invasive fungal infections occurred in 64% and 8% patients respectively.

Graft-versus-host disease

Acute GVHD

At day 100, the CI of grade II-IV aGVHD was 27.5%. Grade III-IV aGVHD occurred in 7 (14%) patients (table 3). Using PBSC graft and ATG prophylaxis, grade II-IV aGVHD and grade III-IV aGVHD occurred in 5/31 (16%) and 3/31 (10%) patients, respectively. All patients with aGVHD grade ≥2 were treated with methylprednisolone 1-2 mg/kg/day as standard first line therapy. Eight patients (16%) were steroid-refractory and received second-line treatment consisting of weekly low dose methotrexate (n=6) and/or extracorporeal photopheresis (n=3) according to the GVHD organ involvement. A third line of treatment with anti-CD25 monoclonal antibody (inolimomab) was administered to 2 patients with refractory GVHD.

In univariate analysis (table 5), the CI of grade II-IV aGVHD was significantly lower in patients receiving ATG (21% versus 50%, p=0.03). There was with no significant difference according to the dose of ATG (p=0.09). The CI of grade II-IV aGVHD was significantly reduced with 5 mg thiotepa as compared to 10 mg (16% vs. 57%, p=0.005). Patients who underwent transplant after 2015 had also a lower CI of aGVHD (p=0.02). No factor significantly influenced aGVHD in multivariate analysis (table 6).

Chronic GVHD

The overall CI of cGVHD was 29% at 2 years. Six patients (12%) had moderate to severe cGVHD forms (table 3). The organs affected were: skin (n=12), mucosa (n=11), genitals (n=3), gastrointestinal tract (n=2), eyes (n=6), liver (n=1) or lungs (n=4). Four patients died during the follow-up period, 2 from relapse and 2 from infection. No significant benefit of ATG in reducing the CI of cGVHD was observed. The CI of cGVHD was significantly reduced in intermediate-DRI patients compared to high-and very high-risk patients (18% vs. 47%, p=0.02) and in patients who received 5 mg/kg thiotepa as compared to 10 mg/kg (13% vs. 57%, p=0.002). However, these factors were not significant in multivariate analysis (table 6).

Relapse and NRM

The relapse incidence (RI) was 4% at day+100 and 23% at 2 years. Relapse or disease progression The CI of NRM was 6% at day+100 and 20% at 2 years. At last-follow-up, 17 patients died. The main causes of death were disease relapse (n=7), GVHD (n=7), infection (n=2) and VOD (n=1). ATG was not significantly associated with the RI or the NRM, both in univariate and multivariate analyses.

Patients in CR had a lower NRM (HR 5.67, 95%CI 1.52-21.16, p=0.01) in multivariate analysis (table 6).

Survival, DFS and GRFS

With a median follow-up of 25 months (range, 12 -62 months), the GRFS, DFS and OS were 51%, 58% and 67%, respectively (figure 1). In univariate analysis, the DFS and OS were 63% and 74% in patients with acute leukemia as compared to 47% and 53% in the patients with other diseases, respectively (p=0.09 and p=0.06). Overall survival and DFS were significantly higher in patients in CR at transplant compared to those who were not (76% vs. 43%, p=0.003 and 63% vs. 43%, p=0.04, respectively) (figure 2). In multivariate analysis (table 6), CR disease status was independently associated with a better GRFS (HR 3.04, 95%CI 1.27-7.26, p=0.01), DFS (HR 3.32, 95%CI 1.31-8.42, p=0.01) and OS (HR 5.19, 95%CI 1.90-14.17, p=0.001). Patients who underwent transplant after 2015 had also a higher DFS (HR 3.64 95%CI 1.14-11.63, p=0.003).

DISCUSSION

Findings from this study confirm that the TBF conditioning regimen is feasible in a haplo setting with PT-Cy and allows for sustained engraftment, manageable toxicity and efficient disease control. The use of ATG reduced the CI of aGVHD and did not seem to increase the incidences of infectious complications or relapse. Disease status before HSCT remained the most important factor associated with GRFS, DFS and OS.

Neutrophil engraftment occurred in all patients, which compares favorably to the reported 13% graft failure rate with the Baltimore RIC regimen [START_REF] Luznik | HLA-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide[END_REF]. The median time to neutrophil recovery was similar to Bacigalupo et al.'s study on TBF conditioning regimen in haplo using BM graft (median 17 days, range 13-32) [START_REF] Bacigalupo | Unmanipulated haploidentical bone marrow transplantation and post-transplant cyclophosphamide for hematologic malignanices following a myeloablative conditioning: an update[END_REF], and shorter than with the Baltimore RIC regimen using PBSC graft (median 20 days, range 14-27) (29).

Our results on bacterial and fungal infections are in line with previous reports in a haplo setting with PT-Cy [START_REF] Luznik | HLA-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide[END_REF][START_REF] Bacigalupo | Unmanipulated haploidentical bone marrow transplantation and post-transplant cyclophosphamide for hematologic malignanices following a myeloablative conditioning: an update[END_REF][START_REF] Raiola | Unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning[END_REF][START_REF] Crocchiolo | Infections after T-replete haploidentical transplantation and high-dose cyclophosphamide as graft-versus-host disease prophylaxis[END_REF][START_REF] Castagna | Bone marrow compared with peripheral blood stem cells for haploidentical transplantation with a nonmyeloablative conditioning regimen and post-transplantation cyclophosphamide[END_REF][START_REF] Cieri | Post-transplantation Cyclophosphamide and Sirolimus after Haploidentical Hematopoietic Stem Cell Transplantation Using a Treosulfan-based Myeloablative Conditioning and Peripheral Blood Stem Cells[END_REF][START_REF] Stasi | Similar transplantation outcomes for acute myeloid leukemia and myelodysplastic syndrome patients with haploidentical versus 10/10 human leukocyte antigen-matched unrelated and related donors[END_REF][START_REF] Solomon | Haploidentical Transplantation Using T Cell Replete Peripheral Blood Stem Cells and Myeloablative Conditioning in Patients with High-Risk Hematologic Malignancies Who Lack Conventional Donors is Well Tolerated and Produces Excellent Relapse-Free Survival: Results of a Prospective Phase II Trial[END_REF]. However, viral reactivations seemed to have occurred more frequently in our cohort, with CMV and EBV reactivations receiving preemptive therapy in 67% and 59% of patients, respectively. The incidence of PTLD was 4%. Previous reports described incidences of CMV reactivation of 38% to 81%, EBV reactivation of 0% to 34% and PTLD of 0% to 3% [START_REF] Luznik | HLA-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide[END_REF][START_REF] Bacigalupo | Unmanipulated haploidentical bone marrow transplantation and post-transplant cyclophosphamide for hematologic malignanices following a myeloablative conditioning: an update[END_REF][START_REF] Raiola | Unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning[END_REF][START_REF] Crocchiolo | Infections after T-replete haploidentical transplantation and high-dose cyclophosphamide as graft-versus-host disease prophylaxis[END_REF][START_REF] Castagna | Bone marrow compared with peripheral blood stem cells for haploidentical transplantation with a nonmyeloablative conditioning regimen and post-transplantation cyclophosphamide[END_REF][START_REF] Cieri | Post-transplantation Cyclophosphamide and Sirolimus after Haploidentical Hematopoietic Stem Cell Transplantation Using a Treosulfan-based Myeloablative Conditioning and Peripheral Blood Stem Cells[END_REF][START_REF] Stasi | Similar transplantation outcomes for acute myeloid leukemia and myelodysplastic syndrome patients with haploidentical versus 10/10 human leukocyte antigen-matched unrelated and related donors[END_REF][START_REF] Solomon | Haploidentical Transplantation Using T Cell Replete Peripheral Blood Stem Cells and Myeloablative Conditioning in Patients with High-Risk Hematologic Malignancies Who Lack Conventional Donors is Well Tolerated and Produces Excellent Relapse-Free Survival: Results of a Prospective Phase II Trial[END_REF].

The incidence of BK virus hemorrhagic cystitis (47%), which required bladder irrigation in half of these patients, was in line with the incidence of 11% to 75% reported in other studies [START_REF] Ruggeri | Incidence and risk factors for hemorrhagic cystitis in unmanipulated haploidentical transplant recipients[END_REF][START_REF] Crocchiolo | Infections after T-replete haploidentical transplantation and high-dose cyclophosphamide as graft-versus-host disease prophylaxis[END_REF][START_REF] Castagna | Bone marrow compared with peripheral blood stem cells for haploidentical transplantation with a nonmyeloablative conditioning regimen and post-transplantation cyclophosphamide[END_REF][START_REF] Solomon | Haploidentical Transplantation Using T Cell Replete Peripheral Blood Stem Cells and Myeloablative Conditioning in Patients with High-Risk Hematologic Malignancies Who Lack Conventional Donors is Well Tolerated and Produces Excellent Relapse-Free Survival: Results of a Prospective Phase II Trial[END_REF].

The use of ATG in our study may explain the higher incidence of viral reactivations found in this population. Indeed, ATG is known to increase the risk of infection after HSCT, especially EBV reactivations [START_REF] Baron | Anti-thymocyte globulin as graft-versus-host disease prevention in the setting of allogeneic peripheral blood stem cell transplantation: a review from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation[END_REF][START_REF] Devillier | Impact of antithymocyte globulin doses in reduced intensity conditioning before allogeneic transplantation from matched sibling donor for patients with acute myeloid leukemia: a report from the acute leukemia working party of European group of Bone Marrow Transplantation[END_REF][START_REF] Storek | Rabbit anti-T cell globulin in allogeneic hematopoietic cell transplantation[END_REF]. Nevertheless, no significant difference was observed in our study between patients receiving ATG and those who did not in terms of incidence of viral reactivations.

The CI of cGVHD in our study was in line with previous studies, which reported a CI ranging from 7% to 56% [START_REF] Luznik | HLA-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide[END_REF][START_REF] Bashey | Comparison of Outcomes of Hematopoietic Cell Transplants from T-Replete Haploidentical Donors Using Post-Transplantation Cyclophosphamide with 10 of 10 HLA-A, -B, -C, -DRB1, and -DQB1 Allele-Matched Unrelated Donors and HLA-Identical Sibling Donors: A Multivariable Analysis Including Disease Risk Index[END_REF][START_REF] Ciurea | Haploidentical transplant with posttransplant cyclophosphamide vs matched unrelated donor transplant for acute myeloid leukemia[END_REF][START_REF] Bacigalupo | Unmanipulated haploidentical bone marrow transplantation and post-transplant cyclophosphamide for hematologic malignanices following a myeloablative conditioning: an update[END_REF][START_REF] Raiola | Unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning[END_REF][START_REF] Castagna | Bone marrow compared with peripheral blood stem cells for haploidentical transplantation with a nonmyeloablative conditioning regimen and post-transplantation cyclophosphamide[END_REF][START_REF] Cieri | Post-transplantation Cyclophosphamide and Sirolimus after Haploidentical Hematopoietic Stem Cell Transplantation Using a Treosulfan-based Myeloablative Conditioning and Peripheral Blood Stem Cells[END_REF][START_REF] Stasi | Similar transplantation outcomes for acute myeloid leukemia and myelodysplastic syndrome patients with haploidentical versus 10/10 human leukocyte antigen-matched unrelated and related donors[END_REF][START_REF] Solomon | Total Body Irradiation-Based Myeloablative Haploidentical Stem Cell Transplantation Is a Safe and Effective Alternative to Unrelated Donor Transplantation in Patients Without Matched Sibling Donors[END_REF][START_REF] Solomon | Total Body Irradiation-Based Myeloablative Haploidentical Stem Cell Transplantation Is a Safe and Effective Alternative to Unrelated Donor Transplantation in Patients Without Matched Sibling Donors[END_REF][START_REF] Ruggeri | Bone marrow versus mobilized peripheral blood stem cells in haploidentical transplants using posttransplantation cyclophosphamide[END_REF][START_REF] O'donnell | Comparable outcomes with marrow or peripheral blood as stem cell sources for hematopoietic cell transplantation from haploidentical donors after non-ablative conditioning: a matched-pair analysis[END_REF]. However, the CI of grade II-IV aGVHD was higher in our patients than in the study by Bacigalupo et al. on MAC in a haplo setting with PT-Cy (27.5% vs. 18%). There may be two explanations: [START_REF] Luznik | HLA-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide[END_REF] we used PBSC in the majority of our patients and (2) we used only TBF conditioning regimen, whereas fludarabine-TBI was used in 38% of patients in Genova [START_REF] Bacigalupo | Unmanipulated haploidentical bone marrow transplantation and post-transplant cyclophosphamide for hematologic malignanices following a myeloablative conditioning: an update[END_REF]. Indeed, our results are in line with the reported CI of grade II-IV aGVHD in MAC haplo with PBSC, which ranges from 15% to 43% [START_REF] Bashey | Comparison of Outcomes of Hematopoietic Cell Transplants from T-Replete Haploidentical Donors Using Post-Transplantation Cyclophosphamide with 10 of 10 HLA-A, -B, -C, -DRB1, and -DQB1 Allele-Matched Unrelated Donors and HLA-Identical Sibling Donors: A Multivariable Analysis Including Disease Risk Index[END_REF][START_REF] Cieri | Post-transplantation Cyclophosphamide and Sirolimus after Haploidentical Hematopoietic Stem Cell Transplantation Using a Treosulfan-based Myeloablative Conditioning and Peripheral Blood Stem Cells[END_REF][START_REF] Solomon | Haploidentical Transplantation Using T Cell Replete Peripheral Blood Stem Cells and Myeloablative Conditioning in Patients with High-Risk Hematologic Malignancies Who Lack Conventional Donors is Well Tolerated and Produces Excellent Relapse-Free Survival: Results of a Prospective Phase II Trial[END_REF][START_REF] Solomon | Total Body Irradiation-Based Myeloablative Haploidentical Stem Cell Transplantation Is a Safe and Effective Alternative to Unrelated Donor Transplantation in Patients Without Matched Sibling Donors[END_REF]. Three retrospective studies have confirmed that the use of PBSC increases the risk of aGVHD in haplo HSCT with PT-Cy [START_REF] Ruggeri | Bone marrow versus mobilized peripheral blood stem cells in haploidentical transplants using posttransplantation cyclophosphamide[END_REF][START_REF] O'donnell | Comparable outcomes with marrow or peripheral blood as stem cell sources for hematopoietic cell transplantation from haploidentical donors after non-ablative conditioning: a matched-pair analysis[END_REF][START_REF] Bashey | Mobilized Peripheral Blood Stem Cells Versus Unstimulated Bone Marrow As a Graft Source for T-Cell-Replete Haploidentical Donor Transplantation Using Post-Transplant Cyclophosphamide[END_REF], and the CI was higher with PBSC, but not significantly different than with BM in another study [START_REF] Castagna | Bone marrow compared with peripheral blood stem cells for haploidentical transplantation with a nonmyeloablative conditioning regimen and post-transplantation cyclophosphamide[END_REF]. Interestingly, a low thiotepa dose at 5 mg/kg reduced significantly the risk of aGVHD and cGVHD without compromising the outcomes. Based on these data, we found no benefit of a thiotepa dose at 10 mg/kg compared to 5 mg/kg. Our results are in line with previous reports with melphan-based conditioning regimen, which used thiotepa dose at 5 mg/kg and showed that thiotepa dose at 10mg/kg is not needed to allow for disease control [START_REF] Majolino | Reduced intensity conditioning with thiotepa, fludarabine, and melphalan is effective in advanced multiple myeloma[END_REF][START_REF] Brammer | Outcomes of Haploidentical Stem Cell Transplantation for Lymphoma with Melphalan-Based Conditioning[END_REF].

Our data show for the first time the benefit of ATG on aGVHD in a haplo setting with PT-Cy. Patients receiving both ATG and PT-Cy in our study had a similar CI of aGVHD with PBSC than in studies reporting BM haplo without ATG. In contrast with our study, ATG had no impact on GVHD and a detrimental role on NRM, leukemia-free survival and OS in a trial including patients with secondary AML undergoing unmanipulated haplo HSCT, compared to patients receiving PT-Cy without ATG.

However, patients receiving both ATG and PT-Cy were excluded from that study [START_REF] Li | Haploidentical transplantation outcomes for secondary acute myeloid leukemia: ALWP of the EBMT study[END_REF]. We recently published a series of patients undergoing HSCT for the treatment of refractory hematologic malignancies after sequential conditioning with thiotepa and ATG [START_REF] Duléry | Sequential Conditioning with Thiotepa in T Cell-Replete Hematopoietic Stem Cell Transplantation for the Treatment of Refractory Hematologic Malignancies: Comparison with Matched Related, Haplo-Mismatched, and Unrelated Donors[END_REF]. There was no significant difference in terms of toxicity and outcomes between haplo with PT-Cy and MRD patients. Compared with MUD, the outcomes were improved in haplo in terms of incidence of aGVHD and GVHD-free, relapse-free survival. Despite the use of ATG in patients with progressive disease, the 2-year RI was 36% in haplo patients. This is comparable to the reported RIs of 33% in patients with active disease who received MAC Haplo without ATG and 37% in those treated with the FLAMSA regimen [START_REF] Raiola | Unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning[END_REF][START_REF] Schmid | Long-term survival in refractory acute myeloid leukemia after sequential treatment with chemotherapy and reduced-intensity conditioning for allogeneic stem cell transplantation[END_REF].

Overall, ATG in haplo with PT-Cy seems to reduce the risk of aGVHD without compromising outcomes. Due to the limited number of patients who did not receive ATG in our study, these results need to be validated in larger cohorts and in well-designed prospective randomized trials.

The NRM in our study is in the higher range of the reported NRM in previous studies on haplo with PT-Cy, in which 1-year NRM ranged from 10% to 22% [START_REF] Luznik | HLA-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide[END_REF][START_REF] Bashey | Comparison of Outcomes of Hematopoietic Cell Transplants from T-Replete Haploidentical Donors Using Post-Transplantation Cyclophosphamide with 10 of 10 HLA-A, -B, -C, -DRB1, and -DQB1 Allele-Matched Unrelated Donors and HLA-Identical Sibling Donors: A Multivariable Analysis Including Disease Risk Index[END_REF][START_REF] Raiola | Unmanipulated haploidentical transplants compared with other alternative donors and matched sibling grafts[END_REF][START_REF] Ciurea | Haploidentical transplant with posttransplant cyclophosphamide vs matched unrelated donor transplant for acute myeloid leukemia[END_REF][START_REF] Ciurea | Haploidentical transplant with posttransplant cyclophosphamide vs matched unrelated donor transplant for acute myeloid leukemia[END_REF][START_REF] Bacigalupo | Unmanipulated haploidentical bone marrow transplantation and post-transplant cyclophosphamide for hematologic malignanices following a myeloablative conditioning: an update[END_REF][START_REF] Raiola | Unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning[END_REF][START_REF] Castagna | Bone marrow compared with peripheral blood stem cells for haploidentical transplantation with a nonmyeloablative conditioning regimen and post-transplantation cyclophosphamide[END_REF][START_REF] Cieri | Post-transplantation Cyclophosphamide and Sirolimus after Haploidentical Hematopoietic Stem Cell Transplantation Using a Treosulfan-based Myeloablative Conditioning and Peripheral Blood Stem Cells[END_REF][START_REF] Stasi | Similar transplantation outcomes for acute myeloid leukemia and myelodysplastic syndrome patients with haploidentical versus 10/10 human leukocyte antigen-matched unrelated and related donors[END_REF][START_REF] Bashey | Mobilized Peripheral Blood Stem Cells Versus Unstimulated Bone Marrow As a Graft Source for T-Cell-Replete Haploidentical Donor Transplantation Using Post-Transplant Cyclophosphamide[END_REF][START_REF] Kasamon | Outcomes of Nonmyeloablative HLA-Haploidentical Blood or Marrow Transplantation With High-Dose Post-Transplantation Cyclophosphamide in Older Adults[END_REF]. However, our study included only 53% patients in CR1. The NRM was significantly reduced to 14% in patients in CR. Our results are in line with the 26% incidence NRM at 6-months reported with TBF haplo in patients who were not in CR [START_REF] Raiola | Unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning[END_REF]. For the whole patient group, the outcomes compare favorably to the results of haplo with PT-Cy using the Baltimore RIC regimen [START_REF] Luznik | HLA-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide[END_REF]. The TBF conditioning regimen allowed for favorable outcomes in patients in CR, in line with previous studies on MAC haplo. However, our data in patients in partial response or progressive disease, and the results of recent studies, suggest that those patients would benefit more from a sequential approach including early post-transplantation immune-intervention combining prophylactic DLI with preventive targeted therapies [START_REF] Duléry | Sequential Conditioning with Thiotepa in T Cell-Replete Hematopoietic Stem Cell Transplantation for the Treatment of Refractory Hematologic Malignancies: Comparison with Matched Related, Haplo-Mismatched, and Unrelated Donors[END_REF][START_REF] Battipaglia | Efficacy and feasibility of sorafenib as a maintenance agent after allogeneic hematopoietic stem cell transplantation for Fms-like tyrosine kinase 3-mutated acute myeloid leukemia[END_REF][START_REF] Goodyear | Azacitidine augments expansion of regulatory T cells after allogeneic stem cell transplantation in patients with acute myeloid leukemia (AML)[END_REF].

In conclusion, our study confirms that the TBF conditioning regimen is an appealing platform in a haplo setting with PT-Cy. Engraftment was sustained in all patients, toxicities were manageable and similar to those reported with other MAC in haplo. A thiotepa dose of 10 mg/kg was more toxic than 5 mg/kg, mainly because of the induction of acute GVHD, with no apparent benefit in terms of disease control. Although the use of PBSC may have increased the CI of aGVHD, the CI of cGVHD and the outcomes were similar to those with BM. Most notably, the addition of ATG seems effective for reducing the CI of aGVHD without increasing the RI or the risk of infectious complications. Abbreviations: DFS: disease free survival, OS: overall survival, RI: cumulative incidence of relapse, 95% CI: confidence interval of 95%, GVHD: graft-versus-host disease, CI: cumulative incidence, NRM: non-relapse mortality, CR: complete response, DRI: disease risk index, ATG: antithymocyte globulin, BM: bone marrow, PBSC: peripheral blood stem cell, HSCT: hematopoietic stem cell transplantation, NA: not applicable.

  occurred in 11 patients, within a median time of 171 days (range, 20-559): 5 AML patients with high risk features, 2 ALL, 1 HTLV1+ acute T-cell lymphoma, 1 Hodgkin lymphoma in 3rd partial remission, 1 Sezary syndrome with progressive disease at transplantation and 1 refractory double hit diffuse large B-cell lymphoma. At last follow-up, 4 out of the 11 patients experiencing relapse were in remission and 7 died. Ten patients received prophylactic therapy to prevent relapse: azacytidine in 2 patients with AML, sorafenib in 4 AML patients with internal tandem duplications of the Fms-like tyrosine kinase 3, dasatinib in one patient with AML secondary to myeloid chronic leukemia, nilotinib in one patient with Philadelphia positive ALL and DLI in two other patients (refractory Hodgkin's lymphoma and biclonal ALL). One out of these 10 patients has experienced further relapse during the follow-up period after sorafenib withdrawal.

  (range) 5 (1.02-9.74) Abbreviations: AML: acute myeloid leukemia, ALL: acute lymphoblastic leukemia, NHL: Non-Hodgkin lymphoma, DLBCL: Diffuse large B-cell lymphoma, MCL: Mantle cell lymphoma, T NOS: lymphoma T not otherwise specified, ATLL: acute T-cell leukemia/lymphoma, CR: complete remission, HSCT: hematopoietic stem cell transplantation, CMV: cytomegalovirus, PBSC: peripheral blood stem cells, BM: Bone marrow.

  150mg/m2); T1B3F (thiotepa 5mg/kg/day for 1 day, Busulfan 3.2mg/kg/day for 3 days, fludarabine 150mg/m2); T2B3F (thiotepa 5mg/kg/day for 2 days, Busulfan 3.2mg/kg/day for 3 days, fludarabine 150mg/m2); ATG: Antithymocyte globulin, PT-Cy: post-transplant cyclophosphamide, D: Day.

  DFS, % (confidence interval) 58% (51-65) Median follow-up (range) 25 months (12-62) Abbreviations: GVHD: graft-versus-host disease, CI: cumulative incidence, OS: overall survival, DFS: disease free survival.

Abbreviations:

  Abbreviations: DFS: disease free survival, OS: overall survival, RI: cumulative incidence of relapse, 95% CI: confidence interval of 95%, GVHD: graft-versus-host disease, NRM: non-relapse mortality, CR: complete remission, ATG: antithymocyte globulin, HSCT: hematopoietic stem cell transplantation.

Figure 1 :Figure 2 :

 12 Figure 1: Kaplan-Meier estimates of overall survival (A) and disease-free survival (B) Figure 2: Kaplan-Meier estimates of overall survival according to disease status at transplant (CR: complete remission).

  Figure 1

  Figure 2

Table 1 .

 1 Patient and donor characteristics at transplant

	Total (n=51)

Table 2 .

 2 Conditioning regimen

	n (%)

Table 3 .

 3 Engraftment and clinical outcomes

	Total (n=51)

Table 5 .

 5 Univariate analysis

				DFS at 24 months		OS at 24 months		2-year RI
	Variables	Value	%	Error +/-	p	%	Error +/-	p	%	95% CI	p
	Recipient age	<=54.9 years >54.9 years	49 68	10 9	0.2	65 68	10 10	0.57	28 16	14.5 -53.8 0.35 6.4 -40.2
	Recipient sex	male female	57 58	9 11	0.93	72 62	8 11	0.85	24 21	12 -47.7 8.5 -52	0.86
	Diagnosis	others Acute leukemia 63 47	12 9	0.09	53 74	12 8	0.06	24 22	9.6 -57.5 11.2 -44	0.72
	Disease status	others CR	43 63	13 8	0.04	43 76	13 8	0.003	21 23	7.5 -61.5 12.2 -43.7	0.96
	DRI	intermediate high/very-high	63 48	8 13	0.37	70 61	8 13	0.3	20 27	10.2 -39.3 0.71 10.9 -65.8
	ATG	no yes	50 60	14 8	0.67	56 71	15 8	0.57	25 22	8.8 -71.2 11.5 -41.1	0.84
	ATG dose (mg/kg)	0 2.5 5	50 67 40	14 9 15	0.34	56 79 50	15 8 16	0.46	25 19 30	8.8 -71.2 8.4 -44.8 10.7 -83.9	0.74
	Thiotepa dose (mg/kg)	5 10	65 43	8 13	0.37	76 50	7 13	0.11	19 29	9.6 -37.3 11.8 -68.9	0.62
	Graft source	BM PBSC	56 58	17 8	0.97	56 71	17 7	0.73	22 23	5.9 -83.2 12.6 -41.8	0.97
	Year of HSCT	<=2015 >2015	44 75	10 8	0.05	56 83	10 8	0.07	29 13	16.3 -53.9 4.2 -37	0.19
			100 day CI of aGVHD	2-year CI of cGVHD	2-year CI of NRM
	Variables	Value	%	95% CI	p	%	95% CI	p	%	95% CI	p
	Recipient age	<=54.9 years >54.9 years	35 20	20.1 -59.5 0.21 9 -44.6	39 22.6 -66.6 0.08 13 4.33 -8.3	23 16	11.2 -47.5 0.57 6.4 -40.1
	Recipient sex	male female	25 32	13.6 -46.1 0.71 15.9 -62.5	26 13.2 -49.7 0.88 30 13.3 -66.3	19 21	9 -39.1 8.6 -51.7	0.75
	Diagnosis	others Acute leukemia 27 29	13.7 -63.1 0.83 15 -46.8	26 10.5 -65.9 0.94 27 14.6 -50.1	29 15	13.6 -63.5 0.19 6.5 -33.5
	Disease status	others CR	36 24	17.1 -74.5 0.51 13.7 -43.3	NA 28 15.6 -49.7 NA	0.88	36 14	17 -75.2 5.9 -30.9	0.06
	DRI	intermediate high/very-high	31 19	19.1 -51.7 0.39 6.5 -53.9	18 47 26.2 -83.8 7.8 -41.7 0.02	17 25	8.2 -36 10.3 -60.5	0.48
	ATG	no yes	50 21	27.4 -91.3 0.03 11 -38.4	31 10.7 -90.9 0.88 25 14 -44.8	25 18	8.9 -70.1 9.1 -35.5	0.65
	ATG dose (mg/kg)	0 2.5 5	50 17 30	27.4 -91.3 7.6 -38.9 11 -82	0.09	31 10.7 -90.9 20 8.7 -44.5 40 17.5 -91.5	0.44	25 14 30	8.9 -70.1 5.4 -34.9 10.8 -83.3	0.52
	Thiotepa dose (mg/kg)	5 10	16 57	7.7 -34.1 0.005 35.3 -92.4	13 57 35.1 -93.2 4.9 -32.8 0.002	16 29	7.7 -34.1 12 -68	0.38
	Graft source	BM PBSC	33 26	12.4 -89.7 0.81 15.7 -43.8	44 23 12.4 -44.4 20 -98.7	0.15	22 19	6 -82.7 10.1 -35.9	0.90
	Year of HSCT	<=2015 >2015	41 13	25.6 -64.9 4.2 -36.9	0.02	33 19.2 -57.8 NA NA	0.22	26 13	13.5 -49.9 4.2 -37	0.25

Table 6 .

 6 Multivariate analysis

				DFS			OS		Relapse
	Variables	p	HR	95% CI Lower Upper	p	HR	95% CI Lower Upper	p	HR	95% CI Lower Upper
	Disease status	0.011 3.324 1.312 8.418 0.001 5.185 1.897 14.173 0.344 1.957 0.487 7.869
	(CR vs. others)
	ATG	0.733 1.189 0.439 3.224 0.581 1.370 0.448 4.190 0.960 1.036 0.261 4.105
	(yes vs. no)
	Thiotepa dose	0.535 1.384 0.495 3.872 0.697 0.799 0.258 2.476 0.608 1.443 0.356 5.852
	(10 vs. 5 mg/kg)
	Year of HSCT	0.029 3.639 1.139 11.626 0.121 3.022 0.747 12.222 0.104 3.675 0.764 17.672
	(>2015 vs. ≤2015)
			acute GVHD		chronic GVHD		NRM
				95% CI			95% CI			95% CI
	Variables	p	HR	Lower Upper	p	HR	Lower Upper	p	HR	Lower Upper
	Disease status	0.236 2.052 0.625 6.732 0.400 1.676 0.503 5.584 0.010 5.669 1.519 21.157
	(CR vs. others)
	ATG	0.058 3.086 0.962 9.898 0.752 0.821 0.241 2.795 0.608 1.473 0.335 6.476
	(yes vs. no)
	Thiotepa dose	0.133 0.357 0.093 1.368 0.080 0.246 0.051 1.181 0.750 1.286 0.273 6.064
	(10 vs. 5 mg/kg)
	Year of HSCT	0.479 1.806 0.352 9.278 0.957 0.955 0.182 5.023 0.171 3.388 0.591 19.437
	(>2015 vs. ≤2015)

Acknowledgements

We thank the nursing staff for providing excellent care for our patients. The study was supported by the "Association for Training, Education and Research in Hematology, Immunology and Transplantation" (ATERHIT).

Author contributions

M Mohty: designed the study, supervised research, analyzed data, and wrote the manuscript; J Bastos and R Dulery: designed the study, collected, assembled and analyzed data, performed statistical analysis, and wrote the manuscript; A Ruggeri and A Paviglianiti: analyzed data, performed statistical analysis and commented on the manuscript; F Malard, E Brissot, C Médiavilla, F Giannotti, G Battipaglia, A Banet, Z Van de Wyngaert, T Ledraa, R Belhocine, S Sestili, A Vekhoff, R Adaeva, F Isnard, O Legrand, MT Rubio: recruited patients and helped writing and commenting on the manuscript; All authors approved submission of the manuscript for publication purposes. Abbreviations: VOD: veno-occlusive disease, TMA: thrombotic microangiopathy, CMV: cytomegalovirus, EBV: Epstein-Barr virus, HHV6: human herpesvirus 6, ICU: intensive care unit.