WO2020058749A1

WO2020058749A1 - An antibody drug fusion based on asparaginase

Info

Publication number: WO2020058749A1
Application number: PCT/IB2018/057259
Authority: WO
Inventors: Maristella MAGGI; Greta PESSINO; Claudia Scotti
Original assignee: Universita' Degli Studi Di Pavia
Priority date: 2018-09-20
Filing date: 2018-09-20
Publication date: 2020-03-26

Abstract

The present invention discloses a fusion protein comprising a targeting antibody fragment and a bacterial asparaginase.

Description

AN ANTIBODY DRUG FUSION BASED ON ASPARAGINASE

Field of the invention

The present invention finds application in the medical field and in particular for the treatment of oncological diseases dependent on L-asparaginase for proliferation.

Background

Asparaginase (ASNase, EC 3.5.1.1) is a protein drug capable of inhibiting tumor growth by removing the extracellular supply of the non-essential amino acids L-asparagine (L-asn) and L-glutamine (L-gln) [1]. Type II ASNases derived from Escherichia coli or Erwinia chrysanthemi have been used since 1970 for the treatment of pediatric Acute Lymphoblastic Leukemia (ALL) [2, 3] . Combinatorial therapy comprising ASNase, glucocorticoids and vincristine, as established by the BMF protocol, accounts for roughly 90% therapy success rate in ALL patients [1, 4] . In vitro studies have proved that ASNases have a great potential for the treatment of a variety of other cancers, also comprising solid tumors like pancreatic carcinoma, breast cancer and lung cancer [5] . Nevertheless, clinical use of asparaginase for the treatment of solid tumors is still unadvisable as ASNase-based therapy has high systemic toxicity and immunogenicity, mainly in adult patients [6] . Systemic toxicity of ASNase affects mainly the liver, pancreas and central nervous system and it is related to removal of L-asn and L-gln from the patient sera, a phenomenon that reduces the non- essential amino acid availability not only for the tumor cells but also for healthy tissues [7] [8] . The drug immunogenicity is a major issue for therapy because production of anti-asparaginase antibodies can lead to immunoreactions but also to silent inactivation of the drug [ 9 ] .

In the last decades, use of antibody drug conjugates (ADCs) has proved that it is possible to direct a specific drug activity only where needed reducing the drug systemic toxicity and, at the same time, concentrating its activity on target cells.

For this purpose, cell surface proteins specifically present on tumor cells can be used as targets.

Cluster of Differentiations (CDs, https://www.uniprot.org/docs/cdlist) are particularly useful in this respect, as they identify receptors specific for different phases of white blood cells maturation, many of which are retained in tumor development .

The production and characterization of a recombinant scFv-ASNase fusion protein carrying a non-inhibitory anti-ASNase scFv without targeting capability [10-11] has been reported, with the aim of improving its resistance to trypsin.

Summary of the invention The authors of the present invention have surprisingly found, produced and characterized a new fusion protein obtained by linking a targeting scFv fragment to E. coli type II L-Asparaginase (EcAII) .

Brief disclosure of the figures

Figure 1 shows Western blot analysis of scFv-Ecall asparaginase. Numbers refer to elution fractions;

figure 2 shows the ELISA results for scFv-Ecall fusion. Abs : absolute relative units;

figure 3 shows the Flow cytometry results obtained by staining Raj i cells with an anti-CD19 antibody. In black (left) : blank sample; in green (right) : stained cells ;

figure 4 shows the results of Flow cytometry results obtained by staining RS4;11 cells with an anti-CD19 antibody. In black (left) : blank sample; in green (right) : stained cells;

figure 5 shows: (a) RS4;11 and (b) Raj i cells viability upon treatment with wt and scFv-Ecall;

figure 6 shows the Immunofluorescence staining of Ra i and RS4; 11 cells. Beyond nuclei, stained by Hoechst, the surface is visible, stained with an anti asparaginase antibody for surface labeling.

Object of the invention

According to a first object, the present invention discloses a fusion protein comprising a targeting antibody fragment and a moiety endowed with asparaginase activity. In a preferred embodiment, the targeting antibody fragment is a scFv fragment.

In a second object, it is disclosed a process for the preparation of a fusion protein comprising a targeting antibody fragment and a moiety endowed with asparaginase activity.

According to an even further object, the present invention discloses a pharmaceutical preparation comprising the fusion protein of the invention.

According to a third object, it is disclosed a fusion protein comprising a targeting antibody fragment and a moiety endowed with asparaginase activity for use as a medicament .

In a preferred embodiment, said fusion protein is disclosed for use as a medicament for the treatment of a neoplastic disease.

A further object of the invention is represented by a method for the treatment of neoplastic diseases comprising the administration of a fusion protein comprising a targeting antibody fragment and a moiety endowed with asparaginase activity to a patient in need thereof .

Detailed description of the invention

According to a first object, the present invention discloses a fusion protein comprising a targeting antibody fragment and a moiety endowed with asparaginase activity. In a preferred embodiment of the present invention, the targeting antibody fragment is a scFv fragment.

In a more preferred embodiment, the targeting antibody fragment is an anti-CD19 scFv fragment.

Particularly, the antigen CD19 is expressed on the surface of ALL cells.

In alternative embodiment of the invention, other CDs can be used.

In a particular embodiment, the moiety endowed with asparaginase activity is represented by a protein having bacterial origin.

In a more preferred aspect, the protein is an E. coli type II L-Asparaginase (EcAII) .

Alternatively, a protein from Erwinia chrysanthemi can also be used.

The amino acid sequence of the fusion protein is here below reported and corresponds do SEQ. ID. N. 1, while the nucleotide sequence encoding the fusion protein of the invention corresponds to SEQ. ID. N. 2:

Each one of the two sequences represents a further object of the present invention.

As per the second object, it is disclosed a process for the preparation of a fusion protein comprising a targeting antibody fragment and a moiety endowed with asparaginase activity.

Said process comprises the step of culturing a host cell, i.e. a preparation of host cells, which has been transformed with the vector above disclosed in an appropriate medium, followed by the recovery and the purification of the single domain antibody.

More in detail, said process comprises the steps of: preparing the sequence encoding for the antibody fragment and preparing the sequence encoding for the asparaginase moiety,

- cloning both genes into a suitable vector,

- transforming a suitable host cell with the vector comprising the two genes,

- expressing the fusion protein, and

- recovering and purifying the fusion protein.

In a preferred embodiment, the sequences, the vector and the host cell are those above disclosed.

A vector comprising the sequence encoding for the targeting antibody fragment of the invention and the asparaginase moiety of the invention represent further embodiments of the invention.

A host cell transformed with the vector disclosed by the present invention represents a further embodiment of the invention.

Preferably, the host cell is selected in the group comprising bacteria, yeast or mammalian cells.

According to the present invention, a preferred host cell is Escherichia coli and even more preferably is Escherichia coli ORIGAMI (DE3 ) .

In a preferred embodiment, therefore, the host cell preparation is a preparation of Escherichia coli cells and, preferably, of Escherichia coli ORIGAMI (DE3) .

As per a further object of the invention, it is disclosed a pharmaceutical preparation comprising the disclosed fusion protein of the invention together with one of more pharmaceutically acceptable carriers and/or excipients and/or preservatives.

In a preferred embodiment, the pharmaceutical preparation of the invention may further comprise a chemotherapeutic drug.

In particular, the chemotherapeutic drug is selected in the group comprising: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin bortezomib buserelin busulfan campothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, demethoxyviridin, dichloroacetate, dienestrol, diethylstilbestrol , docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil , fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, lenalidomide, letrozole, leucovorin, leuprolide, levamisole, lomustine, lonidamine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, metformin, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole octreotide, oxaliplatin, paclitaxel, pamidronate, pemetrexed, pentostatin, perifosine, plicamycin, pomalidomide, porfimer, procarbazine, raltitrexed, rituximab, sorafenib, streptozocin, sunitinib suramin, tamoxifen, temozolomide, temsirolimus , teniposide, testosterone, thalidomide, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, or vinorelbine. The pharmaceutical preparation of the invention can be administered intravenously or intramuscularly.

In particular, the fusion protein is the fusion protein disclosed above for the purposes of the present invention .

Said medicament may find application in the medical or in the veterinary field for the treatment of humans or animals .

In a preferred embodiment, said fusion protein is disclosed for use as a medicament for the treatment of oncological diseases dependent on asparagine, and particularly on L-asparagine, for proliferation.

According to an even more preferred embodiment, said diseases are oncological diseases.

In a particular aspect, said diseases are represented by Acute Lymphoblastic Leukemia (ALL) .

Preferably, the fusion protein of the invention is used for the treatment of diseases in a paediatric patient (<18 years ) .

A further object of the invention is represented by a method for the treatment of diseases comprising the administration of a fusion protein comprising a targeting antibody fragment and a moiety endowed with asparaginase activity to a patient in need thereof. The fusion protein of the invention may find application in a method for treatment in the medical or in the veterinary field for the treatment of human or animal .

In a preferred embodiment, the fusion protein is represented by the molecule detailed in the above disclosure .

According to a preferred embodiment, the diseases treated as per the present invention are oncological diseases dependent on asparagine, and particularly on L-asparagine, for proliferation.

According to a particular embodiment, the method for the treatment of tumors comprises the administration of the fusion protein of the invention in combination with a chemotherapeutic drug.

Preferably, said chemotherapeutic drug can be selected in the group comprising: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, bortezomib, buserelin, busulfan, campothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, demethoxyviridin, dichloroacetate, dienestrol, diethylstilbestrol , docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil , fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, lenalidomide, letrozole, leucovorin, leuprolide, levamisole, lomustine lonidamine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, metformin, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pemetrexed, pentostatin, perifosine, plicamycin, pomalidomide, porfimer, procarbazine, raltitrexed, rituximab, sorafenib, streptozocin, sunitinib, suramin, tamoxifen, temozolomide, temsirolimus , teniposide, testosterone, thalidomide, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, or vinorelbine. For the purposes of the method of the invention, the disclosed pharmaceutical preparation may be administered intravenously or intramuscularly.

The invention is disclosed in more details in the following experimental section.

MATERIALS AND METHODS

Construct design

In order to obtain the fusion gene encoding both for the antibody fragment (scFv) and the L-Asparaginase (EcAII) proteins (namely, scFv-EcAII), the sequence of a published scFv fragment targeted onto the human CD19 (hCD19) surface receptor and the sequence of the wild type ansB gene, respectively, were used as a template. The anti-hCD19 scFv sequence was amplified by Polymerase Chain Reaction (PCR) inserting the restriction sequences recognized by Ncol and BamHI at the gene 5' and 3', respectively. A sequence encoding for 6 histidines was inserted at the gene 5'. The ansB gene encoding for E. coli type II L-Asparaginase was obtained by PCR inserting the restriction sequences recognized by Sad and AvrII at the gene 5' and 3', respectively. The obtained genes were then cloned into the pET45b(+) expression vector previously modified in order to contain a sequence encoding for 6 repeats of the standard GGGGS (G4S) linker sequence between BamHI and Sad restriction sites. The primers used for the cloning are:

The full scFv-EcAII fusion gene inserted in the expression vector was sequenced by Sanger sequencing (Genewiz Europe) in order to exclude the presence of mutations .

Protein expression and purification

The pET45b(+) vector containing the fusion gene was used to transform chemically competent E. coli ORIGAMI (DE3) cells previously transformed with the pTfl6 plasmid (Takara) encoding for the tig factor by heat-shock. After transformation, the cells were selected in solid medium containing ampicillin (100 pg/ml), chloramphenicol (20 pg/ml), tetracyclin (12.5 pg/ml) and kanamycin (15 pg/ml) . A single clone was diluted into 50 ml LB medium containing the above mentioned selection antibiotics and incubated at 37°C shaking overnight. The subsequent day, 10 ml of pre- culture was inoculated in 500 ml of selective LB medium added with 0.05% w/v Arabinose and incubated at 37°C shaking at 250 rpm until OD600nm reached 0.8. At this point, 1 mM IPTG was added and the temperature was lowered to 10°C maintaining the culture shaking at 250 rpm. After 24 h induction, the cells were collected by centrifugation (11000 g, 4°C, 15 min) and resuspended in resuspension buffer (RB, 50 mM Tris-HCl, 200 mM NaCl, 10 mM imidazole, pH 8.0) using a culture volume : buffer volume ratio of 1000 ml: 50 ml.

Resuspended cells were disrupted by sonication (4 cycles at 60% power, 2 min each) . Cell lysate was clarified by centrifugation (20000 g, 4°C, 15 min, and

39000 g, 4°C, 45 min,) and by 0.22 pm filtration. Clear cell lysate was loaded onto a 5 ml HisTrap (GE Healthcare) column for immobilized metal ion affinity chromatography. Bound proteins were eluted by an imidazole steps gradient. After elution, fractions positive for proteins were analyzed by SDS-PAGE.

Asparaginase activity evaluation

scFv-EcAII asparaginase and glutaminase activity were evaluated by a coupled, continuous spectrophotometric assay as reported in Maggi et al . [12}.

Surface CD19 evaluation by FITC

Evaluation of the presence of the target antigen CD19 on the cell surface was evaluated on two cell lines, a type B lymphoma cell line (Raji) and a type T acute lymphoblastic leukemia cell line (Rs4;ll) . Cells to be stained were collected by centrifugation (1300 rpm, 3 min) and 1x10^s cells were resuspended in 5 ml ice-cold PBS. Cells were washed twice in 5 ml ice-cold PBS added with 3% w/v bovine serum albumin (BSA) . Subsequently, aspecific binding sites were blocked by incubating the cells in 0.1 ml PBS added with 3% BSA on ice for a total of 1 h. After incubation, cells were washed twice in 5 ml ice-cold PBS. After the last wash, cells were incubated in the presence of primary anti-human CD19 mAb diluted 1:100 in 100 mΐ PBS added with 1% w/v BSA. The incubation was carried out on ice, shaking the tube at regular intervals for a total of 1 h. Afterwards, cells were washed 5x in 5 ml ice cold PBS. After the last wash the cells were incubated with a secondary Alexa488-conj ugated anti-mouse antibody diluted 1:100 in 100 mΐ PBS added with 1% w/v BSA on ice for 30 min. Cells were then washed 5 times on ice-cold PBS and resuspended in 0.5 ml PBS for analysis by flow- cytometry. Cells stained only with the secondary antibody were systematically used as specificity control .

FACS analysis was performed using a CyFlow SL flow cytometer (Sysmex Partec GmbH) .

Cytotoxici ty evaluation

Dose-response experiments were conducted in order to evaluate scFv-EcAII cytotoxicity on Raj i and Rs4;ll cells. 5xl0⁴ cells were seeded in 100 mΐ RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine in 96-well plates. Cells were treated with different concentrations of scFv-EcAII (ranging from 1 to 0.005 U/ml) and incubated 72 h at 37°C, 5% CO2. Cell viability was evaluated by MTT, for which 15 mΐ of a 5 mg/ml solution of 3- ( 4 , 5-Dimethylthiazol-2-yl ) -2 , 5- Diphenyltetrazolium Bromide were added to each well and cells were incubated at 37°C, 5% CO2 for 4 h. Afterwards, cells were collected by centrifugation and resuspended in 100% DMSO. Absorption at 540 nm was measured using a plate reader. Data were elaborated as percentage of living cells with respect to the untreated control. IC50 was calculated using the 4- parameter logistic curve and indicated the concentration of scFv-EcAII at which cell viability was half of the control. Experiments were conducted in triplicates and repeated at least twice.

Immunostaining

In order to evaluate the capability of scFv-EcAII to bind to surface CD19 expressed on target cells, Raj i and Rs4;ll cells were stained and analyzed by fluorescence microscopy.

Cells to be stained were collected by centrifugation. 1x10^s cells were resuspended in 1 ml RPMI 1640 medium added with 10 % fetal bovine serum and 2 mM L-glutamine and seeded in a 6-well plate. Cells were incubated in the presence of 100 pg scFv-EcAII or PBS (control) at 37°C, 5% CO2 for 2 h. After incubation, cells were collected from the well and transferred to a 15 ml tube. Fixation was obtained by adding paraformaldehyde to a final concentration of 4% v/v. After 15 min fixation at room temperature, 10 ml PBS containing 0.1% Triton X-100 were added to the tube and the solution was briefly vortexed. Cells were incubated at room temperature for 30 min. Afterwards, cells were washed twice in 10 ml PBS added with 0.5 % w/v BSA. Cells were permeabilized by adding 1 ml of 50% v/v ice-cold methanol in PBS. After 15 min incubation on ice, cells were washed twice in 10 ml PBS added with 0.5 % w/v BSA. After the last wash, cells were resuspended in 100 mΐ rabbit anti-EcAII antibody diluted 1:100 in PBS added with 1% w/v BSA. Cells were incubated at room temperature with mild shaking overnight. The subsequent day, cells were washed 5 times in PBS added with 0.5% w/v BSA. Afterwards, cells were resuspended in 100 mΐ Alexa488-conj ugated anti-rabbit secondary antibody diluted 1:100 in PBS added with 1 % w/v BSA and incubated at room temperature with mild shaking for 1 h. After incubation, cells were washed 5 times in PBS added with 0.5 % w/v BSA. Nuclei were stained using Hoechst 33342. Cells were mounted on coverslips using low melting point agarose (1% w/v) and Mowiol.

ELISA

Enzyme-linked immunosorbent assay (ELISA) was done by a sandwich method and using a recombinant human CD19 (exons 1-4)- human Fc fusion (hCDl 9 ( exl-4 ) ) as antigen. The coating was done using 100 pg of scFv-EcAII in PBS in a 96-well ELISA plate incubated at +4°C in a humid chamber overnight. Blocking was done by using a 2% w/v BSA solution in PBS and incubating at room temperature with mild shaking for 2 h. Afterwards, the immobilized svFv-EcAII was incubated with the hCD19(exl-4) antigen at room temperature with mild shaking for 1 h. Signal was developed by secondary anti-human Fc HRP-conj ugated monoclonal antibody diluted 1:2000 in PBS and using the 3, 3 ' , 5, 5 ' -Tetramethylbenzidine (TMB) substrate.

Signal quantification was adjusted versus the reaction blank .

RESULTS

Protein expression, purification and enzymatic activity

The pET45b (+ ) -scFvEcA.il construct containing the gene coding for the anti-CD19 scFv fused by a (G4S) 6 linker to the ansB gene was sequenced for the insert and no mutations with respect to the original sequence were found. The construct was transformed into competent E. coli ORIGAMI (DE3 ) -pTfl 6 cells and protein expression was obtained by IPTG induction at 10°C. Protein purification resulted in a yield of 50 pg/l of culture. The obtained protein presented a 62 kDa band and a second band of roughly 27 kDa. Western blot analysis using an anti-asparaginase antibody revealed that both species were reactive (Fig. 1) . The enzymatic activity of the protein was evaluated in the presence of 1 mM asparagine and resulted to be 90.9112.9 U/mg.

Protein binding capabili ty ELISA was used to determine scFv-EcAII capability of binding to its hCD19 antigen in vitro. According to the obtained results, the fusion protein binds to its antigen in a dose-dependent manner as reported in Fig.

2.

Surface CD19 evaluation by FITC

Fig. 3 and 4 show flow cytometry results demonstrating the positivity of Raj i and RS4;11 cells to CD19. Tables 1 and 2 report the corresponding raw data of Median Fluorescence Intensity (MFI) .

Table 1. RAJI cells flow cytometry data

Table 2. RS4;11 cells flow cytometry data

Cytotoxicity evaluation

Cytotoxicity data for RS4;11 and Raj i cells obtained by the MTT assay are shown in Fig. 5.

The IC50s for both cell lines are very close for the wt and the fusion protein: 0.002 U/ml for the wt and 0.004 U/ml for the fusion in RS4;11 and 0.48 U/ml and 0.50

U/ml for Raj i cells.

Immunostaining

Immunostaining of Raj i and RS4;11 cells using the fusion protein shows a different pattern of distribution of the fluorescence. Fig. 6 shows a superposition of Hoechst-stained nuclei (blue) onto the cells stained with the fusion (green) . In the case of Raj i cells, the staining appears more uniformly concentrated on the membrane surface, while in RS4;11 it has a spotted pattern spread all over the cell.

From the description above reported, the advantages of the present invention will be apparent to those skilled in the art .

In particular, the fusion protein can be produced with high yield.

Also, it has shown to retain its peculiar properties, as it is endowed with both asparaginase activity and cytotoxicity in vitro, which is comparable to those original unfused protein.

Moreover, the fusion protein can actually target the tumoural cells expressing the antigen against which the scFv antibody fragment has been designed to.

The antigen CD19 above disclosed is expressed on the surface of ALL cells; other CDs can be used, depending on those expressed on the specific blood tumor under consideration . Thanks to a good selectivity property, the fusion protein of the invention does not target the normal healthy cells and produce less or negligible systemic side effects; therefore, therapy-related risks are reduced to the patient advantage.

These features are critical to allow a better cytoreduction in affected patients.

Moreover, the targeting by conjugation will allow many of the protein-drug epitopes to be shielded further reducing its immunogenicity .

Overall, the fusion protein disclosed by the present invention represents a promising molecule for the development of an effective drug.

SEQUENCE LISTING

<110> Universita degli Studi di Pavia

<120> An antibody drug conjugate based on asparaginase

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<220>

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Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Thr Gly Ser Gly Gly Gly

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Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

260 265 270

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Leu Pro Asn

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lie Thr lie Leu Ala Thr Gly Gly Thr lie A1a Gly Gly Gly Asp Ser 290 295 300

Ala Thr Lys Ser Asn Tyr Thr Val Gly Lys Val Gly Val Glu Asn Leu 305 310 315 320

Val Asn Ala Val Pro Gin Leu Lys Asp lie A1a Asn Val Lys Gly Glu

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Thr Leu Ala Lys Lys lie Asn Thr Asp Cys Asp Lys Thr Asp Gly Phe

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Val lie Thr His Gly Thr Asp Thr Met Glu Glu Thr Ala Tyr Phe Leu 370 375 380

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Ala Val Val Thr Ala Ala Asp Lys Ala Ser Ala Asn Arg Gly Val Leu

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Val Val Met Asn Asp Thr Val Leu Asp Gly Arg Asp Val Thr Lys Thr

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Asn Thr Thr Asp Val Ala Thr Phe Lys Ser Val Asn Tyr Gly Pro Leu 450 455 460

Gly Tyr lie His Asn Gly Lys lie Asp Tyr Gin Arg Thr Pro Ala Arg 465 470 475 480

Lys His Thr Ser Asp Thr Pro Phe Asp Val Ser Lys Leu Asn Glu Leu

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Pro Lys Val Gly lie Val Tyr Asn Tyr Ala Asn A1a Ser Asp Leu Pro

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ggtgtaagct ggattcgcca gcctccacga aagggtctgg agtggctggg agtaatatgg 180

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aactccaaga gccaagtttt cttaaaaatg aacagtctgc aaactgatga cacagccatt 300

tactactgtg ccaaacatta ttactacggt ggtagctatg ctatggacta ctggggtcaa 360

ggaacctcag tcaccgtctc gagtggtgga ggcggttcag gcggaggtgg ctctggcggt 420

ggcggtagtg tcgacatcca gatgacacag actacatcct ccctgtctgc ctctctggga 480

gacagagtca ccatcagttg cagggcaagt caggacatta gtaaatattt aaattggtat 540

cagcagaaac cagatggaac tgttaaactc ctgatctacc atacatcaag attacactca 600

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720

acgttcggag gggggaetaa gcttgaaata acaggatccg gcggaggtgg ctctggcggt

780

ggtggctctg gtggaggcgg ttcaggcgga ggtggctctg gcggtggtgg ctctggtgga

840

ggcggttcag agctcttacc caatatcacc attttagcaa ccggcgggac cattgccggt

900

ggtggtgact ccgcaaccaa atctaactac acagtgggta aagttggcgt agaaaatctg

960

gttaatgcgg tgccgcaact aaaagacatt gcgaacgtta aaggcgagca ggtagtgaat

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atcggctccc aggacatgaa cgataatgtc tggctgacac tggcgaaaaa aattaacacc

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gactgcgata agaccgacgg cttcgtcatt acccacggta ccgacacgat ggaagaaact

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gcttacttcc tcgacctgac ggtgaaatgc gacaaaccgg tggtgatggt cggcgcaatg

1200

cgtccgtcca cgtctatgag cgcagacggt ccattcaacc tgtataacgc ggtagtgacc

1260

gcagctgata aagcctccgc caaccgtggc gtgctggtag tgatgaatga caccgtgctt

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gatggccgtg acgtcaccaa aaccaacacc accgacgtag cgaccttcaa gtctgttaac

1380

tacggtcctc tgggttacat tcacaacggt aagattgact accagcgtac cccggcacgt

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aagcatacca gcgacacgcc attcgatgtc tctaagctga atgaactgcc gaaagtcggc

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1560

tatgatggca tcgttagcgc tggtgtgggt aacggcaacc tgtataaatc tgtgttcgac

1620

acgctggcga ccgccgcgaa aaccggtact gcagtcgtgc gttcttcccg cgtaccgacg

1680

ggcgctacca ctcaggatgc cgaagtggat gatgcgaaat acggcttcgt cgcctctggc

1740

acgctgaacc cgcaaaaagc gcgcgttctg ctgcaactgg ctctgacgca aaccaaagat

1800

ccgcagcaga tccagcagat cttcaatcag tactaataat aa

1842

<210> 3 <211> 49

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer scFvfusion s

<400> 3

ccatgggtca tcatcatcat catcatgagg tgaaactgca ggagtcagg 49

<210> 4

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer scFvfusion as

<400> 4

ggatcctgtt atttcaagct tagtcccccc tccg

34

<210> 5

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer EcAIIfusion s

<400> 5

gagctcttac ccaatatcac cattttagc

29

<210> 6

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer EcAIIfusion as

<400> 6

cctaggttat tattagtact gattgaagat ctgc

34 References

[1]D.a.M.M.a.T.S.a.B.O.a.C.L.R.a.P.M.V.a.V.L.a.V.G.a.S. C. Covini, Expanding Targets for a Metabolic Therapy of Cancer: L-Asparaginase . , Topics in anti-cancer research 3 (2015) 418--445.

[2] D.a.T.D.A. Douer, New developments in acute lymphoblastic leukemia, Clin Adv Hematol Oncol 12(6 Suppl 12) (2014) 13—22.

[3] A. a . Z . H . a . S . E .A. Emadi, Asparaginase in the treatment of non-ALL hematologic malignancies, Cancer Chemotherapy and Pharmacology 73(5) (2014) 875—883.

[4] J.M.a.R. J.a.L.E.a.K.A.a.M.A.a.H.R.W. Hill, L- asparaginase therapy for leukemia and other malignant neoplasms. Remission in human leukemia, Jama 202(9)

(1967) 882—888.

[5] E. Dufour, F. Gay, K. Aguera, J.Y. Scoazec, F. Horand, P.L. Lorenzi, Y. Godfrin, Pancreatic tumor sensitivity to plasma L-asparagine starvation, Pancreas 41(6) (2012) 940-8.

[6] A. Emadi, H. Zokaee, E.A. Sausville, Asparaginase in the treatment of non-ALL hematologic malignancies, Cancer Chemother Pharmacol 73(5) (2014) 875-83.

[7] J .A. Distasio, D.L. Durden, R.D. Paul, M. Nadji, Alteration in spleen lymphoid populations associated with specific amino acid depletion during L- asparaginase treatment, Cancer Res 42(1) (1982) 252-8.

[8] U. K. a . K. S . S . a .A. W . Narta, Pharmacological and clinical evaluation of L-asparaginase in the treatment of leukemia, Crit Rev Oncol Hematol 61(3) (2007) 208--

221.

[9]

R.a.H.S.P.a.B.J.a.R.C.a.S.L.a.B.A.a.G.N.a.S.M.a.P.C.

Pieters, L-asparaginase treatment in acute lymphoblastic leukemia: a focus on Erwinia asparaginase, Cancer 117(2) (2011) 238--249.

10. Li Guo, Xiyun Yan, Shijun Qian and Guangzhen Meng Selecting and expressing protective single-chain Fv fragment to stabilize L-asparaginase against inactivation by trypsin, Biotechnol Appl Biochem, 2000, 31, 21-27.

11. Guo L, Yan X, Qian S, Meng G. Selecting and expressing protective single-chain Fv fragment to stabilize L-asparaginase against inactivation by trypsin. Biotechnol Appl Biochem. 2000 Feb; 31 (Pt 1) : 21-7.

12. Maggi M, Mittelman SD, Parmentier JH, Colombo G, Meli M, Whitmire JM, Merrell DS, Whitelegge J, Scotti C. A protease-resistant Escherichia coli asparaginase with outstanding stability and enhanced anti-leukaemic activity in vitro. Sci Rep. 2017 Nov 3 ; 7 ( 1 ) : 14479. doi: 10.1038/s41598-017-15075-4.

Claims

1. A fusion protein comprising a moiety represented by a targeting antibody fragment and a moiety having asparaginase activity.

2. The fusion protein according to claim 1, wherein the targeting antibody fragment is represented by a scFv fragment.

3. The fusion protein according to claim 1 or 2, wherein the targeting antibody fragment is represented by an anti-CD19 scFv fragment.

4. The fusion protein according to claim 3 characterized by having the sequence of SEQ. ID. n.l.

5 . The isolated nucleotide sequence encoding for the fusion protein according to claim 4 having a sequence corresponding to the SEQ. ID. n.2.

6. A vector comprising the isolated nucleotide sequence of the preceding claim.

7. A host cell transformed with the vector according to the preceding claim.

8. The host cell according to the preceding claim, which is selected in the group comprising bacteria, yeast or mammalian cells.

9. The host cell according to the preceding claim 7 or 8, which is Escherichia coli .

10 . The host cell according to the preceding claim, which is Escherichia coli ORIGAMI (DE3 ) .

11 . A process for the preparation of the fusion protein according to any one of claims 1 to 4 comprising the step of culturing Escherichia coli cells which have been transformed with the vector of claim 6 in an appropriate medium, followed by the recovery and the purification of the single domain antibody.

12. The fusion protein according to any one of claims 1 to 4 for use as a medicament.

13. The fusion protein according to claim 12 for use as a medicament in the medical or in the veterinary field.

14. The fusion protein according to claim 12 or 13 for use as a medicament for humans, wherein said patient is a paediatric patient with an age of less than 18 years.

15. The fusion protein according to any one of the preceding claim 12 to 14 for use as a medicament in the treatment of solid tumors, both primary and secondary.

16. The fusion protein according to any one of the preceding claim 12 to 15 for use in the treatment of neoplasia selected in the group comprising: gastrointestinal, lung, breast, prostatic, ovarian, liver, kidney, thyroid, nasopharyngeal tumors, sarcomas, lymphomas and multiple myeloma.

17. The fusion protein according to any one of the preceding claims 12 to 16 for use as a medicament in the treatment of Acute Lymphoblastic Leukeamia (ALL) .

18. A method for the treatment of tumors comprising the step of administering a suitable amount of the fusion protein according to claim 1 to a patient in need thereof .

19. The method for the treatment of tumors according to the preceding claim, wherein said tumor is selected in the group comprising: gastrointestinal, lung, breast, prostatic, ovarian, liver, kidney, thyroid, nasopharyngeal tumors, sarcomas, lymphomas and multiple myeloma .

20. The method for the treatment of tumors according to claim 18 or 19 wherein said tumor is Acute Lymphoblastic Leukeamia (ALL) .

21. The method for the treatment of tumors according to any one of claims 18 to 20, wherein said patient is a paediatric patient with an age of less than 18 years.

22. The method for the treatment of tumors according to any one of claims 18 to 21, wherein said patient is a human or an animal .

23. The method for the treatment of tumors according to any one of claims 18 to 21, wherein the fusion protein according to claim 1 is administered in combination with a chemotherapeutic drug.

24. The method for the treatment of tumors according to the preceding claim, wherein said chemotherapeutic drug is selected in the group comprising: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, bortezomib, buserelin, busulfan, campothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, demethoxyviridin, dichloroacetate, dienestrol, diethylstilbestrol , docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil , fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, lenalidomide, letrozole, leucovorin, leuprolide, levamisole, lomustine, lonidamine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, metformin, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole octreotide, oxaliplatin, paclitaxel, pamidronate, pemetrexed, pentostatin, perifosine, plicamycin, pomalidomide, porfimer, procarbazine, raltitrexed, rituximab, sorafenib, streptozocin, sunitinib suramin, tamoxifen, temozolomide, temsirolimus , teniposide, testosterone, thalidomide, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, or vinorelbine.

25. A pharmaceutical preparation comprising the fusion protein according to claim 1 and one of more carriers and/or excipients and/or preservatives pharmaceutically acceptable .

26 . The pharmaceutical preparation according to the preceding claim further comprising a chemotherapeutic drug .

27 . The pharmaceutical preparation according to the preceding claim, wherein said chemotherapeutic drug is selected in the group comprising: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, bortezomib, buserelin, busulfan, campothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, demethoxyviridin, dichloroacetate, dienestrol, diethylstilbestrol , docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil , fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, lenalidomide, letrozole, leucovorin, leuprolide, levamisole, lomustine, lonidamine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, metformin, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pemetrexed, pentostatin, perifosine, plicamycin, pomalidomide, porfimer, procarbazine, raltitrexed, rituximab, sorafenib, streptozocin, sunitinib, suramin, tamoxifen, temozolomide, temsirolimus , teniposide, testosterone, thalidomide, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, or vinorelbine.

28 . The pharmaceutical preparation according to any one of claims 25 to 27 for intravenous or intramuscular administration .