Recombinant dra (AFAE) protein conjugate for treating tumors

Abstract

The present invention describes a recombinant DrA (AfaE) protein conjugate for treating tumors.

Description

[0001] Recombinant DrA (AfaE ) protein conjugate for treating tumors

[0002] DESCRIPTION

[0003] In the literature , CEACAM5 is often used as a synonym for carcinoembryonic antigen ( CEA) , a known biomarker of many types of malign tumors , such as colorectal cancer and non-small cell lung cancer .

[0004] The primary function thereof in the embryonic intestine and in colon tumors is adhesion between epithelial cells .

[0005] Moreover, it carries out a signi ficant role in inhibiting di f ferentiation and apoptosis in colon cells .

[0006] There is evidence that high CEACAM5 expression is closely associated with CD133-positive colorectal cancer stem cells .

[0007] High CEACAM5 expression has also been identi fied in about 25% of patients with advanced non-squamous (NSq) non-small cell lung cancer (NSCLC ) detectable by IHC ( immunohistochemistry) .

[0008] In early experimental studies , about 25% of patients with advanced non-squamous (NSq) non- small cell lung cancer had high CEACAM5 expression .

[0009] Therefore , CEACAM5 is a potential target for treating various forms of cancer .

[0010] Summary of the invention The inventors of the present patent application have developed a recombinant DrA (AfaE) protein which can be used for preparing a conjugate for treating tumors.

[0011] Brief description of the drawings

[0012] Figure 1: SDS-PAGE of IMAC-eluted AfaE.

[0013] Figure 2: Chromatogram of the SEC assay conducted on Superdex 200 10 / 300 column (top) .

[0014] Figure 3: SDS-PAGE analysis with protein samples stored at different temperatures.

[0015] Figure 4: SDS-PAGE analysis on protein samples eluted from the SEC column.

[0016] Figure 5: SDS-PAGE analysis with protein samples conjugated at different protein-daunorubicin molar ratios .

[0017] Figure 6: SEC chromatograms and corresponding SDS- PAGE analyses.

[0018] Figure 7 : Results of the pre-concentration scouting assay. The optimum immobilization pH is pH=5.

[0019] Figure 8: SPR analysis of AfaE bonding with CEACAM5. After the immobilization of CEACAM5 on the sensor, AfaE solution was injected at increasing concentrations, from 1.25 pM to 20 pM. AfaE binds CEACAM5 with a dissociation constant (KD) given by the Koff / Konratio of 73 pM.

[0020] Figure 9: SPR analysis of AfaE bonding with CEACAM5. Equilibrium affinity analysis revealed a KD of 21 pM.

[0021] Figure 10: SPR analysis of Af aE-daunorubicin (1:6) bonding to CEACAM5. After the immobilization of CEACAM5, Af aE-daunorubicin (1:6) was injected at increasing concentrations, from 0.67 gM to 10 gM. Af aE-daunorubicin binds CEACAM-5 with a KD given by the Koff / Konratio of 31 gM.

[0022] Figure 11: SPR analysis of Af aE-daunorubicin (1:6) bonding to CEACAM5. Equilibrium affinity analysis revealed a KD of 21 gM. This demonstrates that the affinity of AfaE for CEACAM5 is unchanged, even adding a ligand.

[0023] Figure 12: SEC chromatogram as generated by the FPLC system, which demonstrates the oligomerization state and confirms that the sample is pure and monodisperse.

[0024] Figure 13: Superdex 200 10 / 300 size exclusion chromatography column calibration curve.

[0025] Figure 14: SDS-page analyses: M (marker) , Oh - 24h - 48h - 54h - 72h (samples incubated with plasma) .

[0026] Figure 15: Western blot analysis: M (marker) , 6h - 24h - 48h - 72h (samples incubated with plasma) .

[0027] Object of the invention

[0028] In a first object, the present invention describes a recombinant DrA (AfaE) protein.

[0029] In a second object, the present invention describes a conjugate of the recombinant bacterial protein with a ligand .

[0030] In a third object, the present invention describes the recombinant DrA (AfaE) protein and the conjugates thereof with a ligand for the medical use. In a particular aspect, the recombinant DrA (AfaE) protein of the invention and the conjugates thereof with a ligand are described for the medical use in the diagnosis and treatment of tumors.

[0031] Detailed description of the invention

[0032] In a first object, the present invention describes a recombinant DrA (AfaE) protein.

[0033] In particular, such a protein has the following amino acid sequence (SEQ. ID no. 2) :

[0034] For the purposes of the present invention, the recombinant protein described comprises the GSS residues, in addition to methionine residue, at the N- terminal before the His-tag.

[0035] According to an aspect of the present invention, the protein can be modified by inserting a cysteine residue in the N-terminal direction before the His-tag (SEQ. ID no . 3 ) : or by replacing the first glutamic acid residue with a cysteine residue in the C-terminal direction after the His-tag (SEQ. ID no. 4) :

[0036] According to another aspect of the invention, the protein can be modi fied by replacing all the lysine residues , with the exception of the lysine residue falling within the receptor binding site , with a glycine residue .

[0037] For the purposes of the present invention, the lysine residue involved in the receptor binding site is the residue in position 86 (Korotkova N . et al , Molecular Microbiology, Volume 67 , I ssue 2 , January 2008 Pages 420-434 ) .

[0038] Advantageously, this modi fication allows a single ligand to be bound to the protein .

[0039] For the purposes of the present invention, said recombinant DrA (AfaE ) protein is in the dimeric conformation .

[0040] In particular, said protein is produced by means of a technological platform involving the expression of the protein of interest in a host cell .

[0041] For the purposes of the present invention, said host cell is for example represented by Escheri chia coli .

[0042] In a second obj ect , a conj ugate of the recombinant protein of the invention with a ligand is described .

[0043] For the purposes of the present invention, such a ligand is represented by a compound with diagnostic or therapeutic activity . According to an aspect, such a ligand is represented by the compound sulfo-SPDB-DM4 (Molecular formula: C46H63CIN4O17S3, CAS No. 1626359-59-8) .

[0044] According to a preferred aspect, the conjugate of the invention comprises said recombinant protein and said ligand in a ratio between 1:1 and 1:14 or between 1 : 1 and 1:6.

[0045] For the purposes of the present invention, the conjugation between the recombinant DrA (AfaE) protein and said ligand is obtained by covalent bonding, for example using succinic anhydride.

[0046] According to an aspect of the present invention, the conjugation between the recombinant DrA (AfaE) protein and said ligand is obtained by means of a cleavable linker .

[0047] The conjugate described by the present invention behaves as a CEACAM-5 receptor binder.

[0048] In accordance with a third object, the present invention describes recombinant DrA (AfaE) protein and a conjugate thereof with a ligand for medical use.

[0049] According to a particular aspect of the invention, recombinant DrA (AfaE) protein conjugate with the ligand is described for medical use in the diagnosis and treatment of tumors.

[0050] In an even more particular aspect, said tumor is selected from the group comprising: breast, pancreas, esophagus, colorectum, stomach, lungs. According to another aspect of the invention, recombinant DrA (AfaE) protein conjugate with a ligand is described for medical use in the treatment of tumors in combination with one or more other anti-tumor agents.

[0051] For the purposes of the present invention, the term "in combination" is to be understood as a treatment simultaneously with or within the same therapeutic protocol .

[0052] The present invention will be further described with reference to the following Experimental Part.

[0053] Subcloning strategy

[0054] In order to express the target protein in a heterologous system (E. coll) , appropriate constructional DNAs were designed, including codon optimization for the host strain. The resulting DNA sequence is described below (SEQ. ID no. 1) .

[0055] (Optimized sequence length: 444, GC% : 57.16.

[0056] (the His-tag in bold, the two stop codons underlined, the restriction sites in italics') The nucleotide sequence was subcloned in the vector pET28a_TEV vector under the Ndel and Xhol restriction sites .

[0057] The nucleotide sequence translated into amino acid sequence, including restriction sites and poly-histidine tags is shown below (SEQ. ID no. 2) .

[0058] Expression and purification steps

[0059] In-house E. coli BL21 (DE3) cells were transformed by heat shock with pET28a-TEV_Af aE vectors. The cells were cultured in 1 liter of 2XTY medium at 37 °C to achieve an optical density (OD600) of 0.6 and then induced by adding IPTG to the final concentration of 0.5 mM for 12 hours at 20°C. The culture cells were harvested by centrifugation at 8000 rpm and 4°C for 10 minutes. The bacterial pellet was then washed with phosphate buffer and stored at -80°C.

[0060] Purification method

[0061] The induced E. coli BL21 (DE3) cells were resuspended in lysis buffer (0.05 M sodium phosphate pH 8; 300 mM NaCl) and killed by ultrasound. After the addition of a protease inhibitor cocktail, the insoluble material of the lysate was removed by centrifugation (14,000 xg for 45 minutes at 4 °C) and the recombinant protein was purified from the supernatant by Immobilized-Metal Affinity Chromatography (IMAC) , eluted with a step gradient of 200 mM to 500 mM imidazole as the final concentration .

[0062] During the entire procedure, the recombinant protein was monitored by standard SDS-PAGE analysis (Figure 1) and the protein concentration was determined by the Bradford assay, using bovine serum albumin as a standard.

[0063] The IMAC-eluted fractions were further purified by PD- 10 desalting column for imidazole removal and final buffer exchange into 50 mM M sodium phosphate pH 8; 150 mm NaCl .

[0064] Final yield = 3.5 ml of concentrated AfaE protein at 3.8 mg / ml .

[0065] Biochemical characterization

[0066] To assess the oligomeric state of the recombinant protein, a size exclusion chromatography (SEC) experiment was carried out on a pre-calibrated Superdex 200 10 / 300 column. As shown in Figure 2, the protein eluted at 15.64 ml as an elution volume corresponding to a calculated molecular weight of 35 kDa . Considering the molecular weight of the monomer unit, it can be concluded that the recombinant AfaE behaves as a dimer in solution. The SEC experiment was performed in 50 mM sodium phosphate pH 8; 150 mM NaCl as a mobile phase.

[0067] Protein stability was studied by storing the AfaE samples at three different temperatures for 10 days, i.e., at 20°C, 4°C and -80°C. The protein integrity and degradation profile was assessed by electrophoretic analysis on SDS-PAGE. As shown in Figure 3, for the two samples stored at low temperature (4°C and -80°C) no degradation phenomena are observed; while the protein stored at room temperature is partially degraded, probably lacking the N-terminal tag.

[0068] Figure 12 shows the original SEC chromatogram with the standard proteins used for column calibration with reference to the following Table, in which Ve= elution volume, V0= exclusion volume, Vc= total column volume, Kav= proportion of pores available for the protein.

[0069] Protein purification - scale up

[0070] Following the procedure described above, we increased protein production by adjusting the volume of the medium broth to 2 L. The cell lysate was subjected to IMAC- based purification followed by size exclusion chromatography which allowed obtaining a pure, homogeneous and monodisperse preparation of the AfaE Protein for the bio-conjugation experiment (Figure 4) .

[0071] Bioconjugation with daunorubicin

[0072] Synthesis of succinic anhydride modified daunorubicin (SAMDAU) .

[0073] In a round-bottomed flask, a solution of: 50 mg of daunorubicin hydrochloride (8.88 10-2 mmol) + 11 mg of succinic anhydride (0.11 mmol) + 5 ml of anhydrous DMF, was stirred under argon in the dark, then 40 pl of TEA are added slowly. The reaction mixture was stirred for 72 hours at room temperature, then the solvent was removed under vacuum at 40-45°C.

[0074] 1 ml of CHCI3 and 20 ml of H2O were then added and, after 1 hour, the resulting red / brownish solid product was filtered, washed a few times with water and dried under vacuum at room temperature.

[0075] Yield: 83.6% (46mg SAMDAU)

[0076] SAMDAU-NHS Synthesis

[0077] A solution of: 46 mg SAMDAU (0.0733 mmol) + 11 mg NHS (0.08 mmol) + 22 mg EDC (0.11 mmol) + 1 ml DMF was stirred under argon in the dark. After 12h the solvent was removed under vacuum at 40-45°C.

[0078] A stock solution was then obtained, consisting of a quarter of the solid product dissolved in anhydrous DMSO. The biocon ugation experiment was carried out by incubating 1 mL of protein solution (2.5 mg / mL as the concentration) with daunorubicin synthesized as described below. The mixture was prepared with a molar excess of daunorubicin at different protein-daunorubicin ratios (i.e., 1:2; 1:4; 1:6; 1:10; 1:12; 1:14; 1:16) and then incubated at 4°C under vigorous stirring. After 2 hours of reaction, the samples were subjected to centrifugation to separate the soluble fraction. As shown in Figure 5, the protein biocon ugation resulted in a band shift with respect to the electrophoretic mobility of the ligand-free protein. The amount of band shift remains constant along the different molar ratios tested .

[0079] Samples corresponding to 1:6 and 1:10 and 1:14 were selected to proceed with a further purification by size exclusion chromatography so as to remove the bioconjugation reagents and purify the protein for the studies of CEACAM-5 receptor bonding by surface plasmon resonance (SPR) . The 1:6 and 1:10 samples were eluted with a defined absorbance peak, while no protein was detected in the 1:14 ratio likely due to the high concentration of DMSO compromising protein stability and folding. The fractions corresponding to the absorbance peak were analyzed by SDS-PAGE, showing the predicted band shift with respect to the ligand-free protein (Figure 6) .

[0080] Interaction studies of AfaE protein and the CEACAM-5 receptor by SPR The bonding interactions were analyzed using a Biacore T100 instrument (GE Healthcare) and the CM5 chip, following standard procedures.

[0081] The CEACAM-5 receptor was purchased from ACROBiosys terns (see instructions provided) . 1. CECAM-5 immobilization

[0082] In order to determine the proper immobilization conditions without permanently changing the sensor chip surface, a pre-concentration analysis was carried out for which the ligand was diluted in several immobilization buffers differing by half or one pH unit (i.e., pH range: 4, 4.5, 5, 5.5) , obtaining an indication of the suitability of the conditions. The electrostatically bound ligand was washed off the surface with 50 mM NaOH. As shown in Figure 1, pH 5, pH 4.5 and pH 4 gave a rapid and high pre-concentration result (Figure 7) .

[0083] CEACAM-5 (pH 5) was immobilized on the surface of a single channel of the CM5 sensor chip. Taking into account the average molecular weight (MW) of the ligand and analytes, the appropriate density of the ligand (RL) on the chip was calculated according to the Equation:

[0084] RL= (PM ligand / PM analyte) X Rmax X (1 / Sm) where Rmax is the maximum bonding signal and Sm corresponds to the bonding stoichiometry. The target capture level of the CEACAM-5 protein on the CM5 surface was of bout 800 / 900 resonance units (RU) (table below) .

[0085] 2. Ligand- free AfaE bonding analysis

[0086] In a single cycle kinetic experiment, AfaE solution was injected in five washes at increasing concentrations, from 1.25 pM to 20 pM, onto the surface of the CEACAM5 functionalized sensor, using HBS-EP+ (Cytiva) as a running buffer, with a contact time of 120 seconds. The reference flow cell was used as a control surface for refractive index modification and non-specific bonding. A long dissociation step (600 seconds) and a single regeneration step followed the last sample injection (10 mM glycine, pH 1.5, 30 seconds contact time) , with no regeneration between each sample injection.

[0087] See Figures 8 and 9.

[0088] The resulting SPR sensorgrams revealed good bonding between AfaE and CEACAM-5 with an equilibrium dissociation constant (KD) of 73 pM and a kinetic profile showing very rapid association and dissociation from the immobilized receptor. The steady-state analysis revealed a KD of 21 pM.

[0089] 3. Analysis of AfaE-daunorubicin bonding (1:6) .

[0090] In a single cycle kinetic experiment, AfaE-daunorubicin (1:6) solution was injected in five washes at increasing concentrations, from 0.67 pM to 10 pM, onto the surface of the CEACAM5 functionalized sensor, using HBS-EP+ (Cytiva) as a running buffer, with a contact time of 120 seconds. The reference flow cell was used as a control surface for refractive index modification and nonspecific bonding. A long dissociation step (600 seconds) and a single regeneration step followed the last sample injection (10 mM glycine, pH 1.5, 30 seconds contact time) , with no regeneration between each sample in ection .

[0091] See Figures 10 and 11.

[0092] The resulting SPR sensorgrams revealed a good bonding between Af aE-daunorubicin and CEACAM-5 with a dissociation constant (KD) of 31 pM and a kinetic profile showing a slower but comparable association with respect to the ligand-free protein. The steady-state analysis revealed a KD of 21 pM.

[0093] Analysis of AfaE stability in plasma

[0094] 25 pL of AfaE (2.5 mg / mL) was diluted in 225 pL of human plasma (human plasma recovered with ava anticoagulants) to a total volume of 250 pL .

[0095] The solution was incubated at 37 °C in a water bath with stirring. The solution was then sampled (20 pL) seven times, at Oh - 6h - 24h - 30h - 48h - 54h - 72h from the beginning of the analysis, and directly frozen at -80°C.

[0096] SDS-page analysis First, the obtained samples were analyzed on SDS-page (15% acrylamide gel) . 1 pL of plasma, 15 pL of AfaE (2.5 mg / mL) and 1 pL of each sample (AfaE concentration 0.25 mg / mL) , respectively, were loaded onto gel.

[0097] The results of the SDS-page analysis are shown in figure 14.

[0098] As expected, due to the low protein concentration, the AfaE-related bands were not clearly visible on the gel for the plasma samples (0 hours to 72 hours) .

[0099] Western blot analysis

[0100] A Western blot analysis was then carried out to identify AfaE and confirm the stability thereof in plasma.

[0101] 5 pL of AfaE, 1 pL of plasma and 1 pL of the samples were loaded onto a 15% acrylamide gel and the electrophoretic run was conducted at 140V for about 1.30 h. At the end of the run, the gel was immersed in the transfer buffer (25 mM Trizma base, 190 m glycine and 20% methanol) for 15 minutes.

[0102] To transfer the proteins from the gel to the nitrocellulose membrane, once the sandwich was assembled, the transfer was carried out at 100V for 1 hour with an ice block in the tank.

[0103] After the transfer, the nitrocellulose membrane was incubated with 5% skim milk in TBST (10 mL TBS lOx, 100 pL Tween, and 90 mL H2O) . The membrane was then washed three times for 5 minutes with TBST. Incubation with the primary antibody 6x HIS (diluted 1:1000 in 5% skim milk) was carried out overnight at 4°C under gentle stirring.

[0104] The subsequent morning, the membrane was washed 5 times for 5 minutes with TBST and incubated with a secondary antibody (diluted 1:8000 in 5% skimmed milk) for 1 hour at room temperature. After incubation, the membrane was washed 5 times for 5 minutes with TBST.

[0105] The chemiluminescent substrate mixture (buffer A + buffer B, in 1 : 1 ratio) was applied to the membrane and the chemiluminescence was captured using a CCD camera imager .

[0106] The results of the Western blot analysis are shown in figure 15.

[0107] The bands for His-marked AfaE protein are present in all the samples analyzed and show the same intensity features, with no degradation byproducts, confirming that AfaE is stable in plasma for at least 72 hours.

Claims

CLAIMS1. A recombinant form of DrA (AfaE) protein having an amino acid sequence corresponding to SEQ. ID no. 2.

2. A recombinant form of DrA (AfaE) protein according to claim 1, wherein in the N-terminal position before the His-tag an additional amino acid residue represented by cysteine is included.

3. A recombinant form of DrA (AfaE) protein according to claim 1, wherein in the C-terminal position after the His-tag the first glutamic acid residue is substituted by a cysteine residue.

3. A recombinant form of DrA (AfaE) protein according to claim 1 or 2, wherein all the lysine residues are replaced by a glycine residue except the lysine residue involved in the binding site.

4. A recombinant form of DrA (AfaE) protein according to any one of the preceding claims 1 to 3, which is in the dimeric form.

5. A conjugate of the recombinant form of DrA (AfaE) protein according to any one of the preceding claims, with a ligand.

6. A conjugate according to the preceding claim, wherein said ligand is represented by the compound sulfo- SPDB-DM4.

7. A conjugate according to claim 5 or 6, wherein said recombinant DrA (AfaE) protein and said ligand are in a ratio between 1:1 and 1:14.

8. A conjugate according to any one of the preceding claims 5 to 7, wherein said recombinant DrA (AfaE) protein and said ligand are conjugated by an optionally cleavable, covalent bonding.

9. A recombinant form of DrA (AfaE) protein according to any one of claims 1 to 4, or a conjugate according to any one of claims 5 to 8, for the medical use.

10. A recombinant form of DrA (AfaE) protein according to any one of claims 1 to 4, or a conjugate according to any one of claims 5 to 9, for the medical use in the diagnosis or treatment of tumor.

11. A conjugate according to the preceding claim, wherein said tumor is selected from the group comprising: breast, pancreas, esophagus, colorectum, stomach, lung tumors .

12. A conjugate according to any one of the preceding claims, for medical use in the treatment of tumor in combination with one or more other antitumor agents.

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