Methods of treatment utilizing biocompatible nanoparticles and therapeutic agents

Abstract

The invention pertains to a therapeutic, prophylactic or diagnostic method comprising, administering a pharmaceutical compound followed by administering a biocompatible nanoparticle comprising an oligomer of albumin, wherein the longest or largest dimension of the nanoparticle is between about 4 nm and about 500 nm. In preferred embodiments, administering the biocompatible nanoparticles is performed between more than 5 minutes and about 72 hours after administering the pharmaceutical compound. In particular embodiments, the pharmaceutical compound is a pharmaceutical antibody, such as a monoclonal antibody, a drug conjugated antibody, an engineered antibody and a multispecific antibody. According to the methods of the invention, administering the biocompatible nanoparticle comprising oligomers of albumin after administering the pharmaceutical compound maintains or increases the therapeutic benefit and reduces toxicity of the pharmaceutical compound when compared to the therapeutic benefit and toxicity induced by administering the pharmaceutical compound alone.

Claims

1. A therapeutic method comprising a step of administering at least one pharmaceutical compound to a subject having a disease or disorder selected from a cardiovascular disease, a central nervous system disease, a cancer, an infectious disease or a metabolic disorder with said at least one pharmaceutical compound and a distinct step of administering at least one biocompatible nanoparticle to said subject, wherein the at least one pharmaceutical compound comprises an IgG antibody or an antigen binding fragment thereof, a crystallizable IgG-fragment (IgG Fc), or a fusion protein; the at least one biocompatible nanoparticle consists of albumin monomers conjugated to each other to form at least one oligomer of albumin (n≥2) through direct interaction or via a linker that consists of maleimide, glutaraldehyde or ethylenediaminocarbodiimide (EDC), the longest or largest dimension of the at least one biocompatible nanoparticle is between about 4 nm and about 500 nm, the at least one biocompatible nanoparticle is distinct from the pharmaceutical compound and said at least one nanoparticle is administered to the subject between 5 minutes and about 72 hours after the pharmaceutical compound.

2. The method according to claim 1, wherein the at least one oligomer of albumin is a dimer of albumin (n=2), a trimer of albumin (n=3), or consists of at least 4 monomers of albumin (n≥4).

3. The method according to claim 1, wherein the at least one oligomer of albumin consists of less than 50 monomers of albumin.

4. The method according to claim 1, wherein the biocompatible nanoparticles consist of a mixture of albumin oligomers, wherein the mixture of albumin oligomers consists of at least two distinct oligomers selected from dimers of albumin (n=2), trimers of albumin (n=3) and albumin oligomers that are at least 4 monomers of albumin (n≥4).

5. The method according to claim 1, wherein the at least one pharmaceutical compound is a fusion protein, an IgG antibody or an antigen binding fragment thereof conjugated to a cytotoxic agent, an engineered antibody or a multispecific antibody.

6. The method according to claim 5, wherein the at least one pharmaceutical compound is selected from abatacept, abciximab, adalimumab, aflibercept, alefacept, alemtuzumab, basiliximab, belimumab, bevacizumab, brentuximab, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, eculizumab, efalizumab, etanercept, gemtuzumab ozogamicin, golimumab, infliximab, ipilimumab, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, rituximab, tocilizumab, trastuzumab and ustekinumab.

7. The method according to claim 1, whereby the administration of the at least one biocompatible nanoparticle and of the pharmaceutical compound maintains or increases the therapeutic benefit of the pharmaceutical compound and reduces toxicity, when compared to therapeutic benefit and toxicity induced by the standard therapeutic dose of said pharmaceutical compound in the absence of biocompatible nanoparticle.

8. The method according to claim 1, wherein the at least one oligomer of albumin consists of less than 25 monomers of albumin.

Description

LEGEND OF THE FIGURES

(1) FIG. 1: Structure of IgG molecules [Pharmacokinetics, Pharmacodynamics and physiologically-based pharmacokinetic modelling of monoclonal antibodies. Dostalek M, Gardner I, Gurbaxani B M, Rose R H, Chetty M. Clin Pharmacokinet (2013) 52:83-124].

(2) FIG. 2: Representation of the at least one biocompatible nanoparticle comprising, or consisting of, at least a dimer of albumin (a/b/c/−n=2) or an oligomer of albumin (d/e/f/g/h/i/j/k/l/m/n/o, n>2).

(3) FIG. 3: Gradient polyacrylamide electrophoresis gel with:

(4) (a): a suspension of mouse serum albumin (MSA), i.e., a suspension of biocompatible nanoparticles No. 1;

(5) (b): a suspension of MSA prepared according to example 1, i.e., a suspension of biocompatible nanoparticles No. 2; and

(6) (c): HiMark Protein standard 31-460 kDa.

(7) FIG. 4: Transmission electron microscopy (TEM) images of biocompatible nanoparticles comprising at least one oligomer of albumin (n≥2) or consisting of an oligomer of albumin, with mean particle size typically above 50 nm (scale bar 500 nm).

(8) FIG. 5: Schematic representation of the treatment schedule for the pharmaceutical composition comprising (i) the biocompatible nanoparticles from example 1 (i.e., the suspension of biocompatible nanoparticles No. 1 or the suspension of biocompatible nanoparticles No. 2) and (ii) the DC101 compound in FaDu xenografts.

(9) FIG. 6: Tumor re-growth delay of the pharmaceutical composition comprising the biocompatible nanoparticles from example 1 and the DC101 compound in FaDu xenografts (mean RTV±SD).

(10) FIG. 7: Gradient polyacrylamide electrophoresis gel with:

(11) (a): a suspension of mouse serum albumin (MSA), i.e., a suspension of biocompatible nanoparticles No. 1 (see Example 2);

(12) (b): a suspension of MSA prepared according to example 1, i.e., a suspension of biocompatible nanoparticles No. 2 (see Example 2);

(13) (c): a suspension of MSA prepared according to example 6, i.e., a suspension of biocompatible nanoparticles No. 3 (see Example 7); and

(14) (d): HiMark Protein standard 31-460 kDa.

EXAMPLES

Example 1

Synthesis of the at Least One Biocompatible Nanoparticle Consisting of at Least an Oligomer (n≥2) of Albumin, for Example a Dimer (n=2) of Albumin, with Mean Particle Size Typically Below 50 nm

(15) Mouse serum albumin (1 g) was dissolved in 77 mL of 100 mM phosphate-buffered saline (PBS) pH8.5. The solution was mixed with 77 mL of a freshly aqueous 2-iminothiolane (reagent) solution (3 mM). After adjusting the pH to 8.5 with sodium hydroxide solution (NaOH), the suspension was incubated at room temperature during 48 h.

(16) Subsequently, the resulting suspension was diluted in phosphate buffer and adjusted to pH 7.3. Elimination of excess reagent was performed and re-concentration of the as prepared suspension was achieved using a polyethersulfone membrane (50 kDa). Final concentration of albumin was determined using the Bradford method and found equal to about 65 g/L.

Example 2

Characterization of the at Least One Biocompatible Nanoparticle Consisting of an Oligomer of Albumin, for Example a Dimer (n=2) of Albumin in the Suspension of Example 1

(17) Presence of nanoparticles consisting of at least an oligomer of albumin was checked by electrophoresis. 7.54, of suspension of mouse serum albumin (MSA) (albumin 0.5 g/L), or of suspension from Example 1 (albumin 0.5 g/L) were mixed with 2.5 μL of a solution of Lithium Dodecyl Sulfate (LDS) sample loading buffer (4×). A non-denaturating 4-12% gradient polyacrylamide gel was cast in the XCell SureLock vertical electrophoresis and run at 200V for 1h50. Typically, the proportion of oligomer is evaluated by separation by size exclusion chromatography on a superose 6 column followed by subsequent dosage of albumin in each fraction with the Bradford method, size evaluation in each fraction by dynamic light scattering (DLS) and molecular weight evaluation on each fraction by gel electrophoresis on a non-denaturating 4-12% gradient polyacrylamide gel.

(18) The results presented in FIG. 3 show that: the MSA suspension (FIG. 3, line a) as such contains a low fraction of oligomers (n≥2) of albumin (suspension of biocompatible nanoparticles No. 1). About 10% of the collected albumin was identified as oligomers of albumin with molecular weights equal to about 120 kDa (n=2) (of note albumin monomer molecular weight is equal to about 66 kDa). The mean particle size of the biocompatible nanoparticles in suspension was measured by dynamic light scattering (DLS) using a zetasizer NanoZS (Malvern 5 Instrument) with a 633 nm HeNe laser at an angle of 173°. The mean particle size of the biocompatible nanoparticles in suspension was equal to about 9 nm with a polydispersity index (PDI) of 0.207. The suspension from Example 1 (FIG. 3, line b) contains a larger proportion of oligomers of albumin (suspension of biocompatible nanoparticles No. 2) when compared to the MSA suspension. About 50% of the collected albumin was identified as oligomers of albumin with molecular weights comprised between about 120 kDa (n=2) and about 1300 kDa (n=20) (of note albumin monomer molecular weight is equal to about 66 kDa). The mean particle size of the biocompatible nanoparticles in suspension was measured by dynamic light scattering (DLS) using a zetasizer NanoZS (Malvern Instrument) with a 633 nm HeNe laser at an angle of 173°. The mean particle size of the biocompatible nanoparticles in suspension was equal to about 30 nm with a polydispersity index (PDI) of 0.250.

(19) For the in vivo experiments described in Examples 4 and 5, both suspensions of biocompatible nanoparticles No. 1 and No. 2 were used to prepare the pharmaceutical composition comprising the combination of (i) at least one biocompatible nanoparticle, said biocompatible nanoparticle comprising at least one oligomer of albumin (n≥2) or consisting of an oligomer of albumin, and (ii) a pharmaceutical compound of interest.

Example 3

Preparation of the at Least One Biocompatible Nanoparticle, Said Biocompatible Nanoparticle Comprising at Least One Oligomer of Albumin (n≥2) (See FIG. 2m) or Consisting of an Oligomer of Albumin (See FIGS. 2a-l), with Mean Particle Size Typically Above 50 nm

(20) The albumin nanoparticles were prepared by the desolvation technique (desolvation process and surface characterization of protein nanoparticles. C. Weber, C. Coester, J. Kreuter, K. Langer, International Journal of Pharmaceutics, V 194; 2000; pp 91-102).

(21) Bovine serum albumin (BSA) (100 mg) was dissolved in 2 mL distilled water at pH 7. A desolvating agent, acetone (5 mL), was added dropwise into the BSA solution, until the solution became turbid. The solution was stirred overnight. The solvent was subsequently eliminated by evaporation. The albumin nanoparticles were observed by Transmission Electronic Microscopy using JEOL JEM 100CX II HR (see FIG. 4).

Example 4

Tumor Re-Growth Delay of the Pharmaceutical Composition Comprising the Biocompatible Nanoparticles from Example 1 and the DC101 Compound in FaDu Xenografts (Mean RTV±SD)

(22) This study was performed to investigate the efficacy of the pharmaceutical composition comprising the biocompatible nanoparticles from Example 1 and DC101 (rat anti-mouse VEGF receptor 2 monoclonal antibody that replaces Bevacizumab (Avastin®) for mice studies) as the therapeutic compound of interest, in FaDu tumor model xenografted on NMRI nude mice.

(23) The human pharyngeal carcinoma FaDu cell line was purchased from LGC Standard (Molsheim, France). Cells were cultured in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum (Gibco), with 5% CO.sub.2.

(24) NMRI nude mice, 6-7 weeks (20-25 g) were ordered from Janvier Labs (France). The mice were maintained under specific pathogen free conditions (sterilized food and water available ad libitum) and kept one week for acclimatization before starting the experiment.

(25) FaDu tumors were obtained by subcutaneous injection of 2.10.sup.6 cells in 50 μL in the lower right flank of the mouse. The tumors were grown until reaching volume around about 100 mm.sup.3. Tumor diameters were measured using digital caliper and the tumor volume in mm.sup.3 was calculated using the following formula:

(26) $\mathrm{Tumor}\mathrm{volume}\left({\mathrm{mm}}^3\right)=\frac{\mathrm{length}\left(\mathrm{mm}\right)\times {\left(\mathrm{width}\right)}^2\left({\mathrm{mm}}^2\right)}{2}$

(27) Mice were randomized into separate cages and identified by a number (paw tattoo). Seven groups were treated as illustrated in FIG. 5 and Table 3.

(28) TABLE-US-00003 TABLE 3 Schedule and dose conditions for the control groups (group 1: vehicle NaCl 0.9%; group 6: biocompatible nanoparticles No. 2) and the treatment groups (group 2: DC101 alone; group 3: injection of biocompatible nanoparticles No. 2 4 h before DC101; group 4: injection of biocompatible nanoparticles No. 2 4 h after DC101; group 5: injection of biocompatible nanoparticles No. 2 24 h after DC101; group 7: injection of biocompatible nanoparticles No. 1 4 h after DC101). bio- Biocompatible compatible nanoparticles nano- DC101 from particles Mice DC101 Adm. DC101 example 1 from number/ Adm. Volume Treatment injection example 1 Groups Treatment group route (dose/adm.) schedule schedule (dose/adm) 1 0.9% NaCl 5 IP Equivalent 1 injection at — — to Days 1-3 and 5 treatment for 3 weeks 2 DC101 alone 5 IP 800 μg/dose 1 injection at Days 1-3 and 5 for 3 weeks 3 DC101 + 5 IP 800 μg/dose 1 injection at IV injection 4 h 10 ml/kg biocompatible Days 1-3 and 5 before treatment nanoparticles for 3 weeks adm at D1-D3-D5 No. 2, 4 h before for 3 weeks 4 DC101 + 5 IP 800 μg/dose 1 injection at IV injection 4 h 10 ml/kg biocompatible Days 1-3 and 5 after treatment nanoparticles for 3 weeks adm at D1-D3-D5 No. 2, 4 h after for 3 weeks 5 DC101 + 5 IP 800 μg/dose 1 injection at IV injection 24 h 10 ml/kg biocompatible Days 1-3 and 5 after treatment nanoparticles for 3 weeks adm at D1-D3-D5 No. 2, 24 h after for 3 weeks 6 biocompatible 5 IV injection in the 10 mL/kg nanoparticles same timing as No. 2 group 4 with adm at D1-D3-D5 for 3 weeks 7 DC101 + 4 IP 800 μg/dose 1 injection at IV injection 4 h 10 ml/kg biocompatible Days 1-3 and 5 after treatment nanoparticles for 3 weeks adm at D1-D3-D5 No. 1, 4 h after for 3 weeks

(29) Group 1: Sterile NaCl 0.9% (control vehicle group).

(30) Five (5) mice were intraperitoneally (IP) injected with a sterile NaCl 0.9% solution (volume equivalent to DC101 injection) on day 1, day 3 and day 5 each week during three consecutive weeks.

(31) Group 2: DC101 at 800 μg/dose (treatment group).

(32) Five (5) mice were intraperitoneally (IP) injected with a sterile DC101 solution (800 μg/dose) on day 1, day 3 and day 5 each week during three consecutive weeks.

(33) Group 3: Pharmaceutical composition, i.e., the combination of the biocompatible nanoparticles No. 2 (65 g/L: 10 mL/kg of animal) and of DC101 (800 μg/dose) (treatment group).

(34) Five (5) mice were intraperitoneally (IP) injected with a sterile DC101 solution (800 μg/dose) on day 1, day 3 and day 5 each week during three consecutive weeks. Each time (day), the intravenous (IV) injection of sterile suspension of biocompatible nanoparticles No. 2 was performed 4 hours before the injection of DC101.

(35) Group 4: Pharmaceutical composition, i.e., the combination of the biocompatible nanoparticles No. 2 (65 g/L: 10 mL/kg of animal) and of DC101 (800 μg/dose) (treatment group).

(36) Five (5) mice were intraperitoneally (IP) injected with a sterile DC101 solution (800 μg/dose) on day 1, day 3 and day 5 each week during three consecutive weeks. Each time (day), the intravenous (IV) injection of sterile suspension of biocompatible nanoparticles No. 2 was performed 4 hours after the injection of DC101.

(37) Group 5: Pharmaceutical composition, i.e., the combination of the biocompatible nanoparticles No. 2 (65 g/L: 10 mL/kg of animal) and of DC101 (800 μg/dose) (treatment group).

(38) Five (5) mice were intraperitoneally (IP) injected with a sterile DC101 solution (800 μg/dose) on day 1, day 3 and day 5 each week during three consecutive weeks. Each time (day), the intravenous (IV) injection of sterile suspension of biocompatible nanoparticles No. 2 was performed 24 hours after the injection of DC101.

(39) Group 6: Biocompatible nanoparticles No. 2 (65 g/L: 10 mL/kg of animal) (control group).

(40) Five (5) mice were intravenously (IV) injected with a sterile suspension of biocompatible nanoparticles No. 2 (65 g/L: 10 mL/kg of animal) on day 1, day 3 and day 5 each week during three consecutive weeks.

(41) Group 7: Pharmaceutical composition, i.e., the combination of the biocompatible nanoparticles No. 1 (65 g/L: 10 mL/kg of animal) and of DC101 (800 μg/dose) (treatment group).

(42) Four (4) mice were intraperitoneally (IP) injected with a sterile DC101 solution (800 μg/dose) on day 1, day 3 and day 5 each week during three consecutive weeks. Each time (day), the intravenous (IV) injection of sterile suspension of biocompatible nanoparticles No. 1 was performed 4 hours after the injection of DC101.

(43) DC101 product (BioXcell—4.83 mg/ml at pH 7, in PBS) was diluted at 4.6 mg/mL in NaCl 0.9% before injection of 1744, to obtain a dose of 800 μg per injection.

(44) Suspension of biocompatible nanoparticles No. 1 and suspension of biocompatible nanoparticles No. 2 (Albumin content equal 65 g/L in PBS buffer) from Example 1 were injected without additional dilution at 10 mL/kg of animal.

(45) DC101 was administrated by intraperitoneal injection (IP) with a 100U (0.3 ml) insulin syringe with a 29G needle (TERUMO, France). Suspension of biocompatible nanoparticles No. 1 and suspension of biocompatible nanoparticles No. 2 from Example 1 were injected by intravenous (IV) injection via lateral tail vein with a 1 mL syringe with a 26G needle (TERUMO, France).

(46) Mice were followed up for clinical signs, body weight and tumor size.

(47) FIG. 6 shows the mean relative tumor volume (mean RTV) for all groups as obtained (in the conditions previously described) after injections of: Vehicle (sterile NaCl 0.9%) intraperitoneally injected on days 1, 3 and 5 of each week during 3 consecutive weeks (group 1); DC101 (800 μg/dose) intraperitoneally injected on days 1, 3 and 5 of each week during 3 consecutive weeks (group 2); DC101 (800 μg/dose) intraperitoneally injected on days 1, 3 and 5 of each week during 3 consecutive weeks with biocompatible nanoparticles of suspension No. 2 intravenously injected 4 hours before DC101 injection (group 3); DC101 (800 μg/dose) intraperitoneally injected on days 1, 3 and 5 of each week during 3 consecutive weeks with biocompatible nanoparticles of suspension No. 2 intravenously injected 4 hours after DC101 injection (group 4); DC101 (800 μg/dose) intraperitoneally injected on days 1, 3 and 5 of each week during 3 consecutive weeks with biocompatible nanoparticles of suspension No. 2 intravenously injected 24 hours after DC101 injection (group 5); Biocompatible nanoparticles No. 2 intravenously injected on days 1, 3 and 5 of each week during 3 consecutive weeks (group 6); and DC101 (800 μg/dose) intraperitoneally injected on days 1, 3 and 5 of each week during 3 consecutive weeks with biocompatible nanoparticles of suspension No. 1 intravenously injected 4 hours after DC101 injection (group 7).

(48) As shown in FIG. 6, a marked tumor growth inhibition is observed after 7 days of treatment for the groups including DC101 alone (group 2) or in combination with biocompatible nanoparticles of suspension No. 2 (groups 3, 4, 5) or in combination with biocompatible nanoparticle of suspension No. 1 (group 7), when compared to vehicle group (group 1) and biocompatible nanoparticles of suspension No. 2 (group 6). This marked tumor growth inhibition is similar between groups 2, 3, 4, 5 and 7.

(49) Overall, those results show that the tumor growth delay obtained by DC101 treatment is not modified when using the pharmaceutical composition of the present invention (corresponding to the combination of the biocompatible nanoparticles from Example 1 and of the DC101 (800 μg/dose)). This tumor growth delay was observed when the biocompatible nanoparticles from Example 1 and the compound of interest (the DC101) were administered sequentially.

Example 5

Toxicity Evaluation of the Pharmaceutical Composition Comprising the Biocompatible Nanoparticles from Example 1 and the DC101 Antibody in FaDu Xenografts

(50) This study was performed to investigate the impact of the combination of the biocompatible nanoparticles from Example 1 with DC101 (rat anti-mouse VEGF receptor 2 monoclonal antibody that replaces Bevacizumab (Avastin®) for mice studies) on the toxicity of the DC101 treatment on FaDu tumor model xenografted on NMRI nude mice from Example 4.

(51) Each mouse of the different groups (groups 1 to 7) of the tumor growth delay experiment (Example 4) was necropsied after mouse euthanasia when tumor volume exceeded 1000 mm.sup.3 or presented any sign of necrosis. For each mouse, the following organs were observed during the necropsy for any sign of toxicity: liver, spleen, kidneys, skin, brain, stomach, intestines, lungs and heart. Table 4 shows the observations made during the necropsy of mice for each group from Example 4.

(52) TABLE-US-00004 TABLE 4 Observations at necropsy made on the different groups of mice from Example 4. Group observations group 1: 0.9% All organs were normal NaCl (5 mice) during necropsy for 5/5 mice group 2: Compared to control group (group 1): DC101 alone kidneys were colorless (clear brown) (5 mice) for 5/5 mice a splenomegaly was present for 3/5 mice liver was colorless (clear brown) for 2/5 mice other organs (intestines, heart, skin, lungs, stomach, brain) were similar to control group group 3: DC101 + Compared to control group (group 1): biocompatible kidneys were colorless (clear brown) nanoparticles for 5/5 mice No. 2, a splenomegaly was present for 2/5 mice injection 4 h liver was colorless (clear brown) for 5/5 mice before (5 mice) other organs (intestines, heart, skin, lungs, stomach, brain) were similar to control group group 4: DC101 + Compared to control group (group 1): biocompatible a splenomegaly was present for 1/5 mice nanoparticles No. liver was little colorless for 2/5 mice 2, injection 4 h other organs (kidneys, intestines, heart, skin, after (5 mice) lungs, stomach, brain) were similar to control group group 5: DC101 + all organs were normal during biocompatible necropsy for 5/5 mice, i.e., similar nanoparticles to control group No. 2, injection 24 h after (5 mice) group 6: all organs were normal during necropsy biocompatible for 5/5 mice, i.e., similar to control nanoparticles No. 2 group (5 mice) group 7: DC101 + Compared to control group (group 1): biocompatible a splenomegaly was present for 1/4 mice nanoparticles No. 1, other organs (liver, kidneys, intestines, injection 4 h heart, skin, lungs, stomach, brain) after (4 mice) were similar to control group

(53) Group 2 (DC101 alone) presents visual signs of toxicity on the liver, spleen and kidneys. Of note, blood sampling performed during the tumor growth delay assay showed an increase of blood viscosity for all mice of group 2.

(54) Interestingly, it has been observed that VEGFR2 (vascular endothelial growth factor receptor 2) selective blockage by DC101 induces an increase of erythropoietin (EPO) expression by liver (Nature Medicine vol. 12, N 7, July 2006, pp 793-800). Increase of EPO production leads to an increase of the production of red blood cells and subsequently to an increase of hematocrit. The increase of number of red blood cells as well as hematocrit increase have been correlated with VEGFR2 inhibition by DC101 (Nature Medicine vol. 12, N 7, July 2006, pp 793-800).

(55) Hematocrit increase with an increase of EPO production is correlated with a secondary erythrocytosis (Nature Medicine vol. 12, N 7, July 2006, pp 793-800). It is established that cases of erythrocytosis, due to the increased red blood cell number, present most of time a splenomegaly (Clin. Lab. Haem. Vol. 21, pp 309-316, 1999). Another cause of secondary erythrocytosis can be renal lesions such as cysts (Clin. Lab. Haem. Vol. 21, pp 309-316, 1999). It has been shown that inhibition of VEGFR2 by DC101 leads to renal cyst formation in mice (Kidney Inter. Vol. 69, pp 1741-1748, 2006) in addition to other renal failure such as proteinuria occurring with anti-angiogenic antibodies (Jpn J. Clin. Oncol. Vol. 43, No. 6, pp 587-595, 2013).

(56) Based on available DC101 literature, observations made during necropsy on group 2 (DC101 alone) can be related to the off-target toxicity of the anti-angiogenic monoclonal antibody DC101. This conclusion is supported by control vehicle group (group 1) and group 6 (biocompatible nanoparticles No. 2) for which no signs of toxicity were observed during necropsy.

(57) Surprisingly inventors observed a marked decrease of toxicity (as assessed by clinical observation of the organs during necropsy of animal) in animals of groups 4 and 7. Even more surprising, no toxicity is observed in all animal of group 5.

(58) Conclusion: The combination of DC101 with biocompatible nanoparticles from Example 1, intravenously injected 4 hours after DC101 injection, is able to preserve DC101 anti-tumor efficacy with a marked decrease of toxicity of DC101 treatment. Of utmost interest, the combination of DC101 with biocompatible nanoparticles from Example 1, intravenously injected 24 hours after DC101 injection, is able to preserve DC101 anti-tumor efficacy and to completely abolish the toxicity of DC101 treatment (when evaluated through visual observation of the main organs of the animals at the time of necropsy).

(59) Therefore the biocompatible nanoparticle of the invention, comprising at least one oligomer of albumin (n≥2) or consisting of an oligomer of albumin, can efficiently reduce unwanted normal tissue toxicities of antibodies (such as DC101), when said at least one oligomer is administered after the antibody in a subject in need of said antibody compound.

Example 6

Preparation of the at Least One Biocompatible Nanoparticle, Said Biocompatible Nanoparticle Comprising at Least One Oligomer of Albumin (n≥2) (See FIG. 2m), with Mean Particle Size Typically of about 50 nm

(60) Mouse Serum Albumin (0.8 g) was dissolved in 10 mL of 100 mM phosphate-buffered saline (PBS) pH 7.4. The solution was mixed with 2.4 mL of a freshly aqueous 4arm-Poly (Ethylene glycol)-Maleimide 11 kDa solution (5 mM). After adjusting the pH to 8.2, the sample was incubated at room temperature during 24 h. Subsequently, the resulting suspension was conserved at pH 9. Final concentration of albumin was determined using the BCA Assay and found equal to about 64 g/L.

Example 7

Characterization of the at Least One Biocompatible Nanoparticle Consisting of an Oligomer of Albumin (n≥2) in the Suspension of Example 6

(61) Presence of nanoparticles consisting of at least one oligomer of albumin was checked by electrophoresis. 7.5 μL of suspension of mouse serum albumin (MSA) (albumin 0.5 g/L), of suspension from Example 1 (albumin 0.5 g/L), and of suspension from Example 6 were mixed with 2.5 μL of a solution of Lithium Dodecyl Sulfate (LDS) sample loading buffer (4×). A non-denaturating 4-12% gradient polyacrylamide gel was cast in the XCell SureLock vertical electrophoresis and run at 200V for 1h50.

(62) Typically, the proportion of oligomer is evaluated by separation by size exclusion chromatography on a superose 6 column followed by subsequent dosage of albumin in each fraction with the Bradford method, size evaluation in each fraction by dynamic light scattering (DLS) and molecular weight evaluation on each fraction by gel electrophoresis on a non-denaturating 4-12% gradient polyacrylamide gel.

(63) The results presented in FIG. 7 show that:

(64) The MSA suspension (FIG. 7, line a) as such contains a low fraction of oligomers (n≥2) of albumin (suspension of biocompatible nanoparticles No. 1). About 10% of the collected albumin was identified as oligomers of albumin with molecular weights comprised equal to about 120 kDa (n=2) (of note albumin monomer molecular weight is equal to about 66 kDa). The mean particle size of the biocompatible nanoparticles in suspension was measured by dynamic light scattering (DLS) using a zetasizer NanoZS (Malvern 5 Instrument) with a 633 nm HeNe laser at an angle of 173°. The mean particle size of the biocompatible nanoparticles in suspension was equal to about 9 nm with a polydispersity index (PDI) of 0.207.

(65) The suspension from Example 1 (FIG. 7, line b) contains a larger proportion of oligomers of albumin (suspension of biocompatible nanoparticles No. 2) when compared to the MSA suspension. About 50% of the collected albumin was identified as oligomers of albumin with molecular weights comprised between about 120 kDa (n=2) and about 1300 kDa (n=20) (of note albumin monomer molecular weight is equal to about 66 kDa). The mean particle size of the biocompatible nanoparticles in suspension was measured by dynamic light scattering (DLS) using a zetasizer NanoZS (Malvern Instrument) with a 633 nm HeNe laser at an angle of 173°. The mean particle size of the biocompatible nanoparticles in suspension was equal to about 30 nm with a polydispersity index (PDI) of 0.250.

(66) The suspension from Example 6 (FIG. 7, line c) contains a larger proportion of oligomers of albumin (suspension of biocompatible nanoparticles No. 3) when compared to the MSA suspension and the suspension of biocompatible nanoparticles No. 2. About 70% of the collected albumin was identified as oligomers of albumin with molecular weights comprised between about 120 kDa (n=2) and about 1300 kDa (n=20) (of note albumin monomer molecular weight is equal to about 66 kDa). The mean particle size of the biocompatible nanoparticles in suspension was measured by dynamic light scattering (DLS) using a zetasizer NanoZS (Malvern Instrument) with a 633 nm HeNe laser at an angle of 173°. The mean particle size of the biocompatible nanoparticles in suspension was equal to about 50 nm with a polydispersity index (PDI) of 0.475.