Pharmaceutical formulation comprising a protein and functionalized chitosan for subcutaneous administration

Abstract

The present invention relates to pharmaceutical formulations comprising a therapeutically effective amount of a protein, and functionalized chitosan with at least one functionalizing moiety comprising at least one acidic function. The formulations of the invention are suitable for forming hydrogels in physiological conditions, and they are thus suitable for delivering proteins. The invention further relates to pharmaceutical formulations of the invention for use as a medicament, preferably for use in the treatment of a cancer.

Description

[0001] PHARMACEUTICAL FORMULATION COMPRISING A PROTEIN AND

[0002] FUNCTIONALIZED CHITOSAN FOR SUBCUTANEOUS ADMINISTRATION

[0003] TECHNICAL FIELD

[0004] The invention relates to the administration of therapeutic proteins, such as antibodies. Especially, it relates to new chitosan formulations that are suitable for forming hydrogels in vivo that are suitable for delivering therapeutic proteins.

[0005] TECHNICAL BACKGROUND

[0006] The pharmaceutical use of therapeutic proteins such as antibodies has increased over the past years. Such therapeutic proteins are mostly administered via the intravenous (IV) route. Alternative administration pathways include subcutaneous injection. The advantage of subcutaneous injection is that it allows the medical practitioner to perform it in a rather short intervention with the patient. Moreover, the patient can be trained to perform the subcutaneous injection by himself. Such selfadministration is particularly useful during maintenance dosing because no hospital care is needed and could have a positive impact on the patient’s quality of life.

[0007] Chitosan-based hydrogels have been demonstrated to be suitable for administration of proteins. Chitosan is a linear polysaccharide composed of randomly distributed P-(1^4)-linked D-glucosamine (deacetylated unit) and N-acetyl -D-glucosamine (acetylated unit).

[0008] WO2023148351 and WO2023057712 disclose chitosan-based gels for the sustained delivery of proteins, which comprise a mixture of two different polysaccharides. Polysaccharide B comprises chelating groups. Said chelating groups are useful for chelating metals, especially pathogen metals when the aim is to maintain homeostasis. The chelating groups further confer a strong hydrophilic behavior to the formulation, which implies good swelling properties.

[0009] The presence of such chelating groups is undeniably an asset in certain patients, for instance those having Wilson disease, and those subject to hemochromatosis. However, chelating and / or sequestering oligo-metals such as copper, iron and / or zinc may trigger deregulations and undesirable effects for other patients, especially for oncology patients. Cancerous patients’ body is generally weakened by heavy and complex treatments, and deregulating their local metal homeostasis should be avoided.

[0010] In addition, the simultaneous presence of two different polysaccharides in the formulations disclosed in WO2023148351 and WO2023057712 allows initiating gel formation in physiological conditions, and also affords the possibility to adjust the rheological properties of the gel by adjusting the ratio between both polysaccharides. However, the need for mixing two polysaccharides complexifies the preparation process and creates the risk of defects in the final formulation, such as insufficient mixing and / or partial inhomogeneity, that could also impact the long-term stability of the formulation. A heterogeneous mixing may for instance locally create in the gel small clusters of chitosan A, that may trigger undesirable inflammatory reactions and / or induce the formation of low resorbability inclusions.

[0011] Thus, there remains a need to provide pharmaceutical polysaccharides formulations that are suitable for forming gels in vivo, that are suitable for sustained delivery of active agents, such as proteins, and that would have no deleterious effect, preferably no effect, on local metal homeostasis in patients. Advantageously, the formulations should comprise a single type of polysaccharide in order to simplify their production and limit the risk of defects.

[0012] SUMMARY OF THE INVENTION

[0013] In this respect, the inventors have evidenced that chitosan-based formulations with satisfying gelling properties could be obtained by using a specific functionalized chitosan in absence of any other polysaccharide. The obtained gels allow sustained delivery of proteins, and have no effect on local metal homeostasis as they do not comprise chelating groups.

[0014] Thus, the present invention first relates to a pharmaceutical formulation comprising:

[0015] - a therapeutically effective amount of at least one protein, and

[0016] - functionalized chitosan, wherein at least part of the primary amine groups of the glucosamine units of chitosan are functionalized with a functionalizing moiety comprising at least one acidic function, wherein the functionalized chitosan represents more than 90 wt% of the total weight of polysaccharides in the formulation.

[0017] In some embodiments, at least one of the acidic functions has a pKa comprised between 4.5 and 8, and / or the basic form of at least one of the acidic functions is negatively charged.

[0018] In some embodiments, the functionalizing moiety comprises at least two acidic functions.

[0019] In some embodiments, the functionalizing moiety comprises at least one unsaturated bond, such as at least one double bond or triple bond, at least one hydrophilic moiety different from the acidic functions, such as an acetyl function and / or a hydroxylic function, and / or at least one ester function.

[0020] In some embodiments, the functionalizing moiety is linked to the primary amine groups of the glucosamine units of chitosan via an amide bond.

[0021] In some embodiments, the functionalizing moiety is obtained by coupling the primary amine groups of the glucosamine units of chitosan with an anhydride, said anhydride being preferably selected from the group consisting of cis -aconitic anhydride, (+)-diacetyl-L-tartaric anhydride, 1,2,4-benzenetricarboxylic anhydride, pyromellitic dianhydride and 3,3’,4,4’-biphenyltetracarboxylic dianhydride. In some embodiments, the protein is selected from the group consisting of an antibody or a fragment thereof, an antibody-drug conjugate, an enzyme, a peptide, a fusion protein and any mixture thereof, preferably an antibody-drug conjugate, an antibody or a fragment thereof, more preferably Trastuzumab or trastuzumab-emtansine.

[0022] In some embodiments, the protein is an antibody-drug conjugate, and the functionalizing moiety preferably comprises at least one ester function and / or one acetyl function.

[0023] In some embodiments, the protein concentration in the formulation is comprised between 10 and 200 g / L, preferably between 50 and 150 g / L.

[0024] In some embodiments, the average molar mass (molecular weight) in weight of the functionalized chitosan is comprised between 100 and 1000 kDa.

[0025] In some embodiments, the acetylation rate of the functionalized chitosan is 20% or lower, preferably 10% or lower.

[0026] In some embodiments, from 5 to 40%, preferably from 10 to 30%, of the primary amine groups of the glucosamine units of chitosan are functionalized with the functionalizing moiety.

[0027] In some embodiments, the pH of the pharmaceutical formulation is comprised between 4.5 and 7.5, preferably between 5 and 5.5.

[0028] In some embodiments, the functionalized chitosan concentration in the pharmaceutical formulation is comprised between 10 and 100 g / L, preferably between 20 and 50 g / L, more preferably between 20 and 30 g / L.

[0029] Another object of the invention is a process for the preparation of a pharmaceutical formulation according to the invention, comprising the steps of: a) providing a solution containing a functionalized chitosan as defined above; b) providing a solution containing a protein or a combination of protein ; c) mixing the solution of step a) and the solution of step b) to obtain a homogenous solution; d) optionally, adjusting the osmolarity of the solution obtained in step c) to provide an osmolarity comprised between 50 and 300 mOsm / L, preferably between 50 and 250 mOsm / L; e) optionally, adjusting the pH of the solution obtained in step c) between 5.0 and 6.5, preferably between 5.0 and 6.4, more preferably between 5.5 and 6.0.

[0030] Another object of the present invention is a kit comprising:

[0031] - at least one first container containing a functionalized chitosan, and

[0032] - at least a second container comprising a protein, wherein the functionalized chitosan and the protein are as defined above.

[0033] Another object of the invention is a hydrogel comprising a formulation according to the invention and water.

[0034] In some embodiments, the hydrogel is injectable.

[0035] Another object of the invention is a pharmaceutical formulation according to the invention; a kit according to the invention or a hydrogel according to the invention, for use as a medicament in a patient, preferably a patient having a cancer and / or an autoimmune disease.

[0036] Another object of the invention is a pharmaceutical formulation according to the invention; a kit according to the invention or a hydrogel according to the invention, for use in the treatment of a cancer.

[0037] FIGURES

[0038] Figure 1 presents the Fourier transform infrared (FTIR) spectrum of raw chitosan and chitosan polymer modified with cis-aconitic anhydride using a ratio of 0.7 equivalent of cis-aconitic anhydride per N- Glucosamine moiety of chitosan.

[0039] Figure 2 presents the zeta potential titration of chitosan polymer modified with cis-aconitic anhydride using a ratio of 0. 18 equivalent of cis-aconitic anhydride per N-Glucosamine moiety of chitosan.

[0040] Figure 3 presents the FTIR spectrum of raw chitosan and chitosan polymer modified with (+)-diacetyl- L-tartaric anhydride using a ratio of 0.35 equivalent of (+)-diacetyl-L-tartaric anhydride per N- Glucosamine moiety of chitosan.

[0041] Figure 4 presents the zeta potential titration of chitosan polymer modified with (+)-diacetyl-L-tartaric anhydride using a ratio of 0.35 equivalent of (+)-diacetyl-L-tartaric anhydride per N-Glucosamine moiety of chitosan.

[0042] Figure 5 presents the 'H nuclear magnetic resonance (NMR) spectrum of chitosan polymer modified with (+)-diacetyl-L-tartaric anhydride using a ratio of 0.35 equivalent of (+)-diacetyl-L-tartaric anhydride per N-Glucosamine moiety of chitosan.

[0043] Figure 6 presents the solubility screening at various pH of chitosan polymer modified with (+)-diacetyl- L-tartaric anhydride using a ratio of 0.35 equivalent of (+)-diacetyl-L-tartaric anhydride per N- Glucosamine moiety of chitosan. From left to right: pH = 3.39; 4.39; 5.15; 6.38; 6.81; 7.18; 8.11; 9.79; 11.76.

[0044] Figure 7 presents the shear measurements (G’ and G”) in sweep frequency mode for formulations A, B and C described in example 3. Figure 8 presents the solubility screening at various pH of chitosan polymer modified with cis-aconitic anhydride using a ratio of 0.18 equivalent of cis-aconitic anhydride per N-Glucosamine moiety of chitosan. From left to right: pH = 3.55; 4.19; 5.51; 6.11; 6.70; 7.08; 7.43; 8.22; 9.49; 11.56.

[0045] Figure 9 presents the shear measurements (G’ and G”) for formulations D, E and F described in example 5.

[0046] Figure 10 presents the shear measurements (G’ and G’ ’) for formulations G and H described in example 6 after gelation (15 min in phosphate buffer).

[0047] Figure 11 present the mice body weight change (as percentage of initial weight) after subcutaneous injection of 100 pL of Al-Ctar gel loaded with 100 mg / mL of trastuzumab (n=15).

[0048] Figure 12 presents the pharmacokinetic profile of trastuzumab administered in mice by IV or by SC injection in co-formulation with Al-Ctar or enzymatic formulation (rHuPH20).

[0049] Figure 13 presents the dynamic oscillatory frequency sweep (left) and strain amplitude sweep measurements (right) of T-DM1 formulation for determination of storage (G’) and loss (G”) modulus.

[0050] Figure 14 presents the rotational shear rate measurements of three Al-Ctar formulations for determination of flowability.

[0051] Figure 15 presents the self-healing properties of Al-Ctar hydrogels formulated with trastuzumab (A) or T-DM1 (B) during dynamic oscillatory time sweep measurements with alternating intervals of low strain (ylow = 1 % and co = 10 rad s ' ) and high strain (yhigh =1000 % and co = 10 rad s ').

[0052] Figure 16 presents the mice body weight change (as percentage of initial weight) after subcutaneous injection of Al-Ctar gel loaded with 50 mg / mL of T-DM1 compared to subcutaneous or intravenous injection of T-DM1 alone.

[0053] Figure 17 presents the H&E staining of Hyposkin models (human explant biopsies) 6 days post SC injection with PBS (control), T-DM1, Al-Ctar or Al-Ctar loaded with T-DM1.

[0054] Figure 18 presents the pharmacokinetic profile of T-DM1 administered in mice by IV or by SC injection in co-formulation with Al-Ctar co-formulation.

[0055] Figure 19 presents the mice body weight change (+ / - SEM) after randomization and multi-dose trastuzumab treatment in a BT-474 tumor model. Arrows indicate treatment administration.

[0056] Figure 20 presents the mean tumor size fold change (+ / - SEM) after randomization and multi-dose trastuzumab treatment in a BT-474 tumor model. Arrows indicate treatment administration.

[0057] Figure 21 presents the mice body weight change (+ / - SEM) after randomization and single dose T-DM1 treatment in a BT-474 tumor model. Arrows indicate treatment administration. Figure 22 presents the mean tumor size fold change (+ / - SEM) after randomization and single dose T- DM1 treatment in a BT-474 tumor model. Arrows indicate treatment administration.

[0058] Figure 23 presents the dynamic oscillatory frequency sweep (left) and strain amplitude sweep measurements (right) of T-Dxd formulation for determination of storage (G’) and loss (G”) modulus.

[0059] Figure 24 presents the rotational shear rate measurements of T-Dxd formulation for determination of flowability and injectability.

[0060] Figure 25 presents the mice body weight change (as percentage of initial weight) after subcutaneous injection of Al-Ctar gel loaded with 50 mg / mL of T-Dxd.

[0061] Figure 26 presents the MRI longitudinal images in mice (T2w axial) over 1 month after subcutaneous injection of Al-Ctar gel loaded with 50 mg / mL of T-DM1.

[0062] Figure 27 presents the hematoxylin and eosin (H&E) staining of tissue sample at the injection site 21 days after the subcutaneous injection in mice of Al-Ctar gel loaded with 50 mg / mL of T-DM1.

[0063] Figure 28 presents (A) the rotational shear rate measurements of Al-Ctar / POl formulations for determination of flowability and (B) the injection force required to inject the formulations through a 23G needle at a speed of 1 mm.s1.

[0064] Figure 29 represents the in vitro release of peptide P01 overtime in a physiological buffer.

[0065] DETAILED DESCRIPTION OF THE INVENTION

[0066] A first object of the invention is a pharmaceutical formulation, comprising:

[0067] - a therapeutically effective amount of at least one protein, and functionalized chitosan, wherein at least part of the primary amine groups of the glucosamine units of chitosan are functionalized with a functionalizing moiety comprising at least one acidic function.

[0068] The functionalized chitosan preferably represents more than 90 wt%, preferably at least 95 wt%, at least 96 wt%, at least 97 wt%, at least 98 wt%, at least 99 wt%, at least 99.5 wt% of the total weight of polysaccharides in the formulation.

[0069] The functionalized chitosan is preferably the only polysaccharide present in the formulation.

[0070] When the functionalized chitosan is not the only polysaccharide in the formulation, the other polysaccharide may be for instance non-fimctionalized chitosan or another biocompatible polysaccharide, glucosaminoglycan, hyaluronic acid, keratan sulfate, condroitin sulfate, or any mixture thereof. In some embodiments, the functionalized chitosan represents more than 95wt%, preferably about 100wt% of the chitosan species, ie of the total of non-functionalized chitosan and of functionalized chitosan. In other words, in some embodiments, the pharmaceutical formulation comprises less than 5wt%, preferably about 0 wt%; of non-functionalized chitosan relative to the total of the chitosan species.

[0071] Definitions

[0072] A ''pharmaceutical formulation" refers to a formulation comprising an active ingredient in association with a pharmaceutically acceptable vehicle or excipient. A pharmaceutical formulation is for therapeutic use, and relates to health. By "pharmaceutically acceptable", it is meant that the ingredients of the pharmaceutical formulation are compatible with each other and not deleterious to the subject to which it is administered.

[0073] A "primary amine" group refers to a group of formula NH2.

[0074] A "therapeutically effective amount" is the least concentration required to effect a measurable improvement or prevention of any symptom or a particular condition or disorder, to effect a measurable enhancement of life expectancy, or to generally improve patient quality of life. The therapeutically effective amount is dependent upon the specific biologically active molecule and the specific condition or disorder to be treated.

[0075] A “protein” is a polymer of natural or non-natural amino acid residues linked together by peptide bonds. Proteins include naturally occurring proteins and recombinant proteins. The protein can be functionally linked (e.g., by chemical coupling, noncovalent association or otherwise) to one or more other molecules such as small molecules, polymers (e.g. polyethylene glycol) or other proteins. The term "protein" may refer to a single molecule or to a multimolecular complex such as a dimer, a trimer or a tetramer. Proteins include single chain or multichain proteins such as antibodies and / or protein scaffolds. Disulfide linkages are commonly found in multichain proteins. The term protein may also apply to amino acid polymers in which one or more amino acids residues are an artificial chemical analogue or a corresponding naturally amino acid. Proteins can be naturally occurring or non-naturally occurring, synthetic, or semisynthetic. The protein or combination of proteins may in particular be derived from the human plasma. “Peptides” are proteins comprising less than 50 amino acids linked together by peptide bonds.

[0076] The term "antibody" refers to immunoglobulin molecules, i. e. , molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term “antibody” encompasses both whole antibody molecules and antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments.

[0077] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population bind the same epitope, except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are uncontaminated by other immunoglobulins. The term "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0078] "Chimeric" monoclonal antibodies are antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as long as they exhibit the desired biological activity. Chimeric antibodies of interest include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. baboon, rhesus or cynomolgus monkey) and human constant region sequences. An example of a chimeric antibody is rituximab.

[0079] "Humanized" forms of non-human (e.g. murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence, except for FR substitution(s) as noted above. The humanized antibody may optionally comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. An example of a humanized antibody is trastuzumab.

[0080] A "full length antibody" is an antibody which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CHI , CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the full length antibody has one or more effector functions. Rituximab, and trastuzumab are examples of full-length antibodies. An "antibody fragment" comprises a portion of a full-length antibody, including the antigen binding and / or the variable region of the full-length antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single-domain antibodies. F(ab')2, Fab, Fab' and Fv are antigen-binding fragments that can be generated from the variable region of IgG and IgM. F(ab')2 fragments contain two antigen-binding regions joined at the hinge through disulfides. F(ab')2 fragments are generally void of most, but not all, of the Fc region. Fab' fragments can be formed by the reduction of F(ab')2 fragments. Fab' fragments contain a free sulfhydryl group that may be alkylated or utilized in conjugation with an enzyme, toxin or other protein of interest. Fab' fragments are derived from F(ab')2; therefore, they may contain a small portion of Fc. Fab is a monovalent fragment that is produced from IgG and IgM, consisting of the VH, CHI and VL, CL regions, linked by an intramolecular disulfide bond. Fv fragments refers to the smallest fragments produced from IgG and IgM that contains a complete antigenbinding site. Fv fragments have the same binding properties and similar three-dimensional binding characteristics as Fab. The VH and VL chains of the Fv fragments are held together by non-covalent interactions. Fv fragments include single chain variable fragments (scFv). A “ Single -domain antibody". also known as “nanobody”, is a monomeric antigen-binding fragment of an antibody which provides various advantages over other antibody fragments as “building blocks” for bsAbs. They occur in nature as the antigen-binding portion of heavy chain antibodies in camelid species (called VHH) and cartilaginous fish (called VNAR) or can be generated from conventional IgGs by obtaining or engineering monomeric, stable VH or VL domains.

[0081] Protein scaffolds are small monomeric proteins with stable tertiary structures and mutable residues. Examples of protein scaffolds include affibodies, adnectins, anticalin proteins, DARPins, and knottins.

[0082] An "acidic function” is the function of a chemical group that is capable of giving protons.

[0083] Protein

[0084] The pharmaceutical formulation comprises a therapeutically effective amount of a protein or of a combination of proteins.

[0085] The content of protein or of combination of proteins in the pharmaceutical formulation is typically comprised between 1 g / L and 1000 g / L, preferably between 1 g / L and 500 g / L, more preferably between 10 g / L and 200 g / L, for example between 50 and 150 g / 1.

[0086] In the present invention, unless otherwise specified, values that are expressed in g / L are equivalent to values expressed in g / kg., as well known in the art.

[0087] The protein molecular weight may be determined using standard methods known to one skilled in the art, including, but not limited to, mass spectrometry (e.g., ESI, MALDI, SDS page technique) or calculation from known amino acid sequences and glycosylation. The protein may have a molecular weight comprised between 1 kDa and 500 kDa, preferably between 10 kDa and 500 kDa, preferably between 20 kDa and 300 kDa, preferably between 40 kDa and 250 kDa preferably between 100 kDa and 250 kDa, preferably between 120 kDa and 250 kDa, preferably between 150 kDa and 250 kDa.

[0088] In specific preferred embodiments, the protein is selected from the group consisting of an antibody, an enzyme, a peptide, a fusion protein, such as an antibody-cytokine fusion protein, and a combination thereof.

[0089] In specific preferred embodiments, the protein is selected from the group consisting of an antibody, an enzyme, a fusion protein, and a combination thereof.

[0090] In some embodiments, the protein is an antibody.

[0091] In some embodiments, the protein is an antibody-drug conjugate.

[0092] The pharmaceutical formulation of the disclosure is preferably devoid of recombinant human hyaluronidase PH20 (rHuPH20), preferably is devoid of enzyme able to degrade hyaluronan.

[0093] Antibody

[0094] The protein or combination of proteins is preferably an antibody or a combination of antibodies.

[0095] The combination of antibodies may be derived from the human plasma. In an embodiment, the antibodies are derived from the plasma of unselected blood donors. Such antibodies are known as human normal (i.e. nonspecific) immunoglobulins usually abbreviated HNI or HNIg. Pharmaceutical formulations comprising such antibodies may be used to provide antibodies to patient having primary immunodeficiency disorders (PIDs), hypogammaglobulinemia, primary immune thrombocytopenia, Guillain Barre syndrome, Kawasaki disease, multifocal motor neuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, or secondary (i.e. acquired) immunodeficiency disorders. In another embodiment, the antibodies are derived from the plasma of selected blood donors. Such antibodies are known as “hyperimmune immunoglobulins”. They are prepared in a similar way as for normal human immunoglobulins, except that the blood donors have high titers of antibodies against a specific organism or antigen in their plasma. Some agents against which hyperimmune globulins may be used include hepatitis B, rabies, tetanus toxin, and varicellazoster.

[0096] The antibody may be a monoclonal or a polyclonal antibody, preferably a monoclonal antibody. Specific examples of monoclonal antibodies herein include chimeric antibodies, humanized antibodies, and human antibodies.

[0097] Exemplary antibodies which can be formulated according to the present invention include, but are not limited to Abatacept, Adalimumab, Alemtuzumab, Alirocumab, Amivantamab, Anifrolumab, Atezolizumab, Avelumab, Balstilimab, Basiliximab, Belatacept, Belimumab, Benralizumab, Besilesomab, Bevacizumab, Bezlotoxumab, Bimekizumab, Blinatumomab, Brodalumab, Burosumab, Canakinumab, Carotuxomab, Cemiplimab, Cetuximab, Concizumab, Crizanlizumab, Daratumumab, Denosumab, Dinutuximab Beta, Dostarlimab, Dupilumab, Durvalumab, Eculizumab, Elotuzumab, Epratuzumab, Erenumab, Evolocumab, Fab Ig Antidigitalique Ovin, Fremanezumab, Galcanezumab, Golimumab, Guselkumab, Ibalizumab, Ig Anti Human Lymphocyte (Lapine), Ig Anti Humane Thymocyte (Equine), Ig Anti Human Thymocyte (Lapine), Infliximab, Iph 4102, Ipilimumab, Isatuximab, Ixekizumab, Lacutamab, Lag525, Lanadelumab, Mcla-128, Mepolizumab, Natalizumab, Naxitamab, Nimotuzumab, Nivolumab, Obinutuzumab, Ocrelizumab, Ofatumumab, Omalizumab, Palivizumab, Panitumumab, Pembrolizumab, Pertuzumab, Ramucirumab, Ranibizumab, Reslizumab, Risankizumab, Rituximab, Sarilumab, Secukinumab, Seribantumab, Siltuximab, Spartalizumab, Tafasitamab, Teprotumumab, Tildrakizumab, Tocilizumab, Tralokinumab, Trastuzumab, Urelumab, Ustekinumab, Vedolizumab, Zalifrelimab, and combinations thereof. Exemplary combination of antibodies which can be formulated according to the present disclosure include, but are not limited to Casirivimab and Imdevimab, a combination of Pertuzumab and Trastuzumab or a combination of Tixagevimab and Cilgavimab.

[0098] The antibody can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecules to increase half-life or stability or otherwise improve the antibody.

[0099] For example, the antibody may be linked to one of a variety of non-proteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The antibody may be functionally linked to polyethylene glycol, for example the antibody is Certolizumab Pegol. The antibody can be functionally linked to chelating agents such as DOTA (1 ,4,7,10-tetraazacyclododecane- N,N’,N”,N”’ -tetracetic acid), NOTA (1 ,4,7- triazacyclononane-1 ,4,7-triacetic acid), NODAGA (1 , 4, 7-triazacyclononane-l -glutaric acid-4, 7- diacetic acid, DOTAGA (2-(4,7,10- tris(carboxymethyl)-l ,4,7,10-tetraazacyclododecan-l - yl)pentanedioic acid), DOTAM (1 ,4,7,10-tetrakis(carbamoylmethyl)-l ,4,7,10 tetraazacyclododecane), NOTAM (1 ,4,7- tetrakis(carbamoyhnethyl)-l ,4,7-triazacyclononane), DOTP (1 ,4,7,10- tetraazacyclododecane 1 ,4,7,10-tetrakis(methylene phosphonate), NOTP (1 ,4,7-tetrakis(methylene phosphonate)- 1 ,4,7-triazacyclononane), TETA (1 ,4,8,11 -tetraazacyclotetradecane-N,N’,N”,N”’- tetraacetic acid), TETAM (1 ,4,8,11 -tetraazacyclotetradecane-N,N’,N”,N”’-tetrakis(carbamoyl methyl), DTPA (diethylene triaminopentaacetic acid), DFO (deferoxamine), Bz-DFO, bispitine ([3,7]- diazabicyclo[3.3.1]nonane), preferably it is functionally linked to a chelating agent selected from DOTAGA, DFO, Bz-DFO, DOTAM, DTPA and bispitine,

[0100] The antibody may be functionally linked to one or more antibodies or antibody fragments, to generate a bispecific or a multi-specific molecule. Exemplary bispecific antibodies which can be formulated according to the present disclosure include, but are not limited to Glofitamab, KN046, IB 13 18. IB 13 18. Emicizumab, Epcoritamab, Tebotelimab, Tebentafusp, Teclistamab, Faricimab, Amivantamab, Mosunetuzumab, Zanidatamab, Flotetuzumab, APVO436, Zenocutuzumab, TNB383B, and combinations thereof. In an embodiment, the bispecific antibody that is formulated is Epcoritamab.

[0101] In an embodiment, the protein or combination of proteins is an antibody or combination of antibodies which binds an antigen selected from the group of HER2 and CD20. CD20 antibodies may be used for therapy of B cell malignancies (such as non-Hodgkin's lymphoma or chronic lymphocytic leukemia) or autoimmune diseases (such as rheumatoid arthritis and vasculitis); HER2 antibodies may be used for cancer therapy (such as breast cancer or gastric cancer). The phrases antibody which binds an antigen". "an antibody recognizing an antigen" and "an antibody having specificity for an antigen" have the same meaning and will be used equally herein.

[0102] In one embodiment, the antibody which is formulated binds HERZ. In one preferred embodiment, the antibody which is formulated is trastuzumab. In one preferred embodiment, the antibody which is formulated is pertuzumab.

[0103] In one embodiment, the combination of antibodies which is formulated is a combination of antibodies which bind two distinct epitopes of HERZ. In one preferred embodiment, it includes a combination of trastuzumab and pertuzumab.

[0104] In one embodiment, the antibody which is formulated binds CD20. In one preferred embodiment, the antibody which is formulated is rituximab.

[0105] In one embodiment, the antibody which is formulated binds CD38. In one preferred embodiment, the antibody which is formulated is daratumumab.

[0106] Antibody-drug conjugates

[0107] In some embodiments, the protein is an antibody-drug conjugate. An “ antib ody-drug conjugate" (or ADC) is a molecule conjugating a drug, such as an anticancer agent, with an antibody targeting specific antigens.

[0108] Examples of ADC include but are not limited to Gemtuzumab ozogamicin, Brentuzimab vedotin, Ado- Trastuzumab emtansine, Inotuzumab ozagomicin, Polatuzumab vedotin, Enfortumab vedotin, Fam- Trastuzumab deruxtecan, Sacituzumab govitecan, Belantamab mafodotin, Loncastuximab Tesirine, Tisotumab vedotin, and Mirvetuximab soravtansine-gynx.

[0109] The anticancer agent which in functionally linked to the antibody may be a cytotoxic agent such as calicheamicin, monomethyl auristatin F (MMAF also known as mafodotin), monoethyl auristatin E (MMAE also known as vedotin), Emtamsine (DM1), Exatecan derivative (Dxd) or N-acetyl -gamma calicheamicin. Exemplary antibody-drug conjugates (ADCs) which can be formulated according to the present disclosure include, but are not limited to, Belantamab Mafodotine, Brentuximab Vedotin, Depatuxizumab Mafodotine, Enfortumab Vedotin, Gemtuzumab Ozogamicine, Inotuzumab Ozogamicin, Moxetumomab Pasudotox, Sacituzumab Govitecan, Trastuzumab Deruxtecan, Trastuzumab Emtansine, preferably Trastuzumab emtansine.

[0110] In some embodiments, the ADC is an ADC with a non-cleavable linker, such as T-DM1. In other embodiments, the ADC is an ADC with a cleavable linker, such as T-Dxd.

[0111] In some embodiments, the ADC is an antibody-oligonucleotide conjugate (AOC), a radioimmunoconjugate or an antibody polymer conjugate (APC).

[0112] The ADC may comprise either a single-drug payload, or a multi -drug payload, such as a dual payload.

[0113] In some embodiments, the ADC is a multi-specific ADC, preferably a bi, tri or quadri-specific ADC. A multi-specific ADC can target several distinct antigens or epitopes, enhancing the therapeutic index of ADC by increasing selectivity and reducing off-tumor toxicity.

[0114] Functionalized chitosan

[0115] In the present invention, the functionalized chitosan is a chitosan wherein at least part of the primary amine groups of the glucosamine units are functionalized with a functionalizing moiety (Fm) comprising at least one acidic function.

[0116] In some embodiments, the functionalized chitosan is of formula (I) below, wherein x ranges from 0 to 0.5 and y ranges from 0.05 to less than 1. Preferably, x is lower than 0.2, more preferably lower than 0.1.

[0117] Formula (I)

[0118] Chitosan is a natural polymer of polysaccharide type of D-glucosamine (GlcN) or co-polysaccharide type, consisting of a random distribution (statistic copolysaccharide) or not (block copolysaccharide) of D-glucosamine and N-acetyl-D-glucosamine (GIcNAc), linked by glycosidic bonds of P(l— >4) type.

[0119] The functionalization rate of the functionalized chitosan may vary in a wide range. The “functionalization rale" is the amount of the primary amine groups of the chitosan that are functionalized with a functionalizing moiety comprising at least one acidic function relative to the total amount of primary amine groups of the chitosan. Preferably, the functionalization rate is such that the desired rheological properties are obtained for the pharmaceutical formulation. In some embodiments, from 5 to 40%, preferably from 10 to 30%, of the primary amine groups of the glucosamine units of chitosan are functionalized with the functionalizing moiety.

[0120] The functionalizing moieties are covalently bound to the primary amine (-NH2) groups of at least part of the non-acetylated glucosamine units of chitosan. The binding of the functionalizing moiety to the primary amine group may be of any suitable type known in the art. In some embodiments, the functionalizing moiety is linked to the primary amine groups of the glucosamine units of chitosan via an amide bond. Such amide bond may typically be formed by reacting the chitosan with an acyl chloride or an anhydride in suitable conditions.

[0121] The functionalized chitosan may be of any molecular weight suitable for implementing the present invention. In some embodiments, the average molecular weight in weight of the functionalized chitosan is comprised between 100 and 1000 kDa, preferably between 200 and 800 kDa, more preferably between 200 and 600 kDa, in particular between 400 and 600 kDa. In some embodiments, the average molecular weight in weight of the functionalized chitosan is comprised between 200 and 400 kDa. In some embodiments, the average molecular weight in weight of the functionalized chitosan is comprised between 100 and 250 kDa. Lower molecular weight chitosans are generally less viscous, which can help injectability.

[0122] The mean molar weight Mw of polysaccharides such as the functionalized chitosan may be measured by any suitable method known in the art. It may be for instance measured by steric exclusion chromatography, the method being described in “Physico-chemical studies of the gelation of chitosan in a hydroalcoholic medium” A. MONTEMBAULT, C. VITON, A. DOMARD, Biomaterials, 26(8), 933-943, 2005.

[0123] In some embodiments, the chitosan is medical -grade chitosan or GMP (Good Manufacturing Parctices)- grade chitosan.

[0124] The functionalized chitosan may comprise any proportion of N-acetyl-glucosamine units suitable for implementing the present invention. In some embodiments, the acetylation rate of the functionalized chitosan is 20% or lower, preferably 10% or lower. The "acetylation rate" refers to the amount of N- acetyl-glucosamine units relative to the total of N-acetyl-glucosamine units and glucosamine units in the chitosan. It means that the proportion of N-acetyl-glucosamine units relative to the total of N-acetyl- glucosamine units and glucosamine units (functionalized or not) of the functionalized chitosan is 20% or lower, preferably 10% or lower.

[0125] The proportion of N-acetyl-D-glucosamine in the chitosan may be determined by any suitable method known in the art. It may be for instance calculated by using ’H NMR, following the Hirai’s methodology (A. HIRAI, H ODANI, A. NAKAJIMA, Polymer Bulletin, 26 (1 ), 87-94, 1991). The concentration of the functionalized chitosan in the pharmaceutical formulation may vary in a wide range. In some embodiments, the functionalized chitosan concentration in the pharmaceutical formulation is comprised between 10 and 100 g / L, preferably between 20 and 50 g / L, more preferably between 20 and 30 g / L.

[0126] Functionalizing moiety

[0127] The chitosan is functionalized with functionalizing moieties comprising at least one acidic function, preferably at least two acidic functions. In some embodiments, the functionalizing moiety comprises 1, 2, 3, 4 or 5 acidic functions, preferably 1 or 2 acidic functions.

[0128] In some embodiments, the pKa of at least one acidic function is comprised between 4.5 and 7. Preferably, the pKa of all acidic functions of the functionalized moieties are comprised between 2 and 8, more preferably between 3 and 8.

[0129] The acidic function may be any acidic function known in the art. In some embodiments, the acidic function (or each acidic function) is selected from the group consisting of carboxyl groups -COOH, sulfonic groups -SO2OH, phosphonate groups -PO(OH)2, thiol groups -SH and alcohol groups -OH. Preferably, the acidic function is a carboxyl group.

[0130] In some embodiments, the basic form of at least one acidic function is negatively charged.

[0131] In some embodiments, the functionalizing moiety comprises at least one unsaturated bond, such as at least one double bond or triple bond. This may allow further functionalization of the functionalized chitosan with desired moieties of functionalities, for instance further functionalization with hydrophilic moieties. Hydrophilic moieties may be for instance selected from the group consisting of hydroxylic groups, sulfonate groups, phosphate groups, thiol groups, ester groups and carboxyl groups. The choice of the functional groups depends on the desired application, so as to improve solubility, interaction with water, and / or other specific properties.

[0132] In some embodiments, the functionalizing moiety comprises at least one ester function, particularly in the case of co-formulation with antibody-drug conjugates such as those bearing an esterase-sensitive linker. The ester function in the modified chitosan might act as a decoy to avoid cleavage of the linker due to esterase activity.

[0133] In some embodiments, the functionalizing moiety comprises at least one hydrophilic moiety different from the acidic functions, such as an acetyl function and / or a hydroxylic function, preferably an acetyl function.

[0134] In some embodiments, the functionalizing moiety does not comprise any chelating group ie no group suitable for chelating cations, as defined for instance in WO2023148351.

[0135] The functionalizing moiety may be bound to the primary amine via an amide bond obtained by reacting the chitosan with an acyl chloride or an anhydride in suitable conditions. In some embodiments, the anhydride is selected from the group consisting of cA-aconitic anhydride , (+)-diacetyl-L-tartaric anhydride

[0136] 1 ,2,4-benzenetricarboxylic anhydride , pyromellitic dianhydride and 3,3’,4,4’-biphenyltetracarboxylic dianhydride , preferably (+)- diacetyl -L-tartaric anhydride.

[0137] In some embodiments, the functionalizing moiety is bound to the primary amine via an amide bond obtained by reacting the chitosan with an anhydride different from DOTAGA in suitable conditions.

[0138] In some embodiments, the functionalized chitosan comprises a single type of functionalizing moiety. In other embodiments, the functionalized chitosan comprises at least two different functionalizing moieties.

[0139] Process for preparing the functionalized chitosan

[0140] The functionalized chitosan may be prepared by any suitable technique known in the art.

[0141] The functionalized chitosan may be prepared with a process comprising the two following successive steps:

[0142] - Step 1: solubilizing a chitosan in an acidic solution at a pH between 4 and 5;

[0143] - Step 2: functionalization of at least a part of the primary amine functions of said chitosan, solubilized at step 1 with a functionalizing moiety.

[0144] The acetylation rate of the chitosan is preferably 20% or lower, more preferably 10% or lower

[0145] Pharmaceutical formulation

[0146] The pharmaceutical formulation may comprise, in addition to the protein and the functionalized chitosan, any other suitable constituent, such as a pharmaceutically acceptable support and / or suitable additives.

[0147] In some embodiments, the pharmaceutical formulation comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient is selected from the group consisting of water, such as sterile water for injection (WFI) or bacteriostatic water for injection (BWFI), a pH buffered solution, such as phosphate -buffered saline PBS, a sterile saline solution, Ringer's solution, a dextrose solution, and combinations thereof. Preferably, the pharmaceutically acceptable excipient is sterile water or bacteriostatic water for injection. A “pharmaceutically acceptable excipient” is an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, for example, FDA Office or EMA.

[0148] In some embodiments, the pharmaceutical formulation comprises at least one additive, such as stabilizers, surfactants, buffering agents, preservatives such as antimicrobial preservatives, protectants, antioxidants, chelating agents and bulking agents.

[0149] According to the present disclosure, stabilizers are compounds increasing protein stability, especially against unfolding and aggregation. Preferably, the stabilizer is admitted by the authorities as a suitable additive in pharmaceutical formulations.

[0150] The stabilizer may be a saccharide. A "saccharide" herein comprises the general composition (CH2O)nand derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, nonreducing sugars, etc. Examples of saccharides herein include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, dextran, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, mellibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, iso- maltulose, etc.

[0151] Preferably, the formulation comprise a non-reducing disaccharide as a stabilizing agent, such as a saccharide selected from the group of trehalose (e.g. in the form of a,a-trehalose dihydrate) and sucrose.

[0152] In some embodiments, the stabilizer is not a polysaccharide.

[0153] The concentration of the stabilizer in the pharmaceutical formulation may vary in a wide range and is preferably comprised between 1 and 500 mM, preferably between 15 and 250 mM, more preferably between 150 and 250 mM. In an embodiment, it is about 210 mM. According to the present disclosure, a "surfactant" refers to a surface-active agent. Surfactants are generally added in protein formulations in order to reduce the exposure of hydrophobic regions and so decreasing protein-protein interactions and interface -induced aggregation, also prevented by competition for adsorption sites.

[0154] Examples of surfactants herein include poloxamers (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl- sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetylbetaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl- betaine (e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; polyethyleneglycol, polypropyleneglycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc). Other examples of pharmaceutically acceptable surfactants include polyoxyethylenesorbitan fatty acid esters (Tween), polyethylenepolypropylene glycols, polyoxyethylenestearates, polyoxyethylene alkyl ethers, e.g. polyoxyethylene monolauryl ether, alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulphate (SDS). Most suitable polyoxyethylenesorbitan fatty acid esters are polysorbate 20 (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Most suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Most suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Most suitable alkylphenol-poly oxyethylene ethers are sold under the trade name Triton-X.

[0155] The surfactant is preferably a nonionic surfactant, preferably a polysorbate, e.g. selected from the group of polysorbate 20, polysorbate 80 and polyethylene polypropylene copolymer.

[0156] The concentration of surfactant in the pharmaceutical formulation is preferably comprised between 0.01 and 0.1 % (w / v), or 0.01 an 0.08 % (w / v), or 0.025 and 0.075 % (w / v).

[0157] According to the present disclosure, the term "buffering agent" refers to an agent which provides that the solution comprising it resists changes in pH by the action of its acid / base conjugate components. Examples of buffering agents that will control the pH in this range include acetate, succinate, gluconate, histidine, citrate, glycylglycine and other organic acid buffers.

[0158] A suitable buffer in the present disclosure is a histidine buffer. A "histidine buffer" is a buffer comprising the amino acid histidine. Examples of histidine buffers include histidine chloride (e.g. L-histidine hydrochloride monohydrate), histidine acetate, histidine phosphate, histidine sulfate.

[0159] A "preservative" is a compound which can be added to the formulation to reduce contamination by and / or action of bacteria, fungi, or another infectious agent. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chained), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohols, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and cresol.

[0160] A "protectant" is a substance which, when combined with a protein, significantly reduces chemical and / or physical instability of the protein upon lyophilization and / or subsequent refrigerated storage.

[0161] Exemplary protectants include sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids, such as arginine or histidine; lyotropic salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, polyethylene glycol, or polypropylene glycol; and combinations thereof. Additional examples of protectants include gelatin, dextrins, modified starch, and carboxymethyl cellulose. Preferred sugar alcohols are those compounds obtained by reduction of mono- and di-saccharides, such as lactose, trehalose, maltose, lactulose, and maltulose. Additional examples of sugar alcohols are glucitol, maltitol, lactitol and isomaltulose.

[0162] In some embodiments, the protectants are selected from the group consisting of sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids, such as arginine, lysine, proline or histidine, and amino acids derivatives; lyotropic salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, polyethylene glycol), or polypropylene glycol); and combinations thereof.

[0163] The protectant may be added to the pre -lyophilized formulation in a lyoprotecting amount. This means that, following lyophilization of the protein in the presence of lyoprotecting amount of the protectant, the protein essentially retains its physical and chemical stability and integrity.

[0164] An “antioxidant” is a pharmaceutically acceptable additive generally used to limit oxidation reactions and maintain the stability and safety of proteins. Examples of antioxidants are ascorbic acid, sodium metabisulfite, histamine, methionine, ascorbic acid, glutathione, vitamin E and polyethylenimine. The antioxidant is preferably methionine, in particular L-methionine.

[0165] The antioxidant concentration in the pharmaceutical formulation is preferably comprised between 5 and 25 mM, more preferably between 5 and 15 mM.

[0166] A “chelating agent” is a pharmaceutically acceptable additive generally used to maintain the stability of proteins.

[0167] Examples of chelating agents include edetate disodium, diethylenetriamine penta-acetic acid, citric acid, hexaphosphate, thioglycolic acid and zinc.

[0168] A "bulking agent" is a pharmaceutically acceptable additive generally used to add mass to a lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g. facilitating the production of an essentially uniform lyophilized cake which maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, lactose, modified starch, polyethylene glycol) and sorbitol.

[0169] The pharmaceutical formulation of the disclosure preferably has a pH and / or an osmolarity close, but different from, the physiological conditions, and in particular lower than the physiological conditions. When placed under physiological conditions, in contact with body fluids, the pharmaceutical formulation of the disclosure forms a gel by a change in pH and / or osmolarity by equilibration with physiological media. The pharmaceutical formulation preferably has an osmolarity comprised between 50 and 600 mOsm / L, preferably comprised between 100 and 600 mOsm / L, more preferably between 250 and 450 mOsm / L, or between 50 and 300 mOsm / L, preferably between 50 and 250 mOsm / L. The “osmolarity” (also known as osmotic concentration) refers to the solute concentration per unit volume of solution. Osmolarity is similar to molarity but includes the total number of moles of dissolved species in solution. Osmolarity is defined as the number of osmoles (Osm) of solute per litre (L) of solution (osmol / L or Osm / L). An osmole is the amount of substance, which must be dissolved in order to produce an Avogadro's number of particles. For substances which do not dissociate, the molarity and the osmolarity will be the same, whereas for substances that are ionized the osmolarity will be the molarity multiplied by the number of dissociated parts, eg. for sodium chloride the osmolarity will be doubled. Osmolality is the number of osmoles of solute per kilogram of solvent. Physiological osmolarity is typically in the range of about 280 mOsm / L to about 310 mOsm / L, typically is about 300 mOsm / L.

[0170] The osmolarity of the pharmaceutical formulation is typically measured using an osmometer, such as a Loser Micro Osmometer MOD200 Plus from Camlab. Prior to measurement, zero is set using 50 pL of distilled water, and the instrument is then calibrated against 25pL of a 300mOsm / kg water standard. Samples are measured by taking an identical volume of 25 pL frozen to -6.0 °C. Alternatively, zero is set using 15 pL of distilled water, and the instrument is then calibrated against 15pL of a 300mOsm / kg water standard. Samples are measured by taking an identical volume of 15 pL frozen to -6.2 °C. pH

[0171] The pH of the pharmaceutical formulation may vary in a wide range. In some embodiments, the pH of the pharmaceutical formulation is comprised between 4.5 and 7.5, preferably between 5.0 and 6.0.

[0172] The pH is measured according to methods known to those skilled in the art, for example using a pH meter such as a Mettler Toledo SevenCompact S210 pH meter. The expression “physiological pH” means a pH comprised between 6.8 and 7.6, typically of about 7.4.

[0173] In some embodiments, the pharmaceutical formulation comprises:

[0174] - from 10 to 200 g / L, more preferably from 50 g / L to 150 g / L, of at least one protein, preferably at least one antibody;

[0175] - from 10 to 100 g / L, preferably from 20 g / L to 50 g / L, more preferably from 20 g / L to 30 g / L, of functionalized chitosan; and

[0176] - at least one pharmaceutically acceptable excipient, preferably water.

[0177] Administration

[0178] The pharmaceutical formulation of the disclosure can easily be injected in a tissue due to its rheological properties while being able to form, in physiological conditions, a biodegradable hydrogel that entraps the proteins and enables their controlled and prolonged release in the tissue.

[0179] The pharmaceutical formulation of the disclosure is preferably under the form of a liquid formulation, more preferably of a liquid injectable formulation. According to the present disclosure, a "liquid injectable formulation” means a liquid formulation having viscosity sufficiently low to allow a good injectability trough a gauge needle.

[0180] The term ''injectability'' or ''syringeability'' refers to the injection performance of the pharmaceutical formulation through a syringe equipped with a 18-32 gauge needle, preferably a 20-27 gauge needle.

[0181] The viscosity of the pharmaceutical formulation may vary in a wide range. In some embodiments, the pharmaceutical formulation has a shear-thinning behavior, ie its viscosity decreases with shear rate.

[0182] The pharmaceutical formulation may have a viscosity comprised between 10 and 500 Pa.s, preferably between 10 and 300 Pa.s., more preferably between 15 and 250 Pa.s, as measured at room temperature by rotational rheometry in plane cone geometry at a shear rate of between 0.01 and 0.001 s’1, for example of 0.001 s’1or 0.01 s’1, preferably of 0.01 s’1.

[0183] In some embodiments, the pharmaceutical formulation has a viscosity comprised between 10 and 10 000 Pa.s, preferably between 10 and 1000 Pa.s, more preferably between 50 and 500 Pa.s, as measured at 25 °C by rotational rheometry in parallel plate geometry at a shear rate of between 0,1 and 1 s1, for example 0,1 or 1 s1, preferably 1 s1.

[0184] In an embodiment, the viscosity of the pharmaceutical formulation is comprised between 10 and 80 Pa.s, preferably between 10 and 50 Pa.s, more preferably between 10 and 30 Pa.s, or a viscosity comprised between 80 and 250 Pa.s, preferably between 80 and 120 Pa.s, for example of about 100 Pa.s.

[0185] In an embodiment, the viscosity of the pharmaceutical formulation is comprised between 10 and 1 000 Pa.s, preferably between 10 and 500 Pa.s, more preferably between 50 and 150 Pa.s, for example of about 100 Pa.s. as measured at 25°C by rotational rheometry in parallel plate geometry at a shear rate of 1 s’1.

[0186] The term "viscosity" refers to the resistance of a substance (typically a liquid) to flow. Viscosity is related to the concept of shear force; it can be understood as the effect of different layers of the fluid exerting shearing force on each other, or on other surfaces, as they move against each other. The units of viscosity are Ns / m2 , known as Pascal -seconds (Pa.s). The viscosity of the pharmaceutical formulation of the disclosure is typically measured by rotational rheometry in plane cone geometry using a rheometer with imposed deformation, such as an Advanced Rheometer AR2000, or a rheometer with imposed stress, such as HR-10 Discovery from TA instruments. Briefly, the fluid is subjected to shear between two plane surfaces, one fixed and one rotating around its axis. The shear gradient is determined by the geometry of the surface on the one hand, and the speed of rotation on the other hand. The shear stress is calculated from the measurement of the torque transmitted by the sample to be characterized. A detailed description of this method can be found in El Kissi et al, Rheology, 10,13-39 (2006). In the present invention, measurements are performed at room temperature (e.g. about 20°C, preferably about 25 °C) and are typically made using a 20 mm sand-blasted plane geometry at a continuous shear rate of between 0.01 and 10 s’1, for example of 0.01 s’1or 10 s’1, in particular of 0.01 s’1(viscosity measurement method (i)). Method (i) may also be implemented in the same conditions using a 4° and 25 mm cone plane geometry. Alternatively, measurements may be performed at room temperature (e.g. about 20°C, preferably about 25 °C) using a C35 / 2° Ti L cone plate geometry with a flow sweep linear study conducted by scanning shear rate from IO-2to 103s1(viscosity measurement method (ii)). Viscosity measurement method (i) is preferably performed using an Advanced Rheometer AR2000 from TA instruments with a 4° and 25mm planar cone geometry. Viscosity measurement method (ii) is preferably performed using a RheoStress 600 rheometer from Thermo Scientific HAAKE using a C35 / 2° Ti L cone plate geometry. In viscosity measurement method (i), the viscosity values may be measured at relatively low shear rates. However, it can be noted that for low viscosity solutions, e.g. for solutions having a viscosity lower than 20 Pa.s, the viscosity values measured according to viscosity measurement method

[0187] (i) at shear rate values of about 0.001 s1can in some cases be distorted because it corresponds to the measurement limit of the machine . For such low viscosity solutions, viscosity measurement is preferably carried out according to viscosity method (i) at shear rates higher than 0.001 s1, typically at shear rates of about of 0.01 s1, preferably at a shear rate of 0.01 s1, or according to viscosity measurement method

[0188] (ii).

[0189] Preferably, viscosity is measured according to method (i).

[0190] Prior to the measurement, the zero deviation is set and the inertia and rotation mapping of the instrument is calibrated. The samples are then spread out on the plate and, when a cone plane geometry is used, the deviation from the cone plane geometry is set to 116 microns in viscosity measurement method (i) or to 105 microns in viscosity measurement method (ii). Advantageously, the excess sample is removed with a spatula to limit edge effects.

[0191] The higher the viscosity of the pharmaceutical formulation, the more localized the pharmaceutical formulation is in the tissue at the site of injection before forming a hydrogel. In the pharmaceutical formulation of the disclosure, the functionalized chitosan is preferably present in the pharmaceutical formulation in dissolved form, meaning that at least 90%, preferably at least 95 % of the functionalized is in dissolved form, % expressed by weight of functionalized chitosan in dissolved form, relative to the total weight of functionalized chitosan in the pharmaceutical formulation.

[0192] The presence of the functionalized chitosan in dissolved form in the pharmaceutical formulation enables the pharmaceutical formulation to be under the form of a viscous solution sufficiently liquid to be injected via a wide range of needles commonly used in the medical field.

[0193] In other words, the pharmaceutical formulation of the disclosure is preferably not under the form of a solid hydrogel, as it is in the case of implantable hydrogels.

[0194] In an embodiment, the pharmaceutical formulation of the disclosure is in the form of a ready- to-use (i.e. ready-to-administer) injectable formulation containing the functionalized chitosan, the protein or combination of proteins and optionally one or more pharmaceutically acceptable excipients. In such embodiment, the pharmaceutical formulation may be supplied in a prefdled device, such as a prefdled syringe. Such prefdled device is another object of the present invention.

[0195] Process for preparing the pharmaceutical formulation

[0196] The pharmaceutical formulation of the invention may be prepared by any suitable process known in the art. Preferably, the process comprises a step of mixing a functionalized chitosan, a protein or a combination of proteins and one or more pharmaceutical excipients as defined above

[0197] Another object of the present invention is thus a process for the preparation of a pharmaceutical formulation, preferably comprising the steps of: a) providing a solution containing a functionalized chitosan as defined above; b) providing a solution containing a protein or a combination of protein ; c) mixing the solution of step a) and the solution of step b) to obtain a homogenous solution; d) optionally, adjusting the osmolarity of the solution obtained in step c) to provide an osmolarity comprised between 50 and 300 mOsm / L, preferably between 50 and 250 mOsm / L; e) optionally, adjusting the pH of the solution obtained in step c) between 4.5 and 7.5, preferably between 5.0 and 6.0.

[0198] The solutions of step a) and step b) are preferably aqueous solution.

[0199] The solution of step b) may be prepared by dissolving a protein in powder form e.g. lyophilized form, typically by mixing the powder and a solvent in particular a solvent suitable for injection to form a solution containing a protein or a combination of proteins.

[0200] Step c) may be carried out by centrifugation. For example, the centrifugation may be carried out between 2000 and 8000 rpm during between 5 and 30 minutes, preferably about 5000 rpm during 10 minutes or between 50 and 200 rpm during 1 and 4 hours, preferably about 100 rpm during 1 hour. Alternatively, centrifugation may be carried out after step c) to remove air bubbles from the solution obtained in step c).

[0201] In an embodiment, step c) is be carried out with a static mixer. In that embodiment, the solution of step a) is contained in a first container and the solution of step b) is contained in a second container, said containers being connected to a static mixer, to provide a system for mixing the solution of step a) and the solution of step b) by injection of the contents of one solution into the other and vice versa. Such injection may be repeated between 100 and 500 times, typically between 100 and 300 times. Step c) is preferably carried out in a static mixer.

[0202] In an embodiment, step c) is carried out by a static mixer, by mixing directly the solution of step a) and the solution of step b) in a reactor. The optional osmolarity adjustment of step d) may be carried out by appropriate dialysis of the solution obtained in step c), for example against water or a solution containing a salt such as NaCl, optionally maintaining the pH by adding a base solution.

[0203] The optional pH adjustment of step e) may be carried out by appropriate addition of a base or an acid, preferably of a base such as NaOH.

[0204] Steps d) and e) may be carried out in any order or may be performed concomitantly. For example, the osmolarity may be adjusted by dialysis while maintaining the pH or vice versa.

[0205] In some embodiments, the pharmaceutical formulation is sterilized before being administered for instance by injection. Sterilization may affect the viscosity of the formulation, but the formulation preferably remains injectable after sterilization.

[0206] Hydrogel

[0207] The Inventors evidenced that the pharmaceutical formulations according to the present invention form hydrogels when they are contacted with aqueous solutions, such as aqueous saline solutions, especially in physiological conditions. This is unexpected in view of the functionalized chitosan formulations known in the art which require the presence of two different types of chitosan so as to induce gelling in physiological conditions.

[0208] The functionalized chitosan is capable of gelling in situ under physiological conditions, in particular at physiological pH and osmolarity, thus forming a hydrogel. Gelling of the pharmaceutical formulation triggers encapsulation of the protein or combination of proteins in the hydrogel.

[0209] As the hydrogel comprises only one type of functionalized chitosan, it is homogeneous.

[0210] The hydrogel is biocompatible and biodegradable, and allows protein release in a controlled, prolonged, and virtually constant manner, especially while avoiding a phenomenon of rapid initial release known as “burst release”.

[0211] The hydrogel results from the gelation of a solution comprising a formulation according to the invention and water.

[0212] In some embodiments, the hydrogel is injectable,

[0213] According to the present disclosure, the term "gel" means a nonfluidic polymeric network swollen by a solvent.

[0214] According to the present disclosure, the expression “hydrogel” means a gel, wherein the swelling solvent is water. The hydrogel comprises approximately from 50% to 99% by weight of water, preferably from 50% to 80% by weight of water, relative to the total weight of hydrogel.

[0215] The phrase "biodegradable hydrogel" means that the hydrogel naturally breaks down under physiological conditions, and in particular under the action of macrophages. The reactions involved during biodegradation may include hydrolysis reactions, ie the breaking of covalent bonds by reaction with water. These reactions may be catalyzed by the action of enzymes naturally present at the site of injection.

[0216] In an embodiment, more than 50 %, preferably more than 80 %, preferably more than 95% by weight of the hydrogel deteriorates in a period of time comprised between 10 and 100 days, preferably between 20 and 65 days, when the hydrogel is placed in physiological conditions, typically after subcutaneous injection in a subject.

[0217] In particular, the hydrogel may virtually release the protein over a period comprised between 10 and 100 days, preferably between 20 and 65 days from the day of administration of the pharmaceutical formulation of the invention.

[0218] A long degradation time advantageously enables to extend the time between two injections. This is particularly advantageous compared to prior art formulations for example with hyaluronidases, which must preferably be injected approximately every two weeks.

[0219] The phrase "biocompatible hydrogel” means a hydrogel having the ability not to degrade the biological environment it is placed into. In particular, the hydrogel produces little or no inflammatory reactions nor necrosis even during a prolonged contact with biological environment.

[0220] In an embodiment, the hydrogel causes little or no inflammation over a period between 10 and 100 days, preferably between 20 and 65 days from the day of administration of the pharmaceutical formulation of the invention.

[0221] Ku

[0222] Another object of the invention is a kit comprising:

[0223] - at least one first container containing a functionalized chitosan, and

[0224] - at least a second container comprising a protein, wherein the functionalized chitosan and the protein are as defined above.

[0225] The kit is suitable for preparing and / or reconstituting a pharmaceutical formulation or a hydrogel according to the invention.

[0226] In some embodiments, the kit further comprises a third container comprising a solvent.

[0227] In some embodiments, the kit further comprises instructions for preparing and / or reconstituting a pharmaceutical formulation or a hydrogel.

[0228] Therapeutical use

[0229] The Inventors evidenced that the formulations and hydrogels according to the present invention are suitable for being subcutaneously injected and allow prolonged release of the protein of combination of proteins. The Inventors further evidenced that the pharmaceutical formulations according to the invention may be efficiently subcutaneously injected as such, or even if it is at least partially under the form of a hydrogel.

[0230] A further object of the invention is thus the pharmaceutical formulation, the kit or the hydrogel according to the invention, for use as a medicament in a patient, preferably a patient having a cancer.

[0231] The terms "cancer" and "cancerous" refer to the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies (including waldenstrom macroglobulinemia), multiple myeloma or myeloid neoplasms. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, nonsmall cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, tumors of the biliary tract, as well as head and neck cancer.

[0232] The pharmaceutical formulation or the kit according to the invention may also be for use in the treatment of nonmalignant diseases in particular nonmalignant diseases selected from autoimmune disease (e.g. psoriasis); endometriosis; scleroderma; restenosis; polyps such as colon polyps, nasal polyps or gastrointestinal polyps; fibroadenoma; respiratory disease; cholecystitis; neurofibromatosis; polycystic kidney disease; inflammatory diseases; skin disorders including psoriasis and dermatitis; vascular disease; conditions involving abnormal proliferation of vascular epithelial cells; gastrointestinal ulcers; Menetrier’s disease, secreting adenomas or protein loss syndrome; renal disorders; angiogenic disorders; ocular diseases such as presumed ocular histoplasmosis syndrome, retinal neovascularization from proliferative diabetic retinopathy, retinal vascularization, diabetic retinopathy, or age related macular degeneration; bone associated pathologies such as osteoarthritis, rickets and osteoporosis; damage following a cerebral ischemic event; fibrotic or edemia diseases such as hepatic cirrhosis, lung fibrosis, carcoidosis, throiditis, hyperviscosity syndrome systemic, Osier Weber-Rendu disease, chronic occlusive pulmonary disease, or edema following bums, trauma, radiation, stroke, hypoxia or ischemia; hypersensitivity reaction of the skin; Guillain-Barre syndrome; graft versus host disease or transplant rejection; Paget’ s disease; bone or joint inflammation; photoaging (e.g. caused by UV radiation of human skin); benign prostatic hypertrophy; certain microbial infections including microbial pathogens selected from adenovirus, hantaviruses, Borrelia burgdorferi, Yersinia spp. and Bordetella pertussis; thrombus caused by platelet aggregation; reproductive conditions such as endometriosis, ovarian hyperstimulation syndrome, preeclampsia, dysfunctional uterine bleeding, or menometrorrhagia; synovitis; atheroma; acute and chronic nephropathies (including proliferative glomerulonephritis and diabetes-induced renal disease); eczema; hypertrophic scar formation; endotoxic shock and fungal infection; familial adenomatosis polyposis; neurodegenerative diseases (e.g. Alzheimer’s disease, AIDS-related dementia, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration); myelodysplastic syndromes; aplastic anemia; ischemic injury; fibrosis of the lung, kidney or liver; T-cell mediated hypersensitivity disease; infantile hypertrophic pyloric stenosis; urinary obstructive syndrome; psoriatic arthritis; and Hasimoto’s thyroiditis. Exemplary nonmalignant indications for therapy herein include psoriasis, endometriosis, scleroderma, vascular disease (e.g. restenosis, atherosclerosis, coronary artery disease, or hypertension), colon polyps, fibroadenoma or respiratory disease (e.g. asthma, chronic bronchitis, bronchiectasis or cystic fibrosis), inflammatory bowel diseases (IBD), primary immunodeficiency disorders (PIDs), hypogammaglobulinemia, primary immune thrombocytopenia, Guillain-Barre syndrome, Kawasaki disease, multifocal motor neuropathy, chronic inflammatory demyelinating polyradiculoneuropathy.

[0233] A further object of the invention is the pharmaceutical formulation, the kit or the hydrogel according to the invention, for use in the treatment of a cancer or of a nonmalignant disease as defined above. In some embodiments, the kit or the hydrogel according to the invention is for use in the treatment of a cancer.

[0234] "Treating" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully "treated" for an infection if, after receiving a therapeutic amount of an antibody according to the methods of the present invention, the patient shows observable and / or measurable reduction in or absence of one or more of the following: reduction in the number of pathogenic cells; reduction in the percent of total cells that are pathogenic; and / or relief to some extent, one or more of the symptoms associated with the specific disease or condition; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

[0235] A further object of the invention is a method for the treatment of cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition or of a hydrogel according to the invention. The term "subject” refers to a mammal, preferably a human. In some embodiments, the subject is a "patient", i.e. a warm-blooded animal, more preferably a human, who is awaiting the receipt of, or is receiving, medical care or was / is / will be the object of a medical procedure, or is monitored for the development of a disease.

[0236] In some embodiments, the subject is an adult (for example a subject above the age of 18). In other embodiments, the subject is a child (for example a subject below the age of 18). In some embodiments, the subject is a male. In other embodiments, the subject is a female.

[0237] One skilled in the art is able to adapt the dose to be administered depending among others on the constituents of the pharmaceutical composition, including the protein, the subject, and the administration route. In some embodiments, the protein is administered at a dose comprised between 0.1 pg / kg and 1000 mg / kg, preferably between 0. 1 mg / kg and 100 mg / kg, even more preferably between 1 mg / kg and 50 mg / kg.

[0238] Administration is preferably subcutaneous injection.

[0239] A “subcutaneous injection” means an injection administered into the subcutis, the layer of skin directly below the dermis and epidermis, collectively referred to as the cutis.

[0240] The subcutaneous injection may be performed at any area suitable for subcutaneous injection, such as the thighs, the abdomen particularly in its umbilical region, the upper arm, in particular the posterior or lateral aspect of the lower part of the upper arm, the back, the lower loins, and the buttocks, in particular the upper outer area of the buttocks.

[0241] Several subcutaneous injections can be done at the same time in different areas suitable for subcutaneous injection. The subcutaneous injection volume may be comprised between 1 and 20 mL, preferably between 2 and 15 mL.

[0242] In one embodiment, the subcutaneous injection volume is comprised between 1 and 5 mL, preferably between 1 and 3 mL, preferably it is about 2 mL. In such embodiment, the subcutaneous injection is preferably performed in the subcutaneous tissue of the thighs, the upper arm, the back and the lower loins.

[0243] In one embodiment, the subcutaneous injection volume is comprised between 5 and 20 mL, preferably between 5 or 10 mL or between 10 and 20 mL, more preferably it is about 15 ml. In such embodiment, the subcutaneous injection is preferably performed in the subcutaneous tissue of the abdomen.

[0244] The pharmaceutical formulation or hydrogel may also be administrated by intraperitoneal injection, intraarticular injection, intrathecal injection, or intra ocular injection.

[0245] “Intraocular injection” (also known as intravitreal injections) means a route of administration via an injection inside of the eye via the vitreous i.e. the gel-like substance that fdls the eye. "Intra-articular injection” means a route of administration via an injection into a joint. Intra-articular injection may be useful in chemotherapy, as well as in treating arthritis.

[0246] “ Intrathecal administration” means a route of administration via an injection into the spinal canal or into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF). Intrathecal administration may be useful chemotherapy.

[0247] The pharmaceutical formulation may also be administered by intratumoral injection.

[0248] “Intratumoral injection” means a direct injection into a tumor. Intratumoral injections can be considered for any tumor where the primary lesion or its metastases are accessible either percuteanously (i.e. needlepuncture of the skin) via direct injection or via specific procedures such as colonoscopy, cystoscopy, bronchoscopy, thoracoscopy, coelioscopy, or even surgery.

[0249] The injection may be administered with a 18-32-gauge needle, preferably a 20-27 gauge needle. The formulations can be administered using a small gauge needle, for example, between 20 and 30 gauge, typically 27, 28 29, or 30 gauge.

[0250] In an embodiment, the pharmaceutical formulation causes no significant inflammation when administered not more than twice daily, once daily, twice weekly, once weekly, once every two weeks or once monthly. The pharmaceutical formulation of the disclosure is preferably administered once every two weeks causing no significant inflammation at the site of injection

[0251] In an embodiment, the pharmaceutical formulation or the kit according to the invention comprises an antibody or combination of antibodies.

[0252] In an embodiment, the pharmaceutical formulation or the kit according to the invention comprises an antibody or combination of antibodies which binds an antigen selected from the group of HER2 antigens, such as trastuzumab, pertuzumab or a combination thereof, and is for use in the treatment of breast cancer, gastric cancer, B cell malignancies (such as non-Hodgkin's lymphoma or chronic lymphocytic leukemia) or autoimmune diseases (such as rheumatoid arthritis and vasculitis).

[0253] In an embodiment, the pharmaceutical formulation or the kit according to the invention comprises an antibody or combination of antibodies which binds an antigen selected from the group of CD20 antigens, such as rituximab, and is for use in the treatment of rheumatoid arthritis, non-Hodgkin's lymphoma (NHL), leukemia such as chronic lymphocytic leukemia, granulomatosis with polyangiitis (Gpa or Wegener's disease), microscopic polyangiitis (Mpa) or Pemphigus vulgaris.

[0254] In an embodiment, the pharmaceutical formulation or the kit according to the invention comprises an antibody or combination of antibodies which binds an antigen selected from the group of CD38 antigens, such as daratumumab, and is for use in the treatment of multiple myeloma or myeloid neoplasms. A further object of the invention is a use of a pharmaceutical composition, a kit or a hydrogel according to the invention in the manufacture of a medicament, preferably a medicament for the treatment of cancer.

[0255] In some embodiments, the pharmaceutical formulation or the hydrogel according to the present invention is used in combination with at least one other therapeutical treatment. Said at least one other therapeutical treatment includes for instance chemotherapy treatments, radiotherapy treatments and immunotherapy treatments. Combination may refer to simultaneous and / or sequential administration.

[0256] Unless otherwise specified, the terms “ comprised between” A and B refers to the ‘A to B’ range, the values of A and B being included in said range. The terms "from A to B” refer to the same range.

[0257] Unless otherwise specified, the % values in the present invention are weight% values.

[0258] The term "about" when referring to a measurable value is meant to encompass variations of + / -5% or less, more preferably + / -2%, and more preferably + / -!% or less of and from the specified value, insofar such variations are appropriate to perform the invention.

[0259] The invention will also be described in further detail in the following examples, which are not intended to limit the scope of this invention, as defined by the attached claims.

[0260] EXAMPLES

[0261] Example 1: Synthesis of chitosan functionalized with cis-aconitic acid (i.e. AI-CACO polymers)

[0262] AI-CACO was obtained after chemical modification of a chitosan polymer with cis-aconitic anhydride. The raw chitosan used for the synthesis was a medical grade chitosan, extracted from Alaska snow crab and was characterized in terms of its size properties, including the number average molar mass (Mn) of 110 ± 3 kg mol1, weight average molar mass (Mw) of 216 ± 3 kg mol1, polydispersity index (1.96 ± 0.05), and gyration radius (46 ± 2 nm). The degree of acetylation of this raw chitosan was determined by ' H NMR spectroscopy using the Hirai method (Hirai et al., Polymer Bulletin, 26, 87-94.1991) and was estimated to be 6±0.5%. Cis-aconitic acid (i.e. cis-Propene-l,2,3-tricarboxylic acid) is a tricarboxylic acid intermediate of the citric acid cycle, with 3 pKa of approximately 2.80, 4.40 and 6.39.

[0263] Various AI-CACO polymers were obtained by varying the molar ratio between cis-aconitic anhydride and chitosan. The following ratios (of cis-aconitic anhydride versus amine moieties of chitosan) have been tested: 0.18, 0.35, 0.70 and 1.05.

[0264] An example of protocol synthesis (with a ratio of 0.7) is the following. 250 mb of ultrapure water and 3.75 g of raw chitosan (i.e. 23.2 mmol of NH2 moieties) as previously described were introduced in a round-bottom flask and the mixture is stirred at 500 rpm for 30 min. Then 3.125 mb of glacial acetic acid was added to solubilize the chitosan and the mixture was kept under stirring at 500 rpm for 5h, until complete solubilization. 250 mL of 1,2 propanediol was added under stirring and after Ih of homogenization, 2.55 g of cis-aconitic anhydride (i.e. 16.36 mmol) was added and the reaction mixture is stirred at 500 rpm for 18h. The functionalized chitosan polymer was purified by tangential filtration against 20L of 0.1M acetic acid solution then 20 L of acetic acid 5mM, using the Sartoflow® Smart device with a Sartocon Slice 200 PESU cassette (polyethersulfone membranes; cut-off: 100 kDa; filtration area: 0.1 m2). The purified AI-CACO polymer was then freeze-dried and characterized.

[0265] The degree of acetylation of AI-CACO was identical to that of the starting chitosan and was measured by1H NMR spectroscopy using the Hirai method (Hirai et al., Polymer Bulletin, 26, 87-94. 1991).

[0266] FTIR spectra from raw chitosan and chitosan polymer modified with cis-aconitic anhydride (ratio 0.70) were compared (Figure 1). FTIR experiments were carried out using an IRAffinity-lS spectrophotometer (Shimadzu). An atmosphere correction was applied on all spectra to remove peaks from CO2. By comparison of both raw and modified chitosan, a strong band at 1550 cm'1is observed, corresponding to the formation of an amide H bond between chitosan and cis-aconitic anhydride. The sharp increase in the intensity of this band highlights effective grafting of chitosan by the anhydride.

[0267] Zeta potential was measured with Zetasizer MPT-2 Malvern and measurement were performed by pH increments of 0.5. Three different polymers obtained by varying the molar ratio of cis-aconitic anhydride versus amine moieties of chitosan showed a similar zeta potential profile with an isoelectric point between 7.67 and 7.87. For example, chitosan modified with cis-aconitic anhydride using a ratio of 0. 18 had an isoelectric point of 7.87 (Figure 2).

[0268] Example 2: Synthesis of chitosan functionalized with (+)-diacetyl-L-tartaric acid (i.e. AI-CTar polymers)

[0269] AI-CTar were obtained after chemical modification of a chitosan polymer with (+)-diacetyl-L-tartaric anhydride. The raw chitosan used for the synthesis was the same medical grade chitosan as described in example 1. (+)-diacetyl-L-tartaric acid (i.e (2R,3R)-2,3-diacetoxysuccinic acid) is a diacid, with pKa of approximately 2.98 and 4.34.

[0270] The synthesis protocol was the following.125 mL of ultrapure water and 1.875 g of raw chitosan (i.e. 11.6 mmol of NH2moieties) as previously described were introduced in a round-bottom flask and the mixture is stirred at 500 rpm for 30 min. Then 1.156 mL of glacial acetic acid was added to solubilize the chitosan and the mixture was kept under stirring at 500 rpm for 5h, until complete solubilization. 125 mL of 1,2 propanediol was added under stirring and after Ih of homogenization, 0.8840 g of (+)- diacetyl-L-tartaric anhydride (i.e. 4.09 mmol) was added and the reaction mixture was stirred at 500 rpm for 18h. The medium was then filtered on filter paper and the functionalized chitosan polymer was purified by tangential filtration against 20 L of 0.1 M acetic acid solution then 20 L of acetic acid 5 mM, using the Sartoflow® Smart device with a Sartocon Slice 200 PESU cassette (polyethersulfone membranes; cut-off: 100 kDa; filtration area: 0.1 m2). The purified AI-CTar polymer was then freeze- dried and characterized.

[0271] Figure 3 represents the compared FTIR spectra from raw chitosan and chitosan polymer modified with (+)-diacetyl-L-tartaric anhydride (with a molar ratio of 0.35). FTIR experiments were carried out using an IRAffmity-lS spectrophotometer (Shimadzu). An atmosphere correction was applied on all spectra to remove peaks from CO2. By comparison of both raw and modified chitosans, it is possible to confirm the successful grafting of (+)-diacetyl-L-tartaric anhydride onto N-Glucosamine units. A strong band at 1541 cm'1is observed, corresponding to the formation of an amide II bond between chitosan and (+)- diacetyl -L-tartaric anhydride. The sharp increase in the intensity of this band highlights effective grafting of chitosan by the anhydride. In addition, the bands at 1718 cm'1and 1224 cm'1are characteristic of the C=O elongation of the ester present in the acetate groups of the (+)-diacetyl-L-tartaric anhydride.

[0272] Zeta potential was measured with Zetasizer MPT-2 Malvern and measurements were performed by pH increments of 0.5. AI-CTar (ratio 0.35) has an isoelectric point of 8.9 (Figure 4).

[0273] The degree of acetylation of AI-CTar was measured by1H NMR spectroscopy using the Hirai method (Hirai et al., Polymer Bulletin, 26, 87-94. 1991) and was similar to that of the starting chitosan.

[0274] Knowing the acetylation degree of the modified chitosan, the degree of substitution of Al-Ctar (proportion of N-glucosamine units functionalized with (+)-diacetyl -L-tartaric acid) was determined by1H NMR. Briefly, N-glucosamine and N-acetyl-glucosamine units of chitosan are each constituted of 7 protons, covalently bonded to carbon atoms, among which the CH3 protons of the acetylated units have a very distinguishable chemical shift 2.0 ppm. On the other side, the diacetyl-L-tartaric acid units is constituted of 8 protons linked to carbon atoms, with two CH3 of acetyl groups (chemical shift of 2.1 ppm) and two protons of CH groups (chemical shift of 5.3 ppm) linked to both the acetate groups and carboxylic groups or amide group (once the (+)-diacetyl-L-tartaric acid anhydride has reacted with the NH2 of chitosan). Figure 5 represents the ' H NMR spectrum of chitosan functionalized with (+)- diacetyl-L-tartaric anhydride with a ratio of 0.35. Knowing the acetylation degree, noted x, the degree of substitution of AI-CTar, noted y, which also corresponds to the functionalization rate, may be calculated using the following equation:

[0275] Where HCH3 chitosanis the integration of the CH3 protons of the acetylated units of chitosan (peak at 2.0 ppm), HCH3 Ctaris the integration of the CH3 protons of the acetylated units of (+)-diacetyl-L-tartaric acid (peak at 2.1 ppm), x is the acetylation degree and y the functionalization rate (i.e. the degree of substitution). The degree of substitution of Al-Ctar is calculated to be 19± 2%. Example 3: Preparation of solutions containing AI-CTar polymer and rheological studies

[0276] Formulations A, B and C were prepared with conventional techniques with the functionalized chitosan obtained in example 2 with 0.35 equivalent of (+)-diacetyl-L-tartaric acid, Trastuzumab antibody, and water.

[0277] Functionalized chitosan obtained in example 2 (AI-CTar) was easily solubilized in ultrapure water up to 10% (w / v) with a pH of 5.3. The solution obtained was recovered and introduced into a suitable fluid dispenser, then centrifuged at 3500 rpm for 5 minutes to obtain an air bubble-free solution.

[0278] A solubility study at 1 g / L of polymer was performed by increasing the pH of the polymer solution from 3.4 to 11.8 (Figure 6). Gelation of the polymer was visually observed around pH 7.2.

[0279] Rheological properties of various formulations were compared.

[0280] Formulation A comprises 100 g / L functionalized chitosan according to example 2.

[0281] Formulation B comprises 50 g / L functionalized chitosan according to example 2.

[0282] Formulation C comprises 50 g / L functionalized chitosan according to example 2 and 100 g / L Trastuzumab. Briefly, one volume of 100 g / L functionalized chitosan according to example 2 was mixed with the same volume of 200 g / L solution of trastuzumab (redispersed with water from lyophilized powder, Trazimera®).

[0283] Shear values (G’ and G”) were measured with a Rheostress 600 rheometer from Thermo Scientific HAAKE by a dynamic frequency sweep test from 0.05 rad / s to 100 rad / s at a constant strain of 1% at 25°C.

[0284] Figure 7 represents the obtained G’ and G” values for formulations A, B and C. When G’ curve is higher than G’ ’ curve, the formulation is a gel. When G’ ’ curve is higher than G’ curve, the formulation is a solution. The loss tangent, noted tan 5. is the ratio between G” and G’ and was calculated at an angular frequency of 1 rad / s. In general, when tan 8 < 1 the material is considered as a viscoelastic solid at that frequency, whereas when tan 8 > 1 the material is considered as a viscoelastic liquid at that frequency. The value of tan 5 indicates the density of the network; the closer it is to 0, the denser the network and therefore the more difficult it is to inject. Figure 7 and Table 1 show that all tested formulations are injectable solutions.

[0285] Table 1: Rheological properties of each Al-Ctar formulation at 1 rad / s.

[0286] Storage modulus G’ Loss modulus G” tan 5

[0287] Formulation A 67.4 100.0 1.48

[0288] Formulation B 3.1 9.0 2.90

[0289] Formulation C 34.2 53.5 1.56 Example 4: Gel formation of Al-Ctar polymers

[0290] Formulations A, B and C described in example 3 were injected in a 10 mM PBS solution using a 23G needle. Immediate gelling was observed, which could be assessed by the formation of small gel threads.

[0291] Example 5: Preparation of solutions containing AI-CACO polymer and rheological studies

[0292] Functionalized chitosan obtained in example 1 (AI-CACO) was easily solubilized in a 1% (w / w) acetic acid solution up to 75 g / L for the ratio of 0.18 (between cis-aconitic anhydride and chitosan in the reaction) and up to 50 g / L for the higher ratios tested. The pH was between 4.80 and 5.20.

[0293] A solubility study at 1 g / L of polymer was performed by increasing the pH of the polymer solution from 3.6 to 9.5 (Figure 8). Gelation of the polymer was visually observed around 7.1, which is coherent with the zeta potential measurement in Figure 2.

[0294] Formulations D, E and F were prepared with conventional techniques with the functionalized chitosan obtained in example 1 with 0.18 equivalent of cis-aconitic acid, Trastuzumab antibody, 1% (w / w) acetic acid solution and water.

[0295] Rheological properties of various formulations were compared.

[0296] Formulation D comprises 75g / L functionalized chitosan according to example 1 with 0.18 equivalent of cis-aconitic acid.

[0297] Formulation E comprises 50 g / L functionalized chitosan according to example 1 with 0.18 equivalent of cis-aconitic acid.

[0298] Formulation F comprises 25 g / L functionalized chitosan according to example 1 with 0.18 equivalent of cis-aconitic acid, and 100 g / L Trastuzumab. Briefly, one volume of 50 g / L functionalized chitosan according to example 1 was mixed with the same volume of 200 g / L solution of trastuzumab (redispersed with water from lyophilized powder, Trazimera®).

[0299] Shear values (G’ and G”) were measured with a Rheostress 600 rheometer from Thermo Scientific HAAKE by a dynamic frequency sweep test from 0.05 rad / s to 100 rad / s at a constant strain of 1% at 25°C.

[0300] Figure 9 presents the obtained G’ and G” values for formulations D, E and F. When G’ curve is higher than G” curve, the formulation is a gel. When G” curve is higher than G’ curve, the formulation is a solution. The loss tangent, noted tan 5. is the ratio between G” and G’ and was calculated at an angular frequency of 1 rad / s. In general, when tan 8 < 1 the material is considered as a viscoelastic solid at that frequency, whereas when tan 8 > 1 the material is considered as a viscoelastic liquid at that frequency. The value of tan 5 indicates the density of the network; the closer it is to 0, the denser the network and therefore the more difficult it is to inject. Figure 9 and Table 2 show that formulation D is a gel whereas formulations E and F are solutions, and all three formulations are easily injectable. In particular, the felt compressive strength for all three formulations is below the clinical standard of 64N.

[0301] Table 2: Rheological properties of each AI-CACO formulation at 1 rad / s.

[0302] Storage modulus G’ Loss modulus G” tan 5

[0303] Formulation D 267 166 0.62

[0304] Formulation E 23.9 31 1.29

[0305] Formulation F 4.53 7.40 1.63

[0306] Example 6: Gel formation of AI-CACO polymers

[0307] Formulation F described in example 5 was injected in a 10 mM PBS solution using a 23G needle. Immediate gelling was observed, which could be assessed by the formation of small gel threads.

[0308] Rheological properties of various formulations 15 min after injection in a Na2HPO4 / NaH2PC>4 buffer were compared.

[0309] Formulation G comprises 25 g / L functionalized chitosan according to example 1 with 0.25 equivalent of cis-aconitic acid.

[0310] Formulation H comprises 25 g / L functionalized chitosan according to example 1 with 0.25 equivalent of cis-aconitic acid and 100 g / L Trastuzumab. Briefly, one volume of 50 g / L functionalized chitosan according to example 1 was mixed with the same volume of 200 g / L solution of trastuzumab (redispersed with water from lyophilized powder, Trazimera®).

[0311] Shear values (G’ and G”) were measured with a MCR 502 Anton Paar rheometer using a parallel plate (PP) measuring geometry of 20 mm diameter, by a dynamic frequency sweep test from 0. 1 rad / s to 100 rad / s at a constant strain of 1% at 25°C.

[0312] Figure 10 represents the obtained G’ and G” values for formulations G and H, 15 min after their injection in phosphate buffer. When G’ curve is higher than G” curve, the formulation is a gel. When G’ ’ curve is higher than G’ curve, the formulation is a solution. The loss tangent, noted tan 5. is the ratio between G” and G’ and was calculated at an angular frequency of 1 rad / s. Figure 10 and Table 3 show that formulations G and H can form a gel after 15 min in Na2HPO4 / NaH2PC>4 buffer.

[0313] Table 3: Rheological properties of each AI-CACO hydrogel at 1 rad / s.

[0314] Storage modulus G’ Loss modulus G” tan 6

[0315] Formulation G 2591.0 278.15 0.12

[0316] Formulation H 1843.6 194.74 0.11 Example 7: In vivo safety study of AI-CACO / Trastuzumab formulations

[0317] Two formulations of AI-CACO polymers, synthetized as described in example 1 and mixed with Trastuzumab solutions, were tested. Briefly, AI-CACO solutions were prepared as described in example 5 and trastuzumab solution was reconstituted in water at 200 g / L from the freeze-dried powder (420 mg Trazimera® vial). Formulations were prepared by mixing an equivalent volume of polymer and trastuzumab solutions.

[0318] 2 mice (Balb c) were injected subcutaneously with 100 pL of 25 g / L AI-CACO (ratio 0.35) with 100 g / L trastuzumab and 2 mice (Balb c) were injected subcutaneously with 100 pL of 15 g / L AI-CACO (ratio 0.35) with 100 g / L trastuzumab.

[0319] After 3 weeks, no adverse effects were observed in all mice despite the large dose injected. Degradation of hydrogel was assessed by visual inspection at the injection site. The formulation with 25 g / L of AI- CACO was not degraded after 3 weeks whereas the formulation with 15 g / L AI-CACO was not visible at all at the injection site after 3 weeks (Table 4).

[0320] Table 4: Hydrogel degradation after SC injection in mouse by visual inspection

[0321] (+: presence of a hydrogel lump under the skin, -: no visual lump)

[0322] TO 3 weeks

[0323] 25 g / L AI-CACO (ratio 0.35) + +

[0324] + 100 g / L trastuzumab

[0325] 15 g / L AI-CACO (ratio 0.35) +

[0326] + 100 g / L trastuzumab

[0327] Example 8: In-vivo study of sustained release of trastuzumab by ALCtar gel

[0328] 15 mice (Balb c) were injected subcutaneously (in the flank) with 100 pL of Al-Ctar formulation loaded with 100 mg / mL of trastuzumab, prepared according to example 3, corresponding to a dose of 500 mg / kg.

[0329] Body weight of mice was monitored for 28 days and results showed that no macroscopic toxicity occurred to the mice (Figure 11).

[0330] Antibody release was compared to previous studies using standard of care treatments (normalized to the same injection volume): IV Trastuzumab (Trazimera, Pfizer), corresponding to a dose of 500 mg / kg of antibody and SC enzymatic formulation of Trastuzumab (Herceptin SC, Roche), corresponding to a dose of 600 mg / kg.

[0331] Following AI-Ctar / Trastuzumab formulation administration, plasmatic concentration of trastuzumab antibody was measured by Time-resolved fluorescence energy transfer (TR-FRET) quantification. Plasma level of trastuzumab was measured according to the manufacturers’ instructions (HTRF Human IgG Detection kit from Revity).

[0332] Figure 12 presents the pharmacokinetic profde of Al-Ctar co-formulation with trastuzumab and shows a more controlled and sustained release of trastuzumab compared to standard treatment (with a more homogenous release and a limited burst effect).

[0333] Example 9: Formulation and rheological studies of Trastuzumab emtansine and AI-CTar polymer

[0334] Formulations were prepared with conventional techniques with the functionalized chitosan obtained in example 2 with 0.35 equivalent of (+)-diacetyl-L-tartaric acid, Trastuzumab emtansine (T-DM1) antibody drug conjugate (ADC), and water.

[0335] Functionalized chitosan obtained in example 2 (AI-CTar) was easily solubilized in ultrapure water up to 10% (w / v) with a pH of 5.3. The solution obtained was recovered and introduced into a suitable fluid dispenser, then centrifuged at 3500 rpm for 5 minutes to obtain an air bubble-free solution.

[0336] Formulation comprises 50 g / L functionalized chitosan according to example 2 and 50 mg / mL T-DM1. Briefly, one volume of 100 g / L functionalized chitosan according to example 2 was mixed with the same volume of 100 mg / mL solution of T-DM1 (redispersed with water from lyophilized powder, Kadcyla®).

[0337] Storage modulus (G’) and loss modulus (G’ ’) were measured with HR10 Discovery rheometer from TA Instrument by a dynamic frequency sweep test from 0.1 rad / s to 100 rad / s at a constant strain of 1% at 25 °C. Dynamic oscillatory strain amplitude sweep measurements were preliminary performed with constant angular frequency (co = 10 rad s ') to investigate the linear-viscoelastic region of formulations.

[0338] Figure 13 represents the obtained G’ and G” values during frequency sweep (left figure) and strain sweep (right figure). When G’ curve is higher than G” curve, the formulation is a gel. When G” curve is higher than G’ curve, the formulation is a solution. The loss tangent, noted tan 5. is the ratio between G” and G’ and was calculated at an angular frequency of 1 rad / s. In general, when tan 8 < 1 the material is considered as a viscoelastic solid at that frequency, whereas when tan 8 > 1 the material is considered as a viscoelastic liquid at that frequency. Figure 13 and Table 5 show that all tested formulations are soft hydrogels.

[0339] Table 5: Rheological properties of each Al-Ctar formulation at 1 rad / s.

[0340] Storage modulus G’ Loss modulus G” tan 5 50 / L A| Ctar +

[0341] 17.8 6.4 0.36

[0342] 50 mg / mL T-DM1 Example 10: Injectability of Al-Ctar formulations with therapeutic antibodies

[0343] Formulations were prepared with conventional techniques with the functionalized chitosan obtained in example 2 with 0.35 equivalent of (+)-diacetyl-L-tartaric acid, Trastuzumab or Trastuzumab emtansine (T-DM1) and water.

[0344] Three formulations were prepared:

[0345] • A formulation comprising 50 g / L functionalized chitosan according to example 3.

[0346] • A formulation comprising 50 g / L functionalized chitosan and 100 mg / mL of trastuzumab according to example 3.

[0347] • A formulation comprising 50 g / L functionalized chitosan and 50 mg / mL T-DM1 according to example 9.

[0348] The flowability was investigated with HR10 Discovery rheometer from TA Instrument by performing rotational shear rate measurements, in which the shear rate was increased from 0.01 s ' to 100 s ' .

[0349] Figure 14 represents the viscosity obtained during the flow sweep experiments for the three formulations and all formulations exhibit a shear-thinning behavior, i.e. viscosity decreases with shear rate, indicating a favorable injectability at high shear rate (typically around 104s1for injection through a syringe).

[0350] The physicochemical properties of Al-Ctar hydrogel after shearing were investigated with a HR10 Discovery rheometer from TA Instrument by performing dynamic oscillatory time sweep measurements with alternating intervals of low strain (ylow = 1 % and co = 10 rad s ' ) and high strain (yhigh =1000 % and co = 10 rad s ' ). The high strain time interval is supposed to simulate an injection process through a needle.

[0351] Figure 15 represents the storage and loss modulus (G’ and G” respectively) over time for trastuzumab (Figure 15 A) and T-DM1 formulations (Figure 15B) with Al-Ctar. Both formulations exhibit a gel structure at t=0 (G’>G”) and when a high shear strain is applied, the modulus decreases, and the formulation is a viscoelastic liquid (G’<G”). However, when the shear strain decreases back to 1%, the hydrogel regains its structure over time. This example highlights that Al-Ctar formulations can be injectable and regain their hydrogel structure after injection.

[0352] Example 11: Toxicity study in mice of Al-Ctar gel loaded with T-DM1 vs T-DM1 alone

[0353] 15 mice (Balb c) were injected subcutaneously (in the flank) with either:

[0354] • 7 pL of Al-Ctar formulation loaded with 50 mg / mL of Trastuzumab emtansine (T-DM1), prepared according to example 9, corresponding to a dose of 17.5 mg / kg of T-DM1. 12 pL of Al-Ctar formulation loaded with 50 mg / mL of Trastuzumab emtansine (T-DM1), prepared according to example 9, corresponding to a dose of 30 mg / kg of T-DM1.

[0355] 15 mice (Balb c) were injected intravenously with the clinically approved IV formulation of T-DM1 (Kadcyla, Roche) at a dose of 15 mg / kg.

[0356] 3 mice (Balb c) were also injected subcutaneously (in the flank) with 100 pL the T-DM1 (Kadcyla, Roche) at different doses: 15 mg / kg, 22.5 mg / kg or 30 mg / kg.

[0357] Body weight of mice was monitored, and results showed no macroscopic toxicity to the mice treated with IV T-DM1 and AI-Ctar / TDMl co-formulations at all tested doses. (Figure 16). Subcutaneous injection of T-DM1 showed no toxicity at 15 and 22.5 mg / kg but at the highest dose (30 mg / kg), a significant weight loss was observed (above ethical criteria), demonstrating a general toxicity at this dose whereas T-DM1 (at the same dose) combined with Al-Ctar hydrogel did not indicate any toxicity.

[0358] Example 12: Biocompatibility of Al-Ctar gel and Al-Ctar gel loaded with T-DM1 on human skin explants

[0359] The study aimed at evaluating the toxicity of the Al-Ctar hydrogel alone or used for the formulation of T-DM1 on human skin explants (Hyposkin models from Genoskin, Toulouse). Al-Ctar hydrogel was prepared according to example 3 (formulation B) and Al-Ctar formulation with T-DM1 (50 mg / mL) was prepared as described in example 9.

[0360] Hyposkin models were subcutaneously injected with Al-Ctar, T-DM1 and AI-Ctar / T-DMl on Day 1 and cultured for up to 7 days (n=3 for each condition). The dose of T-DM1 injected in each biopsy was equivalent between T-DM1 alone and AI-Ctar / TDMl co-formulation (i.e. 2 mg T-DM1 per model). At the end of the incubation period, Hematoxylin & Eosin (H&E) staining was conducted to assess tissue integrity and cell viability.

[0361] On Day 7, Hematoxylin and Eosin (H&E) Staining shows that subcutaneous administration of Al-Ctar alone did not cause any alterations in skin structure (Figure 17). By contrast, models treated with T- DM1 displayed a generalized and strong skin alterations on Day 7, characterized by many pyknotic / eosinophilic cells and loss of nuclei in the epidermis. This was associated with a thickening of the upper layer of the epidermis. For AI-Ctar / T-DMl co-formulation, some skin alterations on Day 7 were also observed (pyknotic / eosinophilic cells and loss of nuclei in the epidermis) but to a lesser extend as T-DM1 alone (no thickening of epidermis observed). H&E staining of the skin models subcutaneously injected with the hydrogel Al-Ctar, with or without T- DM1, revealed the presence of the hydrogel on Day 1, Day 3 and Day 7 confirming the stability of the compound in the dermis for at least 6 days post injection.

[0362] Example 13: In-vivo study of sustained release of T-DM1 by Al-Ctar gel

[0363] As described in example 10, 15 mice (Balb c) were injected subcutaneously (in the flank) with either 7 pL (corresponding to a T-DMldose of 17.5 mg / kg) or 12 pL (corresponding to a T-DMldose of 30 mg / kg) of Al-Ctar formulation loaded with 50 mg / mL of Trastuzumab emtansine (T-DM1), prepared according to example 9.

[0364] To compared antibody release to standard of care treatment, 15 mice were injected with IV T-DM1 (Kadcyla, Roche), corresponding to a dose of 15 mg / kg of T-DM1.

[0365] Plasmatic concentration of T-DM1 antibody was measured by Time-resolved fluorescence energy transfer (TR-FRET) quantification. HTRF Human IgG Detection kit was purchased from Revity . Plasma level of T-DM1 was measured according to the manufacturers’ instructions.

[0366] Figure 18 presents the pharmacokinetic profile of Al-Ctar co-formulation with T-DM1 at two different doses and shows a more controlled release of T-DM1 compared to standard treatment (with a more homogenous release and a limited burst effect).

[0367] Example 14: Therapeutic efficacy of multiple administrations of Trastuzumab in combination with Al-Ctar hydrogel on BT-474 tumor mice model

[0368] Female Athymic nude mice were subcutaneously injected (right flank) with 2xl07BT-474 cells (human breast carcinoma, HER2+) at day 1 (DI). Tumor growth in this model was monitored twice a week and mice were randomized according to tumor size at D13 and treated from D14 to D35. The treatment groups (n=8 per group) are presented in table 6 below:

[0369] Table 6: Treatment plan for therapeutic efficacy study of trastuzumab

[0370] Dose of trastuzumab Group Treatmentz„ , , Route Schedule

[0371] (mg / kg / adm)

[0372] 1 PBS (control) NA SC Single dose

[0373] 2 Al-Ctar NA SC Single dose

[0374] 3 Herceptin SC 15 SC 1 / week for 4w

[0375] 4 Trastuzumab 10 IV 1 / week for 4w

[0376] 5 Al-Ctar + Trastuzumab 15 SC 1 / week for 4w

[0377] 6 Al-Ctar + Trastuzumab 30 SC 1 / week for 4w

[0378] Tumor volume and mice bodyweight were monitored two times a week.

[0379] Figure 19 presents no toxicity (i.e. no body weight loss) associated with Al-Ctar formulations, at all tested doses. However, two mice were euthanized in group 4 (IV Trastuzumab) from D22 post- randomization due to >10% body weight loss, above ethical criteria and two mice were euthanized in group 1 (PBS control) from D42 post-randomization due to tumor necrosis.

[0380] Figure 20 shows that mean fold change tumor size for all trastuzumab treated groups was similar and significantly (p < 0,0001) reduced compared to untreated groups (PBS control and Al-Ctar alone). This study confirms that Trastuzumab in combination with Al-Ctar hydrogel exhibits similar therapeutic efficiency as the clinically approved treatments (Trastuzumab IV or Herceptin SC).

[0381] Example 15: Therapeutic efficacy of single administration of T-DM1 in combination with Al-Ctar hydrogel on BT-474 tumor mice model

[0382] Female Athymic nude mice were subcutaneously injected (right flank) with 2xl07BT-474 cells (human breast carcinoma, HER2+) at day 1 (DI). Tumor growth in this model was monitored twice a week and mice were randomized according to tumor size at D13 and treated from D14 to D35. The treatment groups (n=8 per group) are presented in table 7 below:

[0383] Table 7: Treatment plan for therapeutic efficacy study of T-DM1

[0384] _ Dose of trastuzumab „

[0385] Group Treatmentz„ , , Route

[0386] (mg / kg / adm)

[0387] 1 PBS (control) NA SC

[0388] 2 Al-Ctar NA SC

[0389] 3 T-DM1 17.5 IV

[0390] 4 AI-Ctar + T-DMl 17.5 SC

[0391] 5 AI-Ctar + T-DMl 30 SC

[0392] All treatments were administered as a single injection on D14. Tumor volume and mice bodyweight were monitored two times a week.

[0393] Figure 21 presents no toxicity (i.e. no body weight loss) associated with Al-Ctar formulations, at all tested doses. Two mice were euthanized in group 1 (PBS control) from D42 post-randomization due to tumor necrosis.

[0394] Figure 22 shows that mean fold change tumor size for all T-DM1 treated groups was similar and significantly (p < 0,0001) reduced compared to untreated groups (PBS control and Al-Ctar alone). This study confirms that T-DM1 in combination with Al-Ctar hydrogel exhibits similar therapeutic efficiency as the clinically approved T-DM1 IV formulation (Kadcyla, Roche).

[0395] Example 16: Formulation and rheological studies of Trastuzumab deruxtecan (T-Dxd) and AI- CTar polymer

[0396] Functionalized chitosan obtained in example 2 (AI-CTar) was easily solubilized at 100 g / L as described in example 3.

[0397] Formulation comprises 50 g / L functionalized chitosan according to example 2 and 50 mg / mL Trastuzumab-deruxtecan (T-Dxd). Briefly, one volume of 100 g / L functionalized chitosan according to example 2 was mixed with the same volume of 100 mg / mL solution of T-Dxd (redispersed with water from lyophilized powder, Enhertu®).

[0398] Storage modulus (G’) and loss modulus (G’ ’) were measured with HR10 Discovery rheometer from TA Instrument by a dynamic frequency sweep test from 0.1 rad / s to 100 rad / s at a constant strain of 1% at 25 °C. Dynamic oscillatory strain amplitude sweep measurements were preliminary performed with constant angular frequency (co = 10 rad s ') to investigate the linear-viscoelastic region of formulations.

[0399] Figure 23 represents the obtained G’ and G” values during frequency sweep (left figure) and strain sweep (right figure). Figure 23 and Table 8 show that all tested formulations are soft hydrogels. Table 8: Rheological properties of T-Dxd / AI-Ctar co-formulation at 1 rad / s.

[0400] Storage modulus G’ Loss modulus G” tan 6

[0401] 50 g / L Al-Ctar +

[0402] 52,5 31,4 0.60

[0403] 50 mg / mL T-Dxd

[0404] Figure 24 represents the viscosity behavior of the formulation, when subjected to an increasing shear rate. Al-Ctar coformulation with T-Dxd is injectable as it exhibits a shear-thinning behavior, meaning that the viscosity decreases when the formulation is subject to high shear stress (as could happen during injection though a needle).

[0405] Example 17: Toxicity study in mice of Al-Ctar gel loaded with T-Dxd vs T-Dxd alone

[0406] One mouse was injected subcutaneously (in the flank) with 9 pL of Al-Ctar formulation loaded with 50 mg / mL of Trastuzumab-deruxtecan (T-Dxd), prepared according to example 16, corresponding to a dose of 17 mg / kg of T-Dxd.

[0407] Body weight was monitored, and results showed no macroscopic toxicity to the mouse treated with AI- Ctar / T-Dxd co-formulation (Figure 25).

[0408] Example 18: Biodegradability in mice of Al-Ctar gel loaded with T-DM1 by MRI imaging

[0409] 4 mice (Balb c) were injected subcutaneously (in the flank) with 12 pL (corresponding to a T-DMldose of 30 mg / kg) of Al-Ctar formulation loaded with 50 mg / mL of Trastuzumab emtansine (T-DM1), prepared according to example 9.

[0410] MRI imaging was performed (T2 signal) over a month to follow the degradation of the hydrogel. Figure 26 shows a reduction of the hydrogel volume over time (up to 85%) at 1 month. Noteworthy, the hydrogel was not detectable in 3 out of the 4 mice at 1 month, confirming the biodegradability of the AI-Ctar / TDMl hydrogel.

[0411] Example 19: Biodegradability in mice of Al-Ctar gel loaded with T-DM1 by H&E staining

[0412] 3 mice (Balb c) were injected subcutaneously (in the flank) with 12 pL (corresponding to a T-DMldose of 30 mg / kg) of Al-Ctar formulation loaded with Trastuzumab emtansine (T-DM1).

[0413] Formulation comprises 25 g / kg functionalized chitosan according to example 2 and 50 mg / mL T-DM1. Briefly, one volume of 50 g / kg functionalized chitosan according to example 2 was mixed with the same volume of a 100 mg / mL solution of T-DM1 (redispersed with water from lyophilized powder, Kadcyla®).

[0414] Mice were euthanized after 21 days, and a biopsy of the injection site was performed. Figure 27 represents the hematoxylin and eosin (H&E) staining, highlighting the structure and composition of tissue samples. Arrows indicate the presence of immune cells infiltrate inside the hydrogel structure, confirming an immune cell recruitment and on-going degradation of the hydrogel at day 21.

[0415] Example 20: Formulation of AI-CTar polymer with a peptide

[0416] Formulations were prepared with conventional techniques with the functionalized chitosan obtained in example 2 with 0.35 equivalent of (+)-diacetyl-L-tartaric acid, a peptide P01 having a molecular weight comprised between 1 and 2 kDa, and water.

[0417] Three formulations were tested:

[0418] • Formulation P01-A comprises 25 g / kg functionalized chitosan according to example 2 and 25 g / kg P01. One volume of 100 g / kg functionalized chitosan according to example 2 was mixed with the three volumes of the previous solution of POl / trehalose.

[0419] • Formulation P01-B comprises 25 g / kg functionalized chitosan according to example 2, 50 g / kg P01 and 50 g / kg of trehalose as pharmaceutical excipient. P01 peptide is solubilized at 66 g / kg directly into a trehalose solution (66 g / kg). Then one volume of 100 g / kg functionalized chitosan was mixed with the three volumes of the previous solution of POl / trehalose.

[0420] • Formulation P01-C comprises 50 g / kg functionalized chitosan according to example 2 and 25 g / kg P01. Briefly, one volume of 100 g / kg functionalized chitosan was mixed with the same volume of 50 g / kg solution of peptide P01.

[0421] Figure 28A show that formulations exhibit a shear-thinning behavior, meaning that the viscosity decreases when the formulation is subject to high shear stress.

[0422] The injectability of the samples was evaluated using a Shimadzu AGX plus force machine equipped with a 10 kN load cell to measure the force required to depress the plunger at a controlled rate. Syringes (1 mb BD Hylok™ glass pre-fillable) containing the formulation and fitted with 23G needles were placed in a chamber matching the syringe barrel's outer dimensions. The injection force was measured at a constant plunger speed of 1 mm s-1. Figure 28B also highlights that the 3 formulations are easily injectable as the felt compressive strength is below the clinical standard of 64N.

[0423] The release of P01 was also quantified in vitro. 800 pL of formulation P01-A (25 g / kg Al-Ctar, 25 g / kg P01) were injected in 200 mb of physiological buffer. Over time, 1 mb of supernatant was sampled for quantification of P01 peptide by HPLC-UV and replaced with fresh buffer. Figure 29 confirms that 40% of peptide can be released by diffusion mechanism in 48h from the formulation.

Claims

1. CLAIMS1. Pharmaceutical formulation comprising:- a therapeutically effective amount of at least one protein, and- functionalized chitosan, wherein at least part of the primary amine groups of the glucosamine units of chitosan are functionalized with a functionalizing moiety comprising at least one acidic function, wherein the functionalized chitosan represents more than 90 wt% of the total weight of polysaccharides in the formulation.

2. The pharmaceutical formulation according to claim 1, wherein at least one of the acidic functions has a pKa comprised between 4.5 and 8, and / or wherein the basic form of at least one of the acidic functions is negatively charged.

3. The pharmaceutical formulation according to claim 1 or claim 2, wherein the functionalizing moiety comprises:- at least one unsaturated bond, such as at least one double bond or triple bond,- at least one hydrophilic moiety different from the acidic functions, such as an acetyl function and / or a hydroxylic function, and / or- at least one ester function.

4. The pharmaceutical formulation according to any one of claims 1 to 3, wherein the functionalizing moiety is linked to the primary amine groups of the glucosamine units of chitosan via an amide bond.

5. The pharmaceutical formulation according to any one of claims 1 to 4, wherein the functionalizing moiety is obtained by coupling the primary amine groups of the glucosamine units of chitosan with a anhydride, said anhydride being preferably selected from the group consisting of cis -aconitic anhydride, (+)-diacetyl-L-tartaric anhydride, 1,2,4-benzenetricarboxylic anhydride, pyromellitic dianhydride and 3,3 ’,4, 4 ’-biphenyltetracarboxylic dianhydride.

6. The pharmaceutical formulation according to any one of claims 1 to 5, wherein the protein is selected from the group consisting of an antibody or a fragment thereof, an antibody-drug conjugate, an enzyme, a fusion protein and any mixture thereof, preferably an antibody-drug conjugate or an antibody or a fragment thereof, more preferably Trastuzumab or trastuzumab emtansine.

7. The pharmaceutical formulation according to any one of claims 1 to 6, the protein is an antibody-drug conjugate, and wherein the functionalizing moiety preferably comprises at least one ester function and / or one acetyl function.

8. The pharmaceutical formulation according to any one of claims 1 to 7, wherein the protein concentration in the formulation is comprised between 10 and 200 g / L, preferably between 50 and 150 g / L.

9. The pharmaceutical formulation according to any one of claims 1 to 8, wherein the acetylation rate of the functionalized chitosan is 20% or lower, preferably 10% or lower.

10. The pharmaceutical formulation according to any one of claims 1 to 9, wherein from 5 to 40%, preferably from 10 to 30%, of the primary amine groups of the glucosamine units of chitosan are functionalized with the functionalizing moiety.

11. The formulation according to any one of claims 1 to 10, wherein the functionalized chitosan concentration in the pharmaceutical formulation is comprised between 10 and 100 g / L, preferably between 20 and 50 g / L, more preferably between 20 and 30 g / L.

12. Process for the preparation of a pharmaceutical formulation according to any one of claims 1 to 11, comprising the steps of: a) providing a solution containing a functionalized chitosan as defined above; b) providing a solution containing a protein or a combination of protein ; c) mixing the solution of step a) and the solution of step b) to obtain a homogenous solution; d) optionally, adjusting the osmolarity of the solution obtained in step c) to provide an osmolarity comprised between 50 and 300 mOsm / L, preferably between 50 and 250 mOsm / L; e) optionally, adjusting the pH of the solution obtained in step c) between 5.0 and 6.5, preferably between 5.0 and 6.4, more preferably between 5.5 and 6.0.

13. A kit comprising:- at least one first container containing a functionalized chitosan, and- at least a second container comprising a protein, wherein the functionalized chitosan and the protein are as defined in any one of claims 1 to 11.

14. A hydrogel comprising a formulation according to any one of claims 1 to 11 and water, wherein the hydrogel is preferably injectable.

15. The pharmaceutical formulation according to any one of claims 1 to 11; the kit according to claim 13 or the hydrogel according to claim 14, for use as a medicament in a patient, preferably a patient having a cancer or an autoimmune disease.

16. The pharmaceutical formulation according to any one of claims 1 to 11, the kit according to claim 13 or the hydrogel according to claim 14, for use in the treatment of a cancer.

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