Alginate-based hydrogel compositions and uses thereof

Abstract

The present invention relates to hydrogel compositions comprising oxidized and chemically modified alginate. Such hydrogel is suitable for encapsulating active ingredients, such as T cells, with optimal rheological and injectability properties, hence they can be advantageously used in the treatment of solid tumors.

Description

[0001] ALGINATE-BASED HYDROGEL COMPOSITIONS AND USES THEREOF

[0002] Technical field

[0003] The present invention relates to the field of a hydrogel formulations for drug and cell delivery applications, in particular for controlled release of antitumoral agents and treatment of solid tumors.

[0004] Background

[0005] Cell therapy involves the delivery of cell-based therapies to specific sites, such as tissues or organs, ensuring that the administered cells fulfill their intended function, which is crucial for the success of the treatment. T-cell transfer therapy, a type of immunotherapy, enhances the patient’s own immune cells to target cancer more effectively. This approach involves isolating immune cells from the patient, expanding them in large numbers, and reintroducing them into the bloodstream. However, systemic administration of T cells entails several challenges, including dose-limiting toxicities, off-target effects, and inefficient T-cell trafficking and infiltration. Locoregional T cell delivery methods have emerged as promising alternatives to address these limitations, offering a more controlled delivery to the tumor microenvironment (Sagnella et al., Pharmacological Research 2022, 182; D0l:10.1016 / J.PHRS.2022.106329).

[0006] In this context, biomaterials have gained significant attention fortheir potential to improve the local delivery of adoptive cell therapy (ACT). A key advantage of using biomaterials is their ability to enable localized treatments with lower doses, minimizing systemic toxicity while improving therapeutic efficacy. Indeed, injectable hydrogels offer potential for minimally invasive delivery techniques compared to traditional non-injectable hydrogels, which typically require more invasive placement methods. Several recent studies highlight the effectiveness of this approach. For example, Apple et al. explored the use of advanced hydrogels for CAR-T cell delivery in solid tumors. Their study demonstrated that: (i) local co-injection of CAR-T cells with immunostimulatory cytokines within a hydrogel creates a synthetic immunostimulatory niche; (ii) high local concentrations of cytokines are retained in the hydrogel, avoiding systemic toxicity that would occur with intravenous administration; (iii) CAR-T cell expansion and activation are promoted in this environment, resulting in anti-tumor effects in a medulloblastoma model; and (iv) distal application of the hydrogel also triggered systemic immune responses (Grosskopf et al., Science Advances 2022, 8(14), 8264; doi: 10.1126 / sciadv.abn8264). Another study used thermoresponsive chitosan-polyethylene glycol (PEG) hydrogels for localized and sustained delivery of CAR-T cells in aretinoblastoma (RB) model. This study demonstrated that CAR-T cells targeting the GD2 ganglioside, overexpressed in the RB, together with local release of interleukin-15, successfully eliminated tumor cells without affecting the vision of treated mice (Wang et al., Nature Cancer2020, 7(10), 990-997; doi: 10.1038 / s43018-020-00119-y). In another study by Hu and colleagues, hyaluronic acid scaffolds were loaded with: (i) CAR-T cells directed against human chondroitin sulfate proteoglycan 4, (ii) nanoparticles encapsulating interleukin-15, and (iii) platelets conjugated to the checkpoint inhibitor PDL1 (Hu et al., Nature Biomedical Engineering 2021, 5(9), 1038-1047. doi:10.1038 / s41551-021-00712-1). Their approach inhibited local tumor recurrence and reduced distant tumor growth in a postoperative melanoma model. This localized immunotherapy, delivered via biocompatible hydrogel scaffolds, presents a promising strategy to prevent cancer recurrence after tumor resection.

[0007] Alginate, a natural biomaterial derived from algae, is commonly used in the fabrication of hydrogels due to its biocompatibility, biodegradability, and cross-linking flexibility. Cross-linking stability and hydrogel porosity are important features influencing the degradation rate and cellular release profiles. Ionically cross-linked alginate hydrogels often lack thermal stability and degrade rapidly under physiological conditions (Desai et al., Biomaterials 2015, 50(1), 30-37; doi: 10.1016 / j. biomaterials.2015.01.048; J. Tan et al., International Journal of Biological Macromolecules 2023, 239, 124275. doi:10.1016 / J. IJBIOMAC.2023.124275). However, covalently cross-linked alginates, although less biodegradable, can be modified to increase their degradability by oxidation (Lueckgen et al., Biomaterials 2018, 181, 189-198. doi: 10.1016 / j. biomaterials.2018.07.031). The porosity of hydrogels affects the transport of molecules, cells, and fluids within the matrix, and recent advances allow the mesh size of the alginate hydrogel to be tuned by modifying the polymer concentration, molecular weight, degree of functionalization, and cross-linking density. No many examples are known of biocompatible and naturally-derived hydrogels encapsulating antitumoral agents. A study by Luo and colleagues demonstrated the effectiveness of alginate microsphere as an injectable immune microchip (i-G / MC) system to intratumorally deliver CAR-T cells and enhance their therapeutic efficacy in ovarian adenocarcinomas (Luo et al., ACS Appl. Mater. Interfaces 2020, 12, 51, 56712-56722). The microchips not only served as cell carriers but also created an immune niche to enhance the survival and potency of intratumorally delivered CAR-T cells with Hemo and IL-15. The combination of Hemo and IL-15 promoted the cytotoxicity and memory of CAR-T cells. They also demonstrated that i-G / MC could deliver Heme and IL-15 in a sustained manner and retain them for a prolonged period, much longer than direct injection of the two drugs.There is a need in the art for new biocompatible, injectable materials which allow the controlled release of active ingredients, in particular antitumoral agents.

[0008] Brief description of the invention

[0009] To solve the above-mentioned drawback of prior art, the inventors have carefully devised a new injectable hydrogel composition based on alginate which promotes the viability, proliferation and release of T lymphocytes. In particular, the hydrogel comprises low molecular weight alginate, which is partially oxidized, then reduced and crosslinked through appropriate moieties attached to the alginate chain, at low concentration (with respect to the hydrogel composition).

[0010] Rheological characterization and injectability tests have shown that the new hydrogel per se has desirable and advantageous mechanical properties for injection into patients. In addition, when encapsulating an antitumoral agent such as T cells (e.g. CAR T cells), dendritic cells, cytokines, etc. into the crosslinked alginate polymer, this has shown high viability and proliferation after gel injection. Notably, the release of antitumoral agents from within the gel into a given medium was studied and a controlled and steady release was observed during 10 days. The cytotoxic capacity of the antitumoral agent encapsulated in the hydrogel was also demonstrated, since their release for at least 7 days eliminated up to 95% of tumor cells.

[0011] Thus, a first aspect of the invention relates to an injectable hydrogel composition for use in the treatment or therapy of a solid tumor, said hydrogel comprising a covalently crosslinked alginate polymer comprising:

[0012] - a first alginate polymer modified with a first covalently crosslinkable moiety; and - a second alginate polymer modified with a second covalently crosslinkable moiety,

[0013] wherein the modified first and second alginate polymers are covalently crosslinked through the first and second crosslinkable moieties;

[0014] and wherein:

[0015] from 2% to 10 % of the saccharide units of each of the first and second alginate polymers are oxidized;

[0016] each of the first and second alginate polymer has a molecular weight ranging from 50 to 100 kDa;the concentration of the covalently cross-linked alginate polymer in the hydrogel composition ranges from 0.5 to 1.25% w / v; and

[0017] - the hydrogel further comprises at least an antitumoral agent encapsulated within the covalently crosslinked alginate polymer.

[0018] In a second aspect, the invention refers to an injectable hydrogel composition comprising a covalently crosslinked alginate polymer comprising:

[0019] - a first alginate polymer modified with tetrazine moieties;

[0020] - a second alginate polymer modified with norbornene moieties, and wherein the first alginate polymer is covalently crosslinked with the second alginate polymer through [4+2] adducts of tetrazine moieties and norbornene moieties; and wherein:

[0021] - about 5% of the saccharide units of each of the first and second alginate polymer are oxidized;

[0022] - the first and second alginate polymer have each a molecular weight ranging from 50 to 100 kDa;

[0023] - the molar ratio of norbornene and tetrazine moieties ranges from 1.5:1 to 2:1 ; preferably said ratio is 1.8:1; and

[0024] - the concentration of the covalently crosslinked alginate polymer in the hydrogel ranges from 0.5 to 1.25% w / v.

[0025] In a third aspect, the invention refers to a cell therapy kit comprising:

[0026] - a first alginate polymer comprising tetrazine moieties;

[0027] - a second alginate polymer comprising norbornene moieties;

[0028] - a water buffered solution; and

[0029] - at least an antitumoral agent;

[0030] wherein:

[0031] - about 5% of the saccharide units of each of the first and second alginate polymer are oxidized;

[0032] - the first and second alginate polymer have each a molecular weight ranging from 50 to 100 kDa; preferably from 60 to 90 kDa; more preferably from 70 to 80 KDa; even more preferably is about 75 KDa;- the molar ratio between norbornene and tetrazine groups moieties ranges from 1.5: 1 to 2: 1 ; preferably said ratio is 1.8:1; and

[0033] - the amount of first alginate polymer, second alginate polymer and water buffered solution are such as to form a hydrogel comprising a covalently crosslinked alginate polymer with a concentration from 0.5 to 1.25% w / v.

[0034] Brief description of figures

[0035] Fig. 1. Rheological characterization of hydrogels. Flow curve (A), time sweep of 1% alginate hydrogels (according to the invention) (B), time sweep of 2% alginate hydrogels (comparative) (C), frequency sweep (D), elastic modulus (E), mesh size estimation (F) stress relaxation curves (G) and relaxation time (H).

[0036] Fig. 2. Evaluation of hydrogel injectability. Injection force (A), post-injection cell viability (B), gel status (C) and images of encapsulated and injected live Jurkat cells (D).

[0037] Fig. 3. Injection force during the gelation of HG1 hydrogels using low-molecular-weight (LMW, ~75 KDa) and high-molecular-weight (HMW, >200 KDa) alginate.

[0038] Fig. 4. T cell encapsulation, viability, growth and release from hydrogel to media. Live and dead cell quantification (A), DAPI / phalloidin staining (B), quantification of cells outside the hydrogels (C) and microscopic images of released Jurkat-TOM cells where hydrogels periphery is marked (D).

[0039] Fig. 5. Experimental design for evaluating the viability and cytotoxic capacity of CAR-T cells encapsulated in HG1 hydrogel.

[0040] Fig. 6. Cytotoxic capacity of CAR-T cells measured by tumor-cell death using bioluminescence. All bioluminescence signals were normalized to the positive control (group -) for each time point. Statistical differences relative to the - control group are indicated with *, while statistical differences between different experimental groups within the same time point are indicated with #.

[0041] Detailed description of the invention

[0042] As mentioned above, a first aspect of the invention refers to an injectable hydrogel composition for use in the treatment or therapy of a solid tumor, said hydrogel comprising a covalently crosslinked alginate polymer comprising:

[0043] - a first alginate polymer modified with a first covalently crosslinkable moiety; and- a second alginate polymer modified with a second covalently crosslinkable moiety,

[0044] wherein the modified first and second alginate polymers are covalently crosslinked through the first and second crosslinkable moieties;

[0045] and wherein:

[0046] from 2% to 10 % of the saccharide units of each of the first and second alginate polymers are oxidized;

[0047] each of the first and second alginate polymer has a molecular weight ranging from 50 to 100 kDa;

[0048] the concentration of the covalently cross-linked alginate polymer in the hydrogel composition ranges from 0.5 to 1.25% w / v; and

[0049] the hydrogel further comprises at least an antitumoral agent encapsulated within the covalently crosslinked alginate polymer.

[0050] As used throughout the specification and claims, the singular forms “a”, “an” and “the” include the plural form, unless the context clearly indicates otherwise.

[0051] The use of the terms “comprising”, “containing”, “having”, “including”, and variations thereof herein are meant to encompass the items listed thereafter, and equivalents thereof as well as additional items. The terms “comprising”, “comprises” and similar terms encompass the meaning of “consisting of”, “consists of”, etc.

[0052] For the purposes of the invention, any given ranges include both the lower and the upper end-points of the range. Ranges or values given should be considered approximate when they are defined by the term “about” (i.e. with a 5% margin of variation around indicated point).

[0053] As described herein, “C1-6 alkylene” refers to a linear or branched divalent hydrocarbon group containing 1 to 6 carbon atoms and their branched isomers. Preferably, the “C1-6 alkylene” is a linear divalent hydrocarbon group. More preferably, the “C1-6 alkylene” is a linear C1-3 alkylene, e.g. methylene (-CH2-), ethylene (-CH2CH2-), or propylene (-CH2CH2CH2-).

[0054] As described herein, “Ce arylene” refers to a phenylene (-C6H4-) group, with two available bonding sites that can be in the ortho, meta, or para positions, i.e. o-phenylene (ortho isomer), m-phenylene (meta isomer) and p-phenylene (para isomer).

[0055] As described herein, the term “C5-10 N-heteroaryl” designates a 5- to 10-membered ring system, comprising at least one heteroaromatic ring containing at least one N atom, butoptionally also O and / or S heteroatoms; preferably the C5-10 N-heteroaryl is a Ge N-heteroaryl (the ring is a 6-membered ring).

[0056] As used herein, the term “halogen” refers to -F, -Cl, -Br, or -I.

[0057] The term “injectable” as used herein means that the hydrogel composition is suitable for administration via parenteral routes, including but not limited to intravenous (IV), intramuscular (IM), subcutaneous (SC), intradermal (ID) injection or intratumoral (IT). It is implied that the hydrogel possesses physicochemical properties, such as sterility, biocompatibility, stability, and viscosity, which makes it appropriate to enable safe and effective administration using a syringe, infusion system, or any other injectable delivery device.

[0058] As used herein, the term “effective amount” refers to a quantity of antitumoral agent or combination of antitumoral agents that is sufficient to produce a desired therapeutic effect in a subject in need thereof. The effective amount may vary depending on factors such as the specific condition being treated, the severity of the condition, the subject's age, weight, and overall health, as well as the type of injection of the specific hydrogel formulation. An effective amount does not necessarily imply a complete cure but includes amounts sufficient to reduce, alleviate, or manage symptoms or to delay the progression of the solid tumor.

[0059] As defined herein, a “water-buffered solution” refers to an aqueous solution that contains a buffering system designed to maintain a stable pH within a specified range, ensuring an optimal environment for biological or chemical processes. It is known in the art that, when a cell culture is added to the water-buffered solution, this may also include cell culture media necessary to support cell growth and proliferation (such as amino acids, vitamins, glucose, salts and electrolytes to maintain osmotic balance, growth factors, hormones, and other signaling molecules, among others).

[0060] The term “alginate” refers to a natural, biodegradable polysaccharide derived from brown seaweed, composed primarily of mannuronic acid (M) and guluronic acid (G) units arranged in varying sequences according to the general formula below (m and n are the number of repeating units):

[0061]

[0062] The most common source of commercial alginate is brown algae (Phaeophyceae), which contain alginate in their cell walls to provide structural support and flexibility, although certain bacteria naturally may also produce alginate as part of their exopolysaccharide matrix (e.g. Pseudomonas aeruginosa, Pseudomonas fluorescens, Azotobacter vinelandii). The molecular weight (MW) of alginate may vary significantly depending on its source, extraction method, and processing conditions. It typically falls within the range of 10 kDa to over 1,000 kDa, however a “low molecular weight alginate” (as defined in the first aspect, i.e. between 50 to 100 kDa) was employed in the present invention. It has to be noted that the molecular weight of the first and second alginate polymer refers to the alginate polymer prior to oxidation and modification with crosslinkable groups. Alginate may be found in the form of salts (e.g., sodium alginate, calcium alginate, etc.) and other chemically modified forms.

[0063] In an embodiment, the alginate is sodium alginate, preferably high guluronic acid sodium alginate (that is sodium alginate with a content of guluronic acid residues of 60% or more compared to the sum of guluronic and mannuronic acid residues).

[0064] In an embodiment of the first aspect, from 3.5% to 7.5% of the saccharide units of each of the first and second alginate polymers are oxidized; preferably about 5% of the saccharide units of each of the first and second alginate polymers are oxidized. By the term “oxidized” or the expression that a certain % of the saccharide units of alginate are oxidized (according to the embodiments of this invention) it is meant an alginate that has been subjected to an oxidation step to cleave the C2-C3 bond of the uronic acid residues, converting hydroxyl (-OH) groups into aldehydes (-CHO) with subsequent reduction of such aldehydes to hydroxyl groups. This reduction of the aldehyde groups and / introduction of crosslinkable groups in the alginate chain do not change the % of oxidized saccharide units.

[0065] Oxidation may be carried out with different oxidants, in particular with a periodate salt (such as sodium periodate) which cleaves the C2-C3 bond of the uronic acid residues, converting hydroxyl (-OH) groups into aldehydes (-CHO) as stated above. This oxidation step may increase biodegradability and enhance cell adhesion in biomedical applications. However, the free aldehyde groups are then selectively reduced to -OH groups, e.g. with ammonia borane. Thus, this two-step chemical modification of alginate to an oxidized and aldehyde-free alginate makes it more hydrolytically degradable without preventing efficient crosslinking and gel formation. Subsequently, the first and second alginate polymers are modified with a first and second crosslinkable moiety, respectively.In a preferred embodiment of the invention, the first and second alginate polymer have each a molecular weight ranging from 60 to 90 kDa; more preferably from 70 to 80 kDa; even more preferably, the molecular weight of the alginate is about 75 kDa. The molecular weight of the alginate may be determined according to techniques known in the art, among these Size Exclusion Chromatography (SEC) and Gel Permeation Chromatography (GPC) are quite common.

[0066] In the context of the invention, the alginate is modified in its chemical structure by covalently cross-linkable functional groups.

[0067] In a particular embodiment, the first and second alginate polymers are each modified in their chemical structure by first and second covalently crosslinkable moieties, respectively, each crosslinkable moiety having at least an amino group which allows the binding of said moieties to the alginate chain (specifically, to the carboxyl groups of the alginate chain via an amidic bond). In a preferred embodiment, the covalently crosslinkable moieties have each one amino group and are attached to the first and second alginate polymers through said on amino group.

[0068] In an embodiment, the first alginate polymer comprises a diene moiety as first crosslinkable moiety. In a particular embodiment, the diene moiety is attached to the carboxyl group of the first alginate polymer through an amino group in the diene.

[0069] Examples of suitable diene moieties are 1,2,4-triazines (such as 6-methyl-1,2,4-triazine, 6-phenyl-1,2,4-triazine, 6-cyano-1,2,4-triazine, 6-amino-1,2,4-triazine, 6-chloro-1,2,4-triazine, 6-methoxy-1,2,4-triazine, 6-nitro-1,2,4-triazine, and the like), tetrazines (such as 3,6-di-2-pyridyl-1 ,2,4,5-tetrazine, 3,6-diphenyl-1,2,4,5-tetrazine, and the like) isoxazoles 1,2,4-oxadiazoles, 1,4-benzoquinone, 1,4-naphthoquinone, pyrazines (such as 2,3-dicyanopyrazine), pyridazines (such as 3,6-dichloropyridazine), 6-azauracil, 1,4-dicyanobutadiene, 1,4-bis(trifluoromethyl)-butadiene, and maleic anhydride derivatives. In a preferred embodiment, the first alginate polymer comprises a tetrazine moiety, preferably attached to the carboxyl groups of the alginate via an amidic bond with an amino group in the tetrazine moiety.

[0070] In another embodiment, the second alginate polymer comprises a dienophile moiety as second crosslinkable moiety. In a particular embodiment, the dienophile moiety is attached to the carboxyl group of the second alginate polymer through an amino group in the dienophile.

[0071] Examples of suitable dienophile moieties are norbornene, cyclooctene, cyclooctyne, bicyclo[6.1.0]nonyne, dibenzocyclooctyne, ethyl vinyl ether, butyl vinyl ether, allyl ethers, vinyl sulfides, N-methylaniline, methoxystyrene, methyl acrylate, butyl acrylate,acrylamides, trimethylsilyl enol ethers, vinyl trimethylsilane, N-alkyl imines, oxime ethers, furans, indoles, and propargyl ethers. In a preferred embodiment, the second alginate polymer comprises a norbornene moiety, preferably attached to the carboxyl groups of the alginate via an amidic bond with an amino group in the norbornene moiety.

[0072] In an embodiment, the first alginate polymer comprises a diene moiety as first crosslinkable moiety, preferably attached to the carboxyl group of the first alginate polymer through an amino group in the diene, and the second alginate polymer comprises a dienophile moiety as second crosslinkable moiety, preferably attached to the carboxyl group of the second alginate polymer through an amino group in the diene. Thus, in an embodiment, the covalently crosslinked alginate polymer comprises:

[0073] - a first alginate polymer modified with a diene moiety, preferably a tetrazine moiety; and

[0074] - a second alginate polymer modified with a dienophile moiety, preferably a norbornene moiety;

[0075] wherein the modified first and second alginate polymers are covalently cross-linked through formation of [4+2] adducts between the diene and the dienophile. In a preferred embodiment of the latter, the diene and dienophile are attached to the first and second alginate polymer, respectively, through an amino group, preferably through a -CH2NH2 group, in the diene and dienophile (thus, the first and second alginate polymer are modified through amidic bonds with a diene and a dienophile moiety).

[0076] In an embodiment, the diene is one of formula I:

[0077]

[0078] wherein:

[0079] - Ri is a C1-6 alkylene or a (Ce arylene)(Ci-3 alkylene);

[0080] - R2 is selected from H, methyl, phenyl and C5-10 N-heterocyclyl;

[0081] - X and Y are independently selected from N and CH; and

[0082] - the -NH group is attached to the first alginate polymer through the wavy bond.Preferably, Ri is a C1-3 alkylene or a phenylene(Ci-3 alkylene); more preferably is -CH2-or -(p-phenylene)CH2-.

[0083] Preferably, R2 is H, methyl, phenyl, pyridinyl or pyrimidinyl; preferably, H or Me.

[0084] Preferably, X and Y are the same, more preferably they are N.

[0085] In a preferred embodiment, the diene of formula I is:

[0086]

[0087] In an embodiment, the dienophile is one of formula II:

[0088]

[0089] II

[0090] wherein

[0091] - R3 is a C1-6 alkylene or -C(=O)NHCH2CH2O(CH2CH2)n- wherein n is an integer comprised between 1-200; and

[0092] - the -NH group is attached to the second alginate polymer through the wavy bond. In a particular embodiment, R3 is C1-3 alkylene or -C(=O)NHCH2CH2O(CH2CH2)n-wherein n is an integer comprised between 100-200; more preferably R3 is -CH2- thus giving the compound:

[0093] HN^

[0094]

[0095] The compound II can be found as endo or exo isomer and the corresponding enantiomers, preferably compound II is a mixture of the isomers. It is clear to a skilled person that the compound of formula I is a diene while the compound of formula II is a dienophile. They would, therefore, form [4+2] adducts by Inverse Electron Demand Diels-Alder Cycloaddition (IEDDA) releasing N2.The first and second alginate polymers which are provided with crosslinkable moieties undergo covalent crosslinking to give a covalently crosslinked alginate polymer.

[0096] In a particular embodiment, the diene and dienophile, comprised in the first and second alginate polymers, respectively, and according to any of the above embodiments, are covalently crosslinked by forming [4+2] adducts. Particular experimental conditions for the crosslinking, in particular between a diene and a dienophile, would be known to a skilled person and are further detailed below.

[0097] In a preferred embodiment:

[0098] - a diene moiety and a dienophile moiety having each at least an amino group are attached to the first and second alginate polymer, respectively, through amidic bond of the amino group with the carboxyl groups of alginate; and

[0099] - crosslinking is carried out.

[0100] In an embodiment, the first and second alginate polymer are covalently crosslinked through [4+2] adducts of formula III:

[0101] R2

[0102]

[0103] III

[0104] wherein Algi is the first alginate polymer, Alg2 is the second alginate polymer and Ri, R2, R3, X and Y are defined as above. As it has been already stated, the wavy bond represents an amidic bond between the NH groups in the first and second crosslinkable moiety and carboxyl groups of Algi and Alg2.

[0105] The theoretical degree of substitution of the first and second alginate polymers with the first and second crosslinkable moieties, or any of the diene and dienophile described in the above embodiments, preferably those of formula I and II, may vary substantially. The theoretical degree of substitution (DStheo) is calculated based on the stoichiometry of the reaction and the number of reactive sites per monomer unit. Assuming 100% conversion of functional groups without steric hindrance or incomplete reaction effects, the formula for calculating DStheo would be:

[0106] moles of crosslinkable moieties added

[0107] DStheo = - - - - — - moles of alginateIn a preferred embodiment, the first alginate is modified with the first crosslinkable moiety as defined in any of the above embodiments at a DStheo comprised between 100 and 300, preferably between 150 and 200, more preferably at a DStheo of 170, per alginate polymer chain.

[0108] In a preferred embodiment, the second alginate is modified with the second crosslinkable moiety as defined in any of the above embodiments at a DStheo comprised between 400 and 600, preferably a DStheo of about 500, per alginate polymer chain.

[0109] Once the first and second alginate polymers are modified with the first and second crosslinkable moieties, the actual degree of substitution, DSactual, that is the actual (observed) ratio between the actual moles of crosslinkable moieties divided by the mole of saccharide units of the alginate polymer, can be directly measured (for example, by NMR spectroscopy).

[0110] In an embodiment, each of the first and second alginate polymers comprises between 3% and 15% of first and second crosslinkable moieties, respectively (i.e. moles of crosslinkable group / moles of monomer units in the alginate polymer x 100).

[0111] In an alternative embodiment, each of the first and second alginate polymers comprises from 10 to 100 of first and second crosslinkable moieties, preferably from 15 to 40, respectively.

[0112] From the corresponding values of DSactual for the first and second alginate polymers, a skilled person would be able to readily prepare a covalently crosslinked alginate polymer with a desired ratio of crosslinkable groups by simply adjusting the ratio between moles of modified first alginate polymer and modified second alginate polymer.

[0113] In an embodiment, the ratio between the second and first crosslinkable moieties ranges from 1.5:1 to 2:1, preferably the ratio is 1.8:1.

[0114] In an embodiment, the concentration of the covalently crosslinked alginate polymer in the hydrogel composition ranges from 0.75 to 1.25% w / v, preferably is about 1% w / v. It has been observed that the rheological and injectability properties are even more suitable using about 1 % w / v alginate, since softer and easier to inject gels were obtained. In addition, when cells such as T cells are encapsulated, their proliferation and release improved to a great extent.

[0115] The hydrogel composition is obtained in water medium; preferably, the hydrogel comprises alginate as defined in any of the above embodiments in a water buffered solution, preferably a phosphate-buffered solution (PBS), even more preferably PBS without Ca and Mg. The water buffered solution ensures a suitable pH for the injectablehydrogel composition, said pH being preferably between 7.2 and 7.5. When cells are added to the hydrogel composition, the water buffered solution further comprises cell culture media (such as RPMI, DMEM, OPTIMEM or combinations thereof).

[0116] In embodiment, the hydrogel composition of the first aspect further comprises at least an antitumoral agent selected from T-cells, preferably CAR T-cells; natural killer cells; dendritic cells; cytokines, preferably interferons, tumor necrosis factor-alpha, interleukin-2, interleukin-7, interleukin-15; p53 proteins; RB protein; c5b-c9 complex protein; monoclonal antibodies; checkpoint inhibitors, or a combination thereof. Said at least an antitumoral agent is encapsulated within the covalently crosslinked alginate polymer. In an embodiment, an effective amount of at least an antitumoral agent as defined above is encapsulated within the covalently crosslinked alginate polymer.

[0117] Preferably, the at least an antitumoral agent is a cell selected from T-cells (preferably CAR T-cells); natural killer cells and dendritic cells. In an embodiment, the cell density is comprised between 105and 108cells per ml of injectable hydrogel; preferably, the density is between 105and 5 107cells per ml of injectable hydrogel; more preferably, the density is about 4 107cells per ml of injectable hydrogel.

[0118] Preferably the hydrogel composition of the first aspect further comprises T-cells, more preferably CAR T-cells, encapsulated within the covalently crosslinked alginate polymer. In the context of the present invention, the term ‘T-cells’ refers to a subset of lymphocytes that originate from hematopoietic stem cells in the bone marrow and mature in the thymus. T-cells are characterized by the expression of T-cell receptors (TCRs) on their surface, which recognize specific antigens presented by major histocompatibility complex (MHC) molecules. T-cells include, but are not limited to, cytotoxic T lymphocytes (CD8+ T-cells), which are capable of directly killing target cells, and helper T-cells (CD4+ T-cells), which secrete cytokines to modulate immune responses. The term encompasses both natural T-cells and genetically modified T-cells, such as chimeric antigen receptor (CAR) T-cells. CAR T-cells are designed to recognize specific target antigens on the surface of cells in an MHC-independent manner. CAR constructs typically consist of an extracellular antigen-binding domain (e.g., derived from a monoclonal antibody), a transmembrane domain, and one or more intracellular signaling domains that activate T cell function upon antigen engagement. CAR T cells have targeted cytotoxic activity against malignant cells expressing the designated antigen. In an embodiment, the solid tumor is selected from brain tumor, breast cancer, lung cancer, colorectal cancer, pancreatic cancer, liver cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, esophagealcancer, stomach cancer, head and neck cancer, melanoma, sarcoma, and thyroid cancer; preferably the solid tumor is breast cancer.

[0119] In a second aspect, the invention relates to an injectable hydrogel composition comprising a covalently crosslinked alginate polymer comprising:

[0120] - a first alginate polymer modified with tetrazine moieties;

[0121] - a second alginate polymer modified with norbornene moieties, and wherein the first alginate polymer is covalently crosslinked with the second alginate polymer through [4+2] adducts of tetrazine moieties and norbornene moieties; and wherein:

[0122] - about 5% of the saccharide units of each of the first and second alginate polymer are oxidized;

[0123] - the first and second alginate polymer have each a molecular weight ranging from 50 to 100 kDa;

[0124] - the molar ratio of norbornene and tetrazine moieties ranges from 1.5:1 to 2:1 ; preferably said ratio is 1.8:1; and

[0125] - the concentration of the covalently crosslinked alginate polymer in the hydrogel ranges from 0.5 to 1.25% w / v.

[0126] In an embodiment, the tetrazine and norbornene moieties forming covalently crosslinked [4+2] adducts attached are attached to the carboxylic groups of first and second polymer, respectively, via an amidic bond.

[0127] In a particular embodiment, the tetrazine and norbornene moieties are those of formula I and II, respectively, as described for the first aspect and any of the embodiments thereof. In a particular embodiment, the [4+2] adducts in the covalently crosslinked alginate polymer are those of formula III.

[0128] In an embodiment, the concentration of the oxidized and modified alginate in the hydrogel composition ranges from 0.75 to 1.25% w / v, preferably is about 1% w / v.

[0129] In an embodiment, the molecular weight of the alginate, preferably sodium alginate, ranges from 60 to 90 kDa; preferably from 70 to 80 KDa; more preferably the molecular weight of alginate is about 75 KDa.

[0130] The hydrogel composition is obtained in water medium; preferably in a water buffered solution as defined above.In an embodiment of the second aspect, the hydrogel composition further comprises an active ingredient encapsulated within the covalently crosslinked alginate polymer. The term "active ingredient" refers to a chemical compound or biologic entity that provides the intended therapeutic or prophylactic effect when injected into a subject through the injectable composition. The covalently crosslinked alginate polymer as described in the second aspect is suitable for encapsulation of active ingredients and their controlled release.

[0131] In an embodiment, an effective amount of the active ingredient is encapsulated. The active ingredient may be selected from live cells (T cells, Mesenchymal Stem Cells (MSCs), Hematopoietic Stem Cells (HSCs), natural killer cells; dendritic cells, etc.); cytokines, preferably interferons, tumor necrosis factor-alpha, interleukin-2, interleukin-7 and interleukin-15; p53 proteins; RB protein; c5b-c9 complex protein; monoclonal antibodies; checkpoint inhibitors, viruses / viral vectors (oncolytic viruses, adenovirus vectors, lentiviral vectors, bacteriophages, etc.); bacteria and probiotics (Lactobacillus spp., Bifidobacterium spp., Escherichia coli Nissle 1917, Clostridium butyricum, etc.); fungi (saccharomyces boulardii, Candida oleophila, aspergillus oryzae, etc.); extracellular vesicles (EVs) and exosomes (stem cell-derived exosomes, dendritic cell-derived exosomes, mesenchymal stem cell (MSC) exosomes, etc.); or a combination thereof.

[0132] In a preferred embodiment, the active ingredient is at least an antitumoral agent, preferably selected from T-cells, preferably CAR T-cells; natural killer cells; dendritic cells; cytokines, preferably interferons, tumor necrosis factor-alpha, interleukin-2, intereukin-7, interleukin-15; p53 proteins; RB protein; c5b-c9 complex protein; monoclonal antibodies; checkpoint inhibitors or a combination thereof.

[0133] Preferably, the at least an antitumoral agent is a cell selected from T-cells (preferably CAR T-cells); natural killer cells and dendritic cells. In an embodiment, the cell density is comprised between 105and 108cells per ml of injectable hydrogel; preferably, the density is between 105and 5 107cells per ml of injectable hydrogel; more preferably, the density is about 4- 107cells per ml of injectable hydrogel.

[0134] In a preferred embodiment, T-cells, more preferably CAR T-cells, are encapsulated within the covalently crosslinked alginate polymer.

[0135] In a third aspect, the invention refers to a cell therapy kit for injectable administration comprising:

[0136] - a first alginate polymer comprising tetrazine moieties;

[0137] - a second alginate polymer comprising norbornene moieties;- a water buffered solution; and

[0138] - at least an antitumoral agent;

[0139] wherein:

[0140] - about 5% of the saccharide units of each of the first and second alginate polymer are oxidized;

[0141] - the first and second alginate polymer have each a molecular weight ranging from 50 to 100 kDa; preferably from 60 to 90 kDa; more preferably from 70 to 80 KDa; even more preferably is about 75 KDa;

[0142] - the molar ratio between norbornene and tetrazine moieties ranges from 1.5:1 to 2:1; preferably said ratio is 1.8:1; and

[0143] - wherein the amount of first alginate polymer, second alginate polymer and water buffered solution are such as to form a hydrogel comprising a covalently crosslinked alginate polymer with a concentration from 0.5 to 1.25% w / v.

[0144] Embodiments of the first alginate polymer comprising tetrazine moieties and second alginate polymer comprising norbornene moieties have been already described (in particular, see formulas I and II above). In an embodiment, the concentration of first alginate polymer is between 10%-25% w / v in the water buffered solution, the concentration of second alginate polymer is between 20%-35% w / v in the water buffered solution while the remainder is water buffered solution; preferably, the amount of water buffered solution is between 40-70 wt.% compared to the total weight first and second alginate polymer and water buffered solution.

[0145] The water buffered solution is preferably a phosphate buffer solution (PBS), even more preferably PBS without Ca and Mg, as defined above. When cells are added to the hydrogel composition, the water buffered solution further comprises cell culture media (such as RPMI, DMEM, OPTIMEM or combinations thereof).

[0146] In an embodiment, the at least an antitumoral agent selected from T-cells, preferably CAR T-cells; natural killer cells; dendritic cells; cytokines, preferably interferons, tumor necrosis factor-alpha, interleukin-2, intereukin-7, interleukin-15; p53 proteins; RB protein; c5b-c9 complex protein; monoclonal antibodies; checkpoint inhibitors or a combination thereof. In an embodiment, an effective amount of the at least an antitumoral agent as defined above is used in the third aspect.

[0147] More preferably, the at least antitumoral agent is a cell selected from T-cells (preferably CAR T-cells); natural killer cells and dendritic cells. In an embodiment, the cell density is comprised between 105and 108cells per ml of injectable hydrogel; preferably, thedensity is between 105and 5 107cells per ml of injectable hydrogel; more preferably, the density is about 4 107cells per ml of injectable hydrogel. In a preferred embodiment of the third aspect, the antitumoral agent is T-cells, preferably CAR T-cells. The density of the T-cells, preferably CAR T-cells, can be any one already described above.

[0148] In an embodiment of the third aspect, the first and second alginate polymer are dissolved in separate water buffered solutions (for example, in different parts or compartments of the kit); in this embodiment, the at least an antitumoral agent may be dissolved together with the first alginate polymer, with the second alginate polymer or with both; preferably is dissolved with the water buffered solution of the first or second alginate polymer. This kit facilitates storage or transportation of all the ingredients necessary for administration. The separate water buffered solutions of the firstand second alginate polymer comprised in the kit may be then mixed (prior to administration or at the injection site) at a temperature of about 35-40 °C, thus forming a covalently crosslinked alginate polymer, the concentration of which ranges from 0.5 to 1.25% w / v, more preferably from 0.5 to 1.25% w / v, more preferably said concentration is about 1% w / v.

[0149] In another embodiment, the kit comprises an already crosslinked alginate polymer obtained by covalent crosslinking of the first and second alginate polymer in the water buffer solution at a temperature comprised between 35-40 °C (thus forming a hydrogel composition), said crosslinked alginate polymer having a concentration from 0.5 to 1.25% w / v, preferably from 0.75 to 1.25% w / v, more preferably of about 1% w / v with respect to the hydrogel composition. According to this embodiment, the cell therapy kit is advantageous as the waiting time for gelation is avoided and the administration of the hydrogel can be done without any delay.

[0150] A more preferred temperature for formation of the hydrogel is about 37 °C.

[0151] In an embodiment of the third aspect, the cell therapy kit further comprises a syringe, wherein the syringe comprises the first and second alginate polymer, water buffered solution and T-cells. As stated above, the first and second alginate polymer may be already in the form of covalently crosslinked polymer within the water buffered solution (thus, as an already formed hydrogel) or in separate parts of the kit / separate compartments of the syringe and are mixed prior to administration (such as between 10 min and 6 hours before the administration) or directly at the injection site.

[0152] In a further aspect, the invention refers to a method for preparing an injectable hydrogel composition according to the second aspect, said method comprising the steps of: i) providing a first alginate polymer and a second alginate polymer, each having a molecular weight ranging from 50 to 100 kDa;ii) oxidizing about 5% of the saccharide units of the first and second alginate polymer, thus obtaining a partially oxidized first alginate polymer and a partially oxidized second alginate polymer, respectively;

[0153] iii) reducing the partially oxidized first and second alginate polymer, thus obtaining a reduced first alginate polymer and a reduced second alginate polymer;

[0154] iv) covalently attaching norbornene moieties to the reduced first alginate polymer with and tetrazine moieties to the reduced second alginate polymer, in presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, so to obtain a first alginate polymer modified with tetrazine moieties and a second alginate polymer modified with norbornene moieties;

[0155] v) mixing the first alginate polymer modified with tetrazine moieties and the second alginate polymer modified with norbornene moieties in aqueous medium with a norbornene:tetrazine moieties ratio comprised between 1.5:1 and 2:1 such as to form the injectable hydrogel composition of the second aspect at a concentration of covalently crosslinked alginate comprised between 0.5 and 1.25 w / v%.

[0156] In a particular embodiment of step i), the first and second alginate polymer are the same; more particularly, the molecular weight of each the first and second alginate polymer ranges from 60 to 90 kDa, preferably from 70 to 80 KDa, more preferably is about 75 KDa.

[0157] The oxidation of step ii) may be carried out with different oxidants, preferably with a periodate salt (such as sodium periodate). Step ii) is preferably carried out in water. The concentration of the first and second alginate in water may vary between 0.5 and 1.5 w / v%; preferably is 1.0 w / v%. The water solution of partially oxidized first and second alginate may be further purified by dialysis. In an embodiment, the purified partially oxidized first and second alginate polymers are lyophilized. As already explained above, the oxidant cleaves the C2-C3 bond of the uronic acid residues, converting hydroxyl (-OH) groups into aldehydes (-CHO). This oxidation step may increase biodegradability and enhance cell adhesion in biomedical applications.

[0158] The free aldehyde groups formed in step ii) may be detrimental to subsequent crosslinking, thus they are selectively reduced to -OH groups in step iii). Preferably, the reduction of step iii) is carried out with ammonia borane; water may be a suitable medium for the reduction of step iii).

[0159] The two-step oxidation-reduction of the first and second alginate polymers makes them more hydrolytically degradable without preventing efficient crosslinking and gel formation through the crosslinkable moieties.The reduced first and second alginate polymer which are essentially free of aldehyde groups are then reacted, respectively, with tetrazine and norbornene moieties in step iv). The tetrazine and norbornene moieties preferably comprise -NH2 groups, more preferably -CH2NH2 groups, which form amidic bonds with the carboxyl groups of the reduced first and second alginate polymer through

[0160] In an embodiment of step iv), the first alginate polymer is modified with a tetrazine moiety according to formula I. In a preferred embodiment, the first alginate is modified by adding the first crosslinkable moiety as defined above at a DStheo comprised between 100 and 300, preferably between 150 and 200, per alginate polymer chain.

[0161] In an embodiment of step iv), the second alginate polymer is modified with a norbornene moiety according to formula II. In a preferred embodiment, the second alginate is modified by adding the second crosslinkable moiety as defined above at a DStheo comprised between 400 and 600, preferably a DStheo of about 500, per alginate polymer chain.

[0162] In an embodiment of step iv), DSactual may vary between 15-100 crosslinkable moieties per (first or second) alginate polymer chain. In an alternative embodiment, each of the first and second alginate polymers comprises between 3% and 15% of first and second crosslinkable moieties, respectively (i.e. moles of crosslinkable group / moles of monomer units in the alginate polymer x 100).

[0163] In a preferred embodiment of step iv), the first alginate polymer modified with tetrazine moieties and a second alginate polymer modified with norbornene moieties are each lyophilized.

[0164] In an embodiment of step v), the first alginate polymer modified with tetrazine moieties and a second alginate polymer modified with norbornene moieties are mixed in PBS without Ca and Mg.

[0165] In a preferred embodiment of step v), the norbornene:tetrazine moieties ratio is of 1.8:1. In a preferred embodiment of step v), the concentration of the covalently crosslinked alginate is comprised between 0.5 and 1.25 w / v%, preferably is about 1 w / v%.

[0166] In a preferred embodiment, step v) is carried out at 37 °C.

[0167] The time in step v) required to form the injectable hydrogel composition is not particularly limited. In an embodiment is at least 10 min, at least 20 min, at least 30 min or at least 1 hour. Preferably, the time is comprised between 10 min and 6 hours, more preferably between 20 min and 5 hours, even more preferably between 30 min and 4 hours; mostpreferably the time is between 2 and 3 hours. Preferably conditions for gelation are a temperature of 27 °C and a time between 30 min and 4 hours.

[0168] The injectable hydrogel composition may be formed in presence of at least an active ingredient as defined above, preferably at least an antitumoral agent as defined above, more preferably a cell for solid tumor treatment or therapy as defined above. Preferably, the at least antitumoral agent is a cell selected from T-cells (preferably CAR T-cells); natural killer cells and dendritic cells. In an embodiment, the cell density is comprised between 105and 108cells per ml of injectable hydrogel; preferably, the density is between 105and 5 107cells per ml of injectable hydrogel; more preferably, the density is about 4 107cells per ml of injectable hydrogel.

[0169] Any of the above active ingredients may therefore be encapsulated within the crosslinked alginate polymer.

[0170] The injectable hydrogel composition formation of step v) may take place in any vessel or mold. Preferably, it may take place in a cylindrical mold or in a syringe for direct injection. The hydrogel composition may be formed as a ready-to-use injectable composition; alternatively, the mixing of steps v) may take place during administration at the injection site.

[0171] Further embodiments

[0172] 1. An injectable hydrogel composition for use in the treatment or therapy of a solid tumor, said hydrogel comprises a covalently crosslinked alginate polymer comprising:

[0173] - a first alginate polymer modified with a first crosslinkable moiety; and

[0174] - a second alginate polymer modified with a second crosslinkable moiety, wherein the modified first and second alginate polymers are covalently crosslinked through the first and second crosslinkable moieties;

[0175] and wherein:

[0176] from 2% to 10 % of the saccharide units of each of the first and second alginate polymers are oxidized;

[0177] each of the first and second alginate polymer has a molecular weight ranging from 50 to 100 kDa;

[0178] the concentration of the covalently cross-linked alginate polymer in the hydrogel composition ranges form 0.5 to 2% w / v; andthe hydrogel further comprises at least an antitumoral agent encapsulated within the covalently crosslinked alginate polymer.

[0179] 2. The injectable hydrogel composition for use according to embodiment 1, wherein each of the first and second alginate polymer is sodium alginate.

[0180] 3. The injectable hydrogel composition for use according to embodiment 1 or 2, wherein from 3.5% to 7.5% of the saccharide units of each of the first and second alginate are oxidized, preferably about 5% of the saccharide units of each of the first and second alginate are oxidized.

[0181] 4. The injectable hydrogel composition for use according to any one of embodiments 1 to 3, wherein each of the first and second alginate polymer has a molecular weight ranging from 60 to 90 kDa; preferably from 70 to 80 KDa; more preferably, wherein the molecular weight of each of the first and second alginate polymer is about 75 KDa. 5. The injectable hydrogel composition for use according to any one of embodiments 1 to 4, wherein the first and second alginate polymers are crosslinked through [4+2] adducts of, respectively, a diene and a dienophile as first and second crosslinkable moiety.

[0182] 6. The injectable hydrogel composition for use according to embodiment 5, wherein the diene is one of formula I:

[0183]

[0184] wherein:

[0185] - Ri is a C1-6 alkylene or a (Ce arylene)(Ci-3 alkylene);

[0186] - R2 is selected from H, methyl, phenyl and C5-10 N-heterocyclyl;

[0187] - X and Y are independently selected from N and CH; and

[0188] - the -NH group is attached to first alginate polymer through the wavy bond.

[0189] 7. The injectable hydrogel composition for use according to embodiment 5 or 6, wherein the dienophile is one of formula II:

[0190]

[0191] II

[0192] wherein:

[0193] - R3 is a C1-6 alkylene or a -C(=O)NHCH2CH2O(CH2CH2)n- wherein n is an integer comprised between 1-200; and

[0194] - the -NH group is attached to the second alginate polymer through the wavy bond. 8. The injectable hydrogel composition for use according to any one of the preceding embodiments, wherein the concentration of the covalently crosslinked alginate polymer in the hydrogel composition ranges from 0.5 to 1.25% w / v, preferably is about 1% w / v.

[0195] 9. The injectable hydrogel composition for use according to any one of the preceding embodiments, wherein the at least an antitumoral agent is selected from T-cells, preferably CAR T-cells; natural killer cells; dendritic cells; cytokines, preferably interferons, tumor necrosis factor-alpha, interleukin-2, interleukin-7 and interleukin-15; p53 proteins; RB protein; c5b-c9 complex protein; monoclonal antibodies; checkpoint inhibitors, or a combination thereof; preferably wherein the at least an antitumoral agent is T-cells, more preferably CAR T-cells.

[0196] 10. The injectable hydrogel composition for use according to any one of the preceding embodiments, wherein the solid tumor is selected from brain tumor, breast cancer, lung cancer, colorectal cancer, pancreatic cancer, liver cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, esophageal cancer, stomach cancer, head and neck cancer, melanoma, sarcoma, and thyroid cancer; preferably the solid tumor is breast cancer.

[0197] 11. An injectable hydrogel composition comprising a covalently crosslinked alginate polymer comprising:

[0198] - a first alginate polymer modified with tetrazine moieties;

[0199] - a second alginate polymer modified with norbornene moieties, and wherein the first alginate polymer is covalently crosslinked with the second alginate polymer through [4+2] adducts of tetrazine moieties and norbornene moieties; and wherein:

[0200] - about 5% of the saccharide units of each of the first and second alginate polymer are oxidized;- the first and second alginate polymer have each a molecular weight ranging from 50 to 100 kDa;

[0201] - the molar ratio of norbornene and tetrazine moieties ranges from 1.5:1 to 2:1 ; preferably said ratio is 1.8:1; and

[0202] - the concentration of the covalently crosslinked alginate polymer in the hydrogel ranges from 0.5 to 1.5% w / v.

[0203] 12. The injectable hydrogel composition according to embodiment 11, wherein the molecular weight of each of the first and second alginate, preferably sodium alginate, ranges from 60 to 90 kDa; preferably from 70 to 80 KDa; more preferably is about 75 KDa.

[0204] 13. The injectable hydrogel composition according to any one of embodiments 11 or 12, wherein the concentration of the covalently crosslinked alginate polymer, preferably sodium alginate polymer, in said injectable hydrogel composition ranges from 0.5 to 1.25% w / v, preferably is about 1 % w / v.

[0205] 14. The injectable hydrogel composition according to any one of embodiments 11 to 13, wherein an antitumoral agent is encapsulated within the covalently crosslinked alginate polymer; preferably, wherein the antitumoral agent is selected from T-cells, preferably CAR T-cells; natural killer cells; dendritic cells; cytokines, preferably interferons, tumor necrosis factor-alpha, interleukin-2, intereukin-7, and interleukin-15; p53 proteins; RB protein; c5b-c9 complex protein; monoclonal antibodies; checkpoint inhibitors, or a combination thereof.

[0206] 15. A cell therapy kit for injectable administration comprising:

[0207] - a first alginate polymer comprising tetrazine moieties;

[0208] - a second first alginate polymer comprising norbornene moieties;

[0209] - a water buffered solution; and

[0210] - at least an antitumoral agent, preferably selected from T-cells, preferably CAR T-cells; natural killer cells; dendritic cells; cytokines, preferably interferons, tumor necrosis factoralpha, interleukin-2, intereukin-7, interleukin-15; p53 proteins; RB protein; c5b-c9 complex protein; monoclonal antibodies; checkpoint inhibitors or a combination thereof; wherein:

[0211] - about 5% of the saccharide units of each of the first and second alginate polymer are oxidized;- the first and second alginate polymer have each a molecular weight ranging from 50 to 100 kDa; preferably from 60 to 90 kDa; more preferably from 70 to 80 KDa; even more preferably is about 75 KDa;

[0212] - the molar ratio between norbornene and tetrazine groups moieties ranges from 1.5: 1 to 2: 1 ; preferably said ratio is 1.8:1; and

[0213] - wherein the amount of first alginate polymer, second alginate polymer and water buffered solution are such as to form a hydrogel comprising a covalently crosslinked alginate polymer with a concentration from 0.5 to 1.5% w / v.

[0214] Examples

[0215] Materials. High guluronic acid sodium alginate (MW 75 kDa Pronova UP VLVG; NovaMatrix), sodium periodate (ACS reagent, 99.8%; Sigma-Aldrich), dialysis membranes (Spectra / Por 6, MWCO 3.5 kDa; Spectrum), ammonia borane (97%; Sigma-Aldrich), Ultra Shield Plus 500 MHz spectrometer (Bruker), 2-(N-morpholino)-ethanesulfonic acid (MES; Sigma-Aldrich), N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC; Sigma-Aldrich), N-hydroxysuccinimide (NHS; Sigma-Aldrich), 5-norbornene-2-methylamine (herein also named “N”, TCI Chemicals, #N0907), 3-(p-benzylamino)-1,2,4,5 tetrazine (herein also named “T”, Conju-probe, #CP-6021), hydroxylamine (Sigma-Aldrich), activated charcoal (Sigma-Aldrich), Dulbecco's Phosphate-Buffered Saline (PBS, without Ca++, Mg++, and phenol red; Gibco™), Sigmacote (Sigma-Aldrich), ARES-G2 rheometer (TA Instruments), Texture Analyzer TA.XTPIus (Stable Micro Systems), pCDH-EF1-Luc2-P2A-tdTomato plasmid (Luciferase / tdTomato plasmid, Addgene), RPMI-1640 (Gibco™), DMEM (Gibco™), packaging plasmids (pMDLg-pRRE, pRSV-REV and pMD2.G-VSVG), Turbofect Transfection Reagent (Thermo Fisher Scientific), Sorter SH800S (SONY), Calcein AM (Biomol Bioquest #ABD-22002), ethidium homodimer-1 (Sigma #E1903), Axio Observer 7 (Carl Zeiss Microscopy), Bovine serum albumin (BSA, Sigma-Aldrich, #A7906), Triton X-100 (Sigma-Aldrich), Phalloidin 405 Conjugate (PHA, CruzFluor™, #SC363790), 4', 6-diamidino-2-phenylindole (DAPI; Roche), LSM900 confocal microscope (Carl Zeiss Microscopy), 24-well transwell (Corning, #353097), CytoFLEX flow cytometer (Beckman Coulter).

[0216] Example 1: Synthesis and characterization of hydrogels

[0217] Oxidation of alginate. Hydroxyl groups of 5% of the monomers of low molecular weight (LMW, MW 75 kDa) alginate were oxidized by dissolving the polymer at 1% w / v in double-distilled water (ddH2O) and reacting with sodium periodate (1 mol per 1 mol ofoxidized monomer). The reaction mixture was stirred at 250 rpm in the dark at room temperature overnight. The solution was purified by dialysis in ddH2O for 3 days with 3-4 water changes per day for 3 days. Finally, the purified oxidized alginate was lyophilized. Reduction of oxidized alginate. The 5% oxidized alginate was then reduced using ammonia borane (1 mol per 1 mol of oxidized monomer). The polymer was dissolved at 1% w / v in ddH2O and reacted with ammonia borane (BH3NH3) by stirring at 250 rpm for 16 h at room temperature in dark. The resulting solution was dialyzed and lyophilized as before.

[0218] Alginate functionalization by carbodiimide chemistry. Oxidized and reduced alginate was dissolved at 1% w / v in MES. Then, EDC and NHS were added drop-wise to the alginate solution at 5000 molar equivalents each. EDC and NHS were allowed to react with alginate for 5 minutes at room temperature with gentle stirring.

[0219] For the addition of norbornene groups in alginate chain, N (5-norbornene-2-methylamine) was introduced at a degree of substitution (DStheo) of 500 norbornene molecules per alginate chain. For tetrazine modification, T (3-(p-benzylamino)-1,2,4,5 tetrazine) was added at a DStheo of 170. Both reactions proceeded at room temperature for 18 hours with constant stirring at 700 rpm. Then, hydroxylamine was used for quenching. The resulting solutions were first purified by dialysis as described previously, and then treated with activated charcoal for 30 minutes. Finally, the solutions were sterile-filtered and lyophilized.

[0220] Hydrogels fabrication. Lyophilized norbornene-modified (N-AIg) and tetrazine-modified (T-AIg) alginates were dissolved in PBS without Ca and Mg. The dissolved N-AIg and T-Alg were then mixed maintaining a norbornene:tetrazine (N:T) ratio of 1.8 and final alginate concentrations of 1% or 2% (w / v %). The so-obtained hydrogels were named HG1 (1 w / v%) (according to the invention) and HG2 (2 w / v%) (comparative), respectively.

[0221] For the generation of cylindrical hydrogels, the mixed solution was pipetted into a metallic mold with holds of 8 mm diameter and 2 mm high and a silanized glass slide on bottom and top. After 2.5 hours of casting at 37°C, the glass slides and the mold were removed to obtain the shaped hydrogels.

[0222] To prepare hydrogels for injection, N-AIg and T-AIg were mixed and loaded into a syringe, and allowed to crosslink at 37°C for 2.5 hours.2. characterization

[0223] Nuclear Magnetic Resonance (NMR) characterization of modified alginates. To confirm the oxidation and reduction of the alginate chain, and determine the actual degree of substitution (DSactual) values of N and T, NMR analysis were performed. For this purpose, samples were dissolved in deuterium oxide at 1.5% w / v concentration, and 64 scans were acguired for each measurement. Then, Spectra were analyzed using MestreNova Software (v12.03). A DSactual of 27.73 and 18.71 for N-AIg and T-AIg were obtained, respectively.

[0224] Rheological characterization The rheological properties of the hydrogels precursor solutions and crosslinked hydrogels were characterized using an ARES-G2 rheometer (TA Instruments). For the analysis, N-AIg and T-AIg solutions were mixed and immediately pipetted onto the bottom plate of the rheometer. Then, hydrogels flow behaviour, crosslinking kinetics, and viscoelastic properties were characterized. The viscosity of non-crosslinked hydrogel was measured with a shear rate ranging from 1 to 10 s'1. Then, crosslinking process was analyzed by the time sweep experiment at a constant freguency of 1 Hz. Once gelation of the hydrogels was complete, a freguency sweep was conducted from 0.1 to 1 Hz. Finally, a stress relaxation test was conducted by applying a 50% strain for 5 hours. All measurements were carried out at 37 °C, under linear viscoelastic conditions, using a 40 mm parallel plate geometry with a gap of 0.05 mm and mineral oil in the periphery to prevent dehydration.

[0225] Data analysis was performed using the TA Instruments Trios software (v5.1.1). The crossover points of G' and G" in the time sweep was used to determine the gelation point. To obtain the elastic modulus, first the complex shear modulus (G*) was derived from the storage (G’) and loss (G”) modulus in the plateau using Rubber’s elasticity theory |G*| = G'2+ G"2. Then, the elastic modulus (E) was calculated using the average of G* values and the approximation of Poisson’s ratio (i9) egual to 0.5, E = 2G*(1 + i9). The mesh size was estimated by using the formula: = \J6RT / G'nNavas previously described (Dupont et al., 2011). The stress relaxation data were analyzed to determine the characteristic time reguired for the material to relax 50 % its initial modulus.

[0226] The rheological characterization is further shown in fig. 1. Before gelation, both 1% w / v and 2% w / v hydrogels exhibited very low viscosity (<10 Pa s) (Fig. 1A). The gelation point is defined as the crossover point between the storage modulus (G’) and the loss modulus (G”), which was shown in the time sweep to be of about 12 min and 50 min, respectively, for 1% w / v and 2% w / v hydrogels (Fig. 1B, C). In addition, in case of the 1% w / v concentration, it took longer for the hydrogel to fully form, as more time wasneeded to achieve constant G’ values. The frequency and strain sweep showed that both hydrogels had a G’ greater than G”, i.e. both hydrogels were mostly elastic solids (Fig.

[0227] 1 D). Additionally, in the case of the 1% w / v concentration, the Elastic modulus’ was lower than in the case of 2% w / v concentration (=40 Pa and 550 Pa, respectively), thus having the former hydrogel a lower stiffness (Fig. 1E). The mesh size was significantly higher in 1% w / v concentration (91.36 ± 3.11 nm) compared to 2% w / v (35.66 ± 2.67 nm) suggesting higher porosity and better suitability for cell migration (Fig. 1F). Finally, the stress relaxation test revealed the decrease in modulus in 2 hours of 1% w / v gels, while those with 2% w / v showed no significant relaxation even after 5 hours (Fig. G). The normalized modulus for 1% w / v decreased from 1 to 0.5 in 133.4 ± 9.5 minutes, while for 2% w / v, it remained close to 1 throughout the 5-hour test duration (Fig. H).

[0228] Assessment of injectability. The injectability of both materials (HG1 and HG2) was assessed taking into account i) the mechanical properties (injection force), ii) the effect of injection on cell viability and iii) the visual assessment of the state of both gels after injection.

[0229] Injection force measurements were conducted using a Texture Analyzer TA.XTPIus (Stable Micro Systems) equipped with a 50 N load cell. N-AIg and T-AIg were mixed and immediately loaded into 1 mL syringe with 25G 5 / 8" needle. At 0, 15, 60, 120, and 180 minutes, to assess the effect of crosslinking time on injectability, the plunger was compressed at a constant rate of 1 mm / s to simulate injection. The injection force was calculated as the average force within the plateau region of the force-displacement curve during extrusion. For each time point, measurements were performed in triplicate.

[0230] Using a live / dead test, the possible effect of injection forces on cell viability was assessed.

[0231] Using the visual assessment, the state of the gels was observed after injection. In the mechanical and injectability tests, the gels were injected at different times (0 min, 12 min, 60 min, 120 min and 180 min) and the different assessments were performed. The visual assessment test ended after two hours.

[0232] In both gels, during gelation, a greater injection force was found (Fig 2 A). In HG2, the injection force increased significantly to the maximum from 60 onwards, which coincides with the time at which the gel has completely gelled. In addition, the highest injection force was 11.55 ± 8.84 N; however, in HG1, the injection force increases significantly at 180 minutes (time for complete gelation for this sample) and the highest force was 2.25 ± 0.84 N. According to literature, an injectable material should present an injection force of less than 20 N (S. Park et al., Molecules 2023, 28(13), DOI:10.3390 / molecules28135222), thus both HG1 and HG2 are injectable, albeit HG1 showed better injection features than comparative sample HG2 since much less force was required for injection (Fig. 2A).

[0233] The effect of injection on cell viability was evaluated, since the shear forces during injection of the material can affect its viability. Jurkat cells were encapsulated in HG1 and HG2 and 100pl of each gel were injected at different times (t=0, 12, 60, 120 and 180 min). The viability of the Jurkat cells was then measured using a live / dead assay with calcein (live) and ethidium homodimer-1 (dead). The results showed that in the comparative hydrogel HG2, cell viability significantly decreases as they became more rigid. However, in the hydrogel according to the invention HG1 there was no decrease in viability (live >75%). Finally, the visual evaluation of HG1 and HG2 was carried out at different points in the gelation process.

[0234] The change in the texture of the gels was observed as they gelled (Fig. 20). The difference in the texture of both gels is notable, since once gelled, HG1 had a smoother texture compared to HG2, which had a lumpier texture (Fig. 2C). Fig. 2D shows images of encapsulated and injected live Jurkat cells (D).

[0235] Effect of the alginate molecular weight on injectability. Figure 3 shows the injectability during the gelation process of HG1 hydrogels prepared with commercially available alginates of different molecular weights, in particular, a low-molecular-weight alginate (LMW, =75 kDa) and a high molecular weight alginate (HMW, >200 KDa) sample were used as starting materials to obtain the corresponding hydrogels by following the procedure described in Example 1. As shown in Figure 3, the increase in polymer molecular weight causes the 1% formulation to become non-injectable once gelled (after 60 minutes) for the subjects that received the injection (regardless of age or sex). This is because the hydrogel resistance exceeds the previously described injection-force limit of 12 N (T.E. Robinson, E.A.B. Hughes, A. Bose, E.A. Cornish, J.Y. Teo, N.M. Eisenstein, L.M. Grover, S.C. Cox, Filling the Gap: A Correlation between Objective and Subjective Measures of Injectability, Adv Healthc Mater 9 (2020) 1901521. https: / / doi.org / 10.1002 / adhm.201901521). Thus, only HG1 after gelation using low molecular weight alginate was suitable for injection.

[0236] Example 3. Encapsulation of Jurkat T cells in hydrogels, viability, proliferation and morphology.

[0237] The human T lymphocyte cell line (Jurkat) was obtained from Dr. Asis Palazon Lab, in Cic Biogune and was cultured in RPMI-1640 medium supplemented with 10% fetalbovine serum (FBS) and 1% P / S. For cell tracking, a Luciferase / tdTomato dual-reporter gene was introduced into the Jurkat cell line (Jurkat-TOM) using lentiviral vectors. For lentivirus generation, HEK-293T cells were cultured in DM EM supplemented with 10% FBS and 1% penicillin-streptomycin.

[0238] Jurkat-TOM generation. In order to generate a trackable Jurkat cell line, tdTomato and luciferase positive Jurkat-TOM cell line was generated using a dual reporter plasmid. Briefly, Turbofect Transfection Reagent was used following manufacturer's protocol for lentivirus production employing HEK-293T and standard packaging plasmids system. After 24 hours of incubation, the supernatant containing lentiviruses with Luciferase / tdTomato plasmid was collected and filtered using 0.45 pm filter before adding to the Jurkat cells. 48 hours later, transduced Jurkat-TOM cells were purified using the sorter.

[0239] Cell encapsulation and injectability. To evaluate the injectability of our hydrogels by analyzing its impact on cell survival after injection, Jurkat cells were encapsulated within the hydrogels by adding the cell suspension into the N-AIg prior to mixing with T-AIg at a final concentration of T106cells / mL. After mixing well, at 0, 15, 60, 120, and 180 minutes, 50 pl of the hydrogel were injected through a 25G 5 / 8 " into a Live / Dead solution containing 1:2000 Calcein AM and 1:1000 ethidium homodimer-1. Following 15 minutes of incubation at room temperature in the dark, images were taken. 100 mm z-stack with 15 mm step size was acquired at each hydrogel for n=3 hydrogels per group using an inverted fluorescence microscope. The z-stacks were then collapsed to 2D projections for quantification using Dragonfly. Cell viability was quantified by calculating the percentage of live cells to total cells (sum of live and dead cells).

[0240] Evaluation of viability and proliferation. Jurkat T cells were encapsulated in HG1 and HG2 at a density of 1x106cells / ml. Cell viability and morphology were assessed using calcein / ethidium homodimer-1 and DAPI / phalloidin stains, respectively. The increase in cell number was then quantified to assess proliferation over 10 days. On days 1, 3, 7, and 10, Live / Dead and DAPI / PHA staining were performed and the percentage of live cells and total cell number were quantified to assess cell viability and proliferation. Jurkat cells showed high viability over the 10 days in both gels, although greater proliferation occurred in HG1 (Fig. 4A). In addition, DAPI / phalloidin staining showed remarkable proliferation in HG1, as cell clusters within the gel were visible from day 3 onwards (Fig. 4B). Cell clusters were also observed in HG2, although in smaller numbers and sizes (Fig. 4B).To identify live and dead cells, calcein (1:200) and ethidium homodimer-1 (1:1000) were used to stain encapsulated live and dead cells, respectively. They were incubated for 15 min at room temperature in the dark and imaged under fluorescence microscopy. For evaluating morphology and proliferation, DAPI / PHA staining was used. First, hydrogels were fixed in 4% paraformaldehyde (PFA) for 30 min at room temperature and then washed twice with 3% BSA in PBS. Hydrogels were then permeabilized using 0.3% Triton X-100 and leaving for 10 minutes in the rocker. Once washed again with BSA, PHA (1:40) was added and incubated overnight at 4 °C. After incubation and another wash with BSA, DAPI (1:1000) was added and incubated for 30 minutes at room temperature. Finally, the gels were placed in PBS before imaging under the confocal microscope.

[0241] For Live / Dead and DAPI / Phalloidin staining imaging, 50 mm z-stack with 15 mm step size was acquired at each of the 2 positions per hydrogel for n=3 hydrogels per group. As previously explained, z-stacks were collapsed to 2D projections for quantification and cell viability was quantified by calculating the ratio of live cells to total cells (sum of live and dead cells). Due to the formation of dense cellular clusters after 7 days, the number of viable cells was determined by subtracting the count of dead cells from the total cell population, as identified by DAPI+ nuclei. Quantification of cell numbers to evaluate viability and proliferation at each time point was carried out using Dragonfly.

[0242] T cell release. To assess the cell release properties of HG1 and HG2 over time, Jurkat cells were encapsulated and the number of released cells from the hydrogels to the media was quantified until 10 days (fig. 4C-4D).

[0243] First, as described before, Jurkat cells were encapsulated in HG1 and HG2 by mixing the cells in N-AIg before adding T-Alg. After leaving to crosslink for 2.5h, 50 pl of both HG1 and HG2 were injected through a 25G 5 / 8 " needle into a 24-well and cultured in 1.5 ml of complete RPMI-1640 medium. All media was collected on days 1, 3, 7 and 10, and centrifuged at 500 ref for 5 minutes to obtain the cells. Then, the cell pellet was resuspended in 250 pl of PBS (1 mM EDTA) and incubated with DAPI (1:1000) for 15 minutes in the dark, at room temperature, to stain non-viable cells. The number of cells that were release from the gel to the media was quantified using the cytometer. DAPI-positive cells were classified as non-viable, and the number of released viable cells was determined by subtracting DAPI-positive events from the total cell count. Data were analyzed using CytExpert software (v2.6).

[0244] In order to visualize the cell release process over time, Jurkat-TOM were encapsulated in cylindrical hydrogels and cultured under the same conditions. Imaging using thefluorescence microscope was performed in day 1, 3, 7 and 10, focusing on the well bottom to visualize the hydrogels and the released cells.

[0245] Cells were found outside the gel in both HG1 and HG2, although significantly more cells were found in HG1 as early as day 3. Up to 14 days, the release of cells from within the gel into the culture medium was assessed using the microscope. The periphery of each gel was identified and traced (in white), and the presence of Jurkat outside the gel was detected by tdTomato detection. Visually, more cells were observed outside the gels in HG1.

[0246] Example 4. Encapsulation of CAR-T cells in hydrogels and characterization of the cytotoxic effect on tumor cells.

[0247] In an additional test to evaluate the viability and cytotoxic capacity of CAR-T cells encapsulated in HG1, the following cell lines were employed: the target tumoral cells were MDA-MB-231 CD19+ breast cancer cells (GFP+ and luc+) and the effector cells were anti-CD19 CAR-T cells. The cytotoxic capacity of the CAR-T cells was assessed by measuring tumor-cell death using bioluminescence assays. Two variables were included: the presence or absence of the stimulating cytokine IL-2, and the method of administering the CAR-T cells - either added directly to the culture medium (without gel) or encapsulated in HG1. The following experimental groups were created:

[0248] 1. **-**: positive control consisting of MDA-MB-231 CD19+ cells;

[0249] 2. **CART**: MDA-MB-231 CD19+ cells with CAR-T cells (administered without hydrogel);

[0250] 3. **CART IL-2**: MDA-MB-231 CD19+ cells with CAR-T cells and IL-2 (administered without hydrogel);

[0251] 4. **CART GEL**: MDA-MB-231 CD19+ cells with CAR-T cells encapsulated in HG1; 5. **CART GEL IL-2**: MDA-MB-231 CD19+ cells with CAR-T cells encapsulated in HG1 plus IL-2.

[0252] Regarding the timeline, after 24 hours of the seeding of the MDA-MB-231 cells in the wells, CAR-T cells were added with or without IL-2 and with or without hydrogel (day 0). In the case of CAR-T cells encapsulated in the hydrogel, each hydrogel was placed in a transwell with a membrane containing 8-pm pores, separating the upper and lower compartments (Fig. 5). Tumor breast-cancer cells were seeded in the lower compartment; in the upper compartment, HG1 hydrogels containing encapsulatedCAR-T cells were placed. On days 1, 3, 7, and 10, the bioluminescence signal was measured by adding 150 ng / mL luciferin and reading the plate in the Glomax. To evaluate tumor-cell death, the bioluminescence signal of each well was normalized to the average bioluminescence signal of the positive control (group

[0253]

[0254] with MDA cells alone.

[0255] The results showed that from day 3 onward, both CAR-T cells added with or without hydrogel and with or without IL-2 were capable of eliminating 50% of the tumor cells. From day 7 onward, CAR-T cells encapsulated in HG1 killed significantly more tumor cells than CAR-T cells administered without hydrogel (Figure 6). This demonstrates that the CAR-T cells were able to survive within the hydrogel, exit the hydrogel, and progressively attack tumor cells for at least 10 days.

Claims

34CLAIMS1. An injectable hydrogel composition for use in the treatment or therapy of a solid tumor, said hydrogel comprises a covalently crosslinked alginate polymer comprising:- a first alginate polymer modified with a first crosslinkable moiety; and- a second alginate polymer modified with a second crosslinkable moiety, wherein the modified first and second alginate polymers are covalently crosslinked through the first and second crosslinkable moieties;and wherein:from 2% to 10 % of the saccharide units of each of the first and second alginate polymers are oxidized;each of the first and second alginate polymer has a molecular weight ranging from 50 to 100 kDa;the concentration of the covalently cross-linked alginate polymer in the hydrogel composition ranges from 0.5 to 1.25% w / v; andthe hydrogel further comprises at least an antitumoral agent encapsulated within the covalently crosslinked alginate polymer.

2. The injectable hydrogel composition for use according to claim 1 , wherein each of the first and second alginate polymer is sodium alginate.

3. The injectable hydrogel composition for use according to claim 1 or 2, wherein from 3.5% to 7.5% of the saccharide units of each of the first and second alginate are oxidized, preferably about 5% of the saccharide units of each of the first and second alginate are oxidized.

4. The injectable hydrogel composition for use according to any one of claims 1 to 3, wherein each of the first and second alginate polymer has a molecular weight ranging from 60 to 90 kDa; preferably from 70 to 80 KDa; more preferably, wherein the molecular weight of each of the first and second alginate polymer is about 75 KDa.

5. The injectable hydrogel composition for use according to any one of claims 1 to 4, wherein the first and second alginate polymers are crosslinked through [4+2] adducts of, respectively, a diene and a dienophile as first and second crosslinkable moiety.

6. The injectable hydrogel composition for use according to claim 5, wherein the diene is one of formula I:wherein:- R1 is a C1-6 alkylene or a (Ce arylene)(Ci-3 alkylene);- R2 is selected from H, methyl, phenyl and C5-10 N-heterocyclyl;- X and Y are independently selected from N and CH; and- the -NH group is attached to first alginate polymer through the wavy bond.

7. The injectable hydrogel composition for use according to claim 5 or 6, wherein the dienophile is one of formula II:HNiZ^R3wherein:- R3is a C1-6 alkylene or a -C(=O)NHCH2CH2O(CH2CH2)n- wherein n is an integer comprised between 1-200; and- the -NH group is attached to the second alginate polymer through the wavy bond.

8. The injectable hydrogel composition for use according to any one of the preceding claims, wherein the concentration of the covalently crosslinked alginate polymer in the hydrogel composition ranges from 0.75 to 1.25% w / v, preferably is about 1% w / v.

9. The injectable hydrogel composition for use according to any one of the preceding claims, wherein the at least an antitumoral agent is selected from T-cells, preferably CAR T-cells; natural killer cells; dendritic cells; cytokines, preferably interferons, tumor necrosis factor-alpha, interleukin-2, interleukin-7 and interleukin-15; p53 proteins; RB protein; c5b-c9 complex protein; monoclonal antibodies; checkpoint inhibitors, or a combination thereof; preferably wherein the at least an antitumoral agent is T-cells, more preferably CAR T-cells.

10. The injectable hydrogel composition for use according to any one of the preceding claims, wherein the solid tumor is selected from brain tumor, breast cancer, lung cancer, colorectal cancer, pancreatic cancer, liver cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, esophageal cancer, stomach cancer, head and neck cancer, melanoma, sarcoma, and thyroid cancer; preferably the solid tumor is breast cancer.

11. An injectable hydrogel composition comprising a covalently crosslinked alginate polymer comprising:- a first alginate polymer modified with tetrazine moieties;- a second alginate polymer modified with norbornene moieties, and wherein the first alginate polymer is covalently crosslinked with the second alginate polymer through [4+2] adducts of tetrazine moieties and norbornene moieties; and wherein:- about 5% of the saccharide units of each of the first and second alginate polymer are oxidized;- the first and second alginate polymer have each a molecular weight ranging from 50 to 100 kDa;-the molar ratio of norbornene and tetrazine moieties ranges from 1.5:1 to 2:1; preferably said ratio is 1.8:1; and- the concentration of the covalently crosslinked alginate polymer in the hydrogel ranges from 0.5 to 1.25% w / v.

12. The injectable hydrogel composition according to claim 11, wherein the molecular weight of each of the first and second alginate, preferably sodium alginate, ranges from 60 to 90 kDa; preferably from 70 to 80 KDa; more preferably is about 75 KDa.

13. The injectable hydrogel composition according to any one of claims 11 or 12, wherein the concentration of the covalently crosslinked alginate polymer, preferably sodium alginate polymer, in said injectable hydrogel composition ranges from 0.75 to 1.25% w / v, preferably is about 1 % w / v.

14. The injectable hydrogel composition according to any one of claims 11 to 13, wherein an antitumoral agent is encapsulated within the covalently crosslinked alginate polymer; preferably, wherein the antitumoral agent is selected from T-cells, preferably CAR T-cells; natural killer cells; dendritic cells; cytokines, preferably interferons, tumor necrosis factor-alpha, interleukin-2, intereukin-7, and interleukin-15; p53 proteins; RB protein;c5b-c9 complex protein; monoclonal antibodies; checkpoint inhibitors, or a combination thereof.

15. A cell therapy kit for injectable administration comprising:- a first alginate polymer comprising tetrazine moieties;- a second first alginate polymer comprising norbornene moieties;- a water buffered solution; and- at least an antitumoral agent, preferably selected from T-cells, preferably CAR T-cells; natural killer cells; dendritic cells; cytokines, preferably interferons, tumor necrosis factoralpha, interleukin-2, intereukin-7, interleukin-15; p53 proteins; RB protein; c5b-c9 complex protein; monoclonal antibodies; checkpoint inhibitors or a combination thereof; wherein:- about 5% of the saccharide units of each of the first and second alginate polymer are oxidized;- the first and second alginate polymer have each a molecular weight ranging from 50 to 100 kDa; preferably from 60 to 90 kDa; more preferably from 70 to 80 KDa; even more preferably is about 75 KDa;- the molar ratio between norbornene and tetrazine groups moieties ranges from 1.5: 1 to 2: 1 ; preferably said ratio is 1.8:1; and- wherein the amount of first alginate polymer, second alginate polymer and water buffered solution are such as to form a hydrogel comprising a covalently crosslinked alginate polymer with a concentration from 0.5 to 1.25% w / v.

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