In situ recruitment, reprogramming and release of CAR-T cells

Abstract

The present invention relates to in situ recruitment, reprogramming and release of CAR-T cells. The present invention discloses a hydrogel matrix comprising one or more chemical attractants wherein the one or more chemical attractants comprise a C-C motif chemokine ligand (CCL) 1 (CCL1), a CCL5, a CCL19, a CCL21, a CCL22, a CCL28, a C-X-C motif chemokine ligand (CXCL) 1 (CXCL1), CXCL9, CXCL10, CXCL11, CXCL12, M-CSF, GM-CSF, MCP-1, MCP-3, CCL2, CCL3, CCL7, CCL20, CX3CL1, BRAK, IL-12, S1P, and / or MCP2. Also disclosed are a method of treating cancer in a subject and a method of transferring immune cells in a subject.

Description

[0001] This application is a divisional application of a Chinese patent application filed on June 18, 2020, with application number 202080045567.4 and invention name “In situ recruitment, reprogramming and release of CAR-T cells”. Background Art

[0002] Adoptive transfer of chimeric antigen receptor (CAR) redirected T lymphocytes (CAR-T cells) has produced dramatic clinical responses against B-cell malignancies and is also effective against other cancer types. Current methods for manufacturing CAR-T cells on a clinical scale require extensive cell manipulation in vitro: isolation of autologous T cells, transduction with CAR-encoding viral vectors, and expansion of CAR-T cells in vitro before infusion back into the patient. The complex and time-consuming in vitro procedures require significant expense: the procedure takes 3-4 weeks and costs approximately $500,000. These costs limit the potential to expand this technology to other cancers and patients. What is needed are new methods for recruiting and modifying T cells that avoid the problems associated with current CAR T cells. Summary of the invention

[0003] Disclosed herein are methods and compositions involving hydrogels, including chemoattractants.

[0004] In one aspect, disclosed herein is a hydrogel matrix comprising one or more chemoattractants, wherein the one or more chemoattractants comprise CC motif chemokine ligand (CCL) 1 (CCL1), CCL5, CCL19, CCL21, CCL22, CCL28, CXC motif chemokine ligand (CXCL) 1 (CXCL1), CXCL9, CXCL10, CXCL11, CXCL12, M-CSF, GM-CSF, MCP-1, MCP-3, CCL2, CCL3, CCL7, CCL20, CX3CL1, BRAK, IL-12, S1P and / or MCP2.

[0005] Also disclosed herein is a hydrogel matrix of any of the aforementioned aspects, further comprising a viral vector (such as, for example, a lentivirus, a retrovirus, an adenovirus, or an adeno-associated virus) encoding a chimeric antigen receptor (CAR), an NK cell receptor (including but not limited to anti-CD3 antibodies, CD1d, an Fc fragment of an immunoglobulin, or an anti-Fcγ receptor (FcγRIII) antibody), an NK T cell receptor, and / or a T cell receptor (e.g., an antigen-specific T cell receptor).

[0006] On the one hand, the hydrogel matrix of any of the aforementioned aspects is also disclosed herein, which further comprises one or more antibodies, cytokines and / or co-stimulatory molecules, which activate T cells, natural killer (NK) cells, NK T cells, tumor infiltrating lymphocytes (TIL), bone marrow infiltrating lymphocytes (MIL), tumor infiltrating NK cells (TINK), dendritic cells or macrophages. For example, antibodies can include anti-CD3, anti-CD28, anti-inducible co-stimulators (ICOS), anti-CD40L, anti-DAP10; and cytokines can include IL-2, IL-7, IL-15, IL-21, TNF-α or IFN-γ.

[0007] Also disclosed herein is the hydrogel matrix of any of the foregoing aspects, further comprising a chemotherapeutic agent.

[0008] In one aspect, disclosed herein is a method of treating, preventing, inhibiting and / or attenuating cancer or metastasis in a subject, the method comprising administering to the subject the hydrogel matrix of any of the aforementioned aspects. For example, disclosed herein are methods of treating cancer in a subject, comprising administering to the subject a hydrogel matrix comprising one or more chemoattractants, wherein the one or more chemoattractants include C-C motif chemokine ligand (CCL) 1 (CCL1), CCL5, CCL19, CCL21, CCL22, CCL28, C-X-C motif chemokine ligand (CXCL) 1 (CXCL1), CXCL9, CXCL10, CXCL11, CXCL12, M-CSF, GM-CSF, MCP-1, MCP-3, CCL2, CCL3, CCL7, CCL20, CX3CL1, BRAK, IL-12, S1P and / or MCP2; and wherein the chemoattractant attracts and retains immune cells (such as, for example, T cells, NK cells, NK T cells, TILs, MILs, TINKs, dendritic cells and / or macrophages) to the hydrogel.

[0009] Also disclosed herein are methods of treating, preventing, inhibiting and / or reducing cancer or metastasis of any of the aforementioned aspects, wherein the hydrogel matrix further comprises an immunoblocking inhibitor and / or a chemotherapeutic agent.

[0010] On the one hand, disclosed herein is a method for treating, preventing, inhibiting and / or reducing cancer or metastasis in any of the aforementioned aspects, wherein the hydrogel further comprises a viral vector encoding a chimeric antigen receptor (CAR), an NK cell receptor (including but not limited to anti-CD3 antibodies, CD1d, Fc fragments of immunoglobulins, or anti-Fcγ receptor (FcγRIII) antibodies), NK T cell receptors and / or T cell receptors (TCR) (including antigen-specific T cell receptors). On the one hand, the viral vector transduces immune cells; and the transduced immune cells are released from the hydrogel to the cancer. It should be understood and considered herein that the viral vector can be introduced into the hydrogel matrix before administration to the subject or about 1 day to about 14 days after the hydrogel matrix is ​​administered to the subject. That is, the viral vector can be introduced into the hydrogel in vivo.

[0011] Also disclosed herein is a method of treating, preventing, inhibiting and / or reducing cancer or metastasis of any of the aforementioned aspects, wherein the immune cells are released about 1 week to about 12 weeks after administration of the hydrogel.

[0012] In one aspect, disclosed herein are methods of treating, preventing, inhibiting and / or reducing cancer or metastasis of any of the aforementioned aspects, wherein one or more chemoattractants are released from about 1 hour after application of the hydrogel to about 12 weeks after application of the hydrogel.

[0013] In one aspect, disclosed herein is a method of transducing immune cells (such as, for example, T cells, NK cells, NK T cells, TILs or MILs) in a subject, the method comprising administering to the subject a hydrogel comprising one or more chemoattractants (such as, for example, CCL1, CCL5, CCL19, CCL21, CCL22, CCL28, CXCL1, CXCL9, CXCL10, CXCL11, CXCL12, M-CSF, GM-CSF, MCP-1, MCP-3, CCL2, CCL3, CCL7, CCL20, CX3CL1, BRAK, IL-12, S1P and / or MCP2) and a transgene encoding, for example, a CAR, a NK cell receptor (including but not limited to anti-CD3 antibodies, CD1d, an Fc fragment of an immunoglobulin or an anti-Fcγ receptor (FcγRIII) antibody), a NK A viral vector (such as a lentivirus, a retrovirus, an adenovirus, or an adeno-associated virus) that expresses a T cell receptor and / or a TCR (including but not limited to an antigen-specific TCR). It should be understood and contemplated herein that the viral vector can be introduced into the hydrogel matrix prior to administration to the subject or about 1 day to about 14 days after the hydrogel matrix is ​​administered to the subject. That is, the viral vector can be introduced into the hydrogel in vivo.

[0014] Also disclosed herein is a method of transducing immune cells of any of the aforementioned aspects, wherein the one or more chemoattractants are released from about 1 hour after administration of the hydrogel to about 12 weeks after administration of the hydrogel.

[0015] In one aspect, disclosed herein is a method of transducing immune cells of any of the aforementioned aspects, wherein the hydrogel further comprises one or more antibodies, cytokines and / or co-stimulatory molecules that activate T cells, NK cells, NK T cells, TILs or MILs. For example, the antibody may include anti-CD28, CD3, B7-1, B7-2, anti-inducible co-stimulator (ICOS), ICOS ligand, anti-CD27, CD70, 4-1BBL, anti-41-BB, anti-CD40L, CD40, anti-DAP10, anti-CD30, CD30L, anti-TIM-1, anti-TIM-2, anti-TIM-3, anti-CD44, anti-NK1.1, lectin-like transcript-1 (LLT-1), anti-CD137, CD48, MICA, anti-2B4, anti-glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR); and the cytokine may include IL-2, IL-7, IL-15, IL-21, TNF-α or IFN-γ. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and, together with the description, illustrate the disclosed compositions and methods.

[0017] Figure 1 Schematic diagram of in situ generation of CAR-T cells. The implanted scaffold releases chemokines to recruit host T cells. The viral vector reprograms the T cells with tumor-specific CAR constructs, and the CAR-modified T cells migrate out to fight cancer.

[0018] Figure 2 A schematic diagram of the preparation of macroporous gels by cryogelation is shown.

[0019] Figure 3 Shown is a SEM picture of the lyophilized gel (-20°C).

[0020] Figure 4A and Figure 4B CXCL10 mediated T cell recruitment to implanted scaffolds. Figure 4A shows the experimental timeline. Figure 4B Shown are FACS analyses of CD3+ T cells recruited to blank (control) or CXCL10-releasing scaffolds.

[0021] Figure 5A Figure 5B Figure 5C and Figure 5DDemonstrating that pre-seeded and recruited T cells can be transduced within implanted scaffolds. Experimental timeline of GFP+ T cells pre-seeded into scaffolds or recruited into scaffolds by CXCL10 ( Figure 5A Figure 5C) and FACS analysis ( Figure 5B Figure 5D ). D shows the additional effects of CD3 / CD28 and IL-2 in the scaffold.

[0022] Figure 6 In situ generation of CAR T cells is shown.

[0023] Figure 7 In vivo transduction of recruited T cells with CD19 CAR virus is shown.

[0024] Figure 8 A schematic diagram of creating a mouse Burkitt lymphoma tumor model is shown.

[0025] Fig.9A Luminescent images of tumor size at 19, 24, 29, 33, and 43 days after tumor inoculation are shown for animals that received no treatment, intravenous injection of CD19 CAR T cells, or CCI-alginate scaffolds (CCI-Alg).

[0026] Fig. 9B Shown are total flux and percent body weight change at various time points after tumor inoculation in animals that received no treatment, intravenous CD19 CAR T cells, or CCI-alginate scaffolds (CCI-Alg).

[0027] Fig.10 The number of CAR+ cells per 100ul of blood is shown in mice implanted with CCI scaffolds or injected intravenously with CAR-T cells. Mice were bled by cheek bleeding, red blood cells were lysed, and cells were stained with Hu-CD45, Hu-CD3, and CAR.19 antibodies and analyzed by flow cytometry. DETAILED DESCRIPTION

[0028] Before disclosing and describing the compounds, compositions, articles, devices and / or methods of the present invention, it should be understood that, unless otherwise specified, they are not limited to specific synthetic methods or specific recombinant biotechnology methods, or, unless otherwise specified, they are not limited to specific reagents, and thus, they may of course vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting.

[0029] A. Definition

[0030] Throughout this specification and the claims that follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0031] As used in the specification and the appended claims, the singular forms "a," "an," "the," and "said" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0032] Ranges can be expressed herein as from "about" a particular value and / or to "about" another particular value. When such ranges are expressed, another embodiment includes from a particular value and / or to another particular value. Similarly, when the value is expressed as an approximation using the preceding word "about", it should be understood that the particular value forms another embodiment. It should also be understood that the endpoints of each range are significant relative to another endpoint and independently of another endpoint. It should also be understood that many values ​​are disclosed herein, and each value is disclosed herein as "about" the particular value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. It should also be understood that when "less than or equal to" a value, "greater than or equal to" a value is disclosed, the possible range between values ​​as properly understood by those skilled in the art is also disclosed. For example, if the value "10" is disclosed, "less than or equal to 10" and "greater than or equal to 10" are also disclosed. It should also be understood that throughout the application, data is provided in a variety of different formats, and the data represents endpoints and starting points, as well as the range of any combination of data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as between 10 and 15 are disclosed. It is also understood that every unit between the two specific units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0033] "Administering" to a subject includes any approach of introducing or delivering a medicament to a subject. Can be administered by any suitable approach, including oral, topical, intravenous, subcutaneous, percutaneous, transdermal, intramuscular, intraarticular, parenteral, intraarteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, by implanted reservoirs, parenteral (e.g., subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional and intracranial injection or infusion techniques) etc. As used herein, "parallel administration", "co-administration", "simultaneous administration (simultaneous administration or administered simultaneously)" means that the compound is administered at the same time point or is substantially administered immediately thereafter. In the latter case, the administration time of the two compounds is sufficiently close, so that the observed result is indistinguishable from the result obtained when the compound is administered at the same time point. "Systemic administration" refers to the introduction or delivery of an agent to a subject by a route that introduces or delivers the agent to a wide area of ​​the subject's body (e.g., more than 50% of the body), such as by entering the circulatory or lymphatic system. In contrast, "local administration" refers to the introduction or delivery of an agent to a subject by a route that introduces or delivers the agent to one or more areas in close proximity to the point of administration and does not introduce the agent systemically in therapeutically significant amounts. For example, a locally administered agent is easily detected in the vicinity of the local point of administration, but is not detectable or the amount detected is negligible in distal portions of the subject's body. Administration includes self-administration and administration by others.

[0034] "Biocompatible" generally means that the material and any of its metabolites or degradation products are generally non-toxic to the subject and do not cause significant adverse effects to the recipient.

[0035] "Comprising" means that the composition, method, etc. includes the elements mentioned, but does not exclude other elements. When used to define compositions and methods, "essentially consisting of..." shall mean including the elements mentioned, but not including other elements that are of any significance to the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from separation and purification methods and pharmaceutical carriers, such as phosphate-buffered saline, preservatives, etc. "Consisting of..." shall mean excluding trace elements more than other ingredients and substantial method steps for administering the compositions of the invention. Embodiments defined by each of these transition terms are within the scope of the invention.

[0036] A "control" is an alternative subject or sample used for comparison purposes in an experiment. A control can be a "positive control" or a "negative control."

[0037] "Controlled release" or "sustained release" refers to the release of an agent from a given dosage form in a controlled manner in order to achieve a desired pharmacokinetic profile in vivo. One aspect of "controlled release" drug delivery is the ability to manipulate the formulation and / or dosage form to establish desired agent release kinetics.

[0038] The "effective amount" of a medicament refers to a sufficient amount of the medicament to provide the desired effect. The amount of an "effective" medicament will vary between different subjects, depending on many factors such as the age and general condition of the subject, specific one or more medicaments, etc. Therefore, it is not always possible to specify a quantitative "effective amount". However, the appropriate "effective amount" in any subject case can be determined by a person of ordinary skill in the art using conventional experimental methods. In addition, as used herein, and unless otherwise specifically stated, the "effective amount" of a medicament may also refer to the amount covering a therapeutically effective amount and a preventive effective amount. The "effective amount" of the medicament required to achieve the therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. The dosage regimen may be adjusted to provide the best therapeutic response. For example, several separate doses may be administered daily, or the dosage may be reduced in proportion to the urgency of the treatment situation.

[0039] "Increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. A decrease can be any individual, median, or average increase in a condition, symptom, activity, composition of matter that is a statistically significant amount. Thus, an increase can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, as long as the increase is significant. An increase can also be referenced to a multiple increase. For example, an increase can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 - doubled.

[0040] "Reduction" can refer to any change that causes less gene expression, protein expression, symptom quantity, disease, composition, illness or activity. When the genetic yield of the gene product containing the substance is smaller than the genetic yield of the gene product without the substance, the substance is also understood to reduce the genetic yield of the gene. In addition, for example, reduction can be a change in a disorder symptom, so that the symptom is less than previously observed. Reduction can be any individual, median or average reduction in the illness, symptom, activity, composition of a statistically significant amount. Therefore, as long as the reduction is very significant, the reduction can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. Reduction can also be referenced by multiple reduction. For example, a reduction may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 -fold reduction.

[0041] "Inhibit," "inhibiting," and "inhibition" mean a decrease in an activity, response, condition, disease, or other biological parameter. This may include, but is not limited to, complete ablation of an activity, response, condition, or disease. This may also include, for example, a 10% decrease in an activity, response, condition, or disease compared to an untreated or control level. Thus, the decrease may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any decrease therebetween compared to an untreated or control level.

[0042] As used herein, the terms "prevent," "preventing," or "prevention" and grammatical variations thereof refer to a method of partially or completely delaying or preventing the occurrence or recurrence of a disease and / or one or more of its attendant symptoms, or preventing a subject from acquiring or reacquiring a disease or reducing the risk of a subject acquiring or reacquiring a disease or one or more of its attendant symptoms.

[0043] A "pharmaceutically acceptable" component may refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the present invention and administered to a subject as described herein without causing significant adverse biological effects or interacting in a deleterious manner with any other component of the formulation containing the component. When used in reference to administration to humans, the term generally means that the component has met the required standards of toxicology and manufacturing testing, or it is included in the inactive ingredient guide established by the U.S. Food and Drug Administration.

[0044] "Pharmaceutically acceptable carrier" (sometimes referred to as "carrier") means a carrier or excipient that can be used to prepare a generally safe and non-toxic pharmaceutical or therapeutic composition, and includes acceptable carriers for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" may include, but is not limited to, phosphate buffered saline solution, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. As used herein, the term "carrier" includes, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, or other material well known in the art for pharmaceutical formulations, and as further described herein.

[0045] "Pharmacologically active" (or just "active"), as used in "pharmacologically active" derivatives or analogs, may refer to derivatives or analogs (e.g., salts, esters, amides, conjugates, metabolites, isomers, fragments, etc.) that have the same type of pharmacological activity as the parent compound and to approximately the same extent.

[0046] "Therapeutic agent" refers to any composition having a beneficial biological effect. Beneficial biological effects include therapeutic effects and preventive effects, wherein therapeutic effects are such as the treatment of disorders or other adverse physiological conditions, and preventive effects are such as the prevention of disorders or other adverse physiological conditions (e.g., non-immunogenic cancers). The term also encompasses pharmacologically active derivatives of the beneficial agents specifically mentioned herein in pharmaceutical use, including but not limited to salts, esters, amides, pro-agents, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, or when a specific agent is clearly identified, it should be understood that the term includes the agent itself as well as pharmacologically active salts, esters, amides, pro-agents, conjugates, active metabolites, isomers, fragments, analogs, and the like in pharmaceutical use.

[0047] A "therapeutically effective amount" or "therapeutically effective dose" of a composition (e.g., a composition comprising a medicament) refers to an amount that is effective to achieve a desired therapeutic outcome. In some embodiments, the desired therapeutic outcome is control of type I diabetes. In some embodiments, the desired therapeutic outcome is control of obesity. The therapeutically effective amount of a given therapeutic agent will generally vary according to factors such as the type and severity of the disorder or disease being treated and the age, sex, and weight of the subject. The term may also refer to the amount of the therapeutic agent or the delivery rate (e.g., amount over time) of the therapeutic agent that effectively promotes a desired therapeutic effect (such as pain relief). The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the medicament to be administered and / or the medicament formulation (e.g., the efficacy of the therapeutic agent, the concentration of the medicament in the formulation, etc.), and various other factors understood by those of ordinary skill in the art. In some cases, a desired biological or medical response may be obtained after multiple doses of the composition are administered to the subject for several consecutive days, weeks, or years.

[0048] Throughout this specification and the claims that follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0049] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0050] Throughout this application, various publications are cited. The entire disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this application relates. The disclosed references are also individually and specifically incorporated by reference herein, and the materials contained in the references are discussed in the sentences on which the references are based.

[0051] B. Composition

[0052] The present invention discloses compositions for preparing the compositions disclosed herein, as well as the compositions themselves used in the methods disclosed herein. These and other materials are disclosed herein, and it should be understood that when the present invention discloses combinations, subsets, interactions, groups, etc. of these materials, although specific references to various individual and collective combinations and arrangements of these compounds may not be explicitly disclosed, each of them is specifically considered and described herein. For example, if a specific hydrogel matrix containing a chemoattractant is disclosed and discussed, and many modifications that can be made to many molecules including the hydrogel matrix containing the chemoattractant are discussed, then each and every combination and arrangement of the hydrogel matrix is ​​specifically considered, which contains the chemoattractant and possible modifications, unless there is a specific indication to the contrary. Thus, if a class of molecules A, B and C and a class of molecules D, E and F and examples of combined molecules are disclosed, AD is disclosed, then even if each of them is not cited separately, it is considered that the meaning combinations AE, AF, BD, BE, BF, CD, CE and CF considered individually and collectively are disclosed. Likewise, any subset or combination of these combinations is also disclosed. Thus, for example, a subgroup of AE, BF and CE will be considered disclosed. This concept applies to all aspects of this application, including but not limited to steps in methods of making and using the compositions disclosed herein. Therefore, if there are various additional steps that can be performed, it should be understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods disclosed herein.

[0053] Chimeric antigen receptor (CAR) immunotherapy is a cancer immunotherapy that involves genetically modifying the patient's autologous T cells, NK cells, and / or macrophages into CAR-T cells, CAR-NK cells, CAR-NK-T cells, or CAR macrophages (CARMA). These CAR cells express antibody-based chimeric antigen receptors that can target specific tumor antigens. The process includes the separation of autologous T cells, NK cells, NK T cells, or macrophages, transduction with CAR-encoding viral vectors, and in vitro expansion of CAR-T cells, CAR-NK cells, CAR-NK-T cells, or CARMA and then infusion back into the patient. Despite significant clinical success, the laborious, time-consuming, and expensive procedures associated with the generation of clinical-grade CAR immune cells (e.g., CAR T cells) according to strict regulatory standards remain the main obstacle to the implementation of CAR immune cell therapy as a standard of care for cancer treatment. Methods that can eliminate the time and cost associated with generating CAR immune cells have great clinical significance and can significantly expand the availability of CAR T cells, CAR-NK cells, CAR-NK-T cells, and / or CARMA therapies.

[0054] Generating tumor-specific CAR immune cells in situ can overcome many of the challenges associated with generating CAR-T cells in vitro. Chimeric antigen receptors (CARs) are synthetic receptors that specifically redirect immune cells (e.g., CAR T cells, CAR-NK cells, CAR-NK-T cells, or CARMA) to tumor-associated antigens. Infusion of CD19. CAR-T cells showed significant anti-tumor effects in the first CD19 clinical trial. CAR-T therapy was approved by the FDA in 2017. T cell collection, in vitro activation, genetic modification and expansion of cells, and subsequent infusion into patients are both time-consuming (approximately 4 weeks) and expensive (approximately $500,000). Given the burden that cancer has placed on the healthcare system, providing CAR immune cell therapy to millions of cancer patients is a huge challenge. This article shows that CAR immune cells can be generated in situ in a macroporous scaffold loaded with T cell recruitment factors and CAR-encoding viral vectors. This approach eliminates the time and cost associated with generating CAR immune cells (e.g., CAR T cells, CAR-NK cells, CAR-NK-T cells, or CARMA), has enormous clinical significance, and could significantly expand the availability of CAR immune cell therapies.

[0055] This article discloses a strategy for generating CAR immune cells in situ in a patient using a bioactive macroporous scaffold. The scaffold recruits immune cells, activates and reprograms them into CAR immune cells, and releases the CAR immune cells into the body to find tumors ( Figure 1 ). The controlled release of chemokines from the scaffold creates a gradient that attracts T cells (including but not limited to TIL and MIL) NK cells (including but not limited to TINK), NK T cells, dendritic cells and / or macrophages to the scaffold. The macroporous scaffold promotes chemokine-mediated infiltration of T cells, NK cells, NK T cells, dendritic cells and / or macrophages, and together with embedded cytokines and antibodies, promotes the survival and expansion of T cells, NK cells, NK T cells, dendritic cells and / or macrophages, and provides an interface for the transduction of viral particles. Once the chemokine is fully released, its depletion promotes the migration of CAR-T cells, CAR-NK cells, CAR-NK-T cells or CARMA out of the scaffold. In this method, T cells, NK cells, NK T cells, dendritic cells and / or macrophages are recruited to the biomaterial scaffold, reprogrammed with CAR genes via viral vectors, and then released from the scaffold into the circulation, thereby achieving the in situ seamless generation of CAR-T cells, CAR-NK cells, CAR-NK-T cells or CARMA. Thus, one aspect disclosed herein is a hydrogel matrix comprising one or more chemoattractants.

[0056] It is understood and contemplated herein that the purpose of the chemoattractant is to attract immune cells (e.g., T cells, natural killer (NK) cells, NK T cells, tumor infiltrating lymphocytes (TIL), bone marrow infiltrating lymphocytes (MIL), tumor infiltrating NK cells (TINK), dendritic cells and / or macrophages) to the hydrogel. Examples of one or more chemoattractants for the disclosed hydrogels include, but are not limited to, CC motif chemokine ligand (CCL) 1 (CCL1), CCL5, CCL19, CCL21, CCL22, CCL28, CXC motif chemokine ligand (CXCL) 1 (CXCL1), CXCL9, CXCL10, CXCL11, CXCL12, M-CSF, GM-CSF, MCP-1, MCP-3, CCL2, CCL3, CCL7, CCL20, CX3CL1, BRAK, IL-12, S1P and / or MCP2.

[0057] The hydrogel disclosed herein can be made using any suitable biodegradable polymer. "Polymer" refers to a natural or synthetic organic compound of relatively high molecular weight, whose structure can be represented by repeated small units, monomers. Non-limiting examples of polymers include polyethylene, rubber, cellulose. Synthetic polymers are usually formed by addition or polycondensation of monomers. The term "copolymer" refers to a polymer formed by two or more different repeating units (monomer residues). By way of example and without limitation, a copolymer can be an alternating copolymer, a random copolymer, a block copolymer or a graft copolymer. It is also conceivable that in some aspects, the various block segments of a block copolymer may themselves comprise a copolymer. The term "polymer" includes all forms of polymers, including but not limited to natural polymers, synthetic polymers, homopolymers, heteropolymers or copolymers, addition polymers, etc.

[0058] In one aspect, the hydrogel may include a biocompatible polymer (such as, for example, alginate). Such polymers can also be used to slowly release CAR T cells, CAR NK cells, CAR NK T cells, CARMA, TIL, MIL, TINK and / or MIL into tissues. As used herein, biocompatible polymers include, but are not limited to, polysaccharides such as alginate, chitosan, hyaluronic acid; hydrophilic polypeptides; proteins such as collagen, fibrin, and gelatin; poly(amino acids) such as poly-L-glutamic acid (PGS), γ-polyglutamic acid, poly-L-aspartic acid, poly-L-serine, or poly-L-lysine; polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG), polypropylene glycol (PPG), and poly(ethylene oxide) (PEO); poly(oxyethylated polyols); poly(olefin alcohols); polyvinyl pyrrolidone; poly(hydroxyalkyl methacrylamide); poly(hydroxyalkyl methacrylic acid); poly(saccharides); poly(hydroxy acids); poly(vinyl alcohol), polyhydroxy acids such as poly(lactic acid), poly( (glycolic acid) and poly(lactic-co-glycolic acid); polyhydroxyalkanoates such as poly 3-hydroxybutyric acid or poly 4-hydroxybutyric acid; polycaprolactones; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(lactide-caprolactone); polycarbonates such as tyrosine polycarbonate; polyamides (including synthetic and natural polyamides), polypeptides and poly(amino acids); polyesteramides; polyesters; poly(dioxanones); poly(alkylenes); hydrophobic polyethers; polyurethanes; polyetheresters; polyacetals; polycyanoacrylates; polyacrylates; polymethyl methacrylate; polysiloxanes; poly(oxyethylene) / poly(oxypropylene) copolymers; polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acid) and copolymers thereof.Biocompatible polymers may also include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkyl terephthalates, polyvinyl alcohol (PVA), methacrylate PVA (m-PVA), polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinyl pyrrolidone, polyethylene glycol, polysiloxanes, polyurethanes and copolymers thereof, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, ethyl cellulose, polymers of acrylic acid and methacrylate, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(methacrylic acid),

[0013] Exemplary biodegradable polymers include polyesters, poly(ortho-esters), poly(vinylamines), poly(caprolactones), poly(hydroxybutyrates), poly(hydroxyvalerates), polyanhydrides, poly(acrylic acids), polyethylene glycols, poly(urethanes), polycarbonates, polyphosphates, polyphosphazenes and derivatives thereof, linear and branched copolymers and block copolymers thereof (including triblock copolymers), and blends thereof.

[0059] In some embodiments, the particles comprise biocompatible and / or biodegradable polyesters or polyanhydrides, such as poly(lactic acid), poly(glycolic acid), and poly(lactic-glycolic acid). The particles may comprise one / more of the following polyesters: homopolymers comprising glycolic acid units (referred to herein as "PGA") and lactic acid units (such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide 5, collectively referred to herein as "PLA") and caprolactone units (such as poly(caprolactone), collectively referred to herein as "PCL"); and copolymers comprising lactic acid and glycolic acid units (such as various forms of poly(lactic-co-glycolic acid) and poly(lactide-glycolide), collectively referred to herein as "PLGA", characterized by a ratio of lactic acid to glycolic acid; and polyacrylates, and derivatives thereof. Exemplary polymers also include copolymers of polyethylene glycol (PEG) and the above polyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as "PEGylated polymers". In certain embodiments, the PEG region can be covalently associated with a polymer to produce a "PEGylated polymer" via a cleavable linker. In one aspect, the polymer comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% acetal side groups.

[0060] The triblock copolymers disclosed herein comprise a core polymer such as, for example, polyethylene glycol (PEG), polyvinyl acetate, polyvinyl alcohol, polyvinyl pyrrolidone (PVP), polyethylene oxide (PEO), poly(vinyl pyrrolidone-vinyl acetate), polymethacrylate, polyoxyethylene alkyl ether, polyoxyethylene castor oil, polycaprolactam, polylactic acid, polyglycolic acid, poly(lactic acid-glycolic acid), poly(lactic acid-co-glycolic acid) (PLGA), cellulose derivatives (such as hydroxymethyl cellulose, hydroxypropyl cellulose, etc.).

[0061] In one aspect, the hydrogel matrix can further comprise immune activating and / or maintaining antibodies, chemokines, and cytokines, such as, for example, CD28, CD3, IL-2, IL-7, IL-15, IL-21, IFN-γ, and TNF-α.

[0062] It is understood and contemplated herein that the disclosed hydrogel matrices may further comprise one or more immune checkpoint inhibitors and / or chemotherapeutic agents. Chemotherapeutic agents that can be used in the disclosed hydrogel matrix may include any chemotherapeutic agent known in the art, including but not limited to pomacillin, abiraterone acetate, Abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Bentuximab Vedotine), ADE, Ado-Trastuzumab Emtansine, Adriamycin (doxorubicin hydrochloride), afatinib dimaleate, Afinitor (everolimus), Akynzeo (netopitant and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, Alecensa (alectinib), alectinib, alemtuzumab, Alimta (pemetrexed disodium), Aliqopa (coupanib hydrochloride), Aloxicam (melphalan hydrochloride), Aloxicam tablets (melphalan), and tadalafil (tadalafil). (palonosetron hydrochloride), Alunbrig (brigatinib), Ambochlorin (chlorambucil), Amboclorin (chlorambucil), Amifostine, aminolevulinic acid, Anastrozole, Aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Arranon (nelarabine), Arsenic trioxide, Arzerra (ofatumumab), Aspartame Amidase Erwinia chrysanthemi, atezolizumab, Avastin (bevacizumab), avelumab, axitinib, azacitidine, Bavencio (avelumab), BEACOPP, Becenum (carmustine), Beleodaq (belinostat), belinostat, bendamustine hydrochloride, BEP, Besponsa (inostatin-ozogamicin), bevacizumab, bexarotene, Bexxar (tositumomab and iodine I 131 tositumomab), Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab, Brigatinib, BuMel, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometyx (Cabozantinib Malate), Cabozantinib Malate, CAF, Campath (Alemtuzumab), Camptosar, (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carac (Fluorouracil - Topical), Carboplatin, Carboplatin-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine Transplant, Casodex (Bicalutamide), CEM, Ceritinib, Cerubidine (Doxorubicin Hydrochloride),Cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Clolar (clofarabine), CMF, cabozantinib, Cometriq (cabozantinib malate), coupanisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (actinomycin D), Cotellic (cabozantinib), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine liposomal, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), Actinomycin D, Daratumumab, Darzalex (daratumumab), dasatinib, doxorubicin hydrochloride, doxorubicin hydrochloride and cytarabine liposomal, decitabine, defibrotide sodium, Defitelio (defibrotide sodium), Degarelix, denileukin, denosumab, DepoCyt (cytarabine liposomal), dexamethasone, dexrazoxane hydrochloride, denoximab, docetaxel, Doxil (doxorubicin hydrochloride liposomal), doxorubicin hydrochloride, doxorubicin hydrochloride liposomal, Dox-SL (doxorubicin hydrochloride liposomes), DTIC-Dome (dacarbazine), durvalumab, Efudex (fluorouracil -- topical), Elitek (rasburicase), Ellence (epirubicin hydrochloride), elotuzumab, Eloxatin (oxaliplatin), eltrombopag, Emend (aforepitant), Empliciti (elotuzumab), etanercept mesylate, enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinaze (asparaginase Erwinia chrysanthemi), Ethyol (amifortin), Etopophos (phosphophosphodiesterase Etoposide, Etoposide, Etoposide phosphate, Evacet (liposomal doxorubicin hydrochloride), Everolimus, Evacet, (raloxifene hydrochloride), Evomela (melphalan hydrochloride), Exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil - topical), Fareston (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), Filgrastim, Fludara (fludarabine phosphate), Fludarabine phosphate, Fluoroplex (fluorouracil - topical), Fluorouracil injection, Fluorouracil - topical, Flutamide, Folex (methotrexate), Folex PFS (methotrexate),FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRINOX, FOLFOX, Folotyn (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV nine-valent vaccine), Gazyva (obituzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), Gliadel (carmustine transplant), wafer (carmustine implant), glutaraldehyde, goserelin acetate, Halaven (eribulin mesylate), Hemangeol (propranolol hydrochloride), Herceptin (trastuzumab), recombinant HPV secondary vaccine, recombinant HPV nine-valent vaccine, recombinant HPV four-valent vaccine, Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper-CVAD, Ibrance (palbociclib), isothiocarbamazine, ibrutinib ANI, ICE, Iclusig (Panatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalis, Idhifa (Ematinib Mesylate), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Ointuzumab Ogamycin, Recombinant Interferon Alpha-2b, Interleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alpha-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (gefitinib), Irinotecan hydrochloride, Irinotecan liposome hydrochloride, Istodax (romidepsin), Ixabepilone, Ixazomib citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate), JEB, Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Kymriah (tisagenlecleucel), Kyprolis (cafilomib), Lanreotide acetate, Lapatinib ditosylate, Lartruvo (olalimumab), Lenalidomide, Lenvatinib mesylate, Lenvima (lenvatinib mesylate), Letrozole,Calcium folinate, Leukeran (chlorambucil), leuprolide acetate, Leustatin (cladribine), Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (liposomal doxorubicin hydrochloride), lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lynparza (olaparib), Marqibo (liposomal vincristine sulfate), Matulane (methylprocarbazine hydrochloride), nitrogen mustard hydrochloride, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, mesna, Mesnex (mesna), Methazolastone (temozolomide), methotrexate, methotrexate LPF (methotrexate), methylnaltrexone bromide, Mexate (methotrexate), Mexate- AQ (methotrexate), midostaurin, mitomycin C, mitoxantrone hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (plerixafor), Mustargen (nitrogen mustard hydrochloride), Mutamycin (mitomycin C), Myleran (busulfan), Mylosar (azacytidine), Mylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine tartrate), necituzumab, nelarabine, Neosar (cyclophosphamide), maleic acid Neratinib, Nerlynx (neratinib maleate), netopitant and palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nilandron (nilutamide), nilotinib, nilutamide, Ninlaro (ixazomib citrate), niraprol monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, Odomzo (sonidegi), OEPA, ofatumumab, OFF, Odomzo Lapanib, Olalumumab, Homoharringtonine, Oncaspar (pegaspargase), Ondansetron hydrochloride, Onivyde (liposomal irinotecan hydrochloride), Ontak (denileukin), Opdivo (nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel albumin-stabilized nanoparticle formulation, PAD, Palbociclib, Palifermin, Palonosetron hydrochloride, Palonosetron hydrochloride and Netupitant, Pamidronate disodium, Panitumumab, Panobinostat, Paraplat (carboplatin), Paraplatin (carboplatin), Pazopanib hydrochloride, PCV, PEB,Pegaspargase, pegfilgrastim, peginterferon alfa-2b, PEG-Intron (peginterferon alfa-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor, pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, Portrazza (nesituzumab), pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag), propranolol hydrochloride, Provenge (Sip uleucel-T), Purinethol (mercaptopurine), Purixan (mercaptopurine), radium 223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nine-valent vaccine, recombinant human papillomavirus (HPV) four-valent vaccine, recombinant interferon alpha-2b, regorafenib, Relistor (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, Rituxan (rituximab), Rituxan Hycela (rituximab and human hyaluronidase), Rituximab, Rituximab and human hyaluronidase, Rolapitant hydrochloride, Romidepsin, Romiplostim, Rubidomycin (doxorubicin hydrochloride), Rubraca (recaparib camphorsulfonate), Recaparib camphorsulfonate, Ruxolitinib phosphate, Rydapt (midostaurin), Intrapleural sclerosant aerosol (Talc), Certuinimab, Sipuleucel-T, Somatuline Depot (lanreotide acetate), Sonidegi, Sorafenib tosylate, Sprycel (dasatinib), STANFORD V, Sterile Talc (Talc), Steritalc (Talc), Stivarga (regorafenib), Sunitinib Malate, Sutent (sunitinib malate), Sylatron (pegylated interferon alpha-2b), Sylvant (Cetuximab), Synribo (homoharringtonine), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel),Taxotere (docetaxel), Tecentriq, (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), thioguanine, thiotepa, Tisagenlecleucel, Tolak (fluorouracil - topical), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab, and iodine I 131 Tositumomab, Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (denotuximab), uridine triacetate, VAC, vandetanib, VAMP, Varubi (rolapitant hydrochloride), Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), vemurafenib, Venclexta (venetola), venetola, Verzenio (pomacicillin), Viadur (leuprolide acetate), Vidaza (azacytidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, liposomal vincristine sulfate, vinorelbine tartrate, VIP, vismodegib, Vistogard (uridine triacetate), Voraxaze (glutarcapase), Vorinostat, Votrient (pazopanib hydrochloride), Vyxeos (liposomal doxorubicin hydrochloride and cytarabine), Wellcovorin (leucovorin), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium 223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab) , Yondelis (trabectedin), Zaltrap (ziv-aflibercept), Zarxio (filgrastim), Zejula (nirapril toluenesulfonate monohydrate), Zelboraf (vemurafenib), Zevalin (isbetumomab), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalis), Zykadia (ceritinib), and / or Zytiga (abiraterone acetate). Checkpoint inhibitors include, but are not limited to, antibodies that block PD-1 (nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475),PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHIgM12B7), CTLA-4 (ipilimumab (MDX-010), tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016).

[0063] In one aspect, the present invention also discloses a hydrogel matrix as disclosed herein, which further comprises one or more antibodies, cytokines and / or co-stimulatory molecules, which activate T cells, natural killer (NK) cells, NK T cells, dendritic cells, macrophages, tumor infiltrating NK cells (TINK), tumor infiltrating lymphocytes (TIL) or bone marrow infiltrating lymphocytes (MIL). For example, the antibody may include anti-CD28, CD3, B7-1, B7-2, anti-inducible co-stimulator (ICOS), ICOS ligand, anti-CD27, CD70, 4-1BBL, anti-41-BB, anti-CD40L, CD40, anti-DAP10, anti-CD30, CD30L, anti-TIM-1, anti-TIM-2, anti-TIM-3, anti-CD44, anti-NK1.1, lectin-like transcript-1 (LLT-1), anti-CD137, CD48, MICA, anti-2B4, anti-glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR); and the cytokine may include IL-2, IL-7, IL-15, IL-21, TNF-α or IFN-γ.

[0064] One advantage of the disclosed hydrogels is that once immune cells are contacted with the hydrogel, they can be transduced to contain transgenes, such as chimeric antigen receptors (e.g., antibodies or scFvs targeting tumor-specific antigens), T cell receptors (e.g., antigen-specific T cell receptors), NK cell receptors (including but not limited to anti-CD3 antibodies, CD1d, Fc fragments of immunoglobulins, or anti-Fcγ receptor (FcγRIII) antibodies), NK T cell receptors. Tumor antigens are proteins produced by tumor cells that can elicit an immune response, especially a T cell-mediated immune response. The additional antigen binding domains can be antibodies, or natural ligands of tumor antigens, or molecules that recognize peptides derived from tumor antigens presented by MHC molecules. The choice of additional antigen binding domains will depend on the specific type of cancer to be treated.Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), ALK, CD19, CD123, cyclin B1, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAX5, SART3, CLL-1, fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, sialic acid, PLAC1, RU1, RU2 (AS), intestinal carboxylesterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostate enzymes, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53, p53 mutants, Ras mutants, gplOO, prostaglandins, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-β, survivin and telomerase, legumain, HPV E6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, MelanA / MART 1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin.

[0065] 1. Delivery of the composition to cells

[0066] The transduction of immune cells can occur by any means known in the art. On the one hand, the transduction of immune cells can occur by a viral vector encoding a transgene (such as, for example, a CAR). Therefore, on the one hand, disclosed herein is any disclosed hydrogel, further comprising a viral vector encoding a transgene, such as, for example, a chimeric antigen receptor (CAR), a T cell receptor, a NK cell receptor and / or a NK T cell receptor (such as, for example, a lentivirus, a retrovirus, adenovirus or adeno-associated virus).

[0067] There are many compositions and methods that can be used to deliver nucleic acid to cells in vitro or in vivo. These methods and compositions can be roughly divided into two categories: viral-based delivery systems and non-viral-based delivery systems. For example, nucleic acid can be delivered by many direct delivery systems, such as electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or by the transfer of genetic material in cells or carriers such as cationic liposomes. Suitable transfection methods, including viral vectors, chemical transfection agents or physical and mechanical methods, such as direct diffusion of electroporation and DNA, are described in Wolff, JA, et al., Science, 247, 1465-1468, (1990); and Wolff, JA Nature, 352, 815-818, (1991). Such methods are well known in the art and are easily applicable to compositions and methods described herein. In some cases, these methods will be modified to specifically bind to large DNA molecules. Furthermore, by exploiting the targeting features of the vectors, these approaches can be used to target certain diseases and cell populations.

[0068] (1) Retroviral vectors

[0069] Retroviruses are animal viruses belonging to the Retroviridae virus family, including any type, subfamily, genus or tropism. In general, retroviral vectors are described by Verma, IM, Retroviral vectors for gene transfer.

[0070] Retrovirus is essentially a package in which nucleic acid cargo is packaged. Nucleic acid cargo carries a packaging signal with it, which can ensure that the replicated submolecule is effectively packaged in the packaging shell. In addition to the packaging signal, cis also requires many molecules to replicate and package the replicated virus. Typically, the retroviral genome includes gag, pol and env genes, which are related to the preparation of the protein shell. It is gag, pol and env genes that are usually replaced by exogenous DNA and transferred to the target cell. Retroviral vectors usually include a packaging signal for incorporation into the packaging shell, which indicates the start of the gag transcription unit, the necessary elements for reverse transcription, including the primer binding site for binding the reverse transcription tRNA primer, the terminal repeat sequence for the conversion of the RNA chain during the DNA synthesis process, the purine-rich 5' to 3' LTR sequence (used as the starting site of the second chain of synthetic DNA synthesis), and the specific sequence near the end of the LTR, which can realize the insertion of the retroviral DNA state to insert into the host genome. Removal of the gag, pol and env genes allows about 8 kb of foreign sequences to be inserted into the viral genome, become reverse transcribed, and packaged into new retroviral particles after replication. Depending on the size of each transcript, this amount of nucleic acid is sufficient to deliver one to multiple genes. Positive or negative selectable markers and other genes are preferably included in the insert.

[0071] Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol and env), vectors are usually produced by placing them into packaging cell lines. Packaging cell lines are cell lines that have been transfected or transformed with retroviruses that contain replication and packaging machinery but do not have any packaging signals. When the vector carrying the selected DNA is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles through the machinery provided by the helper cells in cis. The genomes used for the machinery are not packaged because they lack the necessary signals.

[0072] (2) Adenovirus vector

[0073] The construction of replication-deficient adenoviruses has been described (Berkner et al., J. Virology 61: 1213-1220 (1987); Massie et al., Mol. Cell. Biol. 6: 2872-2883 (1986); Haj-Ahmad et al., J. Virology 57: 267-274 (1986); Davidson et al., J. Virology 61: 1226-1239 (1987); Zhang "Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis" BioTechniques 15: 868-872 (1993)). The benefit of using these viruses as vectors is that the extent to which they can spread to other cell types is limited because they can replicate within the initially infected cells but cannot form new infectious viral particles. Recombinant adenovirus has been shown to achieve efficient gene transfer in vivo following direct delivery to airway epithelium, hepatocytes, vascular endothelium, central nervous system parenchyma, and many other tissue sites (Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092 (1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 5:154-159 (1993); 4:461-476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993) and Ragot, J. Gen. Virology 74:501-507 (1993)).Recombinant adenovirus achieves gene transduction by binding to specific cell surface receptors, and the virus is then internalized by receptor-mediated endocytosis in the same manner as wild-type or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell 73:309-319(1993)).

[0074] The viral vector may be a vector based on an adenovirus from which the E1 gene has been removed, these virions being produced in a cell line such as the human 293 cell line. In another preferred embodiment, both the E1 and E3 genes are removed from the adenoviral genome.

[0075] (3) Adeno-associated virus vector

[0076] Another type of viral vector is based on adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is non-pathogenic to humans. AAV-type vectors can transport about 4 to 5 kb, and it is known that wild-type AAV can be stably inserted into chromosome 19 (such as, for example, at AAV integration site 1 (AAVS1)). Vectors containing this site-specific integration property are preferred. A particularly preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene HSV-tk and / or a marker gene, such as a gene encoding green fluorescent protein GFP.

[0077] In another type of AAV virus, AAV contains a pair of inverted terminal repeats (ITRs) flanking at least one cassette containing a promoter directing cell-specific expression operably linked to a heterologous gene. In this context, heterologous refers to any nucleotide sequence or gene that is not native to AAV or B19 parvovirus.

[0078] Typically, the AAV and B19 coding regions have been deleted to form a safe, non-cytotoxic vector. The AAV ITR or its modification confers infectivity and site-specific integration, but does not confer cytotoxicity, and the promoter directs cell-specific expression. For materials related to AAV vectors, U.S. Patent No. 6,261,834 is incorporated herein by reference.

[0079] Thus, the disclosed vectors provide DNA molecules that are capable of integrating into mammalian chromosomes without substantial toxicity.

[0080] Genes inserted in viruses and retroviruses usually contain promoters and / or enhancers to help control the expression of the desired gene product. Promoters are usually one or more DNA sequences that function when they are in a relatively fixed position relative to the transcription start site. Promoters contain core elements required for the basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

[0081] (4) Large Payload Viral Vectors

[0082] Molecular genetic experiments carried out with large human herpesviruses provide a means by which large heterologous DNA fragments can be cloned, propagated and established in cells that allow herpesvirus infection (Sun et al., Nature genetics 8: 33-41, 1994; Cotter and Robertson, Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses (herpes simplex virus (HSV) and Epstein Barr virus (EBV)) have the potential to deliver>150kb human heterologous DNA fragments to specific cells. EBV recombinants can keep the bulk DNA in infected B cells as episomal DNA. Single clones carrying up to 330kb human genome inserts seem to have genetic stability. The maintenance of these episomes requires specific EBV nucleoprotein EBNA1, which is constitutively expressed during EBV infection. In addition, these vectors can be used for transfection, wherein a large amount of protein can be produced transiently in vitro. The herpesvirus amplicon system is also used to package>220kb DNA fragments, and infects cells that can stably keep DNA as episomes.

[0083] Other useful systems include, for example, replication-competent and host-restricted non-replicating vaccinia virus vectors.

[0084] 2. Expression system

[0085] The nucleic acid delivered to the cell generally comprises an expression control system. For example, the gene inserted in the virus and retroviral system generally comprises a promoter and / or an enhancer to help control the expression of the desired gene product. A promoter is generally one or more DNA sequences that work when being in a relatively fixed position relative to the transcription start site. A promoter comprises the core elements required for the basic interaction of RNA polymerase and transcription factor, and may comprise an upstream element and a response element.

[0086] a) Viral promoters and enhancers

[0087] Preferred promoters for controlling transcription of vectors in mammalian host cells can be obtained from a variety of sources, such as the genome of a virus, such as polyoma virus, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, and most preferably cytomegalovirus, or from a heterologous mammalian promoter, such as hepatitis B virus and most preferably cytomegalovirus, or from a heterologous mammalian promoter, such as the β-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment, which also contains the SV40 viral replication origin (Fiers et al., Nature, 273: 113 (1978)). The immediate early promoter of human cytomegalovirus can be conveniently obtained in the form of a HindIII E restriction fragment (Greenway, PJ et al., Gene 18: 355-360 (1982)). Of course, promoters from host cells or related species can also be used herein.

[0088] Enhancers generally refer to DNA sequences that function at variable distances from the transcription start site and may be 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78:993 (1981)) or 3' (Lusky, ML, et al., Mol. Cell Bio. 3:1108 (1983)) relative to the transcription unit. In addition, enhancers can be located within introns (Banerji, JL et al., Cell 33:729 (1983)) and within the coding sequence itself (Osborne, TF, et al., Mol. Cell Bio. 4:1293 (1984). Their length is usually between 10 and 300 bp, and they act in cis. Enhancers act to increase transcription from nearby promoters. Enhancers also typically contain response elements that mediate transcriptional regulation. Promoters may also contain response elements that mediate transcriptional regulation. Enhancers generally determine the regulation of gene expression. Although many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin), enhancers from eukaryotic cell viruses are generally used for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0089] Promoters and / or enhancers can be specifically activated by light or specific chemical events that trigger their function. The system can be regulated by agents such as tetracycline and dexamethasone. There are also methods to enhance viral vector gene expression by exposure to radiation (e.g., gamma radiation) or alkylating chemotherapy drugs.

[0090] In certain embodiments, promoter and / or enhancer region can be used as constitutive promoter and / or enhancer, to maximize the expression of the transcription unit region to be transcribed. In some constructs, promoter and / or enhancer region is active in all eukaryotic cell types, even if it is only expressed in a specific type of cell at a specific time. The preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are the SV40 promoter, cytomegalovirus (full-length promoter) and retroviral vector LTR.

[0091] It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors for selective expression in specific cell types, such as melanoma cells.The glial fibrillary acetate protein (GFAP) promoter has been used to selectively express genes in cells of glial origin.

[0092] The expression vector used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans or nucleated cells) may also contain sequences necessary for termination of transcription, which may affect the expression of mRNA. These regions are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' non-translated region also includes a transcription termination site. Preferably, the transcription unit also includes a polyadenylation region. A benefit of this region is that it increases the likelihood that the transcription unit is processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs have been well established. Preferably, homologous polyadenylation signals are used in transgenic constructs. In some transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed unit contains other standard sequences alone or in combination with the above sequences to improve the expression or stability of the construct.

[0093] b) Markers

[0094] The viral vector may include a nucleic acid sequence encoding a marker product. The marker product is used to determine whether the gene has been delivered to the cell and is expressed once delivered. A preferred marker gene is the E. coli lacZ gene, which encodes beta-galactosidase and green fluorescent protein.

[0095] In some embodiments, the marker can be a selective marker. Examples of suitable selective markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin and puromycin. When such a selective marker is successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used different categories of selection systems. The first category is based on the metabolism of the cell and the use of mutant cell lines that lack the ability to grow independently of supplemented culture medium. Two examples are: CHO DHFR-cells and mouse LTK-cells. These cells lack the ability to grow without adding nutrients such as thymidine or hypoxanthine. Since these cells lack certain genes necessary for the complete nucleotide synthesis pathway, they will not survive unless the missing nucleotides are provided in the supplemented culture medium. An alternative to supplementing the culture medium is to introduce a complete DHFR or TK gene into cells lacking the corresponding gene, thereby changing its growth requirements. Individual cells that are not transformed with DHFR or TK genes will not survive in non-supplemented culture medium.

[0096] The second class is dominant selection, which refers to the selection scheme used in any cell type, and does not require the use of mutant cell lines. These schemes usually use drugs to prevent the growth of host cells. Those cells with new genes will express proteins that transmit drug resistance and make the selection survive. The examples of this dominant selection use drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan, RC and Berg, P. Science 209: 1422 (1980)) or hygromycin (Sugden, B. et al., Mol. Cell. Biol. 5: 410-413 (1985)). These three examples use bacterial genes under eukaryotic control to transmit resistance to appropriate drugs G418 or neomycin (Geneticin), xgpt (mycophenolic acid) or hygromycin respectively. Others include neomycin analogs G418 and puromycin.

[0097] 3. Drug carrier / drug delivery

[0098] As described above, these compositions can also be administered to the body in a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject together with a nucleic acid or a vector without causing any adverse biological effect or interacting in a deleterious manner with any other component of the pharmaceutical composition containing it. As is well known to those skilled in the art, the carrier will naturally be selected to minimize any degradation of the active ingredient and minimize any adverse side effects in the subject.

[0099] The composition can be administered orally, parenterally (e.g., intravenously), intramuscularly, intraperitoneally, transdermally, in vitro, topically, etc., including topical intranasal administration or administration by inhalation. As used herein, "topical intranasal administration" means delivery of the composition to the nasal cavity and nasal passages through one or both nostrils, and may include delivery by a spray mechanism or a drop mechanism, or by atomization of a nucleic acid or a vector. Administration of the composition by inhalation is delivered via the nasal cavity or oral cavity, by a spray or drop mechanism. Delivery can also be directly delivered to any area of ​​the respiratory system (e.g., lungs) through intubation. The exact amount of the composition required varies from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the allergic disorder being treated, the specific nucleic acid or vector used, its mode of administration, etc. Therefore, it is impossible to specify an exact amount for each composition. However, the appropriate amount can be determined by a person of ordinary skill in the art by routine experiments using only the teachings given herein.

[0100] Parenteral administration of the composition, if used, is usually characterized by injection. Injections can be prepared in conventional forms, either as liquid solutions or suspensions, or as solid forms suitable for dissolving the suspension in a liquid prior to injection, or as emulsions. Recently revised parenteral administration methods include the use of sustained release or sustained release systems to maintain a constant dose. See, e.g., U.S. Patent No. 3,610,795, which is incorporated herein by reference.

[0101] The materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells). They may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of using this technology to target specific proteins to tumor tissues (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Immunol. Res., 13:57-81, (1992); and Roffler, et al., Immunol. Res., 13:57-81, (1992). al., Biochem. Pharmacol, 42: 2062-2065, (1991)). "Stealth" and other antibody-conjugated liposomes (including lipid-mediated drugs for colon cancer), receptor-mediated DNA targeting through cell-specific ligands, lymphocyte-mediated tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of using this technology to target specific proteins to tumor tissues (Hughes et al., Cancer Research, 49: 6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104: 179-187, (1992)). In general, receptors participate in the pathway of endocytosis, whether constitutive or ligand-induced. These receptors gather in the pits coated with clathrin, enter the cell through the vesicles coated with clathrin, pass through the acidified endosomes that classify the receptors, and then circulate to the cell surface, store in the cell, or degrade in the lysosome. The internalization pathway has multiple functions, such as nutrient absorption, removal of activated proteins, removal of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligands, and regulation of receptor levels. Depending on the cell type, receptor concentration, ligand type, ligand valence and ligand concentration, many receptors follow more than one intracellular pathway. The molecular and cellular mechanisms of receptor-mediated endocytosis are reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0102] a) Pharmaceutically acceptable carrier

[0103] The compositions include antibodies and can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0104] Suitable carriers and their formulations are described in the following literature: Remington: The Science and Practice of Pharmacy (19th edition), A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Carriers also include sustained-release formulations, such as semipermeable membrane matrices of solid hydrophobic polymers containing antibodies, the matrix of which is in the form of a molded article, for example, a film, a liposome, or a microparticle. It will be apparent to those skilled in the art that certain carriers may be more desirable, for example depending on the route of administration and the concentration of the composition administered.

[0105] Pharmaceutical carriers are known to those skilled in the art. These are generally standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffer solutions at physiological pH. These compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0106] In addition to the selected molecule, the pharmaceutical composition may include carriers, thickeners, diluents, buffers, preservatives, surfactants, etc. The pharmaceutical composition may also include one or more active ingredients, such as antibacterial agents, anti-inflammatory agents, anesthetics, and the like.

[0107] The pharmaceutical composition can be administered in a variety of ways, depending on whether local or systemic treatment is needed and the area to be treated. Administration can be topical (including ocular, vaginal, rectal, intranasal), oral, inhaled, or parenteral, such as intravenous, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavitary or transdermally.

[0108] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including physiological saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous carriers include liquid and nutritional supplements, electrolyte supplements (such as supplements based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as, for example, antibacterial agents, antioxidants, chelating agents and inert gases, etc.

[0109] Topical formulations may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, water, powder or oily bases, thickeners and the like may be necessary or desirable.

[0110] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0111] Some compositions can potentially be administered as pharmaceutically acceptable acid or base addition salts and are formed by the reaction of inorganic acids (such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid and phosphoric acid) and organic acids (such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid and fumaric acid), or inorganic bases (such as sodium hydroxide, ammonium hydroxide, potassium hydroxide) and organic bases (such as mono-, di-, tri-alkyl and aryl amines and substituted ethanolamines).

[0112] b) Therapeutic use

[0113] The effective dose and schedule for administering the composition can be determined empirically, and it is within the technical scope of the art to make such determinations. The dosage range for administering the composition should be large enough to produce the desired effect, thereby affecting the symptoms of the disorder. The dose should not be so large as to cause adverse side effects, such as undesirable cross-reactions, allergic reactions, etc. Generally, the dose will vary with the patient's age, condition, gender, and degree of disease, route of administration, or whether other drugs are included in the regimen, and can be determined by those skilled in the art. If there are any contraindications, the dose can also be adjusted by an individual physician. The dose can vary and can be administered in one or more doses per day for one or more days. For a given class of drugs, guidance for appropriate doses can be found in the literature. For example, guidance for selecting an appropriate dose of antibody can be found in the literature on the therapeutic use of antibodies, for example, Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. Depending on the above factors, a typical daily dose of an antibody used alone may be in the range of about 1 μg / kg to 100 mg / kg body weight or more per day.

[0114] C. Methods for Transducing Immune Cells

[0115] It is understood and contemplated herein that the disclosed hydrogels can be used to attract immune cells (such as, for example, T cells, NK cells, NK T cells, dendritic cells, macrophages, TINK, TIL and / or MIL) to the hydrogel, where immune cell transduction can be performed in vivo. In one aspect, disclosed herein is a method of transducing immune cells (such as, for example, T cells, NK cells, NK T cells, dendritic cells, macrophages, TINK, TIL or MIL) in a subject, the method comprising administering to the subject a hydrogel comprising one or more chemoattractants (such as, for example, CCL1, CCL5, CCL19, CCL21, CCL22, CCL28, CXCL1, CXCL9, CXCL10, CXCL11, CXCL12, M-CSF, GM-CSF, MCP-1, MCP-3, CCL2, CCL3, CCL7, CCL20, CX3CL1, BRAK, IL-12, S1P and / or MCP2) and encoding a transgene (such as, for example, a CAR, an NK cell receptor (including but not limited to an anti-CD3 antibody, CD1d, an Fc fragment of an immunoglobulin or an anti-Fcγ receptor (FcγRIII) antibody), a T cell receptor (e.g., an antigen-specific T cell receptor) or a NK cell receptor. T cell receptor)-expressing viral vector (e.g., lentivirus, retrovirus, adenovirus, or adeno-associated virus).

[0116] It should be understood and considered herein that the viral vector can be introduced into the hydrogel matrix before administration to the subject or about 1 day to about 14 days after the hydrogel matrix is ​​administered to the subject. That is, the viral vector can be introduced into the hydrogel in vivo. For example, the viral vector can be introduced during the gelation of the hydrogel; after gelation, but before the hydrogel is administered to the subject; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days after the hydrogel is administered to the subject.

[0117] It is understood and expected herein that the disclosed chemoattractants will leach out of the hydrogel or release from the hydrogel over time and attract immune cells to the hydrogel, wherein the immune cells (e.g., T cells, NK cells, NK T cells, dendritic cells, macrophages, TINK, TIL or MIL) can be transduced with transgenes such as chimeric antigen receptors, NK T cell receptors, NK cell receptors (including but not limited to anti-CD3 antibodies, CD1d, Fc fragments of immunoglobulins or anti-Fcγ receptor (FcγRIII) antibodies) or T cell receptors (including but not limited to antigen-specific T cell receptors). In one aspect, the release of the chemoattractant can be from 1 hour after the hydrogel is applied to about 12 weeks after the hydrogel is applied. For example, the release of the chemoattractant can be from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 28, 30, 36, 42, 48, 60, 72 hours, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 30, 31 days, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after the hydrogel is applied. Thus, in one aspect, disclosed herein is a method of transducing immune cells, wherein one or more chemoattractants are released from about 1 hour after the hydrogel is applied to about 12 weeks after the hydrogel is applied. The release time of the chemoattractant can be modified by applying the chemoattractant to the hydrogel polymer before or after gelation, with application after gelation resulting in a faster release time and application of the chemoattractant to the polymer during or before gelation resulting in a slower release.

[0118] On the one hand, it is recognized that immune cells may benefit from additional stimulation to maintain the cells or activate the immune cells for treatment. Therefore, on the one hand, disclosed herein is a transduction method, wherein the hydrogel further comprises one or more antibodies, cytokines and / or co-stimulatory molecules that activate T cells, NK cells, NK T cells, dendritic cells, macrophages, TINK, TIL or MIL. For example, the antibody may include anti-CD28, CD3, B7-1, B7-2, anti-inducible co-stimulator (ICOS), ICOS ligand, anti-CD27, CD70, 4-1BBL, anti-41-BB, anti-CD40L, CD40, anti-DAP10, anti-CD30, CD30L, anti-TIM-1, anti-TIM-2, anti-TIM-3, anti-CD44, anti-NK1.1, lectin-like transcript-1 (LLT-1), anti-CD137, CD48, MICA, anti-2B4, anti-glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR); and the cytokine may include IL-2, IL-7, IL-15, IL-21, TNF-α or IFN-γ.

[0119] D. Methods of treating cancer

[0120] In one aspect, disclosed herein are methods of treating, preventing, inhibiting and / or attenuating cancer or metastasis in a subject, the method comprising administering to the subject any of the hydrogel matrices disclosed herein. For example, disclosed herein are methods of treating cancer in a subject, comprising administering to the subject a hydrogel matrix comprising one or more chemoattractants, wherein the one or more chemoattractants include C-C motif chemokine ligand (CCL) 1 (CCL1), CCL5, CCL19, CCL21, CCL22, CCL28, C-X-C motif chemokine ligand (CXCL) 1 (CXCL1), CXCL9, CXCL10, CXCL11 or CXCL CXCL12, M-CSF, GM-CSF, MCP-1, MCP-3, CCL2, CCL3, CCL7, CCL20, CX3CL1, BRAK, IL-12, S1P and / or MCP212; and wherein the chemoattractant attracts and retains immune cells (such as, for example, T cells, NK cells, NK T cells, dendritic cells, macrophages, TINK, TIL or MIL) to the hydrogel.

[0121] On the one hand, the hydrogel used in the method for treating, preventing, inhibiting and / or reducing cancer or metastasis disclosed herein may further include a viral vector encoding a chimeric antigen receptor (CAR), an NK cell receptor (including but not limited to anti-CD3 antibodies, CD1d, Fc fragments of immunoglobulins, or anti-Fcγ receptor (FcγRIII) antibodies), an NK T cell receptor, or a T cell receptor (TCR) (including but not limited to antigen-specific T cell receptors). On the one hand, the viral vector transduces immune cells; and the transduced immune cells are released from the hydrogel to the cancer. It should be understood and considered herein that the viral vector can be introduced into the hydrogel matrix before administration to the subject or about 1 day to about 14 days after the hydrogel matrix is ​​administered to the subject. That is, the viral vector can be introduced into the hydrogel in vivo. For example, the viral vector can be introduced during the gelation of the hydrogel; after gelation but before administering the hydrogel to a subject; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after administering the hydrogel to a subject.

[0122] It is understood and expected herein that the disclosed chemoattractants will leach out of the hydrogel or release from the hydrogel over time and attract immune cells to the hydrogel, wherein the immune cells (e.g., T cells, NK cells, NK T cells, dendritic cells, macrophages, TINK, TIL or MIL) can be transduced with a transgene such as a chimeric antigen receptor, a T cell receptor, a NK cell receptor and / or a NK T cell receptor. In one aspect, the release of the chemoattractant can be from 1 hour after the hydrogel is applied to about 12 weeks after the hydrogel is applied. For example, the release of the chemical attractant can be from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 28, 30, 36, 42, 48, 60, 72 hours, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 30, 31 days, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after the application of the hydrogel. Thus, in one aspect, disclosed herein is a method of treating, preventing, inhibiting and / or reducing cancer or metastasis, wherein one or more chemical attractants are released from about 1 hour after the application of the hydrogel to about 12 weeks after the application of the hydrogel. The release time of the chemoattractant can be modified by applying the chemoattractant to the hydrogel polymer before or after gelation, with application after gelation resulting in a faster release time and application of the chemoattractant to the polymer during or before gelation resulting in a slower release.

[0123] Similarly, it should be understood and considered herein that one advantage of the disclosed hydrogels over conventional CAR therapies is that CAR T cells, CAR NK cells, CAR NK T cells, CARMA, TINK, MIL or TIL are slowly released into the tumor microenvironment. It should be understood and considered herein that the release of immune cells (such as, for example, T cells, NK cells, NK T cells, dendritic cells, macrophages, TINK, TIL or MIL) can be from 1 hour to about 12 weeks after the hydrogel is applied. For example, the release of the chemoattractant can be from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 28, 30, 36, 42, 48, 60, 72 hours, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 30, 31 days, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after the hydrogel is applied. Thus, in one aspect, disclosed herein is a method of treating, preventing, inhibiting and / or reducing cancer or metastasis, wherein the immune cells are released from about 1 week to about 12 weeks after the hydrogel is applied.

[0124] It is understood and contemplated herein that the disclosed hydrogel matrices used in the disclosed methods of treating, preventing, inhibiting and / or reducing cancer or metastasis may further comprise one or more immunoblocking inhibitors and / or chemotherapeutic agents. Chemotherapeutic agents that can be used in the disclosed hydrogel matrix may include any chemotherapeutic agent known in the art, including but not limited to pomacillin, abiraterone acetate, Abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Bentuximab Vedotine), ADE, Ado-Trastuzumab Emtansine, Adriamycin (doxorubicin hydrochloride), afatinib dimaleate, Afinitor (everolimus), Akynzeo (netopitant and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, Alecensa (alectinib), alectinib, alemtuzumab, Alimta (pemetrexed disodium), Aliqopa (coupanib hydrochloride), Aloxicam (melphalan hydrochloride), Aloxicam tablets (melphalan), and tadalafil (tadalafil). (palonosetron hydrochloride), Alunbrig (brigatinib), Ambochlorin (chlorambucil), Amboclorin (chlorambucil), Amifostine, aminolevulinic acid, Anastrozole, Aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Arranon (nelarabine), Arsenic trioxide, Arzerra (ofatumumab), Aspartame Amidase Erwinia chrysanthemi, atezolizumab, Avastin (bevacizumab), avelumab, axitinib, azacitidine, Bavencio (avelumab), BEACOPP, Becenum (carmustine), Beleodaq (belinostat), belinostat, bendamustine hydrochloride, BEP, Besponsa (inostatin-ozogamicin), bevacizumab, bexarotene, Bexxar (tositumomab and iodine I 131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, brigatinib, BuMel, busulfan, Busulfex (busulfan), cabazitaxel, Cabometyx (cabozantinib malate), cabozantinib malate, CAF, Campath (alemtuzumab), Camptosar, (irinotecan hydrochloride), capecitabine, CAPOX, Carac (fluorouracil -- topical), carboplatin, carboplatin-TAXOL, carfilzomib, Carmubris (carmustine), carmustine, carmustine transplant, Casodex (bicalutamide),CEM, Ceritinib, Cerubidine (doxorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), Cetuximab, CEV, Chlorambucil, Chlorambucil-prednisone, CHOP, Cisplatin, Cladribine, Clafen (cyclophosphamide), Clofarabine, Clofarex (clofarabine), Clolar (clofarabine), CMF, Cabozantinib, Cometriq (cabozantinib malate), Copanisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (actinomycin D), Cotellic (cabozantinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (ifosfamide), C yramza (ramucirumab), cytarabine, cytarabine liposomal, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), actinomycin D, daratumumab, Darzalex (daratumumab), dasatinib, doxorubicin hydrochloride, doxorubicin hydrochloride and cytarabine liposomal, decitabine, defibrotide sodium, Defitelio (defibrotide sodium), Degarelix, denileukin, denosumab, DepoCyt (cytarabine liposomal), dexamethasone, dexrazoxane hydrochloride, denoximab, docetaxel, Doxil (doxorubicin hydrochloride liposomal), doxorubicin hydrochloride Star, liposomal doxorubicin hydrochloride, Dox-SL (liposomal doxorubicin hydrochloride), DTIC-Dome (dacarbazine), durvalumab, Efudex (fluorouracil -- topical), Elitek (rasburicase), Ellence (epirubicin hydrochloride), elotuzumab, Eloxatin (oxaliplatin), eltrombopag, Emend (affinity), Empliciti (elotuzumab), etanercept, enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinaze (asparaginase), Et Hyol (amifotine), Etopophos (etoposide phosphate), Etoposide, Etoposide phosphate, Evacet (liposomal doxorubicin hydrochloride), Everolimus, Evacet, (raloxifene hydrochloride), Evomela (melphalan hydrochloride), Exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil -- topical), Fareston (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), Filgrastim, Fludara (fludarabine phosphate), Fludarabine phosphate, Fluoroplex (fluorouracil -- topical), Fluorouracil injection, Fluorouracil -- topical,Flutamide, Folex (methotrexate), Folex PFS (methotrexate), FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRINOX, FOLFOX, Folotyn (pralatrexate), FU-LV, Fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV nine-valent vaccine), Gazyva (obituzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), Gliadel (carmustine transplant), Gliadel wafer (carmustine implant), glutaraldehyde, goserelin acetate, Halaven (eribulin mesylate), Hemangeol (propranolol hydrochloride), Herceptin (trastuzumab), recombinant HPV secondary vaccine, recombinant HPV nine-valent vaccine, recombinant HPV four-valent vaccine, Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper-CVAD, Ibrance (palbociclib), isobutazolamide, ibrutinib, ICE, Iclusig (panatinib hydrochloride), Idamycin (idaminergic acid hydrochloride), Darubicin), Idarubicin hydrochloride, Idelalis, Idhifa (etanercept mesylate), Ifex (ifosfamide), Ifosfamide, Ifosfamidum (ifosfamide), IL-2 (aldesleukin), Imatinib mesylate, Imbruvica (ibrutinib), Imfinzi (durvalumab), Imiquimod, Imlygic (talimogene laherparepvec), Inlyta (axitinib), Inotuzumab ozogamicin, recombinant interferon alpha-2b, interleukin-2 (aldesleukin), Intron A (recombinant interferon alpha-2b), iodine I 131 tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposome, Istodax (romidepsin), ixabepilone, ixazomib citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate), JEB, Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Kymriah (tisagenlecleucel), Kyprolis (cafilomib), lanreotide acetate, lapatinib ditosylate, Lartruvo (olalimumab),Lenalidomide, Lenvatinib mesylate, Lenvima (lenvatinib mesylate), Letrozole, leucovorin, Leukeran (chlorambucil), Leuprolide acetate, Leustatin (cladribine), Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (liposomal doxorubicin hydrochloride), Lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lynparza (olaparib), Marqibo (liposomal vincristine sulfate), Matulane (methylprocarbazine hydrochloride), nitrogen mustard hydrochloride, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, mesna, Mesnex (mesna), Methazolastone (temozolomide), methotrexate, methotrexate LPF (methotrexate), methylnaltrexone bromide, Mexate (methotrexate), Mexate-AQ (methotrexate), aminopterin), midostaurin, mitomycin C, mitoxantrone hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (plerixafor), Mustargen (nitrogen mustard hydrochloride), Mutamycin (mitomycin C), Myleran (busulfan), Mylosar (azacytidine), Mylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine tartrate), nexitozumab, nelarabine, Neos ar (cyclophosphamide), neratinib maleate, Nerlynx (neratinib maleate), netopitant and palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nilandron (nilutamide), nilotinib, nilutamide, Ninlaro (ixazomib citrate), niraprol monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, O domzo (sonidegi), OEPA, ofatumumab, OFF, olaparib, olalimumab, homoharringtonine, Oncaspar (pegaspargase), ondansetron hydrochloride, Onivyde (liposomal irinotecan hydrochloride), Ontak (denileukin), Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, palifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat,Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, PCV, PEB, pegaspargase, pegfilgrastim, pegylated interferon alfa-2b, PEG-Intron (pegylated interferon alfa-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor, pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, Portrazza (necituzumab), pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltropol ), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nine-Valent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon α-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE, Rituxan (Rituximab), Rituxan Hycela (rituximab and human hyaluronidase), Rituximab, Rituximab and human hyaluronidase, Rolapitant hydrochloride, Romidepsin, Romiplostim, Rubidomycin (doxorubicin hydrochloride), Rubraca (recaparib camphorsulfonate), Recaparib camphorsulfonate, Ruxolitinib phosphate, Rydapt (midostaurin), Intrapleural sclerosant aerosol (Talc), Certuinimab, Sipuleucel-T, Somatuline Depot (lanreotide acetate), Sonidegi, Sorafenib tosylate, Sprycel (dasatinib), STANFORD V, Sterile Talc (Talc), Steritalc (Talc), Stivarga (regorafenib), Sunitinib Malate, Sutent (sunitinib malate), Sylatron (pegylated interferon alpha-2b), Sylvant (Cetuximab), Synribo (homoharringtonine), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride),Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Tecentriq, (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), thioguanine, thiotepa, Tisagenlecleucel, Tolak (fluorouracil -- topical), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab, and iodine I 131 Tositumomab, Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (denotuximab), uridine triacetate, VAC, vandetanib, VAMP, Varubi (rolapitant hydrochloride), Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), vemurafenib, Venclexta (venetola), venetola, Verzenio (pomacicillin), Viadur (leuprolide acetate), Vidaza (azacytidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposomal, vinorelbine tartrate, VIP, Vimodegib, Vistogard (uridine triacetate), Vora Xaze (glutaric acid serotonin), Vorinostat, Votrient (pazopanib hydrochloride), Vyxeos (doxorubicin hydrochloride and cytarabine liposome), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium 223 chloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yondelis (trabectedin), Za ltrap (Ziv-aflibercept), Zarxio (filgrastim), Zejula (nirapril toluenesulfonate monohydrate), Zelboraf (vemurafenib), Zevalin (isbetumomab), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalis),Zykadia (ceritinib) and / or Zytiga (abiraterone acetate). Checkpoint inhibitors include, but are not limited to, antibodies that block PD-1 (nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHIgM12B7), CTLA-4 (ipilimumab (MDX-010), tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016).

[0125] The disclosed compositions can be used to treat any disease in which uncontrolled cell proliferation occurs, such as cancer. The disclosed compositions can be used to treat a representative but not limited list of cancers: lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, small cell lung cancer and non-small cell lung cancer and other lung cancers, neuroblastoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, oral squamous cell carcinoma, pharyngeal squamous cell carcinoma, laryngeal cancer and lung squamous cell carcinoma, cervical cancer, uterine cervix cancer, breast cancer, and epithelial cancer, kidney cancer, genitourinary tract cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colon cancer, rectal cancer, prostate cancer or pancreatic cancer.

[0126] E. Examples

[0127] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to prepare and evaluate the compounds, compositions, articles, devices and / or methods claimed herein, and are intended to be purely exemplary and not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, temperatures are ° C or at ambient temperature, and pressures are at or near atmospheric pressure.

[0128] Example 1: Preparation of microporous gel by cryogel

[0129] Alginate is cross-linked with calcium gluconate and frozen at -20°C. Gelation occurs around ice crystals, which act as pore-forming agents ( Figure 2 ). The macroporosity of the scaffold promotes chemokine-mediated T cell infiltration and provides an interface for transduction of viral particles. Figure 3 Scanning electron micrographs of gels prepared in this way are shown.

[0130] Example 2: Mediated recruitment and reprogramming of host T cells

[0131] Here we demonstrate that implanted CXCL10-releasing macroporous scaffolds effectively recruit transplanted T cells ( Figure 4A and Figure 4B ). We also demonstrated that viral vectors transduced approximately 20% of activated human T cells preloaded on alginate scaffolds (Figures 5A and 5B), and 1-2% of the 2-3 million transplanted human PBMCs recruited from the circulation when combined with CD3 / CD28 antibodies and IL-2 within the gel (Figure 5A and 5B). Figure 5C and Figure 5D ). CD3 / CD28 antibodies and IL-2 provide environmental factors that promote T cell activation.

[0132] Example 3: Optimization of scaffold-mediated T cell reprogramming parameters.

[0133] The results show that controlled release of the chemoattractant CXCL10 leads to the recruitment of circulating T cells to the implanted scaffolds. In addition, this paper shows that scaffolds preloaded with activated T cells can be virally transduced. The efficiency of viral vectors in vivo transduction of recruited T cells using GFP and CAR-T vectors.

[0134] Example 4: Optimization and characterization of in vivo transduction of T cells by a viral vector encoding GFP.

[0135] The ability of encapsulated retroviral vectors to transduce recruited T cells can be tested. Human PBMCs (1 x 10 7 Cells / mouse) can be transplanted for 20 days. Alginate scaffolds loaded with CXCL10 (4 μg / mg alginate) and GFP encoding retroviral vectors (two different concentrations) can be implanted in the subcutaneous space of mice (n=4). At different time points (4, 6, 8 days after scaffold implantation), the scaffolds can be removed and the frequency of CD45+CD3+GFP+ cells can be assessed by flow cytometry. Controls can include: 1) blank gel without chemokines and 2) administration of pseudovirus.

[0136] Example 5: In situ generation of CAR-T cells:

[0137] CAR transduction can be done in situ using a chemokine-loaded scaffold and a CAR-encoding viral vector. Optimal recruitment and reprogramming conditions for T cells can be tested using CAR-encoding viral constructs. n=10 can be used (see the statistics section). The scaffold can be removed and the percentage of cell recruitment and CD19. CAR+ cells can be assessed by flow cytometry. Controls can include: 1) blank gel without chemokines and 2) administration of pseudovirus. Viral vectors may degrade in vivo over time. In this case, the virus can be administered by one or more slow intra-scaffold infusions. Similar results were observed from GFP and CAR-encoding viral vectors.

[0138] Hu-PBMC-NSG mice were implanted with scaffolds (CCI-Alg) bearing CXCL10. Human CD19.CAR encoding gamma retrovirus was administered by slow intra-scaffold injection. Three days after viral administration, the scaffolds were removed, digested, and the isolated cells were stained with CD3 and CD19 antibodies for analysis by flow cytometry ( Figure 6 ). Figure 7 In vivo transduction of recruited T cells with CD19 CAR virus is shown.

[0139] Example 6: Characterization of transduced T cell migration:

[0140] Following T cell recruitment and reprogramming, efficient release of the reprogrammed cells into the circulation is critical for therapeutic function. The efficiency and kinetics of transduced T cell migration into the systemic circulation can be characterized.

[0141] To evaluate the migration of transduced T cells into the bloodstream, GFP+ or CAR+ T cells can be measured in mice (n=12) carrying scaffolds for T cell recruitment and transduction. Mice can be sacrificed at different time points (5, 10, 15 days after scaffold implantation) and GFP or CAR expression in systemic T cells can be quantified. Optimal conditions can be used for T cell recruitment and reprogramming. At different time points, T cells can be isolated from blood, spleen, bone marrow, drainage, and peripheral lymph nodes, and the frequency and number of CD45+CD3+GFP+ or CD45+CD3+CAR+ cells can be assessed by flow cytometry. Controls can include: 1) scaffolds without viruses 2) scaffolds pre-seeded with GFP+ T cells. The macroporous nature of the scaffold promotes the migration of CAR-T cells from the scaffold into the blood. If migration is slow or T cells are trapped in the scaffold, the scaffold can be modified with collagen mimetic peptides, because lymphocytes are known to migrate along collagen fibers. In addition, alginate can be chemically modified to carefully adjust the biodegradation rate, and the degradation of the scaffold can be used to increase the release of CAR-T.

[0142] Example 7: Statistical Analysis

[0143] The number of animals for the GFP experiment was determined based on 80% power to detect the above differences at a 2-sided alpha level of 0.05. The number of animals for orthotopic generation of CAR T cells was determined based on a 2-sided 95% confidence interval (CI) of the mean percentage of CD19, with a half-width of the CI of approximately 3.6%, assuming an SD of approximately 5%.

[0144] Example 8: Tumor Model

[0145] NSG mice were used to develop a mouse model of human Burkitt lymphoma (a B-cell lymphoma) using Daudi.ffluc cells (CD19+ tumor cells). Figure 8 ). Mice were monitored at 19, 24, 29, 33, and 43 days after tumor inoculation. Fig.9A As shown, tumor size in mice was observed by luminescence and compared between untreated controls and mice treated with alginate scaffolds or intravenous injections. Weight gain and total flux ( Fig. 9B ), showing no observable differences between treatment groups. In addition, in mice implanted with CCI scaffolds or intravenously injected with CAR-T cells, the number of CAR-T cells per 100ul of blood at different time points after tumor inoculation in animals that received intravenous CD19 CAR T cells or CCI-alginate scaffolds (CCI-Alg). Mice were bled by cheek bleeding, red blood cells were lysed, and cells were stained with Hu-CD45, Hu-CD3, and CAR.19 antibodies and analyzed by flow cytometry ( Fig.10 ).

[0146] F. References

[0147] Brentjens, RJet al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblasticleukemia. Sci. Transl. Med. 5, 177ra38 (2013).

[0148] Dotti, G., Gottschalk, S., Savoldo, B. & Brenner, MK Design and development of therapies using chimeric antigen receptor-expressing Tcells. Immunol. Rev. 257, 107-126 (2014).

[0149] Enblad,G.et al.A PHASE I / IIa TRIAL USING CD19-TARGETED THIRDGENERATION CAR T CELLS FOR LYMPHOMA AND LEUKEMIA.Clin.Cancer Res.(2018).doi:10.1158 / 1078-0432.CCR-18-0426Grupp,S.A.et al.Chimeric Antigen Receptor-Modified T Cells for Acute Lymphoid Leukemia.N.Engl.J.Med.368,1509-1518(2013).

[0150] Kalos,M.et al.T cells with chimeric antigen receptors have potentantitumor effects and can establish memory in patients with advancedleukemia.Sci.Transl.Med.3,95ra73(2011).

[0151] Kochenderfer,J.N.&Rosenberg,S.A.Treating B-cell cancer with T cellsexpressing anti-CD19 chimeric antigen receptors.Nat.Rev.Clin.Oncol.10,267-276(2013).

[0152] Kochenderfer,J.N.et al.Eradication of B-lineage cells and regressionof lymphoma in a patient treated with autologous T cells geneticallyengineered to recognize CD19.Blood 116,4099-4102(2010).

[0153] Lee,D.W.et al.T cells expressing CD19 chimeric antigen receptors foracute lymphoblastic leukaemia in children and young adults:a phase 1 dose-escalation trial.Lancet 385,517-528(2015).

[0154] Maude,S.L.et al.Chimeric antigen receptor T cells for sustainedremissions in leukemia.N.Engl.J.Med.371,1507-1517(2014).

[0155] Prasad,V.Immunotherapy:Tisagenlecleucel-the first approved CAR-T-celltherapy:implications for payers and policy makers.Nat.Rev.Clin.Oncol.15,11-12(2018).

[0156] Ramos,C.A.,Savoldo,B.&Dotti,G.CD19-CAR trials.Cancer J.20,112-118(2014).

[0157] Sadelain,M.,Brentjens,R.&Rivière,I.The promise and potential pitfallsof chimeric antigen receptors.Curr.Opin.Immunol.21,215-223(2009).

[0158] Sharma,P.,King,G.T.,Shinde,S.S.,Purev,E.&Jimeno,A.Axicabtageneciloleucel for the treatment of relapsed / refractory B-cell non-Hodgkin’slymphomas.Drugs Today 54,187-198(2018).

Claims

1. A hydrogel matrix comprising one or more chemoattractants, wherein the one or more chemoattractants comprise C-C motif chemokine ligand (CCL) 1 (CCL1), CCL5, CCL19, CCL21, CCL22, CCL28, C-X-C motif chemokine ligand (CXCL) 1 (CXCL1), CXCL9, CXCL10, CXCL11, CXCL12, M-CSF, GM-CSF, MCP-1, MCP-3, CCL2, CCL3, CCL7, CCL20, CX3CL1, BRAK, IL-12, S1P and / or MCP2.

2. The hydrogel matrix of claim 1, further comprising a viral vector encoding a chimeric antigen receptor (CAR), a NK cell receptor, a NK T cell receptor, or a T cell receptor.

3. The hydrogel matrix of claim 2, wherein the viral vector comprises a lentivirus, a retrovirus, an adenovirus, or an adeno-associated virus.

4. The hydrogel matrix according to any one of claims 1 to 3, further comprising one or more antibodies, cytokines and / or co-stimulatory molecules, which activate T cells, macrophages, natural killer (NK) cells, NK T cells, tumor infiltrating NK cells (TINK), tumor infiltrating lymphocytes (TIL) or bone marrow infiltrating lymphocytes (MIL).

5. The hydrogel matrix of any one of claim 4, wherein the antibodies comprise anti-CD3, CD28, B7-1, B7-2, anti-inducible co-stimulator (ICOS), ICOS ligand, anti-CD27, CD70, 4-1BBL, anti-41-BB, anti-CD40L, CD40, anti-DAP10, anti-CD30, CD30L, anti-TIM-1, anti-TIM-2, anti-TIM-3, anti-CD44, anti-NK1.1, lectin-like transcript 1 (LLT-1), anti-CD137, CD48, MICA, anti-2B4, and anti-glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR).

6. The hydrogel matrix of any one of claims 4, wherein the cytokine comprises IL-2, IL-7, IL-15, IL-21, TNF-α, or IFN-γ.

7. The hydrogel matrix of any one of claims 1-6, further comprising a chemotherapeutic agent.

8. A method of treating cancer in a subject, comprising administering to the subject the hydrogel matrix of any one of claims 1-7.

9. A method of treating cancer in a subject, comprising administering to the subject a hydrogel matrix comprising one or more chemoattractants, wherein the one or more chemoattractants include C-C motif chemokine ligand (CCL) 1 (CCL1), CCL5, CCL19, CCL21, CCL22, CCL28, C-X-C motif chemokine ligand (CXCL) 1 (CXCL1), CXCL9, CXCL10, CXCL11, CXCL12, M-CSF, GM-CSF, MCP-1, MCP-3, CCL2, CCL3, CCL7, CCL20, CX3CL1, BRAK, IL-12, S1P and / or MCP2; and wherein the chemoattractants attract and retain immune cells to the hydrogel.

10. The method for treating cancer according to claim 9, wherein the immune cells include T cells, NK cells, NKT cells, macrophages, dendritic cells, TINK, TIL or MIL.

11. The method for treating cancer according to any one of claims 9 or 10, wherein the hydrogel matrix further comprises an immunoblocking inhibitor.

12. The method for treating cancer according to any one of claims 9-11, wherein the hydrogel matrix further comprises a chemotherapeutic agent.

13. The method for treating cancer according to any one of claims 9-12, wherein the hydrogel further comprises a viral vector encoding a chimeric antigen receptor (CAR), a NK cell receptor, a NK T cell receptor, or a T cell receptor.

14. The method of treating cancer according to claim 13, wherein the viral vector transduces the immune cells; and wherein the transduced immune cells are released from the hydrogel to the cancer.

15. The method of treating cancer according to claim 14, wherein the viral vector is introduced into the hydrogel in vivo about 1 day to about 14 days after the hydrogel is administered to the subject.

16. The method for treating cancer according to claim 14, wherein the viral vector is introduced into the hydrogel before administering the hydrogel to the subject.

17. The method for treating cancer according to any one of claims 14-16, wherein the immune cells are released from about 1 week to about 12 weeks after administration of the hydrogel.

18. The method for treating cancer according to any one of claims 9-17, wherein the one or more chemoattractants are released from about 1 hour after administration of the hydrogel to about 12 weeks after administration of the hydrogel.

19. A method of transducing immune cells in a subject, the method comprising administering to the subject a hydrogel comprising one or more chemoattractants and a viral vector encoding a transgene.

20. The method of transducing immune cells according to claim 19, wherein the viral vector is introduced into the hydrogel in vivo about 1 day to about 14 days after the hydrogel is administered to the subject.

21. The method of transducing immune cells according to claim 19, wherein the viral vector is introduced into the hydrogel before administering the hydrogel to the subject.

22. A method for transducing immune cells according to any one of claims 19-21, wherein the one or more chemoattractants include CC motif chemokine ligand (CCL) 1 (CCL1), CCL5, CCL19, CCL21, CCL22, CCL28, CXC motif chemokine ligand (CXCL) 1 (CXCL1), CXCL9, CXCL10, CXCL11, CXCL12, M-CSF, GM-CSF, MCP-1, MCP-3, CCL2, CCL3, CCL7, CCL20, CX3CL1, BRAK, IL-12, S1P and / or MCP2.

23. The method of transducing immune cells according to any one of claims 19-22, wherein the one or more chemoattractants are released from about 1 hour after administration of the hydrogel to about 12 weeks after administration of the hydrogel.

24. The method for transducing immune cells according to any one of claims 19-23, wherein the transgene encoded by the viral vector comprises a CAR, an NK cell receptor, an NK T cell receptor, or a T cell receptor.

25. The method for transducing immune cells according to any one of claims 19-24, wherein the viral vector comprises a lentivirus, a retrovirus, an adenovirus or an adeno-associated virus.

26. The method for transducing immune cells according to any one of claims 19-25, wherein the immune cells comprise T cells, NK cells, NK T cells, macrophages, dendritic cells, TINK, TIL or MIL.

27. The method for transducing immune cells according to any one of claims 19-26, wherein the hydrogel further comprises one or more antibodies, cytokines and / or co-stimulatory molecules, which activate T cells, NK cells, NKT cells, macrophages, dendritic cells, TINK, TIL or MIL.

28. The method of transducing immune cells according to claim 27, wherein the antibodies comprise anti-CD28, CD3, B7-1, B7-2, anti-inducible co-stimulator (ICOS), ICOS ligand, anti-CD27, CD70, 4-1BBL, anti-41-BB, anti-CD40L, CD40, anti-DAP10, anti-CD30, CD30L, anti-TIM-1, anti-TIM-2, anti-TIM-3, anti-CD44, anti-NK1.1, lectin-like transcript 1 (LLT-1), anti-CD137, CD48, MICA, anti-2B4, and anti-glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR).

29. The method for transducing immune cells according to claim 27, wherein the cytokines include IL-2, IL-7, IL-15, IL-21, TNF-α or IFN-γ.

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