Semi-synthetic biopolymers used to stimulate the immune system

Abstract

This invention generally relates to a method for stimulating the activation of antigen-presenting cells. The method comprises activating the antigen-presenting cells by contacting the cells with an effective amount of a GC polymer with a molecular weight less than 420 kDa, and subsequently determining whether the antigen-presenting cells have been activated by measuring the amount of the co-stimulatory marker CD40 expressed by the cells. Furthermore, this invention relates to an injectable pharmaceutical composition for stimulating the activation of antigen-presenting cells.

Description

[0001] Cross-reference of related applications

[0002] This application claims priority to U.S. Patent Application Serial No. 16 / 367,233, filed March 27, 2019, which has a prior filed continuation-in-part (CIP) application serial number US 16 / 028,221 filed July 5, 2018, which has a national phase entry 371 filed July 16, 2014, derived from PCT application serial number PCT / US2013 / 021903 filed January 17, 2013, and claims priority herein to U.S. Patent Application Serial No. 61 / 588,783, filed January 20, 2012, entitled “Chitosan-Derived Biomaterials and Applications Thereof”. 14 / 372,586, the entire contents of the aforementioned document are incorporated herein by reference in their entirety. Technical Field

[0003] This article generally relates to semi-synthetic glycosylated biopolymers and their use in pharmaceutical compositions for the treatment of proliferative diseases (vesicles). Other uses include, but are not limited to, preventative or therapeutic vaccines. More specifically, the semi-synthetic glycosylated biopolymers described herein can be used to treat solid cancers in various tissues, such as carcinoma, sarcoma, and melanoma, including malignant lung, colon, liver, breast, prostate, pancreas, skin, thyroid, and kidney vesicles, as well as other types of malignant vesicles. Background Technology

[0004] Proliferative diseases such as cancer can develop at any age in any organ and in any tissue. Once a definitive diagnosis of cancer is made, treatment decisions become crucial. While there is no single treatment for all cancers, successful therapies must focus on both the primary tumor and its metastases, if present. Historically, local and regional therapies, such as surgery, ablation, or irradiation, have been used in cancer treatment in conjunction with systemic therapies, such as chemotherapy drugs, or immunotherapy. Despite some success, conventional treatments are not always as effective as desired, and the search for more effective therapies continues (see, for example, Farkona et al., “Cancer immunotherapy: the beginning of the end of cancer?” in BMC Medicine (016) 14:73). Therefore, the medical need for more effective cancer therapies is clearly unmet.

[0005] Certain biopolymers and their derivatives, which can be produced by and isolated from living organisms such as animals, plants, or fungi, exhibit interesting chemical and biological properties that have led to a wide and ever-expanding range of industrial and medical applications. One such biopolymer derivative is chitosan, a structural component found in the exoskeletons of many organisms, such as crustaceans and insects, as well as in the cell walls of fungi. The biopolymer chitosan is a linear homopolymer composed of N-acetylglucosamine units linked by β1→4 glycosidic bonds. Chitosan, as a partially deacetylated form of chitosan, is the most studied compound among biopolymer sources of this class. The presence of primary amino groups in chitosan facilitates various methods for chemical modification, primarily designed to achieve its solubility and impart specific properties for particular applications.

[0006] One such chemical modification is achieved by synthesizing saccharified chitosan (GC) and producing GC, wherein chitosan and reducing sugars are starting materials for producing GC compounds by reductive amination of the free amino groups of chitosan with the carbonyl groups of reducing monosaccharides and / or oligosaccharides.

[0007] Conventional GC, as described in U.S. Patent 5,747,475 (“Chitosan-Derived Biomaterials”) and PCT Application No. PCT / US13 / 021903, has shown efficacy in treating metastatic tumor models in animals, but the correlation between the chemical structure and composition of GC and immune stimulation has not been fully explored.

[0008] However, such conventional GCs are difficult to manufacture, purify, and ultimately use in humans. Furthermore, conventional GCs, as described in U.S. Patent 5,747,475, are virtually impossible to aseptically filter, thus making them unsuitable for industrial manufacturing according to Current Good Manufacturing Practices (cGMP) and therefore unsuitable for human use. Summary of the Invention

[0009] The weight-average molecular weight (M) of the semi-synthetic biopolymer of Formula 1 shown below. W Less than 420 kDa, and has the same characteristics as M W Larger conventional semi-synthetic biopolymers exhibit significantly different properties. In fact, M was unexpectedly found to have properties significantly different from those of conventional GCs taught in U.S. Patent 5,747,475 and PCT Application No. PCT / US13 / 021903. W Semi-synthetic biopolymer compounds of Formula 1 with a strength less than 420 kDa can provide significant activation of dendritic cells (DCs), as indicated by increased CD40 expression. DC activation is a crucial component in inducing effective anti-tumor T-cell responses. It is believed that this activation of DCs can be extrapolated to the use of semi-synthetic biopolymers to treat certain proliferative disorders in human subjects.

[0010] Therefore, in one embodiment, a polymer of formula 1GC is provided:

[0011]

[0012] Where n is the number of subunits, and (a), (b), and (c) indicate that GC is included. mon The number of each of the following monomer subunits:

[0013]

[0014] as well as

[0015]

[0016] Where R = substitution resulting from glycosylation; where for M less than 420 kDa W (n = 3–1933, (a) = 1–986, (b) = 1–386, (c) = 1–560); and DG (degree of saccharification) is at most, but does not include, 30%.

[0017] In one embodiment, the GC polymer is formulated to produce a sterile filtered aqueous mixture with a pH of 5 to about 7; and the sterile filtered aqueous mixture contains about one wt% of the GC polymer, such that the viscosity of the sterile filtered aqueous mixture is about one centiliter to about one hundred centiliters when measured at about 25°C.

[0018] In one example, the molecular weight of the GC polymer is less than 420 kDa.

[0019] In one example, the GC polymer has a molecular weight of approximately 250 kDa.

[0020] In one instance, the DG of the GC polymer is at most, but does not include, 30%.

[0021] In one example, the GC polymer, wherein for M less than 420 kDa... W , n=3–1933, (a)=1–986, (b)=1–386, (c)=1–560.

[0022] In one example, the M of the GC polymer W It has a value of 250 kDa, a DG value of 5%, and a DDA value of 80%.

[0023] In one instance, the GC polymer comprises at least one subunit of each of the different subunits [(a), (b), and (c)].

[0024] Therefore, in another embodiment, a composition for treating growths using tandem ablation therapy is provided, the composition comprising: an immunostimulant, the immunostimulant being a polymer of formula 1GC as described above; and wherein the immunostimulant is conjugated to a tumor-specific antigen.

[0025] Therefore, in another embodiment, a composition for treating growths using tandem ablation therapy is provided, the composition comprising an immunostimulant, the immunostimulant being a polymer of formula 1GC as described above; and wherein the immunostimulant is conjugated with cytokines.

[0026] Therefore, in another embodiment, a composition for conditioning vegetations using tandem ablation therapy is provided, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated with a TLR agonist, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0027] In one example, the composition described above, wherein the tandem ablation therapy is a physical method. The physical method includes heating or freezing the vegetation. The physical method includes electroporation or embolization of the vegetation. In the composition described above, wherein the tandem ablation therapy includes immunotherapy.

[0028] Therefore, in one embodiment, a composition for conditioning vegetations using tandem irradiation therapy is provided, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated to a tumor-specific antigen, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0029] Therefore, in one embodiment, a composition for conditioning vegetations using tandem irradiation therapy is provided, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated with a cytokine, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0030] Therefore, in one embodiment, a composition for conditioning vegetations using tandem irradiation therapy is provided, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated with a TLR agonist, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0031] In one example, the composition as described above, wherein the tandem irradiation therapy comprises photon beam therapy or particle beam therapy, wherein the photon beam is X-rays and gamma rays, the particle beam is a proton beam, and wherein the tandem irradiation therapy comprises immunotherapy.

[0032] Therefore, in one embodiment, a composition for conditioning vegetations using tandem physical and immunological treatments is provided, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated with tumor-specific antibodies and cytokines, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0033] Therefore, a composition is provided for conditioning vegetations using tandem cytotoxic therapy and immunotherapy, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated to a tumor-specific antigen, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0034] Therefore, in one embodiment, a composition is provided for conditioning vegetations using tandem cytotoxic therapy and immunotherapy, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated with a cytokine, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0035] Therefore, in one embodiment, a composition is provided for conditioning vegetations using tandem cytotoxic therapy and immunotherapy, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated with a TLR agonist, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0036] Therefore, in one embodiment, a composition is provided for conditioning growths using tandem physical and immunological treatments, the composition comprising a combination of a chromophore and an immunostimulant, wherein the chromophore and the immunostimulant are conjugated to a tumor-specific antibody, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0037] In one example, the composition described above, wherein the GC polymer is used as an immunostimulant for cancer treatment.

[0038] Therefore, in one embodiment, a method for treating growths in human or other animal hosts is provided, the method comprising:

[0039] a) Select an immunostimulant, wherein the immunostimulant comprises a polymer of formula 1GC as described above;

[0040] b) Ablating or irradiating the selected vegetation, thereby inducing vegetation cell destruction and immunogenic cell death, resulting in fragmented vegetation tissue and cellular molecules; and

[0041] c) Introducing the immunostimulant into or around the vegetation stimulates the host's autoimmune defense system to process the fragmented vegetation tissue and cellular molecules, such as tumor antigens, and thus generate immunity against the vegetation.

[0042] Therefore, in one embodiment, a method for generating tumor-specific antibodies in a tumor-bearing host is provided, the method comprising:

[0043] a) Absorbing or irradiating the tumor to a degree sufficient to induce destruction of the vegetation cells and produce fragmented vegetation tissue and cellular molecules; and

[0044] b) An immunostimulant is introduced into or around the vegetation by injection, thereby stimulating the host's immune system to interact with and process the fragmented vegetation tissue and cellular molecules, thereby inducing a systemic antitumor antibody response, wherein the immunostimulant is a polymer of formula 1GC as described above.

[0045] Therefore, in one embodiment, a method for generating antigen-specific T cells in a tumor-bearing host is provided, the method comprising:

[0046] a) Absorbing or irradiating the tumor to a degree sufficient to induce destruction of the vegetation cells and produce fragmented vegetation tissue and cellular molecules; and

[0047] b) An immunostimulant is introduced into or around the vegetation by injection, thereby stimulating the host's immune system to interact with and process the fragmented vegetation tissue and cellular molecules, thereby inducing a systemic antitumor T-cell response, wherein the immunostimulant is a polymer of formula 1GC as described above.

[0048] Therefore, in one embodiment, a method is provided to destroy vegetations and generate anti-tumor T-cell responses in a tumor-bearing host in parallel, the method comprising:

[0049] (a) Select an immunostimulant, wherein the immunostimulant is a polymer of formula 1GC as described above;

[0050] b) Ablate the vegetation or irradiate the vegetation to a degree sufficient to cause destruction of the vegetation cells and produce fragmented vegetation tissue and cellular molecules;

[0051] c) Introducing the immunostimulant into the vegetation via intratumoral injection, wherein fragmented tissue and cellular molecules are formed at the injection site as an amalgam of the immunostimulant; and

[0052] d) Stimulate T-cell responses against the host's neoplasm cells.

[0053] Therefore, in one embodiment, T provides a method for destroying vegetations and generating an anti-tumor T cell response in a tumor-bearing host in parallel, the method comprising:

[0054] a) Select a chromophore and an immunostimulant, wherein the immunostimulant is a polymer of formula 1GC as described above, and the chromophore is suitable for generating heat or reactive oxygen species when activated in the visible, near-infrared or infrared wavelength range.

[0055] b) Introducing the chromophore into the growth via intratumoral injection;

[0056] c) Irradiate the vegetation with a laser with a wavelength in the visible, near-infrared, or infrared range at a power sufficient to activate the chromophore for a duration to produce a photothermal or photochemical reaction, thereby inducing the destruction of the vegetation cells and producing fragmented vegetation tissue and cellular molecules.

[0057] d) Introducing the immunostimulant into the growth via intratumoral injection, wherein fragmented tissue and cellular molecules are formed as a mixture of the immunostimulant; and

[0058] e) to stimulate a systemic antitumor immunological response within the host.

[0059] In one example, the method further comprises conjugating the immunostimulant to a tumor-specific antibody, thereby forming a conjugate, and administering the conjugate to the host. The method further comprises conjugating the immunostimulant to a tumor-specific antigen, thereby forming a conjugate, and administering the conjugate to the host. The conjugate is selected from the group consisting of cytokines, chemokines, TLR agonists and proteins, cytotoxic agents; or any combination thereof.

[0060] Therefore, in one embodiment, an injectable pharmaceutical composition is provided for stimulating the activation of antigen-presenting cells, the stimulation comprising:

[0061] Antigen-presenting cells are activated by contacting the cells with an effective amount of Formula 1GC polymer:

[0062]

[0063] Where n is the number of subunits, and (a), (b), and (c) indicate that GC is included. mon The number of each of the following monomer subunits:

[0064]

[0065] as well as

[0066]

[0067] Where R = substitution resulting from glycosylation; where for M less than 420 kDa W (n = 3–1933, (a) = 1–986, (b) = 1–386, (c) = 1–560); and DG is at most but does not contain 30%; wherein the pH of the sterile filtered aqueous mixture is from 5 to about 7; and

[0068] The aseptically filtered aqueous mixture contains about one wt% of the GC polymer, such that the viscosity of the aseptically filtered aqueous mixture is about one centiliter to about one hundred centiliters when measured at about 25°C.

[0069] In one instance, the aseptically filtered aqueous mixture of the GC polymer is an immunostimulant.

[0070] In one instance, the injectable pharmaceutical composition is formulated for use in conjunction with tumor ablation, irradiation therapy, or other methods to achieve immunogenic tumor cell death in order to treat growths.

[0071] In one instance, the injectable pharmaceutical composition is formulated for use in combination with tumor ablation, irradiation therapy, or other methods to achieve immunogenic tumor cell death to treat vegetations and is further combined with the administration of checkpoint inhibitors.

[0072] In one instance, the immunostimulant is conjugated to a tumor-specific antigen.

[0073] In one instance, the immunostimulant was conjugated to a TLR agonist.

[0074] In one instance, the immunostimulant is conjugated with a cytokine.

[0075] In one instance, the immunostimulant is conjugated with a chemokine.

[0076] In one instance, the immunostimulant is conjugated with a cytotoxic agent.

[0077] In one instance, the antigen-presenting cell is a macrophage.

[0078] In one instance, the antigen-presenting cell is a dendritic cell.

[0079] In one instance, the effectiveness of the formula is measured by the amount of a co-stimulatory marker, namely CD40.

[0080] Therefore, in one embodiment, a method for stimulating the activation of antigen-presenting cells is provided, the method comprising:

[0081] Antigen-presenting cells are activated by contacting the cells with an effective amount of Formula 1GC polymer:

[0082]

[0083] Where n is the number of subunits, and (a), (b), and (c) indicate that GC is included. mon The number of each of the following monomer subunits:

[0084]

[0085] as well as

[0086]

[0087] Where R = substitution resulting from glycosylation; where for M less than 420 kDaW (n = 3–1933, (a) = 1–986, (b) = 1–386, (c) = 1–560); and DG is at most but not more than 30%; and the activation of the antigen-presenting cells is determined by measuring the amount of the co-stimulatory marker CD40 expressed by the cells.

[0088] In one instance, the antigen-presenting cell is a macrophage.

[0089] In one instance, the antigen-presenting cell is a dendritic cell.

[0090] In one instance, CD40 expression upregulated other costimulatory markers, including the B7 costimulatory marker.

[0091] In one instance, activation of the antigen-presenting cells initiates an anti-tumor T-cell response.

[0092] In one example, the molecular weight of the GC polymer is less than 420 kDa.

[0093] In one example, the GC polymer has a molecular weight of approximately 250 kDa.

[0094] In one instance, the DG of the GC is at most, but does not include, 30%.

[0095] In one instance, where M is less than 420kDa W , n=3–1933, (a)=1–986, (b)=1–386, (c)=1–560.

[0096] In one instance, the GC's M W It has a value of 250 kDa, a DG value of 5%, and a DDA value of 80%.

[0097] In one instance, the GC comprises at least one subunit from each of the different subunits [(a), (b), and (c)].

[0098] Therefore, in another embodiment, there is provided the use of an immunostimulant comprising a polymer of formula 1GC as described above, for treating a human or other animal host of a growth by ablating or irradiating the selected growth, thereby inducing growth cell destruction and immunogenic cell death of the growth, thereby producing fragmented growth tissue and cellular molecules. The immunostimulant is introduced into or around the growth, which stimulates the host's autoimmune defense system to process the fragmented growth tissue and cellular molecules, such as tumor antigens, and thus generate immunity against the growth.

[0099] Therefore, in another embodiment, there is provided the use of an immunostimulant, wherein the immunostimulant is a polymer of formula 1GC as described above, the method being used to generate tumor-specific antibodies in a tumor-bearing host after: ablating or irradiating the tumor to a degree sufficient to induce destruction of vegetation cells and produce fragmented vegetation tissue and cellular molecules; introducing the immunostimulant into or around the vegetation by injection, thereby stimulating the host's immune system to interact with and treat the fragmented vegetation tissue and cellular molecules, thereby inducing a systemic antitumor response.

[0100] Therefore, in another embodiment, there is provided the use of an immunostimulant, wherein the immunostimulant is a polymer of formula 1GC as described above, the method being used to generate tumor-specific T cells in a tumor-bearing host after: ablating or irradiating the tumor to a degree sufficient to induce destruction of vegetation cells and produce fragmented vegetation tissue and cellular molecules; introducing the immunostimulant into or around the vegetation by injection, thereby stimulating the host's immune system to interact with and process the fragmented vegetation tissue and cellular molecules, at which point a systemic anti-tumor T cell response is induced.

[0101] Therefore, in another embodiment, there is provided the use of an immunostimulant, wherein the immunostimulant is a polymer of formula 1GC according to claim 1, which is used to destroy the vegetation by ablating or irradiating the vegetation to a sufficient degree to produce vegetation cell destruction and generate fragmented vegetation tissue and cellular molecules, and in parallel to generate an anti-tumor T cell response in a tumor-bearing host; the immunostimulant is introduced into the vegetation by intratumoral injection, wherein a mixture of the fragmented tissue and cellular molecules and the immunostimulant is formed at the injection site; the T cell response against the vegetation cell tissue within the host is stimulated.

[0102] Therefore, in another embodiment, a use is provided for a chromophore and an immunostimulant, the immunostimulant being a polymer of formula 1GC, for destroying vegetations and, in parallel, generating an anti-tumor T-cell response in a tumor-bearing host, the chromophore being adapted to generate thermal or reactive oxygen species upon activation in the visible, near-infrared, or infrared wavelength range; the chromophore is introduced into the vegetation via intratumoral injection; the vegetation is irradiated with a laser of wavelength in the visible, near-infrared, or infrared range at a power and duration sufficient to activate the chromophore to generate a photothermal or photochemical reaction, thereby inducing the destruction of vegetation cells and generating fragmented vegetation tissue and cellular molecules; the immunostimulant is introduced into the vegetation via intratumoral injection, wherein a mixture of the fragmented tissue and cellular molecules and the immunostimulant is formed; and an anti-tumor immunological response is systemically stimulated in the host.

[0103] In one instance, the use further comprises conjugating the immunostimulant to a tumor-specific antibody, thereby forming a conjugate, and administering the conjugate to the host.

[0104] In one instance, the use further comprises conjugating the immunostimulant to a tumor-specific antigen, thereby forming a conjugate, and administering the conjugate to the host.

[0105] In one instance, the use wherein the conjugate is selected from the group consisting of cytokines, chemokines, TLR agonists and proteins, cytotoxic agents; or any combination thereof.

[0106] Therefore, in one embodiment, a method for stimulating the activation of antigen-presenting cells, such as dendritic cells, is provided, the method comprising:

[0107] Antigen-presenting cells are activated by contacting the cells with an effective amount of Formula 1GC polymer:

[0108]

[0109] Where n is the number of subunits, and (a), (b), and (c) indicate that GC is included. mon The number of each of the following monomer subunits:

[0110]

[0111] as well as

[0112]

[0113] Where R = substitution resulting from glycosylation; where for M less than 420 kDa W(n = 3–1933, (a) = 1–986, (b) = 1–386, (c) = 1–560); and the degree of saccharification (DG) is at most, but does not include, 30%; and

[0114] Whether the antigen-presenting cells are activated is determined by measuring the amount of the co-stimulatory marker CD40 expressed by the cells.

[0115] Therefore, in another aspect, an injectable pharmaceutical composition is provided for stimulating the activation of antigen-presenting cells, the stimulation comprising:

[0116] Antigen-presenting cells are activated by contacting the cells with an effective amount of Formula 1GC polymer:

[0117]

[0118] Where n is the number of subunits, and (a), (b), and (c) indicate that GC is included. mon The number of each of the following monomer subunits:

[0119]

[0120] as well as

[0121]

[0122] Where R = substitution resulting from glycosylation; where for M less than 420 kDa W (n = 3–1933, (a) = 1–986, (b) = 1–386, (c) = 1–560); and DG is at most but not more than 30%, wherein the pH of the aseptically filtered aqueous mixture is from 5 to about 7; and the aseptically filtered aqueous mixture contains about one wt% of the GC polymer, such that the viscosity of the aseptically filtered aqueous mixture is from about one centiliter to about one hundred centiliters when measured at about 25°C.

[0123] Therefore, in another embodiment, a composition for treating growths using tandem ablation therapy is provided, the composition comprising: an immunostimulant, the immunostimulant being a polymer of formula 1GC as described above; and wherein the immunostimulant is conjugated to a tumor-specific antigen.

[0124] Therefore, in another embodiment, a composition for treating growths using tandem ablation therapy is provided, the composition comprising an immunostimulant, the immunostimulant being a polymer of formula 1GC as described above; and wherein the immunostimulant is conjugated with cytokines.

[0125] Therefore, in another embodiment, a composition for conditioning vegetations using tandem ablation therapy is provided, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated with a TLR agonist, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0126] In one instance, the tandem ablation therapy is a physical method.

[0127] In one instance, the physical method includes heating or freezing the growth.

[0128] In one instance, the physical method includes electroporation or embolization of the vegetation.

[0129] In another instance, the tandem ablation therapy includes immunotherapy.

[0130] Therefore, in another embodiment, a composition for conditioning vegetations using tandem irradiation therapy is provided, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated to a tumor-specific antigen, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0131] Therefore, in another embodiment, a composition for conditioning vegetations using tandem irradiation therapy is provided, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated with a cytokine, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0132] Therefore, in another embodiment, a composition for conditioning vegetations using tandem irradiation therapy is provided, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated with a TLR agonist, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0133] In one instance, the tandem irradiation therapy comprises photon beam therapy or particle beam therapy, wherein the photon beam is X-rays and gamma rays, the particle beam is a proton beam, and wherein the tandem irradiation therapy comprises immunotherapy.

[0134] Therefore, in another embodiment, a composition for conditioning vegetations using tandem physical and immunological treatments is provided, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated with tumor-specific antibodies and cytokines, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0135] Therefore, in another embodiment, a composition is provided for conditioning vegetations using tandem cytotoxic therapy and immunotherapy, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated to a tumor-specific antigen, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0136] Therefore, in another embodiment, a composition for conditioning vegetations using tandem cytotoxic therapy and immunotherapy is provided, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated with a cytokine, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0137] Therefore, in another embodiment, a composition is provided for conditioning vegetations using tandem cytotoxic therapy and immunotherapy, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated with a TLR agonist, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0138] Therefore, in another embodiment, a composition for conditioning growths using tandem physical and immunological therapy is provided, the injectable solution comprising a combination of a chromophore and an immunostimulant, wherein the chromophore and the immunostimulant are conjugated to a tumor-specific antibody, and wherein the immunostimulant is a polymer of formula 1GC as described above.

[0139] In one instance, the GC polymer is used as an immunostimulant for treating cancer.

[0140] Therefore, in another embodiment, an immunostimulant is provided comprising a polymer of formula 1GC as described above, the immunostimulant being used in a method for treating a growth in a human or other animal host, the method comprising:

[0141] a) Ablating or irradiating the selected vegetation, thereby inducing vegetation cell destruction and immunogenic cell death, resulting in fragmented vegetation tissue and cellular molecules; and

[0142] b) Introducing the immunostimulant into or around the vegetation stimulates the host's autoimmune defense system to process the fragmented vegetation tissue and cellular molecules, such as tumor antigens, and thus generate immunity against the vegetation.

[0143] Therefore, in another embodiment, an immunostimulant is provided, said immunostimulant being a polymer of formula 1GC as described above, said immunostimulant being used in a method for generating tumor-specific antibodies in a tumor-bearing host, said method comprising:

[0144] a) Absorbing or irradiating the tumor to a degree sufficient to induce destruction of the vegetation cells and produce fragmented vegetation tissue and cellular molecules; and

[0145] b) The immunostimulant is introduced into or around the vegetation by injection, thereby stimulating the host's immune system to interact with and process the fragmented vegetation tissue and cellular molecules, thereby inducing a systemic antitumor antibody response.

[0146] Therefore, in another embodiment, an immunostimulant is provided, said immunostimulant being a polymer of formula 1GC as described above, said immunostimulant being used in a method for generating antigen-specific T cells in a tumor-bearing host, said method comprising:

[0147] a) Absorbing or irradiating the tumor to a degree sufficient to induce destruction of the vegetation cells and produce fragmented vegetation tissue and cellular molecules; and

[0148] b) The immunostimulant is introduced into or around the vegetation by injection, thereby stimulating the host's immune system to interact with and process the fragmented vegetation tissue and cellular molecules, thereby inducing a systemic antitumor T-cell response.

[0149] Therefore, in another embodiment, an immunostimulant is provided, said immunostimulant being a polymer of formula 1GC as described above, said immunostimulant being used in a method for destroying vegetations and, in parallel, generating anti-tumor T cells in a tumor-bearing host, said method comprising:

[0150] a) Ablate the vegetation or irradiate the vegetation to a degree sufficient to cause destruction of the vegetation cells and produce fragmented vegetation tissue and cellular molecules;

[0151] b) Introducing the immunostimulant into the vegetation via intratumoral injection, wherein fragmented tissue and cellular molecules are formed at the injection site as a mixture of the immunostimulant and the tissue; and

[0152] c) Stimulate T-cell responses against the host's neoplasm cells.

[0153] Therefore, in another embodiment, a chromophore and an immunostimulant are provided for use in a method of destroying vegetations and, in parallel, generating an anti-tumor T-cell response in a tumor-bearing host, wherein the chromophore is adapted to generate thermal or reactive oxygen species upon activation in the visible, near-infrared, or infrared wavelength range; and the immunostimulant is a polymer of formula 1GC as described above, the method comprising:

[0154] a) The chromophore is introduced into the growth via intratumoral injection;

[0155] b) Irradiate the vegetation with a laser of wavelength in the visible, near-infrared or infrared range at a power and duration sufficient to activate the chromophore, to produce a photothermal or photochemical reaction, thereby inducing the destruction of vegetation cells and producing fragmented vegetation tissue and cellular molecules.

[0156] c) Introducing the immunostimulant into the vegetation via intratumoral injection, wherein fragmented tissue and cellular molecules are formed as a mixture of the immunostimulant; and

[0157] d) Stimulate an antitumor immunological response systemically within the host.

[0158] In one example, the method further comprises conjugating the immunostimulant to a tumor-specific antibody, thereby forming a conjugate, and administering the conjugate to the host. The method further comprises conjugating the immunostimulant to a tumor-specific antigen, thereby forming a conjugate, and administering the conjugate to the host. The conjugate is selected from the group consisting of cytokines, chemokines, TLR agonists and proteins, cytotoxic agents; or any combination thereof.

[0159] Therefore, in another embodiment, an injectable pharmaceutical composition is provided for stimulating the activation of antigen-presenting cells, the stimulation comprising:

[0160] Antigen-presenting cells are activated by contacting the cells with an effective amount of Formula 1GC polymer:

[0161]

[0162]

[0163] Where n is the number of subunits, and (a), (b), and (c) indicate that GC is included. mon The number of each of the following monomer subunits:

[0164]

[0165] as well as

[0166]

[0167] Where R = substitution resulting from glycosylation; where for M less than 420 kDa W(n = 3–1933, (a) = 1–986, (b) = 1–386, (c) = 1–560); and DG is at most but does not contain 30%; wherein the pH of the sterile filtered aqueous mixture is from 5 to about 7; and

[0168] The aseptically filtered aqueous mixture contains about one wt% of the GC polymer, such that the viscosity of the aseptically filtered aqueous mixture is about one centiliter to about one hundred centiliters when measured at about 25°C.

[0169] In one example, the sterile-filtered aqueous mixture of the GC polymer is an immunostimulant. The injectable pharmaceutical composition is formulated for use in conjunction with tumor ablation, irradiation therapy, or other methods to achieve immunogenic tumor cell death to treat vegetations. The injectable pharmaceutical composition is formulated for use in conjunction with tumor ablation, irradiation therapy, or other methods to achieve immunogenic tumor cell death to treat vegetations and is further combined with the administration of checkpoint inhibitors. The immunostimulant is conjugated to a tumor-specific antigen. The immunostimulant is conjugated to a TLR agonist. The immunostimulant is conjugated to a cytokine. The immunostimulant is conjugated to a chemokine. The immunostimulant is conjugated to a cytotoxic agent. The antigen-presenting cells are macrophages. The antigen-presenting cells are dendritic cells. The effectiveness of the formula is measured by the amount of a co-stimulatory marker, CD40.

[0170] Therefore, in another embodiment, a GC polymer as described above is provided for use in therapy.

[0171] Therefore, in another embodiment, a polymer of formula 1GC is provided:

[0172]

[0173] It is used in methods for stimulating the activation of antigen-presenting cells, where n is the number of subunits, and (a), (b), and (c) indicate the inclusion of GC. mon The number of each of the following monomer subunits:

[0174]

[0175] as well as

[0176]

[0177] Where R = substitution resulting from glycosylation; where for M less than 420 kDa W(n = 3–1933, (a) = 1–986, (b) = 1–386, (c) = 1–560); and DG is at most but not exceeding 30%; the method includes:

[0178] Antigen-presenting cells are activated by contacting the cells with an effective amount of the GC polymer; and

[0179] Whether the antigen-presenting cells are activated is determined by measuring the amount of the co-stimulatory marker CD40 expressed by the cells.

[0180] In one example, the antigen-presenting cell is a macrophage. The antigen-presenting cell is a dendritic cell. The expression of CD40 upregulates other co-stimulatory markers. These other co-stimulatory markers include the B7 co-stimulatory marker. Activation of the antigen-presenting cell initiates an anti-tumor T cell response. The molecular weight of the GC polymer is less than 420 kDa. Alternatively, the molecular weight of the GC polymer is approximately 250 kDa. The DG of the GC is at most, but does not exceed, 30%. In the GC polymer of Formula 1, for a molecular weight less than 420 kDa... W n = 3–1933, (a) = 1–986, (b) = 1–386, (c) = 1–560. Ideally, the inventors have envisioned the M of GC. W The GC has a value of 250 kDa, a DG value of 5%, and a DDA value of 80%. The GC contains at least one subunit of each of the different subunits [(a), (b), and (c)] (described above).

[0181] Further aspects and / or advantages discovered will be set forth in part in the following description and in part learned through the practice of the methods described herein. Attached Figure Description

[0182] These and / or other aspects and advantages discovered will become apparent and more readily understood from the following description taken in conjunction with the accompanying drawings, in which:

[0183] Figure 1 A small molecular weight example of GC is described, namely galactochitosan (molecular weight = 1.88 kDa), in which DG is 10% and the degree of deacetylation (DDA) is 80%.

[0184] Figure 2 It shows the source from M W A table of recirculating data for Equation 1GC with a capacity of less than 420 kDa (#VAL-AM-000754-B).

[0185] Figure 3 M is shown as a 1% solution for GC. WA graph comparing the various values ​​of the filter rate data.

[0186] Figure 4 Showing Figure 3 Particle size data for three GC solutions.

[0187] Figure 5 The bar graph shows the expression of CD40 by DC2.4 stimulated with different concentrations of the compound of Formula 1 for 18 to 24 hours.

[0188] Figure 6 It is a bar chart that shows the result of M W DC2.4 was stimulated with 420 kDa conventional GC at different concentrations for 18 to 24 hours to reduce CD40 expression.

[0189] Figure 7 It is a bar chart that shows the comparison with from Figure 6 M W A regular GC of 420kDa is used from Figure 5 Compound of formula 1 stimulated the expression of CD40 in DC2.4 for 18 to 24 hours at different concentrations.

[0190] Figure 8 The figure shows the efficacy of compound 1 in combination with tumor ablation in a double flank experiment of a B16-F10 mouse melanoma tumor model.

[0191] Figure 9 This is a diagram showing the growth of the first tumor on the right flank after direct treatment.

[0192] Figure 10 This is a diagram showing the growth of the second tumor on the contralateral flank (left) without direct treatment.

[0193] Figure 11 This diagram illustrates the growth of a Panc02-H7 tumor injected orally into the head of the pancreas. (Left) Primary tumor in the pancreas. (Right) Mesenteric metastasis.

[0194] Figure 12 This is a diagram showing the local retention of subcutaneously injected antigen OVA (labeled with Texas Red) in mice.

[0195] Figure 13 The figure shows the survival rate of B16-F10 carrier animals after treatment, which demonstrates the efficacy of the compound of formula 1 when combined with tumor ablation alone (G4) or tumor ablation and anti-PD1 (G6) administration.

[0196] Figure 14Two graphs are shown comparing the growth of contralateral untreated tumors in non-survivors in G4 (ablation + compound 1) and G6 (ablation + compound 1 + anti-PD1).

[0197] Figure 15 It is a bar chart showing the percentage of animal survival protected against re-attacks against the same B16-F10 tumors from G4 (ablation + Formula 1 compound) and G6 (ablation + Formula 1 compound + anti-PD1). Detailed Implementation

[0198] The inventors unexpectedly discovered the following M W Glycated biopolymer compounds of Formula 1 with values ​​less than 420 kDa (n = 3–2362, (a) = 1–1977, (b) = 1–495, (c) = 1–561) and M W Higher values ​​of conventional GC can stimulate dendritic cells, as measured by CD40 expression, and initiate anti-tumor T cell responses. Data can be extrapolated to the use of pharmaceutical compositions including compounds of Formula 1 to treat proliferative disorders in subjects such as humans. It should be understood that all references cited herein are incorporated herein by reference in their entirety.

[0199] definition

[0200] Unless otherwise stated, the following definitions apply:

[0201] Unless the context clearly indicates otherwise, the singular forms “a”, “an”, and “the” contain the corresponding plural indicator.

[0202] As used herein, the term "comprising" is intended to mean that the element listed following the word "comprising" is necessary or mandatory, but other elements are optional and may or may not be present. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0203] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0204] As used herein, the term “consisting of” is intended to include and be limited to anything that follows the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that other elements may be absent.

[0205] As used herein, the term “substantially constitutes” (and its grammatical variations) is intended to cover the enumerated materials or steps “as well as those that do not materially affect the fundamental and novel properties.” See In re Herz, 537F. 2nd ed. 549, 551-52, 190 USPQ461, 463 (CCPA 1976) (emphasis on originality); also see MPEP section 2111.03. Therefore, as used herein, the term “substantially constitutes” should not be construed as equivalent to “includes.”

[0206] As used herein, the terms “glycated chitosan” or “GC” are intended to refer to the glycosylation of the free amino groups of chitosan, i.e., non-enzymatic glycosylation, and the product subsequently stabilized by reduction. In general, glycosylation (or non-enzymatic glycosylation) is intended to refer to the process that occurs without the contribution of enzymes when sugar molecules such as fructose or glucose bind to substrates such as proteins or lipids. One example of this is the non-enzymatic reaction of the amino groups of sugars and proteins to form glycoproteins. Furthermore, the terms “GC” and “compound of Formula 1” are used interchangeably throughout this document.

[0207] As used herein, the term "physicochemical property" is intended to refer to, but is not limited to, any physical, chemical, or physicochemical property of molecular structures such as GC. Several examples of these properties, as further described herein, are: (i) the M... W (ii) Deacetylation degree of GC (DDA); and (iv) Glycation degree of GC (DG).

[0208] As used herein, the term “about” is intended to refer to a measurable value, such as amount or concentration (etc.), and is intended to cover a variation of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0209] As used herein, the symbol “~” is intended to refer to a measurable value, such as amount or concentration (etc.), and is intended to cover variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0210] As used herein, the phrase "physiologically compatible" is intended to refer to materials that are harmless to the body's tissues when in contact with them. In this context, the term is intended to include, but is not limited to, aqueous formulations (e.g., solutions) that are approximately isotonic with the physiological environment of concern. Non-isotonic formulations (e.g., solutions) may sometimes be used clinically, for example, as dehydrating agents. Additional components of the solutions of the present invention may comprise various salts, such as NaCl, KCl, CaCl2, MgCl2, and Na-based buffers.

[0211] As used herein, the term "immunostimulant" is intended to refer to any molecule, composition, or substance that enhances the immune system's ability to respond to antigens; for example, GC that enhances the immune system's ability to respond to tumor antigens.

[0212] As used herein, the term “substantially aqueous” is intended to indicate that a formulation or preparation may contain, in some respects, a certain percentage of one or more non-aqueous components and one or more pharmaceutically acceptable excipients.

[0213] As used herein, the term "checkpoint inhibitor" is intended to refer to a molecule that inhibits the interaction between a checkpoint molecule and its ligand, in one instance being a monoclonal antibody. The binding of certain checkpoint molecules to T cells is a natural mechanism for suppressing / shutting down effector T cell function. Therefore, checkpoint inhibitors can be interpreted as releasing immunosuppression or at least inducing immunosuppression.

[0214] compound

[0215] The inventors made a surprising and unexpected discovery of M as described above and below. W Semi-synthetic biopolymers of Formula 1 with values ​​less than 420 kDa (n = 3–2362, (a) = 1–1977, (b) = 1–495, (c) = 1–561) and M W Larger values ​​of conventional GC stimulated dendritic cells, leading to CD40 expression and subsequently a robust antitumor response. These data are believed to be extrapolable to the use of semi-synthetic biopolymers to treat certain proliferative disorders in human subjects.

[0216] General structural formula 1 is shown above and below and will be used throughout this specification to describe various GCs. GCs are semi-synthetic polymers containing at least one of each of three different monomers, including but not limited to: glucosamine [monomer (a)]; N-acetylglucosamine [monomer (b)]; and N-glycated glucosamine [monomer (c)]. Formula 1 provides a general polymeric structure of GCs containing n number of monomers, wherein the group of monomers comprises a specified number of individual monomers (a), (b), and (c). Furthermore, the description of a particular compound will also include a weight-average molecular weight (M). W The value of M is an industry standard used to report the molecular weight of polymer mixtures. Additional descriptors may include the degree of saccharification (DG) and degree of deacetylation (DDA), which are percentage values ​​of all monomers, respectively, for saccharified glucosamine and non-N-acetylglucosamine. Since polymer mixtures typically contain polymer chains of varying molecular weights, and determining M... W The method relies on the secondary and tertiary structural characteristics of the polymer chain, therefore it can be assumed that MW The reported values ​​vary by ±15% (consistent with the United States Pharmacopeia guidelines).

[0217] nuclear:

[0218] Generally, the core of a semi-synthetic biopolymer as shown in Formula 1 above contains a series of monomers (GCs) shown in parentheses. mon ), which includes the total number of individuals defined by the integer n:

[0219]

[0220] Where n is the number of subunits, and (a), (b), and (c) indicate that GC is included. mon The number of each of the following monomer subunits:

[0221]

[0222] as well as

[0223]

[0224] Where R = substitutions produced by saccharification.

[0225] In one respect, GC of Equation 1 mon Each component contains one or more monomer subunits of each monomer subunit (a), (b), and (c). Generally, semi-synthetic biopolymers contain varying numbers of monomers (a), (b), and (c) (Equation 1).

[0226] Any and each individual definition of a kernel as described herein can be combined with any and each individual definition of n, Mw, DDA, and DG as described herein.

[0227] Integer n:

[0228] Equation 1 represents the general formula for semi-synthetic biopolymers, where "n" is an integer representing the number of monomers. Figure 1 The paper provides a small molecular weight example of galactosylated chitosan (1.88 kDa) as a specific example of Formula 1 and to demonstrate the connectivity of the polymer chains.

[0229] for Figure 1 The structure in the text, n=10, indicates 10 monomers.

[0230] for Figure 1 The structure in the text, (a) = 8, indicates 8 glucosamine monomers.

[0231] for Figure 1 The structure in (b) = 2 indicates 2 N-acetylglucosamine monomers and 80% DDA.

[0232] for Figure 1 The structure in the figure, (c) = 1, R = galactosyl, indicates 1 N-glycated glucosamine monomer and 10% DG.

[0233] Glycated chitosan (GC) is a polymer composed of only three different subunits [(a), (b), and (c), Formula 1].

[0234] GC must contain at least one subunit from each of the different subunits [(a), (b) and (c)].

[0235] In one instance, GC's M W Less than 420kDa.

[0236] In another instance, GC's M W It is approximately 250 kDa.

[0237] In another instance, the GC's DG is at most, but does not include, 30%.

[0238] The integer “n” defines the singleton (GC) as shown in Equation 1. mon The quantity of ).

[0239] Any and each individual definition of “n” as described herein can be combined with any and each individual definition of kernel, Mw, DDA, and DG as described herein.

[0240] Weight-average molecular weight (Mw):

[0241] M W This is a polymer sample, preferably the average measured molecular weight of a higher molecular weight chain. For samples with reported M... W A sample with a value of M has molecules of equal mass distributed around that value. W It is most commonly measured using light scattering techniques that are sensitive to molecular size.

[0242] It should be understood that compounds of Formula 1 contain one or more asymmetric centers, chiral axes, and chiral planes, and thus can produce enantiomers, diastereomers, and other stereoisomers, which can be defined according to absolute stereochemistry, such as (R)- or (S)- of amino acids, or (D)- or (L)-. This invention aims to encompass all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S) or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques such as reversed-phase HPLC. Racemic mixtures can be prepared and then separated into individual optical isomers, or these optical isomers can be prepared by chiral synthesis. Enantiomers can be resolved by methods known to those skilled in the art, for example by forming diastereomeric salts, which can then be separated by crystallization, gas-liquid or liquid chromatography, or a selective reaction of an enantiomer with an enantiomer-specific reagent. Those skilled in the art will also understand that when a desired enantiomer is converted into another chemical entity by separation techniques, additional steps are required to form the desired enantiomeric form. Alternatively, a particular enantiomer can also be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts, or solvents, or by converting one enantiomer into another via asymmetric transformation.

[0243] Some Formula 1 compounds may exist as mixtures of epimers. An epimer is a diastereomer that has an opposite configuration at only one of the two or more stereocenters present in the corresponding compound.

[0244] Furthermore, some Formula 1 compounds may exist in zwitterionic form, and the present invention includes these compounds and mixtures thereof in zwitterionic form.

[0245] Any and each individual definition of Mw as described herein can be combined with any and each individual definition of kernel, n, DDA, and DG as described herein.

[0246] GC's Mw

[0247] The weight-average molecular weight (Mb) of GC can be reliably and accurately determined using any number of suitable techniques in the field of chemistry. W ).

[0248] One example of GC was prepared as an injectable formulation comprising GC with a weight-average molecular weight (MW) of less than 420 kDa.

[0249] In some specific aspects, "n" is M ranging from approximately 3 to approximately 2362. W Integers with a range less than 420kDa.

[0250] Degree of deacetylation (DDA) of GC

[0251] Another property of GC, as represented by Equation 1, is the degree of deacetylation (DDA). The degree of deacetylation of GC can be reliably and accurately determined using any number of suitable techniques in the field of chemistry.

[0252] NMR is a technique that can be used to determine the DDA of GC.

[0253] Any and each individual definition of DDA as described herein can be combined with any and each individual definition of kernel, n, Mw, and DG as described herein.

[0254] Degree of GC glycation (DG)

[0255] Another property of GC, as represented by Equation 1, is the degree of saccharification (DG). The DG of GC can be reliably and accurately determined using any number of suitable techniques in the field of chemistry.

[0256] NMR is a technique that can be used to determine the localization of GC. Furthermore, NMR can be used to characterize other chemical properties of GC.

[0257] Carbon / nitrogen (C / N) elemental combustion analysis is another technique that can be used to determine the DG of GC by comparing the C / N ratio of GC to chitosan starting material.

[0258] Enzymatic digestion combined with HPLC is another technique that can be used to determine the DG of GC.

[0259] It should be understood that other suitable analytical methods and instruments can also be used to simultaneously detect, measure and identify multiple components in a sample.

[0260] DG can be determined using colorimetric measurements of GC derivatives, such as by the ninhydrin reaction.

[0261] Therefore, M with the expected value has been discovered. W The GCs of DDA and DG offer unexpected and advantageous improvements in bioactivity and sterile filtration.

[0262] The specific examples mentioned above include the following:

[0263] Monomer a-monomer a-monomer-a is chitosan; and

[0264] Monomer b-monomer-b-monomer-b is chitin.

[0265] Any and every individual definition of DG as described herein can be combined with any and every individual definition of kernel, n, Mw, and DDA as described herein.

[0266] Exemplary methods for determining viscosity, independent of aseptic filterability

[0267] The viscosity of GC formulations can be reliably and accurately determined using any number of suitable techniques in the field of chemistry.

[0268] It should be understood that various types of instruments, such as viscometers and rheometers, can be used to reliably measure viscosity. Rheometers are used for fluids that cannot be defined by a single viscosity value, and therefore require setting and measuring more parameters compared to viscometers. Close temperature control of the fluid is essential for accurate measurements, especially in materials such as lubricants whose viscosity can double with a change of only 5°C.

[0269] Therefore, the viscosity of GC can be determined by any suitable method known in the art.

[0270] For example, viscosity can be reliably measured in centipoise. Poise is the unit of dynamic viscosity in the centigram second unit system. Centipoise is one-hundredth of a poise and is one millipascal-second (mPa·s) when expressed in SI (1 cP = 10⁻⁶). -2 P = 10 -3 Centipoise (cP) is the correct abbreviation, but alternative abbreviations such as cps, cp, and cPs are also common. Centipoise can be measured using a viscometer. When determining centipoise, all other fluids are typically calibrated against the viscosity of water.

[0271] Example of determining viscosity

[0272] Besides molecular weight, many factors influence the viscosity of solutions, specifically polymer solutions. In the case of GC, the injectability of GC solutions is highly dependent on the viscosity and rheological properties of GC in solution. These properties, in turn, are highly dependent on the molecular weight of GC, DG, and DDA. These properties affect the secondary and tertiary solution structure of GC molecules, thus significantly contributing to the viscosity and rheological properties of the solutions prepared thereby.

[0273] It has been noted that the improved viscosity and rheological properties of GC are, in turn, highly dependent on the specific chemical properties of GC.

[0274] Synthesis method

[0275] Semi-synthetic biopolymers, such as those mentioned above, can be synthesized via reductive amination reactions involving the free amino group of chitosan and the carbonyl group of reducing monosaccharides and / or oligosaccharides. This reaction is a two-step process involving the initial formation of an imine between chitosan and the reducing sugar, followed by the reduction of the imine to an amine using a wide range of reducing agents. The products of the first step of the reaction, primarily a mixture of Schiff bases (where the carbon atom from the carbonyl group is now doubly bonded to the nitrogen atom from the free amine, thereby releasing a molecule of water) and Amadori products (where the carbonyl carbon atom is monobonded to the nitrogen atom of the amino group, while the adjacent carbon atom is doubly bonded to an oxygen atom), can be used as is or after the second step of the reaction—i.e., reduction using hydrides such as borohydride reducing agents, such as NaBH4, NaBH3CN, NaBH(OAc)3, or stabilization by exposure to hydrogen in the presence of a suitable catalyst.

[0276] "GC" refers to the product of free amino glycosylation (i.e., non-enzymatic glycosylation) of chitosan, followed by reductive stabilization. Glycosylation imparts favorable solubility and viscosity properties to chitosan, which facilitates the use of this derivative in conjunction with laser-assisted immunotherapy and other applications of the derivative.

[0277] Chitosan and reducing sugars are starting materials for the manufacture of compounds of formula 1. The presence of primary amino groups in chitosan facilitates various methods for chemical modification, which are primarily designed to achieve its solubility and impart specific properties for particular applications.

[0278] The dissolution of the starting material chitosan can be achieved by dissolving it in aqueous acidic solutions, both organic and inorganic, resulting in the formation of water-soluble chitosan salts through the protonation of free amino groups. Modification of the amino groups in chitosan includes the introduction of chemical groups such as carboxymethyl, glycerol, and N-hydroxybutyl. Glycosylation, i.e., the non-enzymatic glycosylation of the free amino groups in chitosan, followed by reductive stabilization, provides a desirable method for preparing the various pharmaceutical formulations used herein.

[0279] The GCs described herein are in the form of Schiff bases, Amadori products, or, in one example, their reduced secondary amines or alcohols. In another example, the GCs contain a carbonyl reactive group. It is desirable that the GCs described herein are obtained by reacting chitosan with monosaccharides and / or oligosaccharides, in one example, in the presence of an acidifying agent, with a reaction sufficient to achieve a time-dependent Schiff base formation between the carbonyl group of the sugar and the primary amino group of the chitosan (also referred to herein as glycosylation of the amino group), in one embodiment, subsequently stabilized by reducing the Schiff base and its rearranged derivative (Amadori product) to a secondary amine or alcohol, in one embodiment to provide stabilization containing at most 30% DG.

[0280] This disclosure is the first demonstration of achieving, thus, a chitosan polymer containing at most, but not 30%, of saccharification. Thus, according to one example, a GC formulation is substantially composed of a GC polymer having a molecular weight of less than 420 kDa, and further, wherein the GC polymer has at most, but not 30%, of saccharification.

[0281] Therefore, the products generated by the non-enzymatic glycosylation of the free amino groups of chitosan are mainly a mixture of Schiff bases and Amadori products. The Schiff base is the initial carbonyl carbon atom doubly bonded to the nitrogen atom of the amino group (also known as an imine functional group), and the Amadori product is the initial carbonyl carbon atom bonded to the nitrogen atom of the amino group via a single bond, while the adjacent carbon atom doubly bonds to an oxygen atom to form a ketone group. These products (generated by the non-enzymatic glycosylation process) can be used as is or after reduction with hydrides such as borohydride reducing agents, for example, NaBH4, NaBH3CN, NaBH(OAc)3, or after stabilization with hydrogen in the presence of a suitable catalyst.

[0282] The various products obtained by chitosan saccharification will be used as is or reacted with other natural or synthetic materials, such as aldehyde derivatives of GC with substances containing two or more free amino groups, such as on the side chains of amino acids enriched in lysine residues in collagen, on hexosamine residues in chitosan and deacetylated glycoconjugates, or on natural and synthetic diamines and polyamines. This is expected to result in crosslinking through Schiff base formation and subsequent rearrangement, condensation, dehydration, etc. The stabilization of modified GC materials can be achieved spontaneously or by curing involving rearrangement, condensation, or dehydration, either through chemical reduction or by cultivation under various temperature, humidity, and pressure conditions. The chemistry of the Amadori rearrangement, Schiff bases, and the Leukart-Wallach reaction is detailed in The Merck Index, 9th edition (1976), pp. ONR-3, ONR-55, and ONR-80, Library of Congress Card No. 76-27231, which is incorporated herein by reference. The chemistry of the nucleophilic addition reactions applicable to this invention is detailed in Morrison and Boyd, Organic Chemistry, 2nd edition (8th printing, 1970), Library of Congress Card No. 66-25695, Chapter 19, which is incorporated herein by reference.

[0283] As further described herein, it has been surprising to find that specific types (e.g., specific types of reducing sugars) and degrees of glycosylation endow GC with unexpected and advantageous properties for promoting GC binding to tumor ablation, irradiation therapy, cytotoxic agents, checkpoint inhibitors such as anti-PD-1 and PD-L1 antibodies, adoptive immune transfer, cytokine therapy and other therapeutic applications.

[0284] D-galactose derivatives of GC are particularly desirable because the open-chain form of D-galactose has a relatively high rate of natural occurrence. GC can be prepared in any number of suitable formulations, including, for example, solid forms such as viscous formulations or in any other suitable form.

[0285] According to the present invention, chitosan can be non-enzymatically glycosylated using any of a variety of the same or different reducing sugars, such as the same or different monosaccharides and / or oligosaccharides. Examples of such monosaccharide glycosylators include the following D and L-isomers: triose, tetroose, pentose, hexose, heptose, etc., such as glucose, galactose, fructose, mannose, allose, atroose, idole, tarose, fucose, arabinose, gulose, witch hazel, lysolose, ribose, rhamnose, threose, xylose, allulose, sorbose, tagatose, glyceraldehyde, dihydroxyacetone, erythrose, threose, erythritolose, mannoheptanose, sedoheptulose, etc. Suitable oligosaccharides include fructooligosaccharides (FOS), galactosyloligosaccharides (GOS), mannoheptanose (MOS), etc.

[0286] Combination therapy

[0287] Compound of Formula 1 can be combined with any of a variety of other therapies targeting cancer, including, but not limited to, adoptive T-cell transfer therapy, tumor-infiltrating cell therapy, oncolytic viruses, cancer vaccines / dendritic cell-based therapies, and checkpoint therapy blockade.

[0288] Adoptive T-cell transfers, such as chimeric antigen receptor T-cell (CAR T) or TCR-modified T-cell therapies, include the following non-limiting examples of potential “targets” or “receptors” that can interact agonistically or antagonistically with one or more known drug entities.

[0289] ErbB dimer IL4; CD19; GPC3; CD133; BCMA; κ light chain; CD30; IL13Ra2; NY-ESO-1 and HLA-A2:E6; and MAGE-A10.

[0290] The following are non-limiting examples of tumor-infiltrating cell therapies: TIL and MIL.

[0291] The following are non-limiting examples of cancer vaccines that target specific cancers or tumor-associated antigens / receptors, as well as the following dendritic cell-based therapies:

[0292] Prostate cancer; lung adenocarcinoma cells; gastric cancer cells; melanoma antigen; VEGFR-2, prostate cancer antigen; human telomerase; PAP; E7 antigen; MAGE-A3; Her2; NY-ESO01; short-tailed tumors; BPX101; WT-1-expressing tumors; susceptin-expressing tumors; HSP70 and GPC3; HSP96; URLC10, CDCA1, KOC1; MDA-5 and NOXA; and Gp 100; and neo-individual tumor antigens that can be identified on a case-by-case basis.

[0293] The following are non-restrictive examples of oncolytic viruses:

[0294] T-VEC; Coxsackievirus A21 (CVA21–CAVATAK); Pelareorep (Reolysin); DNX-2401; Enadenotucirev (EnAd); LOAd703; GL-ONC1; and Pexa-Vec.

[0295] The following is a non-restrictive list of checkpoint inhibitor drug types, which includes PD-1 inhibitors; PD-L1 inhibitors; and CTLA-4 inhibitors.

[0296] The following are non-limiting examples of other potential targets in anticancer therapy, in which drugs designed to antagonize (inhibit) or agonize (stimulate) such targets can be combined with compounds of Formula 1 described herein:

[0297] IDO1; LAG-3 (CD223); TIM-3; TIGIT; VISTA; B7-H3 (CD276); KIR; A2aR; TGF-β; PI3Kγ; CD47; CD73; OX40; GITR; ICOS; 4-1BB (CD137); CD27-CD70; and CD40.

[0298] Adhesion and blending

[0299] It should also be understood that compounds of Formula 1 may be conjugated or incorporated with any of the various agonists of TLRs, IDOs, arginases, STING, and mature pathways that stimulate antigen-presenting cells, including non-limiting examples of TLRs:

[0300] TLR1-TLR2; TLR2-TLR6; TLR9; TLR3: TLR-4; TLR7: TLR5; TLR7-TLR8;

[0301] TLR104; IDO; and arginase.

[0302] Furthermore, compounds of Formula 1 can be conjugated or blended with any of many other immune adjuvants or immune stimulants, non-limiting examples of which include:

[0303] Delivery systems, such as alum adjuvants, calcium phosphate, liposomes, virions / virus-like particles; emulsions; squalene; saponin-based chemicals; mineral salts; polymeric microspheres / nanoparticles; carbohydrate-based adjuvants; bacterial products / components; and combinations thereof.

[0304] Compounds of Formula 1 may be conjugated or incorporated with any of a variety of cytokines or cytokine derivatives / gene therapies selected from the following non-limiting examples:

[0305] IL-1a, IL1-b, IL-1Ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17A, IL-17B, C, D, IL-17-F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25 (IL- 17E), IL-26, IL-27(p281EB13), IL-28A / B / IL29, IL-30 (p28 subunit of IL-27), IL-31, IL-32, IL-33, IL-34, IL-35 (p351EB13), IL-36, IL-37, IL-38, IFNa, IFNb, IFNg, TGFb, TNFa, GM-CSF, M-CSF, Ad-RTS-hIL-12 and NKTR-214.

[0306] Compounds of Formula 1 may also be conjugated or blended with any of a variety of chemokines selected from the following non-limiting examples:

[0307] CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, Cxcl15, CXCL16, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, XCL1, XCL2, and CX3CL1.

[0308] Compound of Formula 1 can be conjugated or mixed with any of a variety of antigens.

[0309] Major-level tumor antigens include, but are not limited to: tissue differentiation antigens, such as MART-1, gp100, CEA, and CD19; tumor lineage (tumor / testis) antigens, such as NY-ESO-1 and MAGE-A3; normal proteins overexpressed by cancer cells, such as hTERT, EGFR, and mesothelin; viral proteins, such as HPV, EBV, and MCC; tumor-specific mutation antigens, such as Mum-1, β-catenin, CDK4, and ERBB2IP; and tumor-associated carbohydrate antigens, such as GM2, GD2, sTn, MUC-1, and globo-H.

[0310] Selected examples of tumor antigens are listed below (excluding synonyms and antigen sources):

[0311] ERBB2, BIRC5, CEACAM5, WDR46, BAGE, CSAG2, DCT, MAGED4, GAGE1, GAGE2, GA GE3、GAGE4、GAGE5、GAGE6、GAGE7、GAGE8、IL13RA2、MAGEA1、MAGEA2、MAGEA3 MAGEA4, MAGEA6, MAGEA9, MAGE10, MAGE12, MAGEB1, MAGEB2, MAGEC2, TP5 3, TYR, TYRP1, SAGE1, SYCP1, SSX2, SSX4, KRAS, PRAME, NRAS, ACTN4, CTNNB1 CASP8, CDC27, CDK4, EEF2, FN1, HSPA1B, LPGAT1, ME1, HHAT, TRAPPC1, MUM3. MYO1B, PAPOLG, OS9, PTPRK, TPI1, ADFP, AFP, AIM2, ANXA2, ART4, CLCA2, CPSF 1, PPIB, EPHA2, EPHA3, FGF5, CA9, TERT, MGAT5, CEL, F4.2, CAN, ETV6, BIRC7 CSF1, OGT, MUC1, MUC2, MUM1, CTAG1, CTAG2, CAMEL, MRPL28, FOLH1, RAGE, SF MBT1, KAAG1, SART1, TSPYL1, SART3, SOX10, TRG, WT1, TACSTD1, SILV, SCGB2 A2, MC1R, MLANA, GPR143, OCA2, KLK3, SUPT7L, ARTC1, BRAF, CASP5, CDKN2A. UBXD5, EFTUD2, GPNMB, NFYC, PRDX5, ZUBR1, SIRT2, SNRPD1, HERV-K-MEL, CX orf61, CCDC110, VENTXP1, SPA17, KLK4, ANKRD30A, RAB38, CCND1, CYP1B1, MD M2, MMP2, ZNF395, RNF43, SCRN1, STEAP1, 707-AP, TGFBR2, PXDNL, AKAP13, P.S RTN3, PSCA, RHAMM, ACPP, ACRBP, LCK, RCVRN, RPS2, RPL10A, SLC45A3, BCL2L1 DKK1, ENAH, CSPG4, RGS5, BCR, BCR-ABL, ABL-BCR, DEK, DEK-CAN, ETV6-AML 1. LDLR-FUT, NPM1-ALK1, PML-RARA, SYT-SSX1, SYT-SSX2, FLT3, ABL1, AML1.LDLR、FUT1、NPM1、ALK、PML1 RARA SYT、SSX1、MSLN UBE2V1、HNRPL、WHSC2、EIF4EBP1、WNK2、OAS3、BCL-2、MCL1、CTSH、ABCC3、BST2、MFGE8、TPBG、FMOD、XAGE1、RPSA、COTL1、CALR3、PA2 G4、EZH2、FMNL1、HPSE、APC、UBE2A、BCAP31、TOP2A、TOP2B、ITGB8、 RPA1、ABI2、CCNI、CDC2、SEPT2、STAT1、LRP1、ADAM17、JUP、DDR1、I TPR2、HMOX1、TPM4、BAAT、DNAJC8、TAPBP、LGALS3BP、PAGE4、PAK2、CDKN1A、PTHLH、SOX2、SOX11、TRPM8、TYMS、ATIC、PGK1、SOX4、TOR3 A、TRGC2、BTBD2、SLBP、EGFR、IER3、TTK、LY6K、IGF2BP3、GPC3、SLC 35A4、HSMD、H3F3A、ALDH1A1、MFI2、MMP14、SDCBP、PARP12、MET、CC NB1, PAX3-FKHR, PAX3, FOXO1, XBP1, SYND1, ETV5, HSPA1A, HMHA1, TRIM68, ACSM2A, ATR, USB1, RTCB, C6ORF89, CDC25A, CDK12, CRYBA1, CSNK1A1, DSCAML1, F2R, FNDC3B, GAS7, HAUS3, HERC1, HMGN2, SZT2, LRRC41, MATN2, NIN, PLEKHM2, POLR2A, PPP1R3B, RALGAPB, SF3 B1、SLC46A1、STRAP、SYT15、TBC1D9B、THNSL2、THOC6、WHSC1L1、XP O1、BCL11A、SPEN、VPS13D、SOGA1、MAP1A、ZNF219、SYNPO、NFATC2、 NCBP3、HIVEP2、NCOA1、LPP、ARID1B、SYNM、SVIL、SRRM2、RREB1、EP300、RCSD1、CEP95、IP6K1、RSRP1、MYL9、TBC1D10C、MACF1、MAP7D1 MORC2、RBM14、GRM5、NIFK、TLK1、IRS2、PPP1CA、GPSM3、SIK1、HMGN1、MAP3K11、GFI1、KANSL3、KLF2、CCDC88B、TNS3、N4BP2 TPX2, KMT2A SRSF7 GRK2, GIGYF2, SCAP, MIIP, ZC3H14, ZNF106, SKI, SETD2, ATXN2L, SRSF8, LUZP1, KLF10, RERE, MEF2D, PCBP2 LSP1, MEFV, ARHGAP30, CHAF1A, FAM53C, ARHGAP17, HSPB1, NCOR2ATXN2, RBM15, RBM17 SON, TSC22D4, MYC and ANKRD17. ,

[0312] Conjugation or blending may involve adding an antibody before the compound of Formula 1; adding an antibody after the compound of Formula 1; or adding both an antibody and the compound of Formula 1.

[0313] As is known in the art, antibodies are secreted forms of B cell receptors (BCRs) and therefore possess substantially the same level of diversity as BCRs, which can reach 10. 17 Some antibodies have been isolated and subsequently manufactured into therapeutic agents for human use.

[0314] Compounds of Formula 1 can also be conjugated or blended with any of a variety of antibodies comprising the following non-restricted monoclonal antibodies and bispecific antibodies:

[0315] Monoclonal antibodies and so-called “bispecific” antibodies have been developed as anticancer therapeutics. Antibodies employ various mechanisms of action to induce cancer cell death, including direct tumor killing, immune-mediated tumor cell killing, angiogenesis / stromal ablation, antibody-initiated secondary effector cells / molecules / cytotoxic agents, or other indirect mechanisms. As tumor cells are killed, antigens are released. Not wishing to be bound by theory, administering a compound of Formula 1 to a subject, specifically a human subject, can amplify the resulting immune response through the aforementioned steps.

[0316] The following are non-limiting examples of targets for which monoclonal antibodies have been used in cancer therapies:

[0317] HER2; VEGF; EGFR; CD20; CD30; and CD33.

[0318] The following are non-limiting examples of targets for generating antibody-drug conjugates:

[0319] c-Met; gpNMB; EGFR; folate receptor α (FRα); cohesin-4; Trop-2; CD22; CEACAM; CD56; DLL3; CD25; GCC; HER2; GPNMB; CA-6; LIV-1; tyrosine kinase 7; liver glycoprotein-A4; LAMP-1; P-cadherin 3; HER3; Axl; PMSA; PD-1; PD-L1; CTLA-4.

[0320] Bispecific antibodies are engineered antibodies in which multiple specific antibodies are linked together within the same molecule. They serve a variety of biological functions, including T cell recruitment, delivery of CAR-T cells, targeting toxins to tumors, activation of T cells, blocking receptors necessary for tumor growth, activation of monocytes to achieve tumor-killing activity, retargeting of T cells to tumors, delivery of chemotherapy drugs to local tumors, and radioimmunotherapy.

[0321] There are two main structural formats: IgG-like formats and non-IgG-like formats. IgG-like formats include tetravalent sclerosing structures, button-into-holes dual variable domain Ig IgG single-chain Fv (scFv), two-in-one Fab (or dual-action Fab), hemimolecule exchange, and Kλ bodies; non-IgG-like formats include scFv-based BsAbs, nanobodies, docking and lock methods, and dual-affinity retargeting molecules (DART).

[0322] The following are non-limiting examples of bispecific antibodies:

[0323] CD8×CD19; Anti-DLL4×Anti-VEGF; Anti-CD3×Anti-EGFR; Anti-CD3×Anti-GD2; Anti-CD3×Anti-CD19; Anti-CD3×Anti-CD20; Anti-CD3×Anti-EpCAM; Anti-CD3×Anti-CEA; Anti-CD3×Anti-CD123; Anti-CD3×Anti-GPA33; Anti-CD3×Anti-HER2; Anti-CD3×gp100; Anti-CD3×Anti-PSMA; Anti-CD30×Anti-CD16A; Anti-CEA×DTPA-131I; Anti-CE A×HSG; anti-CD3×anti-CD33; anti-angiogenic 2×anti-VEGF-A; anti-Her-1×anti-Her-3; anti-Her2×anti-Her3; anti-IGF1R×anti-Her3; anti-Her1×anti-cMET; anti-CD64×anti-EGFR; B-cell maturation antigen (BCMA); anti-CD19×anti-CD22; anti-CD28×HMV-MAA; anti-CD32B×anti-CD79B; nanoparticles; anti-EGFR×anti-EDV; and radioimmunotherapy.

[0324] Compounds of Formula 1 can be conjugated, blended, or rapidly and continuously used with any of a variety of cytotoxic agents, particularly those that induce immunogenic cell death, or at metronid doses that induce immune stimulation. Non-limiting examples of cytotoxic agents include:

[0325] Acetic acid, ethanol, anhracycline, anti-EGFR mAb 7A7, BK channel agonist, bortezomib, bortezomib plus mitomycin C plus hTert-Ad, cardiac glycoside plus non-ICD inducer, cyclophosphamide, GADD34 / PP1, inhibitor plus mitomycin, irradiation, LV-tSMAC, measles virus, oxaliplatin, PDT containing hypericin, thapsigargin plus cisplatin, doxorubicin, paclitaxel, oncolytic peptide LTX-315, mitoxantrone, oxaliplatin, UVA irradiation, gamma irradiation, shikonin, EGFR-specific antibody 7A7, coxsackievirus B3.

[0326] Compounds of Formula 1 can be conjugated, blended, or rapidly and sequentially applied to any of a variety of agents that reduce systemic or local immunosuppression in cancer patients, which can be achieved by biological agents such as monoclonal antibodies, bispecific antibodies, or small molecule chemotherapy. Immunosuppressants include the following non-limiting examples: (as defined above, examples of immunosuppressants follow "target"):

[0327] T-reg cells (T-reg cells): metronidazole doses of cyclophosphamide, dendritic cell vaccines containing daclizumab, anti-CD25 monoclonal antibodies, tyrosine kinase inhibitors sorafenib, sunitinib, and imatinib.

[0328] Myeloid-derived suppressor cells (MDSCs): fluorouracil and gemcitabine, DS-8273a, and agonist antibodies targeting the TRAIL R2 receptor (DR-5).

[0329] Compounds of Formula 1 can be conjugated, incorporated, or rapidly and sequentially applied to any of the molecular modes associated with DAMP disruption, most of which are induced during immunogenic cell death processes induced by different agents. Non-limiting examples of DAMP and its receptor include (DAMP followed by its receptor):

[0330] ATP: P2Y2 and P2X7; BCL-2: TLR2; Calcium reticulin: CD91; Cyclophilin A: CD147; F-actin: DNGR1; HSP70, HSP90, HSP60, HSP72, GRP78 and GP96: CD91, TLR2, TLR4, SREC1 and FEEL1; Hepatocellular carcinoma-derived growth factor: unknown receptor; Histone: TLR9; HMGB1: TLR2, TLR4, RAG E and TIM3; HMGN1: TLR4; IL-1α: IL-1R; IL-33: ST2; IL-6: IL-6R and GP130; Mitochondrial DNA: TLR9; Mitochondrial transcription factor A: RAGE and TLR9; Sodium urate: Unknown; N-formyl peptide: FPR1; Reactive carbonyl and oxidation-specific epitopes: CD36, SRA, TLR2, TLR4 and CD14; Ribonucleoprotein, mRNA and genomic DNA: TLR3; S100A8, S100A9 and S100A12: RAGE.

[0331] Desired physicochemical properties

[0332] Conventionally generated GC products are difficult to aseptically filter and produce according to GMP standards when dispersed, suspended, or dissolved in aqueous solutions. In fact, as is known in the art, autoclaving and gamma sterilization can degrade the structure of the final product or alter its structure in some way.

[0333] Some of the aspects described in this article overcome the long-standing unmet need for improved therapeutic GC products by providing improved GC that is unaffected by the shortcomings of conventional methods.

[0334] Manufacturing and filtration

[0335] It demonstrates the effect of M W Sterilization by aseptic filtration of GCs with values ​​below 420 kDa is filterable, while higher molecular weight GCs are not. Furthermore, as described in the prior art, conventional GCs containing, for example, PCT application No. PCT / US13 / 021903 have been shown to be difficult to aseptically filter through a 0.22 μm aseptic filter, making them unsuitable for commercial cGMP manufacturing. In contrast, Formula 1 GCs, which have non-obvious rheological properties, have been found to be highly suitable for aseptic filtration, cGMP manufacturing, and human use.

[0336] Percolation and ultrafiltration are industry-standard methods for purifying and concentrating polymer solutions, respectively. Surprisingly, percolation and ultrafiltration are unexpectedly improved using the modified Formula 1 GC described herein. Conventional GC is difficult for percolation and ultrafiltration, leading to filter clogging or other malfunctions, thus making it unsuitable for commercial cGMP manufacturing. On the other hand, the modified GC is highly suitable for percolation and ultrafiltration, thereby significantly improving the manufacturing process.

[0337] According to one example, the formulation is prepared as an aqueous solution with a pH between approximately 5 and approximately 7.

[0338] The formulation can also be prepared as an aqueous solution comprising a buffered saline solution mainly composed of GC.

[0339] The formulation may also be formulated to consist substantially of a GC polymer, wherein the GC polymer has at most but not 30% saccharification.

[0340] According to one specific example, the glycosylated amino groups are present at less than 29% of the total monomers. According to another example, the GC polymer contains glycosylated amino groups present at 1% to 8% of the total monomers. In yet another example, the GC polymer contains glycosylated amino groups present at 3% to 6% of the total monomers. In still another example, the GC polymer contains glycosylated amino groups present at about 0.5% to about 9.5% of the total monomers.

[0341] In another example, the formulation may be formulated to consist essentially of a GC polymer, wherein the degree of saccharification (DG) of the GC polymer is about five (5)% of its total monomers.

[0342] In another example, the formulation may be formulated to consist substantially of a GC polymer, wherein the M of the GC polymer is... W The value is less than 420 kDa.

[0343] Another example comprises GC including about one (1) wt% of a GC polymer dispersed in an aqueous solution, the viscosity of which, when measured at about 25°C, is between about one (1) centiliter and about one hundred (100) centiliters.

[0344] Another example comprises an aqueous solution having about one percent by weight of GC and a degree of saccharification (DG) of less than twenty-nine (29) percent of the GC, wherein the viscosity of the aqueous solution is from about one (1) centiliter to about one hundred (100) centiliters.

[0345] In yet another example, the formulation may be formulated to consist substantially of a GC polymer, the formulation comprising about or more than one wt% of the GC polymer dispersed in an aqueous solution, wherein the GC polymer has about five (5)% saccharification of its total monomers, and wherein the viscosity of the aqueous solution is suitable for easy injectability and administration to a subject.

[0346] In yet another example, the formulation may be formulated to consist substantially of a GC polymer, the formulation further containing one or more different materials miscible in an aqueous solution. Examples of suitable materials include, but are not limited to, hyaluronic acid, chondroitin sulfate, and carboxymethyl cellulose.

[0347] The formulation may comprise a GC polymer including a monosaccharide bound to an additional free amino group. The GC polymer may take any suitable form, such as a Schiff base, an Amadori product, or a mixture thereof. The GC polymer may also be in the form of a reduced Schiff base (secondary amine), a reduced Amadori product (alcohol), or a mixture thereof.

[0348] The formulation can also be formulated in which the GC polymer has a number of chemically modified monosaccharide or oligosaccharide substituents. In one example, the monosaccharide includes galactose.

[0349] The formulation or preparation may also contain a physiologically compatible carrier containing GC.

[0350] The above and other objectives have now been achieved, some of which involve GCs with specific chemical structures and compositions that impart unexpected and surprisingly beneficial properties.

[0351] The present invention also covers a wide range of uses of GC, which has surprising and unexpected properties, as an immunostimulant, such as in combination with tumor ablation, irradiation therapy, cytotoxic agents, checkpoint inhibitors such as anti-PD-1 and PD-L1 antibodies, adoptive immune transfer, cytokine therapy, and other therapeutic applications as further described herein.

[0352] Immunostimulants comprising injectable GC formulations are provided in certain aspects described herein. Ideally, M W GCs smaller than 420 kDa are used in other therapeutic applications, including as adjuncts to tumor ablation or irradiation therapy or other therapies that can induce immunogenic cell death in tumor cells, and as therapeutic uses as immunostimulants and immunomodulators associated with immunotherapies.

[0353] Application mode

[0354] The findings also encompass various routes of administration of GC immunostimulant formulations, such as intramuscular, subcutaneous, intradermal, and intratumoral injection. In the desired method, the immunostimulant is, in one instance, prepared as a formulation for injection into or around a tumor mass. However, it should be recognized that other methods may be sufficient to localize the immunostimulant to the tumor site. One such alternative delivery method is the conjugation of the immunostimulant to a tissue-specific antibody or tissue-specific antigen, thereby enhancing delivery to the tumor site. Any method or combination of different methods for localizing the immunostimulant to the tumor site is acceptable, provided that the delivery mechanism ensures an adequate concentration of the immunostimulant in or around the vegetation.

[0355] For certain lung cancers, it is envisioned that a desired amount of Formula 1GC is locally applied to one or both lungs via upward exhaust.

[0356] According to some aspects, the findings provide for various pharmaceutical formulations of GC used in conjunction with tumor ablation, including: thermal tumor ablation such as radiofrequency ablation (RFA), photothermal laser ablation (PTT), high-intensity focused ultrasound (HIFU), and microwave ablation (MWA); non-thermal ablation such as irreversible electroporation (IRE), electric field therapy, photodynamic cancer therapy (PDT), and cryoablation; and tumor irradiation therapies such as stereotactic body irradiation therapy (SBRT), photon beam or proton beam therapy, and flash irradiation therapy; and / or other tumor destruction methods, as further described in detail herein. It has been observed that the use of GC having a suitable viscosity that allows it to be used as an injectable formulation or other formulation is desirable as an adjunct to methods for inducing immunogenic cell death of vegetations, such as tumor ablation methods, tumor irradiation methods, and / or other methods, including but not limited to chemotherapy and / or tumor immunotherapy methods. Such applications typically involve injecting the GC formulation into a patient, but other routes of administration are also envisioned by the inventors (e.g., inhalation).

[0357] The immunostimulatory composition may further comprise an antitumor antibody conjugated to GC. The immunostimulatory composition may also comprise one or more tumor-specific antigens conjugated to or incorporated with GC.

[0358] The immunostimulatory composition may further comprise cytokines, chemokines or (targeted Toll-like receptor) TLR agonists, vaccine adjuvants, tumor-associated antigens, antitumor antibodies, and DAMP conjugated to or incorporated with GC.

[0359] The findings provide immunostimulant formulations containing suspensions or solutions of GC. In this instance, the GC is used in conjunction with localized vegetation ablation using thermal or non-thermal ablation methods such as RFA, microwave, laser, HIFU, IRE, PDT, and cryoablation.

[0360] The GC is used in conjunction with irradiation therapy for vegetations, such as SBRT or proton beam therapy.

[0361] As described further in detail herein, immunostimulant formulations may further include suitable chromophores for photodynamic or photothermal therapy. The selection of a suitable chromophore is primarily a matter of harmonizing with an acceptable laser irradiation wavelength. Of course, the irradiation wavelength used must be complementary to the optical properties (i.e., absorption peaks) of the chromophore. Other chromophore selection criteria include the ability to generate heat, induce singlet oxygen and other reactive molecules to escape from their own components, such as the toxicity of cisplatin. In one example, the irradiation wavelength is 805. + / - 0.10 nm. The desired chromophore exhibits strong absorption in the red and near-infrared spectral regions where tissues are relatively transparent. Another advantage of this wavelength is the avoidance of potential mutagenic effects encountered by UV-excited sensitizers. However, wavelengths between 150 nm and 2000 nm may prove effective in individual cases. Examples of chromophores include, but are not limited to, single-walled carbon nanotubes (SWNTs), Buckminster fullerenes (C... 60 Indocyanine green, methylene blue, gold nanorods, DHE (polyhexyporphyrin ester / ether), mm-THPP (tetra(m-hydroxyphenyl)porphyrin), AlPcS4 (aluminum phthalocyanine tetrasulfonate), ZnET2 (zinc violet cyanin), and Bchla (bacterial chlorophyll-α).

[0362] Various treatment options were envisioned, including the following twenty-seven (27) example paragraphs:

[0363] 1. In one example, the immunostimulant composition is formulated as a solution or suspension. The solution or suspension may contain, for example, about 1% (by weight) of GC.

[0364] 2. In another example, a composition for conditioning a growth using, for example, a tandem ablation therapy and immunotherapy via physical methods such as heating and freezing of the growth, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated to a tumor-specific antigen, and wherein the immunostimulant is GC.

[0365] 3. In one example, a composition for conditioning a growth using, for example, a tandem ablation therapy and immunotherapy via physical methods such as heating and freezing of the growth, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated to a cytokine, and wherein the immunostimulant is GC.

[0366] 4. In another example, a composition for conditioning a growth using a tandem ablation therapy and immunotherapy, for example by physical methods such as heating and freezing the growth, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated to a TLR agonist, and wherein the immunostimulant is GC.

[0367] 5. In another example, a composition for conditioning vegetations using, for example, tandem irradiation therapy and immunotherapy via X-rays, gamma rays, or proton beams, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated to a tumor-specific antigen, and wherein the immunostimulant is GC.

[0368] 6. In another example, a composition for conditioning vegetations using, for example, tandem irradiation therapy and immunotherapy via X-rays, gamma rays, or proton beams, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated to a cytokine, and wherein the immunostimulant is GC.

[0369] 7. In yet another example, a composition for conditioning vegetations using, for example, tandem irradiation therapy and immunotherapy via X-rays, gamma rays, or proton beams, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated to a TLR agonist, and wherein the immunostimulant is GC.

[0370] 8. In another example, a composition for conditioning vegetations using tandem physical and immunological treatments, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated to an antigen-specific antibody and a cytokine, and wherein the immunostimulant is GC. The immunostimulant may, in some cases, consist substantially of GC.

[0371] 9. In one example, a composition for conditioning vegetations using tandem cytotoxic therapy and immunotherapy, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated to a tumor-specific antigen, and wherein the immunostimulant is GC.

[0372] 10. In one example, a composition for conditioning vegetations using tandem cytotoxic therapy and immunotherapy, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated to a cytokine, and wherein the immunostimulant is GC.

[0373] 11. In one example, a composition for conditioning vegetations using tandem cytotoxic therapy and immunotherapy, the composition comprising an immunostimulant, wherein the immunostimulant is conjugated to a TLR agonist, and wherein the immunostimulant is GC.

[0374] 12. In one example, a composition for conditioning vegetations using tandem physical and immunological therapy, the composition comprising a combination of a chromophore and an immunostimulant, wherein the chromophore and the immunostimulant are conjugated to a tumor-specific antibody, and wherein the immunostimulant is GC. The immunostimulant may, in some cases, consist substantially of GC.

[0375] 13. In one example, an injectable formulation is provided for conditioning vegetations using physical methods such as tumor ablation or irradiation therapy, or cytotoxic therapy, or any combination thereof, in conjunction with immunotherapy, said injectable formulation comprising an immunostimulant, said immunostimulant being GC. The immunostimulant may, in certain circumstances, be conjugated to another component, said component being, but not limited to, cytokines, chemokines, TLR agonists, antibodies, tumor-specific antigens, or any combination thereof.

[0376] 14. Furthermore, a composition can be prepared for conditioning vegetations using a tandem physical therapy and immunotherapy, such as tumor ablation or irradiation therapy, said composition comprising an immunostimulant, and said immunostimulant being M. W GC less than 420kDa.

[0377] 15. A composition may also be prepared for use in conditioning vegetations in a tandem physical therapy and immunotherapy, such as tumor ablation or irradiation therapy, said composition comprising a combination of an immunostimulant and a cytokine, wherein said immunostimulant is M W GC less than 420kDa.

[0378] 16. Furthermore, an injectable solution can be prepared for conditioning vegetations using a tandem physical therapy and immunotherapy approach, such as tumor ablation or irradiation therapy, wherein the composition comprises an immunostimulant, wherein the immunostimulant is M W GC less than 420kDa.

[0379] 17. An injectable solution can also be prepared for conditioning vegetations against a tandem physical therapy and immunotherapy, such as tumor ablation or irradiation therapy, said injectable solution comprising a mixture of a cytokine or TLR agonist and an immunostimulant, wherein said immunostimulant is M W GC less than 420kDa.

[0380] 18. In one example, the GC composition is used as an immunostimulant in novel cancer treatments. Physical therapy and immunotherapy are combined by directly ablating or irradiating the vegetation and subsequently introducing an immunostimulant into or around the ablated or irradiated vegetation. Following tumor ablation or irradiation sufficient to induce vegetation cell destruction, the immunostimulant component enhances the immune response to the tumor-associated antigens released thereby by enhancing the retention and exposure of tumor antigens, enhancing the uptake of tumor antigens by antigen-presenting cells (APCs) such as dendritic cells (DCs), and by activating APCs to avoid tolerance and ultimately stimulating a systemic anti-tumor T-cell response, wherein the immunostimulant is M W GC less than 420kDa.

[0381] 19. In another example, photodynamic therapy and immunotherapy are combined by introducing both a chromophore and an immunostimulant into the vegetation, wherein the immunostimulant is GC. Following irradiation sufficient to induce destruction of the vegetation cells, the immunostimulant component enhances the immune response to the tumor-associated antigens released therefrom by increasing the retention and exposure of tumor antigens, enhancing the uptake of tumor antigens by APCs such as DCs, activating APCs to avoid tolerance, and ultimately stimulating a systemic anti-tumor T-cell response.

[0382] 20. Immunostimulants can be combined with other components, such as cytokines, chemokines, TLR agonists, cytotoxic compositions, antibodies, or antigens, to form solutions for injection into tumor masses, or they can be injected into tumor masses alone. However, it should be recognized that other methods may be sufficient to localize immunostimulants to the tumor site. One such alternative delivery method is the conjugation of the immunostimulant to a tissue-specific antibody or tissue-specific antigen, thereby enhancing delivery to the tumor site. Any method or combination of different methods for localizing immunostimulants to the tumor site is acceptable, provided that the delivery mechanism ensures an adequate concentration of the immunostimulant in the vegetation.

[0383] 21. According to another example, a method for treating a growth in a human or other animal host, the method comprising: (a) selecting an immunostimulant, wherein the immunostimulant comprises GC; (b) ablating or irradiating the selected growth, thereby inducing destruction of growth cells and immunogenic cell death of the tumor, resulting in fragmented growth tissue and cellular molecules; and (c) introducing the immunostimulant into or around the growth, which stimulates the host's autoimmune defense system to process the fragmented growth tissue and cellular molecules, such as tumor antigens, and thus generate immunity against growth cell proliferation, wherein the immunostimulant is GC.

[0384] 22. In yet another example, a method for generating tumor-specific antibodies in a tumor-bearing host comprises ablating or irradiating the tumor to a degree sufficient to induce destruction of vegetation cells and produce fragmented vegetation tissue and cellular molecules, and then injecting an immunostimulant, wherein the immunostimulant is GC, into or around the vegetation. The host's immune system is stimulated to interact with and process the fragmented vegetation tissue and cellular molecules, thereby inducing a systemic antitumor antibody / B cell b response.

[0385] 23. In another example, a method for generating tumor-specific antibodies in a tumor-bearing host includes ablating or irradiating the tumor to a degree sufficient to induce destruction of vegetation cells and produce fragmented vegetation tissue and cellular molecules, and then introducing an immunostimulant into or around the vegetation by injection, thereby stimulating the host's immune system to interact with and process the fragmented vegetation tissue and cellular molecules, thereby inducing a systemic antitumor T-cell response, wherein the immunostimulant is GC.

[0386] 24. An exemplary method for destroying vegetations and generating anti-tumor T-cell responses in a tumor-bearing host in parallel includes: (a) selecting an immunostimulant; (b) ablating or irradiating the vegetation to a degree sufficient to produce vegetation cell destruction and generate fragmented vegetation tissue and cellular molecules; (c) introducing the immunostimulant into the vegetation by intratumoral injection, wherein a mixture of the fragmented tissue and cellular molecules and the immunostimulant is formed at the injection site; and (d) stimulating a T-cell response against the vegetation cell tissue within the host.

[0387] 25. Another exemplary method for destroying vegetations and generating anti-tumor T-cell responses in a tumor-bearing host in parallel comprises: (a) selecting a chromophore and an immunostimulant, the chromophore being adapted to generate thermal energy upon activation in the near-infrared or infrared wavelength range; (b) introducing the chromophore into the vegetation via intratumoral injection; (c) irradiating the vegetation with a laser of a wavelength in the visible, near-infrared, or infrared range at a power sufficient to activate the chromophore for a duration to generate a photothermal response thereby inducing vegetation cell destruction and generating fragmented vegetation tissue and cellular molecules; (d) introducing the immunostimulant into the vegetation via intratumoral injection, wherein a mixture of the fragmented tissue and cellular molecules and the immunostimulant is formed, wherein the immunostimulant is GC; and (e) stimulating a systemic anti-tumor immunological response in the host.

[0388] 26. As described elsewhere herein, the method may further comprise conjugating the immunostimulant to a tumor-specific antibody, thereby forming a conjugate, and administering the conjugate to the host. Alternatively, the method may further comprise conjugating the immunostimulant to a tumor-specific antigen, thereby forming a conjugate, and administering the conjugate to the host. Furthermore, any number of suitable conjugates may be used, such as cytokines, chemokines, TLR agonists, proteins, cytotoxic agents, or any combination thereof.

[0389] 27. The formulations and formulations described herein, containing GC, may also be used in conjunction with photodynamic therapy (PDT). Photosensitizing compounds exhibit a photochemical reaction upon exposure to light. Photodynamic therapy (PDT) uses such photosensitizing compounds and lasers to induce tumor necrosis. Treatment of solid tumors via PDT typically involves systemic administration of tumor-localizing, photosensitizing compounds, and subsequent activation by laser. Upon absorption of light of an appropriate wavelength, the sensitizer transitions from a stable atomic structure to an excited state. Cytotoxicity and ultimately tumor destruction are mediated through the interaction between the sensitizer and molecular oxygen within the treated tissue, resulting in cytotoxic singlet oxygen.

[0390] The following are various combinations of the paragraphs mentioned above:

[0391] For compounds of Formula 1, tandem ablation therapy may include, for example, a regimen using physical methods such as heating or freezing the vegetation (from paragraphs 2, 3, 4, and 13 above); tandem ablation therapy using X-rays, gamma irradiation, or proton beams (from paragraphs 5, 6, 7, 8, and 13 above); tandem cytotoxic therapy (from paragraphs 9, 10, 11, and 13 above); and tandem physical and immunological therapy including a combination of chromophores and immunostimulants (from paragraph 12 above).

[0392] For compounds of Formula 1 with a molecular weight less than 420 kDa, tandem ablation therapy may include, for example, a protocol using physical methods such as heating or freezing the vegetation (from paragraphs 14, 15 and 17 above); tandem ablation therapy using, for example, X-rays, gamma irradiation or proton beams (from paragraphs 14, 15 and 17 above).

[0393] For chromophores used in PDT, a tandem physical and immunological treatment involving a combination of chromophores and immunostimulants is employed (from paragraph 12 above).

[0394] For tumor antigens, tandem ablation therapy may include protocols such as those using physical methods such as heating or freezing the vegetation (from paragraphs 2 and 13 above); tandem ablation therapy such as those using X-rays, gamma irradiation, or proton beams (from paragraphs 5 and 13 above); and tandem cytotoxic therapy (from paragraphs 9 and 13 above).

[0395] For cytokines, tandem ablation therapy, for example, using physical methods such as heating or freezing the vegetation, may include protocols (from paragraphs 3, 13, 15, and 17 above); tandem ablation therapy, for example, using X-rays, gamma irradiation, or proton beams (from paragraphs 6, 8, 13, 14, 15, and 17 above); and tandem cytotoxic therapy (from paragraphs 10 and 13 above).

[0396] For chemokines, tandem ablation therapy may include protocols such as those using physical methods such as heating or freezing the vegetation (from paragraph 13 above); tandem ablation therapy via X-rays, gamma irradiation, or proton beams (from paragraph 13 above); and tandem cytotoxic therapy (from paragraph 13 above).

[0397] For TLR agonists, tandem ablation therapy may include protocols such as those using physical methods such as heating or freezing the vegetation (from paragraphs 4 and 13 above); tandem ablation therapy via X-rays, gamma irradiation, or proton beams (from paragraphs 7 and 13 above); and tandem cytotoxic therapy (from paragraphs 11 and 13 above).

[0398] For antibodies, tandem ablation therapy may include protocols such as those using physical methods such as heating or freezing the vegetation (from paragraph 13 above); tandem ablation therapy such as those using X-rays, gamma irradiation, or proton beams (from paragraphs 8 and 13 above); tandem cytotoxic therapy (from paragraph 13 above); and tandem physical and immunological therapy including combinations of chromophores and immunostimulants (from paragraph 12 above).

[0399] Cancer treatment through a combination of local tumor destruction and immunostimulants

[0400] The desired outcome is to utilize GC with suitable viscosity as an injectable material for cancer treatment. This can be achieved in any suitable manner, for example, by combining local tumor destruction methods such as thermal or non-thermal tumor ablation with tumor immunotherapy. As used herein, the term cancer is a general term intended to encompass any malignant growth of many different types. Cancer is a cell originating in the body that has acquired at least eight specific markers through genetic and / or epigenetic mutations and / or other mechanisms: 1. resistance to cell death; 2. maintenance of proliferation signaling; 3. avoidance of growth inhibitors; 4. activation of invasion and metastasis; 5. achievement of replicable immortality; 6. induction of angiogenesis; 7. avoidance of immune destruction; and 8. deregulation of cellular energy. Growths may recur after attempts at removal or treatment and can lead to patient death unless adequately treated.

[0401] Certain instances of cancers that can be treated with GC of suitable viscosity as an injectable material include carcinomas, sarcomas, and melanomas, including but not limited to those in the liver, cervix, skin, breast, bladder, colon, rectum, prostate, larynx, endometrium, ovary, oral cavity, kidney, testes (non-seminomatous tumors), and lungs (non-small cell lung tumors).

[0402] Furthermore, treatment can be combined appropriately with other types of cancer treatments, such as radiation therapy. Radiation plays a crucial role in the rescue treatment of conditions such as Hodgkin's disease, nodular and diffuse non-Hodgkin's lymphoma, squamous cell carcinoma of the head and neck, mediastinal germ cell tumors, seminoma, prostate cancer, early-stage breast cancer, early-stage non-small cell lung cancer, and medulloblastoma. Radiation can also be used as palliative therapy for prostate and breast cancer in the presence of bone metastases, advanced lung and esophageal cancers in multiple myeloma, gastric cancer and sarcomas, and brain metastases. Treatable cancers include, to a lesser extent, Hodgkin's disease, early-stage non-Hodgkin's lymphoma, testicular (spermatogonia), prostate, laryngeal, and cervical cancers, as well as, to a lesser extent, cancers of the nasopharynx, sinuses, breast, esophagus, and lungs.

[0403] Treatment can also be administered in combination with other types of anti-vesicle drugs in appropriate ways. Anti-vesicle drugs include those that prevent the cell division (mitosis), development, maturation, or spread of vesicle cells. Ideally, an anti-vesicle drug would destroy cancer cells without side effects or toxicity to normal cells, but such drugs do not yet exist. Certain stages of choriocarcinoma, Hodgkin's disease, diffuse large cell lymphoma, Burkitt's lymphoma, and leukemia have been found to be susceptible to anti-vesicle drugs, such as testicular cancer (non-seminomatous carcinoma) and lung cancer (small cell carcinoma). Common classes of anti-vesicle drugs include, but are not limited to, alkylating agents, antimetabolites, plant alkaloids, antibiotics, nitrosoureas, inorganic ions, enzymes, and hormones.

[0404] Improved outcomes of tumor ablation and ablation irradiation methods

[0405] Therefore, the semi-synthetic biopolymer compositions described herein can be used in a variety of applications, including, for example, as immunostimulants or components that are immunostimulants, as detailed herein. Although other uses exist, the primary use of GC is as an immunostimulant in conjunction with common or noteworthy standard tumor ablation methods such as RFA, microwave, HIFU, laser, cryoablation, IRE, and PDT, or ablation irradiation methods such as SBRT and proton beam, and it is in this context that the compound compositions of Formula 1 are described in detail herein.

[0406] As further described herein, another aspect relates to the use of the Formula 1 compound formulation described herein as an immunostimulant in conjunction with common tumor ablation or irradiation therapies. In one instance, the use of this composition encompasses the introduction of an immunostimulant comprising the GC composition into or around the same vegetation before, during, or after tumor ablation or irradiation therapy. The ablation or irradiation therapy is performed in a manner sufficient to induce vegetation cell destruction, and when combined with injection of the GC of the invention or by other means of delivery of the GC of the invention, a systemic antitumor immune response is induced.

[0407] On one hand, the composition of Formula 1 is used in conjunction with the surgical removal of vegetations.

[0408] In some other respects, the outcomes of tumor ablation and irradiation therapy have been improved, with said improvement including the use of the injectable 1GC described herein. This finding also envisions methods for activating specific components of the immune system in conjunction with a systemic antitumor immune response, methods including treatment with GC.

[0409] As further described herein, it has been established that the application of GC combined with tumor ablation and irradiation therapy overcomes the limitations of current tumor ablation and irradiation therapies. Generally, the two basic principles of improvement are (1) reducing the recurrence rate at the site of local tumor ablation or irradiation, which inactivates the targeted tumor and releases tumor antigens, and (2) targeting the injection of an immunostimulant including GC, which interacts with the released tumor antigens and activates antigen-presenting cells such as dendritic cells to induce a systemic immune response against cancer, also known as the “distant” effect. Therefore, the GC described herein effectively interacts with both tumor antigens released from ablated or irradiated tumor cells or post-surgical tumor remnants, and certain components of the immune system such as dendritic cells, macrophages, neutrophils, and other tumor-infiltrating bone marrow and lymphocytes.

[0410] Another advantage of using the injectable compound formulation described herein in combination with tumor ablation or irradiation therapy is that the GC of the present invention directly activates dendritic cells (DCs), which is an important step in preventing tumor tolerance after exposure to tumor antigens.

[0411] Where M W Compounds of Formula 1 described herein with a content less than 420 kDa also stimulate the immune system and induce antigen-specific immunity by 1) activating dendritic cells, 2) increasing the exposure of ablation-released tumor antigens and dendritic cells, and 3) increasing the uptake of tumor antigens by dendritic cells to initiate a systemic T-cell response against cancer.

[0412] Therefore, according to one example, GC formulations activate one or more components of the immune system, thereby mediating the desired therapeutic effect.

[0413] The unexpected efficacy of injectable GCs lies in inducing a distant effect after tumor ablation and irradiation therapy and subsequent exposure of tumor antigens to antigen-presenting cells, which, among other factors, are based on the activation of antigen-presenting cells (e.g., dendritic cells and macrophages).

[0414] In one experiment, this distant effect was demonstrated in a B16-F10 mouse melanoma model, in which two tumors were implanted in the mice, but only one tumor was ablated and bound to M. W Treatment with 1GC of less than 420 kDa. For example... Figure 8As observed, due to the invasive nature of B16-F10 melanoma tumors, any remaining tumor deposits would gradually grow and lead to animal termination. Therefore, long-term survival was only possible when the tumor on the flank was eliminated due to an antitumor immune response, also known as the distant effect. As expected, all untreated animals reached their endpoint within 40 days (tumor growth to maximum tolerated size / death / termination due to severe health decline). While tumor ablation alone or GC alone did result in a minimum long-term survival of ~14% (GC alone ~9%), injection of M after ablation... W Formula 1GC with a capacity of less than 420 kDa significantly improved efficiency by more than 3 times, resulting in a long-term survival rate of 57%.

[0415] Another advantage of using the injectable GCs described herein in combination with tumor ablation or irradiation therapy or other methods of inducing immunogenic cell death is that, by using this method, an immune response is triggered independently in each individual and is not dependent on the expression of the same specific tumor-specific antigens across the receptor host (as required by conventional antibody immunotherapy and vaccination). Animal studies have revealed that, in addition to improving long-term survival and eliminating both primary tumors and distant metastases, CD4... + IFNγ + and CD8 + IFNγ + T cells infiltrated distant, untreated tumors (metastases) during intratumoral injection of GC-binding agents into the primary tumors of the studied animals undergoing tumor ablation. Furthermore, the study demonstrated that successfully treated animals acquired long-term resistance to tumor re-attack, which, combined with other data, further supports the induction of a Th1-type immune response.

[0416] Therefore, the use of the injectable GC described herein offers several advantages that meet the critical need for providing effective cancer treatment. This is particularly beneficial for cancer patients because the formulation described herein also provides a surprisingly beneficial formulation that is easy to administer by injection and is well-suited to clinical workflows, thus providing an effective adjuvant therapy option for conventional tumor ablation and irradiation therapies, which are ineffective against metastases and susceptible to local recurrence if the tumor margins are not adequately treated. The several advantages offered by the injectable compound 1 described herein meet the critical need for providing effective cancer treatment.

[0417] The GC described herein has been shown to induce dendritic cell maturation (assessed by CD40 expression), enhance T cell proliferation, and increase serum neutralization of IFNγ, TNFα, and IL-12 secretion in restimulated splenocytes of tumor-ablated animals. Furthermore, combined ablation effects (e.g., radiofrequency ablation and GC injection according to the invention) have been shown to induce tumor-specific immunity and CD40 expression. + IFNγ + and CD8 + IFNγ + T cell infiltration and reduction of regulatory T cells in distant, untreated metastases.

[0418] As described in further detail herein, the injection of GC in combination with several methods for inducing immunogenic tumor cell death, such as tumor ablation or irradiation therapy, thus offers numerous advantages over conventional tumor ablation and irradiation therapy, including but not limited to:

[0419] • Enhanced local outcomes of ablated or irradiated tumors

[0420] • Elimination of untreated metastatic tumors by inducing distant effects

[0421] • Induces long-term immunity and survival rate

[0422] • Reduce tumor recurrence

[0423] • Limited toxicity and well-tolerated at therapeutic doses

[0424] As further described herein, the formulation possesses several advantages over other conventional and unconventional treatment modalities. The combination of tumor destruction and injection of the Formula 1 compound is key. The most significant advantage is that the Formula 1 compound effectively transforms local tumor ablation or irradiation therapy into systemic immunotherapy against cancer, which now eliminates distant, unablated or unirradiated metastases. Thus, the Formula 1 compound can induce a prominent distant effect of additional local tumor ablation or irradiation therapy. When local tumor destruction occurs after tumor ablation or irradiation therapy, fragmented tissue and cellular molecules are released locally within the host. Under normal circumstances, these cellular molecules, such as tumor antigens, are rapidly cleared from the treated area through normal physiological mechanisms, meaning that when antigen-presenting cells (APCs) enter the area in the days following the ablation event, their exposure to tumor antigens is limited, and this contributes to inducing only a limited downstream T cell response. However, when the Formula 1 compound is injected into the tumor after tumor ablation, the Formula 1 compound, due to its unique electrostatic and physicochemical properties, interacts with and localizes these tumor antigens, thereby effectively increasing the exposure of tumor antigens to infiltrative APCs. Furthermore, in a key step, the Formula 1 compound activates dendritic cells, as measured by, for example, CD40 expression, which is a crucial step in inducing a systemic antitumor immune response against the ablated cancer.

[0425] In summary, long-term survival and overall cancer eradication can be achieved through the use of compounds of Formula 1. This is due to a combination of local tumor elimination, such as tumor burden reduction through tumor ablation, enhanced immune system response due to the interaction between tumor antigens and compounds of Formula 1, and activation of dendritic cells by compounds of Formula 1 as described in further detail herein.

[0426] The additional examples are provided by way of illustration and are not intended to limit the scope of the findings in any way. Therefore, these embodiments should not be construed as limiting the scope of the findings, but should be regarded as illustrative of certain aspects. Many other variations are possible.

[0427] Activation of dendritic cells

[0428] In one experiment, DCs were activated by the compound of Formula 1 in a dose-dependent manner as previously demonstrated, manifested by upregulation of CD40. On the other hand, conventional GCs with a molecular weight of approximately 500 kDa or greater did not affect DC activation, as measured by CD40 expression. This indicates a significant difference in in vitro function, a key step in initiating downstream T cell responses.

[0429] Not wanting to be bound by theory, I believe the main difference between conventional GC and the compound of formula 1 lies in M. W(The molecular weight of conventional GC is approximately 500 kDa or greater, while the molecular weight of the compound of Formula 1 is less than 420 kDa), and the sterilization method. Any of these factors can lead to differences in the activation capacity of DC.

[0430] Assuming that the compound of Formula 1 possesses a specific receptor, the autoclaving process makes it easy to alter the specific orientation of the molecule, and this no longer fits the receptor pocket on the DC. Another conjecture is that within the compound of Formula 1, the optimal M for activating the DC... W M below the standard GC W The value of .

[0431] In summary, it has been demonstrated that the compound of formula 1 activates DCs, as indicated by increased CD40 expression. The inventors believe this is an important part of the mechanism of action of GC's antitumor properties. Conventional GC does not exhibit the same ability in experimental systems.

[0432] Example

[0433] Example 1: An exemplary process for preparing GC

[0434] GC is obtained by reacting chitosan with monosaccharides and / or oligosaccharides, in one instance, in the presence of an acidifying agent, with a reaction time sufficient to achieve Schiff base formation (also referred to herein as amino glycosylation) between the carbonyl group of the sugar and the primary amino group of the chitosan. This is followed by stabilization by reducing the Schiff base and its rearranged derivative (Amadori product).

[0435] Example 2A): Aseptic filtration

[0436] While conventional 1,500 kDa galactosylchitosan described in U.S. Patent 5,747,475 is reportedly easy to synthesize, it is impossible to sterilize it without compromising the integrity of the filter using, for example, a 0.22-micron filter, thus rendering conventional GC unsuitable for GMP production and human use. Furthermore, conventional GC with a molecular weight greater than 420 kDa fails in attempts at aseptic filtration. In contrast, formulations of the compounds of Formula 1 described herein offer significant advantages in GMP production and aseptic filtration due to their unexpected and beneficial chemical structure and composition. For example, in M... W Aseptic filtration using 0.20–0.22 micron filters at a flow rate of 250 kDa is highly feasible without material loss during filtration at a stable flow rate.

[0437] Example 2B): Demonstration of the sterile filtration capability of compound 1 in Example 2A

[0438] Compound 1 is an illustrative example of GC, a semi-synthetic glucosamine-based polymer. Compound 1 is a novel and non-dominant GC. Specifically, the following data support that Compound 1 can be manufactured in a consistent and readily compliant manner and possesses advantageous and unexpected properties. Compound 1 is prepared as a 1.0% (w / w) solution in water buffered to pH 5-6 with a viscosity of 50-60 cPs and is intended for intratumoral injection. Compound 1 is a variant of GC and has the following molecular properties:

[0439] • Weight-average molecular weight (M W ) is ~250kDa

[0440] • Degree of deacetylation (DDA) is ~80%

[0441] • Degree of saccharification (DG) is ~5%

[0442] One of the main advantages exhibited by the compound of formula 1 is its ability to perform sterile filtration, specifically M W Those less than 420 kDa. Aseptic filtration of pharmaceutical solutions is an industry standard for ensuring patient safety. Specifically, in the field of aseptically injectable solutions, aseptic filtration is often the preferred sterilization method because it is a readily scalable process and does not affect the chemical structure of active pharmaceutical ingredients (APIs) present during sterilization, such as autoclaving or gamma irradiation. Additionally, aseptic filtration offers cost advantages over autoclaving and gamma sterilization in terms of process development, validation, and implementation. Aseptic filtration of polymer solutions adds further complexity because certain chemical structures and components can typically slow down or halt the filtration process. Therefore, filtration conditions, as well as the chemical and physicochemical properties of the polymer, must be carefully considered.

[0443] Regarding compounds of Formula 1, it was unexpectedly found that specific examples of formulations requiring defined ranges including concentration and pH were needed to successfully aseptically filter the formulations and deliver compliant and consistent pharmaceutical products. As shown below, experimental results demonstrate comparable filterability compared to aspects outside the exemplary ranges described above for GC, specifically those with molecular weights less than 420 kDa.

[0444] Under current regulatory and scientific standards, a pharmaceutical solution can be considered sterile after filtration through a filter with an effective pore size of 0.22 microns or smaller. Furthermore, processes and materials must be tested and validated in a GMP-compliant manner. The sterile filtration of the pharmaceutical product of Formula 1 has been carefully studied. The total sterilization process for Formula 1 utilizes a Flurodyne 0.20µm capsule filter (part number KA2DFLP1S) from Pall Corporations in a redundant (serial) manner. The selected filter meets all regulatory requirements and is chemically compatible with Formula 1. Additionally, product-specific validation of the process was performed (Study No. -VAL-AM-000754-B). As part of this study, Formula 1 solutions repeatedly demonstrated their effective ability to undergo sterile filtration.

[0445] refer to Figure 2 (Recirculation data for the pharmaceutical product of Formula 1) The data clearly show that when the Formula 1 solution is recirculated through a sterile-grade membrane at constant pressure for up to 3 hours, the flow rate loss is minimal (this indicates minimal fouling or clogging of the filter). This test represents the extreme stress on the system, since sterile filtration is actually a single pass through one or two filters and not a continuous recirculation of the solution through the membrane. This data strongly supports the fact that GC solutions with a molecular weight less than 420 kDa can be sterilely filtered with little or no loss of polymer solution integrity.

[0446] The process validated by Pall in study VAL-AM-000754-B was subsequently executed multiple times at scale. In one instance, GMP-grade Formula 1 solutions were produced, and the following data were collected:

[0447] • The pre-filtration weight of the pharmaceutical product of Formula 1 compound – 7.602 kg

[0448] • Redundant sterile filtration time – 3 hours

[0449] • Filtered weight of the drug product of Formula 1: 7.384 kg

[0450] • Filtration yield – 97.1%

[0451] To demonstrate the superiority of Compound 1 over other less desirable GC aspects in aseptic filtration, aseptic filtration of one of the aspects of Compound 1 was performed in conjunction with a higher M... W Direct comparison of those with values ​​(>420kDa).

[0452] M was synthesized W This is a 1% solution of a conventional GC with approximately 500 kDa. Conventional GC is sterilized by autoclaving, and it is believed that this process actually affects the polymer's molecular weight (M).W To test this, a standard GC solution was synthesized and autoclaved. The resulting GC had an M... W The value was greater than 420 kDa, and its ability to perform aseptic filtration was tested both before and after autoclaving, and compared with the GC instance reported in this paper.

[0453] Now for reference Figure 3 The figure shows filtration rate data for various 1% GC solutions. (This is in order to generate...) Figure 3 Based on the data, 1 mL of each solution was added to a 2.5 mL syringe equipped with a Luer-fitted digital pressure sensor. A small-scale, representative sterile filter with a Luer fitting was then attached to the outlet of the pressure sensor. The solution was forced through the filter, thus maintaining the pressure between 500 and 600 psi. The resulting flow rate was measured.

[0454] Figure 3 The data clearly shows that M W Compounds of Formula 1 with values ​​below 420 kDa maintained a consistent flow rate before all liquid had been pushed through the filter. In contrast, M... W After autoclaving, solutions containing GC with a value greater than 420 kDa exhibited a steadily decreasing droplet rate, eventually clogging the filter and thus stopping filtration. Furthermore, the data supports that autoclaving GC solutions reduced M... W As demonstrated by the lower pressure and improved flow rate of autoclaved materials compared to unautoclaved materials.

[0455] Now for reference Figure 4 Particle size data were collected from the three samples tested. A convenient estimate of the particle size of the chitosan solution is the radius of gyration (Rg). Although Rg is not the exact radius of the particles, it is usually only slightly smaller than the actual radius of the particles. The radius of gyration of the compound solution of Formula 1 was measured to be ~32 nm, while M... W Two GC solutions with values ​​greater than 420 kDa exhibited Rg values ​​of ~52 nm or larger. When considering the larger end of the polymer range, it was found that conventional GC did not achieve aseptic filtration because the particles clearly approached or became larger than the effective pore size of a sterile filter.

[0456] The data presented herein clearly demonstrate the superiority of the novel, non-dominant Formula 1 compound over conventional GCs with a molecular weight greater than 420 kDa in terms of aseptic filtration. Furthermore, and unexpectedly, Formula 1 represents the optimal form of GC for aseptic filtration. It is known that decreasing the pH of a chitosan solution increases Rg, while increasing the pH of a GC solution causes the material to precipitate from the solution (i.e., precipitation). Therefore, it was unexpectedly found that a key parameter for aseptic filtration of Formula 1 is pH optimization, in contrast to conventional GCs with a molecular weight greater than 420 kDa, which cannot perform aseptic filtration across any pH range. The data presented herein and further development work performed on Formula 1 clearly support the described examples and the significant and unexpected advantages over conventional GCs.

[0457] Example 3: Manufacturing Improvements

[0458] In this exemplary study, it was determined that experimental conditions can be adjusted as needed to improve the overall yield during GC production. Unexpectedly, it was found that GC production can be improved by controlling pH conditions, providing better control over the saccharification percentage of the produced GC. Specifically, it was determined that controlling pH is crucial for regulating the half-life of active sodium borohydride (NaBH4) in solution. The half-life of sodium borohydride is pH-dependent, with lower pH values ​​significantly reducing the presence of active NaBH4 through acid-catalyzed decomposition of the reagent, resulting in lower DG values. Therefore, it was determined that NaBH4 is less effective in stabilizing GC by reducing Schiff bases and Amadori products at lower pH. For example, when the pH is kept below five (pH < 5), the half-life of sodium borohydride is extremely short, and therefore the reduction of Schiff bases and Amadori products is less efficient, resulting in a lower saccharification percentage.

[0459] Furthermore, it was determined that at higher pH values ​​in the reaction mixture, the formulation exhibited "gelling" due to the formation of a non-Newtonian solution. For example, gelling was observed when the pH was maintained above six (pH>6). This gelling of the reaction rendered stirring and sodium borohydride dosage ineffective, thereby halting sodium borohydride reduction. In other words, to achieve the goal of efficiently producing 1GC solutions, the pH was optimized to provide a sufficient half-life of sodium borohydride while maintaining the normal fluid properties of the solution.

[0460] Example 4: Using the Formula 1 composition to enhance local antigen retention

[0461] Ablated and released tumor antigens are more readily available for uptake by APCs compared to those within intact tumor cells. This is the first step in APCs initiating downstream adaptive immunity against tumor cells expressing these antigens. However, as discussed, a significant portion of these freshly released antigens is lost, and injection of the Formula 1 compound preserves this crucial information regarding the stimulation of the patient's immune system. To demonstrate this property, fluorescently labeled antigen OVA protein was subcutaneously injected after mixing with the Formula 1 compound or PBS, and retention of OVA was monitored by whole-body imaging over one week. Figure 12 The results showed that, in the presence of compound 1, local OVA concentrations were three to four times higher than in control animals on the first day after injection. Local antigen concentrations decreased over time but were maintained at two to three times higher levels for up to seven days with the help of compound 1. Thus, compound 1 prolonged the availability of tumor antigens to incoming APCs. This maximizes the chances of the immune system acquiring these antigens because, despite the presence of resident DCs (e.g., Langerhans cells / dermal DCs) at the treatment site, the influx of APCs and monocytes differentiating into tissue DCs / MACs can continue for several days. In other words, compound 1 increased the abundance of tumor antigen waves arriving later in the APC wave.

[0462] Example 5: Using the composition of Formula 1 to enhance the efficacy of tumor ablation

[0463] To further verify the distal effect of the ablation compound of formula 1, a bilateral flank tumor injection experiment was performed in another aggressive metastatic tumor model—B16-F10 melanoma in mice. In this experiment, 2*10 5 One B16-F10 tumor cell was intradermally (id) implanted into the right flank of a C57BL / 6 wild-type mouse. When the first tumor reached an average diameter of approximately ~3 mm, a second tumor (5*10) was implanted. 4 (One cell) was implanted in the left flank. Treatment was performed when the first tumor reached 5.5 mm, while the second tumor remained untreated.

[0464] Due to its aggressive nature, animals could only survive long-term if the tumors in both flanks were removed. As expected, all untreated animals reached the endpoint within 40 days. Figure 8 While ablation alone does result in a minimum long-term survival rate of ~14% (compared to approximately ~9% for compound 1 alone), the addition of compound 1 (ablation + compound 1) significantly improves efficacy by more than 3 times, resulting in a long-term survival rate of 57%. Figure 8 ).

[0465] Closer observation of tumor growth revealed that the ablation of compound 1 enhanced local tumor growth at the treatment site. Figure 9) and untreated distant parts of the whole body ( Figure 10 The elimination of both tumors was observed. In long-term survivors of the Formula 1 compound + ablation group (G4), the contralateral tumor showed growth after regression or no growth at all, while in the untreated controls, the tumor gradually grew. In the group receiving ablation alone (G3), although some contralateral tumors did regress, only 2 / 14 of the animals achieved eventual elimination of both the primary and secondary tumors to achieve long-term survival. Because these secondary tumors were never directly treated, their delayed growth / regression was not caused by the direct killing effect of the ablation, but by a downstream effect of the treatment. It is hoped that this distant effect can be replicated clinically for systemic suppression of metastatic lesions.

[0466] In an invasive in situ pancreatic cancer model, PancO2-H7 was injected into the pancreas and treated with interstitial thermal laser ablation plus a compound of formula 1. The advantage of the in situ model is that it more closely simulates the actual physiological niche from which the tumor under study originates, thus reflecting a more representative response to treatment. Primary tumor burden ( Figure 11 (Left) The number of metastatic lesions was reduced by ablation alone, and further reduced by compound 1. Ablation alone had no significant effect on the extent of metastatic lesions, but ablation plus compound 1 reduced the number of metastatic lesions by almost 3 times. Figure 11 (Right). Interestingly, although it had no effect on the primary tumor, compound 1 alone reduced metastasis by almost 50%. It is possible that compound 1 has some unexplored inhibitory effect on tumor cells that have acquired metastatic potential.

[0467] Example 6: Using the composition of Formula 1 to stimulate T cell responses

[0468] The compounds of Formula 1 described above have been shown to enhance tumor ablation both locally and systemically when administered intratumorally in conjunction with an ablation procedure. This enhancement of efficacy requires a complete adaptive immune compartment, as the benefits are offset in nude mice with impaired T and B cell populations in the thymus. More specifically, CD8 was found in the contralateral tumor of the treated animal in the pancreatic cancer model Pan02-H7. + IFNγ + and CD4 + IFNγ + Increased T cell infiltration, along with elevated serum IFNγ and TNFα levels. These data suggest that the Formula 1 compound works at least in part by enhancing the initiation of anti-tumor T cell responses (specifically CTLs and T-helper 1-tilts). Furthermore, this effect is durable, and cured animals were better protected from re-attacks by the same tumor compared to ablation alone.

[0469] To initiate an effective anti-tumor T-cell response, one of the key steps involves antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs), acquiring sufficient quantities of tumor antigens, being appropriately activated, and presenting these antigens to T and B cells after migrating to draining lymph nodes. With the local injection of a compound of Formula 1 into the ablated tumor, it will encounter the entering APCs during the release of tumor antigens via ablation. Whether the compound of Formula 1 has any direct effect on the function of these APCs has been investigated. In vitro work on the macrophage line RAW264.7 showed that the compound of Formula 1 enhanced macrophage function, including phagocytosis, NO production, TNFα production, and the expression of maturation markers CD80 and 86. In the DC cell line DC2.4, experiments surprisingly showed that the compound of Formula 1 activated dendritic cells, interacting with M… W Conversely, GC greater than 420 kDa can be measured by enhancing the co-stimulatory marker CD40. CD40 signaling can also lead to the upregulation of other co-stimulatory markers, such as those in the B7 family. Taken together, it is possible that a key part of the mechanism of action employed by the compound of formula 1 enhances the activation and function of APCs, which plays a crucial role in initiating downstream anti-tumor T cell responses.

[0470] Example 7: Application of Formula 1 compound in activating dendritic cells

[0471] Completely unexpectedly, compound 1 was found to possess properties significantly different from and superior to those of known GCs. As noted above, these superior properties include sterile filterability and differences in molecular weight. In addition to the improved chemical structure and composition, it was also quite unexpected to find similarities to M... W Compared to conventional GC with a concentration greater than 420 kDa, the compound of Formula 1 was able to activate dendritic cells (DCs). This was determined by measuring CD40 expression after co-culturing DCs with the compound of Formula 1 on conventional GC. Without being bound by theory, it is believed that CD40 expression is a key aspect of the mechanism of action of the compound of Formula 1.

[0472] Experiment Overview:

[0473] 1. In a 96-well U-bottom polystyrene plate, add 1*10 mg of D-10 medium to 0.2 ml of D-10 medium. 5 Culture DC cell line DC 2.4 into individual cells. Divide at least once before use.

[0474] 2. Add GC to the wells and culture the cells overnight for 18-24 hours.

[0475] 3. Harvest cells and stain with anti-CD40 antibody to measure the level of DC activation by flow cytometry.

[0476] Read aloud:

[0477] CD40 expression serves as an indicator of DC activation.

[0478] result:

[0479] i) The expression of CD40 on DC was upregulated by the compound of formula 1.

[0480] Three independent experiments were performed. In all cases, CD40 was upregulated in a dose-dependent manner by the compound of Formula 1 (p<0.05), as previously demonstrated. This demonstrates that the compound of Formula 1 can activate DCs and stimulate their maturation. As expected, the positive control TLR4 ligand LPS induced a ~14-fold increase in CD40 and is not included in the figure for clarity. The isotype control was negative, which ruled out nonspecific binding.

[0481] ii) CD40 expression on DCs was not affected by in vitro stimulation of conventional GCs.

[0482] On the other hand, M W Conventional GC values ​​greater than 420 kDa did not affect CD40 expression within the tested dose range, even at concentrations up to 1000 μg / ml. This indicates that conventional GC cannot activate DCs as the compound of Formula 1 did under these experimental conditions. Figure 5 and Figure 6 The data are plotted together in Figure 7 Above, so as to facilitate visual comparison.

[0483] Furthermore, it is believed that combining GC with methods that induce immunogenic cell death, such as tumor ablation or irradiation therapy, may significantly improve the observable outcomes of checkpoint inhibitors and / or other immunotherapies for T-cell-mediated cancers, and thus provide opportunities to design additional immunotherapies to treat proliferative disorders in human subjects.

[0484] Example 8: Combination with checkpoint inhibitors

[0485] The compounds of Formula 1 described above, when used in combination with checkpoint blocking antibodies against PD-1, have been shown to enhance the generation of memory responses against the same tumor when injected intratumorally during ablation procedures. Figure 13 Best demonstrated in one experiment, two B16-F10 tumors were implanted in C57BL / 6 mice, each located on each flank of the back (the second tumor was implanted when the first tumor was 3 mm). At 5.5 mm, only the first tumor was treated with ablation plus compound 1, group 4 (G4), while the second tumor remained untreated. Anti-PD-1 was administered on days 7, 10, 13, and 16 post-tumor implantation in combination with ablation plus compound 1 (G6). The control group consisted of untreated (G1), compound 1 (G2), ablation (G3), and anti-PD-1 (G5).

[0486] In long-term survivors, both the primary tumor and the secondary tumor must be eliminated. In this respect, ablation plus both of the two formula 1 compounds (G4) and ablation plus the formula 1 compound plus anti-PD-1 (G6) were superior to the other groups. Although long-term survival was the same between the two groups in the experiment, any delay in the growth of the secondary tumor in non-survivors would indicate a stronger systemic antitumor response.

[0487] according to Figure 14 Indeed, this is the case. In combination group G6, 3 / 6 of the second tumors showed delayed growth compared to ablation plus compound 1 alone, and 2 / 6 even regressed (which was not observed in ablation plus compound 1 alone). Because these second tumors had never been directly treated, their delayed growth / regression was a result of the systemic immune response. In other words, ablation plus compound 1 synergizes with the checkpoint inhibitor anti-PD-1 and exhibits a stronger initiating effect on systemic anti-tumor immunity. This combination advantage will be further optimized through protocol modifications.

[0488] Now for reference Figure 13 Then on day 97, the same tumor B16-F10 (7.5*10) was used. 4 The survivors in G4 were re-challenged with a double dose of tumor cells (1.5 x 10^6 cells). Compared to 87.5% (7 / 8) of combination group 6 (G6; ablation + compound 1 + anti-PD1), 75% (6 / 8) of the animals in G4 (ablation + compound 1) withstood the re-challenging and remained tumor-free for 30 days. On day 185, a second dose of doubled tumor cells (1.5 x 10^6 cells) was administered. 5 (1 cell) were re-attacked from the survivors of the first re-attack. Compared with 71.4 (5 / 7) of combination group 6 (G6), 50% (3 / 6) of the animals in G4 survived the re-attack and remained tumor-free for 30 days.

[0489] In summary, both ablation plus a compound of formula 1 and the combination of ablation plus a compound of formula 1 and anti-PD-1 produce durable memory against the tumor. Adding anti-PD-1 to the original treatment thereby enhances the generation of this memory response. Similar data have been found in separate experiments where the anti-CTLA-4 + anti-PD-1 checkpoint blockade combination was used instead of anti-PD-1 alone, such as... Figure 13 As shown.

[0490] Other embodiments

[0491] Based on the foregoing description, it will be apparent to those skilled in the art that changes and modifications can be made to the embodiments described herein to adapt them to various uses and conditions.

Claims

1. A composition for use in treating tumors, said composition comprising a sterile-filtered glycosylated chitosan polymer (GC polymer) and at least one checkpoint inhibitor comprising an anti-PD-1 antibody or an anti-PD-L1 antibody, wherein said glycosylated chitosan polymer is represented by Formula 1: Where n is the number of subunits, and (a), (b), and (c) indicate that GC is included. mon The number of each of the following monomer subunits: ； Monomer (a) ； as well as Monomer (b) Monomer (c); Where R = substitutions resulting from glycosylation; where for molecular weights less than 420 kDa, n = 3 – 1933, (a) = 1 – 986, (b) = 1 – 386, (c) = 1 – 560); and the degree of glycosylation is at most but does not include 30%.

2. The composition used according to claim 1, characterized in that, The GC polymer that has been sterile filtered has a molecular weight of 250 kDa or a degree of saccharification of 5%.

3. The composition used according to claim 1, characterized in that, The GC polymer that underwent sterile filtration had a molecular weight of 250 kDa, a degree of saccharification of 5%, and a degree of deacetylation of approximately 80%.

4. The composition used according to any one of claims 1 to 3, characterized in that, The sterile filtered GC polymer is formulated in the form of a physiologically compatible carrier.

5. The composition used according to any one of claims 1 to 3, characterized in that, The sterile filtered GC polymer is formulated into a pharmaceutical composition for injection administration in a sterile filtered aqueous mixture at a pH of 5 to 7, the sterile filtered aqueous mixture containing 1% by weight of the GC polymer, and the GC polymer dissolved in the sterile filtered aqueous mixture having a viscosity of 1 to 100 centitrate as measured at 25 degrees Celsius.

6. The composition used according to any one of claims 1 to 3, characterized in that, The at least one checkpoint inhibitor is an anti-PD-1 antibody.

7. The composition used according to any one of claims 1-3, wherein the glycosylated chitosan polymer is used for intratumoral administration.

8. The composition according to claim 7, wherein the composition is to be administered in conjunction with tumor ablation or radiotherapy.

9. The composition according to claim 8, wherein the radiotherapy comprises X-ray or gamma-ray photon beam therapy or proton beam therapy.

10. The composition used according to any one of claims 1-3, wherein the GC polymer is combined with, mixed with, or rapidly and continuously used in the cytotoxic agent.

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