Pegylated interleukin-10 for cancer treatment

Abstract

This invention relates to pegylated interleukin-10 for the treatment of cancer. It also relates to a method for modulating the immune response of subjects suffering from oncology and immune-related diseases, conditions, and symptoms by administering IL-10 agents, including pegylated IL-10.

Description

[0001] This application is a divisional application of Chinese patent application 201680040506.2, "Polyethylene glycolated interleukin-10 for the treatment of cancer," filed on May 26, 2016.

[0002] Cross-references to related applications This application claims priority to U.S. Provisional Application Serial No. 62 / 167,699, filed May 28, 2015, which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to a method for modulating immune responses using IL-10 agents in the treatment or prevention of oncology and immune-related diseases, conditions and symptoms. Background Technology

[0004] Interleukin-10 (IL-10) is a pleiotropic cytokine that regulates various immune responses through its effects on T cells, B cells, macrophages, and antigen-presenting cells (APCs). IL-10 can suppress immune responses by inhibiting the expression of IL-1α, IL-1β, IL-6, IL-8, TNF-α, GM-CSF, and G-CSF in activated monocytes and activated macrophages, and it also inhibits the production of IFN-γ by NK cells. Although IL-10 is primarily expressed in macrophages, its expression has also been detected in activated T cells, B cells, mast cells, and monocytes. In addition to suppressing immune responses, IL-10 also exhibits immunostimulatory effects, including stimulating the proliferation of thymocytes treated with IL-2 and IL-4, enhancing B cell viability, and stimulating the expression of MHC class II.

[0005] Human IL-10 is a homodimer that becomes biologically inactive after the non-covalent interaction between the two monomeric subunits is disrupted. Data obtained from the published crystal structure of IL-10 indicate that the functional dimer exhibits some similarities to IFN-γ (Zdanov et al., (1995) Structure (Lond) 3:591-601).

[0006] Due to its pleiotropic activity, IL-10 has been associated with a wide range of diseases, conditions, and symptoms, including inflammatory conditions, immune-related conditions, fibrotic conditions, metabolic conditions, and cancer. Clinical and preclinical evaluations of IL-10 in many of these diseases, conditions, and symptoms have solidified its therapeutic potential. Furthermore, PEGylated IL-10 has been shown to be more effective than non-PEGylated IL-10 in certain therapeutic contexts. Summary of the Invention

[0007] This disclosure contemplates the use of IL-10 agents (e.g., pegylated IL-10) as a component of chimeric antigen receptor-T cell therapy (CAR-T cell therapy). CAR stands for an emerging therapy for cancers (e.g., treating B and T-cell lymphomas) and other malignancies. CAR-T T cells typically comprise patient-derived memory CD8+ T cells modified to express recombinant T-cell receptors specific to, for example, known antigens present on the target tumor. While this disclosure is described generally in the context of using CAR-T cell therapy to treat cancer, it should be understood that such therapies are not limited thereto.

[0008] CAR-T T-cell therapy involves the use of adoptive cell transfer (ACT), a process that utilizes T cells cultured by the patient themselves. In CAR-T cell therapy, T cells are removed from the patient and genetically modified to express a CAR that targets a specific antigen against a known cancer (e.g., a tumor). After sufficient numbers have been expanded in vitro, the autologous cells are infused back into the patient, causing the antigen to be specifically destroyed. In this way, CAR-T T-cell therapy is similar to plasmapheresis, where blood taken from the patient is processed to separate a specific component (e.g., removing malignant leukocytes during leukapheresis), and the remainder is returned to the patient's circulation.

[0009] As discussed further below, CAR-T cell therapy has been limited in several ways: the induction of antigen-specific toxicity in normal tissues expressing the target antigen, and the extremely high efficacy of CAR-T cell therapy leading to life-threatening cytokine release syndrome. Specifically, the interaction of high-affinity T cell receptors with significant antigen loads has been observed to result in activation-induced cell death. Historically, IL-10 has been discussed in the context of enhancing activation-induced cell death (Georgescu et al. (1997) J Clin Invest 100(10):2622-33). However, the data presented in this paper suggest that IL-10 agents can be used in conjunction with CAR-T T cell therapy to prevent or limit activation-induced cell death while enhancing CD8+ T cell function and survival.

[0010] As further discussed below, human IL-10 is a homodimer, and each monomer contains 178 amino acids, wherein the first 18 amino acids comprise a signal peptide. Specific embodiments of this disclosure comprise mature human IL-10 polypeptides lacking the signal peptide (see, for example, U.S. Patent No. 6,217,857) or mature human PEG-IL-10. In other specific embodiments, the IL-10 agent is a variant of mature human IL-10. This variant may exhibit activity that is lower, equivalent to, or higher than mature human IL-10; in some embodiments, the activity is equivalent to or higher than that of mature human IL-10.

[0011] Certain embodiments of this disclosure contemplate modifying IL-10 to enhance one or more properties (e.g., pharmacokinetic parameters, efficacy, etc.). Such IL-10 modifications include PEGylation, glycosylation, albumin (e.g., human serum albumin (HSA)) conjugation and fusion, and hydroxyethyl starchization. In a particular embodiment, IL-10 is PEGylated. In another embodiment, the modification of IL-10 does not produce therapeutically harmful effects on immunogenicity, and in yet another embodiment, the modified IL-10 is less immunogenic than unmodified IL-10. The terms “IL-10,” “IL-10 polypeptide,” “pharmaceutical,” etc., are intended to be broadly interpreted and include, for example, human and non-human IL-10-related polypeptides, including their homologues, variants (including mutant proteins), and fragments, as well as IL-10 polypeptides having, for example, a leader sequence (e.g., a signal peptide), and the aforementioned modified forms. In another specific embodiment, the terms “IL-10,” “IL-10 polypeptide,” and “pharmaceutical” refer to an agonist. Specific embodiments involve polyethylene glycolated IL-10, which is also referred to herein as "PEG-IL-10". This disclosure also considers nucleic acid molecules encoding the aforementioned substances, carriers containing such nucleic acid molecules, and cells expressing IL-10 agents (e.g., transformed cells and host cells).

[0012] This disclosure contemplates methods for modulating T-cell-mediated immune responses to target cell populations in a subject using CAR-T cell therapy and IL-10 agents. One particular embodiment contemplates a method for modulating T-cell-mediated immune responses to target cell populations in a subject, comprising a) introducing into the subject a plurality of therapeutically effective, genetically modified cells expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises at least one antigen-specific targeting region capable of binding to the target cell population, and wherein the binding of the chimeric antigen receptor targeting region to the target cell population induces activation-induced cell death; and b) administering to the subject a therapeutically effective amount of IL-10 agent sufficient to prevent or limit the activation-induced cell death. In a particular embodiment, the CAR comprises an antigen-binding domain that specifically recognizes the target cell population.

[0013] In some embodiments of this disclosure, the IL-10 agent enhances the function of activated memory CD8+ T cells. In other embodiments, the amount of IL-10 agent applied is sufficient to enhance cytotoxic function.

[0014] Embodiments in which an IL-10 agent is administered before, simultaneously with, or after the administration of multiple therapeutically effective cells are considered. In some embodiments of this disclosure, the IL-10 agent is administered subcutaneously.

[0015] In some embodiments, this disclosure contemplates administering an IL-10 agent in an amount sufficient to achieve a serum concentration of 10 to 100 ng / mL. In some embodiments, the IL-10 agent is administered to the subject in an amount sufficient to maintain a mean serum trough concentration of 1 pg / mL to 10.0 ng / mL. In some embodiments, the mean serum trough concentration of 1.0 pg / mL to 10.0 ng / mL is maintained for at least 95% of a defined time period. In other embodiments of this disclosure, the mean serum trough concentration of IL-10 is in the range of 1.0 pg / mL to 100 pg / mL; from 0.1 ng / mL to 1.0 ng / mL; from 1.0 ng / mL to 10 ng / mL; from 0.5 ng / mL to 5.0 ng / mL; from 0.75 ng / mL to 1.25 ng / mL; or from 0.9 ng / mL to 1.1 ng / mL. In specific embodiments of this disclosure, the average serum trough concentration of IL-10 is at least 1.25 ng / mL, at least 1.5 ng / mL, at least 1.6 ng / mL, at least 1.7 ng / mL, at least 1.8 ng / mL, at least 1.85 ng / mL, at least 1.9 ng / mL, at least 1.95 ng / mL, at least 1.97 ng / mL, at least 1.98 ng / mL, at least 1.99 ng / mL, at least 2.0 ng / mL, or greater than 2 ng / mL.

[0016] In another implementation, the aforementioned time period is at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 6 weeks, at least 2 months, at least 3 months, at least 6 months, at least 9 months, or more than 12 months.

[0017] In a specific embodiment of this disclosure, the average serum trough concentration of IL-10 is maintained for a period of at least 85%, at least 90%, at least 96%, at least 98%, at least 99%, or 100%.

[0018] As envisioned, a dosing regimen sufficient to maintain the desired steady-state serum trough concentration (e.g., 1 ng / mL) would result in an initial serum trough concentration higher than the desired steady-state serum trough concentration. Due to the pharmacodynamic and pharmacokinetic characteristics of IL-10 in mammalian subjects, even when dosing parameters (e.g., dose and frequency) remain constant, the initial trough concentration (e.g., achieved by administering one or more loading doses followed by a series of maintenance doses) gradually but steadily decreases over a period of time. After this period, the gradual but steady decrease ends and a steady-state serum trough concentration is maintained.

[0019] For example, mice (e.g., C57BL / 6 mice) require parenteral administration (e.g., SC and IV) of approximately 0.1 mg / kg / day of an IL-10 agent (e.g., mIL-10) to maintain a steady-state serum trough concentration of 2.0 ng / mL. However, the steady-state serum trough concentration is not reached until approximately 30 days after starting administration at 0.1 mg / kg / day (and after any loading dose). Instead, after reaching the initial serum trough concentration (e.g., 2.5 ng / mL), this concentration gradually but steadily decreases over, for example, approximately 30 days, after which the desired steady-state serum trough concentration (2.0 ng / mL) is maintained. Those skilled in the art will be able to determine the dose required to maintain the desired steady-state trough concentration using, for example, ADME and patient-specific parameters.

[0020] This disclosure contemplates methods for comprising at least one modification to form a modified IL-10 agent, wherein the modification does not alter the amino acid sequence of the IL-10 agent. In some embodiments, the modified IL-10 agent is a PEG-IL-10 agent. In other embodiments, the PEG-IL-10 agent may comprise at least one PEG molecule covalently linked to at least one amino acid residue of at least one subunit of IL-10 or a mixture comprising mono- and di-glycolic IL-10. The molecular weight of the PEG component of the PEG-IL-10 agent may be greater than about 5 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In some embodiments, the molecular weight is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, or about 10 kDa to about 30 kDa.

[0021] In some embodiments, the modified IL-10 agent comprises at least one Fc fusion molecule, at least one serum albumin (e.g., HSA or BSA), an HSA fusion molecule, or an albumin conjugate. In other embodiments, the modified IL-10 agent is glycosylated, hydroxyethylated, or comprises at least one albumin-binding domain. Some modified IL-10 agents may contain more than one type of modification. In certain embodiments, the modification is site-specific. Some embodiments include a linker. Modified IL-10 agents will be discussed in detail below.

[0022] This disclosure also considers using CAR-T cell therapy to treat or prevent diseases, conditions, or symptoms (e.g., cancer-related conditions) in subjects, together with the introduction of genetically modified cells expressing IL-10. Due to its direct and local effects, the amount of IL-10 secreted from such cells necessary for the highly effective induction of antigen-specific toxicity in normal tissues expressing target antigens and for the extremely high efficacy of CAR-T cell therapy leading to life-threatening cytokine release syndrome is far lower than the amount of IL-10 administered to subjects in a conventional manner (e.g., subcutaneously). In fact, the amount of secreted IL-10 necessary to achieve the aforementioned effects may be undetectable in serum.

[0023] In some such embodiments, this disclosure contemplates a method for modulating a T-cell-mediated immune response in a subject to a target cell population, comprising introducing to the subject a plurality of therapeutically effective, genetically modified cells expressing: a) a chimeric antigen receptor, wherein the chimeric antigen receptor comprises at least one antigen-specific targeting region capable of binding to the target cell population, and wherein the binding of the chimeric antigen receptor targeting region to the target cell population is capable of inducing activation-induced cell death; and b) an amount of IL-10 agent sufficient to prevent or limit the activation-induced cell death.

[0024] In some embodiments, the chimeric antigen receptor and the IL-10 agent are expressed by the same vector, while in other embodiments, the chimeric antigen receptor and the IL-10 agent are expressed by different vectors. In a particular embodiment, multiple therapeutically effective cells are transfected with a vector expressing an amount of IL-10 agent sufficient to enhance cytotoxic function. The vector may be, for example, a plasmid or a viral vector. This disclosure also contemplates any other means of expressing the IL-10 agent. In a particular embodiment, the expression of the IL-10 agent is regulated by an expression control element.

[0025] In the above embodiments, the plurality of cells are obtained from the subject and genetically modified in vitro. In some embodiments, the plurality of cells are obtained from the subject via plasma exchange. In other embodiments of this disclosure, the plurality of cells are memory CD8+ T cells, while in other embodiments, they are autologous tumor cells.

[0026] This disclosure contemplates a method for modulating a T-cell-mediated immune response in a subject to a target cell population, comprising introducing to the subject a) a first plurality of therapeutically effective cells, said cells being genetically modified to express a chimeric antigen receptor, said chimeric antigen receptor comprising at least one antigen-specific targeting region capable of binding to said target cell population, and said binding of said chimeric antigen receptor targeting region to said target cell population is capable of inducing activation-induced cell death; and b) a second plurality of therapeutically effective cells, said cells being genetically modified to express an amount of IL-10 agent sufficient to prevent or limit said activation-induced cell death.

[0027] In one embodiment, multiple therapeutically effective cells are transfected with a vector expressing an amount of IL-10 sufficient to enhance cytotoxic function. In other embodiments, a second plurality of therapeutically effective cells comprises CD8+ T cells transfected with a vector expressing IL-10.

[0028] In certain embodiments, the first plurality of cells are obtained from a subject and genetically modified in vitro, while in other embodiments, the second plurality of cells are obtained from a subject and genetically modified in vitro. This disclosure contemplates embodiments in which the first plurality of cells and the second plurality of cells are obtained from a subject via plasma exchange. In some embodiments, the first plurality of cells are memory CD8+ T cells, and the second plurality of cells are naive CD8+ T cells. In other embodiments, the first plurality of cells and the second plurality of cells are autologous tumor cells.

[0029] In each of the foregoing embodiments, the target cell population may contain tumor antigens. Vigneron, N. et al. (July 15, 2013, Cancer Immunity 13:15) described a database of T-cell-specific human tumor antigens containing more than 400 tumor antigen peptides. Examples of tumor antigens include, but are not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0030] This disclosure also considers using CAR-T cell therapy to treat or prevent diseases, conditions, or symptoms (e.g., cancer-related conditions) in subjects by combining administration of an IL-10 agent (e.g., PEG-IL-10) or the introduction of a vector expressing an IL-10 agent.

[0031] Specific embodiments include a method of treating a subject suffering from a cancer-related disease, condition, or symptom (e.g., tumor), comprising a) introducing into the subject a plurality of therapeutically effective, genetically modified cells expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises at least one antigen-specific targeting region capable of binding to the target cell population, and wherein the binding of the chimeric antigen receptor targeting region to the target cell population is capable of inducing activation-induced cell death; and b) administering to the subject a therapeutically effective amount of IL-10 agent sufficient to prevent or limit the activation-induced cell death. In a specific embodiment, the treated subject suffers from an immune-related disease, condition, or symptom described herein or another disease, condition, or symptom.

[0032] In some embodiments of this disclosure, such methods are used in treatment regimens for the prevention of cancer-related diseases, conditions, or symptoms in subjects, while in other embodiments, such methods are used in treatment regimens for the prevention of immune-related conditions. Other aspects of the above methods, including dosing parameters and regimens for IL-10 agents and exemplary types of such agents, are described elsewhere herein.

[0033] Further embodiments of this disclosure contemplate a method for treating a subject suffering from a cancer-related disease, condition, or symptom, comprising introducing to the subject a plurality of genetically modified cells that are therapeutically effective in expressing: a) a chimeric antigen receptor, wherein the chimeric antigen receptor comprises at least one antigen-specific targeting region capable of binding to the target cell population, and wherein the binding of the chimeric antigen receptor targeting region to the target cell population is capable of inducing activation-induced cell death; and b) an amount of IL-10 agent sufficient to prevent or limit the activation-induced cell death.

[0034] In some embodiments, the chimeric antigen receptor and the IL-10 agent are expressed by the same vector, while in other embodiments, the chimeric antigen receptor and the IL-10 agent are expressed by different vectors. In a particular embodiment, multiple therapeutically effective cells are transfected with a vector expressing an amount of IL-10 agent sufficient to enhance cytotoxic function. The vector may be, for example, a plasmid or a viral vector. This disclosure also contemplates any other means of expressing the IL-10 agent. In a particular embodiment, the expression of the IL-10 agent is regulated by an expression control element.

[0035] In the above embodiments, the plurality of cells are obtained from the subject and genetically modified in vitro. According to this disclosure, in some embodiments, the plurality of cells are obtained from the subject via plasma exchange. In a particular embodiment, the plurality of cells are memory CD8+ T cells, while in other embodiments they are autologous tumor cells.

[0036] This disclosure contemplates methods in which an IL-10 agent is expressed at an amount sufficient to prevent or limit activation-induced cell death at least one week after introduction into a subject. In other specific embodiments, an IL-10 agent is expressed at an amount sufficient to prevent or limit activation-induced cell death at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, or at least one year or longer after introduction into a subject.

[0037] Other embodiments of this disclosure contemplate methods for treating a subject suffering from a cancer-related disease, condition, or symptom, comprising introducing to the subject a) a first plurality of therapeutically effective cells, said cells being genetically modified to express a chimeric antigen receptor, said chimeric antigen receptor comprising at least one antigen-specific targeting region capable of binding to said target cell population, and said binding of said chimeric antigen receptor targeting region to said target cell population capable of inducing activation-induced cell death; and b) a second plurality of therapeutically effective cells, said cells being genetically modified to express an amount of IL-10 agent sufficient to prevent or limit said activation-induced cell death. Examples of the duration for which IL-10 agent is expressed in an amount sufficient to prevent or limit activation-induced cell death are described elsewhere herein.

[0038] In some implementations, the above methods are used in treatment regimens to prevent diseases, conditions, or symptoms (including cancer or immune-related diseases, conditions, or symptoms) in subjects.

[0039] This disclosure considers methods in which IL-10 agents are expressed for a period of time in an amount sufficient to prevent or limit activation-induced cell death (described elsewhere herein).

[0040] In one embodiment, the first plurality of therapeutically effective cells are transfected with a vector expressing an amount of IL-10 sufficient to enhance cytotoxic function. In other embodiments, the second plurality of therapeutically effective cells comprise CD8+ T cells transfected with a vector expressing IL-10.

[0041] In certain embodiments, the first plurality of cells are obtained from a subject and genetically modified in vitro, while in other embodiments, the second plurality of cells are obtained from a subject and genetically modified in vitro. This disclosure contemplates embodiments in which the first plurality of cells and the second plurality of cells are obtained from a subject via plasma exchange. In some embodiments, the first plurality of cells are memory CD8+ T cells, and the second plurality of cells are naive CD8+ T cells. In other embodiments, the first plurality of cells and the second plurality of cells are autologous tumor cells.

[0042] In each of the aforementioned embodiments, the target cell population may contain tumor antigens, examples of which are described elsewhere in this document.

[0043] This disclosure contemplates nucleic acid molecules encoding the IL-10 agents described herein. In some embodiments, the nucleic acid molecule is operatively linked to an expression control element that confers expression of the nucleic acid molecule encoding the IL-10 agent. In some embodiments, a vector (e.g., a plasmid or viral vector) contains the nucleic acid molecule. Transformation or host cells expressing the IL-10 agent are also contemplated herein.

[0044] In other embodiments, this disclosure provides methods for enhancing the function of CAR-T T cells, comprising a) genetically engineering T cells to express CAR, thereby generating CAR-T T cells; and b) regulating CAR-T T cells with an agent (e.g., small interfering RNA (siRNA)) that reduces the amount of at least one cytokine secreted by the CAR-T T cells. Examples of cytokines include, but are not limited to, members of the tumor necrosis factor family or the transforming growth factor β superfamily (e.g., TGF-β). Embodiments in which reducing the amount of TGF-β reduces the proliferation of regulatory T cells are considered.

[0045] Other embodiments will be apparent to those skilled in the art based on the teachings of this disclosure. Attached Figure Description

[0046] Figure 1 The fold increase in PD-1 and LAG3+ peripheral T cells after 29 days of treatment with PEG-rHuIL-10 was depicted.

[0047] Figure 2 This indicates that PEG-IL-10 preferentially increases IFNγ production in memory CD8+ T cells (CD45RO+) compared to naive CD8+ T cells.

[0048] Figure 3 This indicates that PEG-IL-10 can limit activation-induced cell death in CD8+ T cells (CD45RO+). Detailed Implementation

[0049] Before further describing this disclosure, it should be understood that this disclosure is not limited to the specific embodiments set forth herein, and it should also be understood that the terminology used herein is for the purpose of describing the specific embodiments only and is not intended to be limiting.

[0050] When numerical ranges are provided, it should be understood that, unless the context explicitly indicates otherwise, every intermediate value (to one-tenth of the lower limit unit) between the upper and lower limits of the range and any other specified value or intermediate value within the specified range is covered within this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also covered within this invention, subject to any specifically excluded limits within the specified range. When a specified range includes one or two limits, the range excluding any one or both of those included limits is also included in this invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0051] It must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise. It should also be noted that claims can be drafted to exclude any optional elements. Therefore, this statement is intended as a basis for the use of exclusionary terms such as “only,” “merely,” etc., in conjunction with the narration of claim elements or the use of the limiting word “negative.”

[0052] The publications discussed herein are provided only for information available prior to the filing date of this application. Furthermore, the publication dates provided may differ from the actual publication dates and may require separate verification.

[0053] Overview CAR-T T-cell therapy is a promising treatment for cancer-related (e.g., B- and T-cell lymphomas) and immune-related malignancies. CAR-T T cells typically comprise patient-derived memory CD8+ T cells modified to express recombinant T-cell receptors specific to, for example, known antigens present on the target tumor. While this disclosure is generally described in the context of using CAR-T cell therapy to treat cancer, it should be understood that such therapies may also be used to treat other indications.

[0054] As further discussed in this article, high-affinity T-cell receptor interactions with significant antigen loads have been observed to lead to activation-induced cell death when CAR-T cell therapy has been used to treat certain cancers (e.g., non-B-cell malignancies). Although IL-10 has previously been linked to enhanced activation-induced cell death, the data presented in this article suggest that IL-10 agents can be used in conjunction with CAR-T T-cell therapy to prevent or limit activation-induced cell death while enhancing CD8+ T-cell function and survival.

[0055] It should be noted that any reference to "human" in relation to the polypeptides and nucleic acid molecules of this disclosure is not intended to limit the manner or source of obtaining the polypeptides or nucleic acids, but only to relate to the sequence, as it may correspond to the sequence of naturally occurring human polypeptides or nucleic acid molecules. In addition to human polypeptides and the nucleic acid molecules encoding them, this disclosure also considers IL-10-related polypeptides and corresponding nucleic acid molecules from other species.

[0056] definition Unless otherwise stated, the following terms are intended to have the meanings described below. Other terms are defined elsewhere in the specification.

[0057] The terms “patient” or “subject” are used interchangeably to refer to humans or non-human animals (e.g., mammals).

[0058] The term "application," when applied to, for example, a subject, cell, tissue, organ, or biological fluid, refers to the contact between a subject, cell, tissue, organ, or biological fluid and a substance such as IL-10 or PEG-IL-10, a nucleic acid (e.g., a nucleic acid encoding natural human IL-10), a pharmaceutical composition comprising the above substances, or a diagnostic agent. In the case of cells, application includes contact between the reagent and the cell (e.g., in vitro or ex vivo) and contact between the reagent and a fluid, wherein the fluid is in contact with the cell.

[0059] The term "treatment," etc., refers to a process of action (e.g., administration of IL-10 or a pharmaceutical composition containing IL-10) initiated after a disease, symptom, or condition or its symptoms have been diagnosed, observed, etc., in order to temporarily or permanently eliminate, alleviate, inhibit, mitigate, or improve at least one underlying cause of the disease, symptom, or condition afflicting a subject, or at least one symptom associated with the disease, symptom, or condition afflicting the subject. Therefore, treatment includes inhibiting (e.g., preventing the development or further development of a disease, symptom, or condition or its associated clinical symptoms) an active disease. The term can also be used in other contexts, such as when IL-10 or PEG-IL-10 is in contact with IL-10 receptors, for example, in a fluid or colloidal phase.

[0060] As used in this article, "need for treatment" refers to a judgment made by a physician or other caregiver that a subject needs or will benefit from treatment. This judgment is based on a variety of factors within the physician's or caregiver's area of ​​expertise.

[0061] The term "prevention," etc., refers to a process of action (e.g., administration of IL-10 or a pharmaceutical composition containing IL-10) that, in order to temporarily or permanently prevent, inhibit, suppress, or reduce the risk of a subject developing a disease, condition, or symptom (as determined by, for example, the absence of clinical symptoms) or delay its onset, typically initiated in subjects predisposed to a particular disease, condition, or symptom. In some cases, the term also refers to slowing the development of a disease, condition, or symptom or inhibiting its development into a harmful or other undesirable state.

[0062] As used in this article, "need for prevention" refers to a judgment made by a physician or other caregiver that a subject needs or will benefit from preventive healthcare. This judgment is based on a variety of factors within the physician's or caregiver's area of ​​expertise.

[0063] The phrase "therapeutic effective dose" refers to an amount of a drug, alone or as part of a pharmaceutical composition, administered to a subject in a single dose or as part of a series of doses, such that it has a detectable, positive effect on any symptom, aspect, or characteristic of a disease, condition, or ailment when administered to the subject. Therapeutic effective doses can be determined by measuring the associated physiological effects and can be adjusted in conjunction with the dosing regimen and diagnostic analysis of the subject's condition. For example, measuring the amount of inflammatory cytokines produced after administration can indicate whether a therapeutic effective dose has been used.

[0064] The phrase “a quantity sufficient to achieve change” refers to a detectable difference between the levels of an indicator measured before (e.g., baseline levels) and after the administration of a particular therapy. Indicators include any objective parameter (e.g., serum IL-10 concentration) or a subjective parameter (e.g., subject well-being).

[0065] The term "small molecule" refers to chemical compounds with a molecular weight of less than approximately 10 kDa, less than approximately 2 kDa, or less than approximately 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, and synthetic molecules. In treatment, small molecules can penetrate cells more easily than large molecules, are less prone to degradation, and are less likely to trigger an immune response.

[0066] The term "ligand" refers to peptides, polypeptides, membrane-associated molecules, or membrane-bound molecules or complexes thereof that can act as receptor agonists or antagonists. "Ligand" encompasses both natural and synthetic ligands, such as cytokines, cytokine variants, analogs, mutant proteins, and antibody-derived binding compositions. "Ligand" also includes small molecules, such as peptide mimics of cytokines and peptide mimics of antibodies. The term also includes agents that are neither agonists nor antagonists, but which can bind to receptors without significantly affecting their biological properties, such as signal transduction or adhesion. Furthermore, the term includes membrane-bound ligands that have been modified, for example, by chemical or recombinant methods, into soluble forms of membrane-bound ligands. Ligands or receptors can be entirely intracellular, meaning they can be located in the cytoplasm, nucleus, or some other intracellular compartment. A complex of a ligand and a receptor is called a "ligand-receptor complex."

[0067] The terms “inhibitor” and “antagonist” or “activator” and “agonist” refer to, for example, molecules that inhibit or activate, such as ligands, receptors, cofactors, genes, cells, tissues, or organs. An inhibitor is a molecule that reduces, blocks, prevents, delays, inactivates, desensitizes, or downregulates, such as genes, proteins, ligands, receptors, or cells. An activator is a molecule that increases, activates, promotes, enhances, sensitizes, or upregulates, such as genes, proteins, ligands, receptors, or cells. An inhibitor can also be defined as a molecule that reduces, blocks, or inactivates constitutive activity. An “agonist” is a molecule that interacts with a target to cause or promote an increase in the activation of the target. An “antagonist” is a molecule that counteracts the action of an agonist. Antagonists prevent, reduce, inhibit, or neutralize the activity of agonists, and antagonists can also prevent, inhibit, or reduce the constitutive activity of a target (e.g., a target receptor), even in the absence of an identified agonist.

[0068] The term "regulatory agent" refers to the ability of a molecule (e.g., an activator or inhibitor) to directly or indirectly increase or decrease the function or activity of IL-10 agents (or the nucleic acid molecules encoding them); or to enhance the ability of a molecule to produce an effect equivalent to that of IL-10 agents. The term "regulatory agent" is broadly intended to refer to molecules that can affect the aforementioned activities. For example, a regulator of a gene, receptor, ligand, or cell is a molecule that alters the activity of a gene, receptor, ligand, or cell, where the activity can be activated, inhibited, or its regulatory properties altered. Regulators can act alone or with the help of cofactors, such as proteins, metal ions, or small molecules. The term "regulatory agent" includes agents that operate through the same mechanism of action as IL-10 (i.e., regulate the same signaling pathway as IL-10 in a similar manner) and are capable of evoking a biological response equivalent to (or stronger than) that of IL-10.

[0069] Examples of modulators include small molecule compounds and other bioorganic molecules. Numerous libraries of small molecule compounds (e.g., combinatorial libraries) are commercially available and can serve as a starting point for identifying modulators. Those skilled in the art can develop one or more assays (e.g., biochemical or cell-based assays) in which such libraries can be screened to identify one or more compounds with desired properties; subsequently, a skilled medicinal chemist can optimize such one or more compounds by, for example, synthesizing and evaluating their analogues and derivatives. Synthetic and / or molecular modeling studies can also be used to identify activators.

[0070] The "activity" of a molecule can describe or refer to its binding to ligands or receptors; catalytic activity; the ability to stimulate gene expression or cell signaling, differentiation, or maturation; antigenic activity; regulation of the activity of other molecules; and so on. The term can also refer to the activity that regulates or maintains intercellular interactions (e.g., adhesion) or the activity that maintains cellular (e.g., cell membrane) structure. "Activity" can also mean specific activity, such as [catalytic activity] / [mg protein] or [immune activity] / [mg protein], concentration in a biological compartment, etc. The term "proliferative activity" encompasses activities necessary for or specifically associated with promoting (e.g.) normal cell division, as well as cancer, tumors, developmental abnormalities, cell transformation, metastasis, and angiogenesis.

[0071] As used herein, terms such as “comparable,” “comparable activity,” “activity comparable to,” “comparable effect,” and “effect comparable to” are relative terms that can be observed quantitatively and / or qualitatively. The meaning of these terms generally depends on the context in which they are used. For example, two agents activating receptors may be considered to have comparable effects qualitatively, but if, in assays accepted in the art (e.g., dose-response assays) or in animal models accepted in the art, one agent achieves only 20% of the activity of the other agent, then quantitatively the two agents may be considered not to have comparable effects. When comparing one result to another (e.g., one result to a reference standard), “comparable” generally means that a result deviates from a reference standard by less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 7%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In a particular embodiment, a result is considered comparable to a reference standard if it deviates from a reference standard by less than 15%, less than 10%, or less than 5%. For example, but not as a limitation, activity or effect can refer to efficacy, stability, solubility, or immunogenicity.

[0072] The term "response" in the context of cells, tissues, organs, or organisms encompasses biochemical or physiological behaviors, such as changes in concentration, density, adhesion or migration, gene expression rate, or differentiation state within biological compartments, where these changes are associated with activation, stimulation, or treatment, or with internal mechanisms such as gene programming. In some cases, the terms "activation," "stimulation," etc., refer to cell activation regulated by internal mechanisms and external or environmental factors; while the terms "inhibition," "downregulation," etc., refer to the opposite effect.

[0073] The terms “polypeptide,” “peptide,” and “protein,” used interchangeably herein, refer to any length of amino acid polymer, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a modified polypeptide backbone. The term includes fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences; fusion proteins having heterologous and homologous leader sequences; fusion proteins with or without N-terminal methionine residues; fusion proteins having immunolabeled proteins; and so on.

[0074] It should be recognized that throughout this publication, references are made to amino acids based on either single-letter or three-letter codes. For the reader's convenience, the single-letter and three-letter amino acid codes are provided below: .

[0075] As used herein, the term “variant” encompasses both naturally occurring and non-naturally occurring variants. Naturally occurring variants include homologues (peptides and nucleic acids differing from each other in amino acid or nucleotide sequences) and allelic variants (peptides and nucleic acids differing from each other in amino acid or nucleotide sequences within the same species). Non-naturally occurring variants include peptides and nucleic acids that contain variations in amino acid or nucleotide sequences, where the sequence changes are artificially introduced (e.g., mutant proteins); for example, the change is produced in a laboratory through human intervention (“artificial”). Therefore, “mutant protein” as used herein broadly refers to a mutated recombinant protein, which typically carries one or more amino acid substitutions and often originates from a cloned gene that has undergone site-directed or random mutagenesis or from a fully synthesized gene.

[0076] The terms “DNA,” “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” are used interchangeably in this document and refer to a polymer of nucleotides (deoxyribonucleotides or ribonucleotides or their analogues) of any length. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers, etc.

[0077] As used in the context of polypeptide structure herein, “N-terminus” (or “amino terminus”) and “C-terminus” (or “carboxyl terminus”) refer to the amino and carboxyl ends of the polypeptide, respectively, while the terms “N-terminus” and “C-terminus” refer to the relative positions in the amino acid sequence of the polypeptide toward the N-terminus and C-terminus, respectively, and may include residues at the N-terminus and C-terminus, respectively. “Immediately following the N-terminus” or “immediately following the C-terminus” refers to the position of the first amino acid residue relative to the second amino acid residue, wherein the first and second amino acid residues are covalently linked to provide a continuous amino acid sequence.

[0078] In the context of an amino acid sequence or polynucleotide sequence, “derived from” (e.g., “derived from” the amino acid sequence of an IL-10 polypeptide) is intended to indicate that the polypeptide or nucleic acid has a sequence based on a reference polypeptide or nucleic acid (e.g., a naturally occurring IL-10 polypeptide or a nucleic acid encoding IL-10), and is not intended to limit the source or method of preparing the protein or nucleic acid. For example, the term “derived from” includes homologues or variants of the reference amino acid or DNA sequence.

[0079] In the context of peptides, the term "isolated" refers to a target peptide, if naturally occurring, in an environment different from its natural environment. "Isolated" is intended to include peptides substantially enriched in the target peptide and / or partially or substantially purified within a sample. In cases where the peptide is not naturally occurring, "isolated" indicates that the peptide has been isolated from an environment prepared by synthetic or recombinant means.

[0080] "Enrichment" means that a sample is subjected to non-natural manipulation (e.g. by scientists) such that the target peptide is present at a higher concentration (e.g., at least 3, 4, 8, 64 or more) than the peptide concentration in the original sample, such as a biological sample (e.g., a sample in which the peptide is naturally present or present after application), or b) a higher concentration than the environment in which the peptide was prepared (e.g., as in bacterial cells).

[0081] "Substantially pure" means that the component (e.g., peptide) constitutes more than about 50% of the total composition, and typically more than about 60% of the total peptide content. More generally, "substantially pure" means a composition in which at least 75%, at least 85%, at least 90% or more of the total composition is the target component. In some cases, the peptide will constitute more than about 90% or more of the total composition.

[0082] When referring to ligand / receptor, antibody / antigen, or other binding pairs, the terms "specific binding" or "selective binding" indicate a binding reaction that determines the presence of proteins in a heterogeneous population of proteins and other biological agents. Thus, under specified conditions, a specified ligand binds to a specific receptor without binding to other proteins present in the sample in significant quantities. The antibody of the considered method, or a binding composition derived from an antibody's antigen-binding site, binds to its antigen or a variant or mutant protein with an affinity at least two, at least ten, at least 20, or at least 100 times higher than that with any other antibody or a binding composition derived therefrom. In a particular embodiment, as determined by, for example, Scatchard assays, the antibody affinity will be greater than about 10. 9 Liters per mole (Munsen et al., 1980 Analyt. Biochem. 107: 220-239).

[0083] IL-10 and PEG-IL-10 The anti-inflammatory cytokine IL-10, also known as human cytokine synthesis inhibitory factor (CSIF), is classified as a type 2 cytokine, which includes a group of cytokines such as IL-19, IL-20, IL-22, IL-24 (Mda-7) and IL-26, interferons (IFN-α, -β, -γ, -δ, -ε, -κ, -Ω and -τ) and interferon-like molecules (limitin, IL-28A, IL-28B and IL-29).

[0084] IL-10 is a pleiotropic cytokine with pleiotropic effects in immune regulation and inflammation. Produced by mast cells, it counteracts the inflammatory activity of these cells at the site of allergic reactions. While it inhibits the synthesis of pro-inflammatory cytokines such as IFN-γ, IL-2, IL-3, TNFα, and GM-CSF, IL-10 also stimulates certain T cells and mast cells, and promotes B cell maturation, proliferation, and antibody production. IL-10 can block NF-κB activity and participate in the regulation of the JAK-STAT signaling pathway. It also induces cytotoxic activity of CD8+ T cells and antibody production by B cells, and inhibits macrophage activity and pro-tumor inflammation. Regulation of CD8+ T cells is dose-dependent, with higher doses inducing stronger cytotoxic responses.

[0085] Human IL-10 is a homodimer with a molecular weight of 37 kDa, in which each 18.5 kDa monomer contains 178 amino acids, the first 18 of which form a signal peptide, and two cysteine ​​residues form two intramolecular disulfide bonds. The IL-10 dimer becomes biologically inactive after the non-covalent interaction between the two monomeric subunits is disrupted.

[0086] This disclosure contemplates human IL-10 (NP_000563) and mouse IL-10 (NP_034678) with 80% homology and their uses. Additionally, the scope of this disclosure includes orthologs of IL-10 from other mammalian species, including rats (registration number NP_036986.2; GI 148747382); cattle (registration number NP_776513.1; GI 41386772); sheep (registration number NP_001009327.1; GI57164347); dogs (registration number ABY86619.1; GI 166244598); and rabbits (registration number AAC23839.1; GI 3242896) and their modified forms.

[0087] As mentioned above, the terms "IL-10," "IL-10 polypeptide," "IL-10 molecule," and "IL-10 agent" are intended to be broadly interpreted and include, for example, human and non-human IL-10-related polypeptides, including their homologues, variants (including mutant proteins), and fragments, as well as IL-10 polypeptides having, for example, a leader sequence (e.g., a signal peptide), and the aforementioned modified forms. In other specific embodiments, IL-10, IL-10 polypeptides, and IL-10 agents are agonists.

[0088] The IL-10 receptor, a type II cytokine receptor, consists of α and β subunits, also known as R1 and R2, respectively. Receptor activation requires binding to both α and β subunits. One homodimer of the IL-10 polypeptide binds to the α subunit, while the other homodimer binds to the β subunit.

[0089] The practicality of recombinant human IL-10 is generally limited by its relatively short serum half-life, likely due to factors such as renal clearance, proteolytic degradation, and monomerization in the bloodstream. Therefore, various methods have been explored to improve the pharmacokinetic properties of IL-10 without disrupting its dimer structure and thus without adversely affecting its activity. Polyethylene glycolization of IL-10 leads to improvements in certain pharmacokinetic parameters (e.g., serum half-life) and / or enhanced activity.

[0090] As used herein, the terms “PEG-IL-10” and “PEG-IL-10” refer to an IL-10 molecule in which one or more polyethylene glycol molecules are covalently linked to at least one amino acid residue of the IL-10 protein, typically via a linker, resulting in a stable link. The terms “mono-PEG-IL-10” and “mono-PEG-IL-10” indicate that a polyethylene glycol molecule is covalently linked to a single amino acid residue on a subunit of an IL-10 dimer, typically via a linker. As used herein, the terms “di-PEG-IL-10” and “di-PEG-IL-10” indicate that at least one polyethylene glycol molecule is typically linked to a single residue on each subunit of an IL-10 dimer, typically via a linker.

[0091] In some embodiments, the PEG-IL-10 used in this disclosure is mono-PEG-IL-10 in which 1 to 9 PEG molecules are covalently linked via a linker to the α-amino group of an amino acid residue at the N-terminus of a subunit of an IL-10 dimer. Mono-glycolization on an IL-10 subunit typically results in a heterogeneous mixture of non-glycolized, mono-glycolized, and dimerized IL-10 due to subunit shuffling. Moreover, proceeding the glycolization reaction to completion typically yields nonspecific and polyglycolized IL-10, thereby reducing its biological activity. Therefore, specific embodiments of this disclosure include the application of a mixture of mono-glycolized and dimerized IL-10 produced by the methods described herein.

[0092] In certain embodiments, the average molecular weight of the PEG moiety is between about 5 kDa and about 50 kDa. While the method or site of PEG-IL-10 linkage is not critical, in some embodiments, PEGylation does not alter, or only minimally alters, the activity of the IL-10 agent. In some embodiments, the increase in half-life outweighs any decrease in biological activity. The biological activity of PEG-IL-10 is typically measured by assessing the levels of inflammatory cytokines (e.g., TNF-α or IFN-γ) in the serum of subjects challenged with bacterial antigens (lipopolysaccharide (LPS)) and treated with PEG-IL-10, as described in U.S. Patent No. 7,052,686.

[0093] Various objectives can be considered for the preparation of IL-10 variants, including increasing serum half-life, reducing the immune response to IL-10, facilitating purification or preparation, reducing the conversion of IL-10 to its monomeric subunits, improving therapeutic efficacy, and mitigating the severity or incidence of side effects during therapeutic use. Amino acid sequence variants, although some may be post-translational variants such as glycosylated variants, are generally predetermined variants not found in nature. Any variant of IL-10 can be used, provided it maintains an appropriate level of IL-10 activity.

[0094] The phrase "conservative amino acid substitution" refers to a substitution in which an amino acid in a protein is replaced by an amino acid with a side chain having similar acidity, basicity, charge, polarity, or side chain size to maintain protein activity. Conservative amino acid substitution typically requires substitution of amino acid residues within the following group: 1) L, I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D, E. Guidance on substitution, insertion, or deletion can be based on the alignment of amino acid sequences of different variant proteins or proteins from different species. Therefore, in addition to any naturally occurring IL-10 polypeptide, this disclosure also considers substitutions having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, typically not exceeding 20, 10, or 5 amino acids, wherein the substitutions are generally conserved amino acid substitutions.

[0095] The present invention also considers active fragments (e.g., subsequences) of mature IL-10 containing consecutive amino acid residues derived from mature IL-10. The length of the consecutive amino acid residues in the peptide or polypeptide subsequence varies depending on the specific naturally occurring amino acid sequence from which the subsequence originates. Typically, peptides and polypeptides can be about 20 to about 40 amino acids, about 40 to about 60 amino acids, about 60 to about 80 amino acids, about 80 to about 100 amino acids, about 100 to about 120 amino acids, about 120 to about 140 amino acids, about 140 to about 150 amino acids, about 150 to about 155 amino acids, about 155 amino acids, up to full-length peptides or polypeptides.

[0096] Furthermore, the IL-10 peptide can exhibit defined sequence identity with a reference sequence over a defined length of consecutive amino acids (e.g., a "comparison window"). Sequence alignment methods used for comparison are well known in the art. The best sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by executing these algorithms by computer (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics package Madison, Wis), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (edited by Ausubel et al., Supplement 1995)).

[0097] For example, a suitable IL-10 peptide may contain an amino acid sequence that is identical to a sequence of about 20 amino acids to about 40 amino acids, about 40 amino acids to about 60 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 120 amino acids, about 120 amino acids to about 140 amino acids, about 140 amino acids to about 150 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to a full-length peptide or polypeptide having an amino acid sequence identity of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%.

[0098] As discussed further below, IL-10 peptides can be isolated from natural sources (e.g., environments other than those in which they naturally occur) and can also be prepared recombinantly (e.g., in genetically modified host cells such as bacteria, yeast, Pichia pastoris, insect cells, etc.), wherein the genetically modified host cells are modified with nucleic acids containing a nucleotide sequence encoding the peptide. IL-10 peptides can also be synthesized (e.g., through cell-free chemical synthesis).

[0099] This disclosure considers nucleic acid molecules encoding IL-10 agents, including naturally occurring and non-natural isotypes, allelic variants, and splice variants. This disclosure also covers nucleic acid sequences that differ from naturally occurring DNA sequences by one or more bases but, due to the degeneracy of the genetic code, still translate into an amino acid sequence corresponding to an IL-10 polypeptide.

[0100] Chimeric antigen receptor T cells Chimeric antigen receptor T cells (CAR; also known as artificial T cell receptors, chimeric T cell receptors, and chimeric immune receptors) represent an emerging therapy for cancers (e.g., for treating B and T-cell lymphomas) and other malignancies. CAR-T T cells typically comprise patient-derived memory CD8+ T cells modified to express recombinant T cell receptors specific to, for example, known antigens present on the target tumor. Other types of T cells considered herein include naive T cells, central memory T cells, effector memory T cells, or combinations thereof. While this disclosure is described generally in the context of using CAR-T cell therapy to treat cancer, it should be understood that such therapies are not limited thereto.

[0101] CAR-T T-cell therapy includes the use of adoptive cell transfer (ACT). ACT, utilizing patient-derived T cells, has shown promise as a patient-specific cancer therapy (Snook and Waldman (2013) Discov Med 15(81):120-25). The potential of ACT has been greatly expanded by using genetic engineering methods to insert antigen-targeting receptors with defined specificity into T cells. In most cases, these engineered chimeric antigen receptors are used to specifically transplant monoclonal antibodies onto T cells.

[0102] CAR-T cell therapy begins by taking T cells from a patient. These T cells are then genetically engineered to express CARs that target antigens specific to known cancers, such as tumors. After sufficient numbers have been expanded in vitro, the autologous cells are infused back into the patient, causing the antigens to be specifically destroyed.

[0103] CARs are a class of antigen-targeting receptors composed of intracellular T-cell signaling domains fused to single-chain variable fragments (scFvs) that are typically derived from monoclonal antibodies and bind to extracellular tumor sites. CARs directly recognize cell surface antigens, independent of MHC-mediated presentation, allowing the use of a single receptor construct specific to any given antigen in all patients.

[0104] Chimeric antigen receptors typically contain several main components, some of which are described below.

[0105] As used herein, the phrase "antigen-specific target region" (ASTR) refers to a region that guides a CAR to a specific antigen. The target region on the CAR is extracellular. In certain embodiments of this disclosure, the CAR comprises at least two target regions targeting at least two different antigens. In other specific embodiments, the CAR comprises three or more target regions targeting at least three or more different antigens. In some embodiments, the antigen-specific target region comprises an antibody or a functional equivalent thereof, a fragment thereof, or a derivative thereof, and each target region targets a different antigen. The target region may comprise a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody, or a double-chain antibody, each specific to the target antigen. In some aspects of this disclosure, the target region may comprise linked cytokines, ligand-binding domains from naturally occurring receptors, soluble protein-peptide ligands of the receptor, peptides, affinity molecules, and vaccines to induce an immune response. Those skilled in the art know of other molecules that can be used as antigen-specific target regions.

[0106] As used herein, the term "extracellular spacer domain" (ESD) refers to a hydrophilic region between an antigen-specific targeting region and a transmembrane domain. This disclosure contemplates embodiments in which the CAR includes an ESD, examples of which include FcAb fragments or fragments or derivatives thereof; hinge regions of antibodies or fragments or derivatives thereof; CH2 or CH3 regions of antibodies; artificial spacer sequences, including Gly3 or CH1 and CH3 domains of IgG (such as human IgG4); or combinations thereof. Other ESDs are known to those skilled in the art and are also considered herein.

[0107] As used herein, the term "transmembrane domain" (TMD) refers to a CAR region that crosses the plasma membrane. In some embodiments, the transmembrane domain is a transmembrane protein (e.g., a type I transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. Other transmembrane domains that may be used in conjunction with the teachings of this disclosure will be known to those skilled in the art.

[0108] As used herein, the terms "intracellular signal transduction domain" (ISD) and "cytoplasmic domain" refer to the CAR portion that transduces effector functional signals and directs the cell to perform its specialized functions. Examples of ISDs include the ζ chain of the T cell receptor complex or any of its homologues (e.g., the η chain, FcεR1γ and β chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), human CD3 ζ chain, CD3 polypeptides (δ, Δ, and ε), syk family tyrosine kinases (Syk, ZAP70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T-cell transduction, such as CD2, CD5, and CD28. Other ISDs that may be used in conjunction with the teachings of this disclosure will be appreciated by those skilled in the art.

[0109] The term "co-stimulatory domain" (CSD) refers to a portion of the CAR that enhances the proliferation, survival, or development of memory cells. As noted elsewhere herein, the CAR of this disclosure may comprise one or more co-stimulatory domains. In some embodiments of this disclosure, the CSD comprises one or more members of the TNFR superfamily: CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, or combinations thereof. Other co-stimulatory domains that may be used in conjunction with the teachings of this disclosure are known to those skilled in the art.

[0110] When used in conjunction with the CAR-T T-cell technology described herein, the terms "connector," "connector domain," and "connector region" refer to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any domain / region of the CAR of this disclosure. The connector may consist of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. Some embodiments include the use of longer connectors when it is desirable to ensure that two adjacent domains do not interfere with each other spatially. In some embodiments, the connectors are non-cleavable, while in others they are cleavable (e.g., 2A connectors (e.g., T2A), 2A-like connectors or their functional equivalents, and combinations thereof). Embodiments of this disclosure are contemplated in which the connector comprises a microRNA virus 2A-like connector, porcine cyclovir (P2A), a Tetrasovirus micranthae (T2A), or a combination thereof, variants, and functional equivalents thereof. Further embodiments include a connector sequence comprising Asp-Val / Ile-Glu-X-Asn-Pro-Gly. (2A) -pro (2B) The motif leads to the cleavage between 2A glycine and 2B proline. Other linkers will be apparent to those skilled in the art and are contemplated for use in conjunction with the teachings of this disclosure.

[0111] CAR-T T-cell therapy has seen relatively rapid progress (see U.S. Patent Application Publication No. 20150038684). First-generation CARs involve the fusion of an antigen recognition domain with a CD3ζ activating chain of a T-cell receptor (TCR) complex. While these first-generation CARs induce T-cell effector function in vitro, their in vivo efficacy is largely limited by their poor anti-tumor activity. Advances in CAR technology have led to second-generation CARs, which include a CD3ζ activating chain tandem with a CSD, examples of which include intracellular domains from CD28 or various TNF receptor family molecules such as 4-1BB (CD137) and OX40 (CD134). Third-generation CARs have been developed, in addition to the CD3ζ activating chain, including two co-stimulatory signals, with the CSD most commonly derived from CD28 and 4-1BB. Second- and third-generation CARs have significantly improved anti-tumor efficacy. However, whether specific combinations of co-stimulatory molecules benefit other aspects is not fully understood. Moreover, the increased efficacy of second- and third-generation CARs, coupled with the lack of true tumor-specific antigen targets, also increases the risk of severe toxicity. (See, for example, Carpenito et al. (2009) Proc Natl Acad Sci USA 106(9):3360-65; Grupp et al. (2013) N Engl J Med 368(16):1509-18).

[0112] Activation-induced cell death Infusion of genetically modified T cells targeting specific antigens has several potential benefits, including long-term disease control, rapid onset of action similar to cytotoxic chemotherapy or targeted therapy, and circumvention of immune tolerance and MHC restrictions in the T cell repertoire. However, the use of CAR-T cell therapy for certain cancers (e.g., non-B-cell malignancies) has been partially limited by the induction of antigen-specific toxicity in normal tissues expressing the target antigen and the extremely high potency of CAR-T cell therapy, which sometimes leads to life-threatening cytokine release syndromes (Magee (November 2014) Discov Med 18(100):265-71). Specifically, interactions between high-affinity T cell receptors and significant antigen loads have been observed to lead to activation-induced cell death (Song et al. (2012) Blood 119(3):696-706; Hombach et al. (2013) Mol Ther 21(12):2268-77).

[0113] Activation-induced cell death (AICD), a programmed cell death resulting from the interaction of Fas receptors (e.g., Fas, CD95) and Fas ligands (e.g., FasL, CD95 ligands), helps maintain peripheral immune tolerance. AICD effector cells express FasL and induce apoptosis in cells expressing Fas receptors. Activation-induced cell death is a negative regulator of activated T lymphocytes produced by repeated stimulation of their T cell receptors. Alterations in this process may lead to autoimmune diseases (Zhang J et al., (2004) Cell Mol Immunol. 1(3):186-92).

[0114] Mechanistically, the binding of the Fas ligand to the Fas receptor triggers Fas receptor trimerization, and its cytoplasmic domain can then bind the death domain of the adaptor protein FADD (a Fas-associated protein with a death domain). Procysteine ​​8 binds to the death effector domain of FADD and is self-activated by proteolysis; Fas, FADD, and procysteine ​​8 together form a death-inducing signaling complex. Activated caspase 8 is released into the cytosol, where it activates and initiates the caspase cascade of apoptosis (Nagata S. (1997) Cell.: 88(3):355-65s).

[0115] The balance between activation-induced proliferation and death of effector cells is crucial for T cell homeostasis. While quiescent T cells are prone to apoptosis, clonal expansion occurs when T cells are stimulated by TCR / CD3 in the presence of cytokines such as IL-2, IL-4, IL-7, and IL-12. Interestingly, the roles of these molecules in T cell homeostasis are sometimes contradictory. For example, IL-2 is essential for the proliferation and survival of CD4+ T cells, but it is also a prerequisite for activation-induced cell death. Moreover, IL-18 has been shown to promote the expansion and survival of activated CD8+ T cells. IL-18 can influence the immune / inflammatory response by regulating the size of CD8+ T cell populations with specific functions upon exposure to stimulation. Regulation of activated T cell proliferation and activation-induced cell death is closely related to the immune / inflammatory response (Li, W. et al. (July 2007) J Leukocyte Bio 82(1):142-51).

[0116] The impact of IL-10 on CAR-T T cell therapy The characteristics of IL-10 agents (e.g., PEG-IL-10) are described elsewhere in this article. As an anti-inflammatory and immunosuppressive molecule, IL-10 inhibits antigen presentation, CD4+ T cell function, pathogen-specific function of CD8+ T cells (Biswas et al. (2007) J Immunol 179(7):4520-28), viral epitope-specific CD8+ T cell IFNγ response (Liu et al., (2003) J Immunol 171(9):4765-72) and anti-LCMV (lymphocytic choriomeningitis virus) CD8+ T cell response (Brooks et al. (2008) PNAS USA 105(51):20428-433).

[0117] Although IL-10 has been discussed in the context of enhancing activation-induced cell death (Georgescu et al. (1997) J Clin Invest 100(10):2622-33), the in vitro and in vivo data presented in this paper suggest that IL-10 agents (e.g., PEG-IL-10) can be combined with CAR-T T cell therapy to prevent or limit activation-induced cell death while enhancing CD8+ T cell function and survival.

[0118] For example, the findings presented in Example 1 of the Experimental Section demonstrate that PEG-IL-10 administration mediates CD8+ T cell immune activation. As described in Example 1, the number of CD8+ T cells expressing PD-1 and LAG3 was compared in tumor patients before and after treatment with PEG-rHuIL-10 (see Example 1). Both PD-1 and LAG3 are markers of CD8+ T cell activation and cytotoxicity. The number of peripheral CD8+ T cells expressing PD-1 increased approximately 2-fold, while the number of peripheral CD8+ T cells expressing LAG3 increased approximately 4-fold. Overall, these data indicate that PEG-IL-10 administration mediates CD8+ T cell immune activation.

[0119] It was also observed that administration of PEG-IL-10 enhanced the function of activated memory CD8+ T cells (see Example 2). Memory T cells (also known as antigen-experienced T cells) are a class of T lymphocytes (e.g., helper T cells (CD4+) and cytotoxic T cells (CD8+)) that have previously encountered their homologous antigens during infection, cancer exposure, or prior vaccination. In contrast, naive T cells have not yet encountered their homologous antigens in their vicinity; they are typically characterized by the lack of activation markers CD25, CD44, or CD69 and the lack of the memory CD45RO isotype. Memory T cells (typically CD45RO+) are capable of regenerating and generating a faster and stronger immune response than naive T cells.

[0120] Since CAR-T T cells are derived from memory CD8+ T cells, the effect of PEG-IL-10 on memory CD8+ T cells was evaluated in vitro. The data presented in Example 2 are consistent with the effect of PEG-IL-10 on enhancing the function of activated memory CD8+ T cells.

[0121] Methods and Models This disclosure contemplates various methods and models for identifying candidate subject populations (or individual subjects) who have suffered or are suspected of suffering from activation-induced cell death caused by CAR-T cell therapy and who may respond to the therapies described herein. Such therapies include monotherapy with an IL-10 agent (e.g., PEG-IL-10) and combination therapy with an IL-10 agent in combination with one or more different agents that have been shown to exhibit beneficial activity in preventing or limiting activation-induced cell death. In some embodiments, the methods and models allow for determining whether administration of an IL-10 agent achieves a desired reduction in activation-induced cell death or whether a combination of an IL-10 agent with another agent is more beneficial. In other embodiments, the methods and models allow for determining whether administration of a combination results in fewer undesirable effects.

[0122] Some embodiments of this disclosure include the use of in vitro, ex vivo, and in vivo methods and / or models. In some embodiments of this disclosure, the subject population (or individual subject) is a non-human animal (e.g., a rodent) or a human.

[0123] For example, but not as a limitation, one aspect of this disclosure contemplates a method for determining whether a test subject who has suffered or is suspected of suffering activation-induced cell death is a candidate for treatment with an IL-10 agent (e.g., PEG-IL-10), said method comprising a) providing the test subject with a marker of activation-induced cell death, b) administering the IL-10 agent to the test subject in an amount sufficient to achieve a desired response in a reference population, and c) determining whether the test subject exhibits a desired response; wherein determination of a desired response indicates that the test subject is a candidate for treatment. Those skilled in the art can modify such methods for use in combination therapies. A desired response can be any outcome deemed favorable in this case.

[0124] As described above, this disclosure also considers various models. Any model that provides reliable, reproducible results can be used. Those skilled in the art are familiar with models that can be used in conjunction with the subject matter of this disclosure; in one embodiment, an IL-10 agent (e.g., PEG-IL-10) is evaluated in a model containing a non-human subject (e.g., a mouse). Specific embodiments of this disclosure consider models for determining whether an IL-10 agent, with or without combination with another agent, is a candidate for preventing or reducing activation-induced cell death.

[0125] Further embodiments of this disclosure include methods or models for determining the optimal amount of an IL-10 agent, whether or not it is to be combined with another agent. The optimal amount may be, for example, the amount that achieves the best effect in a subject or subject population. By controlling the amount of the agent, clinicians are able to determine the optimal dosing regimen for preventing or reducing activation-induced cell death.

[0126] biomarkers This disclosure also contemplates the use of biomarkers in conjunction with the methods and models described herein. The term "biomarker" refers to a characteristic that is objectively measured and evaluated as an indicator of a normal biological process, a pathogenic process, or a pharmacological response to a therapeutic intervention. An indicator can be any substance, structure, or process that can be measured in vivo or in its products and that affects or predicts outcomes or the incidence of disease.

[0127] In some embodiments of this disclosure, biomarkers are used to predict clinical response to treatment with IL-10 agents (e.g., PEG-IL-10). In some cases, pre-treatment biomarkers may be used in such therapies where the biomarkers have been validated to the point that they can be applied as part of the standard of care treatment decision-making process.

[0128] serum concentration In the methods described herein, plasma IL-10 levels can be characterized in several ways, including: (1) average serum trough concentrations of IL-10 above a specified level or within a certain range; (2) average serum trough concentrations of IL-10 above a specified level over a certain period; (3) steady-state serum IL-10 concentrations above or below a specified level or within a certain range; or (4) C-squared values ​​of concentration curves above or below a specified level or within a certain range. max As described in this article, mean serum trough IL-10 concentrations have been found to be particularly important for efficacy in certain indications.

[0129] In some embodiments of this disclosure, plasma and / or serum level concentration curves that can be generated include: above about 1.0 pg / mL, above about 10.0 pg / mL, above about 20.0 pg / mL, above about 30 pg / mL, above about 40 pg / mL, above about 50.0 pg / mL, above about 60.0 pg / mL, above about 70.0 pg / mL, above about 80.0 pg / mL, above about 90 pg / mL, above about 0.1 ng / mL, above about 0.2 ng / mL, above about 0.3 ng / mL, above about 0.4 ng / mL, above about 0.5 ng / mL, above about 0.6 ng / mL, above about 0.7 ng / mL, above about 0.8 ng / mL, and above about 1.0 pg / mL. Mean plasma and / or serum trough concentrations of IL-10: 0.9 ng / mL, above approximately 1.0 ng / mL, above approximately 1.5 ng / mL, above approximately 2.0 ng / mL, above approximately 2.5 ng / mL, above approximately 3.0 ng / mL, above approximately 3.5 ng / mL, above approximately 4.0 ng / mL, above approximately 4.5 ng / mL, above approximately 5.0 ng / mL, above approximately 5.5 ng / mL, above approximately 6.0 ng / mL, above approximately 6.5 ng / mL, above approximately 7.0 ng / mL, above approximately 7.5 ng / mL, above approximately 8.0 ng / mL, above approximately 8.5 ng / mL, above approximately 9.0 ng / mL, above approximately 9.5 ng / mL, or above approximately 10.0 ng / mL.

[0130] In certain embodiments of this disclosure, the average serum trough concentration of IL-10 is in the range of 1.0 pg / mL to 10 ng / mL. In some embodiments, the average serum trough concentration of IL-10 is in the range of 1.0 pg / mL to 100 pg / mL. In other embodiments, the average serum trough concentration of IL-10 is in the range of 0.1 ng / mL to 1.0 ng / mL. In still other embodiments, the average serum trough concentration of IL-10 is in the range of 1.0 ng / mL to 10 ng / mL. It should be understood that, even if such ranges are not explicitly listed, this disclosure also contemplates any ranges of concentrations incorporated herein by reference. For example, in one embodiment, the average serum IL-10 concentration may be in the range of 0.5 ng / mL to 5 ng / mL. For example, specific embodiments of this disclosure include average serum trough concentrations of IL-10 in the range of about 0.5 ng / mL to about 10.5 ng / mL, about 1.0 ng / mL to about 10.0 ng / mL, about 1.0 ng / mL to about 9.0 ng / mL, about 1.0 ng / mL to about 8.0 ng / mL, about 1.0 ng / mL to about 7.0 ng / mL, about 1.5 ng / mL to about 10.0 ng / mL, about 1.5 ng / mL to about 9.0 ng / mL, about 1.5 ng / mL to about 8.0 ng / mL, about 1.5 ng / mL to about 7.0 ng / mL, about 2.0 ng / mL to about 10.0 ng / mL, about 2.0 ng / mL to about 9.0 ng / mL, about 2.0 ng / mL to about 8.0 ng / mL, and about 2.0 ng / mL to about 7.0 ng / mL.

[0131] In certain embodiments, a mean serum trough concentration of 1-2 ng / mL is maintained throughout the treatment duration. This disclosure also considers embodiments in which the mean serum peak concentration of IL-10 during the treatment duration is less than or equal to about 10.0 ng / mL. Further embodiments consider a mean serum trough concentration of IL-10 greater than or equal to about 1.0 pg / mL. The optimal mean serum concentration is generally the mean serum concentration at which the desired therapeutic effect is achieved without introducing undesirable adverse effects.

[0132] Certain embodiments of this disclosure provide a method for monitoring subjects receiving IL-10 therapy to predict and thus potentially avoid adverse effects, the method comprising: (1) measuring the peak concentration of IL-10 in the subject; (2) measuring the trough concentration of IL-10 in the subject; (3) calculating peak-trough fluctuations; and (4) using the calculated peak-trough fluctuations to predict potential adverse effects in the subject. In a specific subject population, smaller peak-trough fluctuations indicate a lower likelihood that the subject will experience adverse effects related to IL-10. Additionally, in some embodiments, specific dosing parameters are used to determine specific peak-trough fluctuations for the treatment of specific diseases, conditions, and symptoms, and those fluctuations are used as a reference standard.

[0133] For most drugs, plasma drug concentrations decrease in a multi-exponential manner. Immediately following intravenous administration, the drug rapidly distributes throughout the initial space (defined as a minimum of plasma volume), followed by a slower, more equilibrium distribution into extravascular spaces (e.g., certain tissues). Intravenous administration of IL-10 is associated with this type of two-compartment kinetic model (see Rachmawati, H. et al. (2004) Pharm. Res. 21(11):2072-78). The pharmacokinetics of subcutaneous recombinant hIL-10 have also been investigated (Radwanski, E. et al. (1998) Pharm. Res. 15(12):1895-1901). Therefore, the volume of distribution factor is relevant when assessing appropriate dose-related parameters for IL-10. Moreover, efforts have been made to explore how to target IL-10 agents to specific cell types (see, for example, Rachmawati, H. (May 2007) Drug Met. Dist. 35(5):814-21), and the utilization of IL-10 pharmacokinetics and administration mechanisms can attest to the invaluable success of such efforts.

[0134] This disclosure considers any dosage and administration regimen that results in the maintenance of any of the aforementioned serum trough concentrations of IL-10. For example, but not as a limitation, when the subject is human, non-PEGylated hIL-10 may be administered at doses higher than 0.5 μg / kg / day, higher than 1.0 μg / kg / day, higher than 2.5 μg / kg / day, higher than 5 μg / kg / day, higher than 7.5 μg / kg, higher than 10.0 μg / kg, higher than 12.5 μg / kg, higher than 15 μg / kg / day, higher than 17.5 μg / kg / day, higher than 20 μg / kg / day, higher than 22.5 μg / kg / day, higher than 25 μg / kg / day, higher than 30 μg / kg / day, or higher than 35 μg / kg / day. Additionally, for example, but not as a limitation, when the subject is human, dosages can be higher than 0.5 µg / kg / day, higher than 0.75 µg / kg / day, higher than 1.0 µg / kg / day, higher than 1.25 µg / kg / day, higher than 1.5 µg / kg / day, higher than 1.75 µg / kg / day, higher than 2.0 µg / kg / day, higher than 2.25 µg / kg / day, higher than 2.5 µg / kg / day, higher than 2.75 µg / kg / day, and higher than 3.0 µg / kg / day. Doses of PEGylated hIL-10 containing relatively small amounts of PEG (e.g., 5 kDa mono-PEG-hIL-10, di-PEG-hIL-10) are administered at doses above 3.25 µg / kg / day, above 3.5 µg / kg / day, above 3.75 µg / kg / day, above 4.0 µg / kg / day, above 4.25 µg / kg / day, above 4.5 µg / kg / day, above 4.75 µg / kg / day, or above 5.0 µg / kg / day.

[0135] While the preceding discussion of IL-10 serum concentrations, the necessary dosage and treatment regimens to achieve specific IL-10 serum concentrations, etc., pertains to monotherapy with IL-10 agents (e.g., PEG-IL-10), skilled technicians (e.g., pharmacologists) can determine the optimal dosing regimen when IL-10 agents (e.g., PEG-IL-10) are administered in combination with one or more adjunctive therapies.

[0136] Methods for generating IL-10 The polypeptides disclosed herein can be generated by any suitable method, including non-recombinant (e.g., chemical synthesis) and recombinant methods.

[0137] A. Chemical synthesis In the case of chemically synthesized peptides, synthesis can be carried out in either the liquid or solid phase. Solid-phase peptide synthesis (SPPS) allows for the incorporation of non-natural amino acids and / or peptide / protein backbone modifications. Various forms of SPPS, such as 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butyloxycarbonyl (Boc), can be used to synthesize the peptides disclosed herein. Details of the chemical synthesis are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. Chem. 6:3-10; and Camarero JA et al., (2005) Protein Pept Lett. 12:723-8).

[0138] Solid-phase peptide synthesis can be performed as described below. The α-functional group (Nα) and any reactive side chains are protected with acid- or base-labile groups. The protecting groups are stable under conditions where an amide bond is attached, but can be readily cleaved without damaging the already formed peptide chain. Suitable protecting groups for the α-amino functional group include, but are not limited to: Boc, benzyloxycarbonyl (Z), O-chlorobenzyloxycarbonyl, diphenylisopropoxycarbonyl, tert-pentyloxycarbonyl (Amoc), α,α-dimethyl-3,5-dimethoxy-benzyloxycarbonyl, o-nitrosulfinyl, 2-cyano-tert-butoxy-carbonyl, Fmoc, 1-(4,4-dimethyl-2,6-dioxocyclohexyl-1-ethylene)ethyl (Dde), etc.

[0139] Suitable side-chain protecting groups include, but are not limited to: acetyl, allyl (All), allyloxycarbonyl (Alloc), benzyl (Bzl), benzyloxycarbonyl (Z), tert-butoxycarbonyl (Boc), benzyloxymethyl (Bom), o-bromobenzyloxycarbonyl, tert-butyl (tBu), tert-butyldimethylsilyl, 2-chlorobenzyl, 2-chlorobenzyloxycarbonyl, 2,6-dichlorobenzyl, cyclohexyl, cyclopentyl, 1-(4,4-dimethyl-2,6-dioxocyclohexyl-1-yl)ethyl (Dde), isopropyl, 4-methoxy-2,3,6-trimethylbenzylsulfonyl (Mtr), 2,3,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), neopentyl, tetrahydropyran-2-yl, toluenesulfonyl (Tos), 2,4,6-trimethoxybenzyl, trimethylsilyl, and triphenylmethyl (Trt).

[0140] In solid-phase synthesis, C-terminal amino acids are coupled to a suitable support material. A suitable support material is one that is inert to the reagents and reaction conditions of the stepwise condensation and cleavage reactions in the synthesis process and is insoluble in the reaction medium used. Examples of commercially available support materials include styrene / divinylbenzene copolymers modified with reactive groups and / or polyethylene glycol; chloromethylated styrene / divinylbenzene copolymers; hydroxymethylated or aminomethylated styrene / divinylbenzene copolymers; and so on. When it is desired to prepare peptide acids, polystyrene (1%)-divinylbenzene or TentaGel® derivatized with 4-benzyloxybenzyl alcohol (Wang-anchor) or 2-chlorotriphenylmethyl chloride can be used. In the case of peptide amides, polystyrene (1%)-divinylbenzene or TentaGel® derivatized with 5-(4'-aminomethyl)-3',5'-dimethoxyphenoxy)valerate (PAL-anchor) or p-(2,4-dimethoxyphenyl-aminomethyl)phenoxy (Rink amide anchor) can be used.

[0141] The linkage with the polymer carrier can be achieved by adding an activating agent to ethanol, acetonitrile, N,N-dimethylformamide (DMF), dichloromethane, tetrahydrofuran, N-methylpyrrolidone or similar solvents, at room temperature or high temperature (e.g., between 40°C and 60°C) and reaction time, for example, 2 to 72 hours, to react the C-terminus Fmoc-protected amino acid with the carrier material.

[0142] The coupling of Nα-protected amino acids (e.g., Fmoc amino acids) with PAL, Wang, or Rink anchoring agents can be achieved, for example, by means of coupling agents such as N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC) or other carbodiimides, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU) or other urea salts, O-acyl-urea, benzotriazol-1-yl-tripyrrolidinyl-phosphonium hexafluorophosphate (PyBOP) or other phosphonium salts, N-hydroxysuccinimide, other N-hydroxyimides, or oximes, in the presence of... The reaction is carried out in the presence or absence of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole, for example, by means of TBTU with added HOBt, with or without the addition of a base such as diisopropylethylamine (DIEA), triethylamine or N-methylmorpholine (e.g. diisopropylethylamine), for a reaction time of 2-72 hours (e.g., 3 hours in a solvent such as dimethylformamide, N-methylpyrrolidone or dichloromethane, for example, in a 1.5 to 3-fold excess of amino acids and coupling agents, for example, 2-fold excess, and at a temperature between about 10°C and 50°C, for example, 25°C).

[0143] Instead of coupling reagents, active esters (such as pentafluorophenyl, p-nitrophenyl, etc.), symmetrical anhydrides of Nα-Fmoc-amino acids, their acyl chlorides or acyl fluorides can also be used in the above cases.

[0144] Nα-protected amino acids (e.g., Fmoc amino acids) can be coupled with 2-chlorotriphenylmethyl resin in dichloromethane with added DIEA for a reaction time of 10 to 120 minutes, such as 20 minutes, but not limited to the use of this solvent and this base.

[0145] The sequential coupling of protected amino acids can be performed according to conventional methods in peptide synthesis, typically in an automated peptide synthesizer. After cleaving the Nα-Fmoc protecting group of the coupled amino acid on the solid phase by treating it with piperidine (10% to 50%) in dimethylformamide for 5 to 20 minutes, or with 50% piperidine in DMF for 2 × 2 minutes and with 20% piperidine in DMF for 1 × 15 minutes, a 3 to 10-fold excess (e.g., 10-fold excess) of the next protected amino acid is coupled with the previous amino acid in an inert, non-aqueous, polar solvent such as dichloromethane, DMF, or a mixture of both, at a temperature of about 10°C to 50°C (e.g., 25°C). The reagents mentioned above for coupling the first Nα-Fmoc amino acid with PAL, Wang, or Rink anchoring agents are suitable as coupling agents. The active ester of the protected amino acid or its chloride or fluoride, or symmetrical anhydride, can also be used as alternatives.

[0146] At the end of solid-phase synthesis, the peptide is cleaved from the support material, simultaneously cleaving the side-chain protecting groups. Cleavage can be performed in trifluoroacetic acid or other strongly acidic media with the addition of 5%–20% v / v scavengers such as dimethyl sulfide, ethyl methyl sulfide, anisole, thiocresol, m-cresol, anisole dithiol, phenol, or water, for example, 15% v / v 1:1:1 dimethyl sulfide / dithiol / m-cresol, over 0.5 to 3 hours, e.g., 2 hours. Peptides with fully protected side chains are obtained by cleavage of the 2-chlorotriphenylmethyl anchoring agent with 2:2:6 glacial acetic acid / trifluoroethanol / dichloromethane. The protected peptide can be purified by silica gel chromatography. If the peptide is attached to the solid phase via the Wang anchoring agent and if the intention is to obtain a peptide with C-terminal alkylamidation, cleavage can be performed by ammonolysis with alkylamines or fluoroalkylamines. Ammonolysis is carried out at a temperature of about -10°C to 50°C (e.g., about 25°C) and for a reaction time of about 12 to 24 hours (e.g., about 18 hours). Alternatively, the peptide can be cleaved from the support by, for example, re-esterification with methanol.

[0147] The resulting acidic solution can be mixed with 3 to 20 times the volume of cold ether or n-hexane, for example, 10 times the excess of diethyl ether, to precipitate the peptide and thus separate the scavenging agent and cleaved protecting groups remaining in the ether. Further purification can be achieved by reprecipitating the peptide several times from glacial acetic acid. The resulting precipitate can be placed in water or tert-butanol or a mixture of both solvents, such as a 1:1 mixture of tert-butanol and water, and then freeze-dried.

[0148] The obtained peptides can be purified by a variety of chromatographic methods, including ion exchange on a weakly basic resin in acetate form; hydrophobic adsorption chromatography on non-derivative polystyrene / divinylbenzene copolymers (e.g., Amberlite® XAD); adsorption chromatography on silica gel; ion exchange chromatography, for example on carboxymethyl cellulose; partition chromatography, for example on Sephadex® G-25; countercurrent partition chromatography; or high-performance liquid chromatography (HPLC), for example, reversed-phase HPLC on an octyl or octadecylsilyl silica (ODS) phase.

[0149] B. Recombination generation Methods describing the preparation of human and mouse IL-10 can be found, for example, in U.S. Patent No. 5,231,012, which teaches methods for generating proteins with IL-10 activity, including recombinant and other synthetic techniques. IL-10 can be viral, and the cloning and expression of viral IL-10 from Ebola virus (BCRF1 protein) is disclosed in Moore et al. (1990) Science 248:1230. IL-10 can be obtained in a variety of ways using standard techniques known in the art, such as those described herein. Recombinant human IL-10 is also commercially available, for example from PeproTech, Inc., Rocky Hill, NJ.

[0150] When using recombinant technology to generate peptides, any suitable construct and any suitable host cell can be used to generate peptides that are intracellular or secreted proteins. The host cell can be a prokaryotic or eukaryotic cell, such as bacterial (e.g., *E. coli*) or yeast host cells. Other examples of eukaryotic cells that can be used as host cells include insect cells, mammalian cells, and / or plant cells. When using mammalian host cells, these can include human cells (e.g., HeLa, 293, H9, and Jurkat cells); mouse cells (e.g., NIH3T3, L, and C127 cells); primate cells (e.g., Cos 1, Cos 7, and CV1); and hamster cells (e.g., Chinese hamster ovary (CHO) cells).

[0151] Various host-vector systems suitable for expressing peptides can be used according to standard procedures known in the art. See, for example, Sambrook et al., 1989 Current Protocols in Molecular Biology, Cold Spring Harbor Press, New York; and Ausubel et al., 1995 Current Protocols in Molecular Biology, edited by Wiley and Sons. Methods for introducing genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate methods, etc. The transfer method can be selected to provide stable expression of the introduced nucleic acid encoding the peptide. The nucleic acid encoding the peptide can be provided as a heritable episodic element (e.g., a plasmid) or can be integrated into the genome. Various suitable vectors for generating the target peptide are commercially available.

[0152] Vectors can provide extrachromosomal maintenance within host cells or integration into the host cell genome. Expression vectors provide transcriptional and translational regulatory sequences and can provide inducible or constitutive expression, wherein coding regions are operatively linked under transcriptional control of transcription initiation and translation termination regions. Typically, transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. Promoters can be constitutive or inducible and can be strongly constitutive promoters (e.g., T7).

[0153] Expression constructs typically have appropriate restriction sites located near the promoter sequence to allow for the insertion of a nucleic acid sequence encoding the target protein. Operable selection markers may be present in the expression host to facilitate selection of cells containing the vector. Furthermore, expression constructs may include additional elements. For example, the expression vector may have one or two replication systems, thereby enabling its retention in an organism, such as for expression in mammalian or insect cells and for cloning and amplification in prokaryotic hosts. Additionally, expression constructs may contain selection marker genes to allow selection of transformed host cells. Selection genes are well known in the art and will vary depending on the host cell used.

[0154] Protein isolation and purification can be performed according to methods known in the art. For example, proteins can be isolated from cell lysates that have been genetically modified to constitutively and / or express proteins after induction, or from synthetic reaction mixtures by immunoaffinity purification, which typically involves contacting the sample with an anti-protein antibody, washing to remove non-specifically bound substances, and eluting specifically bound proteins. The isolated proteins can be further purified by dialysis and other methods commonly used for protein purification. In one embodiment, metal chelate chromatography can be used to separate proteins. Proteins may be modified to facilitate separation.

[0155] A polypeptide can be prepared in a substantially pure or isolated form (e.g., free from other polypeptides). This polypeptide can be present in a composition that is rich in it relative to other components that may be present (e.g., other polypeptides or other host cell components). For example, a purified polypeptide can be provided such that it is present in a composition substantially free from other expressed proteins, such as less than about 90%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1%.

[0156] Recombinant techniques can be used to generate IL-10 peptides by manipulating various IL-10-associated nucleic acids known in the art to provide constructs capable of encoding IL-10 peptides. It should be recognized that, given a specific amino acid sequence, a person skilled in the art, based on their background and experience, such as molecular biology, will recognize a variety of different nucleic acid molecules encoding such amino acid sequences.

[0157] Amide bond substitution In some cases, IL-10 includes one or more bonds other than peptide bonds, for example, at least two adjacent amino acids linked by a bond other than an amide bond. For example, one or more amide bonds within the IL-10 backbone may be substituted to reduce or eliminate undesirable proteolysis or other degradation pathways, and / or increase serum stability, and / or limit or increase conformational flexibility.

[0158] In another instance, one or more amide bonds (-CO-NH-) in IL-10 may be replaced by isosteric amide bonds, such as -CH2NH-, -CH2S-, -CH2CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, or -CH2SO-. One or more amide bonds in IL-10 may also be replaced by, for example, reduced isosteric pseudopeptide bonds. See Couder et al. (1993) Int. J. Peptide Protein Res. 41:181-184. Such replacements and their implementation methods are known to those skilled in the art.

[0159] Amino acid substitution One or more amino acid substitutions can be made in the IL-10 peptide. The following are non-limiting examples: a) Replacing hydrophobic amino acids substituted with alkyl groups, including alanine, leucine, isoleucine, valine, ortholeucine, (S)-2-aminobutyric acid, (S)-cyclohexylalanine or other simple α-amino acids substituted with aliphatic side chains from C1-C10 carbons, including branched, cyclic and straight-chain alkyl, alkenyl or alkynyl substitutions. b) Replacing aromatically substituted hydrophobic amino acids, including phenylalanine, tryptophan, tyrosine, sulfotyrosine, biphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 2-benzothiophene alanine, 3-benzothiophene alanine, and histidine, including the above aromatic amino acids in forms substituted with amino, alkylamino, dialkylamino, aza, halogenated (fluorine, chlorine, bromine, or iodine), or alkoxy (from C1-C4), illustrative examples being 2-, 3-, or 4-amino... 2-, 3- or 4-chlorophenylalanine, 2-, 3- or 4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or 5-methoxytryptophan, 2'-, 3'- or 4'-amino-, 2'-, 3'- or 4'-chloro-, 2, 3- or 4-biphenylalanine, 2'-, 3'- or 4'-methyl-, 2-, 3- or 4-biphenylalanine and 2- or 3-pyridylalanine; c) Substitution of amino acids containing basic side chains, including arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, including the aforementioned amino acids substituted with alkyl, alkenyl, or aryl groups (C1-C2). 10Branched, straight, or cyclic derivatives, regardless of whether the substitution is on a heteroatom (e.g., on an α-nitrogen or one or more distal nitrogen atoms, or on an α-carbon, e.g., at the pre-R position). Illustrative examples include: N-ε-isopropyl-lysine, 3-(4-tetrahydropyridyl)glycine, 3-(4-tetrahydropyridyl)alanine, and N,N-γ,γ'-diethyl-homogeneous. Also included are compounds in which an alkyl group occupies the pre-R position of the α-carbon, such as α-methyl-arginine, α-methyl-2,3-diaminopropionic acid, α-methyl-histidine, and α-methyl-ornithine. Also included are compounds composed of any of the following: alkyl, aromatic, heteroaromatic (wherein the heteroaromatic group has one or more nitrogen, oxygen, or sulfur atoms, alone or in combination), carboxylic acid, or many known activated derivatives such as acyl chlorides, active esters, active azolides and related derivatives, as well as lysine, ornithine, or 2,3-diaminopropionic acid; d) Alkyl, aryl, aralkyl and heteroaryl sulfonamides and tetrazolium-substituted alkyl amino acids, including aspartic acid, glutamic acid, homoglutamic acid, tyrosine, 2,4-diaminopropionic acid, ornithine or lysine. e) Substituted side-chain amide residues, including asparagine, glutamine, and alkyl or aromatic substituted derivatives of asparagine or glutamine; and f) Substitution of hydroxyl-containing amino acids, including serine, threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromatic derivatives of serine or threonine.

[0160] In some cases, IL-10 contains one or more naturally occurring, non-genetically encoded L-amino acids, synthetic L-amino acids, or D-enantiomers of amino acids. For example, IL-10 may contain only D-amino acids. For example, IL-10 peptides may contain one or more of the following residues: hydroxyproline, β-alanine, o-aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid, m-aminomethylbenzoic acid, 2,3-diaminopropionic acid, α-aminoisobutyric acid, N-methylglycine (sarcosine), ornithine, citrulline, tert-butylalanine, tert-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, ortholeucine, naphthylalanine, pyridylalanine, 3-benzothiophene alanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine. Alanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, β-2-thienylalanine, methionine sulfoxide, arginine, N-acetyllysine, 2,4-diaminobutyric acid, rho-aminophenylalanine, N-methylvaline, homocysteine, homoserine, ε-aminohexanoic acid, ω-aminohexanoic acid, ω-aminoheptanoic acid, ω-aminooctanoic acid, ω-aminodecanoic acid, ω-aminotetradecanoic acid, cyclohexylalanine, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, δ-aminovaleric acid, and 2,3-diaminobutyric acid.

[0161] Other modifications Cysteine ​​residues or cysteine ​​analogs can be introduced into the IL-10 peptide to provide a connection to another peptide via a disulfide bond or to provide cyclization of the IL-10 peptide. Methods for introducing cysteine ​​residues or cysteine ​​analogs are known in the art; see, for example, U.S. Patent No. 8,067,532.

[0162] The IL-10 peptide can be cyclized. One or more cysteine ​​residues or cysteine ​​analogs can be introduced into the IL-10 peptide, wherein the introduced cysteine ​​residue or cysteine ​​analog can form a disulfide bond with a second introduced cysteine ​​residue or cysteine ​​analog. Other cyclization methods include the introduction of an oxime linker or a lanethionine linker; see, for example, U.S. Patent No. 8,044,175. Any combination of amino acids (or non-amino acid moieties) capable of forming cyclization bonds can be used and / or introduced. Amino acids (or amino acids and -(CH2)) with functional groups that allow the introduction of bridges can be used. n -CO- or -(CH2) n Any combination of -C6H4-CO-) can form a cyclic bond. Some examples are disulfides, and disulfide analogs such as -(CH2). n - Carbon bridges, thioacetals, thioether bridges (cystathionine or lanathionine), and bridges containing esters and ethers. In these examples, n can be any integer, but is usually less than ten.

[0163] Other modifications include, for example, N-alkyl (or aryl) substitution (ψ[CONR]), or crosslinking of the main chain with amides and other cyclic structures within the building block. Other derivatives include C-terminal hydroxymethyl derivatives, o-modified derivatives (e.g., C-terminal hydroxymethyl benzyl ethers), and N-terminal modified derivatives, including substituted amides such as alkylamides and hydrazides.

[0164] In some cases, one or more L-amino acids in the IL-10 polypeptide are replaced by one or more D-amino acids.

[0165] In some cases, IL-10 peptides are retroinverso analogs (see, for example, Sela and Zisman (1997) FASEB J. 11:449). Retroinverso peptide analogs are isomers of linear peptides in which the orientation of the amino acid sequence is reversed (reverse), and, for example, the chirality (D or L) of one or more amino acids is reversed (reverse) by using D-amino acids instead of L-amino acids. [See, for example, Jameson et al. (1994) Nature 368:744; and Brady et al. (1994) Nature 368:692].

[0166] IL-10 peptides may include a “protein transduction domain” (PTD), which refers to a peptide, polynucleotide, carbohydrate, or organic or inorganic molecule that facilitates passage through lipid bilayers, micelles, cell membranes, organelle membranes, or vesicle membranes. A PTD attached to another molecule facilitates the molecule's passage across membranes, for example, from the extracellular space to the intracellular space, or from the cytosol to organelles. In some embodiments, the PTD is covalently linked to the N-terminus of the IL-10 peptide, while in other embodiments, the PTD is covalently linked to the C-terminus of the IL-10 peptide. Exemplary protein transduction domains include, but are not limited to, protein transduction domains of peptides containing at least eleven amino acids (corresponding to residues 47-57 of HIV-1 TAT containing YGRKKRRQRRR; SEQ ID NO: 1); polyarginine sequences containing a number of arginine residues sufficient to guide entry into the cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or 10-50 arginines); VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6): 489-96); Drosophila antennal foot protein transduction domain (Noguchi et al. (2003) Diabetes 52(7): 1732-1737); truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21: 1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA). 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO: 2); the transmembrane peptide GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 3); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 4); and RQIKIWFQNRRMKWKK (SEQ ID NO: 5). Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO:1), RKKRRQRRR (SEQ ID NO:6); arginine homopolymers from 3 to 50 arginine residues; exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO:1); RKKRRQRR (SEQ ID NO:7); YARAAARQARA (SEQ ID NO:8); THRLPRRRRRR (SEQ ID NO:9); and GGRRARRRRRR (SEQ ID NO:10).

[0167] The carboxyl group (COR3) of the amino acid at the C-terminus of the IL-10 polypeptide can exist in a free form (R3=OH) or in the form of a physiologically tolerant base or alkaline earth metal salt (e.g., sodium, potassium, or calcium salt). The carboxyl group can also be esterified with primary, secondary, or tertiary alcohols such as methanol, or branched or unbranched C1-C6 alkyl alcohols such as ethanol or tert-butanol. The carboxyl group can also be amidated with primary or secondary amines such as ammonia, or branched or unbranched C1-C6 alkylamines or C1-C6 dialkylamines such as methylamine or dimethylamine.

[0168] The amino group of the amino acid NR1R2 at the N-terminus of the IL-10 peptide can be in a free form (R1=H and R2=H) or in the form of a physiologically tolerant salt (e.g., chloride or acetate). The amino group can also be acid-acetylated, such that R1=H and R2=acetyl, trifluoroacetyl, or adamantyl. The amino group can be protected by amino protecting groups commonly used in peptide chemistry, such as those provided above (e.g., Fmoc, benzyloxy-carbonyl(Z), Boc, and Alloc). The amino group can be N-alkylated, wherein R1 and / or R2=C1-C6 alkyl, C2-C8 alkenyl, or C7-C9 aralkyl. The alkyl group can be straight-chain, branched, or cyclic (e.g., ethyl, isopropyl, and cyclohexyl, respectively).

[0169] Specific modifications to enhance and / or simulate IL-10 functionality Improving one or more physical properties and / or administration methods of the therapeutic modifiers disclosed herein (e.g., IL-10) is generally beneficial and sometimes necessary. Improvements to physical properties include, for example, modulating immunogenicity; increasing water solubility, bioavailability, serum half-life, and / or therapeutic half-life; and / or modulating biological activity. Certain modifications may also be used, for example, to generate antibodies (e.g., epitope tags) for detection assays and to facilitate protein purification. Such improvements must generally be conferred without adversely affecting the biological activity of the therapeutic modifier and / or increasing its immunogenicity.

[0170] Polyglycolization of IL-10 is one specific modification considered in this disclosure, while other modifications include, but are not limited to, glycosylation (N- and O-linked); polysialylation; albumin fusion molecules comprising serum albumin (e.g., human serum albumin (HSA), cyno serum albumin, or bovine serum albumin (BSA)); albumin binding via, for example, conjugated fatty acid chains (acylation); and Fc-fusion proteins.

[0171] Polyethylene glycolationThe clinical efficacy of protein therapeutics is often limited by short plasma half-lives and susceptibility to protease degradation. Studies of various therapeutic proteins (e.g., filgrastim) have demonstrated that these difficulties can be overcome through various modifications, including conjugating or linking polypeptide sequences to any of a variety of non-protein polymers such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyethylene. This is typically achieved by covalently binding the linker portion of the protein and non-protein polymers (e.g., PEG). Such PEG-conjugated biomolecules have been shown to possess clinically useful properties, including improved physical and thermal stability, reduced susceptibility to enzymatic degradation, increased solubility, longer circulating half-life and reduced clearance, decreased immunogenicity and antigenicity, and reduced toxicity.

[0172] In addition to the beneficial effects of PEGylation on pharmacokinetic parameters, PEGylation itself can enhance activity. For example, PEG-IL-10 has been shown to be more effective against certain cancers than unPEGylated IL-10 (see, for example, EP206636A2).

[0173] PEGs suitable for conjugating peptide sequences are generally soluble in water at room temperature and have the general formula R(O-CH2-CH2). n OR, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and where n is an integer from 1 to 1000. When R is a protecting group, it typically has 1 to 8 carbons. The PEG conjugated to the polypeptide sequence can be linear or branched. This disclosure contemplates branched PEG derivatives, "star-PEG", and multi-arm PEG. The molecular weight of the PEG used in this disclosure is not limited to any particular range, and examples are given elsewhere herein; for example, some embodiments have a molecular weight between 5 kDa and 20 kDa, while other embodiments have a molecular weight between 4 kDa and 10 kDa.

[0174] This disclosure also contemplates compositions of conjugates in which PEGs have different n values, and thus various different PEGs are present in specific proportions. For example, some compositions comprise mixtures of conjugates in which n=1, 2, 3, and 4. In some compositions, the percentage of the conjugate in which n=1 is 18-25%, the percentage of the conjugate in which n=2 is 50-66%, the percentage of the conjugate in which n=3 is 12-16%, and the percentage of the conjugate in which n=4 is up to 5%. Such compositions can be generated by reaction conditions and purification methods known in the art. Exemplary reaction conditions are described throughout the specification. Conjugates can be separated using cation exchange chromatography, and fractions containing conjugates with, for example, a desired number of linked PEGs can be identified, purified from unmodified protein sequences, and purified from conjugates with other numbers of linked PEGs.

[0175] Polyglycolation most commonly occurs at the α-amino group at the N-terminus of peptides, the ε-amino group on the lysine side chain, and the imidazole group on the histidine side chain. Since most recombinant peptides have a single α-amino group and multiple ε-amino and imidazole groups, many positional isomers can be generated depending on the linker chemistry. General polyethylene glycolation strategies known in the art can be applied herein.

[0176] Two widely used first-generation activated monomethoxy PEGs (mPEGs) are succinimide carbonate PEG (SC-PEG; see, e.g., Zalipsky et al. (1992) Biotönol. Appl. Biochem 15:100-114; and Miron and Wilcheck (1993) Bio-conjug. Chem. 4:568-569) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., Dolence et al., U.S. Patent No. 5,650,234), which preferentially react with lysine residues to form carbamate bonds, but are also known to react with histidine and tyrosine residues. Bonding to histidine residues on certain molecules (e.g., IFNα) has been shown to be hydrolyzed unstable imidazole carbamate bonds (see, e.g., Lee and McNemar, U.S. Patent No. 5,985,263). Second-generation polyethylene glycolation techniques have been designed to avoid these unstable bonds and the lack of selectivity in terms of residual reactivity. The PEG-aldehyde linker is used to target a single site at the N-terminus of a peptide via reductive amination.

[0177] PEG can be bound to the disclosed polypeptide via a terminal reactive group (“spacer region”) mediating the bond between a free amino or carboxyl group of one or more polypeptide sequences and polyethylene glycol. PEGs having a spacer region that can bind to a free amino group include N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating a succinate of polyethylene glycol with N-hydroxysuccinimide. Another activated polyethylene glycol that can bind to a free amino group is 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine, which can be prepared by reacting polyethylene glycol monomethyl ether with cyanuric chloride. Activated polyethylene glycols that bind to a free carboxyl group include polyoxyethylenediamine.

[0178] The conjugation of one or more polypeptide sequences of this disclosure with PEG having spacer regions can be carried out by a variety of conventional methods. For example, the conjugation reaction can be carried out in a solution with a pH of 5 to 10 at a temperature of 4°C to room temperature, using a reagent with a molar ratio of 4:1 to 30:1 with the protein for 30 minutes to 20 hours. Reaction conditions can be selected to guide the reaction to produce the desired degree of substitution. Generally, low temperature, low pH (e.g., pH=5), and short reaction time tend to reduce the number of PEGs linked, while high temperature, neutral to high pH (e.g., pH≥7), and longer reaction time tend to increase the number of PEGs linked. The reaction can be terminated using a variety of means known in the art. In some embodiments, the reaction is terminated by acidifying the reaction mixture and freezing it, for example, at -20°C. Polyethylene glycolation of various molecules is discussed in, for example, U.S. Patent Nos. 5,252,714, 5,643,575, 5,919,455, 5,932,462, and 5,985,263. PEG-IL-10 is described, for example, in U.S. Patent No. 7,052,686. Specific reaction conditions considered for use in this paper are listed in the experimental section.

[0179] This disclosure also considers the use of PEG mimics. Recombinant PEG mimics have been developed that retain the properties of PEG (e.g., increased serum half-life) while conferring several additional advantageous properties. For example, simple polypeptide chains capable of forming extended conformations similar to PEG (containing, for example, Ala, Glu, Gly, Pro, Ser, and Thr) can be recombined and fused with target peptide or protein drugs (e.g., Amunix's XTEN technology; Mountain View, CA). This avoids the need for additional conjugation steps in the production process. Moreover, the established molecular biology techniques enable control over the side-chain composition of the polypeptide chain, thereby allowing for optimization of immunogenicity and production properties.

[0180] Glycosylation For the purposes of this disclosure, "glycosylation" is intended to broadly refer to the enzymatic process of linking a glycan to a protein, lipid, or other organic molecule. When used in conjunction with this disclosure, the term "glycosylation" is generally intended to indicate the addition or deletion of one or more carbohydrate moieties (by removing potential glycosylation sites or by chemical and / or enzymatic means of deleting glycosylation sites) and / or the addition of one or more glycosylation sites that may or may not be present in the native sequence. Furthermore, the phrase encompasses qualitative changes in the glycosylation of native proteins, involving variations in the characteristics and proportions of the various carbohydrate moieties present.

[0181] Glycosylation can significantly affect the physical properties (e.g., solubility) of peptides such as IL-10, and may also be important in protein stability, secretion, and subcellular localization. Glycosylated peptides can also exhibit enhanced stability or improve one or more pharmacokinetic properties, such as half-life. Furthermore, improved solubility can, for example, enable formulations that are more suitable for drug administration than those containing non-glycosylated peptides.

[0182] The addition of glycosylation sites can be accomplished by altering the amino acid sequence. For example, the polypeptide can be modified by adding or substituting one or more serine or threonine residues (for O-linked glycosylation sites) or asparagine residues (for N-linked glycosylation sites). The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type can differ. One type of sugar commonly found in both is N-acetylneuraminic acid (hereinafter referred to as sialic acid). Sialic acid is typically a terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, can impart acidity to glycoproteins. Specific embodiments of this disclosure include the generation and use of N-glycosylated variants.

[0183] The polypeptide sequence disclosed herein can be optionally altered by changes at the nucleic acid level, particularly by mutating the nucleic acid encoding the polypeptide at a preselected base, thereby generating a codon that will be translated into the desired amino acid.

[0184] Polysialylation This disclosure also considers the use of polysialylation to conjugate peptides with naturally occurring, biodegradable α-(2→8)-linked polysialic acid (“PSA”) to improve peptide stability and in vivo pharmacokinetics. PSA is a biodegradable, nontoxic natural polymer with high hydrophilicity, which imparts a high apparent molecular weight in the blood, increasing its serum half-life. In addition, polysialylation of a range of peptide and protein therapeutics has resulted in a significant reduction in proteolysis, preservation of in vivo activity, and reduction in immunogenicity and antigenicity (see, for example, G. Gregoriadis et al., Int. J. Pharmaceutics 300(1-2):125-30). Various techniques for site-specific polysialylation are available (see, for example, T. Lindhout et al., PNAS 108(18)7397-7402 (2011)).

[0185] Albumin fusion Additional suitable components and molecules used for conjugation include albumins, such as human serum albumin (HSA), macaque serum albumin, and bovine serum albumin (BSA).

[0186] According to this disclosure, albumin can be conjugated to drug molecules (e.g., polypeptides described herein) at the carboxyl terminus, amino terminus, carboxyl terminus and amino terminus, and internally (see, for example, USP 5,876,969 and USP 7,056,701).

[0187] In the HSA-drug conjugates contemplated in this disclosure, various forms of albumin can be used, such as albumin pre-sequences and their variants, fragments and variants thereof, and HSA variants. Such forms typically possess one or more desired albumin activities. In other embodiments, this disclosure relates to fusion proteins comprising peptide drug molecules fused directly or indirectly to albumin, albumin fragments, and albumin variants, wherein the fusion protein exhibits higher plasma stability than the unfused drug molecule, and / or the fusion protein retains the therapeutic activity of the unfused drug molecule. In some embodiments, indirect fusion is achieved via a linker, such as a peptide linker or a modified form thereof.

[0188] As described above, the fusion of albumin with one or more polypeptides of this disclosure can be achieved, for example, through gene manipulation, such that a nucleic acid encoding HSA or a fragment thereof is linked to a nucleic acid encoding the one or more polypeptide sequences.

[0189] Alternative albumin binding strategies: Several albumin-binding strategies have been developed as alternatives to direct fusion and can be used with the IL-10 agents described herein. For example, this disclosure considers albumin binding via conjugated fatty acid chains (acylation) and albumin binding via fusion proteins comprising a polypeptide sequence containing an albumin-binding domain (ABD) and the sequences of one or more polypeptides described herein.

[0190] Combining with other molecules Additional suitable components and molecules used for conjugation include, for example, thyroglobulin; tetanus toxoid; diphtheria toxoid; polyamino acids such as poly(D-lysine:D-glutamic acid); rotavirus VP6 polypeptide; influenza virus hemagglutinin, influenza virus nucleoprotein; keyhole hemocyanin (KLH); and hepatitis B virus core protein and surface antigen; or any combination thereof.

[0191] Therefore, this disclosure contemplates conjugating one or more additional components or molecules, such as another polypeptide (e.g., a polypeptide having an amino acid sequence heterologous to the subject polypeptide) or a carrier molecule, to the N- and / or C-terminus of a polypeptide sequence. Thus, exemplary polypeptide sequences can be provided as conjugates with another component or molecule.

[0192] The IL-10 peptide can also be conjugated with large, slowly metabolized macromolecules such as proteins; polysaccharides such as agarose gels, agarose, cellulose, or cellulose beads; polymeric amino acids such as polyglutamic acid or polylysine; amino acid copolymers; inactivated viral particles; inactivated bacterial toxins, such as toxoids derived from diphtheria, tetanus, cholera, or leukocyte toxin molecules; inactivated bacteria; and dendritic cells. If desired, such conjugations can be used to generate antibodies against the disclosed peptide.

[0193] Additional candidate components and molecules for conjugation include those suitable for isolation or purification. Specific non-limiting examples include binding molecules such as biotin (a biotin-avidin specific binding pair), antibodies, receptors, ligands, lectins, or molecules containing a solid support, including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips, and membranes.

[0194] Fc-fusion molecule In some embodiments, the amino or carboxyl terminus of the disclosed polypeptide sequence may be fused to an immunoglobulin Fc region (e.g., human Fc) to form a fusion conjugate (or fusion molecule). Fc fusion conjugates have been shown to increase the systemic half-life of biologics, and therefore the biologics product may require infrequent administration.

[0195] Fc binds to nascent Fc receptors (FcRn) in the endothelial cells lining blood vessels, and upon binding, the Fc fusion molecule is protected from degradation and re-release into circulation, thus maintaining a longer molecular circulation. This Fc binding is believed to be the mechanism by which endogenous IgG maintains its long plasma half-life. Compared to traditional Fc-fusion conjugates, recent Fc-fusion technologies link a single copy of a biologic to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biologic.

[0196] Other modifications This disclosure contemplates the use of other IL-10 modifications, either currently known or to be developed in the future, to improve one or more properties. Examples include hydroxyethyl starchization, aspects of which are described, for example, in U.S. Patent Applications Nos. 2007 / 0134197 and 2006 / 0258607, and fusion molecules containing SUMO as a fusion tag (LifeSensors, Inc.; Malvern, PA).

[0197] connectorThe linker and its uses have been described above. Any of the aforementioned components and molecules used to modify the polypeptide sequence of this disclosure may optionally be conjugated via a linker. Suitable linkers include “flexible linkers” which are generally of sufficient length to allow some movement between the modified polypeptide sequence and the linked component or molecule. Linker molecules are typically about 6-50 atoms long. Linker molecules may also be, for example, arylaceyne, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. Suitable linkers can be readily selected and can have any suitable length, such as 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50, or more than 50 amino acids.

[0198] Examples of flexible joints include glycine polymers (G). n Glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers (e.g., (GmSo)n, (GSGGS) n (SEQ ID NO:11), (G m S o G m ) n 、(G m S o G m S o G m ) n (SEQ ID NO:12), (GSGGS) m ) n (SEQ ID NO:11), (GSGS) m G) n (SEQ ID NO:12) and (GGGS) m ) n (SEQ ID NO:13) and combinations thereof, wherein m, n, and o are each independently selected from at least 1 to 20 integers, such as 1-18, 2-16, 3-14, 4-12, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) and other flexible joints. Glycine and glycine-serine polymers are relatively unstructured and can therefore serve as neutral linkages between components. Examples of flexible joints include, but are not limited to, GGSG (SEQ ID NO:14), GGSGG (SEQ ID NO:15), GGSG (SEQ ID NO:12), GSGGG (SEQ ID NO:16), GGGSG (SEQ ID NO:17), and GSSSG (SEQ ID NO:18).

[0199] Other examples of flexible joints include glycine polymers (G). n Or glycine-serine polymers (e.g., (GS)). n (GSGGS) n (SEQ ID NO:11), (GGGS) n (SEQ ID NO:13) and (GGGGS) n (SEQ ID NO:19), where n=1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50). Exemplary flexible linkers include, but are not limited to, GGGS (SEQ ID NO:13), GGGGS (SEQ ID NO:19), GGSG (SEQ ID NO:14), GGSGG (SEQ ID NO:15), GGSSG (SEQ ID NO:12), GGSGG (SEQ ID NO:16), GGGSG (SEQ ID NO:17), and GSSSG (SEQ ID NO:18). Polymers of these linker sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) can be linked together to provide flexible linkers that can be used to conjugate heterologous amino acid sequences to the IL-10 agents disclosed herein. As described in this article, heterologous amino acid sequences can be signal sequences and / or fusion chaperones, such as albumin, Fc sequences, etc.

[0200] Therapeutic and preventive uses This disclosure contemplates the use of IL-10 agents (e.g., PEG-IL-10) described herein to prevent or reduce the severity of activation-induced cell death in patients undergoing CAR-T cell therapy. More specifically, the IL-10 agent is used in a method involving the modulation of a T-cell-mediated immune response to a target cell population in a subject, comprising introducing into the subject a plurality of therapeutically effective, genetically modified cells expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises at least one antigen-specific targeting region capable of binding to the target cell population, and wherein the binding of the chimeric antigen receptor targeting region to the target cell population induces activation-induced cell death.

[0201] In a particular embodiment, a therapeutically effective amount of IL-10 sufficient to prevent or limit activation-induced cell death is administered to the subject parenterally (e.g., subcutaneously). In other embodiments, a plurality of genetically modified cells expressing a chimeric antigen receptor and an amount of IL-10 sufficient to prevent or limit activation-induced cell death are introduced into the subject. In yet another embodiment, a therapeutically effective amount of IL-10 sufficient to prevent or limit activation-induced cell death is introduced into the subject via cells genetically modified to express the IL-10 agent, whereby the expression construct exists in cells distinct from those expressing the CAR.

[0202] The genetic material encoding the IL-10 agent can be introduced into cells in any manner known to those skilled in the art. Two main categories of methods are those using recombinant viruses (also known as viral vectors) and those using naked DNA or DNA complexes (non-viral methods). Examples of viruses that can be used include, but are not limited to, retroviruses, adenoviruses, and herpes simplex viruses. Examples of non-viral methods include, but are not limited to, injection of naked DNA, physical methods to enhance delivery (e.g., electroporation), and chemical methods to enhance delivery (e.g., lipoplexes).

[0203] In some embodiments of this disclosure, the vector (e.g., a viral vector) is genetically engineered to deliver a gene. The vector can be administered intravenously or injected directly into a specific tissue in the body, where it is taken up by individual cells. Optionally, a subset of cells from a subject can be removed and exposed to the vector in an ex vivo environment, followed by the return of the vector-containing cells to the patient. In a particular embodiment, the expression of the IL-10 agent is modulated by an expression control element.

[0204] CAR-T cell therapy, used in conjunction with the IL-10 agents described herein, can be used to treat or prevent proliferative diseases, conditions, or symptoms, including cancers such as uterine cancer, cervical cancer, breast cancer, prostate cancer, testicular cancer, gastrointestinal cancers (e.g., esophageal cancer, oropharyngeal cancer, stomach cancer, small bowel cancer, large bowel cancer, colon cancer, or rectal cancer), kidney cancer, renal cell carcinoma, bladder cancer, bone cancer, bone marrow cancer, skin cancer, head and neck cancer, liver cancer, gallbladder cancer, heart cancer, lung cancer, pancreatic cancer, salivary gland cancer, adrenal cancer, thyroid cancer, brain cancer (e.g., glioma), ganglion cancer, cancers of the central nervous system (CNS) and peripheral nervous system (PNS), and cancers of the hematopoietic and immune systems (e.g., spleen or thymus). This disclosure also provides methods for treating or preventing other cancer-related diseases, conditions, or symptoms, including, for example, immunogenic tumors, non-immunogenic tumors, dormant tumors, virus-induced cancers (e.g., epithelial carcinoma, endothelial carcinoma, squamous cell carcinoma, and papillomavirus), adenocarcinoma, lymphoma, carcinoma, melanoma, leukemia, myeloma, sarcoma, teratoma, chemically induced cancer, metastatic cancer, and angiogenesis. This disclosure contemplates reducing tolerance to tumor cell or cancer cell antigens, for example, by modulating the activity of regulatory T cells and / or CD8+ T cells (see, for example, Ramirez-Montagut et al. (2003) Oncogene 22:3180-87; and Sawaya et al. (2003) New Engl. J. Med. 349:1501-09). In certain embodiments, the tumor or cancer is colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma, or leukemia. The terms cancer-related diseases, symptoms, and conditions are used broadly to refer to conditions that are directly or indirectly related to cancer, and include, for example, angiogenesis and precancerous conditions such as developmental abnormalities.

[0205] In other embodiments, CAR-T cell therapy, used in conjunction with the IL-10 agents described herein, can be used to treat or prevent immune / inflammatory-related conditions. As used herein, terms such as “immune disease,” “immune condition,” “immune syndrome,” “inflammatory disease,” “inflammatory condition,” and “inflammatory syndrome” are intended to broadly encompass any immune or inflammatory-related condition (e.g., pathological inflammation and autoimmune diseases). Such conditions are often closely associated with other diseases, conditions, and symptoms. For example, “immune condition” can refer to proliferative conditions such as cancer, tumors, and angiogenesis; including resistance to infections (acute and chronic), tumors, and cancers that are eradicated by the immune system.

[0206] A non-limiting list of diseases, conditions, and symptoms related to immunity and inflammation includes arthritis (e.g., rheumatoid arthritis), kidney failure, lupus, asthma, psoriasis, colitis, pancreatitis, allergies, fibrosis, surgical complications (e.g., where inflammatory cytokines prevent healing), anemia, and fibromyalgia. Other diseases and conditions that may be associated with chronic inflammation include Alzheimer's disease, congestive heart failure, stroke, aortic stenosis, arteriosclerosis, osteoporosis, Parkinson's disease, infections, inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis), allergic contact dermatitis and other eczemas, systemic sclerosis, transplantation, and multiple sclerosis.

[0207] Pharmaceutical Composition When administering an IL-10 agent to a subject, this disclosure contemplates the use of any form of composition suitable for administration to a subject. Typically, such compositions are “pharmaceutical compositions” comprising IL-10 and one or more pharmaceutically or physiologically acceptable diluents, carriers, or excipients. Pharmaceutical compositions can be used in the methods of this disclosure; thus, for example, pharmaceutical compositions can be administered to a subject ex vivo or in vivo to practice the treatment and prevention methods and uses described herein.

[0208] The pharmaceutical compositions disclosed herein can be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are described herein. Furthermore, the pharmaceutical compositions can be used in combination with other therapeutically active agents or compounds described herein to treat or prevent the diseases, conditions, and symptoms considered in this disclosure.

[0209] Pharmaceutical compositions typically comprise a therapeutically effective amount of the IL-10 agent considered in this disclosure and one or more pharmaceutically and physiologically acceptable formulation agents. Suitable pharmaceutically or physiologically acceptable diluents, carriers, or excipients include, but are not limited to, antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methylparaben, ethylparaben, or n-propylparaben, paraben esters), emulsifiers, suspending agents, dispersants, solvents, fillers, fillers, detergents, buffers, mediators, diluents, and / or adjuvants. For example, a suitable mediator may be an aqueous solution of physiological saline or a citrate-buffered saline solution, possibly supplemented with other substances commonly found in pharmaceutical compositions intended for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary mediators. Those skilled in the art will readily recognize the various buffers that may be used in the pharmaceutical compositions and dosage forms considered herein. Typical buffers include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. For example, the buffer component can be a water-soluble substance such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and their salts. Acceptable buffers include, for example, Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), and N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

[0210] After formulation, the pharmaceutical composition can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations can be stored in ready-to-use form, lyophilized form requiring rehydration before use, liquid form requiring dilution before use, or other acceptable forms. In some embodiments, the pharmaceutical composition is provided in a single-use container (e.g., a single-use vial, ampoule, syringe, or auto-injector (similar to, for example, EpiPen®)), while in other embodiments, a multiple-use container (e.g., a multiple-use vial) is provided. Any drug delivery device can be used to deliver IL-10, including implants (e.g., implantable pumps) and catheter systems, slow-infusion pumps, and devices, all of which are well known to those skilled in the art. Accumulated injections, typically administered subcutaneously or intramuscularly, can also be used to release the peptides disclosed herein over a defined time period. Accumulated injections are typically solid- or oil-based and generally contain at least one of the formulation components listed herein. Those skilled in the art are familiar with the possible formulations and uses of accumulated injections.

[0211] Pharmaceutical compositions can be in the form of sterile injectable aqueous or oily suspensions. Such suspensions can be formulated using suitable dispersants or wetting agents and suspending agents mentioned herein, according to known techniques. Sterile injectable formulations can also be sterile injectable solutions or suspensions in non-toxic, parenteral-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable diluents, solvents, and dispersion media include water, Ringer's solution, isotonic sodium chloride solution, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS), ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Additionally, sterile, fixed oils are conventionally used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Furthermore, fatty acids such as oleic acid can be used to prepare injectable formulations. Prolonged absorption of certain injectable formulations can be achieved by including agents that delay absorption (e.g., aluminum monostearate or gelatin).

[0212] Pharmaceutical compositions containing an active ingredient may be in forms suitable for oral administration, such as tablets, capsules, lozenges, tablets, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules or syrups, solutions, microbeads or elixirs. In certain embodiments, the active ingredient of a pharmaceutically effective agent co-administered with the IL-10 pharmaceutical agent described herein is in a form suitable for oral administration. Pharmaceutical compositions intended for oral administration may be prepared according to any method known in the art for producing pharmaceutical compositions, and such compositions may contain one or more pharmaceutical agents, such as sweeteners, flavoring agents, coloring agents, and preservatives, to provide a pharmaceutically refined and palatable formulation. Tablets, capsules, etc., contain an active ingredient mixed with a non-toxic, pharmaceutically acceptable excipient suitable for the production of tablets. These excipients may be, for example, diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrants such as corn starch or alginic acid; binders such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc.

[0213] Tablets, capsules, etc., suitable for oral administration can be uncoated or coated using known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing sustained action. For example, delay-release materials such as glyceryl monostearate or glyceryl distearate can be used. They can also be coated using techniques known in the art to form osmotic therapeutic tablets for controlled release. Other pharmaceutical preparations include biodegradable or biocompatible particles or polymers such as polyesters, polyamines, hydrogels, polyvinylpyrrolidone, polyanhydrides, polyglycolic acid, ethylene-vinyl acetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide / glycolic acid copolymers, polylactide / glycolic acid copolymers, or ethylene-vinyl acetate copolymers to control the delivery of the administered composition. For example, oral pharmaceutical preparations can be encapsulated in microcapsules or colloidal drug delivery systems, with microcapsules prepared by coagulation techniques or by interfacial polymerization, using hydroxymethyl cellulose or gelatin microcapsules or poly(methyl methacrylate) microcapsules, respectively. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, microbeads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Methods for preparing the formulations mentioned above will be readily apparent to those skilled in the art.

[0214] Oral formulations may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate, kaolin, or microcrystalline cellulose, or as soft gelatin capsules in which the active ingredient is mixed with an aqueous or oily medium such as peanut oil, liquid paraffin, or olive oil.

[0215] Aqueous suspensions contain active substances mixed with excipients suitable for their production. Such excipients can be suspending agents, such as sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and gum arabic; dispersants or wetting agents, such as naturally occurring phospholipids (e.g., lecithin), or condensation products of olefinic oxygen and fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide and long-chain fatty alcohols (e.g., heptadecaethyleneoxycetyl alcohol), or condensation products of ethylene oxide and esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitan monooleate), or condensation products of ethylene oxide and esters derived from fatty acids and hexitol anhydrides (e.g., polyvinyl dehydrated sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives.

[0216] Oily suspensions can be formulated by suspending the active ingredient in vegetable oils (such as peanut oil, olive oil, sesame oil, or coconut oil) or mineral oils (such as liquid paraffin). Oily suspensions may contain thickeners such as beeswax, solid paraffin, or cetyl alcohol. Sweeteners, such as those listed above, and flavoring agents may be added to provide a palatable oral formulation.

[0217] Dispersible powders and granules suitable for preparing aqueous suspensions by adding water provide active ingredients that can be mixed with dispersants or wetting agents, suspending agents, and one or more preservatives. Suitable dispersants or wetting agents and suspending agents are illustrated herein.

[0218] The pharmaceutical compositions disclosed herein may also be in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin, or a mixture thereof. Suitable emulsifiers may be naturally occurring gums, such as gum arabic or tragacanth; naturally occurring phospholipids, such as soybean or lecithin, and esters or metaesters derived from fatty acids; hexitanic anhydrides, such as sorbitan monooleate; and condensation products of metaesters and ethylene oxide, such as polyoxyethylene sorbitan monooleate.

[0219] The formulation may also include a carrier to protect the composition from rapid degradation or elimination from the body, such as controlled-release formulations, including implants, liposomes, hydrogels, prodrugs, and microencapsulated delivery systems. For example, a time-delaying material may be employed, such as glyceryl monostearate or glyceryl stearate alone or in combination with waxes.

[0220] This disclosure contemplates the administration of the IL-10 peptide in the form of a rectal suppository. The suppository can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, thus melting in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter and polyethylene glycol.

[0221] The IL-10 formulations (e.g., PEG-IL-10) and other formulations considered in this disclosure may be in the form of any other suitable pharmaceutical composition currently known or to be developed in the future (e.g., a spray for nasal or inhalation use).

[0222] The concentration of the peptide (e.g., IL-10) or fragment thereof in the formulation can vary widely (e.g., from less than about 0.1% by weight, typically or at least about 2% to up to 20% to 50% or more), and will generally be selected primarily based on fluid volume, viscosity, and subject-based factors, depending on, for example, the specific administration method chosen.

[0223] Application route This disclosure contemplates the administration of IL-10 agents (e.g., PEG-IL-10) and combinations thereof in any suitable manner. Suitable routes of administration include parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implantation), intraperitoneal, intracisional, intra-articular, intraperitoneal, intracerebral (within brain parenchyma), and intraventricular), oral, nasal, vaginal, sublingual, intraocular, rectal, local (e.g., transdermal), sublingual, and inhalation. Accumulated injectable formulations, typically administered subcutaneously or intramuscularly, may also be used to release the IL-10 agents disclosed herein over a defined time period.

[0224] In some specific embodiments of this disclosure, the IL-10 agent (e.g., PEG-IL-10) is administered parenterally, and in other specific embodiments, the parenterally administration is transdermal.

[0225] Regarding CAR-T cell therapy, this article describes an alternative approach to introducing a subject with multiple genetically modified cells that are therapeutically effective and express chimeric antigen receptors, wherein the chimeric antigen receptors include at least one antigen-specific targeting region capable of binding to a target cell population, and wherein the binding of the chimeric antigen receptor targeting region to the target cell population can induce activation-induced cell death.

[0226] Combination therapy In conjunction with the CAR-T T-cell therapy described herein, this disclosure contemplates the use of an IL-10 agent (e.g., PEG-IL-10) in combination with one or more active agents (e.g., chemotherapy agents) or other prophylactic or therapeutic nonpharmacological modalities (e.g., local or total radiation therapy). For example, this disclosure contemplates treatment regimens in which treatment is administered before or after a radiation phase with one or more adjunctive therapies (e.g., CAR-T T-cell therapy and administration of an IL-10 agent) or agents as described herein. In some embodiments, this disclosure also contemplates the use of CAR-T T-cell therapy and an IL-10 agent (e.g., PEG-IL-10) in combination with bone marrow transplantation, peripheral blood stem cell transplantation, or other types of transplantation therapy.

[0227] As used herein, “combination therapy” is intended to include therapies that can be administered or introduced alone, such as those formulated for single administration (e.g., those provided in a kit), and therapies that can be administered or introduced together. In some embodiments, the IL-10 agent and other agents are administered or applied sequentially, for example, one agent is administered before one or more other agents. In other embodiments, the IL-10 agent and other agents are administered simultaneously, for example, two or more agents are administered simultaneously or approximately simultaneously; the two or more agents may be present in two or more individual formulations or combined into a single formulation (i.e., a co-formulation). Regardless of whether the agents are administered sequentially or simultaneously, their combined administration is contemplated for the purposes of this disclosure.

[0228] In the stated circumstances, the IL-10 agent of this disclosure can be used in combination with at least one other active agent in any suitable manner. In one embodiment, treatment with the IL-10 agent and other agents is maintained for a period of time. In another embodiment, treatment with the at least one other agent is reduced or interrupted (e.g., when the subject is stable), while treatment with the IL-10 agent of this disclosure (e.g., PEG-IL-10) is maintained at a constant dosing regimen. In another embodiment, treatment with the other agents is reduced or interrupted (e.g., when the subject is stable), while treatment with the IL-10 agent of this disclosure is also reduced (e.g., lower dose, lower frequency, or shorter treatment regimen). In yet another embodiment, treatment with the other agents is reduced or interrupted (e.g., when the subject is stable), and treatment with the IL-10 agent of this disclosure is increased (e.g., higher dose, more frequent dosing, or longer treatment regimen). In yet another embodiment, treatment with the other agents is maintained, and treatment with the IL-10 agent of this disclosure is reduced or interrupted (e.g., lower dose, lower frequency, or shorter treatment regimen). In another embodiment, treatment with the other agents and treatment with the IL-10 agent of this disclosure (e.g., PEG-IL-10) is reduced or interrupted (e.g., with lower doses, lower frequency of administration, or shorter treatment regimens).

[0229] In conjunction with the CAR-T T-cell therapy described herein, this disclosure provides methods for treating and / or preventing proliferative conditions, cancer, tumors, or precancerous lesions, symptoms, or conditions using an IL-10 agent (e.g., PEG-IL-10) and at least one additional therapeutic, prophylactic, or diagnostic agent exhibiting the desired activity. Some embodiments of this disclosure contemplate the use of conventional chemotherapeutic agents (e.g., alkylating agents, nitrogen mustard, nitrosoureas, antibiotics, antimetabolites, folic acid analogs, purine analogs, pyrimidine analogs, anti-hormonal agents, and taxanes). Other embodiments of this disclosure contemplate methods for tumor inhibition or tumor growth, including administration of the IL-10 agent described herein in combination with a signal transduction inhibitor (e.g., GLEEVEC or HERCEPTIN) or an immunomodulator to achieve additive or synergistic inhibition of tumor growth.

[0230] In conjunction with the CAR-T T-cell therapy described herein, this disclosure also provides methods for treating and / or preventing immune and / or inflammation-related diseases, conditions, and symptoms, and related conditions, using an IL-10 agent (e.g., PEG-IL-10) and at least one additional agent or diagnostic agent exhibiting the desired activity. Examples of therapeutic agents used in combination therapies include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase-2 (COX-2) inhibitors, steroids, TNF antagonists (e.g., REMICADE and ENBREL), interferon-β1a (AVONEX), interferon-β1b (BETASERON), and immune checkpoint inhibitors (e.g., YERVOY).

[0231] Dosage The IL-10 formulation disclosed herein (e.g., PEG-IL-10) can be administered to subjects in amounts depending on factors such as the administration target (e.g., desired degree of regression); the age, weight, sex, and health and physical condition of the subject administering the formulation; and the route of administration. Effective doses and dosing regimens can be readily determined from, for example, safety and dose escalation studies, in vivo studies (e.g., animal models), and other methods known to those skilled in the art.

[0232] As discussed in detail elsewhere, this disclosure considers embodiments in which IL-10 is administered to achieve and / or maintain certain mean serum trough concentrations.

[0233] Typically, dosing parameters specify that the dose should be less than the amount that may cause irreversible toxicity to the subject (i.e., the maximum tolerated dose, "MTD") and not less than the amount required to produce a measurable effect in the subject. This amount is determined, for example, by pharmacokinetic and pharmacodynamic parameters related to ADME, taking into account the route of administration and other factors.

[0234] The effective dose (ED) is the amount of a drug that produces a therapeutic response or desired effect in a subset of subjects taking it. The "half-maximal effective dose" or ED 50 is the amount of a drug that produces a therapeutic response or desired effect in 50% of the population administering it. While ED 50 is commonly used as a measure of a reasonable expected value for a drug's effect, clinicians may not necessarily consider this dose appropriate after considering all relevant factors. Therefore, in some cases, the effective dose may be greater than the calculated ED 50; in others, it may be less than the calculated ED 50; and in still others, it may be the same as the calculated ED 50.

[0235] The therapeutically effective dose range of PEG-IL-10 is approximately 0.01 to approximately 100 μg protein / kg body weight / day, approximately 0.1 to 20 μg protein / kg body weight / day, approximately 0.5 to 10 μg protein / kg body weight / day, or approximately 1 to 4 μg protein / kg body weight / day. In some embodiments, PEG-IL-10 is administered by continuous infusion to deliver approximately 50 to 800 μg protein / kg body weight / day (e.g., approximately 1 to 16 μg protein / kg body weight / day of PEG-IL-10). The infusion rate can be varied based on evaluations of, for example, adverse effects and blood cell counts. Other specific dosing parameters for IL-10 formulations are described elsewhere in this document.

[0236] In some embodiments, the dosage of the disclosed IL-10 agent is included in the term "unit dosage form." The phrase "unit dosage form" refers to a physically discrete unit, each unit containing, alone or in combination with one or more additional agents, a predetermined amount of the disclosed IL-10 agent sufficient to produce the desired effect. It should be understood that the parameters of the unit dosage form will depend on the specific agent and the desired effect.

[0237] Reagent test kit This disclosure also contemplates kits comprising IL-10 agents (e.g., PEG-IL-10) and pharmaceutical compositions thereof. Kits are typically in the form of a physical structure containing various components, as described below, and can be used, for example, to practice the methods described above.

[0238] The kit may include the IL-10 agent (e.g., PEG-IL-10) disclosed herein (e.g., provided in a sterile container), which may be in the form of a pharmaceutical composition suitable for administration to a subject. The IL-10 agent may be provided in a ready-to-use form or in a form requiring rehydration or dilution prior to administration. When the IL-10 agent is in a form requiring rehydration by the user, the kit may also include buffers, pharmaceutically acceptable excipients, etc., packaged together with or separately from the IL-10 agent. The kit may also contain the IL-10 agent and / or components of a specific CAR-T T-cell therapy to be used; the kit may contain several agents individually or the agents may be combined in the kit. The kits disclosed herein are designed to maintain the conditions necessary for properly maintaining the components contained therein (e.g., refrigeration or freezing).

[0239] The kit may include labels or packaging inserts containing identification information for its components and instructions for use (e.g., dosing parameters, clinical pharmacological properties of the active ingredient, including mechanism of action, pharmacokinetic and pharmacodynamic properties, adverse effects, contraindications, etc.). Each component of the kit may be encapsulated in a separate container, and all various containers may be contained within a single package. Labels or inserts may include manufacturer information, such as batch number and expiration date. Labels or packaging inserts may, for example, be integrated into the physical structure containing the components, be contained separately in the physical structure, or be affixed to components of the kit (e.g., ampoules, syringes, or vials).

[0240] Labels or inserts may be additionally included or incorporated into computer-readable media such as disks (e.g., hard drives, cards, storage disks), optical discs (e.g., CD-ROM / RAM, DVD, MP3), magnetic tapes, or electronic storage media such as RAM and ROM, or a combination of these such as magnetic / optical storage media, FLASH media, or memory cards. In some embodiments, no actual instructions are included in the kit, but a method for obtaining instructions from a remote source, such as via an Internet website, is provided.

[0241] The present invention also relates to the following embodiments: 1. A method for modulating a T-cell-mediated immune response to a target cell population in a subject, comprising: a) Introduce multiple genetically modified cells that are therapeutically effective and express chimeric antigen receptors (CARs) into the subject. The chimeric antigen receptor comprises at least one antigen-specific targeting region capable of binding to the target cell population, and The binding of the chimeric antigen receptor targeting region to the target cell population can induce activation-induced cell death; and b) Administer to the subject a therapeutically effective amount of IL-10 sufficient to prevent or limit the activation-induced cell death; This regulates the T cell-mediated immune response.

[0242] 2. A method for modulating a T-cell-mediated immune response in a subject to a target cell population, comprising introducing into the subject a plurality of therapeutically effective, genetically modified cells expressing the following substances: a) Chimeric antigen receptor (CAR), The chimeric antigen receptor comprises at least one antigen-specific targeting region capable of binding to the target cell population, and The binding of the chimeric antigen receptor targeting region to the target cell population can induce activation-induced cell death; and b) An amount of IL-10 agent sufficient to prevent or limit the activation-induced cell death; This regulates the T cell-mediated immune response.

[0243] 3. A method for modulating a T-cell-mediated immune response to a target cell population in a subject, comprising introducing into the subject: a) The first multiple cells that respond to treatment, said cells being genetically modified to express chimeric antigen receptors (CARs). The chimeric antigen receptor comprises at least one antigen-specific targeting region capable of binding to the target cell population, and The binding of the chimeric antigen receptor targeting region to the target cell population can induce activation-induced cell death; and b) A second or more cells that respond to the treatment, said cells being genetically modified to express an amount of IL-10 agent sufficient to prevent or limit said activation-induced cell death. This regulates the T cell-mediated immune response.

[0244] 4. The method according to any one of embodiments 1-3, wherein the CAR comprises an antigen-binding domain that specifically recognizes the target cell population.

[0245] 5. The method according to any one of embodiments 1-3, wherein the CAR further comprises a transmembrane structural domain and a signal transduction structural domain.

[0246] 6. The method according to embodiment 5, wherein the signal conduction structure domain includes the CD3ζ signal conduction structure domain.

[0247] 7. The method according to embodiment 5, wherein the signal transduction domain comprises at least one co-stimulatory domain.

[0248] 8. The method according to any one of embodiments 1-3, wherein the IL-10 agent enhances the function of activated memory CD8+ T cells.

[0249] 9. The method according to embodiment 1, wherein the IL-10 agent is administered prior to the administration of the multiple cells to which the treatment is effective.

[0250] 10. The method according to embodiment 1, wherein the IL-10 agent is administered simultaneously with the administration of the multiple cells for which the treatment is effective.

[0251] 11. The method according to embodiment 1, wherein the IL-10 agent is administered after the administration of the multiple cells to which the treatment is effective.

[0252] 12. The method according to embodiment 2, wherein the chimeric antigen receptor and the IL-10 agent are expressed by the same carrier.

[0253] 13. The method according to embodiment 2, wherein the chimeric antigen receptor and the IL-10 agent are expressed by different carriers.

[0254] 14. The method according to embodiment 2, wherein the multiple cells that are effective in the treatment are transfected with a vector expressing an amount of the IL-10 agent sufficient to enhance cytotoxic function.

[0255] 15. The method according to embodiment 3, wherein the second plurality of therapeutically effective cells are transfected with a vector expressing an amount of the IL-10 agent sufficient to enhance cytotoxic function.

[0256] 16. The method according to embodiment 3, wherein the second plurality of cells that are therapeutically effective comprise CD8+ T cells transfected with a vector expressing the IL-10 agent.

[0257] 17. The method according to any one of embodiments 12-16, wherein the vector comprises a plasmid.

[0258] 18. The method according to any one of embodiments 12-16, wherein the vector comprises a viral vector.

[0259] 19. The method according to any one of embodiments 12-16, wherein the expression of the IL-10 agent is regulated by an expression control element.

[0260] 20. The method according to embodiment 1, wherein the amount of the IL-10 agent applied is sufficient to enhance cytotoxic function.

[0261] 21. The method according to embodiment 20, wherein the amount of the IL-10 agent applied is sufficient to achieve a serum concentration of 10-100 ng / mL.

[0262] 22. The method according to embodiment 1, wherein the IL-10 agent is PEG-IL-10.

[0263] 23. The method according to embodiment 22, wherein the PEG-IL-10 comprises at least one PEG molecule covalently linked to at least one amino acid residue of at least one monomer of IL-10.

[0264] 24. The method according to embodiment 22, wherein the PEG-IL-10 comprises a mixture of mono-polyethylene glycolated IL-10 and di-polyethylene glycolated IL-10.

[0265] 25. The method according to embodiment 22, wherein the molecular weight of the PEG component of PEG-IL-10 is from 5 kDa to 20 kDa.

[0266] 26. The method according to embodiment 22, wherein the molecular weight of the PEG component of PEG-IL-10 is at least 20 kDa.

[0267] 27. The method according to embodiment 22, wherein the molecular weight of the PEG component of PEG-IL-10 is at least 30 kDa.

[0268] 28. The method according to embodiment 1, wherein the IL-10 agent is administered subcutaneously.

[0269] 29. The method according to embodiment 1 or 2, wherein the plurality of cells are obtained from the subject and genetically modified in vitro.

[0270] 30. The method according to embodiment 29, wherein the plurality of cells are obtained from the subject by plasma separation and replacement.

[0271] 31. The method according to embodiment 3, wherein the first plurality of cells are obtained from the subject and genetically modified in vitro.

[0272] 32. The method according to embodiment 31, wherein the second plurality of cells are obtained from the subject and genetically modified in vitro.

[0273] 33. The method according to embodiments 31 and 32, wherein the first plurality of cells and the second plurality of cells are obtained from the subject by plasma separation and replacement.

[0274] 34. The method according to embodiment 30, wherein the plurality of cells are memory CD8+ T cells.

[0275] 35. The method according to embodiment 33, wherein the first plurality of cells are memory CD8+ T cells.

[0276] 36. The method according to embodiment 33, wherein the second plurality of cells are naïve CD8+ T cells.

[0277] 37. The method according to embodiment 1 or 2, wherein the plurality of cells are autologous tumor cells.

[0278] 38. The method according to embodiment 3, wherein the first plurality of cells and the second plurality of cells are autologous tumor cells.

[0279] 39. The method according to any one of embodiments 1-3, wherein the target cell population comprises tumor antigens.

[0280] 40. The method according to embodiment 43, wherein the tumor antigen is selected from CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0281] 41. A method for treating a subject suffering from a cancer-related disease, condition, or symptom, comprising: a) Introduce multiple genetically modified cells that are therapeutically effective and express chimeric antigen receptors (CARs) into the subject. The chimeric antigen receptor comprises at least one antigen-specific targeting region capable of binding to the target cell population, and The binding of the chimeric antigen receptor targeting region to the target cell population can induce activation-induced cell death; and b) Administer to the subject a therapeutically effective amount of IL-10 to prevent or limit the activation-induced cell death.

[0282] 42. A method of treating a subject suffering from a cancer-related disease, condition, or symptom, comprising introducing into the subject a plurality of genetically modified cells that are therapeutically effective in expressing the following substances: a) Chimeric antigen receptor (CAR), The chimeric antigen receptor comprises at least one antigen-specific targeting region capable of binding to the target cell population, and The binding of the chimeric antigen receptor targeting region to the target cell population can induce activation-induced cell death; and b) An amount of IL-10 agent sufficient to prevent or limit the activation-induced cell death.

[0283] 43. A method of treating a subject suffering from a cancer-related disease, condition, or symptom, comprising introducing to said subject: a) The first multiple cells that respond to treatment, said cells being genetically modified to express chimeric antigen receptors (CARs). The chimeric antigen receptor comprises at least one antigen-specific targeting region capable of binding to the target cell population, and The binding of the chimeric antigen receptor targeting region to the target cell population can induce activation-induced cell death; and b) A second plurality of cells that respond to treatment, said cells being genetically modified to express an amount of IL-10 agent sufficient to prevent or limit said activation-induced cell death.

[0284] 44. The method according to any one of embodiments 41-43, wherein the CAR comprises an antigen-binding domain that specifically recognizes the target cell population.

[0285] 45. The method according to any one of embodiments 41-43, wherein the CAR further comprises a transmembrane structural domain and a signal transduction structural domain.

[0286] 46. ​​The method according to embodiment 45, wherein the signal conduction structure domain comprises a CD3ζ signal conduction structure domain.

[0287] 47. The method according to embodiment 45, wherein the signal transduction domain comprises at least one co-stimulatory domain.

[0288] 48. The method according to any one of embodiments 41-43, wherein the IL-10 agent enhances the function of activated memory CD8+ T cells.

[0289] 49. The method according to embodiment 41, wherein the IL-10 agent is administered prior to the administration of the multiple cells to which the treatment is effective.

[0290] 50. The method according to embodiment 41, wherein the IL-10 agent is administered simultaneously with the administration of the multiple cells for which the treatment is effective.

[0291] 51. The method according to embodiment 41, wherein the IL-10 agent is administered after the application of the treatment to multiple cells that are effective.

[0292] 52. The method according to embodiment 42, wherein the chimeric antigen receptor and the IL-10 agent are expressed by the same carrier.

[0293] 53. The method according to embodiment 42, wherein the chimeric antigen receptor and the IL-10 agent are expressed by different carriers.

[0294] 54. The method according to embodiment 42, wherein the multiple cells that are effective in treatment are transfected with a vector expressing an amount of the IL-10 agent sufficient to enhance cytotoxic function.

[0295] 55. The method according to embodiment 43, wherein the second plurality of therapeutically effective cells are transfected with a vector expressing an amount of the IL-10 agent sufficient to enhance cytotoxic function.

[0296] 56. The method according to embodiment 43, wherein the second plurality of cells that are therapeutically effective comprise CD8+ T cells transfected with a vector expressing the IL-10 agent.

[0297] 57. The method according to any one of embodiments 52-56, wherein the vector comprises a plasmid.

[0298] 58. The method according to any one of embodiments 52-56, wherein the vector comprises a viral vector.

[0299] 59. The method according to any one of embodiments 52-56, wherein the expression of the IL-10 agent is regulated by an expression control element.

[0300] 60. The method according to embodiment 41, wherein the amount of the IL-10 agent applied is sufficient to enhance cytotoxic function.

[0301] 61. The method according to embodiment 60, wherein the amount of the IL-10 agent applied is sufficient to achieve a serum concentration of 10-100 ng / mL.

[0302] 62. The method according to embodiment 41, wherein the IL-10 agent is PEG-IL-10.

[0303] 63. The method according to embodiment 62, wherein the PEG-IL-10 comprises at least one PEG molecule covalently linked to at least one amino acid residue of at least one monomer of IL-10.

[0304] 64. The method according to embodiment 62, wherein the PEG-IL-10 comprises a mixture of mono-polyethylene glycolated IL-10 and di-polyethylene glycolated IL-10.

[0305] 65. The method according to embodiment 62, wherein the molecular weight of the PEG component of PEG-IL-10 is from 5 kDa to 20 kDa.

[0306] 66. The method according to embodiment 62, wherein the molecular weight of the PEG component of PEG-IL-10 is at least 20 kDa.

[0307] 67. The method according to embodiment 62, wherein the molecular weight of the PEG component of PEG-IL-10 is at least 30 kDa.

[0308] 68. The method according to embodiment 41, wherein the IL-10 agent is administered subcutaneously.

[0309] 69. The method according to embodiment 41 or 42, wherein the plurality of cells are obtained from the subject and genetically modified in vitro.

[0310] 70. The method according to embodiment 69, wherein the plurality of cells are obtained from the subject by plasma separation and replacement.

[0311] 71. The method according to embodiment 43, wherein the first plurality of cells are obtained from the subject and genetically modified in vitro.

[0312] 72. The method according to embodiment 71, wherein the second plurality of cells are obtained from the subject and genetically modified in vitro.

[0313] 73. The method according to embodiments 71 and 72, wherein the first plurality of cells and the second plurality of cells are obtained from the subject by plasma separation and replacement.

[0314] 74. The method according to embodiment 70, wherein the plurality of cells are memory CD8+ T cells.

[0315] 75. The method according to embodiment 73, wherein the first plurality of cells are memory CD8+ T cells.

[0316] 76. The method according to embodiment 73, wherein the second plurality of cells are naïve CD8+ T cells.

[0317] 77. The method according to embodiment 41 or 42, wherein the plurality of cells are autologous tumor cells.

[0318] 78. The method according to embodiment 43, wherein the first plurality of cells and the second plurality of cells are autologous tumor cells.

[0319] 79. The method according to any one of embodiments 41-43, wherein the target cell population comprises tumor antigens.

[0320] 80. The method according to embodiment 43, wherein the tumor antigen is selected from CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR or any combination thereof.

[0321] 81. The method according to embodiment 42 or 43, wherein the IL-10 agent is expressed at an amount sufficient to prevent or limit the activation-induced cell death at least two weeks after introduction into the subject.

[0322] 82. The method according to embodiment 42 or 43, wherein the IL-10 agent is expressed at an amount sufficient to prevent or limit the activation-induced cell death at least one month after introduction into the subject.

[0323] 83. The method according to embodiment 42 or 43, wherein the IL-10 agent is expressed at an amount sufficient to prevent or limit the activation-induced cell death for at least three months after introduction into the subject.

[0324] 84. A nucleic acid molecule encoding an IL-10 agent as described in embodiment 42 or 43.

[0325] 85. The nucleic acid molecule according to embodiment 84, wherein the nucleic acid molecule is operatively connected to an expression control element that imparts expression of the nucleic acid molecule encoding the IL-10 agent.

[0326] 86. A carrier comprising a nucleic acid molecule according to embodiment 84 or 85.

[0327] 87. The vector according to embodiment 86, wherein the vector includes a viral vector.

[0328] 88. The vector according to embodiment 87, wherein the vector comprises plasmids.

[0329] 89. A transformed or host cell that expresses the IL-10 agent described in embodiment 52 or 43.

[0330] 90. A method for enhancing the function of CAR-T T cells, comprising: a) Genetically engineered T cells to express CAR, thereby generating CAR-T T cells; and b) Modify the CAR-TT cells with an agent that reduces the amount of at least one cytokine secreted by the CAR-TT cells. This enhances the function of the CAR-T T cells.

[0331] 91. The method according to embodiment 90, wherein the agent is small interfering RNA (siRNA).

[0332] 92. The method according to embodiment 91, wherein the cytokine is a member of the tumor necrosis factor family or the transforming growth factor β superfamily.

[0333] 93. The method according to embodiment 92, wherein the member of the tumor necrosis factor family is TNFα.

[0334] 94. The method according to embodiment 92, wherein the member of the transforming growth factor β superfamily is TGF-β.

[0335] 95. The method according to embodiment 94, wherein reducing the amount of TGF-β reduces the proliferation of T regulatory cells.

[0336] experiment The following embodiments are provided to provide a complete disclosure and description of how to prepare and use the invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their invention, nor to represent the performance of the experiments described below, or all experiments that can be performed. It should be understood that the exemplary descriptions written in the present tense are not necessarily performed, but can be performed to generate the data described therein, etc. While every effort has been made to ensure accuracy regarding the figures used (e.g., quantities, temperatures, etc.), some experimental errors and biases should be taken into account.

[0337] Unless otherwise specified, parts are by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius (°C), and pressure is at or near atmospheric pressure. Standard abbreviations are used, including: s or sec = second; min = minute; h or hr = hour; aa = amino acid; bp = base pair; kb = kilobase; nt = nucleotide; ng = nanogram; μg = microgram; mg = milligram; g = gram; kg = kilogram; dl or dL = deciliter; μl or μL = microliter; ml or mL = milliliter; l or L = liter; nM = nanomolar; μM = micromolar; mM = millimole; M = mole; kDa = kilodalton; im = intramuscular; ip = intraperitoneal; SC or SQ = subcutaneous; HPLC = high-performance liquid chromatography (HPLC) High-performance liquid chromatography; BW = body weight; U = unit; ns = no statistical significance; PMA = phorbol 12-myristate 13-acetate; PBS = phosphate-buffered saline; DMEM = Dalberg modified Eagle medium; PBMC = primary peripheral blood mononuclear cells; FBS = fetal bovine serum; FCS = fetal bovine serum; HEPES = 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; LPS = lipopolysaccharide; RPMI = Los Angeles Memorial Institute medium; APC = antigen-presenting cells; FACS = fluorescently activated cell sorting.

[0338] Materials and methods.

[0339] When indicated, the following general materials and methods are used, or may be applicable to the following embodiments: Molecular biology proceduresStandard methods in molecular biology are described in the scientific literature (see, for example, Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel et al. (2001) Current Protocols in Molecular Biology, vols. 1–4, John Wiley and Sons, Inc., New York, NY, which describe cloning and DNA mutagenesis in bacterial cells (vol. 1), cloning in mammalian cells and yeast (vol. 2), glycoconjugate and protein expression (vol. 3), and bioinformatics (vol. 4)).

[0340] Antibody-related processes The generation, purification, and fragmentation of polyclonal and monoclonal antibodies are described (e.g., Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., NY); flow cytometry, including fluorescence activated cell sorting (FACS) methods, are available (see, e.g., Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ); and fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, peptides, and antibodies used as, for example, diagnostic reagents, are available (Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, OR.; Sigma-Aldrich (2003) Catalogue, St. Louis, MO.). Further discussion of antibodies appears elsewhere in this article.

[0341] softwareSoftware packages and databases for identifying, for example, antigen fragments, leader sequences, protein folds, functional domains, glycosylation sites, and sequence alignments are available (see, for example, GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); and DeCypher™ (TimeLogic Corp., Crystal Bay, NV).

[0342] Polyethylene glycolation PEGylated IL-10 as described herein can be synthesized in any manner known to those skilled in the art. Exemplary synthetic schemes for generating mono-PEG-IL-10 and mixtures of mono- and di-PEG-IL-10 have been described (see, for example, U.S. Patent No. 7,052,686; U.S. Patent Publication No. 2011 / 0250163; WO 2010 / 077853). Specific embodiments of this disclosure comprise a mixture of selectively PEGylated mono- and di-PEG-IL-10. In addition to utilizing their own PEG production and use technologies (and other drug delivery technologies) suitable for the practices of this disclosure, those skilled in the art are familiar with numerous commercial suppliers of PEG-related technologies (e.g., NOF America Corp (Irvine, CA) and Parchem (New Rochelle, NY)).

[0343] animal Various mouse and other animal strains known to those skilled in the art can be used in conjunction with the teachings of this disclosure. For example, immune-active Balb / C or B-cell-deficient Balb / C mice can be obtained from The Jackson Lab., Bar Harbor, ME and used according to standard procedures (see, for example, Martin et al. (2001) Infect. Immun., 69(11):7067-73 and Compton et al. (2004) Comp. Med. 54(6):681-89).

[0344] IL-10 concentration Serum IL-10 concentration levels and exposure levels can be determined using standard methods used in the art. For example, when the experimental subjects are mice, serum exposure levels are determined by collecting whole blood (~50 μL / mouse) from the tail of the mouse into a regular capillary tube, separating the serum and blood cells by centrifugation, and measuring IL-10 exposure levels using a standard ELISA kit and technique.

[0345] FACS analysisMany protocols, materials, and reagents used for FACS analysis are commercially available and can be used in conjunction with the teachings herein (e.g., Becton-Dickinson, Franklin Lakes, NJ; Cell Signaling Technologies, Danford, MA; Abcam, Cambridge, MA; Affymetrix, Santa Clara, CA). Direct flow cytometry (i.e., using a conjugated primary antibody) and indirect flow cytometry (i.e., using a primary antibody and a conjugated secondary antibody) can be used. An exemplary direct flow protocol is as follows: Wash harvested cells and adjust the cell suspension concentration to 1–5 x 10⁻⁵ cells / mL in ice-cold PBS, 10% FCS, and 1% sodium azide. 6 Cells / mL. Cells can be stored in 12x75mm... 2 Cells are stained in round-bottom polystyrene Falcon tubes. They can be centrifuged thoroughly, allowing for removal of the supernatant with minimal cell loss, but not to the point of being difficult to resuspend. Labeled primary antibody (0.1–10 μg / mL) can be added, and a dilution can be prepared in 3% BSA / PBS if necessary. After incubation at 4°C for at least 30 minutes, cells can be washed three times by centrifugation at 400g for 5 minutes, and then resuspended in 0.5–1 mL of ice-cold PBS, 10% FCS, and 1% sodium azide. Cells can be kept on ice in the dark until analysis (preferably within the same day). Cells can also be fixed using standard methods for preservation for several days; fixation of different antigens may require antigen-specific optimization.

[0346] The analysis described below is representative, not exclusive.

[0347] Gene expression assays of PBMCs and CD8+ T cells. The following protocols provide exemplary assays for examining gene expression.

[0348] Human PBMCs can be isolated according to any standard protocol (see, for example, Fuss et al. (2009) Current Protocols in Immunology, Unit 7.1, John Wiley, Inc., NY). In any standard tissue culture-treated 6-well plate (BD; Franklin Lakes, NJ), 2.5 mL of PBMCs (8 million cells / mL) can be cultured per well with complete RPMI containing RPMI (Life Technologies; Carlsbad, CA), 10 mM HEPES (Life Technologies; Carlsbad, CA), 10% FCS (Hyclone Thermo Fisher Scientific; Waltham, MA), and a penicillin / streptomycin mixture (Life Technologies; Carlsbad, CA). Human PEGylated IL-10 can be added to the wells at a final concentration of 100 ng / mL, followed by a 7-day incubation. CD8+ T cells can be isolated from PBMCs using Miltenyi Biotec's MACS cell isolation technology, following the manufacturer's protocol (Miltenyi Biotec; Auburn, CA). Qiagen's RNeasy kit and RT were used respectively. 2 The First Strand kit, following the manufacturer's instructions (Qiagen NV; Netherlands), can extract RNA and synthesize cDNA from isolated CD8+ T cells and CD8+ T cell-depleted PBMCs. Quantitative PCR of the cDNA template can be performed using the RT² SYBR Green qPCR Mastermix from Qiagen and primers (IDO1, GUSB, and GAPDH) according to the manufacturer's protocol. IDO1 Ct values ​​can be normalized to the average Ct values ​​of the housekeeping genes GUSB and GAPDH.

[0349] Measurement of PBMC and CD8+ T cell cytokine secretion. When treated with PEG-IL-10 followed by an anti-CD3 antibody, activated primary human CD8+ T cells secrete IFN-γ. The following protocol provides an exemplary assay for examining cytokine secretion.

[0350] Human PBMCs can be isolated according to any standard protocol (see, for example, Fuss et al. (2009) Current Protocols in Immunology, Unit 7.1, John Wiley, Inc., NY). In any standard tissue culture-treated 6-well plate (BD; Franklin Lakes, NJ), each well is cultured with 2.5 mL of PBMCs (cell density of 8 million cells / mL) in complete RPMI containing RPMI (Life Technologies; Carlsbad, CA), 10 mM HEPES (Life Technologies; Carlsbad, CA), 10% FCS (Hyclone Thermo Fisher Scientific; Waltham, MA), and a penicillin / streptomycin mixture (Life Technologies; Carlsbad, CA). Human PEGylated IL-10 can be added to the wells at a final concentration of 100 ng / mL, followed by 3 days of incubation. CD8+ T cells can be isolated from PBMCs using Miltenyi Biotec's MACS cell isolation technology, following the manufacturer's protocol (Miltenyi Biotec; Auburn, CA). The isolated CD8+ T cells can then be cultured for 4 hours in any standard tissue culture plate with complete RPMI containing 1 μg / mL anti-CD3 antibody (Affymetrix eBioscience). After 4 hours of incubation, the culture medium can be collected, and IFN-γ can be measured using a commercial ELISA kit, following the manufacturer's protocol (Affymetrix eBioscience).

[0351] TNFα inhibition assay. PMA-stimulation of U937 cells (a human lymphoblastic cell line from the lungs of Sigma-Aldrich (#85011440); St. Louis, MO) induces TNFα secretion, followed by treatment of these TNFα-secreting cells with human IL-10, resulting in a dose-dependent reduction in TNFα secretion. An exemplary TNFα inhibition assay can be performed using the following protocol.

[0352] After culturing U937 cells in RMPI containing 10% FBS / FCS and antibiotics, seed 1 x 10⁵ viable U937 cells in 96-well flat-bottom plates (any plasma-treated tissue culture plate may be used, e.g., Nunc; Thermo Scientific, USA) in triplicate for each condition. Plate cells to provide the following conditions (all in at least triplicate; double the number of wells for 'single medium', as half will be used for viability after incubation with 10 nM PMA): 5 ng / mL LPS alone; 5 ng / mL LPS + 0.1 ng / mL rhIL-10; 5 ng / mL LPS + 1 ng / mL rhIL-10; 5 ng / mL LPS + 10 ng / mL rhIL-10; 5 ng / mL LPS + 100 ng / mL rhIL-10; 5 ng / mL LPS + 1000 ng / mL rhIL-10; 5 ng / mL LPS + 0.1 ng / mL PEG-rhIL-10; 5 ng / mL LPS + 1 ng / mL PEG-rhIL-10; 5 ng / mL LPS + 100 ng / mL PEG-rhIL-10; 5 ng / mL LPS + 100 ng / mL PEG-rhIL-10; and 5 ng / mL LPS + 1000 ng / mL PEG-rhIL-10. Expose each well to 200 µL of 10 nM PMA for 24 hours and incubate at 37 °C in a 5% CO2 incubator until approximately 90% cell adhesion is achieved. Three additional wells can be resuspended, and cell counts can be performed to assess viability (>90% should indicate viability). Wash gently but thoroughly three times with fresh, PMA-free medium to ensure cells remain in the wells. Add 100 μL of medium containing an appropriate concentration (2-fold at 100% dilution) of rhIL-10 or PEG-rhIL-10 to each well and incubate at 37 °C in a 5% CO2 incubator for 30 minutes. Add 100 μL of 10 ng / mL LPS stock solution to each well to achieve a final concentration of 5 ng / mL LPS in each well and incubate at 37 °C in a 5% CO2 incubator for 18–24 hours. Remove the supernatant according to the manufacturer's instructions and perform a TNFα ELISA. Run each conditioned supernatant in duplicate in the ELISA.

[0353] MC / 9 cell proliferation assayAdministration of IL-10 to MC / 9 cells (a mouse cell line with mast cell characteristics, available from Cell Signaling Technology; Danvers, MA) resulted in a dose-dependent increase in cell proliferation. Thompson-Snipes, L. et al. (1991) J. Exp. Med. 173:507-10 described standard assays for supplementing MC / 9 cells with IL3+IL-10 and IL-3+IL-4+IL-10. Suppliers (e.g., R&D Systems, USA; and Cell Signaling Technology, Danvers, MA) use this assay as a batch-release assay for rhIL-10. Those skilled in the art will be able to modify the standard assay described by Thompson-Snipes, L. et al. so that cells are supplemented with IL-10 only.

[0354] Activation-induced cell death assay The following protocol provides an exemplary assay for activation-induced cell death.

[0355] Human PBMCs can be isolated according to any standard protocol (see, for example, Fuss et al. (2009) Current Protocols in Immunology, Unit 7.1, John Wiley, Inc., NY). CD8+ T cells (CD45RO+) can be isolated using Miltenyi Biotec's anti-CD45ROMACS beads and MACS cell isolation technology, according to the manufacturer's protocol (Miltenyi Biotec Inc; Auburn, CA). To activate the cells, 1 mL of isolated cells (at a density of 3 x 10⁻⁶) can be used. 6Cells were cultured for 3 days in AIM V medium in standard 24-well plates (BD; Franklin Lakes, NJ) pre-coated with anti-CD3 and anti-CD28 antibodies (Affymetrix eBioscience, San Diego, CA) (Life Technologies; Carlsbad, CA). Pre-coating was performed by adding 300 μL of carbonate buffer (0.1 M NaHCO3 (Sigma-Aldrich, St. Louis, MO), 0.5 M NaCl (Sigma-Aldrich), pH 8.3) containing 10 μg / mL anti-CD3 and 2 μg / mL anti-CD28 antibodies to each well, incubating at 37°C for 2 hours, and washing each well with AIM V medium. After a 3-day activation period, cells were collected, counted, and re-coated in 1 mL of AIM V medium in standard 24-well plates (density 2 x 10⁻⁶ cells / mL). 6 Cells / mL) were treated with 100 ng / mL PEG-hIL-10 for 3 days. The activation and treatment process with PEG-hIL-10 can be repeated, and viable cells can then be counted according to the manufacturer's (LifeTechnologies) protocol using the trypan blue exclusion method.

[0356] Tumor models and tumor analysis The effects of the IL-10 agents described herein on various tumors can be evaluated using any tumor models, assays, etc., accepted in the art. The tumor models and tumor analyses described below are representative of those that can be utilized. The dosage is 10 IL-10 per tumor inoculation. 4 10 5 Or 10 6 Subcutaneous or intradermal injection of syngeneic mouse tumor cells. Ep2 breast cancer, CT26 colon cancer, PDV6 squamous cell carcinoma of the skin, and 4T1 breast cancer models can be used (see, for example, Langowski et al. (2006) Nature 442:461-465). Immunoactive Balb / C or B-cell-deficient Balb / C mice can be used. PEG 10-mIL-10 can be administered to immunocompetent mice, while PEG-hIL-10 treatment can be administered to B-cell-deficient mice. Tumors should be allowed to reach 100-250 mm before treatment begins. 3The size of the tumor was determined. IL-10, PEG-mIL-10, PEG-hIL-10, or a buffer control were administered subcutaneously at sites away from tumor implantation. Tumor growth was typically monitored twice weekly using electronic calipers. Tumor tissue and lymphoid organs were harvested at various endpoints to measure mRNA expression of numerous inflammatory markers and to perform immunohistochemical analysis of several inflammatory cell markers. Tissue was rapidly frozen in liquid nitrogen and stored at -80°C. Primary tumor growth was typically monitored twice weekly using electronic calipers. Tumor volume could be calculated using a formula (width...). 2 x length / 2), where length is the longer dimension. The tumor is allowed to reach 90-250 mm before treatment begins. 3 The size.

[0357] Example 1 PEG-IL-10-mediated CD8+ T cell immune activation Changes in the number of CD8+ T cells expressing PD-1 and LAG3 were measured in cancer patients before and after 29 days of treatment with PEG-rHuIL-10. Two patients who responded to therapy with sustained partial response showed increased PD1+ CD8T cells in their blood. The first patient (renal cell carcinoma) received 20 μg / kg PEG-rHuIL-10 subcutaneously daily and experienced a 71% reduction in total tumor burden after 22 weeks. The second patient (melanoma) received 40 μg / kg PEG-rHuIL-10 subcutaneously daily and experienced a 57% reduction in total tumor burden after 22 weeks.

[0358] Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of each patient before and during treatment and analyzed using FACS. Figure 1 As shown, the number of peripheral CD8+ T cells expressing PD-1 increased approximately two-fold within 29 days and continued to increase during treatment, while the number of peripheral CD8+ T cells expressing LAG3 increased approximately four-fold within 29 days. Both PD-1 and LAG3 are biomarkers of CD8+ T cell activation and cytotoxicity. These findings suggest that administration of PEG-rHuIL-10 mediates CD8+ T cell immune activation.

[0359] Example 2 PEG-IL-10 enhances the function of activated memory CD8+ T cells. Memory T cells (also known as antigen-experienced T cells) are a type of T lymphocyte that has previously encountered and responded to its homologous antigen during a previous infection, cancer exposure, or prior vaccination (e.g., helper T cells (CD4+) and cytotoxic T cells (CD8+)). In contrast, naive T cells have not yet encountered their homologous antigens in their surroundings; they are typically characterized by the lack of activation markers CD25, CD44, or CD69, and the absence of the memory CD45RO isotype. Memory T cells (usually CD45RO+) can regenerate and generate a faster and stronger immune response than naive T cells.

[0360] Given that CAR-T T cells originate from memory CD8+ T cells, the effect of PEG-IL-10 on memory CD8+ T cells was evaluated in vitro using standard methods (examples of which are described in this article). Figure 2 As shown, PEG-IL-10 preferentially enhances IFNγ production in memory CD8+ T cells (CD45RO+) rather than in naive CD8+ T cells. These data are consistent with the effect of PEG-IL-10 in enhancing the function of activated memory CD8+ T cells.

[0361] Example 3 PEG-IL-10 treatment produces a greater number of activated memory CD8+ T cells. As described in this article, CAR-T cell therapy originates from memory CD8+ T cells. For it to be effective, the infused memory CD8+ T cells must not only exhibit cytotoxicity but also be sustained (Curran KJ, Brentjens RJ. (April 20, 2015) J Clin Oncol pii: JCO.2014.60.3449; Berger et al., (Jan 2008) J Clin Invest 118(1):294-305). However, repeated activation of T cells leads to activation-induced cell death, which reduces the number of cells and thus decreases the overall efficacy.

[0362] Using the procedure described herein, activation-induced cell death in human CD45RO+ memory CD8+ T cells from two donors was measured with and without PEG-IL-10 treatment. Figure 3 As shown, treatment of human CD45RO+ memory CD8+ T cells with PEG-IL-10 after two rounds of TCR and co-stimulation-induced activation resulted in a greater number of viable cells. These data indicate that PEG-IL-10 can limit activation-induced cell death, leading to a greater number of activated memory T cells persisting. These observations suggest that the combined use of PEG-IL-10 with CAR-T cell therapy provides additional clinical benefits.

[0363] This document describes specific embodiments of the invention, including the best mode known to the inventors for carrying out the invention. Upon reading the foregoing, the description of the disclosed embodiments and variations will become apparent to those skilled in the art, and it is anticipated that such variations can be adopted as appropriate. Therefore, the invention is intended to be practiced in ways different from those specifically described herein, and the invention includes all modifications and equivalents to the subject matter set forth in the appended claims as permitted by applicable law. Furthermore, unless otherwise stated herein or otherwise clearly contradicted by the context, the invention covers any combination of the foregoing elements in all its possible variations.

[0364] All publications, patent applications, registration numbers and other references cited in this specification are incorporated herein by reference as if each individual publication or patent application were specifically and individually cited by reference.

Claims

1. A pharmaceutical composition for treating cancer, angiogenesis, or precancerous conditions, comprising a plurality of genetically modified cells that are therapeutically effective in expressing a chimeric antigen receptor (CAR). in: The chimeric antigen receptor (CAR) comprises at least one antigen-specific targeting region (ASTR) capable of binding to a target cell population, a transmembrane domain (TMD), and an intracellular signal transduction domain (ISD) containing a co-stimulatory domain (CSD). The intracellular signal transduction domain (ISD) includes CD3ζ; The co-stimulatory domain (CSD) comprises CD28, CD137 (4-1BB), or a combination thereof; and The binding of the chimeric antigen receptor targeting region to the target cell population can induce activation-induced cell death, and the target cell population is administered a therapeutically effective amount of IL-10 agent sufficient to prevent or limit the activation-induced cell death, wherein the IL-10 agent is IL-10 or PEG-IL-10.

2. The pharmaceutical composition of claim 1, wherein the IL-10 agent is administered prior to the administration of the therapeutically effective plurality of cells.

3. The pharmaceutical composition according to claim 1, wherein the IL-10 agent is administered simultaneously with the treatment of multiple cells.

4. The pharmaceutical composition of claim 1, wherein the IL-10 agent is administered after administration of the multiple cells to which the treatment is effective.

5. The pharmaceutical composition according to any one of claims 1-4, wherein the amount of the IL-10 agent administered is sufficient to enhance cytotoxic function.

6. The pharmaceutical composition according to claim 5, wherein the amount of the IL-10 agent applied is sufficient to achieve a serum concentration of 10-100 ng / mL.

7. The pharmaceutical composition according to any one of claims 1-6, wherein the IL-10 agent is PEG-IL-10.

8. The pharmaceutical composition according to any one of claims 1-7, wherein the PEG-IL-10 comprises at least one PEG molecule covalently linked to at least one amino acid residue of at least one monomer of IL-10.

9. The pharmaceutical composition according to any one of claims 1-8, wherein the PEG-IL-10 comprises a mixture of mono- and di-polyethylene glycolated IL-10.

10. The pharmaceutical composition according to any one of claims 1-9, wherein the PEG-IL-10 has a PEG component with a molecular weight of 5 kDa to 20 kDa.

11. The pharmaceutical composition according to any one of claims 1-9, wherein the PEG-IL-10 has a PEG component with a molecular weight of at least 20 kDa.

12. The pharmaceutical composition according to any one of claims 1-9, wherein the PEG-IL-10 has a PEG component with a molecular weight of at least 30 kD.

13. A pharmaceutical composition for treating cancer, angiogenesis, or precancerous conditions, comprising a plurality of genetically modified cells that are therapeutically effective in expressing: a) Chimeric antigen receptor (CAR), The chimeric antigen receptor (CAR) comprises at least one antigen-specific targeting region (ASTR), a transmembrane domain (TMD), and an intracellular signal transduction domain (ISD) containing a co-stimulatory domain (CSD) capable of binding to a target cell population. The binding of the chimeric antigen receptor targeting region to the target cell population can trigger activation-induced cell death. The intracellular signal transduction domain (ISD) mentioned therein contains CD3ζ; The co-stimulatory domain (CSD) comprises CD28, CD137 (4-1BB), or a combination thereof; and b) An amount of IL-10 agent sufficient to prevent or limit the activation-induced cell death.

14. The pharmaceutical composition of claim 13, wherein the chimeric antigen receptor and the IL-10 agent are expressed by the same carrier.

15. The pharmaceutical composition of claim 13, wherein the chimeric antigen receptor and the IL-10 agent are expressed by different carriers.

16. The pharmaceutical composition according to any one of claims 13-15, wherein the plurality of therapeutically effective cells are transfected with a vector expressing an amount of the IL-10 agent sufficient to enhance cytotoxic function.

17. A pharmaceutical composition for treating cancer, angiogenesis, or precancerous conditions, comprising: a) The first multiple cells that respond to treatment, said cells being genetically modified to express chimeric antigen receptors (CARs). The chimeric antigen receptor (CAR) comprises at least one antigen-specific targeting region (ASTR) capable of binding to a target cell population, a transmembrane domain (TMD), and an intracellular signal transduction domain (ISD) containing a co-stimulatory domain (CSD). The intracellular signal transduction domain (ISD) mentioned therein contains CD3ζ, The co-stimulatory domain (CSD) comprises CD28, CD137 (4-1BB), or a combination thereof, and the binding of the chimeric antigen receptor targeting region to the target cell population induces activation-induced cell death; and b) A second plurality of cells that respond to treatment, said cells being genetically modified to express an amount of IL-10 sufficient to prevent or limit said activation-induced cell death.

18. The pharmaceutical composition of claim 17, wherein the second plurality of therapeutically effective cells are transfected with a vector expressing an amount of the IL-10 agent sufficient to enhance cytotoxic function.

19. The pharmaceutical composition of claim 17, wherein the second plurality of therapeutically effective cells comprise CD8+ T cells transfected with a vector expressing the IL-10 agent.

20. The pharmaceutical composition according to any one of claims 14-16 and 18-19, wherein the carrier comprises a plasmid.

21. The pharmaceutical composition according to any one of claims 14-16 and 18-19, wherein the carrier comprises a viral vector.

22. The pharmaceutical composition according to any one of claims 14-16 and 18-21, wherein the expression of the IL-10 agent is regulated by an expression control element.

23. The pharmaceutical composition according to any one of claims 1-22, wherein the chimeric antigen receptor (CAR) comprises two co-stimulatory domains (CSD).

24. The pharmaceutical composition according to any one of claims 1-23, wherein the IL-10 agent enhances the function of activated memory CD8+ T cells.

25. The pharmaceutical composition according to any one of claims 1-24, wherein the target cell population comprises a tumor antigen, and wherein the tumor antigen is selected from CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

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