Compositions and methods for immunotherapy profiling

Abstract

To provide compositions and methods for immunotherapy profiling.SOLUTION: Compositions and methods for pharmacodynamic monitoring of immunotherapy are provided herein. The compositions include an immunotherapeutic agent linked to protease substrates. Upon administration, the compositions target sites of disease where proteases are upregulated during responsive immunotherapy, and subsequently cleave the attached substrates. Cleavage fragments are detected in a sample from the body, and detection of the fragments is indicative of an effect of the immunotherapeutic agent. In one embodiment, the therapeutic agent is an immune checkpoint inhibitor such as an anti-PD1 or anti-CTLA4 antibody. The protease substrate can also include a quencher molecule and fluorescent molecule flanking the substrate. In one embodiment, the detectable signal is a peptide fragment of the protease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Technical field of the invention The present invention relates generally to immunotherapy and pharmacodynamic monitoring of immunotherapy. [Background technology]

[0002] Background of the Invention Immunotherapy harnesses the immune system to treat a myriad of diseases, including cancer, organ transplant rejection, infectious diseases, allergic diseases, autoimmunity, and chronic inflammation. Immunotherapy employs both humoral and cellular immune responses using therapeutic antibodies (e.g., pembrolizumab / αPD-1), cytokines (e.g., proleukin / IL-2), and cell-based therapies (e.g., Kymriah / CART cells). New techniques that harness T cell immunity, for example, through adoptive transfer of genetically engineered cells or by reactivating endogenous antitumor CD8+ T cells with immune checkpoint blockade antibodies, have placed immunotherapy at the forefront of cancer treatment research. Immunotherapies that attenuate T cell responses through costimulatory blockade (e.g., abatacept / CTLA-4Ig) have also become a major avenue of treatment research for preventing transplant rejection or treating autoimmune and chronic inflammatory disorders.

[0003] Despite the broad potential of immunotherapy, the majority of patients do not achieve clinical benefit, and others may develop immunotherapy resistance during or between treatments due to poorly understood mechanisms. Patients who respond to immunotherapy may exhibit atypical response patterns that can be misinterpreted as disease progression. As a result, the benefits of immunotherapy are under-realized, and techniques for identifying biomarkers of immune response are inadequate. Due to inadequate response monitoring and identification of underlying resistance mechanisms, not only does the disease persist in the population, but drug development and clinical trials also face significant obstacles.

[0004] Tissue biopsy remains the gold standard for diagnosis but is invasive, with samples representing less than 0.1% of all disease sites (Cyll et al., Br J Cancer, 117(3):367-375 (2017)). Liquid biopsies offer a noninvasive approach, but biomarker dilution in blood significantly limits sensitivity (Nagrath, S. et al., Nature, 450(7173):1235-1239 (2007); Hori et al., Sci Transl Med, 3(109):109ra16 (2011)). Imaging techniques can also be limited by low sensitivity and specificity, as well as atypical response patterns commonly associated with immunotherapy, potentially misidentifying responsive patients as treatment failures. The development of better, noninvasive biomarkers could lead to earlier identification of responsive patients and elucidation of novel immunotherapy mechanisms. It is therefore an object of the present invention to provide immune checkpoint compositions and methods for monitoring their effectiveness. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Cyll et al., Br J Cancer,117(3):367-375(2017) [Non-patent document 2] Nagrath, S. et al., Nature, 450(7173):1235-1239(2007) [Non-patent document 3] Hori et al., Sci Transl Med,3(109):109ra16(2011) Summary of the Invention [Means for solving the problem]

[0006] Compositions and methods for pharmacodynamic monitoring of responses during immunotherapy are provided herein. Exemplary compositions include immunotherapeutic agents linked to protease substrates that detect protease activity in immune cells and disease sites and generate a detectable signal in the presence of protease activity. Upon administration, the compositions target disease sites where proteases are upregulated during responsive immunotherapy and then cleave the attached substrate. The cleaved fragments are detected in a body sample, and detection of the fragments indicates the effectiveness of the immunotherapeutic agent.

[0007] In one embodiment, the therapeutic agent is an immune checkpoint inhibitor, such as an anti-PD1 or anti-CTLA4 antibody. The protease substrate can also include a quencher molecule and a fluorescent molecule adjacent to the substrate. In one embodiment, the detectable signal is a peptide fragment of the protease.

[0008] Another embodiment provides a method of treating or preventing a disease in a subject in need thereof by administering to the subject an effective amount of a therapeutic agent linked to a protease substrate that provides a detectable signal responsive to protease activity promoted by the therapeutic agent, detecting and measuring the signal in a sample from the subject, determining the effect of the therapeutic agent on the subject (wherein if a detectable signal is detected, the subject is determined to be responsive to the therapeutic agent, and if no detectable signal is detected, the subject is determined to be non-responsive to the therapeutic agent), and administering the same effective amount of the therapeutic agent to the responsive subject or adjusting the effective amount of the therapeutic agent administered to the non-responsive subject. In one embodiment, the therapeutic agent is an immune checkpoint inhibitor, such as an anti-PD1 or anti-CTLA4 antibody.

[0009] In one embodiment, a subject determined to be non-responsive to an immunotherapeutic agent is administered a different immunotherapeutic agent.

[0010] In another embodiment, detecting and measuring the signal includes collecting a sample, such as a urine sample or a blood sample, from the subject and measuring the detectable signal in the sample. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 is a schematic diagram of an exemplary experimental use of the disclosed compositions and methods. A protease substrate functionalizes the targeting site of a therapeutically active therapeutic agent, where the attached substrate is cleaved by the protease upregulated during the responsive treatment, amplifying the detection signal in urine. The urine sample is analyzed by mass spectrometry.

[0012] [Figure 2-1] Figure 2A is a schematic diagram of amine coupling of granzyme B (GzmB) substrates to αPD-1 to generate "αPD-1 therasensors." Figure 2B is a graph showing PD-1 binding by αPD-1 modified with a GzmB substrate (Therasensor) and unmodified PD-1 (αPD-1). The X-axis represents αPD-1 concentration (μg / mL; Log10), and the Y-axis represents PD-1 binding. Figure 2C is a flow plot of CD8 tumor-infiltrating T cells showing comparable staining with unmodified αPD-1 or αPD-1 modified with a GzmB substrate (Therasensor). Figure 2D is a graphical representation of Figure 2C. Figure 2E is a graph showing the protease cleavage rates of αPD-1 modified with a GzmB substrate (Therasensor) incubated with or without GzmB or with the control protease, thrombin. [Figure 2-2] Same as above.

[0013] [Figure 3-1]Figure 3A is a schematic diagram of amine coupling of a GzmB substrate with CTLA-4 Ig to generate a "CTLA-4 Ig Therasensor." Figures 3B-3C are graphs showing target binding by GzmB substrate-modified CTLA-4 Ig (CTLA4-Ig Therasensor) or unmodified CTLA-4 Ig (CTLA4-Ig) in a CD80 / CD86 antibody competition assay. Figure 3D is a bar graph showing proliferation of Cell Trace Violet (CTV)-labeled BL / 6 CD8+ cells co-incubated with BALB / c CD11c+ dendritic cells in the presence of αCD40L alone, αCD40L + unmodified CTLA4-Ig (αCD40L + CTLA4-Ig), or αCD40L + modified CTLA4-Ig (αCD40L + Therasensor). Figure 3E is a line graph showing the protease cleavage rates of CTLA-4IG modified with GzmB substrates incubated with or without GzmB or with the indicated proteases (abbreviations: CTSB, cathepsin B; MMP2, matrix metalloproteinase 2; MMP9, matrix metalloproteinase 9; MMP15, matrix metalloproteinase 15; C1S, complement component S1; MASP1, mannose-associated serine protease 1). [Figure 3-2] Same as above.

[0014] [Figure 4-1]Figure 4A is a schematic diagram of GzmB cleavage of GzmB substrate-modified αPD-1 in the supernatant of activated T cells but not tumor cells. Figure 4B is a line graph showing the protease cleavage rates when GzmB substrate-modified αPD-1 (GzmB serasensor), control serasensor, or αPD-1 was incubated with the supernatant of activated T cells, CT26 cells, MC38 cells, B16 cells, or vehicle alone. Figure 4C is a schematic diagram of αPD-1 serasensor cleavage during T cell killing of tumor cells. Figure 4D is a bar graph showing the cytotoxicity rate measured by LDH assay. Figure 4E is a bar graph showing GzmB protein secretion determined by ELISA. Increased cell killing and GzmB secretion were observed with increasing effector / target ratios (1:1, 5:1, 10:1). Figure 4F is a bar graph showing the protease activity of control and αPD-1 serasensors across multiple effector to target cell ratios. Figure 4G is a bar graph showing the protease activity of αPD-1 serasensors in cells incubated with P-Mel or OT-1. Figure 4H is a bar graph showing the protease activity of CTLA-4Ig serasensors added to supernatants from cocultures of OT-1 cells with either OVA-expressing EG7 cells or the parental non-OVA-expressing EL4 cell line (E:T ratios of 1:1, 5:1, and 10:1). [Figure 4-2] Same as above. [Figure 4-3] Same as above.

[0015] [Figure 5-1]Figure 5A is a line graph showing MC38 syngeneic tumor volume over time in mice treated with GzmB substrate-modified αPD-1 (αPD-1 therasensor) or an isotype control therasensor. Figure 5B is a panel of flow cytometry plots showing intracellular GzmB staining within CD8+ TILs isolated from MC38 tumors after two treatment doses. Figures 5C and 5D are graphs showing the percentage (Figure 5C) and number (Figure 5D) of GzmB-positive CD8 TILs per tumor. Figure 5E is a schematic diagram of the experimental method for urinary analysis of therasensor in MC38 tumor-bearing mice. Figure 5F is a graph showing renal clearance of peptide fragments in tumor-bearing mice treated with control therasensor or α-PD1 therasensor. Figures 5G-5H are graphs showing tumor volume over time in CT26 tumor-bearing mice treated with α-CTLA4 monotherapy (Figure 5G), α-PD1 / CTLA-4 combination therapy (Figure 5H), or left untreated. The X-axis represents time (days), and the Y-axis represents tumor volume (mm). The gray area represents the treatment window. Figure 5I is a panel of flow cytometry plots showing intracellular GzmB staining within CD8+ TILs isolated from CT26 tumors on day 18. Figures 5J-5K are graphs showing the percentage (Figure 5J) and number (Figure 5K) of GzmB-positive CD8 TILs per tumor. Figure 5L is a schematic diagram of the experimental method for urinary analysis of Therasensor in CT26 tumor-bearing mice. 5M-5N are graphs showing renal clearance of cleaved fluorescent reporter in urine of tumor-bearing mice treated with αCTLA-4, αPD-1 / CTLA-4, or untreated. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above.

[0016] [Figure 6-1]Figure 6A is a timeline showing the test procedure. Figures 6B-6I are photographs showing skin allograft rejection over time. Figure 6J is a plot of immunohistochemistry data showing the percentage of CD8 staining in the graft and healthy skin tissue of mice bearing allografts and syngeneic grafts. Figure 6K is a plot of immunohistochemistry data showing the percentage of GzmB staining in the graft and healthy skin tissue of mice bearing allografts and syngeneic grafts. Figure 6L is a plot of skin graft scores showing the quality of skin allograft grafts in untreated mice and mice treated with CTLA4-Ig and αCD154 that responded poorly ("non-responsive") or strongly ("responsive") to costimulatory blockade treatment. Figure 6I is a graft survival curve showing the survival rates of untreated, non-responsive, and responsive grafts. Figure 6J is a graph showing the rate of renal clearance of cleaved fluorescent reporter in urine on PODs 4, 7, and 15. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above.

[0017] [Figure 7-1]Figure 7A is a schematic diagram of the patient cohort from Riaz et al., 2017. Figure 7B is a graph showing the classification of responders from non-responders using 250 extracellular proteases. Figure 7C is a graph showing the classification of responders from non-responders using 14 extracellular proteases identified as important by the Lasso algorithm. Figure 7D is a graph showing the relative importance weights of the 14 extracellular proteases in Figure 7C. Figures 7E-7F are graphs showing the identification of resistance mechanisms by pathway analysis. Figure 7E shows non-responders predicted by a panel of 12 proteases for reduced expression of the IFNγ pathway. Figure 7F shows the classification of non-responders with reduced MHC I antigen presentation using the same panel of 12 proteases. Figure 7G is a graph showing the percentage of pathways from each molecular process (IFNγ and MHC I antigen presentation) that were lost when comparing gene expression between responders and non-responders. FIG. 7H is a graph showing the relative weight of the Lasso coefficients in classifying non-responders with and without reduced MHC I presentation. [Figure 7-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description of the Invention I. Definition It is to be understood that the present disclosure is not limited to the compositions and methods described herein, and the experimental conditions described, which may themselves vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Any compositions, methods and materials similar or equivalent to those described herein can be used to practice or test the present invention.All publications mentioned are incorporated herein in their entirety by reference.

[0020] Use of the terms "a," "an," "the," and similar referents in the context of describing the presently claimed invention (particularly in the context of the claims) are to be construed as covering both the singular and the plural unless otherwise indicated herein or clearly contradicted by context.

[0021] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each individual value falling within the range, unless otherwise stated herein, and each individual value is incorporated into the specification as if it were individually recited herein.

[0022] The use of the term "about" is intended to describe a value above or below the stated value by approximately + / - 10%; in other embodiments, values ​​may vary above or below the stated value by approximately + / - 5%; in other embodiments, values ​​may vary above or below the stated value by approximately + / - 2%; in other embodiments, values ​​may vary above or below the stated value by approximately + / - 1%. The above ranges are intended to be made clear by context, and no further limitations are implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "etc.") described herein is intended merely to better elucidate the invention and does not impose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0023] As used herein, a molecule is said to be capable of "immunospecifically binding" to a second molecule when such binding exhibits the specificity and affinity of the antibody for its cognate antigen. An antibody is said to be capable of immunospecifically binding to a target region or conformation ("epitope") of an antigen when such binding involves the antigen recognition site of an immunoglobulin molecule. An antibody that immunospecifically binds to a particular antigen may bind other antigens with lower affinity if the other antigens share some sequence or conformational similarity recognized by the antigen recognition site as determined, for example, by immunoassay, BIACORE® assay, or other assay known in the art, but will not bind completely unrelated antigens. However, in some embodiments, antibodies (and antigen-binding fragments thereof) do not cross-react with other antigens. Antibodies may also bind other molecules, such as FcR receptors, in a non-immunospecific manner via binding domains in other regions / domains of the molecule that do not contain the antigen recognition site, such as the Fc region.

[0024] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule having a "variable region" antigen recognition site, including an antigen-binding fragment of an antibody. The term "variable region" is intended to distinguish such domains of an immunoglobulin from domains shared broadly by antibodies (such as the antibody Fc domain). The variable region includes "hypervariable regions," the residues of which are involved in antigen binding. The hypervariable regions include amino acid residues in the "complementarity-determining regions" or "CDRs" (i.e., generally approximately 24-34 residues (L1), 50-56 residues (L2), and 89-97 residues (L3) of the light chain variable domain and approximately 27-35 residues (H1), 50-65 residues (H2), and 95-102 residues (H3) of the heavy chain variable domain; see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Health, Bethesda, MD. (1991)) and / or residues of the "hypervariable loops" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) of the light-chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy-chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.The term antibody includes monoclonal antibodies, polyspecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (see, e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231:25; WO 94 / 04678 and WO 94 / 25591; U.S. Pat. No. 6,005,079), single-chain Fvs (scFvs) (see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, 1999). New York, pp. 269-315 (1994)), single-chain antibodies, disulfide-linked Fvs (sdFv), intrabodies, diabodies, triabodies, tetrabodies, Bis-scFv, minibodies, Fab2, Fab3, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies against an antibody). In particular, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0025] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more portions of an antibody that contain the antibody's complementarity-determining regions ("CDRs") and, optionally, framework residues comprising the antibody's "variable region" antigen recognition site, and that exhibit the ability to immunospecifically bind to an antigen. Such fragments include Fab', F(ab'), Fv, single-chain (ScFv), and variants, naturally occurring variants thereof, as well as fusion proteins comprising the antibody's "variable region" antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor, or a receptor ligand, etc.).

[0026] As used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0027] As used herein, the term "modulate" refers to the ability to alter an effect, outcome, or activity (e.g., signal transduction). Such modulation can be agonistic or antagonistic. Antagonistic modulation can be partial (i.e., attenuating but not eliminating) or can completely eliminate such activity (e.g., neutralization). Modulation can include receptor internalization after antibody binding or a decrease in receptor expression on target cells. Agonistic modulation can enhance or otherwise increase or potentiate an activity (e.g., signal transduction). In yet another embodiment, such modulation can alter the nature of the interaction between a ligand and its cognate receptor, altering the nature of the induced signal transduction. For example, a molecule, by binding to a ligand or receptor, can alter the ability of such molecule to bind to other ligands or receptors, thereby altering their overall activity. In some embodiments, such modulation results in at least a 10% change in a measurable immune system activity, at least a 50% change in such activity, or at least a 2-fold, 5-fold, 10-fold, or at least a 100-fold change in such activity.

[0028] As used herein, the term "polypeptide" refers to a chain of amino acids of any length, regardless of modification (e.g., phosphorylation or glycosylation). The term polypeptide includes proteins and fragments thereof. Polypeptides may be "exogenous," meaning that they are "heterologous," i.e., foreign to the host cell in which they are utilized, such as human polypeptides produced by bacterial cells. Polypeptides are disclosed herein as amino acid residue sequences. These sequences are written from left to right in the direction from amino terminus to carboxy terminus. According to standard nomenclature, amino acid residue sequences are represented by three-letter or one-letter codes, as shown below: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0029] As used herein, the terms "treat," "treating," "treatment," and "therapeutic use" refer to the elimination, reduction, or amelioration of one or more symptoms of a disease or disorder. As used herein, a "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to mediate clinically relevant elimination, reduction, or amelioration of such symptoms. An effect is clinically relevant if its magnitude is sufficient to affect the health or prognosis of the recipient subject. A therapeutically effective amount can refer to an amount of a therapeutic agent sufficient to delay or minimize the onset of a disease, for example, to delay or minimize the spread of cancer. A therapeutically effective amount can also refer to the amount of a therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

[0030] As used herein, the term "prophylactic agent" refers to an agent that can be used to prevent a disorder or disease prior to the detection of symptoms of such a disorder or disease. A "prophylactically effective" amount is the amount of a prophylactic agent sufficient to mediate such protection. A prophylactically effective amount can also refer to the amount of a prophylactic agent that provides a prophylactic benefit in the prevention of disease.

[0031] As used herein, the terms "immune," "immunological," or "immune" response refer to the development in a recipient patient of a beneficial humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secretory products) response to a peptide. Such a response may be an active response elicited by administration of an immunogen, or a passive response elicited by administration of antibodies or primed T cells. A cellular immune response is elicited by presentation of a polypeptide epitope in association with class I or class II MHC molecules and results in the expression of antigen-specific CD4 + T helper cells and / or CD8 + Activates cytotoxic T cells. The response may also include activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglial cells, eosinophils, and activation or recruitment of neutrophils or other components of innate immunity. The presence of a cell-mediated immune response can be determined by proliferation assays (CD4 + The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T cells from an immunized syngeneic animal and measuring the protective or therapeutic effect in a second subject.

[0032] Activated T cells specific for the molecular structure of invading pathogens proliferate and attack the pathogens. These attacks can either directly kill the pathogen or secrete antibodies that enhance phagocytosis of the pathogen and disrupt infection. Some T cells respond to APCs of the innate immune system and indirectly induce immune responses by releasing cytokines.

[0033] As used herein, "immune cells" refers to cells of hematopoietic origin, including, but not limited to, T cells, B cells, monocytes, dendritic cells, and macrophages.

[0034] As used herein, "inflammatory molecule" refers to a molecule that results in an inflammatory response, including, but not limited to, cytokines and metalloproteases, including, but not limited to, IL-1β, TNF-α, TGF-β, IFN-γ, IL-18, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs.

[0035] As used herein, the terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including but not limited to humans, rodents such as mice and rats, and other laboratory animals.

[0036] As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline solutions, water and emulsions (such as oil / water or water / oil emulsions), as well as various types of wetting agents.

[0037] As used herein, the term "immunosuppression" refers to the suppression of the immune system and its ability to fight infection and other diseases. Immunosuppression can be intentionally induced by drugs, or can occur as a side effect of certain diseases, environmental factors, or other drugs such as anti-cancer drugs and steroids.

[0038] As used herein, the terms "immunosuppressive disease" and "immunodeficiency disease" refer to diseases characterized by partial or complete suppression or dysfunction of a subject's immune response.

[0039] As used herein, the term "cancer" refers to a neoplasm or tumor resulting from the abnormal and uncontrolled growth of cells. As used herein, cancer explicitly includes leukemia and lymphoma. The term "cancer" refers to a disease involving cells that have the potential to metastasize to distant sites and exhibit phenotypic traits distinct from non-cancerous cells, such as the formation of colonies in a three-dimensional substrate, such as soft agar, or the formation of tubular networks or web-like matrices in three-dimensional basement membrane or extracellular matrix preparations. Non-cancerous cells do not form colonies in soft agar, but rather form distinct spheroid structures in three-dimensional basement membrane or extracellular matrix preparations. II. Compositions and Methods for Immunotherapeutic Profiling

[0040] Provided herein are immunotherapeutic compositions and methods of use for both treating a disease in a subject in need thereof and profiling a subject's immune response to immunotherapy. Exemplary compositions include an immunotherapeutic agent conjugated to a protease substrate capable of being cleaved from the immunotherapeutic agent by a disease- or tissue-specific protease. In one embodiment, when the immunotherapeutic agent reaches the disease site and exerts its therapeutic effect, increased immunoprotease activity cleaves the attached protease substrate from the immunotherapeutic agent, releasing peptide fragments or detectable signals into the circulation, where they are selectively filtered into the urine. The circulating cleaved fragments or detectable signals can be detected in a sample from the subject, such as a blood sample or a urine sample. A. Immunotherapeutic Agents

[0041] In one embodiment, the immunotherapeutic agent conjugated to the protease substrate is a checkpoint inhibitor. Immune checkpoint inhibitors generally reverse immune suppression within the tumor microenvironment by blocking inhibitory immune checkpoint molecules such as PD-1 using therapeutic antibodies such as pembrolizumab (αPD1) or ipilimumab (αCTLA-4) (Tumeh PC et al., Nature, 515(7528):568-71(2014)).

[0042] In one embodiment, the immunotherapeutic agent is an antibody, antigen-binding fragment, fusion protein, or small molecule. In another embodiment, the immunotherapeutic agent is a T-cell therapy, such as CAR-T cell therapy. In yet another embodiment, the immunotherapeutic agent is an immunosuppressant. The targets of immunotherapeutic agents are described in detail below. 1.PD-1

[0043] Programmed Death-1 (PD-1) is a member of the CD28 family of receptors that, when induced on T cells, delivers a negative immune response. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that reduces T cell proliferation and / or the strength and / or duration of the T cell response. Suitable PD-1 antagonists are described in U.S. Pat. Nos. 8,114,845, 8,609,089, and 8,709,416, the entities of which are specifically incorporated herein by reference, and include compounds or agents that bind to the ligand of PD-1 and block, thereby preventing or inhibiting binding of the ligand to the PD-1 receptor, or that directly bind to and block the PD-1 receptor without inducing inhibitory signaling by the PD-1 receptor.

[0044] In some embodiments, the PD-1 receptor antagonist binds directly to the PD-1 receptor without inducing inhibitory signaling, and also binds to a ligand of the PD-1 receptor, reducing or preventing the ligand from inducing signaling by the PD-1 receptor. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and induce inhibitory signaling, fewer cells are compromised by the negative signals delivered by PD-1 signaling, resulting in a more robust immune response.

[0045] PD-1 signaling is thought to be driven by binding of PD-1 ligands (such as B7-H1 or B7-DC) in close proximity to peptide antigens presented by the major histocompatibility complex (MHC) (see, e.g., Freeman, Proc. Natl. Acad. Sci. USA, 105:10275-10276 (2008)). Therefore, proteins, antibodies, or small molecules that prevent co-ligation of PD-1 and TCR on the T cell membrane are also useful PD-1 antagonists.

[0046] In some embodiments, the PD-1 receptor antagonist is a small molecule antagonist or antibody that reduces or prevents PD-1 receptor signaling by binding to a ligand of PD-1 or PD-1 itself (particularly when co-ligation of PD-1 and TCR does not follow from such binding, thereby causing inhibitory signaling by the PD-1 receptor).

[0047] Other PD-1 antagonists contemplated by the methods of the present invention include antibodies that bind to PD-1 or a ligand of PD-1, and other antibodies.

[0048] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following U.S. Patent Nos. 7,332,582, 7,488,802, 7,521,051, 7,524,498, 7,563,869, 7,981,416, 8,088,905, 8,287,856, 8,580,247, 8,728,474, 8,779,105, 9,067,999, 9,073,994, 9,084,776, 9,205,148, 9,358,289, 9,387,247, 9,492,539, and 9,492,540, all of which are incorporated by reference in their entireties.

[0049] Exemplary anti-B7-H1 (also referred to as anti-PD-L1) antibodies include, but are not limited to, those described in the following U.S. Patent Nos. 8,383,796, 9,102,725, 9,273,135, 9,393,301, and 9,580,507, all of which are specifically incorporated by reference herein in their entirety.

[0050] For anti-B7-DC (also known as anti-PD-L2) antibodies, see U.S. Patent Nos. 7,411,051, 7,052,694, 7,390,888, 8,188,238, and 9,255,147, all of which are specifically incorporated by reference in their entirety.

[0051] Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC polypeptides, including homologs and variants thereof, as well as active fragments of any of the foregoing, and fusion proteins incorporating any of these. In some embodiments, the fusion protein comprises a soluble portion of B7-DC linked to the Fc portion of an antibody, such as human IgG, and does not incorporate all or part of the transmembrane portion of human B7-DC.

[0052] The PD-1 antagonist can also be a fragment of mammalian B7-H1, e.g., from a primate, e.g., mouse, or human, where the fragment binds to and blocks PD-1 but does not result in inhibitory signaling by PD-1. The fragment can also be part of a fusion protein, e.g., an Ig fusion protein.

[0053] Other useful polypeptide PD-1 antagonists include those that bind to the PD-1 receptor ligand. These include PD-1 receptor proteins or soluble fragments thereof that can bind to PD-1 ligands, such as B7-H1 or B7-DC, and prevent binding to endogenous PD-1 receptors, thereby preventing inhibitory signaling. B7-H1 has also been shown to bind to the protein B7.1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such fragments also include soluble ECD portions of the PD-1 protein containing mutations, such as the A99L mutation, that increase binding to the natural ligand (Molnar et al., PNAS, 105:10483-10488 (2008)). B7-1 or soluble fragments thereof that can bind to the B7-H1 ligand and prevent binding to endogenous PD-1 receptors, thereby preventing inhibitory signaling, are also useful.

[0054] PD-1 and B7-H1 antisense nucleic acids (both DNA and RNA) and siRNA molecules can also be PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 in T cells and the production of T cell ligands such as B7-H1, PD-L1, and / or PD-L2. For example, siRNA (e.g., about 21 nucleotides in length, specific to the gene encoding PD-1 or the gene encoding the PD-1 ligand, and the oligonucleotide can be easily purchased commercially) complexed with a carrier such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8):2231-2244 (2009)) can be easily taken up by cells expressing PD-1 and its ligand, reducing the expression of these receptors and ligands, thereby reducing inhibitory signaling in T cells and thereby activating T cells. 2.CTLA4

[0055] Cytotoxic T lymphocyte-associated protein 4 (CTLA4) is a protein receptor that functions as an immune checkpoint and downregulates immune responses. CTLA4 is constitutively expressed in regulatory T cells, but is only upregulated in conventional T cells after activation. CTLA4 transmits inhibitory signals to T cells. In some embodiments, the immunotherapeutic agent is a CTLA4 antagonist, such as an antagonistic anti-CTLA4 antibody. Examples of anti-CTLA4 antibodies contemplated for use in the methods of the present invention include those described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).

[0056] Specific examples of anti-CTLA4 antibodies useful in the methods of the invention include the human anti-CTLA4 antibody ipilimumab, administered at a dose of, for example, about 10 mg / kg, and the human anti-CTLA4 antibody tremelimumab, administered at a dose of, for example, about 15 mg / kg. See also Sammartino et al., Clinical Kidney Journal, 3(2):135-137 (2010), published online in December 2009.

[0057] In another embodiment, the antagonist is a small molecule. A series of small organic compounds have been shown to bind to B7-1 ligand and prevent it from binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368 (2002)). These small organic compounds can be administered alone or together with anti-CTLA4 antibodies to reduce the inhibitory signaling of T cells. 3. Other immune checkpoint inhibitors

[0058] In another embodiment, the immunotherapeutic agent is an immune checkpoint inhibitor that inhibits the activity of other immune checkpoint molecules, including but not limited to, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, NOX2, TIM3, VISTA, SIGLEC7, and SIGLEC9.

[0059] B7-H3, also known as CD276, is an immune checkpoint molecule of the B7 family. B7-H3 is involved in regulating T cell-mediated immune responses. It also plays a role in protecting tumor cells by inhibiting natural killer-mediated cytolysis and serves as a marker for detecting neuroblastoma cells. It is also involved in the development of acute and chronic transplant rejection and in regulating lymphocyte activity at mucosal surfaces. B7-H3 immunotherapeutic agents are known in the art. Exemplary anti-B7-H4 agents include, but are not limited to, those described in the following U.S. Patent Nos. 7,847,081, 8,802,091, and 9,371,395, all of which are specifically incorporated herein by reference in their entirety.

[0060] Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme with immunosuppressive properties. IDO is known to suppress T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. IDO immunotherapeutics are known in the art. Exemplary anti-IDO agents include, but are not limited to, those described in the following U.S. Patent Nos. 7,598,287, 9,598,422, and 10,323,004, all of which are specifically incorporated herein by reference in their entirety.

[0061] Lymphocyte activation gene-3 (LAG3) is an inhibitory receptor of antigen-activated T cells. When LAG3 binds to a ligand such as FGL1, it delivers an inhibitory signal. After TCR engagement, LAG3 associates with CD3-TCR at the immune synapse and directly inhibits T cell activation. LAG3 suppresses immune responses not only through its direct effect on CD8+ T cells but also through its action on Tregs. LAG3 immunotherapeutic agents are known in the art. Exemplary anti-LAG3 agents include, but are not limited to, those described in the following U.S. Patent Nos. 10,188,730 and 10,358,495, all of which are specifically incorporated herein by reference in their entirety.

[0062] V-type immunoglobulin domain-containing suppressor of T cell activation (VISTA) is an immunomodulatory receptor that inhibits T cell response. VISTA is expressed in hematopoietic cells. VISTA immunotherapeutic agents are known in the art. Exemplary anti-VISTA agents include, but are not limited to, those described in the following U.S. Patent Nos. 9,381,244 and 10,273,301, all of which are specifically incorporated herein by reference in their entirety. 4.CAR-T cells

[0063] Another form of immunotherapy contemplated for use in the disclosed compositions and methods is CAR-T cells. Chimeric antigen receptor T cells (CAR-T cells) are T cells genetically engineered to produce an artificial T cell receptor, which gives the engineered T cells the ability to target specific proteins. CAR-T immunotherapy is based on modifying T cells to recognize cancer cells, allowing them to more effectively target and destroy them. T cells are collected from a subject and genetically engineered to express a specific T cell receptor. The resulting CAR-T cells are then infused into the subject to attack their tumors. CAR-T cells can be derived from T cells in the subject's own blood (autologous) or from T cells from another healthy donor (allogeneic). Once isolated from the subject, these T cells are genetically engineered to express a specific CAR and are programmed to target antigens present on the surface of the tumor. For safety, CAR-T cells are engineered to be specific for antigens expressed on tumors that are not expressed on healthy cells.

[0064] In one embodiment, the CAR-T cells are linked to a protease substrate that is cleaved from the CAR-T cells by a protease produced when the CAR-T cells affect diseased cells. In such embodiments, detection of a separated detectable signal in the subject's urine indicates that the CAR-T cells are affecting the subject. 5. Immunosuppressants

[0065] In another embodiment, the immunotherapeutic agent is an immunosuppressant. Immunosuppressants include, but are not limited to, antibodies against other lymphocyte surface markers (e.g., CD40, alpha-4 integrin) or against cytokines, fusion proteins (e.g., CTLA-4-Ig (Orencia®), TNFR-Ig (Enbrel®)), TNF-α blockers (such as Enbrel, Remicade, Cimzia, and Humira), cyclophosphamide (CTX) (i.e., Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune™), methotrexate (MTX) (i.e., Rheumatrex®, Trexall®), belimumab (i.e., Benlysta®), or other immunosuppressants (e.g., cyclosporine A, FK506-like compounds, rapamycin compounds, or steroids), antiproliferative agents, cytotoxic agents, or other compounds that may aid in immunosuppression.

[0066] The immunosuppressant may be a CTLA-4 fusion protein, such as CTLA-4-Ig (abatacept). CTLA-4-Ig fusion proteins compete with the costimulatory receptor CD28 on T cells for binding to CD80 / CD86 (B7-1 / B7-2) on antigen-presenting cells, thus functioning to inhibit T cell activation. In another embodiment, the immunosuppressant is a CTLA-4-Ig fusion protein known as belatacept. Belatacept contains two amino acid substitutions (L104E and A29Y) that significantly enhance its binding activity to CD86 in vivo. In another embodiment, the immunosuppressant is Maxy-4.

[0067] In another embodiment, the immunosuppressant is cyclophosphamide (CTX). Cyclophosphamide (generic name for Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune™), also known as cytophosphan, is a nitrogen mustard alkylating agent of the oxazofurin group. It is used to treat various types of cancer and some autoimmune disorders. Cyclophosphamide (CTX) is the primary drug used to treat diffuse proliferative glomerulonephritis in patients with renal lupus.

[0068] As used herein, the term "rapamycin compound" includes the neutral tricyclic compound rapamycin, rapamycin derivatives, rapamycin analogs, and other macrolide compounds that are believed to have the same mechanism of action as rapamycin (e.g., inhibition of cytokine function). The term "rapamycin compound" includes compounds that are structurally similar to rapamycin, e.g., compounds with similar macrocyclic structures that have been modified to enhance their therapeutic efficacy. Exemplary rapamycin compounds are known in the art (see, e.g., WO 95122972, WO 95116691, WO 95104738, U.S. Pat. Nos. 6,015,809; 5,989,591; 5,567,709; 5,559,112; 5,530,006; 5,484,790; 5,385,908; 5,202,332; 5,162,333; 5,780,462; 5,120,727).

[0069] The term "FK506-like compounds" includes FK506 and FK506 derivatives and analogs, e.g., compounds structurally similar to FK506, e.g., compounds having a similar macrocyclic structure that has been modified to enhance its therapeutic effect. Examples of FK506-like compounds include, for example, those described in International Publication No. 00101385. In some embodiments, the term "rapamycin compounds" as used herein does not include FK506-like compounds. B. Detectable signal molecule

[0070] The disclosed immunotherapeutic agents bind to a protease substrate that is cleaved by the protease, releasing a detectable signal from the peptide fragment or therapeutic agent. In some embodiments, the detectable signal is a cleavage product or peptide fragment of the protease substrate itself. Upon cleavage, the protease fragment is released into the circulation and detected in urine by mass spectrometry.

[0071] In another embodiment, the detection signal is a protease substrate engineered with a quencher molecule before the cleavage site and a fluorescent reporter after the cleavage site. Upon cleavage of the protease substrate, the quencher and fluorescent reporter are separated, releasing the reporter into the circulation. The fluorescent signal is detected in urine using standard methods, such as flow cytometry.

[0072] The protease substrate can be attached to the immunotherapeutic agent using methods known in the art. In one embodiment, the protease substrate is attached to the immunotherapeutic agent by introducing a linker that forms a covalent bond between the protease substrate and the immunotherapeutic agent. Exemplary reactions that can be used to link the protease substrate include, but are not limited to, amine-amine crosslinking using NHS ester, thiol-thiol crosslinking using maleimide, amine-thiol crosslinking using NHS ester and maleimide, and biotin / streptavidin interaction. In one embodiment, the protease substrate is attached to the immunotherapeutic agent by an amine coupling reaction. 1. Protease Substrates

[0073] The disclosed compositions and methods for using them to determine the efficacy of a therapeutic response rely on protease activity to cleave a protease substrate and release a detectable signal from a peptide fragment or therapeutic agent. Proteases are a class of enzymes containing over 550 members encoded in the human genome, many of which have disease-specific roles, including important roles in immunity. For example, cytotoxic T cell-mediated target cell death is a protease-driven process involving 1) signaling of death receptors and activation of caspases, proteases whose activity mediates cell death, and 2) secretion of granzymes, proteases that enter target cells via a perforin-dependent mechanism and activate caspase-mediated cell death. Furthermore, proteases are central to other aspects of immune activity, including cell migration, matrix degradation and repair, and complement activation, while tumor proteases, such as inflammatory and matrix-degrading proteases, are established hallmarks of cancer (Arias et al., Trends Cancer, 3(6):407-422 (2017); Egeblad et al., Nat Rev Cancer, 2(3):161-174 (2002)).

[0074] Given the central role proteases play in immunity, oncology, and the biology underlying the pathophysiology of multiple diseases, proteases offer innovative approaches for immunotherapy response monitoring (Dudani et al., Ann Rev of Cancer Biology, (2018)). For example, a "hot" tumor is marked by effective immune infiltration of cytotoxic T cells, which kill cancer cells primarily through a perforin-dependent granzyme-mediated pathway. Granzymes constitute a family of potent serine proteases (Larimer et al., Cancer Res, 77(9):2318-2327 (2017); Voskoboinik et al., Nat Rev Immunol, 15(6):388-400 (2015)). Tumor expression of proteases, including inflammatory and matrix-degrading proteases, is well established as a hallmark of fundamental tumor biology, including angiogenesis, growth, and metastasis (Dudani et al., Ann Rev of Cancer Biology, (2018)). These protease signatures can be used to stage cancer, monitor progression and regression, and provide early indications of drug response. In one embodiment, the disclosed immunotherapeutic agents have the ability to quantify immune and disease-site specific protease activity early in treatment, predicting therapeutic efficacy and enabling the identification of active biomarkers indicative of resistance to immunotherapy.

[0075] In one embodiment, the catalytic protease amplifies (1000-fold) the detection signal at the disease or treatment site. Following protease cleavage, the immunotherapeutic agents disclosed herein are concentrated in the urine instead of being diluted in the blood, further concentrating the signal by up to 100-fold. This allows for ultrasensitive and early detection of T cell activity prior to radiologically detectable changes at the disease site.

[0076] The protease substrate contains a recognition sequence for cleavage by a protease. Cleavage of the protease substrate bound to the immunotherapeutic agent releases a peptide fragment of the substrate for the detectable signal molecule linked to the substrate from the immunotherapeutic agent. In some embodiments, the protease substrate bound to the immunotherapeutic agent is a tumor-specific protease substrate. Exemplary tumor-associated proteases include, but are not limited to, cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein 1, kallikrein 3 (PSA), kallikrein 10, kallikrein 15, uPA, uPAR, caspases, matrix metalloproteinases such as MMP1, MMP2, MMP8, MMP9, MMP13, MMP14, and ADAM. In another embodiment, the protease substrate is a cell-specific protease substrate, such as a T cell-specific protease substrate. Exemplary cell-specific proteases include, but are not limited to, neutrophil serine proteases such as cathepsin G, neutrophil elastase, and proteinase 3, mucosa-associated lymphoid tissue 1 (MALT1), granzymes, and cysteine ​​proteinases of the caspase family such as caspase-3, -6, -7, and -8. 2. Other detection molecules

[0077] In some embodiments, the detection signal is a protease substrate engineered with a quencher molecule before the cleavage site and a fluorophore or fluorescent reporter after the cleavage site. Quencher molecules are known in the art. Exemplary quencher molecules include, but are not limited to, Deep Dark Quenchers (Eurogentec), DABCYL, TAMRA, BHQ-1®, BHQ-2®, BHQ-3®, BBQ®-650, ECLIPSE, Iowa Black® quencher, and QSY. Exemplary fluorophores or fluorescent reporters include, but are not limited to, 6-FAM™, TET™, JOE™, HEX™, VIC®, Cyanine 3, ROX™, LC Red 640, Cyanine 5, fluorescein isothiocyanate (FITC), rhodamine (tetramethylrhodamine isothiocyanate, TRITC, Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, Texas Red, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 680, and Alexa Fluor 750.

[0078] In some embodiments, the protease substrate is engineered with other detectable molecules, such as avidin, biotin, β-galactosidase, luciferase, alkaline phosphatase (AP), and horseradish peroxidase (HRP). In such embodiments, the detectable molecule is cleaved from the protease substrate, which remains attached to the immunotherapeutic agent, and released into the circulation. The detectable molecule is then detected in the urine sample using an appropriate detection method, such as, but not limited to, ELISA, Western blotting, immunoassay, and bioluminescence assay. C. Pharmaceutical Compositions

[0079] Pharmaceutical compositions containing the disclosed activity-sensing immunotherapeutic agents are provided. The immunotherapeutic agents can be administered parenterally (intramuscularly, intraperitoneally, intravenously (IV), or subcutaneously), transdermally (passively, or using iontophoresis or electroporation), or transmucosally (nasally, vaginally, rectally, or sublingually), or using bioerodible inserts, and can be formulated into dosage forms appropriate for each administration route.

[0080] In some in vivo approaches, the compositions disclosed herein are administered to a subject in a therapeutically effective amount.As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dosage that is sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated, or that is sufficient to provide desired pharmacological and / or physiological effects.The exact dosage will vary depending on a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), disease, and treatment being performed.

[0081] As further research is conducted, information regarding appropriate dosage levels for the disclosed immunomodulatory agents for the treatment of various symptoms in various patients will become apparent. A practitioner of ordinary skill will be able to ascertain appropriate dosing, taking into account the recipient's treatment status, age, and overall health. The selected dosage will depend on the desired therapeutic effect, the route of administration, and the desired duration of treatment. For the disclosed immunomodulatory agents, dosage levels of 0.001 to 20 mg / kg body weight are generally administered to mammals daily. Dosages for anti-PD-1, anti-B7-H1, and anti-CTLA4 antibodies are known in the art and may range, for example, from 0.1 to 100 mg / kg, or from 1 to 50 mg / kg, or even shorter ranges such as 10 to 20 mg / kg. Suitable doses for human subjects may be between 5 and 15 mg / kg, with 10 mg / kg of antibody (e.g., human anti-PD-1 antibody) being a particular embodiment. In general, lower dosages may be administered via intravenous injection or infusion.

[0082] In certain embodiments, the immunomodulatory agent is administered locally, for example, by direct injection at the site to be treated. This injection generally results in a local concentration of the immunomodulatory agent composition that is higher than that achieved by systemic administration. The immunomodulatory agent composition can be combined with a matrix, as described above, to help increase the local concentration of the polypeptide composition by reducing passive diffusion of the polypeptide from the site to be treated. 1. Formulations for parenteral administration

[0083] In some embodiments, compositions disclosed herein, including those containing peptides and polypeptides, are administered in aqueous solution via parenteral injection. Formulations may also be in the form of suspensions or emulsions. Generally, pharmaceutical compositions are provided containing an effective amount of a peptide or polypeptide, and optionally contain pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions optionally contain one or more of the following components: diluents, sterile water, buffered saline solutions of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength; and additives such as detergents and solubilizers (e.g., TWEEN® 20 (polysorbate-20), TWEEN® 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol), and bulking agents (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.Preparation can be lyophilized and redissolved / resuspended immediately before use.Preparation can be sterilized by, for example, filtering through a bacteria-retaining filter, incorporating composition into sterilizing agent, irradiating composition, or heating composition. 2. Formulations for oral administration

[0084] In embodiments, the composition is formulated for oral delivery. Oral solid dosage forms are generally described in Chapter 89 of Remington's Pharmaceutical Sciences, 18th Edition, 1990 (Mack Publishing Co., Easton Pa. 18042). Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules, or particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, or incorporation of the material into liposomes. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the disclosed product. See, for example, Remington's Pharmaceutical Sciences, 18th Edition (1990, Mack Publishing Co., Easton Pa. 18042), pages 1435-1712, incorporated herein by reference. The composition may be prepared in liquid form or in dry powder (e.g., lyophilized) form. The composition may also be formulated using liposome or proteinoid encapsulation. Liposomal encapsulation may be used, and the liposomes may be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556). Marshall, K. In: Modern Pharmaceutics Edited by G.S. Banker and C.R. Hodes Chapter 10, 1979. Generally, formulations include a peptide (or a chemically modified form thereof) and inert ingredients that protect the peptide in the gastric environment and release the biologically active agent in the intestine.

[0085] Drugs can be chemically modified to allow for effective oral delivery of derivatives. Generally, the intended chemical modification is to attach at least one moiety to the component molecule itself, which allows for uptake into the bloodstream from the stomach or intestine, or direct uptake into the intestinal mucosa. Also desired is the overall stability of one or more components and increased circulation time in the body. PEGylation is an example of chemical modification for pharmaceutical applications. Other moieties that can be used include propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1,3-dioxolane, and poly-1,3,6-tioxocane [see, e.g., Abuchowski and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as Drugs. Hocenberg and Roberts, eds. (Wiley-Interscience: New York, NY) pp. 367-383; and Newmark et al. (1982) J. Appl. Biochem. 4:185-189].

[0086] Another embodiment provides liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain inert diluents; adjuvants, such as wetting agents, emulsifying agents, suspending agents; and other ingredients, including sweetening, flavoring, and perfuming agents.

[0087] Controlled-release oral formulations may be desirable. The drug can be incorporated into an inert matrix, such as a gum, that allows release by either diffusion or leaching mechanisms. A slowly degenerating matrix may also be incorporated into the formulation. Another form of controlled release is based on the Oros Therapeutic System (Alza Corp.), i.e., the drug is encapsulated in a semipermeable membrane that allows water to enter and the drug to be pushed out through a single small opening by osmotic effect.

[0088] For oral formulations, the location of release may be the stomach, the small intestine (duodenum, jejunum, or ileum), or the large intestine. In some embodiments, release will avoid the deleterious effects of the gastric environment, either by protecting the drug (or derivative) or by releasing the drug (or derivative) beyond the gastric environment, such as in the intestine. To ensure sufficient gastric resistance, a coating impermeable to at least pH 5.0 is essential. Examples of more common inactive ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP50, HPMCP55, polyvinyl acetate phthalate (PVAP), Eudragit L30D™, Aquateric™, cellulose acetate phthalate (CAP), Eudragit L™, Eudragit S™, and Shellac™. These coatings can be used as mixed films. 3. Formulations for topical administration

[0089] The disclosed immunotherapeutic agents can be applied locally, which does not work well for most peptide formulations, but can be effective, particularly when applied to the lungs, nose, nasal passages (sublingual, buccal), vaginal, or rectal mucosa.

[0090] The composition, when delivered as either an aerosol or spray-dried particles having an aerodynamic diameter of less than about 5 microns, can be delivered to the lungs upon inhalation and travel across the lung epithelial lining to the bloodstream.

[0091] A wide range of mechanical devices designed for pulmonary delivery of therapeutic agents can be used, including, but not limited to, nebulizers, metered-dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Specific examples of commercially available devices include the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin metered-dose inhaler (Glaxo Inc., Research Triangle Park, NC); and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes, and Mannkind all have inhalable insulin powder preparations approved or in clinical trials, and this technology may be applicable to the formulations described herein.

[0092] Formulations for administration to mucosal membranes are generally spray-dried drug particles that can be incorporated into tablets, gels, capsule suspensions, or emulsions. Standard excipients are available from any formulation manufacturer.

[0093] Transdermal formulations can also be prepared. These typically come as ointments, lotions, sprays, or patches, all of which can be prepared using standard techniques. Transdermal formulations may require the inclusion of penetration enhancers. D.How to use

[0094] The disclosed activity-sensing immunotherapeutic agents are useful for predicting and pharmacodynamically monitoring immunotherapeutic responses in subjects receiving immunotherapeutic agents for the treatment of a disease or disorder. In such applications, subjects undergo treatment for a disease or disorder and are noninvasively monitored for response to treatment using a single composition. In one embodiment, the subject is administered an immunotherapeutic agent or a composition containing an immunotherapeutic agent. After a period of time, a sample is taken from the subject. The sample may be blood or urine. The sample is analyzed for the presence of a detectable signal associated with the immunotherapeutic agent. In one embodiment, the detectable signal is analyzed by ELISA, mass spectrometry, flow cytometry, colorimetry, bioluminescence, or immunoassay.

[0095] In one embodiment, if the detectable signal molecule is present in the sample above the detectable limit, the subject is considered to be responsive to treatment, and the remaining treatment regimen is administered at the effective dose initially administered.If the detectable signal molecule is not present in the sample, the subject is considered to be non-responsive, and the treatment regimen is discontinued, or the dosage of the treatment regimen is increased to the next dosage, and the detection process is repeated.If the subject does not continue to show signs of the detectable signal molecule in the urine sample, the subject is discontinued from the treatment regimen.In some embodiments, the subject is switched to a different therapeutic agent disclosed herein, or the subject is switched to a different type of treatment, such as chemotherapy or CAR-T cell therapy.

[0096] In another embodiment, multiple immunotherapeutic agents or a composition comprising multiple immunotherapeutic agents are administered to a subject, and each detectable signal in the subject's urine is analyzed to generate a signal profile. In such an embodiment, a panel of immunotherapeutic agents can be used to distinguish resistance mechanisms in non-responsive subjects. The disclosed immunotherapeutic agents can determine whether a subject has primary or acquired resistance to immunotherapy. In primary resistance, the subject is non-responsive to the immunotherapeutic agent upon the first administration of the immunotherapeutic agent. In some embodiments, a subject has primary resistance due to the absence of tumor antigens that are not recognized by T cells. In other embodiments, cancer cells may possess tumor antigens but may develop mechanisms to avoid presenting the antigens on their MHC-restricted surfaces.

[0097] Acquired resistance is resistance to immunotherapeutic agents when they are administered subsequently.In some embodiments, acquired resistance is caused by the decline of T cell function, the lack of T cell recognition due to the downregulation of tumor antigen presentation, and the occurrence of escape mutants in cancer.In one embodiment, a panel of immunotherapeutic agents is constructed, which can classify subjects into different classes of immunotherapeutic agent resistance according to expression patterns.Common mechanisms of immunotherapeutic resistance include, but are not limited to, decreased sensitivity to IFN-γ, decreased expression of receptors on MHC, co-expression of inhibitory receptors, upregulation of alternative inhibitory checkpoints, and high mutation overload in tumors.

[0098] In one embodiment, cancer resistance proteases are known in the art, and a panel of such proteases can be used to classify resistance. In one embodiment, resistance due to reduced IFN-γ signaling can be determined using a panel of immunotherapeutic agents conjugated with protease substrates, including, but not limited to, all or part of GZMA, PRSS55, PRSS48, KLK15, MMP21, CPA1, MMP23A, CTRB1, MMP24, PRSS3P2, TPSG1, OVCH2, PHEX, and KLK14. In another embodiment, resistance due to reduced beta-2-microglobulin (B2M) expression on MHC I can be determined using a panel of immunotherapeutic agents conjugated with protease substrates, including, but not limited to, all or part of PLAU, ADAM8, CELA2B, CASP4, CPD, MMP25, MME, NUP98, CYLD, ASTL, ECE1, and USP32. 1. Treatment target a. Cancer

[0099] The disclosed compositions and methods can be used to treat cancer. Generally, the agent is used to stimulate or enhance the subject's immune response to cancer by administering to the subject an amount of the disclosed activity-sensing immunotherapeutic agent. The immunotherapeutic agent can promote or enhance the immune response by binding to an inhibitory immune checkpoint molecule or its receptor and inhibiting signal transduction by the immune checkpoint molecule. This method can reduce one or more symptoms of cancer.

[0100] In one embodiment, the disclosed immunotherapeutic agents reverse immune suppression within the tumor microenvironment by blocking inhibitory immune checkpoint molecules.

[0101] During their development, cancer cells acquire a characteristic set of functional capabilities through various mechanisms. Such capabilities include evading apoptosis, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion / metastasis, limitless replicative potential, and persistent angiogenesis. The term "cancer cell" is meant to encompass both pre-malignant and malignant cancer cells. In some embodiments, cancer refers to a benign tumor that remains localized. In other embodiments, cancer refers to a malignant tumor that has invaded and destroyed adjacent body structures and metastasized to distant sites. In still other embodiments, cancer is associated with a specific cancer antigen (e.g., pan-carcinoma antigen (KS1 / 4), ovarian carcinoma antigen (CA125), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), CD19, CD20, HER2 / neu, etc.).

[0102] The methods and compositions disclosed herein are useful in treating the following cancers: carcinomas, including bladder, breast, colon, kidney, liver, lung, ovarian, pancreatic, gastric, cervical, thyroid, and skin cancers (including squamous cell carcinoma); hematopoietic malignancies of the lymphoid system, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, and Burkitt's lymphoma; hematopoietic malignancies of the myeloid system, including acute and chronic myeloid leukemia and promyelocytic leukemia; fibroblastic malignancies; They are useful in the treatment or prevention of a variety of cancers or other abnormal proliferative disorders, including, but not limited to, tumors of mesenchymal origin, including sarcoma and rhabdomyoscarcoma; other tumors, including melanoma, seminoma, teratoma, neuroblastoma, and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannoma; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarama, and osteosarcoma; and other tumors, including melanoma, xenoderma pegmentosum, keratoactanthoma, seminoma, thyroid follicular carcinoma, and teratocarcinoma.

[0103] Cancers caused by abnormalities in apoptosis can also be treated by the disclosed methods and compositions.Such cancers may include, but are not limited to, follicular lymphoma, carcinoma with p53 mutation, hormone-dependent tumors of the breast, prostate, and ovary, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndrome.In certain embodiments, malignant tumors or abnormal proliferation changes (such as metaplasia and dysplasia) or hyperproliferative disorders are treated or prevented by the present methods and compositions in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus.In other specific embodiments, sarcoma, melanoma, or leukemia are treated or prevented by the present methods and compositions.

[0104] Specific cancers and related disorders that can be treated or prevented by the methods and compositions disclosed herein include acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemic leukemia, and myelodysplastic syndromes; chronic leukemias, such as, but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas, such as, but not limited to, Hodgkin's disease or non-Hodgkin's disease lymphoma (e.g., diffuse anaplastic lymphoma kinase (ALK)-negative, large B-cell lymphoma, and the like); anaplastic lymphoma (DLBCL); diffuse anaplastic lymphoma kinase (ALK) positive, large B-cell lymphoma (DLBCL); anaplastic lymphoma kinase (ALK) positive, ALK+ anaplastic large cell lymphoma (ALCL), acute myeloid lymphoma (AML); multiple myeloma, including but not limited to, smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; osteosarcoma and connective tissue sarcomas, including but not limited to, bone marrow sarcoma sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma, etc.; including, but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumors brain tumors, including acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary cerebral lymphoma; breast cancer, including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast carcinoma, mucinous breast carcinoma, tubular breast carcinoma, papillary breast carcinoma, Paget's disease, and inflammatory breast carcinoma; adrenal gland carcinoma, including but not limited to pheochromocytoma and adrenocortical carcinoma;thyroid cancer, such as, but not limited to, papillary or follicular thyroid cancer, medullary thyroid cancer, and histoplastic thyroid cancer; pancreatic cancer, including, but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumors, and carcinoid or islet tumors; pituitary cancer, including, but not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly, and Insipid diabetes mellitus; eye cancer, including, but not limited to, ocular melanoma, such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancer, including, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, including, but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancer, including, but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancer, including, but not limited to, endometrial cancer and uterine sarcoma; ovarian epithelial cancer, borderline tumors, germ cell tumors, and stromal tumors. ovarian cancer, including but not limited to; esophageal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cell carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; gastric cancer, including but not limited to adenocarcinoma, fungus (polypoid) carcinoma, ulcerative carcinoma, superficial spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancer, including but not limited to hepatocellular carcinoma and hepatoblastoma, including but not limited to adenocarcinoma gallbladder cancer, including but not limited to; bile duct carcinoma, including but not limited to papillary, nodular, and diffuse; lung cancer, including but not limited to non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung carcinoma; testicular cancer, including but not limited to germinal tumor, seminoma, undifferentiated, classic (typical), spermatocytic, non-seminoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma (yolk sac tumor), prostate cancer, including but not limited to adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancer, including but not limited to squamous cell carcinoma; basal cell carcinoma; salivary gland carcinoma, including but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, including but not limited to squamous cell carcinoma and verrucous carcinoma;Skin cancer, including but not limited to basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, and acral lentiginous melanoma; kidney cancer, including but not limited to renal cell carcinoma, adenocarcinoma, Grawitz tumor, fibrosarcoma, and transitional cell carcinoma (of the renal pelvis and / or uterus); Wilms' tumor; and bladder cancer, including but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. Additionally, cancers include myxosarcoma, osteogenic sarcoma, mesothelioma, lymphangioendothelial sarcoma, mesothelioma, synovium, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma (reviews of such disorders include Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia, and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America; (See America). b. Infectious diseases

[0105] The disclosed compositions and methods can be used to treat infectious diseases and infectious disorders. Generally, the agents are used to stimulate or enhance a subject's immune response to an infection by administering to the subject an amount of an activity-sensing immunotherapeutic agent that modulates immune checkpoint molecule expression, ligand binding, cross-linking, inhibitory signaling, or a combination thereof. In one embodiment, the immunotherapeutic agent inhibits, reduces, or blocks inhibitory immune signaling by immune checkpoint molecules. In another embodiment, the immunotherapeutic agent induces, promotes, or enhances an immune response by inducing, promoting, or enhancing signaling by immune checkpoint molecules. This method can reduce one or more symptoms of an infectious disease.

[0106] Infection or disease can be caused by bacteria, viruses, protozoa, helminths, or other pathogenic microorganisms that invade and attack cells, ie, by cytotoxic T lymphocytes.

[0107] Infection or disease can be acute or chronic. Acute infection is generally a short-term infection. During acute microbial infection, immune cells begin to express immunomodulatory receptors. Thus, in some embodiments, the method comprises increasing the immune stimulatory response to acute infection.

[0108] The infection may be caused by, for example, but not limited to, Candida albicans, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, or Mycobacterium.

[0109] In some embodiments, the disclosed compositions are used to treat chronic infections, such as infections in which T cell depletion or T cell anergy occurs, causing the infection to persist in the host for an extended period of time.

[0110] Exemplary infections to be treated are chronic infections caused by hepatitis virus, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), herpes virus, Epstein-Barr virus, or human papillomavirus.

[0111] Because viral infections are primarily eliminated by T cells, increased T cell activity is therapeutically useful in situations where more rapid or complete elimination of infectious viral agents would be beneficial to an animal or human subject. Thus, the disclosed compositions can be administered to treat local or systemic viral infections, including, but not limited to, immunodeficiencies (e.g., HIV), papillomas (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza virus A), and the common cold (e.g., human rhinovirus), as well as other viral infections caused by, for example, HTLV, hepatitis virus, respiratory syncytial virus, vaccinia virus, and rabies virus. The molecules can be administered locally to treat viral skin diseases such as herpes zoster or shingles, or genital warts. The molecules can also be administered systemically to treat systemic viral diseases, including, but not limited to, AIDS, influenza, the common cold, or encephalitis.

[0112] The specific strains are Actinomyces, Anabaena, Bacillus, Bacteroides, Bdell ovibrio、Bordetella、Borrelia、Campylobacter、Caulobacter、Chlamydia、Chlor obium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Haemophilus influenza type B(HIB)、Hyphomicrobium、Legionella、Leptspirosis、Listeria、Meningococcus A、BおよびC、Methanobacterium、Micrococcus、Myobacterium、Mycoplasma、Myxococcus、Neisseria 、Nitrobacter、Oscillatoria、Prochloron、Proteus、Pseudomonas、Phodospirillum、Rickettsia 、Salmonella、Shigella、Spirillum、Spirochaeta、Staphylococcus、Streptococcus、Streptomy ces、Sulfolobus、Thermoplasma、Thiobacillus、およびTreponema、Vibrio、Yersinia、Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum、Trypanosoma brucei、Entamoeba histolytica、Toxoplasma gondii、Trichomonas vaginalisおよびSchistosomaInfections caused by microorganisms, including but not limited to, Bacillus subtilis, Bacillus anguilliformis, Bacillus subtil ...

[0113] Other microorganisms that can be treated using the disclosed compositions and methods include bacteria, such as Klebsiella, Serratia, Pasteurella bacteria; pathogens associated with cholera, tetanus, botulism, anthrax, plague, and Lyme disease; or fungal or parasitic pathogens, such as Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix (schenkii), Blastomyces (dermatitidis), Paracoccidioides (brasiliensis), Coccidioides (immitis), and Histoplasma (capsulatuma), Entamoeba, histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondi, etc.), Sporothrix, Blastomyces, Paracoccidioides, Coccidioides, Histoplasma, Entamoeba, Histolytica, Balantidium, Naegleria, Acanthamoeba, Giardia, Cryptosporidium, Pneumocystis, Plasmodium, Babesia, or Trypanosoma. C. graft rejection

[0114] In another embodiment, the disclosed compositions and methods can be used prophylactically or therapeutically to reduce or inhibit transplant rejection or graft-versus-host disease. Transplant rejection occurs when a transplanted organ or tissue is not accepted by the transplant recipient's body. Rejection typically occurs because the recipient's immune system attacks the transplanted organ or tissue. The disclosed methods can be used to promote immune tolerance of a transplant or graft by a recipient by administering to the subject an effective amount of one or more of the disclosed activity-sensing immunotherapeutic agents. In one embodiment, induction of immune tolerance can be measured by analyzing the amount of detectable molecules released in the urine of a subject receiving an immunotherapeutic agent for reducing or inhibiting transplant rejection.

[0115] The transplanted material can be a cell, tissue, organ, limb, finger, or body part, e.g., a human body part. The transplant is usually allogeneic or xenogeneic. The disclosed composition is administered to a subject in an effective amount to reduce or inhibit transplant rejection. The composition can be administered systemically or locally by any acceptable route of administration. In some embodiments, the composition is administered to the transplant site before, during, or after transplantation. In one embodiment, the composition is administered to the transplant site parenterally, such as by subcutaneous injection.

[0116] In other embodiments, the composition is administered directly to the cell, tissue, or organ to be transplanted ex vivo. In one embodiment, the transplant material is contacted with the composition before transplantation, after transplantation, or both.

[0117] In other embodiments, the composition is administered to an immune tissue or organ, such as a lymph node or spleen.

[0118] The transplant material can also be treated with enzymes or other materials that remove cell surface proteins, carbohydrates, or lipids known or suspected to be involved in immune responses such as transplant rejection. i.Cells

[0119] Any kind of cell population can be transplanted into a subject. Cells can be allogeneic or xenogeneic. Xenogeneic means that the cell population contains more than one type of cell. Exemplary cells include progenitor cells such as stem cells and pluripotent cells, which can be collected from a donor and transplanted into a subject. Cells are optionally treated before transplantation as described above. ii.Organization

[0120] Any tissue can be used as a graft. Exemplary tissues include skin, adipose tissue, cardiovascular tissues such as veins, arteries, capillaries, valves, etc.; nervous tissue, bone marrow, lung tissue, eye tissues such as the cornea and lens, cartilage, bone, and mucosal tissue. iii. Organs

[0121] Exemplary organs that can be used for transplantation include, but are not limited to, kidney, liver, heart, spleen, bladder, lung, stomach, eye, tongue, pancreas, intestine, etc. The organ to be transplanted can also be modified prior to transplantation as described above.

[0122] One embodiment provides a method of inhibiting or reducing chronic transplant rejection in a subject by administering an effective amount of a composition to inhibit or reduce chronic transplant rejection relative to a control. iv. Graft-versus-host disease (GVHD)

[0123] The disclosed compositions and methods can be used to treat graft-versus-host disease (GVHD) by administering an effective amount of the composition to alleviate one or more symptoms associated with GVHD. GVHD is a major complication associated with allogeneic hematopoietic stem cell transplantation, in which functional immune cells in the transplanted bone marrow recognize the recipient as "foreign" and launch an immunological attack. It can also occur during blood transfusions under certain circumstances. Symptoms of GVHD include skin rash or changes in skin color or texture, diarrhea, nausea, liver dysfunction, yellowing of the skin, increased susceptibility to infection, dry and irritated eyes, and mouth irritation or dry mouth. d. Autoimmune and chronic infectious diseases

[0124] The disclosed immunotherapeutics can also be used to treat inflammatory or autoimmune diseases and disorders. In such embodiments, the immunotherapeutic agent is an agent that modulates immune checkpoint molecule expression, ligand binding, cross-linking, inhibitory signaling, or a combination thereof. Exemplary inflammatory or autoimmune diseases / disorders include rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disorders, autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue syndrome immune deficiency syndrome (CFS), and others. deficiency, syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Dego's disease, dermatomyositis, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), Iga nephropathy, insulin-dependent diabetes mellitus (type 1), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome syndromes), polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-person syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0125] In some embodiments, the inflammatory or autoimmune disease is caused by a pathogen or is the result of an infection. [Example]

[0126] Example 1: Protease substrate-modified checkpoint blockade immunotherapeutics retain target binding and sense granzyme B activity. material and method:

[0127] To combine therapeutic activity and response monitoring capabilities, the αPD-1 cancer immunotherapy antibody was functionalized with the granzyme B (GzmB) protease-sensing biomarker using amine-reactive chemistry ( Figure 2A ). result:

[0128] αPD-1 maintained its targeting ability when functionalized with the GzmB protease substrate, as determined by a similar EC50 evaluated relative to unmodified αPD-1 (Figure 2B). Functionalized αPD-1 also retained target binding to tumor-infiltrating CD8+ T cells (Figure 2C).

[0129] To determine whether GzmB can access and cleave the antibody-bound substrate, we functionalized αPD-1 with an engineered GzmB substrate with a quencher molecule before the cleavage site and a fluorescent reporter (FAM) after it (Figure 1). After cleavage, the reporter is separated from the quencher, generating a fluorescent signal for quantification. Using an in vitro cleavage assay, functionalized αPD-1 demonstrated specific cleavage by GzmB without cross-cleavage by thrombin, a common serum protease (Figure 2E).

[0130] Example 2. Functionalized costimulatory blockade therapeutic agents are functional and sense Granzyme B activity. material and method:

[0131] To determine the applicability of this approach to other protein biologics, abatacept, a CTLA-4Ig fusion protein that binds CD80 and CD86 to block T cell costimulation, was functionalized with GzmB substrates as described above and shown in Figure 3A. result:

[0132] Functionalized CTLA-4Ig targeted CD80 / CD86 with similar potency to unmodified CTLA-4Ig, as determined by competitive binding with anti-CD80 and CD86 antibodies (Figures 3B-3C). Functionalization with the GzmB protease substrate did not impair the ability of CTLA-4 to attenuate T cell activation and proliferation when assessed against the unmodified protein (Figure 3D). Using an in vitro cleavage assay, modified CTLA-4Ig demonstrated specific cleavage by GzmB without cross-cleavage by matrix, complement, or immune proteases (Figure 3E). These results demonstrate that alternative immunotherapeutic agents (αPD-1 and CTLA-4Ig) can be functionalized with protease-sensing substrates without compromising function.

[0133] Example 3. Immunotherapeutic agents functionalized with GzmB detect CD8+ T cell-mediated cytotoxicity. material and method:

[0134] To determine the ability of the functionalized immunotherapeutic agents to detect T cell activity, αPD-1 functionalized with the GzmB substrate was incubated with supernatants isolated from activated CD8 + T cells or various cancer cell lines (CT26, MC38, or B16 cell lines) ( Figure 4A ). result:

[0135] Functionalized αPD-1 was not cleaved when incubated with the supernatants of either cancer cell line, but showed an increase in fluorescent signal over time when incubated with the supernatants of activated T cells (Figure 4B). Control αPD-1 bound to a control substrate for complement protease C1 (LQRIYK, (SEQ ID NO: 3)) was also not cleaved by the supernatants of activated T cells. Sensing of GzmB activity was tested during co-incubation of CD8+ T cells isolated from Pmel-1 TCR transgenic mice (gp100-specific) with B16 melanoma cells (expressing gp100, recognized by Pmel T cells) (Figure 4C) (Klebanoff et al., Clin Cancer Res, 17(16):5343-5352 (2011); Abad et al., J Immunother, 31(1):1-6 (2008); Overwijk et al., J Exp Med, 198(4):569-580 (2003)). Addition of functionalized αPD-1, but not control αPD-1, significantly increased the fluorescence signal at multiple T cell-to-target cell ratios, corresponding to increased cell killing and GzmB protein secretion (Figures 4D-4F). No increase in signal was observed when co-cultured with OT-IT cells that do not recognize B16 cells, confirming that the measured protease activity corresponds to antigen-specific T cell-mediated cytotoxicity (Figure 4G). Cleavage of CTLA-4Ig functionalized with the GzmB substrate was tested using a transgenic OT-IT cell line that recognizes the chicken ovalbumin (OVA)-derived peptide epitope SIINFEKL (SEQ ID NO: 4) and targets OVA-expressing EG7 cells, but not the parental EL4 cell line, which does not express OVA. Incubation of OT-IT cells with EG7-OVA cells but not with EL4 control cells resulted in increased fluorescent signaling (Figure 4H). These results demonstrate that immunotherapeutic agents (αPD-1 and CTLA-4Ig) functionalized with protease-sensing substrates can sense T cell activity, particularly cytotoxicity.

[0136] Example 4. Granzyme B protease activity corresponds to responsive immunotherapy. result:

[0137] To determine the significance of protease activity as a biomarker of responsive immunotherapy, we characterized the kinetics of GzmB protease expression within tumor-infiltrating CD8+ T cells during immunotherapy treatment in a PD-1-responsive MC38 tumor model (Figure 5A). Responsive immunotherapy during PD-1 blockade corresponded to an increase in the number of CD8+ TILs expressing the cytotoxic mediator GzmB (Figure 5B-5D). Next, MC38 mice were treated with αPD-1 functionalized with a GzmB substrate or an isotype control, allowing quantification of protease activity before treatment (day 11) and early in treatment (days 14 and 17) (Figure 5E-5F). Responsive treatment correlated with increased GzmB activity, as determined by an increase in the urinary signal at day 17 for αPD-1 but not for mice treated with an isotype control. Using the CT26 tumor model, GzmB expression in CD8+ T cells and activity, as detected by urinary secretion of the cleaved biomarker, increased early in treatment during responsive αPD-1 / CTLA-4 combination therapy, but not during unresponsive αCTLA-4 monotherapy (Figures 5G-5N). Taken together, these data indicate that GzmB protease activity may serve as a biomarker of early treatment response to immunotherapy. Future development of this technology will identify protease signatures corresponding to responsive immunotherapies to inform the construction of multiplex biomarker libraries containing GzmB and other highly enriched immune- and disease-specific proteases.

[0138] Example 5. Accumulation of CD8 T cells and expression of granzyme B protease at the transplant site corresponds to the onset of acute cellular rejection. result:

[0139] Histological criteria for staging the severity of ACR include features such as tissue damage and the presence of apoptotic cells, which are downstream effects of anti-graft T cell responses. Measuring the activity of proteases that drive disease pathology may serve as early biomarkers and predict disease trajectories, such as using MMP activity to predict liver fibrosis progression and regression. Therefore, we investigated the feasibility of using a GzmB activity nanosensor, consisting of an iron oxide nanoparticle core (IONP) conjugated with a GzmB protease substrate, for the early detection of ACR (Figure 6A). To quantify the kinetics of skin graft health and rejection, we assigned a score of 4 to healthy allografts, a score of 0 to complete rejection, and intermediate scores based on features such as the ratio of viable to necrotic skin and the presence of ulcers or scabs. According to these metrics, graft scores began to decrease significantly on day 9 post-transplant and reached the endpoint when the allograft was completely rejected within 2 weeks post-transplant (Figure 6B-6H). To identify the earliest time point of GzmB upregulation, we analyzed graft tissue by immunohistochemistry on day 7 and found a significant increase in both graft-infiltrating CD8 T cells and GzmB expression (Figures 6I-6J). Taken together, this data provides evidence that GzmB expression and activity are significantly upregulated in allograft tissue at the onset of acute cellular rejection.

[0140] Example 6. Responding and non-responsive CTLA-4Ig treatment groups can be stratified by granzyme B protease activity. result:

[0141] Abatacept, a CTLA-4Ig fusion protein that binds to CD80 and CD86 to block T cell costimulation, is used in the clinic to prevent transplanted organ rejection and treat various chronic inflammatory and autoimmune diseases. A costimulation blockade therapeutic model was developed in which skin graft recipient mice (BALB / c skin transplanted into BL / 6 recipient mice) were treated with CTLA-4Ig and graft health and survival were monitored. CTLA-4Ig treatment extended the duration of graft engraftment in some animals ("responders"), while other mice remained unresponsive to treatment and ultimately rejected the graft at a rate similar to that of untreated animals ("non-responders") (Figure 6K-6L). Using GzmB-functionalized CTLA-4Ig, significantly increased GzmB activity was observed at POD 15 in untreated and CTLA-4Ig non-responders, but not in the CTLA-4 responder group, corresponding to the prolonged duration of graft engraftment (Figure 6M).

[0142] Example 7. Tumor protease signatures of ICB response and acquired resistance. result:

[0143] A significant proportion of patients who demonstrate an objective response ultimately relapse (e.g., up to one-third in melanoma) despite continued treatment with checkpoint inhibitors. Mechanisms of resistance include impaired T cell recognition (reduced antigen presentation) or activation (insensitivity to IFN-γ signaling). To identify changes in protease expression during checkpoint inhibitor response and resistance, we examined independent studies of serial biopsies from 68 melanoma patients before and early after treatment with αPD-1 (Hugo et al., Cell, 165:35-44 (2016); Riaz et al., Cell, 171:934-949 (2017)) (Figure 7A). Expression levels of 250 extracellular proteases were used as features for binary classification of responders and non-responders using support vector machines (SVM) (Figure 7B). In equally divided training and validation cohorts, we found that protease expression could be used to distinguish responders from non-responders with a near-perfect AUROC (>0.98). Next, by querying the 250 proteases most important for classification and applying the Lasso algorithm, we defined a shortened list of 14 significant proteases that could be used to classify the same patients with an AUROC >0.96 (Figure 7C-7D). These results demonstrate that protease expression can be used to classify patient responders from non-responders.

[0144] Next, we determined whether protease expression could be used to define mechanisms of resistance. We analyzed whole-gene transcripts from non-responders to identify differentially expressed genes (genes with a tscore >100) compared to responders. Using these genes, we performed pathway analysis for frequent mechanisms of resistance to immune checkpoint therapy, focusing specifically on two pathways: IFNγ signaling and MHC I antigen presentation (Figure 7E-F). Using this approach, we identified a panel of proteases that could identify the resistance mechanism of reduced sensitivity to IFNγ in non-responders, as well as a panel of proteases that could identify reduced MHC I antigen presentation (Figure 7H). The proportion of pathways for each resistance mechanism (IFNγ and MHC I presentation) showed loss in distinct patients (Figure 7G).

[0145] In the foregoing specification, the invention has been described in relation to specific embodiments thereof, and while numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that the specific details described herein may be varied considerably without departing from the underlying principles of the invention.

[0146] All references cited herein are incorporated by reference in their entirety. The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, reference should be made to the appended claims, rather than the foregoing specification, as indicating the scope of the invention. The present invention provides, for example, the following items. (Item 1) 1. A method of administering immunotherapy and monitoring a response to immunotherapy in a subject in need thereof, comprising: administering to the subject an effective amount of at least one therapeutic agent linked to a protease substrate, the therapeutic agent providing a detectable signal in response to protease activity promoted by the therapeutic agent; detecting and measuring the signal in a sample from the subject; determining the effect of the therapeutic agent on the subject, wherein if the detectable signal is detected, the subject is determined to be responsive to the therapeutic agent, and if the detectable signal is not detected, the subject is determined to be non-responsive to the therapeutic agent; administering the same effective amount of said therapeutic agent to a responsive subject or adjusting said effective amount of therapeutic agent administered to a non-responsive subject. (Item 2) 2. The method of item 1, wherein the therapeutic agent is an immune checkpoint inhibitor. (Item 3) 3. The method of claim 2, wherein the immune checkpoint inhibitor is an anti-PD-1 or anti-CTLA-4 antibody. (Item 4) Item 10. The method of item 1, wherein the therapeutic agent is an immunosuppressant. (Item 5) 5. The method of item 4, wherein the immunosuppressant is CTLA-4Ig. (Item 6) 2. The method of claim 1, wherein the protease substrate is conjugated to a reporter molecule. (Item 7) 7. The method of claim 6, wherein the reporter molecule is a fluorescent molecule, a bioluminescent molecule, or a mass tag. (Item 8) 2. The method of claim 1, wherein the protease substrate comprises a quencher molecule and a fluorescent molecule adjacent to the substrate. (Item 9) 2. The method of claim 1, wherein the detectable signal is a peptide fragment derived from the protease substrate. (Item 10) 2. The method of claim 1, wherein the detectable signal is a fluorescent reporter. (Item 11) 2. The method of claim 1, wherein the detectable signal is a mass tag. (Item 12) 2. The method of claim 1, wherein the step of adjusting the effective amount of the immunotherapeutic agent further comprises administering a different immunotherapeutic agent. (Item 13) 2. The method of claim 1, wherein the sample comprises a urine sample or a blood sample. (Item 14) Item 10. The method of claim 1, wherein the step of measuring the signal comprises subjecting the sample to mass spectrometry, flow cytometry, or ELISA. (Item 15) 2. The method of claim 1, wherein the non-responsive subject has immune resistance. (Item 16) Item 10. The method of item 1, wherein the subject has cancer. (Item 17) Item 10. The method of item 1, wherein the subject has an infectious disease. (Item 18) Item 10. The method of item 1, wherein the subject has a transplanted organ. (Item 19) A composition comprising a therapeutic agent linked to a protease substrate that provides a detectable signal in response to protease activity promoted by said therapeutic agent. (Item 20) 20. The composition of item 19, wherein the therapeutic agent is an immune checkpoint inhibitor. (Item 21) 21. The composition of claim 20, wherein the immune checkpoint inhibitor is an anti-PD-1 or anti-CTLA-4 antibody. (Item 22) 20. The composition of claim 19, wherein the therapeutic agent is an immunosuppressant. (Item 23) 23. The composition of claim 22, wherein the immunosuppressant is CTLA-4Ig. (Item 24) 20. The composition of claim 19, wherein the detectable signal is a peptide fragment derived from the protease substrate. (Item 25) 20. The composition of claim 19, wherein the protease substrate is conjugated to a reporter molecule. (Item 26) 26. The method of claim 25, wherein the reporter molecule is a fluorescent molecule, a bioluminescent molecule, or a mass tag. (Item 27) 20. The method of claim 19, wherein the protease substrate comprises a quencher molecule and a fluorescent molecule adjacent to the substrate.

Claims

[Claim 1] The invention described in the specification.

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