阅读说明：本技术 用于增强的免疫疗法的生物响应抗体复合物 (Bioresponse antibody complexes for enhanced immunotherapy ) 是由 顾臻 陈倩 于 2020-03-09 设计创作，主要内容包括：本发明公开了包含免疫检查点阻断抑制剂的生物响应蛋白质复合物及其使用方法。(Bioresponse protein complexes comprising an immune checkpoint blockade inhibitor and methods of use thereof are disclosed.)

1. A bioresponsive hydrogel matrix comprising a CD47/sirpa inhibitor and an immune checkpoint blockade inhibitor.

2. The bioresponse hydrogel matrix of claim 1, wherein the CD47/sirpa blocking inhibitor is selected from the group consisting of: hu5F9-G4, CV1, B6H12, 2D3, CC-90002 and TTI-621.

3. The bioresponse hydrogel matrix of claim 1, wherein said immune checkpoint blockade inhibitor is a PD-1/PD-L1 blockade inhibitor.

4. The bioresponse hydrogel matrix of claim 3, wherein said PD-1/PD-L1 blocking inhibitor is selected from the group consisting of: nivolumab, pembrolizumab, pidilizumab, astuzumab, avilumab, Devolumab, and BMS-936559.

5. The bioresponse hydrogel matrix of claim 1, wherein said immune checkpoint blockade inhibitor is a CTLA-4/B7-1/2 blockade inhibitor.

6. The bioresponse hydrogel matrix of claim 5, wherein said CTLA-4/B7-1/2 blocking inhibitor comprises yiprizumab.

7. The bioresponsive hydrogel matrix of any one of claims 1-6, wherein said hydrogel matrix comprises a Reactive Oxygen Species (ROS) degradable hydrogel.

8. The bioresponsive hydrogel matrix of any one of claims 1-7, wherein said bioresponsive hydrogel matrix comprises cross-linked albumin.

9. The bioresponsive hydrogel matrix of any one of claims 1-8, wherein the bioresponsive hydrogel matrix comprises an inner core and an outer shell; and wherein the CD47/SIRPa inhibitor is crosslinked to the outer shell and an immune checkpoint inhibitor is crosslinked to the inner core.

10. The bioresponsive hydrogel matrix of any one of claims 1-8, wherein the bioresponsive hydrogel matrix comprises an inner core and an outer shell; and wherein the CD47/SIRPa inhibitor is crosslinked to the inner core and an immune checkpoint inhibitor is crosslinked to the outer shell.

11. The bioresponse hydrogel matrix of claim 9 or 10, wherein said CD47/SIRPa inhibitor and said immune checkpoint inhibitor are crosslinked to said bioresponse hydrogel matrix by a ROS-responsive crosslinking agent.

12. The bioresponsive hydrogel matrix of claim 11, wherein said cross-linking agent comprises bis-N-hydroxysuccinimide (NHS) modified 2,2' - [ propane-2, 2-diylbis (thio) ] diacetic acid (NHS-IE-NHS).

13. A method of treating cancer in a subject comprising administering to the subject the bioresponsive hydrogel matrix of any one of claims 1-12.

14. A method of treating cancer in a subject comprising administering to the subject a bioresponse hydrogel matrix comprising a CD47/sirpa inhibitor and an immune checkpoint blockade inhibitor.

15. The method of claim 14, wherein the CD47/sirpa blocking inhibitor is selected from the group consisting of: hu5F9-G4, CV1, B6H12, 2D3, CC-90002 and TTI-621.

16. The method of claim 14, wherein the blocking inhibitor is a PD-1/PD-L1 blocking inhibitor.

17. The method of claim 16, wherein the PD-1/PD-L1 blocking inhibitor is selected from the group consisting of: nivolumab, pembrolizumab, pidilizumab, astuzumab, avilumab, Devolumab, and BMS-936559.

18. The method of claim 14, wherein the blocking inhibitor is a CTLA-4/B7-1/2 blocking inhibitor.

19. The method of claim 18, wherein the CTLA-4/B7-1/2 blocking inhibitor comprises yiprizumab.

20. The method of treating cancer according to any one of claims 13-19, wherein the cancer is selected from the group consisting of: lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck squamous cell cancer, kidney cancer, small-cell lung cancer and non-small cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, oral squamous cell cancer, pharyngeal squamous cell cancer, laryngeal squamous cell cancer, lung squamous cell cancer, colon cancer, cervical cancer, breast cancer, epithelial cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, large intestine cancer, hematopoietic cancer, testicular cancer, colon cancer, and rectal cancer.

Background

Immune Checkpoint Blockade (ICB) therapy, particularly blockade of cytotoxic T lymphocyte antigen 4(CTLA-4) and programmed cell death protein 1/programmed cell death ligand 1(PD-1/PD-L1), has achieved exciting clinical advances in a number of malignancies, including non-small cell lung cancer, melanoma, urothelial cancer, renal cell cancer, bladder cancer, head and neck cancer. Despite these achievements in the clinic, ICB still has many challenges to overcome, such as low objective response rates and systemic side effects. In patients with immunogenic tumors characterized by high expression of tumor-associated antigens, activated T cells induce a durable immune response only after CTLA-4 or PD-1/PD-L1 blockade. In addition, side effects, such as autoimmune diseases, often occur during ICB treatment due to off-target binding of antibodies to normal cells. Promotion of ICB response without serious side effects has been one of the central subjects in the field of cancer immunotherapy.

Cancer cells can typically escape immune system recognition through upregulation of integrin-associated proteins, also known as "eat me" signals (CD 47). Blocking CD47 will activate phagocytes to phagocytose cancer cells and promote antigen presentation. These results lay the theoretical basis for the conjecture that CD47 blockade could be used to promote objective responses to CTLA-4 or PD-1/PD-L1 blockade. Furthermore, with respect to the modulated release of immunomodulatory antibodies, it is increasingly desirable to design engineered bioresponse immunotherapeutic formulations for the controlled release of therapeutic substances that can respond to the Tumor Microenvironment (TME) to enhance their retention and efficacy in tumors and minimize systemic antidotes. What is needed are novel methods and compositions for inhibiting endogenous immunosuppressive signaling.

Disclosure of Invention

Methods and compositions relating to bioresponse hydrogel matrices are disclosed.

In one aspect, disclosed herein is a bioresponse hydrogel matrix comprising a CD47/sirpa inhibitor (such as Hu5F9-G4, CV1, B6H12, 2D3, CC-90002, and TTI-621) and an immune checkpoint blockade inhibitor (such as a PD-1/PD-L1 inhibitor and/or a CTLA-4/B7-1/2 inhibitor).

Also disclosed herein is the bioresponse hydrogel matrix of any preceding aspect, wherein the immune checkpoint blockade inhibitor is a PD-1/PD-L1 blockade inhibitor (such as nivolumab, pembrolizumab, pidilizumab, astuzumab, avizumab, devoluumab, and BMS-936559).

Also disclosed herein is the bioresponse hydrogel matrix of any preceding aspect wherein the immune checkpoint blockade inhibitor is a CTLA-4/B7-1/2 blockade inhibitor (such as yiprimab).

In one aspect, disclosed herein is the bioresponsive hydrogel matrix of any preceding aspect, wherein the hydrogel matrix comprises a Reactive Oxygen Species (ROS) degradable hydrogel, such as a hydrogel comprising albumin and a bis-N-hydroxysuccinimide (NHS) -modified 2,2' - [ propane-2, 2-diylbis (thio) ] diacetic acid (NHS-IE-NHS) crosslinker.

Also disclosed herein is the bioresponse hydrogel matrix of any preceding aspect, wherein the bioresponse hydrogel matrix comprises an inner core and an outer shell; and wherein the CD47/SIRPa inhibitor is crosslinked to the outer shell and the immune checkpoint inhibitor is crosslinked to the inner core, or wherein the CD47/SIRPa inhibitor is crosslinked to the inner core and the immune checkpoint inhibitor is crosslinked to the outer shell.

In one aspect, disclosed herein is a method of treating, preventing, inhibiting, alleviating, and/or attenuating cancer and/or metastasis in a subject, the method comprising administering to the subject the bioresponse hydrogel of any preceding aspect. For example, disclosed herein are methods of treating, preventing, inhibiting, and/or attenuating cancer and/or metastasis in a subject, the method comprising administering to the subject a bioresponse hydrogel comprising a CD47/sirpa inhibitor (such as Hu5F9-G4, CV1, B6H12, 2D3, CC-90002, and TTI-621) and an immune checkpoint blockade inhibitor (such as a PD-1/PD-L1 inhibitor and/or a CTLA-4/B7-1/2 inhibitor).

Drawings

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

FIGS. 1A, 1B, 1C, 1D, 1E, 1F and 1G show schematic representations and characterizations of the ROS response [email protected] aCD47 protein complex. Fig. 1A shows a schematic diagram illustrating the use of ROS-sensitive complexes for synergistic immunotherapy of controlled sequential release of aCD47 and aPD1 in the tumor microenvironment. FIGS. 1B and 1C show the average hydrodynamic size of the aPD1 core (1B) and [email protected] aCD47 complex (1C) as determined by DLS. Illustration is shown: aPD1 TEM images (scale bar: 200nm) of the core (B) and [email protected] aCD47 complex (1C). Fig. 1D shows a scanning tem (stem) image of [email protected] aCD47 complex showing gadolinium labelled aCD47 (green) and calcium labelled aPD1 (red) (scale bar: 100 nm). FIG. 1E shows the presence or absence of H as measured by DLS₂O₂Degradation behavior of [email protected] aCD47 complex in PBS (0.5 mM). Illustration is shown: in the presence of H₂O₂(ii) a TEM image (scale bar: 100nm) of [email protected] aCD47 complex in PBS. FIG. 1F shows the dispersion in the presence or absence of H₂O₂(0.5mM) of aCD47 and aPD1 in complex in PBS. Data are expressed as mean ± s.e.m. (n ═ 3). FIG. 1G shows a liquid crystal display device containing H₂O₂In PBS (5) using H of the complex₂O₂And (5) clearing and testing. Data are expressed as mean ± s.e.m. (n ═ 3).

FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H show ROS-responsive protein complexes for scavenging ROS in TME to reverse the immunosuppressive environment. FIGS. 2A and 2B illustrate the useDark red reagent ROS levels, a.u., arbitrary units, in tumors collected from mice with or without complex treatment, measured on day 5 by flow cytometry analysis (2A) and confocal fluorescence imaging (2B). Figure 2C shows expression of NF-kB p65 and MMP2 in B16F10 tumors by western blot analysis. FIG. 2D shows CD45 in B16F10 tumors analyzed by flow cytometry⁺Percentage of cells. FIG. 2E shows M2-like macrophages in B16F10 tumors analyzed by flow cytometry (CD 206)^hiF4/80⁺CDllb⁺) Percentage of (c). FIG. 2F shows flow cytometryCD4 in B16F10 tumors analyzed⁺Foxp3⁺Percentage of T cells. FIG. 2G shows CD8 in B16F10 tumors analyzed by flow cytometry⁺Percentage of T cells. Data are expressed as mean ± s.e.m. (n-4). Fig. 2H shows a schematic diagram showing various immune responses after ROS-sensitive complex treatment. Statistical significance was calculated via the two-tailed student t-test. P value: p<0.05；**P<0.01；***P<0.005。

FIGS. 3A and 3B show intratumoral CD3 following ROS-responsive complex treatment⁺(3A) And CD4⁺Absolute percentage of T cells (3B). Data are expressed as mean ± s.e.m. (n-4). Statistical significance was calculated via the two-tailed student t-test. P value: p<0.05；**P<0.01；***P<0.005。

Fig. 4A, 4B, 4C, 4D, and 4E show CD47 blockade for increasing phagocytosis of cancer cells and activating phagocytic immune cells. Fig. 4A shows a representative confocal image showing that the aCD47 treatment resulted in robust phagocytosis (scale bar, 50pm) of red fluorescently labeled B16F10 cells by green fluorescently labeled BMDM. Fig. 4B shows phagocytosis of cancer cells by BMDM as determined by flow cytometry. Data are expressed as mean ± s.e.m. (n ═ 3). FIG. 4C shows CD45 in tumors following CD47 blockade⁺CD11c on cells⁺Percentage of DC gating. FIGS. 4D and 4E show CD45 in tumors following CD47 blockade⁺CD11c⁺CD80 on cells⁺CD86⁺DC (4D) and CD103⁺Percentage of DC (4E). Data are expressed as mean ± s.e.m. (n-4). Statistical significance was calculated via the two-tailed student t-test. P value: p<0.05；**P<0.01；***P<0.005。

FIGS. 5A, 5B and 5C show the retention behavior of intratumorally injected protein complexes. Figures 5A and 5B show in vivo fluorescence imaging to show retention times of aCD47(5A) and aPD1(5B) in tumors at different time points after injection of free antibody or [email protected] aCD47 complex. Fig. 5C shows confocal immunofluorescence images (scale bar, 200 μm) of tumors collected from mice treated with free antibody or [email protected] aCD47 complex at different time points. The red and green signals represent aPD1 and aCD47, respectively.

Fig. 6A, 6B, 6C, 6D, 6E, 6F and 6G show protein complex-mediated checkpoint blockade for inhibition of B16F10 tumor growth in vivo. Fig. 6A shows in vivo bioluminescence imaging of B16F10 tumors after different treatments. Four representative mice are shown per group. Fig. 6B and 6C show individual (6B) and average (6C) tumor growth curves in different groups. Data are expressed as mean ± s.e.m. (n ═ 6). FIGS. 6D, 6E and 6F show CD3 in tumors after different treatments⁺T cells (6D), CD4⁺T cells (6E) and CD8⁺Absolute percentage of T cells (6F). Data are expressed as mean ± s.e.m. (n-4). Figure 6G shows a representative flow cytometry analysis of T cell infiltration in tumors. Statistical significance was calculated via one-way analysis of variance using a graph-based post-hoc test. P value: p<0.05；**P<0.01；***P<0.001。

Fig. 7A, 7B, 7C, 7D, 7E, 7F, 7G and 7H illustrate protein complex-mediated checkpoint blockade for inhibition of distant tumor growth. Figure 7A shows a schematic showing [email protected] aCD47 complex processing in inhibiting cancer metastasis. The tumors on the right are referred to as "primary tumors" treated with the [email protected] aCD47 complex, and the tumors on the left are referred to as "metastatic tumors" without any treatment. Figure 7B shows in vivo bioluminescence imaging of B16F10 tumor after local injection of [email protected] aCD47 complex. Four representative mice are shown in each group. Fig. 7C and 7D show left and right tumor growth curves (7C) and weights (7D) for untreated and treated mice. Data are expressed as mean ± s.e.m. (n ═ 6). (E-h) representative flow cytometry analysis of T cell infiltration in tumors (7E) and CD3 in tumors after different treatments⁺T cell (7F), CD4⁺T cells (7G) and CD8⁺Absolute percentage of T cells (7H). Data are expressed as mean ± s.e.m. (n-4). Statistical significance was calculated via one-way analysis of variance using a graph-based post-hoc test. P value: p<0.05；**P<0.01；***P<0.001。

Detailed Description

Before the present compounds, compositions, articles of manufacture, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to specific reagents unless otherwise specified, as such, they may, of course, vary. Further, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

A. Definition of

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

Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It will also be understood that a number of values are disclosed herein, and that each value is disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a "less than or equal to" value, a "greater than or equal to" value is disclosed, possible ranges between the values are also disclosed as is well understood by those skilled in the art. For example, if the value "10" is disclosed, then "less than or equal to 10" and "greater than or equal to 10" are also disclosed. It should also be understood that throughout this application, data is provided in a number of different formats, and that the data represents endpoints and starting points, and ranges for any combination of data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than or equal to, and equal to 10 and 15 and between 10 and 15 are disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13 and 14 are also disclosed.

The term "subject" is defined herein to include animals, such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, and the like. In some embodiments, the subject is a human.

"administration" to a subject includes any route of introducing or delivering an agent to a subject. Administration can be by any suitable route, including oral, topical, intravenous, subcutaneous, transdermal, intramuscular, intraarticular (intra-joint), parenteral, intraarteriolar, intradermal, intracerebroventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, by implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intraarticular (intra-articular), intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion techniques), and the like. As used herein, "concurrently administering," "co-administering," "simultaneously administering (or administered simultaneously)" means that the compounds are administered at the same point in time or substantially immediately following. In the latter case, the administration times of the two compounds are close enough that the observed results are indistinguishable from those obtained when the compounds are administered at the same time point. By "systemic administration" is meant the introduction or delivery of an agent to a subject by a route that introduces or delivers the agent to a broad area of the subject's body (e.g., greater than 50% of the body), such as by entering the circulatory or lymphatic systems. In contrast, "topical administration" refers to the introduction or delivery of an agent to a subject by a route that introduces or delivers the agent to one or more areas immediately adjacent to the point of administration, and does not systemically introduce the agent in therapeutically significant amounts. For example, topically applied agents are readily detectable in the vicinity of the point of local application, but are not detectable or are detectable in negligible amounts in distal portions of the subject's body. Administration includes self-administration and others.

By "biocompatible" is generally meant that the material and any metabolites or degradation products thereof are generally non-toxic to the subject and do not cause significant side effects to the recipient.

By "comprising" is meant that the compositions, methods, etc., include the elements mentioned, but not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean including the recited elements, but excluding other elements having any significance to the combination. Thus, a composition consisting essentially of the elements as defined herein does not exclude trace contaminants and pharmaceutically acceptable carriers, such as phosphate buffers, preservatives and the like, from the isolation and purification process. "consisting of … …" shall mean excluding trace elements in excess of other ingredients and the substantial method steps for administering the compositions of the invention. Embodiments defined by each of these transitional terms are within the scope of the present invention.

A "control" is a surrogate subject or sample in an experiment for comparison purposes. The control may be a "positive control" or a "negative control".

By "controlled release" or "sustained release" is meant that the agent is released from a given dosage form in a controlled manner in order to achieve a desired pharmacokinetic profile in vivo. One aspect of "controlled release" agent delivery is the ability to manipulate the formulation and/or dosage form to establish the desired release kinetics of the agent.

An "effective amount" of an agent is an amount of the agent sufficient to provide the desired effect. The amount of "effective" agent will vary from subject to subject, depending on the age and general condition of the subject, the particular agent or agents, and a number of factors. Thus, it is not always possible to specify an "effective amount" for quantification. However, an appropriate "effective amount" in any subject case can be determined by one of ordinary skill in the art using routine experimentation. Furthermore, as used herein, and unless otherwise specifically stated, an "effective amount" of an agent can also be an amount that encompasses both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of an agent required to achieve a therapeutic effect may vary depending on factors such as the age, sex, and weight of the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be reduced proportionally to the exigencies of the therapeutic situation.

"reducing" can refer to any change that results in less gene expression, protein expression, number of symptoms, disease, composition, disorder, or activity. A substance is also understood to reduce the genetic yield of a gene when the genetic yield of a gene product containing the substance is small compared to the genetic yield of a gene product not containing the substance. Further, for example, a reduction may be an alteration in the symptoms of a disorder such that fewer symptoms are observed than previously. The reduction may be a statistically significant amount of the disorder, symptom, activity, any individual in the composition, a median or average reduction. Thus, the reduction may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as long as the reduction is very significant.

By "Inhibit (inhibition, inhibiting and inhibition)" is meant reducing activity, response, condition, disease or other biological parameter. This may include, but is not limited to, complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in activity, response, condition, or disease as compared to an untreated or control level. Thus, the reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any reduction in between compared to the untreated or control level.

As used herein, the term "prevent (preceding, or preceding)" and grammatical variations thereof refers to delaying or preventing, partially or completely, the occurrence or recurrence of a disease and/or one or more of its attendant symptoms, or preventing a subject from acquiring or regaining disease or reducing the subject's risk of acquiring or regaining disease or one or more attendant symptoms.

A "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component can be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein, without causing a significant adverse biological effect or interacting in a deleterious manner with any of the other components of the formulation in which the component is included. When used in reference to administration to a human, the term generally means that the component has met the required standards of toxicological and manufacturing testing, or that it is included in the inactive ingredient guidelines set forth by the U.S. food and drug administration.

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

"pharmacologically active" (or only "active"), as used in reference to a "pharmacologically active" derivative or analog, can refer to a derivative or analog (e.g., salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) that has the same type of pharmacological activity as the parent compound and to an approximately equal degree.

"therapeutic agent" refers to any composition having a beneficial biological effect. Beneficial biological effects include therapeutic effects such as treatment of a disorder or other adverse physiological condition, and prophylactic effects such as prevention of a disorder or other adverse physiological condition (e.g., a non-immunogenic cancer). The term also encompasses pharmacologically active derivatives of the beneficial agents specifically mentioned herein in the context of pharmaceutical use, including but not limited to salts, esters, amides, precursor agents, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, or when a particular agent is explicitly identified, it is understood that the term includes the agent itself as well as pharmaceutically active salts, esters, amides, precursor agents, conjugates, active metabolites, isomers, fragments, analogs, and the like, which are pharmaceutically acceptable.

"Polymer" refers to a relatively high molecular weight natural or synthetic organic compound, the structure of which may be represented by repeating small units, monomers. Non-limiting examples of polymers include polyethylene, rubber, cellulose. Synthetic polymers are typically formed by addition or polycondensation of monomers. The term "copolymer" refers to a polymer formed from two or more different repeating units (monomer residues). By way of example and not limitation, the copolymer may be an alternating copolymer, a random copolymer, a block copolymer, or a graft copolymer. It is also contemplated that, in certain aspects, the various block segments of the block copolymer may themselves comprise the copolymer. The term "polymer" includes all forms of polymers including, but not limited to, natural polymers, synthetic polymers, homopolymers, heteropolymers or copolymers, addition polymers, and the like.

A "therapeutically effective amount" or "therapeutically effective dose" of a composition (e.g., a composition comprising a pharmaceutical agent) refers to an amount effective to achieve a desired therapeutic result. In some embodiments, the desired therapeutic result is control of type I diabetes. In some embodiments, the desired therapeutic outcome is the control of obesity. The therapeutically effective amount of a given therapeutic agent will generally vary depending upon factors such as the type and severity of the disorder or disease being treated, as well as the age, sex, and weight of the subject. The term can also refer to an amount of a therapeutic agent or a rate of delivery (e.g., an amount over time) of a therapeutic agent that is effective to promote a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary depending on the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of the agent in the formulation, etc.), and a variety of other factors as understood by one of ordinary skill in the art. In some cases, a desired biological or medical response can be obtained after administering multiple doses of the composition to a subject for several consecutive days, weeks, or years.

In this specification and the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

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

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this application pertains. The disclosed references are also individually and specifically incorporated by reference herein, and the material contained in the references is discussed in the sentence in which the reference is based.

B. Composition comprising a metal oxide and a metal oxide

The compositions themselves useful for preparing the compositions disclosed herein, as well as for use in the methods disclosed herein, are disclosed. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular bioresponse hydrogel matrix is disclosed and discussed and a number of modifications that can be made to a plurality of molecules comprising the bioresponse hydrogel matrix are discussed, unless the contrary is indicated, various and every combination and permutation of the bioresponse hydrogel matrix are specifically contemplated, as well as possible modifications. Thus, if a class of molecules A, B and C is disclosed as well as a class of molecules D, E and F and examples of combination molecules are disclosed, then A-D is disclosed, and even if each is not individually referenced, individual and collectively contemplated meaning combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E and C-F are considered disclosed. Likewise, any subset or combination of these combinations is also disclosed. Thus, for example, it will be considered that subgroups of A-E, B-F and C-E are disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

Here, an albumin-based complex with anti-PD-1 (aPD1) in the core and anti-CD 47(aCD47) in the shell ([email protected] aCD47 complex) was designed using Reactive Oxygen Species (ROS) responsive linkers for use in combination therapy. It is shown herein that in ROS-enriched TME, the ROS response [email protected] aCD47 complex may first release aCD47 from the coat continuously to activate the recognition of cancer cells by the innate immune system and enhance the T cell response. Subsequently released aPD1 can then exert a PD1 blocking effect in order to effectively increase alloreactive T cells to attack cancer cells. Furthermore, the ROS-responsive complex not only serves as a reservoir for controlled release of the antibody, but also modulates ROS levels in TME (fig. 1A). ROS are important signaling messengers in the immune system, are closely related to immunosuppressive responses, and promote tumor development and progression. Thus, the ROS-degradable complex may contribute to an effective anti-tumor immune response through the controlled sequential release of aCD47 and aPD1, coupled with the down-regulation of ROS-sensitive signals within the TME.

Disclosed herein are bioresponse hydrogel matrices comprising CD47/sirpa inhibitors (such as Hu5F9-G4, CV1, B6H12, 2D3, CC-90002 and TTI-621) and immune checkpoint blockade inhibitors (such as PD-1/PD-L1 inhibitors and/or CTLA-4/B7-1/2 inhibitors).

In one aspect, blocking inhibitors useful in the disclosed bioresponse hydrogel matrices can be any inhibitor of immune checkpoint blocking inhibitors, such as PD-1/PD-L1 blocking inhibitors, and/or CTLA-4/B7-1/2 blocking inhibitors (such as yiprimab). Examples of PD-1/PD-L1 blocking inhibitors for use in the disclosed bioresponse hydrogel matrices may include any PD-1/PD-L1 blocking inhibitor known in the art, including but not limited to nivolumab, pembrolizumab, pidilizumab, astuzumab, avizumab, dewalizumab, and BMS-936559.

As described herein, the disclosed bioresponse hydrogel matrices utilize CD 47/signal-regulatory protein a (sirpa) inhibitors (such as Hu5F9-G4, CV1, B6H12, 2D3, CC-90002, and/or TTI-621) inhibitors to sensitize a subject to immune checkpoint inhibition therapy. It is to be understood and contemplated herein that the CD47/sirpa inhibitors for use in the disclosed bioresponse hydrogel matrices may include any known CD47/sirpa inhibitors, including but not limited to Hu5F9-G4, CV1, B6H12, 2D3, CC-90002, and/or TTI-621.

To facilitate these functions, the bioresponse hydrogel matrix can be designed as a polymer. "Polymer" refers to a relatively high molecular weight natural or synthetic organic compound, the structure of which may be represented by repeating small units, monomers. Non-limiting examples of polymers include polyethylene, rubber, cellulose. Synthetic polymers are typically formed by addition or polycondensation of monomers. The term "copolymer" refers to a polymer formed from two or more different repeating units (monomer residues). By way of example and not limitation, the copolymer may be an alternating copolymer, a random copolymer, a block copolymer, or a graft copolymer. It is also contemplated that, in certain aspects, the various block segments of the block copolymer may themselves comprise the copolymer. The term "polymer" encompasses all forms of polymers including, but not limited to, natural polymers, synthetic polymers, homopolymers, heteropolymers or copolymers, addition polymers, and the like. In one aspect, the gel matrix can include a copolymer, a block copolymer, a diblock copolymer, and/or a triblock copolymer.

In one aspect, the bioresponsive hydrogel matrix can comprise a biocompatible polymer, such as methacrylated hyaluronic acid (m-HA). In one aspect, the biocompatible polymer is crosslinkable. Such polymers may also be used to slowly release fat browning and/or fat regulating agents into tissue. As used herein, biocompatible polymers include, but are not limited to, polysaccharides; a hydrophilic polypeptide; poly (amino acids) such as poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly-L-aspartic acid, poly-L-serine, or poly-L-lysine; polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG), polypropylene glycol (PPG), and poly (ethylene oxide) (PEO); poly (oxyethylenated polyols); poly (alkene alcohols); polyvinylpyrrolidone; poly (hydroxyalkyl methacrylamide); poly (hydroxyalkyl methacrylic acid); poly (saccharides); poly (hydroxy acids); poly (vinyl alcohol), polyhydroxy acids such as poly (lactic acid), poly (glycolic acid), and poly (lactic-glycolic acid); polyhydroxyalkanoates such as poly-3-hydroxybutyric acid or poly-4-hydroxybutyric acid; polycaprolactone; poly (n-ester); a polyanhydride; poly (phosphazenes); poly (lactide-caprolactone); polycarbonates such as tyrosine polycarbonate; polyamides (including synthetic and natural polyamides), polypeptides, and poly (amino acids); a polyester amide; a polyester; poly (dioxanone); poly (alkylen) s; a hydrophobic polyether; a polyurethane; a polyether ester; a polyacetal; polycyanoacrylates; a polyacrylate; polymethyl methacrylate; a polysiloxane; poly (oxyethylene)/poly (oxypropylene) copolymers; polyketal; a polyphosphate salt; a polyhydroxyvalerate salt; polyalkylene oxalates; a polyalkylene succinate salt; poly (maleic acid) and copolymers thereof. Biocompatible polymers may also include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkyl terephthalates, polyvinyl alcohols (PVA), methacrylates (m-PVA), polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinyl pyrrolidones, polyethylene glycols, polysiloxanes, polyurethanes and copolymers thereof, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, ethyl celluloses, polymers of acrylic and methacrylic esters, methyl celluloses, ethyl celluloses, hydroxypropyl methyl celluloses, hydroxybutyl methyl celluloses, cellulose acetates, cellulose propionates, cellulose acetate butyrates, cellulose acetate phthalates, carboxyethyl celluloses, cellulose triacetates, cellulose sulfate sodium salts, poly (methyl methacrylate), poly (vinyl acetate), poly (co-vinyl acetate), poly (co-p-and poly (meth) s), poly (meth) and poly (meth) s) and poly (meth) s, Exemplary biodegradable polymers include polyesters, poly (ortho esters), poly (vinylamines), poly (caprolactone), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), poly (octadecyl acrylate), polyethylene, polypropylene, poly (ethylene glycol), poly (ethylene oxide), poly (ethylene terephthalate), poly (vinyl alcohol), poly (vinyl acetate, polyvinyl chloride polystyrene, and polyvinylpyrrolidone and derivatives thereof, linear and branched copolymers and block copolymers thereof, and blends thereof Poly (hydroxyvalerate), polyanhydrides, poly (acrylic acid), polyethylene glycol, poly (urethane), polycarbonates, polyphosphates, polyphosphazenes and derivatives thereof, linear and branched copolymers and block copolymers thereof, and blends thereof.

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

The triblock copolymers disclosed herein comprise a core polymer such as, for example, polyethylene glycol (PEG), polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), poly (vinylpyrrolidone-vinyl acetate), polymethacrylates, polyoxyethylene alkyl ethers, polyoxyethylene castor oil, polycaprolactam, polylactic acid, polyglycolic acid, poly (lactic-glycolic acid) copolymers (PLGA), cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, and the like. In one aspect, the core copolymer may be flanked by polypeptide blocks.

Examples of diblock copolymers that may be used in the micelles disclosed herein include polymers such as polyethylene glycol (PEG), polyvinyl acetate, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), poly (vinylpyrrolidone-vinyl acetate), polymethacrylates, polyoxyethylene alkyl ethers, polyoxyethylene castor oil, polycaprolactam, polylactic acid, polyglycolic acid, poly (lactic-glycolic acid) (PLGA).

It is to be understood and contemplated herein that the bioresponse hydrogel matrix may be designed to have a biological response to the microenvironment of the tumor and release CD47/sirpa inhibitors (such as Hu5F9-G4, CV1, B6H12, 2D3, CC-90002, and TTI-621) and immune checkpoint blockade inhibitors (such as PD-1/PD-L1 inhibitors and/or CTLA-4/B7-1/2 inhibitors) as well as any other anti-cancer agents into the tumor microenvironment upon exposure to factors within the microenvironment, such as reactive oxygen species, including but not limited to peroxides (e.g., hydrogen peroxide), superoxide, hydroxyl radicals, and singlet oxygen; the presence of acidity; redox potential (glutathione (GSH)); specific tumor-associated enzymes; hypoxia; and adenosine-5' -triphosphate (ATP). Thus, in one aspect, the disclosed bioresponse hydrogel matrices comprise a bioresponse scaffold that releases CD47/sirpa inhibitors (such as Hu5F9-G4, CV1, B6H12, 2D3, CC-90002, and TTI-621) and immune checkpoint blockade inhibitors (such as PD-1/PD-L1 inhibitors and/or CTLA-4/B7-1/2 inhibitors) and/or other anticancer agents into the tumor microenvironment (e.g., Reactive Oxygen Species (ROS) degradable hydrogels) upon exposure to factors within the microenvironment. In one aspect, the hydrogel can include crosslinked polyvinyl alcohol (PVA) and N¹- (4-boronobenzyl) -N³- (4-borono-phenyl) -N¹,N¹,N³,N³-tetramethyl groupPropane-1, 3-diamine (TSPBA). In one aspect, the ROS-responsive hydrogel may be obtained by crosslinking poly (vinyl alcohol) (PVA) with an ROS-labile linker: n is a radical of¹- (4-boronobenzyl) -N³- (4-borono-phenyl) -N¹,N¹,N³,N³-tetramethylpropane-1, 3-diamine (TSPBA) via N¹,N¹,N³,N³-tetramethylpropane-1, 3-diamine with an excess of 4- (bromomethyl) phenylboronic acid. TSPBA contains two phenylboronic acids, which complex with multiple diols on PVA. When exposed to H in the tumor microenvironment₂O₂When used, TSPBA will be oxidized and hydrolyzed, resulting in dissociation of the polymer scaffold and release of PVA and payload. In another aspect, the hydrogel may comprise 2,2' - [ propane-2, 2-diyl bis (thio) modified by bis-N-hydroxysuccinimide (NHS)]Diacetic acid (NHS-IE-NHS) crosslinker to both CD47/SIRPa inhibitor and immune checkpoint blockade inhibitor.

In one aspect, a CD47/sirpa inhibitor (such as Hu5F9-G4, CV1, B6H12, 2D3, CC-90002, and TTI-621) and an immune checkpoint blockade inhibitor (such as a PD-1/PD-L1 inhibitor and/or a CTLA-4/B7-1/2 inhibitor) may be arranged in a biologically responsive hydrogel matrix in a core-shell structure to facilitate sequential release of the CD47/sirpa inhibitor and the immune checkpoint blockade inhibitor such that the core binding inhibitor is released into the tumor microenvironment after release of the shell binding inhibitor. Thus, in one aspect, disclosed herein is a bioresponse hydrogel matrix, wherein the bioresponse hydrogel matrix comprises an inner core and an outer shell; and wherein the CD47/SIRPa inhibitor is crosslinked to the outer shell and the immune checkpoint inhibitor is crosslinked to the inner core, or wherein the CD47/SIRPa inhibitor is crosslinked to the inner core and the immune checkpoint inhibitor is crosslinked to the outer shell.

Anticancer agents that may be used in the disclosed bioresponsive hydrogel matrices may include any anticancer agent known in the art, including, but not limited to, Abetiril, Abitetron acetate, Abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticulate formulation), ABVD, ABVE-PC, AC-T, Adcetris (Bentuximab), ADE, Ado-trastuzumab Emtansine, doxorubicin (doxorubicin hydrochloride), Afatinib maleate, Afinitor (Everolimus), Akynzeo (Netopiratan and Palonosetron hydrochloride), Aldara (imiquimod), Addison, Alecensa (Elletinib), Elletinib, Allen (Pametrexed disodium), Aliqoppa (Copanlisib hydrochloride), Eikeland (Melphalan hydrochloride) for injection, Ekrainian (Ekrainian), Alcalan (Melphalan hydrochloride), Alulox (Alcohoron hydrochloride), Albeman (Albumin hydrochloride), Albumin hydrochloride (Albumin hydrochloride), Albemap, Ambochloririn (chlorambucil), ambocriol chlorambucil), amifostine, aminolevulinic acid, anastrozole, aprepitant, Aredia (disodium pamidronate), Arimidex (anastrozole), Aromasin (exemestane), aranon (nelarabine), arsenic trioxide, Arzerra (ofatumumab), chrysanthemumab asparaginase, altuzumab, avastin (bevacizumab), avizumab, axitinib, azacitidine, Bavencio (avizumab), beacop, Becenum (carmustine), Beleodaq (bevacizumab), bevacizumab, bendamustine hydrochloride, BEP, Besponsa (inozumab) zomab zomicin, bevacizumab, Bexxar (tositumomab and tositumomab 131), borvacizumab, blevacizumab (bleb), bizepinocytomib (bleb), blevacizumab (bizepinib), blevacizumab (blevacizumab), Bexxar (Bexxar and tositumomb), bortezomib (bortezomib), bortezomib (bizidine), bortezomib (bizidinil), bortezomib (bizib), bevacizumab (Bexxar, bexxb, bevacizb, b, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound selected from the group consisting of tobuximab, bugatinib, BuMel, Busulfex, cabazitaxel, Cabometyx malate, cabozantinib malate, CAF, Campath, Camptosar, irinotecan hydrochloride, capecitabine, CAPOX, caroc, carboplatin paclitaxel, carfilzomib, carmubin, carmustine, Casodex, CEM, ceritinib, Cerubidine, cerumubicin hydrochloride, Cervarix, clovir, cetuximab, CEV, chlorambucil-prednisone, CHOP, cisplatin, cladribine, Clafen, clorariranib, clofarabine, Cotifaca, Coxilb, Coxilitinib, Cofava, Coxib, Cochlozimab, Coxib, and C, Cosmegen (dactinomycin), Cotellic (cobicistinib), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine liposome, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, darramucimumab, Darzalex (Darzalex), dasatinib, daunorubicin hydrochloride, Daunombicin hydrochloride and cytarabine liposome, desicitabine, defibrinoside sodium, deffitolilo (defibrinoside sodium), degarelix, dinebilungia-diphtheria toxin linker, dinolizumab, depocyst (cytarabine liposome), dexamethasone, dexrazoxane hydrochloride, dexrazoxane, docetaxel, doxexil (doxycycline hydrochloride), doxycycline hydrochloride, doxorubicin hydrochloride, doxycycline hydrochloride (doxycycline hydrochloride), doxycycline hydrochloride (SL-doxycycline hydrochloride), doxycycline hydrochloride (doxycycline hydrochloride) DTIC-Dome (dacarbazine), Dewar-mab, Efudex (fluorouracil-topical), Elitek (Labrazie), Ellene (epirubicin hydrochloride), Eltuzumab, Eloxitin (oxaliplatin), Eltropophaemine, Emend (aprepitant), Emplicititi (Eltuzumab), Ensidipine mesylate, Enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erive (Virgimod), erlotinib hydrochloride, Erwinazeze (Erwinia chrysanthemi asparaginase), Ethylol (amifostine), Etopophos (etoposide phosphate), etoposide phosphate, Etoponet (Adiposide hydrochloride), Evicolimus, Evisolimus (raloxifene hydrochloride), Evoomela (Mermetan hydrochloride), Esimetiracetam 5-FU (fluorouracil-5-topical), FU (fluorouracil-5 (fluorouracil-topical) Fareston (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil-topical), fluorouracil injection, fluorouracil-topical, flutamide, Folex (methotrexate), Folex PFS (methotrexate), FOLFIRI-Bevacizumab, FOLFIRI-Cexiximab, FOLFIRINOX, FOLFOX, Folotyn (pralatrexate), FU-LV, fulvestrant, gavagaride (recombinant HPV quadrivalent vaccine), Gazyva 9 (recombinant HPV nine valent vaccine), Gazyva (attuzumab), Gefitinib, Germinal hydrochloride, cisplatin-gemcitabine, oxaliplatin, Getumubulin-Germin, Germin-Germinal, Germinal (Germinalfacacetin), Germinal (Gimeracin), Germin), Germinal (Gimeracin hydrochloride), Germin (Gimeracin), Gernit acid (Gimeracin), Gernic acid, Gernit), Gernit (Gimeracin hydrochloride (Gimeracin), Gernic acid, Gernic, Geranilate, Gernit, Geranilate, Geranitidine, Geranilate, C, Geranilate, C, Geranilate, C, and so, C, Geranilate, and so, for example, Geranilate, for example, and so, for example, and so, Gliadel (carmustine implant), Gliadel wafer (carmustine implant), carboxypeptidase, goserelin acetate, Halaven (eribulin mesylate), Hemangeol (propranolol hydrochloride), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant HPV nine-valent vaccine, recombinant HPV tetravalent vaccine, recombinant Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper-CVAD, Ibrance (palbociclib), tiimomab Tiuxetan, ibrutinib, ICE, iclusisig (panatinib hydrochloride), Idamycin (idarubicin hydrochloride), idarubicin hydrochloride, iradriise, idifa (enicilnidipine mesylate), ix (ifosfamide), ifosfamide, ifsfanum (ifosfamide), IL-2 (aldesmuibrutin), imatinib, ibrutinib sulfonate (lasilytica), ibritumomab (tamoxifen), ibritumomab (tamicit), ibritumomab (naftid), ibritumomab (irinotecan), imafosfamide (imsfamide), imab, imazernib (tamicilin), imazelta, tamicine, imazelta, tamicine, tamicit, e, tamicilin (tamicilin, e, tamicilin, imab, tamicilin, e, tamicilin, imahi, tamicilin, e, tamicilin, e, tamicilin, or (tamicilin, or, Inotuzumab ozomicin, interferon alpha-2 b, recombinant interleukin-2 (aldesleukin), Intron A (recombinant interferon alpha-2 b), I131 tositumomab and tositumomab, YIPURIMAb, Iressa (gefitinib), irinotecan hydrochloride liposome, Istodax (Romidexin), ixabepilone, ixazofamid citrate, Ixempla (ixabepilone), Jakafi (ruxolitinib phosphate), JEB, Jentaa (cabazitaxel), Kadcycla (Ado-trastuzumab Emtansine), Keoxifene (Raloxifene hydrochloride), Kepivavamin (Kelviflam), Keytrudida (pemirovax), Kisqali (Ribosini), Kymmerih (Tisageneucil), Kymilva (Kypris), zoledram acetate, Revernatremilat, Lertyme), Lertyme (Lertyme), Leventimab, E, and their derivatives, Calcium folinate, Leukerazine (chlorambucil), leuprorelin acetate, Leustatin (cladribine), Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (Adriamycin hydrochloride liposome), lomustine, Lonsurf (trifluridine and Tibipyridine hydrochloride), Lupron (Leuprolide acetate), Lupron Depit-Ped (Leuprolide acetate), Lynparza (Orlaparib), Marqo (vincristine sulfate liposome), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, megestrol acetate, Mekinist (Trimetinib), Melphalan hydrochloride, mercaptopurine, Melsex (mesna), Methazotone (temozolomide), methotrexate L (AQL), Mexadone bromide, Mexate (Mexate C), methotrexate (methotrexate C), methotrexate, Mitoxantrone hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (plerixafor), Mustargen (mechlorethamine hydrochloride), Mutamicin (mitomycin C), Myleran (busulfan), Mylosar (azacitidine), Mylotarg (gemtuzumab-ozogamicin), nanoparticulate paclitaxel (paclitaxel albumin-stabilized nanoparticulate formulation), Navelbine (vinorelbine tartrate), tolituzumab, nelarabine, Neosar (cyclophosphamide), lenatinib maleate, Nerlynx (lenatinib maleate), Netupidan and palonosetron hydrochloride, Neula Negota (peggefitinib), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nilandron (nimitamide), nilotinib, nimitamide, Wuxalo (citrate), Wulazuride, Mononide, citric acid, Nolvatex (Nolvariex hydrate), Nepaludi (nilutamide), Nepaludix (nimoramide, and nimoramide hydrochloride, Adalimumab, ondomzo (sonedji), OEPA, ofatumumab, OFF, olaparib, olanzab, homoharringtonine, oncocaspar (pemetrexed), ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposome), Ontak (dinil-diphtheria toxin linker), opdiv (nivolumab), OPPA, osetinib, oxaliplatin, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, palifermin, palonosetron hydrochloride and netupitant, disodium pamidronate, panitumumab, panobistat, parasplat (carboplatin), parasplatin (carboplatin), pazopanib hydrochloride, PCV, PEB, pemetrexed, pegfilzumab, peginterferon alpha-2 b, PEG-intron (peginterferon alpha-2 b), pemetrexed alpha-2 b), peginterferon alpha-2 b, Pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor, pomalidomide, Pomalyst (pomalidomide), panatinib hydrochloride, Portrazza (nimotuzumab), pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldeskin), Prolia (dinomab), Promacta (eltopranolamine), propranolol hydrochloride, Provenge (Sipuleucel-T), Purinethol (mercaptopurine), Purixan (mercaptopurine), radium 223 dichloride, Raloxifene hydrochloride, Ramopluromab, Labridase, R-CHOP, R-CVP, recombinant Human Papilloma Virus (HPV) bivalent vaccine, recombinant Human Papilloma Virus (HPV) nine-valent vaccine, recombinant Human Papilloma Virus (HPV) interferon vaccine, recombinant Human Papilloma Virus (HPV) 2-methyl-vor (Regonador), Regonador (Regonador-T) vaccine, Reductura-T, Reductuli (Reductuli-T) vaccine, and Reductuli, R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), Ribocini, R-ICE, Rituxan (rituximab), Rituxan Hycela (rituximab and hyaluronidase human), rituximab and hyaluronidase human, Lapiditan hydrochloride, Romidexin, Romitastin, Rubidomycin hydrochloride, Rubraca (Ricapra camphorsulfonate), Ricapacamo camphorsulfonate, Ruxolitinib phosphate, Rydatypit (midostaurin), Serranolo pleura sol (Talc), Sertuximab, Sipuleucel-T, Somatador type (lanreotide acetate), Sonide, Sorafenib tosylate, Sprycel (Darnotron malic acid), STANFORD V, Stercuit Talc (Talc), Talcitc, Talcityta (Talcitc), sunitinib acid depot, sunitinib (Sulta-2), sunitinib-alpha interferon (Sulta-L-2), sunitinib (Sulta-L-S-L-R-L-S-L, Sylvant (cetuximab), Synribo (homoharringtonine), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (Osteinib), Talc, Talimogen Laherparvec, tamoxifen citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxoe (docetaxel), Teentriq (Attitumomab), Tear mod (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), thioguanine, thiotepa, Tisagegenlecileucel, Tolak (fluorouracil-HCl), topical irinotecan, Torrisel (Torashizimab), tretin, trexisol (Trecetist), Trecil (Trecilacil), Tretricitabine, Toxolimus (Toratonidine hydrochloride), Torasidone hydrochloride, Tourette (Tourette, Toluotreta (Tourette, Toluotreta (D, Tourette, D, Tourette, D, Tourette's, D, Tourette's, D, Tourette's, D, T, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), unitaxin (dituximab), uridine triacetate, VAC, vandetanib, VAMP, Varubi (dolastatin hydrochloride), Vectibix (panitumumab), VelP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), verofenib, venlex (vinetock), vinetock, Verzenio (aberciry), viduru (leuprolide acetate), vida (azacitidine), vinblastine sulfate, vinaspora s (vincristine sulfate), vincristine sulfate liposome, vinorelbine tartrate, VIP, vistogigo, Vistogard (triacetic acid), voraxze (carboxypeptidase), vorinostat (vorinosite), vycotinib (voxolone hydrochloride), doxycycline hydrochloride (oxypercalcin hydrochloride), calcium oxylidoxime (xolone), calcium chloride (xolone (calcium chloride), victorine hydrochloride (xolone (calcium chloride), victorine hydrochloride), victorine (vallec), vallec (valtrex), valtrex (valcanine, vallec), vallec (r), valcanine, vallec (r), and vallec (r) salts of the like, Xelairi, xeloxx, Xgeva (dinolizumab), Xofigo (radium 223 dichloride), xtanid (enzalutamide), yrenvoy (Yipri mab), Yondelis (tratinib), Zaltrap (Ziv-aflibercept), Zarxio (filgrastim), Zejula (nilapanib tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (tiumomatibetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), zta omel (zoledronic acid), Zydelig (idegridiris), Zykadia (rietinib), and/or zybivatlon (acetic acid).

1. Antibodies

(1) Antibodies in general

The term "antibody" is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, fragments or polymers of those immunoglobulin molecules are also included in the term "antibody", and human or humanized forms of immunoglobulin molecules or fragments thereof are also disclosed. Antibodies can be tested for a desired activity using the in vitro assays described herein or similar methods, and then tested for their in vivo therapeutic and/or prophylactic activity according to known clinical testing methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of them may also be divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. One skilled in the art will recognize comparable classes of mice. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies in the population are identical except for naturally occurring mutations that may be present in a small subset of antibody molecules. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remainder of one or more chains is identical with or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity.

Any procedure for producing monoclonal antibodies can be used to prepare the disclosed monoclonal antibodies. For example, the disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature (Nature), 256:495 (1975). In the hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

Monoclonal antibodies can also be prepared by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Antibody libraries or active antibody fragments can also be generated and screened using phage display technology, for example, as described in U.S. Pat. No. 5,804,440 to Burton et al and U.S. Pat. No. 6,096,441 to Barbas et al.

In vitro methods are also suitable for the production of monovalent antibodies. The antibody may be digested to produce fragments thereof, particularly Fab fragments, using conventional techniques known in the art. For example, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 and U.S. Pat. No. 4,342,566, published 12, 22, 1994. Papain digestion of antibodies typically produces two identical antigen binding fragments (called Fab fragments), each having an antigen binding site and a residual Fc fragment. Pepsin treatment produces fragments that have two antigen binding sites and are still capable of cross-linking antigens.

As used herein, the term "antibody or fragment thereof" encompasses chimeric and hybrid antibodies with dual or multiple antigen or epitope specificities, as well as fragments such as F (ab ') 2, Fab', Fab, Fv, scFv, and the like, including hybrid fragments. Thus, antibody fragments are provided that retain the ability to bind to their specific antigen. These Antibodies and fragments can be prepared by techniques known in the art, and can be screened for specificity and activity according to the methods described in the examples and general methods for generating Antibodies and screening Antibodies for specificity and activity (see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, (1988)).

Also included within the meaning of "antibody or fragment thereof" are conjugates of antibody fragments and antigen binding proteins (single chain antibodies).

Fragments, whether or not appended to other sequences, may also include insertions, deletions, substitutions, or other selective modifications of particular regions or particular amino acid residues, provided that the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment. These modifications may provide additional properties such as removal/addition of amino acids capable of forming disulfide bonds, extending their biological life, altering their secretory properties, etc. In any case, the antibody or antibody fragment must have a biologically active property, such as specific binding to its cognate antigen. The functional or active region of the antibody or antibody fragment can be determined by mutagenesis of a particular region of the protein, followed by expression and detection of the expressed polypeptide. These methods will be apparent to those skilled in the art and may include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment (Zoller, MJ. Curr. Opin. Biotechnol.3:348-354, 1992).

As used herein, the term "antibody (antibodies or antibodies)" may also refer to human antibodies and/or humanized antibodies. Many non-human antibodies (e.g., antibodies from mice, rats, or rabbits) are natural antigens in humans and, therefore, cause an adverse immune response when administered to humans. Thus, the use of human or humanized antibodies in these methods helps to reduce the likelihood that administration of the antibody to a human will elicit an adverse immune response.

(2) Human antibodies

The disclosed human antibodies can be prepared using any technique. The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic mutant mice capable of producing a full repertoire of human antibodies in an immune response have been described (see, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al, Nature, 362:255-258 (1993); Bruggemann et al, Yeast in Immunol, 7:33 (1993)). Specifically, in these chimeric and germline mutant mice, homozygous deletion of the antibody heavy chain joining region (j (h)) gene resulted in complete suppression of endogenous antibodies, while successful transfer of the human germline antibody gene array into these germline mutant mice resulted in production of human antibodies upon antigen challenge. Antibodies with the desired activity were selected using the Env-CD4 co-receptor complex as described herein.

(3) Humanized antibodies

Antibody humanization techniques typically involve the use of recombinant DNA techniques to manipulate DNA sequences encoding one or more polypeptide chains of an antibody molecule. Accordingly, a humanized form of a non-human antibody (or fragment thereof) is a chimeric antibody or antibody chain (or fragment thereof, such as an sFv, Fv, Fab ', F (ab') 2, or other antigen-binding portion of an antibody) that comprises a portion of the antigen-binding site from a non-human (donor) antibody integrated into the framework of a human (acceptor) antibody.

To produce a humanized antibody, residues from one or more Complementarity Determining Regions (CDRs) of an acceptor (human) antibody molecule are substituted with residues from one or more CDRs of a donor (non-human) antibody molecule known to have desired antigen binding properties (e.g., certain specificity and affinity for a target antigen). In some cases, Fv Framework (FR) residues of the human antibody are substituted with corresponding non-human residues. Humanized antibodies may also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Typically, humanized antibodies have one or more amino acid residues introduced from a non-human source. In practice, humanized antibodies are typically human antibodies in which some CDR residues are substituted by residues from analogous sites in rodent antibodies, and some FR residues may be substituted by residues from analogous sites in rodent antibodies. Humanized antibodies typically contain at least a portion of an antibody constant region (Fc), typically a human antibody constant region (Jones et al, Nature, 321:522-525(1986), Reichmann et al, Nature, 332:323-327(1988), and Presta, curr, Opin, Structure, biol.,2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be generated by replacing rodent CDR or CDR sequences with the corresponding sequences of a human antibody according to the method of Winter and coworkers (Jones et al, Nature, 321:522-525(1986), Riechmann et al, Nature, 332:323-327(1988), Verhoeyen et al, Science, 239:1534-1536 (1988)). Methods that can be used to generate humanized antibodies are also described in U.S. Pat. No. 4,816,567(Cabilly et al), U.S. Pat. No. 5,565,332(Hoogenboom et al), U.S. Pat. No. 5,721,367(Kay et al), U.S. Pat. No. 5,837,243(De o et al), U.S. Pat. No. 5,939,598(Kucherlapati et al), U.S. Pat. No. 6,130,364(Jakobovits et al), and U.S. Pat. No. 6,180,377(Morgan et al).

2. Drug carrier/drug delivery

As noted above, these compositions may also be administered to the body in the form of a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject with a nucleic acid or vector without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. As is well known to those skilled in the art, the carrier will naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

The compositions can be administered orally, parenterally (e.g., intravenously), intramuscularly, intraperitoneally, transdermally, extracorporeally, topically, etc., including topically intranasally or by inhalation. As used herein, "topical intranasal administration" means delivery of the composition to the nasal cavity and nasal passages through one or both nostrils, and may include delivery by a spray mechanism or a droplet mechanism, or by nebulization of a nucleic acid or vector. Administration of the composition by inhalation is nasal or oral, delivered by a spray or droplet mechanism. Delivery may also be directly to any region of the respiratory system (e.g., the lungs) through intubation. The exact amount of the composition required will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, the mode of administration thereof, and the like. Therefore, it is not possible to specify exact amounts for each composition. However, the appropriate amount can be determined by one of ordinary skill in the art by routine experimentation using only the teachings given herein.

Parenteral administration of the composition (if used) is typically characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for dissolving suspensions in liquids prior to injection, or as emulsions. A more recently revised method of parenteral administration involves the use of slow release or slow release systems to maintain a constant dose. See, for example, U.S. Pat. No. 3,610,795, which is incorporated herein by reference.

The material may be a solution, suspension (e.g., incorporated into microparticles, liposomes, or cells). They may be targeted to specific cell types by antibodies, receptors, or receptor ligands. The following references are examples of the use of this technique to target specific proteins to tumor tissue (Senter et al, Bioconjugate chem., 2:447-451, (1991); Bagshawe, K.D., Br.J.cancer, 60:275-281, (1989); Bagshawe et al, Br.J.cancer, 58:700-703, (1988); Senter et al, Bioconjugate chem., 4:3-9, (1993); Battelli et al, Cancer Immunol.Immunother.,35:421-425, (1992); Pietesz and McKenzie, Immunog.Revieews, 129:57-80, (1992); and Roffler et al, Biomunol.206rmacol, 42: 2062-5, (1991)). "stealth" and other antibody-conjugated liposomes (including lipid-mediated drugs against colon cancer), receptor-mediated targeting of DNA by cell-specific ligands, lymphocyte-mediated targeting of tumors, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technique to target specific proteins to tumor tissue (Hughes et al, Cancer Research,49: 6214-. In general, receptors are involved in pathways of endocytosis, whether constitutive or ligand-induced. These receptors accumulate in clathrin-coated pockets, enter the cell through clathrin-coated vesicles, pass through acidified endosomes that classify the receptors, and then circulate to the cell surface, are stored intracellularly, or are degraded in lysosomes. The internalization pathway has multiple functions, such as nutrient uptake, activated protein removal, macromolecule clearance, opportunistic entry of viruses and toxins, dissociation and degradation of ligands, and modulation of receptor levels. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, ligand type, ligand valency and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6,399-409 (1991)).

a) Pharmaceutically acceptable carriers

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

Suitable carriers and formulations thereof are described in the following documents: remington The Science and Practice of Pharmacy (19 th edition), edited by A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological saline, ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Carriers also include sustained release formulations, such as semipermeable membrane matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., membranes, liposomes or microparticles. It will be apparent to those skilled in the art that certain carriers may be preferable, for example depending on the route of administration and the concentration of the composition being administered.

Pharmaceutical carriers are known to those skilled in the art. These are generally standard carriers for administering drugs to humans and include solutions in sterile water, physiological saline, and buffers at physiological pH. These compositions may be injected intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

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

The pharmaceutical compositions may be administered in a variety of ways depending on whether local or systemic treatment is desired and the area of treatment. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal), oral, inhalation, or parenteral, e.g., intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

Formulations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including physiological saline and buffered media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements (such as ringer's dextrose based supplements), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, water, powdered or oily bases, thickeners and the like may be necessary or desirable.

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

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

b) Therapeutic uses

Effective dosages and schedules for administering the compositions can be determined empirically, and making such determinations is within the skill of the art. The dosage range of the composition administered should be sufficiently large to produce the desired effect, thereby affecting the symptoms of the disorder. The dosage should not be so large as to cause adverse side effects such as unwanted cross-reactions, allergic reactions, and the like. In general, the dosage will vary with the age, condition, sex, and extent of disease of the patient, the route or regimen of administration of the other drug included, and can be determined by one of skill in the art. The dosage may also be adjusted by the individual physician if any contraindications are present. The dosage may vary, and may be administered in one or more doses per day for one or more days. Guidelines for appropriate dosages can be found in the literature for a given class of drugs. For example, guidelines for selecting appropriate dosages of Antibodies can be found in the literature for therapeutic use of Antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al, Nos. Publications, Park Ridge, N.J. (1985) chapter 22 and page 303-357; smith et al, Antibodies in Human diagnostics and Therapy, edited by Haber et al, Raven Press, New York (1977), pp 365-. Depending on the factors mentioned above, a typical daily dosage of antibody used alone may range from about 1. mu.g/kg to 100mg/kg body weight per day or more.

C. Methods of treating cancer

In one aspect, disclosed herein is a method of treating, preventing, inhibiting, and/or attenuating cancer and/or metastasis in a subject, the method comprising administering to the subject any of the bioresponse hydrogel matrices disclosed herein. For example, disclosed herein are methods of treating, preventing, inhibiting, and/or attenuating cancer and/or metastasis (such as a cancer with low PD-L1 expression or a non-immunogenic cancer selected from the group consisting of melanoma, non-small cell lung cancer, kidney cancer, head and neck cancer, and/or bladder cancer) in a subject, the method comprising administering to the subject a bioresponse hydrogel matrix comprising a CD47/SIRPa inhibitor (such as Hu5F9-G4, CV1, B6H12, 2D3, CC-90002, and TTI-621) and an immune checkpoint blockade inhibitor (such as a PD-1/PD-L1 inhibitor and/or a CTLA-4/B7-1/2 inhibitor).

"treating (Treat, treating, or treatment)" and grammatical variations thereof as used herein includes administration of a composition for the purpose of partially or completely preventing, delaying, treating, curing, alleviating, altering, remedying, improving, ameliorating, stabilizing, slowing, and/or reducing the intensity or frequency of one or more diseases or conditions, a symptom of a disease or condition, or the root cause of a disease or condition. The treatment according to the invention can be applied preventively, palliatively or remedially. Prophylactic treatment is administered to a subject prior to onset of disease (e.g., prior to the appearance of overt signs of cancer), during early onset (e.g., after initial signs and symptoms of cancer), or after a defined progression of cancer. Prophylactic administration can occur one or more days to years before symptoms of the infection appear.

In one aspect, the immune checkpoint blockade inhibitors used in the disclosed methods of treating, preventing, inhibiting, and/or attenuating cancer and/or metastasis in a subject include any inhibitor of an immune checkpoint known in the art, such as a PD-1/PD-L1 blockade inhibitor or a CTLA-4/B7-1/2 blockade inhibitor (such as yiprizumab). Examples of PD-1/PD-L1 blocking inhibitors for use in the disclosed bioresponse hydrogel matrices may include any PD-1/PD-L1 blocking inhibitor known in the art, including but not limited to nivolumab, pembrolizumab, pidilizumab, astuzumab, avizumab, dewalizumab, and BMS-936559. Thus, in one aspect, disclosed herein is a method of treating, preventing, inhibiting, and/or attenuating cancer and/or metastasis in a subject, the method comprising administering to the subject a bioresponse hydrogel matrix comprising a CD47/sirpa inhibitor (such as Hu5F9-G4, CV1, B6H12, 2D3, CC-90002, and TTI-621) and an immune checkpoint blockade inhibitor; wherein the blocking inhibitor is a PD-1/PD-L1 blocking inhibitor such as nivolumab, pembrolizumab, pidilizumab, astuzumab, Avermelimumab, Devolumab, and BMS-936559; or a CTLA-4/B7-1/2 inhibitor, such as yiprizumab. It is to be understood and contemplated herein that the bioresponse hydrogel matrix may be designed to contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 blocking inhibitors simultaneously.

In one aspect, the disclosed methods of treating, preventing, inhibiting, and/or attenuating cancer and/or metastasis may comprise administering to a subject a pharmaceutical composition or a bioresponsive hydrogel matrix at any frequency suitable for treating a particular cancer in the subject, the method comprising administering to the subject any of the therapeutic delivery vehicles or pharmaceutical compositions disclosed herein, as well as the bioresponsive hydrogel matrix, including, but not limited to, comprising a CD47/sirpa inhibitor (such as, Hu5F9-G4, CV1, B6H12, 2D3, CC-90002, and TTI-621) and an immune checkpoint blockade inhibitor). For example, the pharmaceutical composition and/or the bioresponse hydrogel matrix may be administered to the patient at least once every 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, every 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, once every 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In one aspect, the pharmaceutical composition and/or bioresponsive hydrogel matrix is administered at least 1, 2, 3, 4, 5, 6, 7 times per week.

As disclosed herein, the bioresponse hydrogel matrix scaffold may be designed to release any CD47/sirpa inhibitor, immune checkpoint blockade inhibitor and/or additional anti-cancer agent encapsulated in the hydrogel when the hydrogel degrades in response to factors in the tumor microenvironment. Thus, disclosed herein are methods of treating, preventing, inhibiting, and/or attenuating cancer and/or metastasis in a subject, wherein a bioresponse hydrogel matrix comprises a bioresponse scaffold that releases a CD47/sirpa inhibitor, immune checkpoint blockade inhibitor, and/or other encapsulated anti-cancer agent into a tumor microenvironment upon exposure to factors within the tumor microenvironment. In one aspect, the bioresponsive hydrogel comprises a Reactive Oxygen Species (ROS) degradable hydrogel. It is understood and contemplated herein that release of the CD47/sirpa inhibitor, the immune checkpoint blockade inhibitor, and/or any other encapsulated anti-cancer agent into the tumor microenvironment by the bioresponsive hydrogel matrix is influenced by the microenvironment. In one aspect, disclosed herein are methods of treating, preventing, inhibiting, and/or attenuating cancer and/or metastasis in a subject, wherein the bioresponse hydrogel matrix releases the CD47/sirpa inhibitor, immune checkpoint blockade inhibitor, and/or any other encapsulated anti-cancer agent into the tumor microenvironment for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days.

In one aspect, the amount of the pharmaceutical composition and/or bioresponse hydrogel matrix disclosed herein administered to a subject for use in the disclosed methods can include any amount determined by a physician to be suitable for treating a particular cancer in a subject. For example, the amount of the pharmaceutical composition and/or the bioresponse hydrogel matrix may be from about 10mg/kg to about 100 mg/kg. For example, the amount of the pharmaceutical composition administered, the bioresponse hydrogel administered may be at least 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, 21mg/kg, 22mg/kg, 23mg/kg, 24mg/kg, 25mg/kg, 30mg/kg, 35mg/kg, 40mg/kg, 45mg/kg, 50mg/kg, 55mg/kg, 60mg/kg, 65mg/kg, 70mg/kg, 75mg/kg, 80mg/kg, 85mg/kg, 90mg/kg, 95mg/kg or 100 mg/kg. Thus, in one aspect, disclosed herein is a method of treating cancer in a subject, wherein the dosage of the pharmaceutical composition and/or the bioresponse hydrogel matrix administered is from about 10mg/kg to about 100 mg/kg.

As described above, it is to be understood and contemplated herein that the disclosed methods of treating, preventing, inhibiting, and/or attenuating cancer and/or metastasis in a subject may further comprise administering any anti-cancer agent (such as gemcitabine) that will further contribute to the attenuation, inhibition, treatment, and/or elimination of cancer and/or metastasis. Anticancer agents that may be used in the disclosed bioresponse hydrogels or as additional therapeutic agents in addition to the disclosed pharmaceutical compositions and/or bioresponse hydrogel matrices for the methods of attenuating, inhibiting, treating and/or eliminating cancer and/or metastasis in a subject disclosed herein may include any anticancer agent known in the art, including, but not limited to, abercillin, abiraterone acetate, Abitrexate (methotrexate), Abraxane (a paclitaxel albumin-stabilized nanoparticulate formulation), ABVD, ABVE-PC, AC-T, Adcetris (bentuximab), ADE, Ado-trastuzumab Emtansine, Adriamycin (doxorubicin hydrochloride), afatinib maleate, Afinitor (everolimus), akzeo (netupitan and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, alensena (exenatide), Alemtinib, alemtuzumab, alimata (disodium pemetrexed), Aliqopa (Copanlisib hydrochloride), ekalan (melphalan hydrochloride) for injection, eflanine tablet (melphalan), Aloxi (palonosetron hydrochloride), allnobrigide (bugatinib), ambochlorrin (chlorambucil), ambocrine, aminoacetylpropionic acid, anastrozole, aprepitant, aridia (disodium pamidronate), Arimidex (anastrozole), Aromasin (exemestane), aranon (nelarabine), arsenic trioxide, Arzerra (oclomab), agrinscriptia asparaginase, altuzumab, avastin (bevacizumab), alxitinib, azacitidine, banciconi (bancioio), abenzumab (oplulu p, bevacizumab), bevacizumab (bevacizumab), bevaciz (e, bevacizumab), bevaciz (e, bevacizumab), bevaciz (e, bevacizumab), bevaciz (e), bevacizumab), bevacizb), bevacizumab), bevaciz (e), bevaciz) for injection, bevaciz (e), bevaciz), bevacizb, bevaciz (e, bevaciz), bevaciz (e, bevacizb, bevaciz) and/e (e), etc.), d, etc.), and/e (e), etc.), and/e (e), etc.), and/or (e) for injection, etc.), and/injection, Bexarotene, Bexxar (tositumomab and iodo I131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, bornatemab, Blincyto (bornaemet), bortezomib, Bosulif (bosutinib), bosutinib, bentuximab, bocatinib, BuMel, busulfan, Busulfex (busulfan), cabazitaxel, Cabometyx (cabozantinib malate), cabozantinib malate, CAF, Campath (almitumab), Camptosar, (irinotecan hydrochloride), capecitabine, CAPOX, Carac (fluorouracil-topical), carboplatin-TAXOL, carfilzomib, Carmubris (carmustine), carmustine implant, Casodex (bicalutamide), cevimentine, cerubine (cebivatine butyrate), puriflam, cebixin (cebivatine), cebixin (cebivatib hydrochloride), cebixin (cebivatib, cebixin, cebivatib, cebivatine, gemini, and c, CHOP, cisplatin, cladribine, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Clolar (clofarabine), CMF, cobitinib, Cometriq (carboplatin malate), Copalisib hydrochloride, COPDA, COPP-ABV, Cosmegen (dactinomycin), Cotellic (cobitinib), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), Cytosine, Cytosamide liposomes, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), Darafenib, dacarbazine, Dacogen (decitabine), dactinomycin, Darzalex (Darzaleumab), Darasatinib, daunorubicin hydrochloride, Daunicidin and cytarabine hydrochloride, Cytosine, dactinomycin, Cytosine, Decitabine hydrochloride, Cytosine, Decantinocine hydrochloride, Decitabine, Cytosine, Decitabine, Cyramucirumicine, Cylitorin (D), Decitabine, Cyrinine, Decitabine, Cytosine, Doxorubiginine, Decitabine, Cytosine, Doxolone (Decitabine), Cytosine, Doxol, Cytosine, Doxol (Deltacrolidine), Cytosine, Doxol (Deltacrolin-L) liposome, Doxol (Deltacroline, Doxol (Delimitsu liposome, Doxol (D) liposome, Doxol (D-D, Doxol-D, Doxol, and D-D, C, Doxol, and D (D-D, C, D-D, D-D, C, D-D, C, D-D, Dexamethasone, dexrazoxane hydrochloride, detoximab, docetaxel, Doxil (doxorubicin HCl liposome), doxorubicin HCl liposome, Dox-SL (doxorubicin HCl liposome), DTIC-Dome (dacarbazine), Dewar mab, Efudex (fluorouracil-topical), Elitek (Labraziase), Ellene (epirubicin HCl), Eltuzumab ozolomide, Eloxitin (oxaliplatin), Eltropophaemine, Emend (aprepitant), Emplicititi (Eltuzumab), Ensidipine mesylate, Enzalutamide, epirubicin HCl, CH, Erbitux (cetuximab), eribulin, Erigel (Virgil), erlotinib HCl, Erwinazeze (Chrysanthemum asparaginase), Etol (amifostine), Etopops (etoposide phosphate), etoposide hydrochloride, etoposide (doxorubicin HCl), etoposide hydrochloride (doxorubicin HCl), Dox (doxorubicin HCl) (fluorouracil hydrochloride (Fluoromycin HCl) (Fluorosa) Everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil-topical), Fareston (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil-topical), fluorouracil injection, fluorouracil-topical, flutamide, Folex (methotrexate), Folex PFS (methotrexate), FOLFIRI, FOIRI-bevacizumab, FOIRI-cetuximab, FOIRINOX, FOOX, Folotyn (Folotrexate), FU-LV, fulvestrant, gavels (recombinant HPV vaccine), Gattva 9 (recombinant HPV), nonazotocytib (gemcitabine), Ab (Invitrogen), Invitrogen (Invitrogen), Fluorox (Invitrogen), Fluorogefitinib (Invitrogen), Fluorogefitinib), vaccine, Foltx-vaccine, Foltuzumab), and vaccine (Invitrogen (vaccine), and vaccine), Gemotabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab-ozolomide, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib maleate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wave (carmustine implant), carboxypeptidase, goserelin acetate, Halaven (eribulin mesylate), Hemangel (propranolol hydrochloride), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant HPV nine-valent vaccine, recombinant HPV, recombinant Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper-CVAD, Ibrance (palbociclib), ibritumomab Tiuxetan, ibrutinib, ILUsigi (pinanib hydrochloride), Idamycin hydrochloride, idarubicin hydrochloride, Ibrix (ifosfamide), ifosfamide hydrochloride), ifosfamide (ifosfamide mesylate), ifosfamide (ifosfamide), ifolin (ifolin hydrochloride), ifolin (ifolin), isosfamide hydrochloride), isoxapride (ifolin), isoxapride (I), gexib (I (isoxaprid), gexib (I (isoxapril), gexib (gexib), gexib (gexib), gexib (gexib) and gexib), gexib (gexib) and gexib (gexib) in (gexib) and gexib) in (gexib), gexib) in, Ifosfamide (ifosfamide), IL-2 (aldesleukin), imatinib mesylate, Imbruvica (ibrutinib), Imfinzi (Dewaruzumab), imiquimod, Imlygic (Talimogen Laherparvec), Inlyta (axitinib), Inotuzumab ozomicin, interferon alpha-2 b, recombinant interleukin-2 (aldesleukin), Intron A (recombinant interferon alpha-2 b), iodine I131 tositumomab and tositumomab, yiprimaumab, Iressa (gefitinib), irinotecan hydrochloride liposome, Istodax (romidepsin), ixabepilone, ixazofam, Ixempla (ixabepilone), Jakanib (phosphate), Paveb, Jettiva (cabazitaxel), Kadcysene (Adhate-laune), Kelvin (Kelvizumab), Kelvin hydrochloride (Kelvin), Kelvin (Kelvin hydrochloride), Rituximab (Jexib), Rituzumab (Rivasicine), Rituximab (Kelvin), Rituximab), Rituzie (Kelvi), Rituximab (Kelvin), Rituzif), Rizif (Kelvia (Kelvi), Rizizan, Rizivu (Kelvi), Rizizan, Rizivue, Rizizan, Rizivue, Rizie, Rizizan, Rizie, Rizizan, Rizie, Rizizan, Rizie, e, Rizizan, e, Pizizan, e, Rituzizane, e, Rituzizan, e, Rituzizan, E, e, E, kymriah (Tisagenleceucel), Kyprolis (Carfilzomib), lanreotide acetate, lapatinib ditosylate, Lartruvo (olanzumab), lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, calcium folinate, Leukeran (chlorambucil), leuprorelin acetate, Leustatine (cladribine), Levulan (aminoacetylpropionic acid), Linfolizin (chlorambucil), LipoDox (Adriamycin hydrochloride), lomustine, Lonsurf (trifluridine and tipyrimidine hydrochloride), Lupron (leuprorelin acetate), Lupron Depot-Ped (leuprorelin acetate), Lyaranza (olapanpanib), Maribo (vincristine sulfate), Matthiolane (melphalan), melphalan hydrochloride), Mekininomycin hydrochloride, Mekininosine hydrochloride, melphalan hydrochloride, Methazolastone (temozolomide), methotrexate LPL (methotrexate), methylnaltrexone bromide, Mexate (methotrexate), Mexate-AQ (methotrexate), midostaurin, mitomycin C, mitoxantrone hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (Prenafil), Mustagen (nitrogen mustard hydrochloride), Mutamycin (mitomycin C), Myleran (busulfan), Mylosar (azacitidine), Mylotarg (Gituzumab-Ozomicin), nanoparticulate paclitaxel (a nanoparticulate formulation stabilized with paclitaxel albumin), Navelbine (vinorelbine tartrate), tolytuzumab, nelarabine, Neosarura (cyclophosphamide), maleic acid lenatinib, Nerlynx (maleic acid), Netupidan and paleon hydrochloride, Nexatilin (Nexatilin), Nexatilin (Nexatine), Nexatilin (Nexatrin), Nexatrin, Nexatilin (Nexatrin), Nexatrin (Nexatrin), Nexatrinexanox, Nexatin (Nexase, Nexatilin, Nexatrin (Niglan), Nexaglibenoxazerumin, Nexaglibenoxatrex (R, Nigerin, Nigerinamide, and Nexazone, Nexaglibenoxazergram), Nexation (R, Nexaglibenoxaziram, Nigerin, Nigerinamide, and Nexaglibenoxaziram, Nigerinamide, Nigerin, Nigerinamide, Nigerinabeninamide, and Nexazone, Nigerin, and Nexabeninabeninabeninamide, and Nexaglibeninabeninabeninabeninazone, and so, Nilotinib, nilutamide, nilaro (isoxazomib citrate), nilapanib tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romidepsin), attuzumab, Odomzo (sonedgi), OEPA, ofatumumab, OFF, olaparib, olamab, homoharringtonine, oncocaspar (pemetrexed), ondansetron hydrochloride, Onivyde (irinotecan liposome hydrochloride), Ontak (dinleukin-diphtheria toxin linker), Opdivo (nivolumab), OPPA, osetinib, oxaliplatin, paclitaxel albumin stabilized nanoparticulate formulations, PAD, palbociclib, palivumin, palonosetron hydrochloride and netupitan, pamidronate disodium pamidronate, panini, bilanostat, parlat (parlat), PCV, carboplatin hydrochloride, PCV, carboplatin, PEB, Pemenadione, polyethylene glycol filgrastim, polyethylene glycol interferon alpha-2 b, PEG-intron (polyethylene glycol interferon alpha-2 b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), Plerixaf, pomalidomide, Pomalyst (Pomalidomide), pinatinib hydrochloride, Portraza (nixituzumab), Prazisha, prednisone, procarbazine hydrochloride, Proleukin (Adiletin), Prolia (dinolizumab), Promacta (Eltrombopamine), propranolol hydrochloride, Provenge (Sipuleucel-T), Purinitetet (mercaptopurine), Purixan (mercaptopurine), radium 223 dichloride, Raloxifen hydrochloride, Relumab, Labub enzyme, Labulase, R-CVR, Human Papilloma Virus (HPV) recombinant HPV vaccine (HPV) and HPV vaccine, Recombinant Human Papilloma Virus (HPV) nine-valent vaccine, recombinant Human Papilloma Virus (HPV) tetravalent vaccine, recombinant interferon alpha-2 b, regorafenib, Relistor (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), Ribocini, R-ICE, Rituxan (rituximab), Rituxan Hycela (rituximab and hyaluronidase human), rituximab and hyaluronidase human, Lapidem hydrochloride, Romidexine, Romidin, Rubidomycin (daunorubicin hydrochloride), Rubraca (Ricapabumanesulfonate), Ricapabum camphorsulfonate, Luxolitinib phosphate, Rydatpt (Midosteilin), Serlangue pleura intraaerosol (Talc), Stitumomab, Sipuucel-T, Marelelin Dulanoline acetate, Reynanid, sorafenib, and a, Sprycel (dasatinib), STANFORD V, sterile Talc (Talc), Steritalc (Talc), Stivarga (Regorafenib), sunitinib malate, Sutent (sunitinib malate), Sylatron (PEG Interferon alpha-2 b), Sylvant (Stituximab), Synribo (homoharringtonine), Tabloid (thioguanine), TAC, Tafinalar (Dalafinil), Tagrisso (Osteinib), Talc, Tamoligene Laherparepvec, Taramoxifene citrate, Tarabine PFS (cytarabine), Tarca (erlotinib hydrochloride), Targretin (Bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotene (docetaxel), Temerinqq (Arthrontartan), Tergtamine (Tetiazamide), Thielalopurine (Thielalopamide hydrochloride), Thielalopyr (Thielagic), Thielagic, Talmorigine (TM), Talcosixolite (Taxol), Taxolite, Tachoxie (docetaxel), Thiela), Thielagic, Tachol (TM), Tachol, Tacholtem-L (Tachoxifrag), Tachoxib, Tachol (Tachoxib), Tachoxifrag), Tachoxib, Tachoxifrag, Tachoxib (Tachoxie, Tachoxifrag, Tachoxib, Tachoxifrag, Tachoxib, Taxifrag, Taxib, Taxifrag, Taxifrage, Taxifrag, Taxifrage, Taxifrag, Torrisel (temsirolimus), tositumomab and iodine I131 tositumomab, Totect (dexrazoxane hydrochloride), TPF, trabectedin, tremetinib, trastuzumab, Trenda (bendamustine hydrochloride), trifluridine and tipyrimidine hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (dexoxib), uridine triacetate, VAC, Vandanib, VAMP, Varubibi (Laxatan hydrochloride), Vebicx (monoclonal), VelP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), Verzenio (Abelix), Vincur (leuprolide acetate), Vidazar (PFazar), Vicingula (Virosartan sulfate), Neotame (S sulfate), Neotame sulfate, Vincephramide sulfate, vincristine sulfate, vinorelbine sulfate, Liposome, VIP, VIIMIDOMODE, Vistogard (uridine triacetate), Voraxaze (carboxypeptidase), Vorinostat, Votrient (pazopanib hydrochloride), Vyxeos (daunorubicin hydrochloride and cytarabine liposome), Wellcovorin (calcium folinate), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (dinolizumab), Xofigo (radium 223 dichloride), Xtandi (enzalutamide), Yervoy (lepril monoclonal), Yondedilis (tradine), Zaltrap (Ziv-Abelicept), Zarxio (filgrastim), Zejuju (zeula (nilapamide tosylate monohydrate), Zelborafafeaf (Virofenib), Zevalin (ibrituitan), Zinecartatin (dexrazol hydrochloride), azurite-azurite (zuelandiol), Zorex (Zorex), Zornia (Zrystax), Zornia (Zrystan), Zornia (Zrystan) and Zrystan (Zrystan) to (Zrystan, Zrystan (Zrystan) to, Zrystan (Zryst) to (Zrystan) to (Zrystan, Zrystan (Zrystan, Zryst) to (Zrystan, Zrystan (Zrystan) to (Zrystan, Zryst (Zrystan, Zryst) and Zrystan (Zrystan, Zryst) and Zryst, Zrystan (Zryst) is (Zryst ) and Zrystan, Zryone (Zrystan (Zryst) is (Zryst) and (Zryst) is (Zrystan) and (Zryst) to (Zryst) is (Zryst, Zrystan, Zryone, Zryst, Z, And/or Zytiga (abiraterone acetate).

The disclosed compositions can be used to treat any disease that undergoes uncontrolled cellular proliferation (such as cancer and metastasis), including but not limited to cancers with low PD-L1 expression or non-immunogenic cancers. Representative, but not limiting, lists of cancers for which the disclosed compositions are useful for treatment are as follows: lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, cancer of the nervous system, head and neck cancer, squamous cell cancer of the head and neck, lung cancer such as small-cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cancer of the oral cavity, squamous cancer of the throat, laryngeal and squamous cancer, cervical cancer, breast cancer, as well as epithelial cancer, kidney cancer, genitourinary tract cancer, lung cancer, esophageal cancer, head and neck cancer, large intestine cancer, hematopoietic cancer, testicular cancer, colon cancer, rectal cancer, prostate cancer or pancreatic cancer.

D. Examples of the invention

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in ° c or at ambient temperature, and pressure is at or near atmospheric.

1. Example 1: active oxygen responsive protein complexes of aPD1 and aCD47 antibodies for enhanced immunotherapy

a) Formation of bioresponse antibody complexes

ROS-responsive antibody complexes via ROS-responsive cross-linkers: bis-N-hydroxysuccinimide (NHS) -modified 2,2' - [ propane-2, 2-diylbis (thio) ] diacetic acid (NHS-IE-NHS) is obtained by cross-linking aPD1 and aCD 47. Briefly, this protein complex is prepared via two steps: 1) mixing aPD1 and albumin with NHS-IE-NHS to form aPD1 (albumin) core complex; 2) add the aCD47, additional albumin and NHS-IE-NHS to allow the application of aCD47 (albumin) on pre-synthesized aPD1 (albumin) cores. The [email protected] aCD47 complex obtained showed a higher antibody binding efficiency (about 90%). The average diameter of the core complex was 96nm, and the final core-shell complex showed an increased size of 220nm (fig. 1B and 1C). Elemental mapping further validated the core-shell distribution of aPD1 (calcium chelated) and aCD47 (gadolinium chelated) in the protein complex (FIG. 1D).

To verify the ROS-responsive dissociation behavior of the complex, the protein complex was dissolved in a solution containing 500. mu. M H₂O₂Phosphate Buffered Saline (PBS). Dissociation of the complexes was observed via dynamic light scattering and TEM imaging (fig. 1E). The release profiles of aPD1 and aCD47 were quantified using an enzyme-linked immunosorbent assay (ELISA). In agreement with expectations, aPD1And aCD47 from containing H₂O₂The complexes in PBS solution released, while the amount of antibody released was minimal in pure PBS solution (fig. 1F). As expected, the aacd 47 was released first, followed by aPD1 release. This differential release behavior of aPD1 and aCD47 facilitates their corresponding roles in TME. In addition, ROS sensitive linker can effectively scavenge H in PBS solution₂O₂(FIG. 1G).

b) ROS scavenging Effect of the Complex

During tumor development, ROS levels in cancer are elevated, which is often associated with immunosuppressive TMEs, increasing the likelihood of tumor migration, invasion, and resistance. Given the ability of the complex to capture ROS, ROS levels in TME and the immune response of different immune cells were studied. As expected, ROS levels in TME were significantly reduced after intratumoral (i.t.) injection of blank complexes (formed from IgG antibodies) (fig. 2A and 2B). ROS-sensitive signals and the redox-sensitive transcription factor NF-kB are known to stimulate abnormal cancer cell proliferation and to elevate Matrix Metalloproteinase (MMP) levels, promoting the invasive and metastatic processes of tumors. Thus, the expression of NF-kB and MMP-2 in tumors was also examined after treatment with ROS sensitive blank complex. Significant down-regulation of both NF-kB and MMP-2 was observed in the tumors compared to the control group (untreated) (FIG. 2C).

To further investigate the immune effects mediated by oxidative stress after deprivation of ROS using the blank complex, different immune cell populations were examined, including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs) and regulatory T cells (tregs). Although MDSC (CD 45)⁺CD11b⁺Gr-1⁺) No significant reduction was observed, but M2 type TAM (CD 45) was observed in tumors injected with the blank complex⁺CD11b⁺F4/80⁺CD206^hl) And Treg (CD 3)⁺CD4⁺Foxp3⁺) Significant reduction in (fig. 2D, 2E and 2F). Furthermore, tumor infiltrating lymphocytes in tumors (CD 3)⁺TIL) and cytotoxic T lymphocytes (CD 3)⁺CD8⁺CTL) also increased slightly (fig. 2G and fig. 3). Taken together, the use of ROS-responsive complexes to scavenge ROS in TME inhibitsExpression of NF-kB and MMP-2 reduced immunosuppressive cells and enhanced infiltration of potent T cells (FIG. 2H).

c) Immune response induced by CD47 blockade

Innate immune cells, including Dendritic Cells (DCs) and macrophages, play an important role in the initiation of the adaptive immune system via phagocytosis and antigen presentation. However, cancer cells can often evade phagocytosis by up-regulating the expression of CD47 ("allowances me" signal). To verify that aCD47 promotes phagocytosis of cancer cells by macrophages, the interaction between cancer cells and macrophages was first studied in vitro. Bone marrow-derived macrophages (BMDM) labeled with green fluorescent signal were incubated with red fluorescently labeled B16F10 cells that had been pre-incubated with IgG or aCD47 antibody. More cancer cells pre-incubated with aCD47 were phagocytosed by BMDM compared to IgG-treated B16F10 cells (fig. 4A and 4B). To verify the ability of aCD47 to activate anti-tumor immune responses, the aCD47 complex was injected intratumorally into tumors. More phagocytic cells including macrophages and DCs infiltrated into the tumor (fig. 4C). To further evaluate the immune response after treatment with aCD47, DC stimulation was studied by flow cytometry. DC maturation was observed (CD 80)⁺CD86⁺) Significant increase and CD103⁺The percentage of DCs increased, which was critical for antigen delivery, T cell activation and expansion, and intact anti-tumor immunity (fig. 4D and 4E).

d) Complexes that extend release of immunomodulatory payloads

Given the unique core-shell structure of the antibody complex, the aCD47 on the surface of the complex can bind to cancer cells, thereby prolonging the retention time of the antibody in the tumor. To verify this, the retention of immunomodulatory antibodies was studied in vivo. aPD1 and aCD47 were labeled with cyanine 5.5(aPD1-Cy5.5) and indocyanine green (aCD47-ICG), respectively. Then, the "free aPD1 and aCD 47" or "aPD [email protected] aCD47 complex" formulations were injected to the tumor site in mice and monitored at various time points post-injection using an in vivo fluorescence imaging system. It was observed that the signal from free aPD1-cy5.5 decreased rapidly over the next three days, demonstrating that aPD1 diffused rapidly out of the tumor, while aPD1-cy5.5 encapsulated in the core of the complex still showed the observed aPD1 signal in the tumor (fig. 5A). The signal of aCD47 was comparable in both groups, confirming efficient binding between aCD47 and cancer cells, which also promoted complex retention (fig. 5B). In addition, confocal imaging of tumor sections further confirmed the prolonged retention of aPD1 in the tumor, with a stronger aPD1 red signal observed in mice injected with [email protected] aCD47 complex (fig. 5C).

e) [email protected] aCD47 Complex antitumor efficacy in vivo

The antitumor activity of the combination therapy based on the [email protected] aCD47 complex was then evaluated in vivo. C57BL6 mice with melanoma B16F10 tumor were randomized into five groups: untreated (G1), aPD1 complex (aPD1 in both core and shell, 100 μ G per mouse) (G2), aCD47 complex (aCD47 in both core and shell, 100 μ G per mouse) (G3), [email protected] aCD47 complex (aCD47 in shell, 50 μ G per mouse; aPD1 in core, 50 μ G per mouse) (G4), and free aPD1 and aCD47(aCD 47: 50 μ G per mouse, aPD 1: 50 μ G per mouse). The growth of the tumor was monitored by bioluminescence imaging for the next few days and measured by a conventional caliper (fig. 6A, 6B and 6C). The tumor growth of mice treated with the [email protected] aCD47 complex was significantly slower than in the other four groups. Compared with the group treated with aPD1 complex or aCD47 complex, [email protected] aCD47 complex treatment achieved a clear synergistic effect. Notably, tumor growth in the free antibody treated group was inhibited only on the first two days due to rapid diffusion of free aPD 1. Furthermore, the body weight of the mice in the different groups was not affected.

To study the immune response in tumors after different treatments, tumors were harvested five days after treatment and analyzed by flow cytometry and immunofluorescence imaging. Greater TIL (CD 3) than the other four treatment groups compared to the untreated group⁺Cells) infiltrated into the tumor of the mouse. Furthermore, CD4 in tumors after treatment with [email protected] aCD47 complex⁺Cells and CD8⁺The absolute number of T cells was significantly increased (fig. 6D, 6E, 6F and 6G).Immunofluorescence imaging visually indicated that there was more CD8⁺Cells were expanded into tumors after treatment with [email protected] aCD47 complex. Collectively, these observations suggest that combination therapy with the [email protected] aCD47 complex triggers an enhanced T cell-mediated anti-cancer immune response.

Next, it was investigated whether local injection of [email protected] aCD47 complex could induce a systemic immune response to inhibit cancer metastasis. B16F10 tumor cells were inoculated in the left and right flank of each mouse. The tumor in the right flank as a primary tumor was injected with [email protected] aCD47 complex, while the distant tumor at the opposite site was untreated to mimic cancer metastasis (fig. 7A). In mice injected with [email protected] aCD47 complex, the bioluminescent signal of the tumor and the size of the tumor were significantly reduced. Notably, for the mice injected with the [email protected] aCD47 complex in their primary tumors, the distant tumors of the mice were also effectively inhibited (FIGS. 7B and 1C). Consistent with these results, the weight of primary and distant tumors was also significantly reduced in mice treated with the [email protected] aCD47 complex, which is consistent with CD3⁺The percentage increase in TIL was consistent (fig. 7D, 7E, 7F). In addition, CD8⁺And CD4⁺The inter-tumor percentage of T cells was also significantly increased in both primary and distant tumors in treated mice (fig. 7G and 7H). Taken together, these results indicate that local injection of [email protected] aCD47 complex activates a systemic immune response to inhibit potential metastasis.

f) Discussion of the related Art

In this study, a protein-based complex comprising aPD1 in the core and aCD47 in the shell was designed using ROS sensitive linkers for enhancing immune checkpoint blockade. ROS produced in TME often play an important role as signaling messengers in the immune system, which are associated with tumor-related immunosuppression and T cell dysfunction. It is shown herein that synergistic therapeutic efficacy can be achieved by bioresponse protein complexes. Given the abundant ROS in TME and the unique core-shell structure, the [email protected] aCD47 complex can sequentially release aCD47 from the outer shell and aPD1 from the inner core. The released aCD47 blocks the "eat me" signal in tumor cells, promoting the recognition of cancer cells by the innate immune system and activating T cell immune responses. Further aPD1 released subsequently blocked PD-1 on the TIL, increasing the population of alloreactive T cells. The distribution of the aCD47 on the surface of the protein complex can prolong the retention time of the antibody in the tumor. Furthermore, the ROS-responsive linker not only contributes to the controlled release of the antibody, but also acts as a scavenger of ROS to reverse immunosuppressive TME. Downregulation of NF-kB and MMP-2 expression, a reduction of immunosuppressive cells including TAM and Treg, and an enhanced infiltration of potent T cells in tumors were observed. In addition, local treatment of ROS-responsive protein complexes can generate a systemic anti-tumor immune response that can not only inhibit primary tumor growth, but also prevent the possibility of cancer metastasis.

In conclusion, bioresponse protein complexes can effectively reverse immunosuppressive TME and promote immune checkpoint blockade. The unique core-shell distribution of the acds 47 and aPD1 in the complex prolongs the retention time of the antibody and enables the sequential release of the antibody in the tumor site. However, parameters associated with the protein complex require further optimization, such as optimizing ROS response profiles and percentages of aCD47 and aPD 1.

g) Materials and methods

(1) [email protected] aCD47 Complex.

To synthesize the ROS-responsive crosslinker, 2' - [ propane-2, 2-diylbis (thio) ] diacetic acid (5.0mg, 1 equivalent), EDC (6.9mg, 2 equivalents), and NHS (5.1mg, 2 equivalents) were mixed in dimethyl sulfoxide (DMSO) and stirred at room temperature for 6 hours. To obtain aPD1 core complex, albumin from mouse serum (20 equiv) and aPD1(1 equiv) were mixed in Phosphate Buffered Saline (PBS) and then slowly added to ROS-responsive cross-linker in DMSO (200 equiv). The mixture was stirred at 4 ℃ overnight. After centrifugation at 20000rpm to remove free albumin or antibodies, the aPD1 core complex obtained was purified. Thereafter, additional albumin (20 equivalents), aCD47(1 equivalent), and ROS-responsive crosslinker (200 equivalents) were added to aPD1 complex solution and stirred overnight at 4 ℃. After centrifugation at 20000rpm to remove free albumin or antibody, the obtained [email protected] aCD47 complex was purified. Control blank complexes were prepared using IgG from rat, instead of the opposing antibody, following the same procedure.

(2) And (5) characterizing.

The size distribution and morphology of the aPD1 complex and [email protected] aCD47 complex were measured by Dynamic Laser Scattering (DLS) and TEM (JEOL 2000FX), respectively. The distribution of the antibodies in the complexes was characterized using analytical TEM (Titan) (for gadolinium and calcium sequestration by aCD47 and aPD1, respectively.) the amount of different antibodies conjugated in the complexes (IgG from rat serum represents aCD47 and IgG from rabbit serum represents aPD1) was measured by ELISA (rat IgG Total ELISA kit, eBioscience, cat. No. 88-50490-22; rabbit IgG Total ELISA kit, Thermo Fisher, cat. No. 15137).

(3) In vitro phagocytosis assay.

Bone marrow-derived macrophages (BMDM) isolated from C57BL/6 mice were stained with CellTracker Green (C7025, Thermo-Fisher Scientific) and B16F10 cells were stained with CellTracker deep (C34565, Thermo-Fisher Scientific, USA). Cancer cells were blocked with IgG or aCD47 and then co-cultured with macrophages in serum-free medium. After 2h incubation at 37 ℃, confocal microscopy (Zeiss LSM 710) and CytoFLEX flow cytometry (Beckman) were used to study the phagocytosis of cancer cells by macrophages.

(4) In vivo tumor models and treatments.

To measure the combined therapeutic effect of [email protected] aCD47 complex, fLuc-B16F10 melanoma cells (1x 10)⁶) Injected subcutaneously (s.c.) into the right flank of each C57BL/6 mouse. Seven days later, mice were randomly divided into five groups (n ═ 6). Mice were injected intratumorally (i.t.) with different formulations including aCD47 complex (aCD 47: 100. mu.g per mouse), aPD1 complex (aPD 1: 100. mu.g per mouse), [email protected] aCD47 complex (aCD 47: 50. mu.g per mouse, aPD 1: 50. mu.g per mouse), free aPD1 and free aCD47(aCD 47: 50. mu.g per mouse, aPD 1: 50. mu.g per mouse). For metastatic tumor models, a total of 1x10 was used⁶Two flank of each C57BL/6 mouse were injected subcutaneously with individual fLuc-B16F10 melanoma cells. One week later, [email protected] aCD47 complex (aCD 47: 50. mu.g per mouse, aPD 1:50 μ g per mouse) was intratumorally injected into the tumor in the right flank of each mouse. The volume of the tumor was measured and calculated according to the following formula: width of²x length x 0.5. Tumor growth was also observed by in vivo imaging instrument (IVIS) Spectrum system (Perkin Elmer Ltd). Mixing d-fluorescein (Thermo Scientific)^TM Pierce^TM) Ten minutes after (0.15mg/g) intraperitoneal injection into each mouse, the mice were imaged for an exposure time of 5 minutes. When showing signs of imperfect health or tumour size exceeding 1.5cm³When needed, animals were euthanized.

(5) Materials, cell lines, and animals.

Comprising 2,2' - [ propane-2, 2-diyl bis (thio)]All chemicals including diacetic acid, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. Albumin from mouse serum, immunoglobulin g (IgG) from rat serum, and IgG from rabbit serum were purchased from Sigma-Aldrich. anti-CD 47 antibody (aCD47) (Cat #127518, Clone: miap301) and anti-PD 1 antibody (aPD1) (Cat #135233, Clone: 29F.1A12) were purchased from Biolegend Inc. Mouse B16F10 melanoma cell line was purchased from UNC tissue culture facilities. B16F10-luc cells were a gift from doctor Leaf Huang, university of North Carolina, Church. At 37 ℃ and 5% CO₂Next, the cells were cultured in Dulbecco's modified eagle's medium (Gibco, Invitrogen) containing 100U/mL of penicillin (Invitrogen) and 10% fetal bovine serum (Invitrogen, Carlsbad, Calif.). Female C57BL/6 mice (6-10 weeks old) were purchased from Jackson laboratories. All mouse studies were performed according to protocols approved by the institutional animal care and use committee at cathedral mountain division and north carolina state university, university of north carolina.

(6) Flow cytometry.

To study the different immune cells in tumors, differently treated tumors collected from mice were cut into small pieces and homogenized in cold staining buffer to form single cells. Cells were stained with the fluorescent-labeled antibodies CD45(Biolegend, catalog No. 103108, Clone:30-F11), CD11b (Biolegend, catalog No. 101212, Clone: Ml/70), F4/80(Biolegend, catalog No. 123128, Clone: BM8), CD80(Biolegend, catalog No. 104708, Clone:16-10A1), CD206(Biolegend, catalog No. 141706, Clone: C068C2), Gr-1(Biolegend, catalog No. 108408, Clone: RB6-8C5), CD 5(Biolegend, catalog No. 100236, Clone:17A 5), CD 5(Biolegend, catalog No. 5, Clone:53-6.7), CD 72 (Biolegend, catalog No. 36406, catalog No. 100K 1.5), CD 5(Biolegend, catalog No. 5, Clone:5, Clone No. 5, Clone:5, catalog No. 5, Biollene catalog No. 5, Clone: 5(Biolegend 5, catalog No. 5, Biollene, catalog No. 5, Clone:5, Biollene, catalog No. 5, Biollene No. 5, see 5, Biollene: 5, see the instructions for Biollene: Biollene < 11, see < BIG < 11, see < BILEX > -5, see < BILEX < BIL > -5, see < BILEX < BIL > (BIL >, < BIL > (BIL >) and BioL > (BIL >) for the specification. All antibodies used here were diluted 200-fold. Stained cells were measured on a CytoFLEX flow cytometer (Beckman) and analyzed by the FlowJo software package (10.0.7 edition; TreeStar, USA, 2014).

(7) And (4) performing immunofluorescence staining.

Tumors were harvested from mice in different groups and frozen in Optimal Cutting Temperature (OCT) medium. Tumors were dissected via cryomicrotome, mounted on slides, and stained with CD8(Abeam, cat ab22378) primary antibody overnight at 4 ℃. Subsequently, a fluorescently labeled goat anti-rat IgG (H + L; Thermo Fisher Scientific, Cat. No. A18866) secondary antibody was added. Confocal microscopy (Zeiss LSM 710) was used to record slides. All these antibodies used in the experiment were diluted 200-fold.

(8) Western blotting.

Each sample with equal amounts of protein as determined by the bicinchoninic acid protein assay kit (BCA) was mixed with an equal volume of 2 × Laemmli buffer and boiled at 95 ℃ for 5 min. After completion of gel electrophoresis and protein conversion, anti-NF-kB p65 antibody (Abeam, cat ab237591), anti-MMP 2 antibody (Abeam, cat ab92536) and anti- β -actin antibody (Abeam, cat ab8226) were used as primary antibodies according to the manufacturer's instructions. Secondary antibodies including goat anti-mouse antibody (Novus Biologicals, cat # NBP1-75151) and goat anti-rabbit antibody (Novus Biologicals, cat # NBP2-30348H) were used for these blots.

(9) And (5) carrying out statistical analysis.

As shown, all results are expressed as mean ± standard error of mean (s.e.m.). Graph-based post hoc tests and one-way analysis of variance (ANOVA) were used for multiple comparisons, and two-tailed student t-test was used for both sets of comparisons. Survival benefit was determined by log rank test. All statistical analyses were performed by the Prism Software package (PRISM 5.0; GraphPad Software, USA, 2007). The threshold for statistical significance was P < 0.05.

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