Methods and compositions for treating cancer using targeted PORE-forming agents

Abstract

Aspects of the disclosure relate to compositions comprising an antigen targeting agent, such as an anti-CD47 targeting agent, conjugated to a pore-forming agent, such as Listeriolysin O (LLO). Further aspects of the disclosure relate to a composition comprising a LLO molecule conjugated to an antigen targeting agent, through a cleavable linker. Further aspects of the disclosure relate to use of said compositions in the manufacture of a medicament and / or methods treating a disease, such as cancer, in a patient comprising, administering an antibody-drug conjugate comprising an antigen targeting agent linked to a pore-forming agent, to the patient.

Description

METHODS AND COMPOSITIONS FOR TREATING CANCER USING TARGETEDPORE-FORMING AGENTSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application Serial No. 63 / 557,335 filed February 23, 2024, and U.S. Provisional Patent Application Serial No. 63 / 736,334 filed December 19, 2024, the contents of each of which are hereby incorporated by reference in their entirety.BACKGROUNDI. Technical Field

[0002] This disclosure relates at least to the fields of molecular biology and medicine.II. Background

[0003] Continual progress towards the development of more powerful antibody-drug conjugates (ADCs) (used herein interchangeably with antibody-toxin conjugates, ATCs) has driven innovation in payload design ranging from cytotoxins to non-chemotherapeutics to immune-stimulating agents1'4. Targeting the cytosolic DNA sensor cGMP-AMP synthase (cGAS) and its downstream effector, the stimulator of interferon genes (STING), within antigen-presenting cells (APCs) is one therapeutic strategy for generating tumour- specific T- cell immunity5'7. Several STING agonists are currently in clinical development with varying degrees of success8'11. Nevertheless, at present, no ADCs in advanced development or approved for clinical use at this time specifically target cytosolic immune sensors or are designed to enhance APC functionalities.

[0004] Antibodies against the myeloid checkpoint CD47 may augment STING signaling in APCs via the escape of engulfed tumour double-stranded DNA from phagolysosomes12. Nevertheless, the described phagolysosomal escape is a highly inefficient, random process, and no specialized endogenous transporters have been identified that can perform such tasks13. Similarly, cross presentation of neoantigens also requires the release of cancer peptides from phagolysosomes into the cytosol, where they can be further processed by proteasomes and loaded onto MHC molecules in the endoplasmic reticulum. Therefore, driving phagolysosomal escape of tumour cell components, whether they are nucleic acids or peptides, may stimulate a more robust and effective antitumour immune response.

[0005] Listeria (L.) monocytogenes survive phagolysosomal destruction to gain access to the cytosol by producing Listeriolysin O (LLO), a non-enzymatic cytolysin activated byreducing agents and inhibited by oxidizing agents with maximum activity at pH 5.514'16. In acidic phagosomes (pH ~5.9), LLO monomers oligomerize into arcs that fuse to form beta barrel channels in phagolysosome membranes17. Upon entry into the cytosol, LLO monomers are deprotonated and inactivated, thereby preserving cellular integrity. Liberated L. monocytogenes can then form a replicative niche in the host cell. Phagosomal release of bacterial nucleic acids and proteins drives Toll-like receptor signalling, cGAS-STING activation, and antigen presentation, which can collectively facilitate T-cell mobilization for pathogen clearance and long-term immunity to reinfection18'20.SUMMARY

[0006] Provided herein are technologies including compositions, methods, and kits for improved treatment of disease states, such as cancer, through promotion of endogenous immune cell targeting to- and / or engulfation-of a target, including but not limited to cancerous cells, and subsequent release of the target or components / constituent parts thereof (e.g., nucleic acids or peptides etc.) from an acidic organelle (e.g., early-late phagosome and / or phagolysosome, etc.) of the immune cell into the cytoplasm of said cell. In some embodiments, such a release of antigens promotes antigen presentation and / or cGAS-STING pathway activation for tumour immunity. In some embodiments, compositions and methods provided herein increase the relative effectiveness of immunotherapies, such as CD47 targeted immunotherapies. In some embodiments, compositions and methods provided herein increase the relative effectiveness of co-therapies, such as but not limited to, immune-check point blockade therapies.

[0007] In some embodiments, provided herein are compositions comprising, a CD47 targeting agent conjugated to a pore-forming agent. In some embodiments, a CD47 targeting agent comprises an antibody or a functional fragment thereof. In some embodiments, an antibody or functional fragment thereof comprises Magrolimab, B6H12, BRIC126, SRF231, MIAP410, AO- 176, CC-90002, GenSci-059, Lemzoparlimab, Letaplimab, Ligufalimab, ZL- 1201, IBI-322, TG-1801, IMC-002, TQB2928, PF-07257876, HX009, and / or STI-6643

[0008] . In some embodiments, an antibody or functional fragment thereof is humanized.In some embodiments, an antigen targeting agent is capable of binding to an antigen expressed on a cancer cell. In some embodiments, an antigen targeting agent neutralizes the target antigen. In some embodiments, a targeted cancer cell comprises a breast cancer, lung cancer, non-small cell lung cancer, brain cancer, glioblastoma, triple-negative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma,colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma cell. In some embodiments, a cancer cell overexpresses CD47.

[0009] In some embodiments, a pore forming agent comprises, consists essentially of, or consists of a bacterial peptide and / or bacterial toxin. In some embodiments, a pore forming agent comprises melittin, proteins from the membrane attack complex / perforin (MACPF) family, cholesterol dependent cytolysin (CDC) family, and / or the Pleurotolysin B pore-forming family, e.g., but not limited to, perfringolysin O (PFO), alveolysin, streptolysin O (SLO), pneumolysin, ivanolysin, hemolysin, pyolysin, flavomodulin, tetanolysin O, MAC / perforin domain containing protein, complement component C8-alpha, complement component C6, complement component C9, complement component C7, complement component C8-beta, perforin-like protein 1, apextrin, hemopexin, photopexin a / b, SpoCl-CIC, toxin AvTX-60A, toxin PsTX-60B, MACPF domain-containing protein CADI, MACPF domain-containing protein NSL1, Macrophage expressed protein, Pleurotolysin, Erylysin, Ostreolysin, Cytolysin, Aegerolysin, intermedilysin (ILY), siulysin, aerolysin, ClyA, colicin, lysenin, CrylAa, Fragaceatoxin C, perforin, Tc holotoxin, a-haemolysin, SmhB, and / or Listeriolysin O (LLO). In some embodiments, a pore forming agent is only active in an acidic environment (e.g., a phagolysosome) with a pH less than or equal to about 6.5. In some embodiments, a pore forming agent is only active in an acidic environment (e.g., a phagolysosome) with a pH less than or equal to about 6.0 or 5.9, and / or is optimally active at a pH of equal to or about 5.5. In some embodiments, a pore forming agent is active in a phagosome. In some embodiments, a pore forming agent is not active in the cytoplasm of a cell. In some embodiments, a pore forming agent is not active at the cell membrane. In some embodiments, a pore forming agent comprises, consists essentially of, or consists of LLO.

[0010] In some embodiments, a CD47 antigen targeting agent and a pore-forming agent are reversibly or irreversibly conjugated together. In some embodiments, a CD47 antigen targeting agent and a pore-forming agent are conjugated through a cleavable linker. In some embodiments, a pore forming agent is active only once cleaved from the antigen targeting agent. In some embodiments, a CD47 antigen targeting agent and the pore-forming agent are conjugated through click chemistry. In some embodiments, click chemistry is copper-free click chemistry. In some embodiments, click chemistry comprises a Polyethylene glycol (PEG),dibenzocyclooctyne (DBCO), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and / or a triazole linker. In some embodiments, a composition comprises at least 90% product showing a molar ration of 1 pore-forming agent molecule per CD47 antigen targeting agent molecule. In some embodiments, a pore-forming agent is conjugated to a primary amine on a CD47 antigen targeting agent. In some embodiments, a pore-forming agent is cleavable from the CD47 antigen targeting agent when exposed to reduced conditions.

[0011] Also provided herein, in certain embodiments, are compositions comprising compositions disclosed herein, such as CD47 antigen targeting agent conjugated to a poreforming agent. In some embodiments, a composition comprises a pharmaceutically acceptable excipient. Also provided herein, in certain embodiments, are methods of making compositions disclosed herein, such methods comprising conjugating an antigen targeting agent (e.g., a CD47 antigen targeting agent) to a pore-forming agent. Also provided herein, in certain embodiments, are methods of treating a disease or disorder in a subject, the methods comprising administering to the subject one or more of the compositions disclosed herein.

[0012] Also provided herein, in certain embodiments, are compositions comprising, a CD47 antigen targeting agent conjugated to Listeriolysin O (LLO). In certain embodiments, a CD47 antigen targeting agent comprises Magrolimab, B6H12, BRIC126, SRF231, MIAP410, AO-176, CC-90002, GenSci-059, Lemzoparlimab, Letaplimab, Ligufalimab, ZL-1201, IBI- 322, TG-1801, IMC-002, TQB2928, PF-07257876, HX009, and / or STI-6643. In certain embodiments, a CD47 antigen targeting agent is humanized. In certain embodiments, a CD47 antigen targeting agent is capable of binding to a CD47 antigen expressed on a cancer cell. In certain embodiments, a CD47 antigen targeting agent neutralizes the CD47 target antigen. In certain embodiments, the composition targets a cancer cell, wherein the cancer cell comprises a breast cancer, lung cancer, non-small cell lung cancer, brain cancer, glioblastoma, triplenegative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma cell. In certain embodiments, LLO permeabilizes phagolysosomes in an acidic environment with a pH less than or equal to about 6.5. In certain embodiments, LLO permeabilizes phagolysosomes in an acidic environment with a pH less than or equal to about 6.0 or 5.9, and / or is optimally active at a pH of equal to or about 5.5. Incertain embodiments, LLO is active in a phagosome. In certain embodiments, LLO is not active in the cytoplasm of a cell. In certain embodiments, LLO is not active at the cell membrane.

[0013] In certain embodiments, a CD47 antigen targeting agent and the LLO are conjugated together through a reversable or irreversible linker. In certain embodiments, a CD47 antigen targeting agent and the LLO are conjugated through a cleavable linker. In certain embodiments, a LLO is active only once cleaved from the CD47 antigen targeting agent. In certain embodiments, a CD47 antigen targeting agent and the LLO are conjugated through click chemistry. In certain embodiments, click chemistry is copper-free click chemistry. In certain embodiments, click chemistry comprises a Polyethylene glycol (PEG), dibenzocyclooctyne (DBCO), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and / or a triazole linker. In certain embodiments, a composition comprises at least 90% product showing a molar ration of 1 LLO molecule per CD47 antigen targeting agent molecule. In certain embodiments, LLO is conjugated to a primary amine on the CD47 antigen targeting agent. In certain embodiments, LLO is cleavable from the CD47 antigen targeting agent when exposed to reduced conditions.

[0014] In certain embodiments, also provided herein are compositions comprising a CD47 antigen targeting agent conjugated to the LLO as described herein. In some embodiments, a composition also comprises a pharmaceutically acceptable excipient. Also provided herein, in certain embodiments, are methods of making compositions, the method comprising conjugating a CD47 antigen targeting agent to LLO. Also provided herein, in certain embodiments, are methods of treating a disease or disorder in a subject, the methods comprising administering one or more of the compositions disclosed herein to the subject in need thereof.

[0015] In certain embodiments, provided herein are compositions comprising a Listeriolysin O (LLO) molecule conjugated to an antigen targeting agent, through a cleavable linker. In certain embodiments, an antigen targeting agent comprises an antibody or functional fragment thereof. In certain embodiments, an antibody or functional fragment thereof targets a CD47, CD24, PDL1, CD19, CD20, CD30, GPRC5D, EGFR, EGFR2, BCMA, c-MET, and / or HER2 antigen. In certain embodiments, an antibody or fragment thereof is humanized. In certain embodiments, an antigen targeting agent is capable of binding to an antigen expressed on a cancer cell. In certain embodiments, an antigen targeting agent neutralizes the target antigen. In certain embodiments, an antigen targeting agent targets a cancer cell, and the cancer cell comprises a breast cancer, lung cancer, non-small cell lung cancer, brain cancer, glioblastoma, triple-negative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectaladenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma cell. In certain embodiments, LLO is only active in an acidic environment with a pH less than or equal to about 6.5. In certain embodiments, an antigen targeting agent and the LLO are conjugated through a cleavable linker that is cleaved upon exposure to reduced conditions. In certain embodiments, an LLO is active only once cleaved from the antigen targeting agent. In certain embodiments, LLO molecule and the antigen targeting agent are conjugated through click chemistry. In certain embodiments, click chemistry is copper-free click chemistry. In certain embodiments, LLO is conjugated to a primary amine on the antigen targeting agent.

[0016] Also provided herein, in certain embodiments, are compositions comprising a LLO molecule conjugated to an antigen targeting agent through a cleavable linker. In some embodiments, a composition comprises a pharmaceutically acceptable excipient. Also provided herein, in some embodiments, are methods of making compositions disclosed herein, the methods comprising conjugating an antigen targeting agent to LLO through use of a cleavable linker.

[0017] In some embodiments, provided herein are methods of treating a disease or disorder in a subject in need thereof, the methods comprising administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, and optionally a therapeutically effective amount of an immune checkpoint inhibitor. In some embodiments, a pore-forming agent is LLO. In some embodiments, na antigen targeting agent targets CD47. In some embodiments, a patient has, has been diagnosed with, has one or more symptoms of, or is suspected of having a cancer. In some embodiments, a cancer comprises breast cancer, lung cancer, non- small cell lung cancer, brain cancer, glioblastoma, triplenegative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma. In some embodiments, a method of treatment comprises administering to a subject a therapeutically effective amount of an antigen targeting agentconjugated to a pore-forming agent, and a therapeutically effective amount of an immune checkpoint inhibitor comprises intratumoral, intraperitoneal, and or / intravenous delivery. In some embodiments, an immune checkpoint inhibitor comprises PD-1 inhibitors (e.g., nivolumab, pembrolizumab, MEDI0680, and / or pidilizumab), PDL2-inhibitors (e.g., AMP- 224, rHIgM12B7), PDL1 inhibitors (e.g., Durvalumab, atezolizumab, avelumab), CTLA-4 inhibitors, B7-1 inhibitors, and / or B7-2 inhibitors. In some embodiments, an immune checkpoint inhibitor comprises a PD1 -antigen targeting agent. In some embodiments, a PD1- antigen targeting agent comprises nivolumab, pembrolizumab, MEDI0680, and / or pidilizumab. In some embodiments, methods provided herein comprise treatment with one or more additional therapies or therapeutic agents, that are co-administered before, after, or during the administration of a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, and a therapeutically effective amount of an immune checkpoint inhibitor. In some embodiments, one or more additional therapies comprise tumor debulking, surgery, chemotherapy, radiotherapy, immunotherapy, and / or other agents.

[0018] In some embodiments, provided herein are methods of treating a disease or disorder in a subject in need thereof, the methods comprising administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent. In some embodiments, a pore-forming agent is LLO. In some embodiments, an antigen targeting agent targets CD47. In some embodiments, a patient has, has been diagnosed with, has one or more symptoms of, or is suspected of having a cancer. In some embodiments, a cancer comprises breast cancer, lung cancer, non- small cell lung cancer, brain cancer, glioblastoma, triplenegative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma. In some embodiments, one or more additional therapies or therapeutic agents are co-administered before, after, or during the administration of a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent. In some embodiments, one or more additional therapies comprise tumor debulking, surgery, chemotherapy, radiotherapy, immunotherapy, and / or other agents.

[0019] Also provided herein, in some embodiments, are methods of treating a disease or disorder in a subject in need thereof, the methods comprising administering to a subject atherapeutically effective amount of a composition according to the disclosure. In some embodiments, a patient has, has been diagnosed with, has one or more symptoms of, or is suspected of having a cancer. In some embodiments, a cancer comprises breast cancer, lung cancer, non-small cell lung cancer, brain cancer, glioblastoma, triple-negative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma. In some embodiments, administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, comprises intratumoral, intraperitoneal, and or / intravenous delivery. In some embodiments, one or more additional therapies or therapeutic agents are co-administered before, after, or during the administration of a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent. In some embodiments, one or more additional therapies comprise tumor debulking, surgery, chemotherapy, radiotherapy, immunotherapy, and / or other agents.

[0020] It is specifically contemplated that any limitation discussed with respect to one embodiment of the inventions disclosed herein may apply to any other embodiment of the inventions disclosed. Furthermore, any composition of the disclosure may be used in any method of the disclosure, and any method of the disclosure may be used to produce or to utilize any composition of the disclosure. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Detailed Description, Drawings, Claims, and Description of the Drawings.

[0021] Certain embodiments of the present disclosure are characterized through the following enumerated aspects.

[0022] Aspect 1 is a composition comprising a CD47 targeting agent conjugated to a poreforming agent.

[0023] Aspect 2 is the composition of aspect 1, wherein the CD47 targeting agent comprises an antibody or a functional fragment thereof.

[0024] Aspect 3 is the composition of aspect 1 or 2, wherein the antibody or functional fragment thereof comprises Magrolimab, B6H12, BRIC126, SRF231, MIAP410, AO-176, CC-90002, GenSci-059, Lemzoparlimab, Letaplimab, Ligufalimab, ZL-1201, IBI-322, TG-1801, IMC-002, TQB2928, PF-07257876, HX009, and / or STI-6643.

[0025] Aspect 4 is the composition of any one of aspects 2 to 3, wherein the antibody or functional fragment thereof is humanized, optionally wherein the antibody or functional fragment thereof is an IgGl, IgG2, IgG2B, or IgG4 isotype.

[0026] Aspect 5 is the composition of any one of aspects 1 to 4, wherein the antigen targeting agent is capable of binding to an antigen expressed on a cancer cell.

[0027] Aspect 6 is the composition of any one of aspects 1 to 5, wherein the antigen targeting agent neutralizes the target antigen.

[0028] Aspect 7 is the composition of aspect 5 or 6, wherein the cancer cell comprises a breast cancer, lung cancer, non- small cell lung cancer, brain cancer, glioblastoma, triplenegative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma cell.

[0029] Aspect 8 is the composition of aspect 7, wherein the cancer overexpresses CD47.

[0030] Aspect 9 is the composition of any one of aspects 1 to 8, wherein the pore forming agent comprises Listeriolysin O (LLO), melittin, proteins from the membrane attack complex / perforin (MACPF) family, cholesterol dependent cytolysin (CDC) family, and / or the Pleurotolysin B pore-forming family, e.g., but not limited to, perfringolysin O (PFO), alveolysin, streptolysin O (SLO), pneumolysin, ivanolysin, hemolysin, pyolysin, flavomodulin, tetanolysin O, MAC / perforin domain containing protein, complement component C8-alpha, complement component C6, complement component C9, complement component C7, complement component C8-beta, perforin-like protein 1, apextrin, hemopexin, photopexin a / b, SpoCl-CIC, toxin AvTX-60A, toxin PsTX-60B, MACPF domain-containing protein CADI, MACPF domain-containing protein NSL1, Macrophage expressed protein, Pleurotolysin, Erylysin, Ostreolysin, Cytolysin, Aegerolysin, intermedilysin (ILY), siulysin, aerolysin, ClyA, colicin, lysenin, CrylAa, Fragaceatoxin C, perforin, Tc holotoxin, a- haemolysin, and / or SmhB.

[0031] Aspect 10 is the composition of any one of aspects 1 to 9, wherein the pore forming agent comprises a bacterial peptide and / or bacterial toxin.

[0032] Aspect 11 is the composition of any one of aspects 1 to 10, wherein the pore forming agent is only active in an acidic environment with a pH of, of less than, or of about 6.5.

[0033] Aspect 12 is the composition of any one of aspects 1 to 11, wherein the pore forming agent is only active in an acidic environment with a pH of, of less than, or of about 6.0 or 5.9, and / or is optimally active at a pH of or of about 5.5.

[0034] Aspect 13 is the composition of any one of aspects 1 to 12, wherein the pore forming agent is active in a phagosome.

[0035] Aspect 14 is the composition of any one of aspects 1 to 13, wherein the pore forming agent is not active in the cytoplasm of a cell.

[0036] Aspect 15 is the composition of any one of aspects 1 to 14, wherein the pore forming agent is not active at the cell membrane.

[0037] Aspect 16 is the composition of any one of aspects 1 to 15, wherein the pore forming agent comprises, consists essentially of, or consists of LLO.

[0038] Aspect 17 is the composition of any one of aspects 1 to 16, wherein the CD47 antigen targeting agent and the pore-forming agent are reversibly or irreversibly conjugated together.

[0039] Aspect 18 is the composition of any one of aspects 1 to 17, wherein the CD47 antigen targeting agent and the pore-forming agent are conjugated through a cleavable linker.

[0040] Aspect 19 is the composition of any one of aspects 1 to 18, wherein the pore forming agent is active only once cleaved from the antigen targeting agent.

[0041] Aspect 20 is the composition of any one of aspects 1 to 19, wherein the CD47 antigen targeting agent and the pore-forming agent are conjugated through click chemistry.

[0042] Aspect 21 is the composition of claim 19, wherein the click chemistry is copper- free click chemistry.

[0043] Aspect 22 is the composition of claim 19 or 21, wherein the click chemistry comprises a Polyethylene glycol (PEG), dibenzocyclooctyne (DBCO), succinimidyl 3-(2- pyndyldi th io) ro ionate (SPDP), and / or a triazole linker.

[0044] Aspect 23 is the composition of any one of aspects 1 to 22, wherein the composition comprises at least 90% product showing a molar ration of 1 pore-forming agent molecule per CD47 antigen targeting agent molecule.

[0045] Aspect 24 is the composition of any one of aspects 1 to 22, wherein the poreforming agent is conjugated to a primary amine on the CD47 antigen targeting agent.

[0046] Aspect 25 is the composition of any one of aspects 1 to 24, wherein the poreforming agent is cleavable from the CD47 antigen targeting agent when exposed to reduced conditions.

[0047] Aspect 26 is the composition comprising, the CD47 antigen targeting agent conjugated to a pore-forming agent of any one of aspects 1 to 25.

[0048] Aspect 27 is the composition of claim 26, also comprising a pharmaceutically acceptable excipient.

[0049] Aspect 28 is a method of making the composition of any one of aspects 1 to 27, the method comprising conjugating a CD47 antigen targeting agent to a pore-forming agent.

[0050] Aspect 29 is a method of treating a disease or disorder in a subject, the method comprising administering the composition of any one of aspects 1 to 27 to the subject.

[0051] Aspect 30 is a composition comprising, a CD47 antigen targeting agent conjugated to Listeriolysin O (LLO).

[0052] Aspect 31 is the composition of claim 30, wherein the CD47 antigen targeting agent comprises Magrolimab, B6H12, BRIC126, SRF231, MIAP410, AO-176, CC-90002, GenSci- 059, Lemzoparlimab, Letaplimab, Ligufalimab, ZL-1201, IBI-322, TG-1801, IMC-002, TQB2928, PF-07257876, HX009, and / or STI-6643.

[0053] Aspect 32 is the composition of any one of aspects 30 to 31, wherein the CD47 antigen targeting agent is humanized.

[0054] Aspect 33 is the composition of any one of aspects 30 to 32, wherein the CD47 antigen targeting agent is capable of binding to a CD47 antigen expressed on a cancer cell.

[0055] Aspect 34 is the composition of any one of aspects 30 to 33, wherein the CD47 antigen targeting agent neutralizes the CD47 target antigen.

[0056] Aspect 35 is the composition of claim 33 or 34, wherein the cancer cell comprises a breast cancer, lung cancer, non-small cell lung cancer, brain cancer, glioblastoma, triplenegative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma cell.

[0057] Aspect 36 is the composition of any one of aspects 30 to 35, wherein the LLO permeabilizes phagolysosomes in an acidic environment with a pH of, of less than, or of about 6.5.

[0058] Aspect 37 is the composition of any one of aspects 30 to 36, wherein the LLO permeabilizes phagolysosomes in an acidic environment with a pH of, of less than, or of about 6.0 or 5.9, and / or is optimally active at a pH of or of about 5.5.

[0059] Aspect 38 is the composition of any one of aspects 30 to 37, wherein the LLO is active in a phagosome.

[0060] Aspect 39 is the composition of any one of aspects 30 to 38, wherein the LLO is not active in the cytoplasm of a cell.

[0061] Aspect 40 is the composition of any one of aspects 30 to 39, wherein the LLO is not active at the cell membrane.

[0062] Aspect 41 is the composition of any one of aspects 30 to 40, wherein the CD47 antigen targeting agent and the LLO are conjugated together through a reversible or an irreversible linker.

[0063] Aspect 42 is the composition of any one of aspects 30 to 41, wherein the CD47 antigen targeting agent and the LLO are conjugated through a cleavable linker.

[0064] Aspect 43 is the composition of any one of aspects 30 to 42, wherein the LLO is active only once cleaved from the CD47 antigen targeting agent.

[0065] Aspect 44 is the composition of any one of aspects 30 to 43, wherein the CD47 antigen targeting agent and the LLO are conjugated through click chemistry.

[0066] Aspect 45 is the composition of claim 44, wherein the click chemistry is copper- free click chemistry.

[0067] Aspect 46 is the composition of claim 44 or 45, wherein the click chemistry comprises a Polyethylene glycol (PEG), dibenzocyclooctyne (DBCO), succinimidyl 3-(2- pyndyldi th io) ro ionate (SPDP), and / or a triazole linker.

[0068] Aspect 47 is the composition of any one of aspects 30 to 46, wherein the composition comprises at least 90% product showing a molar ration of 1 LLO molecule per CD47 antigen targeting agent molecule.

[0069] Aspect 48 is the composition of any one of aspects 30 to 47, wherein the LLO is conjugated to a primary amine on the CD47 antigen targeting agent.

[0070] Aspect 49 is the composition of any one of aspects 30 to 48, wherein the LLO is cleavable from the CD47 antigen targeting agent when exposed to reduced conditions.

[0071]

[0072] Aspect 50 is a composition comprising, the CD47 antigen targeting agent conjugated to the LLO of any one of aspects 30 to 49.

[0073] Aspect 51 is the composition of aspect 50, also comprising a pharmaceutically acceptable excipient.

[0074] Aspect 52 is a method of making the composition of any one of aspects 30 to 51, the method comprising conjugating a CD47 antigen targeting agent to LLO.

[0075] Aspect 53 is a method of treating a disease or disorder in a subject, the method comprising administering the composition of any one of aspects 30 to 52 to the subject.

[0076] Aspect 54 is a composition comprising, a Listeriolysin O (LLO) molecule conjugated to an antigen targeting agent, through a cleavable linker.

[0077] Aspect 55 is the composition of aspect 54, wherein the antigen targeting agent comprises an antibody or functional fragment thereof.

[0078] Aspect 56 is the composition of aspect 54 or 55, wherein the antibody or functional fragment thereof targets a CD47, CD24, PDL1, CD19, CD20, CD30, GPRC5D, EGFR, EGFR2, BCMA, c-MET, and / or HER2 antigen.

[0079] Aspect 57 is the composition of aspect 55 or 56, wherein the antibody or fragment thereof is humanized, optionally wherein the antibody or functional fragment thereof is an IgGl, IgG2, IgG2B, or IgG4 isotype.

[0080] Aspect 58 is the composition of any one of aspects 54 to 57, wherein the antigen targeting agent is capable of binding to an antigen expressed on a cancer cell.

[0081] Aspect 59 is the composition of any one of aspects 54 to 58, wherein the antigen targeting agent neutralizes the target antigen.

[0082] Aspect 60 is the composition of aspect 58, wherein the cancer cell comprises a breast cancer, lung cancer, non- small cell lung cancer, brain cancer, glioblastoma, triplenegative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma cell.

[0083] Aspect 61 is the composition of any one of aspects 54 to 60, wherein the LLO is only active in an acidic environment with a pH of, of less than, or of about 6.5.

[0084] Aspect 62 is the composition of any one of aspects 54 to 60, wherein the LLO is only active in an acidic environment with a pH of, of less than, or of about 6.0 or 5.9, and / or is optimally active at a pH of or of about 5.5.

[0085] Aspect 63 is the composition of any one of aspects 54 to 62, wherein the LLO is active in a phagosome.

[0086] Aspect 64 is the composition of any one of aspects 54 to 63, wherein the LLO is not active in the cytoplasm of a cell.

[0087] Aspect 65 is the composition of any one of aspects 54 to 64, wherein the LLO is not active at the cell membrane.

[0088] Aspect 66 is the composition of any one of aspects 54 to 65, wherein the antigen targeting agent and the LLO are conjugated together.

[0089] Aspect 67 is the composition of any one of aspects 54 to 66, wherein the antigen targeting agent and the LLO are conjugated through a cleavable linker that can be cleaved upon exposure to reduced conditions.

[0090] Aspect 68 is the composition of any one of aspects 54 to 67, wherein the LLO is active only once cleaved from the antigen targeting agent.

[0091] Aspect 69 is the composition of any one of aspects 54 to 68, wherein the LLO molecule and the antigen targeting agent are conjugated through click chemistry.

[0092] Aspect 70 is the composition of aspect 69, wherein the click chemistry is copper- free click chemistry.

[0093] Aspect 71 is the composition of any one of aspects 54 to 70, wherein the LLO is conjugated to a primary amine on the antigen targeting agent.

[0094] Aspect 72 is the composition comprising, the LLO molecule conjugated to an antigen targeting agent through a cleavable linker of any one of aspects 54 to 72.

[0095] Aspect 73 is the composition of aspect 72, also comprising a pharmaceutically acceptable excipient.

[0096] Aspect 74 is a method of making the composition of any one of aspects 54 to 73, the method comprising conjugating an antigen targeting agent to LLO.

[0097] Aspect 75 is a method of treating a disease or disorder in a subject, the method comprising administering the composition of any one of aspects 54 to 74 to the subject.

[0098] Aspect 76. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, and optionally a therapeutically effective amount of an immune checkpoint inhibitor.

[0099] Aspect 77 is the method of aspect 76, wherein the pore-forming agent is or comprises LLO.

[0100] Aspect 78 is the method of aspect 76 or 77, wherein the antigen targeting agent targets CD47.

[0101] Aspect 79 is the method of any one of aspects 76 to 78, wherein the patient has, has been diagnosed with, has one or more symptoms of, or is suspected of having a cancer.

[0102] Aspect 80 is the method of aspect 79, wherein the cancer comprises breast cancer, lung cancer, non-small cell lung cancer, brain cancer, glioblastoma, triple-negative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma.

[0103] Aspect 81 is the method of any one of aspects 76 to 80, wherein administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a poreforming agent, and a therapeutically effective amount of an immune checkpoint inhibitor comprises intratumoral, intraperitoneal, and or / intravenous delivery.

[0104] Aspect 82 is the method of any one of aspects 76 to 81, wherein the immune checkpoint inhibitor comprises PD-1 inhibitors (e.g., nivolumab, pembrolizumab, MEDI0680, and / or pidilizumab), PDL2-inhibitors (e.g., AMP- 224, rHIgM12B7), PDL1 inhibitors (e.g., Durvalumab, atezolizumab, avelumab), CTLA-4 inhibitors, B7-1 inhibitors, and / or B7-2 inhibitors.

[0105] Aspect 83 is the method of any one of aspects 76 to 81, wherein the immune checkpoint inhibitor comprises a PD1 -antigen targeting agent.

[0106] Aspect 84 is the method of aspect 83, wherein the PDl-antigen targeting agent comprises nivolumab, pembrolizumab, MEDI0680, and / or pidilizumab.

[0107] Aspect 85 is the method of any one of aspects 76 to 83, wherein one or more additional therapies or therapeutic agents are co-administered before, after, or during the administration of a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, and a therapeutically effective amount of an immune checkpoint inhibitor.

[0108] Aspect 86 is the method of any one of aspects 76 to 85, wherein the one or more additional therapies comprise tumor debulking, surgery, chemotherapy, radiotherapy, immunotherapy, and / or other agents.

[0109] Aspect 87 is the method of any one of aspects 76 to 86, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, the tumor accumulates intratumoral CD8+ T cells with TCRs specific to tumor antigens.

[0110] Aspect 88 is the method of any one of aspects 76 to 87, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, bone-marrow derived dendritic cells and / or bone-marrow derived macrophages display tumor-associated antigens.

[0111] Aspect 89 is the method of any one of aspects 76 to 88, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, bone-marrow derived dendritic cells and / or bone-marrow derived macrophages exhibit activation of stimulator of interferon genes (STING).

[0112] Aspect 90 is the method of any one of aspects 76 to 89, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent: APCs exhibit increased expression of genes encoding one or more of CD86, IL1, and IL12, and / or lymphocytes exhibit increased expression of genes encoding CD40.

[0113] Aspect 91 is the method of any one of aspects 76 to 90, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, the antigen targeting agent conjugated to a pore-forming agent accumulates within one or more tumor.

[0114] Aspect 92 is the method of any one of aspects 76 to 91, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, the subject exhibits systemic antitumor immune memory.

[0115] Aspect 93 is the method of aspect 92, wherein antitumor immune memory comprises: interferon-gamma production by isolated splenic CD8+ T cells upon contact with (i) one or more tumor cell and / or (ii) one or more tumor- associated antigen.

[0116] Aspect 94 is a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent.

[0117] Aspect 95 is the method of aspect 94 wherein the pore-forming agent is LLO.

[0118] Aspect 96 is the method of aspect 94 or 95, wherein the antigen targeting agent targets CD47.

[0119] Aspect 97 is the method of any one of aspects 94 to 96, wherein the patient has, has been diagnosed with, has one or more symptoms of, or is suspected of having a cancer.

[0120] Aspect 98 is the method of any one of aspects 94 to 97, wherein the cancer comprises breast cancer, lung cancer, non-small cell lung cancer, brain cancer, glioblastoma, triple-negative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma.

[0121] Aspect 99 is the method of any one of aspects 94 to 98, wherein one or more additional therapies or therapeutic agents are co-administered before, after, or during the administration of a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent.

[0122] Aspect 100 is the method of any one of aspects 94 to 99, wherein the one or more additional therapies comprise tumor debulking, surgery, chemotherapy, radiotherapy, immunotherapy, and / or other agents.

[0123] Aspect 101 is a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of a composition according to any one of the preceding aspects.

[0124] Aspect 102 is the method of any one of aspects 94 to 101, wherein the patient has, has been diagnosed with, has one or more symptoms of, or is suspected of having a cancer.

[0125] Aspect 103 is the method of aspect 102, wherein the cancer comprises breast cancer, lung cancer, non-small cell lung cancer, brain cancer, glioblastoma, triple-negative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma.

[0126] Aspect 104 is the method of any one of aspects 94 to 103, wherein administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a poreforming agent, comprises intratumoral, intraperitoneal, and or / intravenous delivery.

[0127] Aspect 105 is the method of any one of aspects 94 to 104, wherein one or more additional therapies or therapeutic agents are co-administered before, after, or during the administration of a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent.

[0128] Aspect 106 is the method of any one of aspects 94 to 105, wherein the one or more additional therapies comprise tumor debulking, surgery, chemotherapy, radiotherapy, immunotherapy, and / or other agents.

[0129] Aspect 107 is the method of any one of aspects 94 to 106, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, the tumor accumulates intratumoral CD8+ T cells with TCRs specific to tumor antigens.

[0130] Aspect 108 is the method of any one of aspects 94 to 107, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, bone-marrow derived dendritic cells and / or bone-marrow derived macrophages display tumor-associated antigens.

[0131] Aspect 109 is the method of any one of aspects 94 to 108, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, bone-marrow derived dendritic cells and / or bone-marrow derived macrophages exhibit activation of stimulator of interferon genes (STING).

[0132] Aspect 110 is the method of any one of aspects 94 to 109, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent,

[0133] APCs exhibit increased expression of genes encoding one or more of CD86, IL1, and IL12, and / or lymphocytes exhibit increased expression genes encoding CD40.

[0134] Aspect 111 is the method of any one of aspects 94 to 110, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, the antigen targeting agent conjugated to a pore-forming agent accumulates within one or more tumor.

[0135] Aspect 112 is the method of any one of aspects 94 to 111, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, the subject exhibits systemic antitumor immune memory.

[0136] Aspect 113 is the method of claim 112, wherein antitumor immune memory comprises: interferon-gamma production by isolated splenic CD8+ T cells upon contact with (i) one or more tumor cell and / or (ii) one or more tumor- associated antigen.

[0137] Other objects, features and advantages of the present inventions will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the inventions, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.DESCRIPTION OF THE DRAWINGS

[0138] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present inventions. The inventions may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0139] FIGs. 1A-1T, Anti-CD47 antibody was conjugated with Listeriolysin O and purified. FIG. 1A shows a schematic of protein- antibody conjugation. As an example, anti- CD47 antibody and Listeriolysin O (LLO) were modified using the click chemistry labelling reagent DBCO-PEG4-NHS ester and the crosslinking reagent SPDP-PEG 11 -azide, respectively. The modified LLO protein was mixed with the modified antibody and allowed to react overnight at room temperature. Unconjugated antibody and Listeriolysin O proteins were removed by affinity and size exclusion chromatographic methods, respectively. Resultant fusions were referred to as “CD47-LLO” or “LLO-CD47”. FIG. IB shows a western blot of purified LLO. The results show successful purification of LLO. FIG. 1C shows a sheep red blood cell (sRBC) hemolysis assay of purified LLO. The results show about 50% hemolysis within about 0.5 ng / mL to 1 ng / mL of LLO. FIG. ID shows a chemical reaction between a CD47 antibody and the click chemistry labeling reagent DBCO-PEG4-NHS ester (Conju- Probe, SKU# CP-2028). FIG. IE shows a chemical reaction between LLO and the click chemistry crosslinking reagent SPDP-PEG 11 -azide (BroadPharm, Cat#BP-25143).FIG. IF shows representative SDS-PAGE results of IgG-LLO conjugate in reductive and non-reductive loading buffers. FIG. 1G shows SDS-PAGE results of azide-labeled LLO, DBCO-labeled anti- CD47, conjugation reaction, affinity column passthrough, and size exclusion input sample. CD47-LLO conjugate is appreciable at about 206 kDa. FIG. 1H shows a size exclusion chromatogram of conjugated CD47-LLO (solid line) and anti-CD47 alone (dashed line). Aclear separation between the elution peaks for unconjugated anti-CD47 and conjugated CD47- LLO was evident. FIG. II shows SDS-PAGE results of conjugate in reductive and non- reductive loading buffers. Reduced conditions efficiently caused dissociation of LLO from anti-CD47. FIG. 1J shows hemolysis assay of LLO, CD47-LLO, and CD47-LLO treated with a reducing agent (5 mM DTT, 30 min). Hemolysis percentage was normalized to 0.1% Triton X-100. Under non-reducing conditions, CD47-LLO did not cause hemolysis. In contrast, reduced CD47-LLO (CD47-LLO+DTT) caused a degree of hemolysis comparable to LLO alone. Data shown represent mean s.d. analyzed by one way analysis of variance with Tukey’s multiple comparisons test. N=3 biologically independent experiments. FIG. IK shows the results of an apoptosis assay of mouse bone marrow-derived macrophages (BMDMs) treated with CD47-LLO or LLO for 24 hours analyzed by flow cytometry. CD47-LLO treatment resulted in a significantly lower level of apoptosis relative to LLO treatment (P<0.0001). Data shown represent mean s.d. analyzed by one way analysis of variance with Tukey’s multiple comparisons test. N=3 biologically independent experiments. FIGs. 1L-1S show in vitro toxicity assays of CD47-LLO with cancer cell lines. Flow cytometry analysis and quantification of apoptosis and necrosis in 4TlBr4 (FIGs. 1L-1M), EO771 (FIGs. 1N-1O), KPC (FIGs. 1P-1Q), and D4M.3A (FIGs. 1R-1S) cells after treatment with 2 ug / mL CD47- LLO for 0, 6, or 24 hours. Data shown represent mean ± s.d. (FIGs IM, IO, IQ, IS) analyzed by one-way analysis of variance with Tukey’s multiple comparisons test. Data show at least n = 3 biologically independent experiments. FIG. IT shows a proposed mechanism of action of CD47-LLO. Tumour cells sidestep phagocytosis by amplifying their expression of the “don’t eat me” signal, CD47 (boxes, left). The anti-CD47 antibody promotes tumour cell phagocytosis by phagocytes, e.g., antigen-presenting cells (APCs), macrophages, and / or dendritic cells (DCs). Inside phagolysosomes, LLO monomers dissociate from anti-CD47 due to reducing conditions and form membrane permeations that allow tumour DNA and antigenic peptides to escape into the cytosol (inset, right). This in turn activates cGAS-STING to produce type I interferons (IFNs) and enhance cross-presentation of neoantigens. LRP, low-density lipoprotein receptor-related protein- 1. SIRPalpha, signal regulatory protein alpha.

[0140] FIGs. 2A-2AR, CD47-LLO promoted phagocytosis, lysosomal permeabilization, and antigen presentation. FIG. 2A shows bone marrow-derived macrophage (BMDM) phagocytosis of EO771 cells (white arrows) as visualized by confocal laser scanning microscopy. Representative images are shown from at least three independent experiments. Scale bars, 20 pm. FIG. 2B shows quantification of BMDMs containing EO771 cells per field of view (BMDMs were collected from n = 3 C57BL6 mice with >200 BMDMscounted per condition). FIGs. 2C-2D show flow cytometry analysis (FIG. 2C) and quantification (FIG. 2D) of phagocytic activity of bone marrow-derived dendritic cells (BMDCs) as evaluated by flow cytometry. BMDCs were collected from n = 3 C57BL6 mice. FIGs. 2E-2F show flow cytometry analysis (FIG. 2E) and quantification of phagocytic activity of BMDMs (FIG. 2F). BMDMs were collected from at least n = 4 C57BL6 mice. FIGs. 2G- 2H show flow cytometry analysis (FIG. 2G) and quantification (FIG. 2H) of CD86 and CD206 expression in BMDMs isolated from EO771 co-cultures. BMDMs were collected from n = 4 C57BL6 mice. FIG. 21 shows transmission electron microscopic (TEM) images of gold nanoparticles engulfed by phagocytic immune cells (e.g., bone marrow-derived macrophages (BMDMs)) either inside phagolysosomes (arrows top panel) or within the cytoplasm (arrows bottom panel) after treatment with anti-CD47 or CD47-LLO, respectively. The central arrow in the bottom right panel shows a double membraned phagolysosome with a wall perforation. FIG. 2J shows quantification of intact vacuoles from TEM images; n = 5 BMDMs quantified per condition. The results showed a significant decrease (P<0.05) in the number of intact vacuoles in BMDMs treated with CD47-LLO relative to anti-CD47 control. FIG. 2K shows confocal images of BMDMs (white arrows) stained with LysoTracker Red. Representative images are shown from at least three independent experiments. FIG. 2L shows quantification of LysoTracker Red puncta per BMDM (BMDMs were collected from n = 3 C57BL6 mice with >115 BMDMs counted per condition). FIG. 2M shows flow cytometry analysis of BMDMs stained with acridine orange. FIGs. 2N-2O show quantification of acridine red (FIG. 2N) and acridine green (FIG. 20). BMDMs collected from n = 4 C57BL6 mice. FIGs. 2P-2Q show flow cytometry analysis (FIG. 2P) and quantification (FIG. 2Q) of BMDCs stained with acridine orange. BMDCs were collected from n = 3 C57BL6 mice. FIGs. 2R-2S show flow cytometry analysis (FIG. 2R) and quantification (FIG. 2S) of cross-presentation of SIINFEKL-H2Kb peptides on the surfaces of BMDMs (n = 4). FIGs. 2T-2U show flow cytometry analysis (FIG. 2T) and quantification (FIG. 2U) of cross-presentation of SIINFEKL-H2Kb peptides on the surfaces of BMDCs (n = 3). FIG. 2V shows representative flow cytometry results of antigen presentation assays from BMDMs co-cultured with EO771- OVA cells for 48 hours with 2 ug / mL CD47-LLO, anti-CD47, IgG-LLO, or IgG. BMDMs were collected from n = 4 mice. These data demonstrate that BMDMs show increased APC- H2Kb bound to SIINFEKL with CD47-LLO. FIGs. 2W-2X show flow cytometry analysis (FIG. 2W) and quantification (FIG. 2X) of the proliferation of CD8+ T cells isolated from transgenic OT-I mice and cocultured with BMDMs with or without cancer cells (n = 5). Data shown represent mean ± s.d. (FIGs. 2B, 2F, 2H, 2L, 2N, 20, 2S, 2X) or mean ± s.e.m. (FIG.2J) analyzed by two-sided unpaired Student’s t tests (FIGs. 2J, 2L) or one-way analysis of variance with Tukey’s multiple comparisons test (FIGs. 2B, 2F, 2H, 2N, 20, 2S, 2X). FIG. 2Y shows a schematic of experiments involving intratumoural (IT) injections of CD47-LLO or anti-CD47 in syngeneic orthotopic 4TlBr4 breast cancer models. FIG. 2Z shows representative immuno staining of Wild-type (WT) mice inoculated with 4TlBr4 breast tumours and treated with anti-CD47 or CD47-LLO as described in the examples and in FIG. 2Y. Representative images showed that levels of phosphorylated STING and macrophages (F4 / 80+), detected by immuno staining in 4TlBr4 breast tumour paraffin-embedded sections, were increased in CD47-LLO treated animals relative to anti-CD47 treated animals. Arrowheads show F4 / 80+and pSTING+co-labeled cells. Scale bar, 20 pm. FIG. 2AA shows quantification of F4 / 80+cells per DAPI+cells per field of view (n = 3 biologically independent tumours per condition, with >5000 DAPI+cells counted per tumour). The results showed a significant increase (P <0.0001) of F4 / 80+cells per DAPI+cells in CD47-LLO treated animals relative to anti-CD47 control animals. FIG. 2AB shows quantification and comparison of pSTING fluorescence intensity (pSTING FLU) per F4 / 80+cells obtained with Icy Bioimage Analysis software from 7 randomly selected images of 3 biologically independent mice per condition. The results showed a significant increase (P <0.0001) of pSTING FLU and % F4 / 80+cells in CD47-LLO treated animals relative to anti-CD47 control animals. FIG. 2AC shows western blotting of proteins associated with the cGAS-STING pathway (pSTING, STING, cGAS, HSP90) in BMDMs from C57BL / 6J mice that were co-cultured with EO771 cells and treated with IgG, anti-CD47 or CD47-LLO; after treatment, the BMDMs were isolated and proteins were extracted for analysis. As evidenced by western blotting, CD47-LLO treatment increased pSTING and cGAS levels in BMDMs co-cultured with EO771 cells compared to IgG or anti-CD47 treated controls without changes to basal STING levels. FIGs. 2AD-2AE show flow cytometry analysis (FIG. 2AD) and quantification (FIG. 2AE) of pSTING levels in CDl lb+BMDMs isolated from co-cultures with EO771 cells. Data show n = 4 technical replicates from n = 2 C57BL6 mice. The results showed a significant increase (P = <0.0001) in pSTING levels in CD1 lb+BMDMs isolated from co-cultures with EO771 cells when treated with CD47-LLO relative to controls treated with anti-CD47. FIGs. 2AF-2AG show flow cytometry analysis (FIG. 2AF) and quantification (FIG. 2AG) of pSTING levels in BMDCs isolated from co-cultures with EO771 cells, n = 4 C57BL6 mice. Data shown represent mean ± s.d. analyzed by one-way analysis of variance with Tukey’s multiple comparisons test. FIG. 2AH shows representative flow cytometry of pSTING assays. BMDMs were co-cultured with EO771 cells for 24 hours with 2ug / mL CD47-LLO, anti-CD47, IgG-LLO, or IgG. BMDMswere collected from n=4 mice. FIG. 2AI shows RT-PCR results of interferon (IFN)-alpha (IFNa) expression levels in BMDMs co-cultured with EO771 cells for 24 hours. BMDMs were collected from n = 3 C57BL6 mice. The results showed a significant increase (P = 0.0471) in IFNa transcripts in CD47-LLO treated conditions relative to controls. FIG. 2AJ shows RT- PCR results of interferon (IFN)-beta (IFNP) expression levels in BMDMs co-cultured with EO771 cells for 24 hours. BMDMs were collected from n=3 C57BL6 mice; experiments were performed in 3 batches. The results showed a significant increase (P = 0.0029) in IFNP transcripts in CD47-LLO treated conditions relative to controls. FIG. 2AK shows tumor necrosis factor (TNF)-alpha (TNFa) levels in cell culture supernatants from cells from FIG. 2AJ measured by enzyme-linked immunosorbent assay (ELISA). The results showed a significant increase in TNFa levels in CD47-LLO treated conditions relative to IgG or anti- CD47 controls. FIG. 2AL shows growth curves for EO771 breast tumours from WT (STING+ / +) or STING7mice inoculated with EO771 breast tumour cells and treated with intratumoural IgG or CD47-LLO. n = 7 for CD47-LLO-treated and IgG-treated WT mice; n = 5 for CD47-LLO-treated and n = 4 for IgG-treated STING7mice. The results showed a significant decrease (P<0.0001) in tumor volume in WT (STING+ / +) mice treated with CD47- LLO relative to IgG treated WT (STING+ / +) mice and CD47-LLO treated STING'7'mice controls. FIG. 2AM shows comparison of tumour volumes at day 12 after intratumoural injection. The results showed a significant decrease in tumor volume in CD47-LLO treated animals relative to IgG controls. Data shown represented mean ± s.e.m. (2B, 2N) or mean ± s.d. (FIGs. 2AA, 2AE, 2AI, 2AJ, 2AK, 2AN, 2AP, 2AQ, and 2AM) analyzed by two-sided unpaired Student’s t tests (FIGs. 2J, 2AA, and 2AE), one-way analysis of variance with Tukey’s multiple comparisons test (FIGs. 2AI, 2AJ, 2AK, 2AN, 2AP, 2AQ, and 2AM), two- way analysis of variance with Tukey’s multiple comparisons test (2AL), or unpaired Mann- Whitney U test (2AB). FIG. 2AN shows quantification of CD86 and CD206 expression in CDllb+BMDMs isolated from EO771 cell co-cultures. BMDMs were collected from n = 3 C57BL6 mice. The results showed a significant increase (P = 0.003 or 0.032, respectively) in the ratio of CD86+to CD206+cells in CD47-LLO treated conditions relative to controls (“NC” = non-cleavable). FIG. 2AO shows representative flow cytometry results of gating strategy for CD86 and CD206 for BMDMs that were cocultured with EO771 cells for 48 hours. The results showed an increase in CD86+ / CD206- cells in the group treated with CD47-LLO relative to the groups treated with IgG, anti-CD47, or CD47-LLO-NC. FIG. 2AP shows a BMDM phagocytosis assay of EO771 cells treated with CD47-LLO. BMDMs were collected from n = 3 C57BL6 mice. The results showed significant increases (P<0.0001) in the phagocytosispercentage as the concentration of CD47-LLO increased relative to untreated control. FIG. 2AQ shows results from antigen presentation assay of BMDMs co-cultured with EO771-OVA cells for 48 hours. BMDMs were collected from n = 6 mice. The results showed a significant increase (P < 0.0001) in H-2Kb bound to SIINFEKL (SINFEKL) in CD47-LLO treated conditions relative to controls. FIG. 2AR shows representative flow cytometry results of antigen presentation assay for BMDMs that were co-cultured with EO771-OVA cells for 48 hours collected from n=6 mice. The results showed an increase in antigen presentation in the group treated with CD47-LLO (LLO-CD47) relative to the groups treated with IgG or anti- CD47.

[0141] FIGs. 3A-3Y, CD47-LLO drove innate and adaptive immune responses in primary breast cancer and melanoma in vivo. FIG. 3A shows WT mice that were inoculated with EO771 breast tumours and treated with intratumoural anti-CD47, CD47-LLO, IgG-LLO, or a non-cleavable form of CD47-LLO (CD47-LLO-NC). Tumour volumes were monitored and analyzed for size for the indicated periods (2 day intervals for up to 12 days). Insets show representative photographs of mice at 14 days after EO771 breast tumour inoculation, n = 6 for IgG-LLO-treated mice, and n = 7 for all remaining groups. The results showed a significant decrease (P<0.001) in tumor volume in CD47-LLO treated animals relative to controls. FIG. 3B shows tumour volume quantification at 12 days after tumour inoculation. The results showed a significant decrease in tumor volume at 12 days post-tumour inoculation in CD47- LLO treated animals relative to anti-CD47 and CD47-LLO-NC treated controls. FIG. 3C shows 4TlBr4 tumour volumes after intratumoural injection of anti-CD47 or CD47-LLO. Mice were treated as described in FIG. 2V. FIG. 3D shows tumour volume of WT mice that were inoculated with D4M.3A melanoma tumours in the right flank and treated with anti-CD47 or CD47-LLO. Tumour volumes were monitored and analyzed for the indicated periods (variable day intervals for up to 14 days), n = 6 for all groups. Insets show representative photographs of tumours at 20 days after D4M.3A melanoma tumour inoculation. The results showed a significant decrease is tumour volume in CD47-LLO treated animals relative to anti-CD47 treated control animals (P<0.0001). FIG. 3E shows survival curves plotted and analyzed for mice in each anti-CD47 or CD47-LLO treatment arm. n = 6 for all groups. The results showed a significant extension of survival in mice treated with CD47-LLO relative to mice treated with anti-CD47 (P<0.0467). FIGs. 3F-3G show flow cytometry analysis (FIG. 3F) and quantification (FIG. 3G) of CD86+ and CD206+ tumour-associated macrophages in 4TlBr4 tumours at day 16 after tumour inoculation in each group, n = 4 biologically independent experiments. FIGs. 3H-3I show flow cytometry analysis (FIG. 31) and quantification (FIG.3H) of CD4+ tumour-associated lymphocytes in EO771 tumours at day 16 after tumour inoculation in each group, n = 4 biologically independent experiments. FIGs. 3J-3K show flow cytometry analysis (FIG. 3K) and quantification (FIG. 3J) of CD8+ tumour-associated lymphocytes in EO771 tumours at day 16 after tumour inoculation in each group. N = 4 biologically independent experiments. FIG. 3L shows a schematic illustrating the mechanism of SIINFEKL-H2Kb tetramer+CD8+ T-cell expansion. FIGs. 3M-3N show flow cytometry analysis (FIG. 3M) and quantification (FIG. 3N) of SIINFEKL-H2Kb tetramer+CD8+ T-cells within the tumour microenvironment (n = 4 biological replicates). FIGs. 3O-3P show flow cytometry analysis (FIG. 30) and quantification (FIG. 3P) of SIINFEKL-H2Kb tetramer+CD8+ T-cells within the spleen (n = 4 biological replicates). FIG. 3Q shows flow cytometry analysis of SIINFEKL-H2Kb tetramer+CD8+ T-cells within the tumour microenvironment. Data shown represent mean ± s.e.m. analyzed by two-way analysis of variance with Tukey’s multiple comparisons test. FIG. 3R shows a schematic illustrating the establishment and treatment of bilateral breast tumours inoculated in C57BL6 mice. FIG. 3S shows treated and untreated tumour volumes were monitored and analyzed for the indicated periods, n = 6 for all groups. FIGs. 3T-3U show flow cytometry analysis (FIG. 3T) and quantification (FIG. 3U) of SIINFEKL-H2Kb tetramer+CD8+ T-cells within the untreated tumour microenvironment (n = 4 biological replicates). FIGs. 3V-3W show representative fluorescence images (FIG. 3V) and quantification (FIG. 3W) of EO771 tumour-bearing mice taken at predetermined times after intratumoural injection of IR800CW-tagged CD47-LLO (25 ug). n = 2 biologically independent mice. Bilateral tumours enclosed in circles. FIGs. 3X-3Y show ex vivo fluorescence images (FIG. 3X) and quantification (FIG. 3Y) of tumour and major organs collected at 24 hours after intratumoural administration.

[0142] FIGs. 4A-4AG, Transcriptome analysis revealed CD47-LLO TAM proinflammatory signatures in vivo. In FIGs. 4A-4K EO771 tumors were harvested on day 3 following intratumoural treatment with IgG (n=3), anti-CD47 (n=4), or CD47-LLO (n=4), and Fluorescent- Activated Cell Sorting (FACS) sorted on CD45+cells. Cells were fixed, and 100,000 CD45+cells / tumour were pooled into respective treatment groups and subjected to scRNA-seq. FIG. 4A shows flow cytometry plots of the percentage of CD45+cells (squares) isolated from EO771 tumours treated with intratumoral anti-CD47, or CD47-EEO. The results showed an increase in the percentage of CD45+cells from tumours treated with CD47-EEO relative to tumour treated with anti-CD47. FIG. 4B shows quantification of CD45+cells isolated from EO771 tumours treated with intratumoral IgG, anti-CD47, or CD47-EEO. The results showed a significant increase in the percentage of CD45+cells from tumours treatedwith CD47-LLO relative to tumours treated with IgG or anti-CD47 (P=0.0102 and P=0.007, respectively). FIGs. 4C-4I show quality control for single-cell RNA sequencing. FIG. 4C shows the distribution of RNA counts by cell density across treatment groups. The results showed an overlapping distributions of RNA counts by cell density between treatment groups. FIG. 4D shows the distribution of RNA features by cell density across treatment groups. The results showed an overlapping distributions of RNA features by cell density between treatment groups. FIG. 4E shows the distribution of mitochondrial ratio by cell density across treatment groups. The results showed an overlapping distributions of mitochondrial ratio by cell density between treatment groups. FIG. 4F shows the distribution of loglO genes per unique molecular identifier (UMI) across treatment groups. The results showed an overlapping distributions of loglO genes per unique molecular identifier (UMI) between treatment groups. FIG. 4G shows a violin plot showing the number of RNA features across treatment groups. The results showed a similar number of RNA features between treatment groups. FIG. 4H shows a violin plot showing the number of RNA counts across treatment groups. The results showed a similar number of RNA counts between treatment groups. FIG. 41 shows a violin plot showing the percent of mitochondrial reads across treatment groups. The results showed a similar number of mitochondrial reads between treatment groups. FIGs. 4J-4L show tumor-associated CD45+cells were detected by single-cell RNA sequencing. FIG. 4J shows a UMAP projection of all single cells from 3 treatment groups color-coded by cluster. The results showed 18 identifiable clusters. FIG. 4K shows a heatmap of 20 differentially expressed genes in clusters, ranked by False Discovery Rate (FDR). The results showed gene expression differences between clusters and between sample groups. FIG. 4L shows a dot plot showing marker expression for different clusters. Dot size indicates the percentage of cells in each cluster expressing the gene and colors indicate the average expression levels. The results showed differences in marker expression between clusters. FIG. 4M shows a Uniform Manifold Approximation and Projection (UMAP) projection by assignment of 13,891 single cells from 3 treatment groups and the composition of different cell types of said cells. The results showed possible identification of NK cells, T cells, B cells, tumour cells, dendritic cells (DCs), macrophages, and granulocytes. FIG. 4N shows a UMAP projection by sample of 13,891 single cells from 3 treatment groups (IgG, anti-CD47, and CD47-LLO) and the composition of different cell types of said cells. The results showed the composition of celltype clusters based on treatment group. FIGs. 4O-4P show tumour-associated neutrophils (TANs) were enriched in CD47-LLO tumors. FIG. 40 shows representative images of Ly-6g+(top) and Ly-6g+ / CDl lb+(bottom) cells detected by immunostaining in 4TlBr4 breast tumourparaffin-embedded sections. The results showed an higher number of Ly-6g+cells in the CD47- LLO treated group relative to the anti-CD47 treated group. FIG. 4P shows a quantification of Ly-6g+cells per DAPI+cells per field of view (n = 3 biologically independent tumours per condition, with >2000 DAPI+cells counted per tumour). The results showed a significantly higher percentage of Ly-6g+cells in the CD47-LLO treated group relative to the anti-CD47 treated group. FIGs. 4Q-4U show differentially-expressed genes were found to define tumour- associated granulocyte clusters. FIG. 4Q shows a UMAP projection of only CD45+granulocytes color-coded by sample (IgG, anti-CD47, or CD47-LLO). The results showed cell clustering based on sample. FIG. 4R shows a UMAP projection of only CD45+granulocytes color-coded by cluster. The results showed six identifiable granulocyte clusters. FIG. 4S shows the number of cells (y-axis) from each cluster (x-axis) color-coded by sample. The results showed a different number of cells per cluster in each sample. FIG. 4T shows the percentage of cells (y-axis) from each cluster (x-axis) color-coded by sample. The results showed a different percentage of cells based on cluster in each sample. FIG. 4U shows a dot plot depicting the top 5 differentially expressed genes per macrophage cluster. The dot size indicates the percentage of cells in each cluster expressing the gene and colors indicate the average expression levels. The results showed different expression levels of the top 5 differentially expressed genes per macrophage cluster between clusters. FIG. 4V shows a UMAP projection of only macrophages color-coded by cluster. The results showed 10 identifiable macrophage clusters. FIG. 4W shows the top 20 differentially expressed genes in ten macrophage clusters ranked by False Discovery Rate (FDR). Gene expression values were centered, scaled, and transformed to a -2 to 2 scale. The results showed gene expression differences based on treatment group and macrophage cluster. FIG. 4X shows UMAP projections of macrophage populations showing the distribution of transcriptional signatures of pre-defined macrophage and monocyte subsets. Inset shows a dot plot depicting the average expression and percent expression of signature genes per macrophage cluster. The results showed differences in the expression of classical, regulatory, resolution phase, and non- classical gene signatures between macrophage clusters. FIGs. 4Y-4Z show differentially- expressed genes defined tumour-associated macrophage clusters. FIG. 4Y shows a dot plot depicting the top 10 differentially expressed genes per macrophage cluster. The results showed different expression levels of the top 10 differentially expressed genes per macrophage cluster between clusters. FIG. 4Z shows a dot plot depicting the differential expression of relevant genes per macrophage cluster. The results showed different expression levels of the relevant genes per macrophage cluster between clusters. FIG. 4AA shows a heatmap depicting 1expression of cGAS-STING pathway related-genes in macrophage clusters by treatment group and cluster. The results showed differences in the expression of cGAS-STING pathway related- genes in macrophages between macrophage clusters and treatment groups. FIG. 4AB shows a heatmap depicting expression of phagocytosis related-genes in macrophage clusters by treatment group and cluster. The results showed differences in the expression of phagocytosis related- genes in macrophages between macrophage clusters and treatment groups. FIG. 4AC shows a UMAP projection of macrophage population clusters by treatment sample. The results showed differences in the types of macrophage clusters identified per treatment group. FIG. 4AD shows the fractions of cells (y-axis) from each cluster (x-axis) color-coded by treatment sample. The results showed differences in the proportion of cells by treatment group that composed each macrophage cluster. FIG. 4AE shows a gene set enrichment analysis utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG) gene set for tumor-associated macrophages with heatmap displaying select upregulated and downregulated pathways in each cluster. The results showed differences in the expression of depicted KEGG gene sets based on macrophage cluster. FIGs. 4AF-4AG show Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology Biological Process (GOBP) analysis of tumour-associated macrophage clusters. FIG. 4AF shows a gene set enrichment analysis utilizing the KEGG gene set for tumor-associated macrophages with heatmap displaying the ten most upregulated and downregulated pathways in each cluster ranked by their normalized enrichment scores (NES). The results showed differences in KEGG pathway enrichment between clusters. FIG. 4AG shows a gene set enrichment analysis utilizing the GOBP gene set for tumor-associated macrophages with heatmap displaying the ten most upregulated and downregulated pathways in each cluster ranked by their NES. The results showed differences in GOBP pathway enrichment between clusters.

[0143] FIGs. 5A-5Q, CD47-LLO drove APC-adaptive immune cell clustering and crosstalk. FIG. 5A shows confocal fluorescence microscopy images of CD8+ cells (asterisks) in 4TlBr4 tumours after intratumoural injection of anti-CD47 or CD47-LLO. The results showed an increase in the number of CD8+ cells at the tumour in CD47-LLO treated animals relative to anti-CD47 treated animals. FIG. 5B shows quantification of CDl lc+ cells per DAPI+ cells per field of view (n = 3 biologically independent tumours per condition, with >5000 DAPI+ cells counted per tumour). The results showed a significant increase in the percentage of CDl lc+ cells at the tumor site in CD47-LLO treated mice compared to anti- CD47 treated control mice (P<0.0001). FIG. 5C shows quantification of CD4+ cells per DAPI+ cells per field of view (n = 3 biologically independent tumours per condition, with>5000 DAPI+ cells counted per tumour). The results showed a significant increase in the percentage of CD4+ cells at the tumour site in CD47-LLO treated animals relative to anti- CD47 treated animals (P=0.0007). FIG. 5D shows quantification of CD8+ cells per DAPI+ cells per field of view (n = 3 biologically independent tumours per condition, with >5000 DAPI+ cells counted per tumour). The results showed a significant increase in the percentage of CD8+ cells at the tumour in CD47-LLO treated animals relative to anti-CD47 treated animals(P=0.0004). FIG. 5E shows quantification of percentages of CDl lc+, CD4+, and CD8+ cells that colocalized in triad clusters. The results showed a significant increase in the percent of CD8+ cells in triads (P=0.0061), CD4+ cells in triads (P=0.0405), and CD1 lc+ cells in triads (P=0.0051) in the tumours of CD47-LLO treated mice relative to anti-CD47 treated mice. FIG. 5F shows a UMAP projection of lymphocyte populations showing the distribution of pre-defined T cell subsets. FIG. 5G shows cell-to-cell communication networks inferred with CellChat software from gene expression of ligands and their receptors in immune cell clusters of interest from CD45+ cells isolated from EO771 tumours. Strength of cell-to-cell interactions is represented in the edge width in CD47-LLO mice and mice treated with anti— CD47. FIG. 5H shows a schematic for generating F4 / 80- or CD8-depleted mice and subsequent treatment with intratumoural CD47-LLO or anti-CD47. FIG. 51 shows the efficiency of F4 / 80+cell depletion, which was evaluated ex vivo, in mouse tumors after treatment with anti-CD47, CD47-LLO, or anti-CSF-IR antibody. The results showed a significant decrease in the percentage of F4 / 80+cells in the LLO-CD47+aCSFlR group relative to the LLO-CD47 treated group (P=0.0005). FIG. 5 J shows tumor volumes for WT mice inoculated with EO771 tumours that were treated with anti-CD47+IgG, anti-CD47+anti- CSF1R, CD47-LLO+IgG, or CD47-LLO+anti-CSFlR antibody as described in the examples and FIG. 5H. Tumour-bearing animals were treated with intratumoural anti-CD47 or CD47- LLO. Tumour volumes were monitored and analyzed for the indicated periods. For anti- CSFlR-treated animals, n = 7 for anti-CD47 and n = 8 for CD47-LLO. For IgG-treated animals, n = 4 for anti-CD47 and n = 7 for CD47-LLO. The results showed a significant decrease in tumour volume in CD47-LLO treated animals compared to controls (P=0.0009). FIG. 5K shows tumour volume quantification at 10 days after treatment initiation as described in FIGs. 5J and 5H. The results showed a significant decrease in tumour volume at 10 days posttreatment initiation in CD47-LLO treated animals compared to anti-CD47 and CD47- LLO+anti-CSFlR treated animals (P=0.036 and P=0.01, respectively). FIG. 5L shows tumour volumes for WT mice inoculated with EO771 tumours that were treated with IgG, anti-CD8 antibody, CD47-LLO, or CD47-LLO+anti-CD8 antibody as described in FIG. 5H. Tumourbearing mice were treated with intratumoural anti-CD47 or CD47-LLO. Tumour volumes were monitored and analyzed for the indicated periods, n = 5 for all treatment groups. The results showed a significant decrease in tumour volume in CD47-LLO treated animals relative to controls (P<0.0001). FIG. 5M shows tumour volume quantification at 10 days after treatment initiation as described in the examples and FIGs. 5L and 5H. The results showed a significant decrease in tumour volume in CD47-LLO treated animals relative to IgG and CD4-LLO+anti- CD8 treated animals (P=0.008 and P=0.01, respectively). Data shown represent mean ± s.e.m. (FIGs. 3A, 3B, 3D, 5E, 5J, 5L) or mean ± s.d. (FIGs. 5B, 5C, 5D, 5K, 5M) analyzed by two- sided unpaired Student’s t test (FIGs. 3E, 5B, 5C, 5D, 5E), one-way analysis of variance with Tukey’s multiple comparisons test (FIGs. 3B, 5K, 5M), two-way analysis of variance with Tukey’s multiple comparisons test (FIGs. 3A, 5J, 5L), or two-sided log-rank (Mantel-Cox) test (3E). FIG. 5N shows the efficiency of CD8 T-cell depletion, which was evaluated ex vivo, in mouse spleens after treatment with IgG, CD47-LLO, or anti-CD8 antibody. The results showed a significant decrease in the percentage of spleen CD8 T cells in the group treated with anti-CD8 antibody relative to the IgG treated group (P<0.0001) as well as in the LLO- CD47+anti-CD8 treated group relative to the LLO-CD47 treated group (P<0.0001). FIG. 50 shows confocal fluorescence microscopy images of CDl lc+ cells (arrowheads) in 4TlBr4 tumours after intratumoural injection of anti-CD47 or CD47-LLO. Scale bar, 10 pm. The results showed an increase in CDl lc+ cells at the tumour site in CD47-LLO treated mice compared to anti-CD47 controls. FIG. 5P shows confocal fluorescence microscopy images of CD4+ cells (arrows) in 4TlBr4 tumours after intratumoural injection of anti-CD47 or CD47- LLO. The results showed an increase in CD4+ cells at the tumour in CD47-LLO treated mice compared to anti-CD47 treated mice. FIG. 5Q shows confocal fluorescence microscopy images of CDl lc+, CD4+, and CD8+ (CDl lc+ / CD4+ / CD8+) cell clusters (dashed circles) in 4TlBr4 tumours after intratumoural injection of anti-CD47 or CD47-LLO. The results showed an increase in the number of CDl lc+ / CD4+ / CD8+ cell clusters at the tumour in CD47-LLO treated animals relative to anti-CD47 treated animals.

[0144] FIGs. 6A-6AE, CD47-LLO drove systemic antitumour immunity to inhibit breast cancer metastasis. FIGs. 6A-6B BALB / cJ mice were inoculated with luciferaseexpressing 4TlBr4 (Luc-4T1) tumour cells and treated with intratumoural CD47-LLO or anti- CD47 as indicated in FIG. 2Y. FIG. 6A shows representative bioluminescent images of Luc- 4TlBr4 breast tumours before tumour debulking (i.e., on day 12 after tumour inoculation) and after tumour debulking (i.e., days 16-36). n = 7 for intratumoural CD47-LLO and n = 7 for intratumoural anti-CD47. The results showed decreased tumor metastases in mice treated withCD47-LLO relative to mice treated with anti-CD47. FIG. 6B shows quantified signal intensity of Luc-4TlBr4 breast tumours before tumour debulking (i.e., on day 12 after tumour inoculation) and after tumour debulking (i.e., days 16-36). n = 7 for intratumoural CD47-LLO and n = 7 for intratumoural anti-CD47. The results showed a significant reduction in tumor radiance over time in CD47-LLO treated mice compared to anti-CD47 treated mice (P=0.0060). FIG. 6C shows survival curves that were plotted and analyzed for mice in each treatment arm (CD47-LLO or anti-CD47). The results showed a significant extension in survival of tumour bearing mice treated with CD47-LLO relative to tumour bearing mice treated with anti-CD47 (P=0.0031). FIG. 6D shows PD1 protein expression levels that were detected by immuno staining in tumour paraffin-embedded sections from anti-CD47 or CD47- LLO treated mice. The representative images represent random fields of view from 1 of 3 biologically independent mice. Arrowheads show CD8+cells co-labeled with anti-PDl. Scale bar, 20 pm. The results showed an increase in the number of CD8+cells expressing PD1 in CD47-LLO treated mice relative to anti-CD47 treated mice. FIG. 6E shows a quantification of CD8+cells that co-labeled with anti-PDl (CD8+ / PD1+; n = 3 biologically independent tumours per condition, with >1400 DAPI+cells counted per tumour from at least 5 randomly selected fields of view). The results showed a significant increase in the percentage of CD8+ / PD1+cells in CD47-LLO treated mice relative to anti-CD47 treated mice (P=0.0104). FIG. 6F shows PDL1 protein expression levels detected by immunostaining in tumour paraffin-embedded sections. Representative images from random fields of view from 1 of 3 biologically independent mice. The results showed an increase in the expression of PDL1 in CD47-LLO treated mice relative to anti-CD47 treated mice. FIG. 6G shows a quantification of DAPI+cells that co-labeled with anti-PDLl (DAPI+ / PD-Ll+;n = 3 biologically independent tumours per condition, with >800 DAPI+cells counted per tumour from 5 randomly selected fields of view). The results showed a significant increase in the percentage of DAPI+ / PD-L1+cells in CD47-LLO treated mice relative to anti-CD47 treated mice (P=0.0367). FIGs. 6H-6I show flow cytometry analysis (FIG. 6H) and quantification (FIG. 61) of CD8+ T cells isolated from 4TlBr4 tumours treated with intraperitoneal CD47-LLO, intraperitoneal anti-CD47, intraperitoneal CD47-LLO plus anti-PDl, or intraperitoneal anti-CD47 + anti-PDl. n = 5 for intraperitoneal anti-CD47 and CD47-LLO, n = 6 for intraperitoneal anti-CD47 + anti-PDl, and n = 8 for intraperitoneal CD47-LLO + anti-PDl. The results showed a significant increase in the relative number of CD8+T cells isolated from CD47-LLO+anti-PD-l treated mice relative to controls (P=0.0016). FIG. 6J shows a schema for generating in vivo syngeneic orthotopic breast cancer models for intraperitoneal injection of CD47-LLO, anti-CD47, and anti-PDl.FIGs. 6K-6L show flow cytometry analysis (FIG. 6K) and quantification (FIG. 6L) of PD-1 negativity in CD8 T cells isolated from 4TlBr4 tumours treated with anti-CD47, CD47-LLO, anti-CD47+ anti-PD-1, or CD47-LLO + anti-PD-1. Data show mean ± s.d. (b) analyzed by one-way analysis of variance with Tukey’s multiple comparisons test. For FIGs. 6M-6N, BALB / cJ mice were inoculated with Luc-4TlBr4 tumours and treated with intraperitoneal CD47-LLO or anti-CD47 with or without anti-PDl as indicated in FIG. 6J. FIG. 6M shows representative bioluminescent images of Luc-4TlBr4 breast tumours before tumour debulking (i.e., day 12 after tumour inoculation) and after tumour debulking (i.e., days 16-36). n = 4 for intraperitoneal CD47-LLO, n = 7 for intraperitoneal anti-CD47. For mice treated with anti- PDl, n = 9 for intraperitoneal anti-CD47 and n = 6 for intraperitoneal CD47-LLO. The results showed a synergistic effect between CD47-LLO and anti-PD-1 antibody in decreasing tumor metastasis relative to monotherapy or controls. FIG. 6N shows quantified signal intensity of Luc-4TlBr4 breast tumours before tumour debulking (i.e., day 12 after tumour inoculation) and after tumour debulking (i.e., days 16-36). n = 4 for intraperitoneal CD47-LLO, n = 7 for intraperitoneal anti-CD47. For mice treated with anti-PDl, n = 9 for intraperitoneal anti-CD47 and n = 6 for intraperitoneal CD47-LLO. The results showed a decrease in tumor radiance over time in mice treated with CD47-LLO+anti-PD-l relative to anti-CD47+anti-PD-l and CD47- LLO controls (P=0.0092 and P<0.0001, respectively). FIG. 60 shows quantification of EO771 tumours taken at predetermined times after intraperitoneal injection of IR800CW-tagged CD47-LLO (100 ug). n = 4 biologically independent mice. FIG. 6P-6Q show ex vivo fluorescence images (FIG. 6P) and quantification of tumor and major organs collected at 36 h after intraperitoneal administration (FIG. 6Q). FIG. 6R shows survival curves that were plotted and analyzed for mice in each treatment arm (anti-CD47, CD47-LLO, anti-CD47+anti- PD-1, or CD47-LLO+anti-PD-l). The results showed a significant extension in survival of mice treated with CD47-LLO+anti-PD-l relative to controls (P=0.0007). Data shown represent mean ± s.e.m. (6B, 6E, 6G, 6N) or mean ± s.d. (61) analyzed by two-sided unpaired Student’s t test (6E, 6G), Welch analysis of variance with Dunnet’s multiple comparisons test (61), two- way analysis of variance with Benjamini, Krieger, and Yekutieli multiple comparisons test (6B, 6N), or two-sided log-rank (Mantel-Cox) test (6C, 6R). FIGs. 6S-6T show in vivo toxicity of CD47-LLO. FIG. 6S shows lymphocyte counts at day 2 and day 9 after intraperitoneal injection of drug (50 pg IgG, 50 pg anti-CD47, 100 pg CD47-LLO, or 100 pg CD47-LLO + 200 pg anti-PDl). The results showed insignificant differences in the number of lymphocytes at day 9 between control treated and CD47-LLO treated (P=0.1021) or CD47-LLO+anti-PD-l treated (P=0.6986). FIG. 6T shows red blood cell (RBC) counts at day 2 and day 9 after druginjection. The results showed insignificant differences in RBC counts between CD47- LLO+anti-PD- 1 treated animals relative to controls at day 2 (P=0.1058) and day 9 (P=0.5750). FIG. 6U shows serum blood urea nitrogen (BUN) levels at day 9 after drug injection. The results showed insignificant differences in the BUN levels between CD47-LLO+anti-PD-l treated animals relative to controls (P=0.3501). FIG. 6V shows Serum aspartate transaminase / alanine transaminase (AST / ALT) levels at day 9 after drug injection. The results showed insignificant differences in the AST / ALT levels between CD47-LLO+anti-PD-l treated animals relative to controls (P=0.0642). FIG. 6W shows body weight changes in mice at day 2 and day 9 after drug injection. The results showed insignificant differences in body weight (% change) between CD47-LLO+anti-PD-l treated animals relative to controls at day 2 (P=0.8670) and day 9 (P=0.5674). FIG. 6X shows HE staining of paraffin sections of major organs two days after intraperitoneal injection of CD47-LLO or CD47-LLO and anti-PD-1. Scale bar, 200 pm. The results showed lack of changes in tissue morphology, necrosis, or inflammation in mice treated with CD47-LLO or CD47-LLO+anti-PD-l relative to control. FIG. 6Y shows serum IL-6 levels at 4 hours after drug injection measured by enzyme-linked immunosorbent assay (ELISA). Data show n = 4 C57BL6 mice per treatment group. FIG. 6Z shows serum IL-ip levels at 4 hours after drug injection measured by ELISA. Data show n = 4 C57BL6 mice per treatment group. FIG. 6AA shows serum concentrations of anti-LLO antibody at 6 weeks after drug injection by sandwich immunogenicity assay. Data show n = 3 mice per IgG treatment group and n = 5 mice per CD47-LLO treatment group. Data shown represent mean ± s.d. (FIGs. 6S-6W, 6Y-6AA) analyzed by one-way analysis of variance with Tukey’s multiple comparisons test (FIGs. 6S-6W) or two-sided unpaired Student’s t test (FIGs. 6Y-6AA). FIG. 6AB shows 4TlBr4-tumour-bearing mice treated with intratumoural CD47-LLO or intraperitoneal CD47-LLO that survived >80 days after tumour inoculation were re-inoculated with 1 million 4TlBr4 tumour cells. Tumour volumes were monitored and analyzed for the indicated periods and compared with 4TlBr4 tumour-naive animals, n = 4 tumour-naive animals, n = 3 intratumoural CD47-LLO animals, n = 4 intraperitoneal CD47- LLO animals. Data shown represent mean ± s.e.m. FIG. 6AC shows a schematic illustrating the isolation of splenic CD8+ T cells from long-term 4TlBr4 tumour survivors and establishment of 4TlBr4 or EO771 co-cultures. FIGs. 6AD-6AE show ELISPOT assay (FIG. 6AD) and quantification (FIG. 6AE) of interferon (IFN)-gamma release from splenic CD8+ T cells co-cultured with EO771 or 4TlBr4 cells, n = 3 biologically independent samples. Data shown represent mean ± s.e.m. (FIGs. 6B, 6E, 6G, 6N, 6AB) or mean ± s.d. (FIGs. 61, 6AE) analyzed by two-sided unpaired Student’s ttest (FIGs. 6E, 6G, 6AE), Welch analysis ofvariance with Dunnet’ s multiple comparisons test (FIG. 61), two-way analysis of variance with Benjamini, Krieger, and Yekutieli multiple comparisons test (FIGs. 6B, 6N), or two-sided logrank (Mantel-Cox) test (FIGs. 6C, 6R).

[0145] FIG. 7 shows how co-therapy of CD47-LLO and anti-PD-1 antibody displayed a synergistic effect in increasing CD8+ T cells relative to monotherapy or controls. Provided are representative flow cytometry plots of the analysis of CD8+ T cells isolated from 4TlBr4 tumours treated with intraperitoneal CD47-LLO, intraperitoneal anti-CD47, intraperitoneal CD47-LLO+ anti-PDl, or intraperitoneal anti-CD47 + anti-PDl. n = 4 for intraperitoneal anti-CD47 and CD47-LLO, n = 5 for intraperitoneal anti-CD47 + anti-PDl, and n = 7 for intraperitoneal CD47-LLO + anti-PDl.

[0146] FIGs. 8A-8F, show the apoptotic effects of CD47-LLO on various tumor cell lines. FIG. 8A shows apoptosis rates for THP1 cells treated with CD47-LLO or Listeriolysin O for 24 hours. The results showed a lower percentage of apoptotic THP1 cells (Annexin V+) in the CD47-LLO treated group relative to the LLO treated group. FIG. 8B shows an MTS assay of a THP1 cell line treated with CD47-LLO for 48 hours. The results showed a stable relative number of THP1 cells with increasing concentrations of CD47-LLO (0 pg / mL to 8 pg / mL). FIG. 8C shows an MTS assay of a D4M.3A cell line treated with CD47-LLO for 48 hours. The results showed a modest decrease in the relative number of D4M.3A cells with increasing concentrations of CD47-LLO (0.125 pg / mL to 8 pg / mL). FIG. 8D shows an MTS assay of an LLC cell line treated with CD47-LLO for 48 hours. The results showed a gradual decrease in the relative number of LLC cells with increasing concentrations of CD47-LLO (0 pg / mL to 8 pg / mL). FIG. 8E shows an MTS assay of an EO771 cell line treated with CD47- LLO for 48 hours. The results showed a marked decrease in the relative number of EO771 cells with increasing concentrations of CD47-LLO (0 pg / mL to 8 pg / mL). FIG. 8F shows an MTS assay of a MDA-MB-468 cell line treated with CD47-LLO for 48 hours. The results showed a modest decrease in the relative number of MDA-MB-468 cells with increasing concentrations of CD47-LLO (0 pg / mL to 8 pg / mL).

[0147] FIGs. 9A-9C, elucidate the in vivo antitumour effect of intratumoural CD47- LLO in a 4TlBr4 Model. FIG. 9A shows 4TlBr4 tumour volumes after intratumoural injection of anti-CD47 or CD47-LLO (LLO-CD47). Mice were treated as described in FIG. 2Y. The results showed a significantly lower tumour volume in mice treated with CD47-LLO relative to mice treated with anti-CD47 (P=0.0005). FIG. 9B shows a flow cytometry plot analysis of CD86+and CD206+tumour-associated macrophages (TAMs) (CD86+ / CD206+) in 4TlBr4 tumours at day 16 after tumour inoculation in each group (anti-CD47 or CD47-LLO).N = 4 biologically independent experiments. The results showed a lower percentage of CD86+ / CD206+TAMs from mice treated with CD47-LLO relative to mice treated with anti- CD47. FIG. 9C shows a quantification of CD86+and CD206+tumour-associated macrophages in 4TlBr4 tumours at day 16 after tumour inoculation in each group, n = 4 biologically independent experiments. The results showed a significantly lower ratio of CD206+to CD86+cells in the group treated with CD47-LLO relative to the group treated with anti-CD47 (P=0.002).

[0148] FIG. 10 shows how mice treated with IT CD47-LLO or IP CD47-LLO+anti- PD-1 had reduced tumour volume relative to untreated mice. 4TlBr4-tumour-bearing mice treated with intratumoural CD47-LLO that survived >60 days after tumour inoculation or intraperitoneal CD47-LLO that survived > 80 days after tumour inoculation that were reinoculated with 1 million 4TlBr4 tumour cells. Tumour volumes were monitored and analyzed for the indicated periods and compared with 4TlBr4 tumour-naive animals. n=4 tumour-naive animals, n=3 intratumoural (IT) CD47-LLO animals, n=4 intraperitoneal (IP) CD47-LLO animals. Data shown represent mean ± s.e.m.

[0149] FIGs. 11A-11E, are in vitro toxicity assays with BMDM and cancer cell lines showing the effects of LLO-CD47 relative to LLO alone. FIG. 11A shows an apoptotic assay by flow cytometry of THP1 cells treated with LLO-CD47 or Listeriolysin O for 24 hours. The results showed a significant lower level of THP1 apoptosis in the CD47-LLO treated group compared to LLO (P<0.0001). FIG. 11B shows an MTS assay for an MDA-MB-468 cell line treated with CD47-LLO (LLO-CD47) or Listeriolysin O for 48 hours. The results showed a difference in the relative cell numbers between LLO and CD47-LLO treatments. FIG. 11C shows an MTS assay for an THP1 cell line treated with LLO-CD47 or Listeriolysin O for 48 hours. The results showed a difference in the relative cell numbers between LLO and CD47- LLO treatments. FIG. 11D shows an MTS assay for an EO771 cell line treated with LLO- CD47 or Listeriolysin O for 48 hours. The results showed a difference in the relative cell numbers between LLO and CD47-LLO treatments. FIG. HE shows an MTS assay for an LLC cell line treated with LLO-CD47 or Listeriolysin O for 48 hours. The results showed a difference in the relative cell numbers between LLO and CD47-LLO treatments.

[0150] FIGs. 12A-12B, are schematic representations of breast cancer model treatment schedules. FIG. 12A shows a schematic of the generation of breast cancer mouse models and treatment schema with IT CD47-LLO (LLO-CD47), anti-CD47, anti-CSFIR, anti- CD8, or a combination thereof. FIG. 12B shows a schematic of the generation of breast cancermouse models and treatment schema with IP CD47-LLO (LLO-CD47), anti-CD47, anti-PD-1, or a combination thereof.

[0151] FIGs. 13A-13B, shows how CD47-LLO increased IFNp expression levels and PE-H-2Kb binding to SIINFEKL relative to controls. FIG. 13A shows RT-PCR results of interferon-beta (IFNP) expression levels in BMDM co-cultured with EO771 for 24 hours. BMDM were collected from n=3 C57BL6 mice, experiments were performed in three batches. The results showed a significant increase in the relative expression of IFNP with treatment of CD47-LLO (LLO-CD47) relative to control (P=0.0082). FIG. 13B shows representative flow cytometry results of antigen presentation assays from BMDMs that were co-cultured with EO771-OVA cells for 48 hours. BMDM were collected from n=6 mice, experiments performed in two batches. The results showed an increase in the PE-H-2Kb bound to SIINFEKL from cells treated with CD47-LLO (LLO-CD47) relative to anti-CD47 (aCD47) or IgG controls.

[0152] FIGs. 14A-14E, are in vivo toxicity data showing a general lack of toxicity of CD47-LLO relative to controls. FIG. 14A shows body weight change of mice after IP injection of 50 pg of IgG, anti-CD47, or CD47-LLO (LLO-CD47). The results showed insignificant differences in body weight (% change) between CD47-LLO treated animals relative to controls at day 2 (P=0.11) and day 9 (P=0.26). FIG. 14B shows red blood cell (RBC) counts after drug injection. The results showed insignificant differences in RBC counts between CD47-LLO treated animals and controls at baseline (P=0.67), day 2 (P=0.07) and day 9 (P=0.67). FIG. 14C shows lymphocyte counts after drug injection. The results showed insignificant differences in lymphocyte counts between CD47-LLO treated animals and controls at baseline (P=0.4281) and day 9 (P=0.282), but significant differences at day 2 (P=0.0003). FIG. 14D shows BUN levels in the serum after 2 and 9 days of drug injection. The results showed insignificant differences in the BUN levels between CD47-LLO treated animals and controls (P=0.42). FIG. 14E shows AST / ALT levels in the serum after 2 and 9 days of drug injection. The results showed insignificant differences in the BUN levels between CD47-LLO treated animals and controls (P=0.26).

[0153] FIGs. 15A-15C, show how CD47-LLO displayed improved in vivo characteristics relative to controls. FIG. 15A shows 4T1 tumor volume after intratumoral injection of anti-CD47 or CD47-LLO (LLO-CD47). The results showed a lower tumour volume over time in CD47-LLO treated animals relative to anti-CD47 treated animals. FIG. 15B shows the efficiency of CD8 T cell depletion that was evaluated ex vivo in mouse spleens following treatment with IgG, CD47-LLO (LLO-CD47), or anti-CD8 antibody. The results showed significant CD8 T cell depletion with anti-CD8 antibody relative to IgG control(P<0.0001) and significant CD8 T cell depletion with anti-CD8 antibody in CD47-LLO treated mice relative to CD47-LLO treated mice (P<0.0001). FIG. 15C shows the efficiency of F4 / 80+cell depletion that was evaluated ex vivo in mouse spleens following treatment with anti-CD47, CD47-LLO (LLO-CD47), or anti-CSF-IR antibody (MD). The results showed a significant depletion of F4 / 80+cells in mice treated with CD47-LLO+anti-CSF-IR antibody (LLO-CD47- MD) relative to CD47-LLO treated mice (P=0.0005).

[0154] FIG. 16 shows flow cytometry gating and analysis of pSTING levels in CDl lb+BMDMs isolated from co-cultures with EO771 cells.

[0155] FIG. 17 shows a BMDM gating strategy for pSTING for FIG. 2AE and FIG. 16.

[0156] FIG. 18 shows a BMDM gating strategy for CD86 and CD206 expression for FIG.2AN and FIG. 2AO.

[0157] FIG. 19 shows a gating strategy for tumor-infiltrating T lymphocytes for FIG. 7.

[0158] FIG. 20 shows a tumor-infiltrating CDl lb+gating strategy for CD86 and CD206 expression for FIG. 9B.

[0159] FIG. 21 shows a tumor-infiltrating CD45+gating strategy for FIGs. 4A-4B, 4M- 4N, 4V-4X, 4AA-4AE, 4J-4L, 4Q-4U, 4Y-4Z, and 4AF-4AG.

[0160] FIGs. 22A-22M show the different gating strategies used herein and the uncropped Western Blot of FIG. 2. FIG. 22A shows the gating strategy for tumour-infiltrating T lymphocytes for FIGs. 3 and 6. FIG. 22B shows the gating strategy for tumour-infiltrating macrophages of FIG. 3. FIG. 22C shows the gating strategy for splenic T-cells of FIG. 3. FIG. 22D shows the gating strategy for tumour-infiltrating CD45+ cells of FIG. 4, FIG. 5. FIG. 22E shows the gating strategy for T-cell proliferation assay of FIG2. FIG. 22F shows the gating strategy for BMDC SIINFEKL-H2Kb of FIG. 2. FIG. 22G shows the gating strategy for BMDC phagocytosis assay of FIG. 2. FIG. 22H shows the gating strategy for BMDM acridine orange of FIG. 2. FIG. 221 shows the gating strategy for BMDM phagocytosis of FIG. 2. FIG. 22J shows the gating strategy for BMDM SIINFEKL-H2KB of FIG. 2. FIG. 22K shows the gating strategy for BMDM pSTING of FIG. 2. FIG. 22L shows the gating strategy for BMDC pSTING of FIG. 2. FIG. 22M shows the uncropped Western Blot of FIG. 2.

[0161] FIGs. 23A-23K show that magrolimab conjugated with LLO (magrolimab-LLO) promotes macrophage polarization, phagocytosis and anti-tumor activity using human cancer cell lines via a mechanism similar to other antibody-LLO conjugates described herein. FIG. 23A shows conjugation and purification of magrolimab antibody (isotype IgG4) with LLO. Lane 1 is Lot #1, magrolimab-LLO, Lane 2 is Lot #2 magrolimab-LLO, Lane 3 is Lot #1magrolimab-LLO reduced with beta-mercaptoethanol, and Lane 4 is Lot #2 magrolimab-LLO reduced with beta-mercaptoethanol. FIGs. 23B-E show in vitro toxicity assays of magrolimab- LLO with human cancer cell lines. Flow cytometry analysis (FIGs. 23B-D) and quantification of apoptosis and necrosis (FIG. 23E) in A549 lung carcinoma cells after treatment with 2 pg / mL magrolimab-LLO for 0, 6, or 24 hours (n = 3 biologically independent experiments). FIGs. 23F-I show that magrolimab-LLO promotes macrophage polarization to an Ml phenotype. Bone marrow-derived macrophages (BMDM) were co-cultured with human A549 lung cancer cells in the presence of IgG, magrolimab, or magrolimab-LLO followed by analysis by flow cytometry (FIGs. 23F-H) and quantification of CD86 and CD206 expression in BMDMs isolated from EO771 co-cultures (FIG. 231). BMDMs were collected from n = 3 C57BL6 mice. FIGs. 23J-K show that magrolimab-LLO increases macrophage phagocytosis. FIG. 23J shows a schematic of experimental design. FIG. 23K shows quantification of human A549 DNA isolated from BMDMs by quantitative PCR. This experiment was repeated independently twice with n = 3 technical replicates shown.DETAILED DESCRIPTION

[0162] Generating antitumor immune responses generally requires the phagocytosis of tumor cells and subsequent cross-presentation of tumor-derived antigens by antigen-presenting cells. However, these processes are impeded by phagocytosis checkpoints and inefficient cytosolic transport of antigenic peptides from phagolysosomes. In some embodiments, the current disclosure provides methods and / or compositions comprising novel antigen targeting drug conjugates, such as antibody-drug conjugates (ADCs) (used herein interchangeably with antibody-toxin conjugates, ATCs), that target an antigen of interest on a diseased cell, such as a cancer cell. In some embodiments, an antigen targeting agent comprises an antibody or antigen binding fragment derived therefrom. In some embodiments, an antigen targeting agent comprises a ligand (natural or modified) of the target antigen. In some embodiments, an antigen of interest comprises, or is, cluster of differentiation 47 (CD47). In some embodiments, an antigen of interest comprises, or is, CD47. In some embodiments, a drug conjugate comprises an antigen binding agent linked to a bacterial toxin. In some embodiments, a bacterial toxin comprises Listeriolysin O (LLO), derived from the intracellular bacterium Listeria monocytogenes. In some embodiments, a drug is conjugated to an antigen binding agent through a cleavable linker. In some embodiments, a drug is conjugated to an antigen binding agent through a non-cleavable linker. In some embodiments, a composition comprises a CD47 binding agent conjugated through a cleavable linker to LLO. In some embodiments, acomposition comprises, or is, an ADC comprising an anti-CD47 antibody or antigen binding fragment derived thereof conjugated, optionally via a cleavable linker, to LLO (“CD47-LLO” or “LLO-CD47”). Also provided herein are methods for treating diseases, such as cancer, comprising administering CD47-LLO conjugates. In some embodiments, CD47-LLO promotes the phagocytosis of cancer cells followed by the release and activation of LLO to form pores on phagolysosomal membranes. In some embodiments, formation of phagolysosomal membrane pores allows cytosolic entry of phagolysosomal contents (e.g., cellular nucleic acids, cellular peptides, LLO, tumor-derived contents, etc.), leading to enhanced antigen cross-presentation of peptides (e.g., tumor-derived peptides, cellular peptides, etc.) and activation of cytosolic immune sensors. In some embodiments, technologies provided herein can be utilized to reduce the rates of cancer metastasis. In some embodiments, technologies provided herein can be utilized to reduce the rates of cancer metastasis following de-bulking of a tumor.

[0163] In some embodiments, provided herein are the first antibody drug conjugate (ADC) specifically designed to promote improved recognition of cancer cell by immune cells and to facilitate the functions of the body's immune system to eliminate cancer. In some embodiments, provided herein are the first antibody drug conjugate (ADC) specifically designed not to kill cancer cells per se, but rather to promote improved recognition of cancer cell by immune cells and to facilitate the functions of the body's immune system to eliminate cancer. In some embodiments, provided herein are compositions and methods that are uniquely different from those previously described, in that instead of directly killing cancer cells, compositions and / or methods described herein are designed to promote cancer cells to be more effective in activating an individual’s antitumor immune responses.

[0164] In some embodiments, provided herein are conjugated antibodies and / or antigen targeting agents that are designed to efficiently activate an individual’s anti-tumor immune response, such as through antigen presentation cells (APCs). In some embodiments, provided herein are conjugated antibodies designed to efficiently activate an anti-tumor immune response in APCs. In some embodiments, provided herein are antigen targeting agents conjugated to a bacterial protein. In some embodiments, provided herein are antigen targeting agents conjugated to a bacterial peptide. In some embodiments, provided herein are antigen targeting agents conjugated to a bacterial toxin. In some embodiments, provided herein are antigen targeting agents conjugated to Listeriolysin O (LLO). In some embodiments, provided herein is a composition comprising an anti-CD47 antibody conjugated to Listeriolysin O (LLO). In some embodiments, conjugation of an anti-CD47 antibody and LLO is through acleavable linker. In some embodiments, provided herein are methods of using compositions to treat a disease for which an improved immune response can be therapeutic, such as, but not limited to cancer. In some embodiments, provided herein are methods of treating cancer using compositions described herein combined with co-therapies such as but not limited to surgery, chemotherapy, radiotherapy, and / or immunotherapy. In some embodiments, a combination of compositions provided herein and a co-therapy result in a synergistic antitumor response.

[0165] In some embodiments, a target antigen comprises, consists essentially of, or consists of CD47. While not being bound by theory, canonically, CD47 is known to act as a “do not eat me” signal expressed on the surface of cancer cells. CD47 overexpression can help cancer cells evade capture and / or recognition by innate immune cells. In some embodiments, the blockade of CD47 can enhance innate immune response against a tumor and induce downstream adaptive immune response. In some embodiments, an anti-CD47 antibody and / or CD47 binding agent is undergoing clinical development. In some embodiments, an anti-CD47 antibody and / or CD47 binding agent is one described in Hua Yang et al., Biomarkers Research (2023), 11:15; which is incorporated herein by reference for the purpose described herein.

[0166] In some embodiments, provided herein are improvements to the potential treatment / therapeutic efficacy of anti-CD47 therapies. In some embodiments, provided herein are improvements to the potential treatment / therapeutic efficacy of treatments comprising LLO. In some embodiments, through conjugation with a pore forming protein, e.g., the bacterial protein LLO, the conjugate (e.g., CD47-LLO) can elicit a significantly stronger anti- tumoral immune response and / or reduced cytotoxicity in an individual when compared with an anti-CD47 agent and / or the pore forming agent (e.g., LLO) utilized alone. In some embodiments, CD47-LLO conjugates maintain tolerably low levels of toxicity and / or reduce toxicity relative to the constituent parts. In some embodiments, following release of an LLO protein from a CD47 conjugate (e.g., through exposure to reduced conditions), LLO can form pores on the phagosomes, letting the contents of the phagosome (e.g., cancer cell components) escape into the cell cytoplasm. In some embodiments, an anti-CD47 antibody blocks the CD47 molecule on the cancer cells (e.g., is a neutralizing antibody) and thus promotes phagocytosis of the cancer cell by immune cells such as but not limited macrophages and / or dendritic cells. In some embodiments, once inside the phagosome of an immune cell (e.g., macrophage, dendritic cell, etc.), a conjugated LLO protein detaches from an antibody and forms pores on the phagosome, which helps the contents in the phagosome to leak to the cytoplasm of the immune cell. As the result, this conjugated antibody can release the phagolysosomal contents, e.g., the cancer cell DNA and cancer cell proteins, from the phagosomes to the cytoplasm ofthe APCs thus activating a robust anti-tumor immune response. In some embodiments, the escaped phagolysosomal contents (e.g., cancer DNA, etc.) can activate the DNA-sensing cGAS-STING pathway and / or the escaped phagolysosomal proteins (e.g., cancer peptides, LLO, etc.) can enhance antigen presentation. In some embodiments, the combination of activation of both the antigen presentation and DNA-sensing pathways can help to elicit priming of T cells to attack tumor cells. In some embodiments, provided herein are compositions and methods for inducing a strong downstream anti-tumoral immune response in an individual in need thereof. In some embodiments, provided herein are novel and surprisingly efficacious compositions with therapeutic functions that are significantly improved over current therapies, such as anti-CD47 therapies (e.g., improved tumor cell control, such as through improved anti-tumoral immune responses).

[0167] In some embodiments, provided herein are improvements to the potential treatment / therapeutic efficacy of ADC therapies. In some embodiments, provided herein are improvements to the potential treatment / therapeutic efficacy of ADC therapies conjugated to a pore forming agent. In some embodiments, provided herein are improvements to the potential treatment / therapeutic efficacy of ADC therapies conjugated to LLO. In some embodiments, provided herein are conjugation sites and linkages designed to make the conjugated drug remain inactive until cleaved. In some embodiments, provided herein are conjugation sites and linkages designed to make the conjugated drug remain inactive until the ADC enters a phagosome. In some embodiments, provided herein are conjugation sites and linkages designed to make the conjugated drug remain bound to the antibody until the ADC enters a phagosome together with a cancer cell. In some embodiments, provided herein are conjugation sites and linkages designed to make the conjugated drug remain generally inactive until the ADC enters a phagosome together with a cancer cell. In some embodiments, provided herein are conjugation sites and linkages designed to make the conjugated drug remain bound to the antibody until cleaved. In some embodiments, provided herein are conjugation sites and linkages designed to make the conjugated drug remain bound to the antibody and generally inactive until cleaved. In some embodiments, LLO protein is released and activated after entering a phagosome. In some embodiments, the toxicity of the ADC is well controlled and / or tolerated by a subject in need thereof. In some embodiments, the ADC is a therapy. In some embodiments, the ADC is an antibody-based therapy. In some embodiments, the ADC therapy is used in a clinical setting.

[0168] In some embodiments, provided herein are compositions comprising an antibody conjugated to LLO. In some embodiments, provided herein are compositions comprising ananti-CD47 antibody conjugated to LLO. In some embodiments, provided herein are compositions comprising an anti-PDLl antibody conjugated to LLO. In some embodiments, provided herein are compositions comprising an anti-HER2 antibody conjugated to LLO. In some embodiments, provided herein are compositions comprising an anti-CD24 antibody conjugated to LLO. In some embodiments, provided herein are compositions comprising an anti-EGFR antibody conjugated to LLO. In some embodiments, provided herein are compositions comprising an anti-EGFR2 antibody conjugated to LLO. In some embodiments, provided herein are compositions comprising an anti-BCMA antibody conjugated to LLO. In some embodiments, provided herein are compositions comprising an anti-CD20 antibody conjugated to LLO. In some embodiments, provided herein are compositions comprising an anti-CD22 antibody conjugated to LLO. In some embodiments, provided herein are compositions comprising an anti-CD38 antibody conjugated to LLO. In some embodiments, provided herein are compositions comprising an anti-CD19 antibody conjugated to LLO. In some embodiments, provided herein are compositions comprising an anti-TROP2 antibody conjugated to LLO. In some embodiments, provided herein are compositions comprising an anti-Nectin-4 antibody conjugated to LLO. In some embodiments, provided herein are compositions comprising an anti-GPRC5D antibody conjugated to LLO. In some embodiments, the composition of an antibody conjugated to LLO drastically enhances immune activation against cancer. In some embodiments, the composition of an antibody conjugated to LLO drastically enhances immune activation against cancer relative to unconjugated antibody therapy. In some embodiments, provided herein are methods of using antibodies conjugated to LLO to enhance immune activation against cancer. In some embodiments, provided herein are methods of using antibodies conjugated to LLO to treat cancer. In some embodiments, the cancer may be, but is not limited to, breast cancer, lung cancer, non-small cell lung cancer, brain cancer, glioblastoma, triple-negative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma. In some embodiments, the cancer may be a solid tumour. In some embodiments, the cancer may be a liquid cancer. In some embodiments, the cancer may be a blood cancer.

[0169] In some embodiments, provided herein are methods of generating and / or using novel antibody-toxin conjugates that do not rely on the toxin directly killing cancer cells, but rather rely on enabled phagocytosis of a cancer cell followed by escape of phagocytosed molecules (e.g., nucleic acid, proteins, cancer cell components, etc.) into the cytoplasm of the phagocyte (e.g., antigen presenting cells) for more effectively activating an immune response. In some embodiments, provided herein are methods of generating novel antibody-toxin conjugates that rely on enabled phagocytosis of a cancer cell followed by escape of phagocytosed molecules (e.g., nucleic acid, proteins, cancer cell components, etc.) into the cytoplasm of the phagocyte (e.g., antigen presenting cells) for more effectively activating an immune response.

[0170] In some embodiments, provided herein are compositions and methods for enhancing anti-CD47 therapy. In some embodiments, compositions of and methods of using anti-CD47 ADC provide drastic improvements against solid tumours relative to unconjugated anti-CD47 therapy. In some embodiments, compositions of and methods of using anti-CD47 ADC induce a strong immune response compared to unconjugated anti-CD47 therapy. In some embodiments, compositions of and methods of using anti-CD47 ADC address an unmet need by improving the efficacy of unconjugated anti-CD47 monotherapy. In some embodiments, provided herein are compositions of and methods of using anti-CD47 ADC in combination with other immunotherapies as a combination therapy. In some embodiments, provided herein are compositions of and methods of using anti-CD47 ADC in combination with tumor resection as a combination therapy. In some embodiments, provided herein are compositions of and methods of using anti-CD47 ADC in combination with anti-PD-1 antibody as a combination therapy. In some embodiments, combination therapy provides a synergistic effect in cancer treatment and / or immune activation.I. Definitions

[0171] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and / or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined.

[0172] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0173] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0174] The term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0175] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein when referring to a gene product.

[0176] “Homology,” or “identity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules share sequence identity at that position. A degree of identity between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non- homologous” sequence shares less than 60% identity, less than 50% identity, less than 40% identity, less than 30% identity, or less than 25% identity, with one of the sequences of the current disclosure.

[0177] The terms “amino portion,” “N-terminus,” “amino terminus,” and the like as used herein are used to refer to order of the regions of the polypeptide. Furthermore, when something is N-terminal to a region it is not necessarily at the terminus (or end) of the entire polypeptide, but just at the N-terminus of the region or domain. Similarly, the terms “carboxy portion,” “C- terminus,” “carboxy terminus,” and the like as used herein is used to refer to order of the regions of the polypeptide, and when something is C-terminal to a region it is not necessarilyat the terminus (or end) of the entire polypeptide, but just at the C-terminus of the region or domain.

[0178] The terms "polynucleotide," “nucleic acid,” and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single- stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double- stranded form.

[0179] Cells or a culture of cells are “substantially free” of certain reagents or elements, such as serum, signaling inhibitors, animal components or feeder cells, exogenous genetic elements or vector elements, as used herein, when they have less than 10% of the element(s), and are “essentially free” of certain reagents or elements when they have less than 1% of the element(s).

[0180] Cells or a culture of cells described herein as “essentially free” of certain reagents or elements, such as serum, signaling inhibitors, animal components or feeder cells, when the culture, matrix or medium respectively, can have a level of these reagents lower than a detectable level using conventional detection methods known to a person of ordinary skill in the art or these agents have not been extrinsically added to the culture, matrix or medium. The serum-free medium may be essentially free of serum.

[0181] The term "immune cell" as used herein refers to a cell that is part of the immune system and helps the body fight infections and other diseases. Immune cells include natural killer cells, invariant NK cells, NK T cells, T cells of any kind (e.g., regulatory T cells, CD4.sup.+ T cells, CDS. sup. + T cells, or gamma-delta T cells), B cells, monocytes,granulocytes, myeloid cells neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, and / or stem cells (e.g., mesenchymal stem cells (MSCs) or induced pluripotent stem (iPSC) cells).

[0182] A “gene,” “polynucleotide,” “coding region,” “sequence,” “segment,” “fragment,” or “transgene” which “encodes” a particular protein, is a nucleic acid molecule which is transcribed and optionally also translated into a gene product, e.g., a polypeptide, in vitro or in vivo when placed under the control of appropriate regulatory sequences. The coding region may be present in either a cDNA, genomic DNA, or RNA form. When present in a DNA form, the nucleic acid molecule may be single- stranded (i.e., the sense strand) or double- stranded. The boundaries of a coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A gene can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the gene sequence.

[0183] The term “optionally” as used herein refers to an element, step, or parameter that may or may not be utilized in any method, composition, or kit of the disclosure.

[0184] The term “cell” is herein used in its broadest sense in the art and refers to a living body which is a structural unit of tissue of a multicellular organism, is surrounded by a membrane structure which isolates it from the outside, has the capability of self-replicating, and has genetic information and a mechanism for expressing it. Cells used herein may be naturally-occurring cells or artificially modified cells (e.g., fusion cells, genetically modified cells, etc.).

[0185] Reference throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "an additional embodiment," or "a further embodiment" or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0186] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease.“Treatment,” as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0187] In some embodiments, the methods are useful for reducing the size and / or cell number of a tumor. In some embodiments, the method of the disclosure are useful for inhibiting the growth of tumors, such as solid tumors, in a subject.

[0188] The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[0189] The term “antibody” includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies and antibody fragments that may be human, mouse, humanized, chimeric, or derived from another species. A “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies that is being directed against a specific antigenic site.

[0190] “Antibody or functional fragment thereof’ or “antibody antigen binding fragment” means an immunoglobulin molecule that specifically binds to, or is immunologically reactive with a particular antigen or epitope, and includes both polyclonal and monoclonal antibodies. The term antibody includes genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies). The antibody may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. The term functional antibody fragment includes antigen binding fragments of antibodies, including e.g., Fab', F(ab')2, Fab, Fv, rlgG, and scFv fragments. The term scFv refers to a single chain Fv antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfidelinkages. The fragment may also optionally be a multimolecular complex. A functional antibody fragment retains the ability to bind its cognate antigen at comparable affinity to the full antibody.

[0191] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this disclosure. In contrast to polyclonal antibody preparations, which typically include several different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0192] The phrases “pharmaceutical composition” or “pharmacologically acceptable composition” refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. The preparation of a pharmaceutical composition comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.

[0193] As used herein, “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, and Ringer's dextrose), non-aqueous solvents (e.g., propylene glycol,polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition may be adjusted according to well-known parameters.

[0194] The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired responses discussed herein in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the effect desired. The actual dosage amount of a composition of the present embodiments administered to a patient or subject can be determined by physical and physiological factors, such as body weight, the age, health, and sex of the subject, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic interventions, idiopathy of the patient, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance. For example, a dose may also comprise from about 1 pg / kg / body weight to about 1000 mg / kg / body weight (this such range includes intervening doses) or more per administration, and any particular dose derivable therein. In non-limiting examples of a range derivable from the numbers listed herein, a range of about 5 pg / kg / body weight to about 100 mg / kg / body weight, about 5 pg / kg / body weight to about 500 mg / kg / body weight, etc., can be administered. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0195] The use of a single chain variable fragment (scFv) is of particular interest. scFvs are recombinant molecules in which the variable regions of light and heavy immunoglobulin chains encoding antigen-binding domains are engineered into a single polypeptide. Generally, the VH and VL sequences are joined by a linker sequence. See, for example, Ahmad (2012) Clinical and Developmental Immunology Article ID 980250, herein specifically incorporated by reference. Described herein are BCMA-specific scFv molecules that comprise the variable regions of light and heavy immunoglobulin chains encoding BCMA-binding domains that are engineered into a single polypeptide. Similarly, the CSl-specific scFv molecules describedherein comprise the variable regions of light and heavy immunoglobulin chains encoding CS1- binding domains that are engineered into a single polypeptide.

[0196] As used herein, the term “binding affinity” refers to the equilibrium constant for the reversible binding of two agents and is expressed as a dissociation constant (Kd). Binding affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4- fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater, or more (or any derivable range therein), than the binding affinity of an antibody for unrelated amino acid sequences. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms "immunoreactive" and "preferentially binds" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0197] The term "binding" refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.

[0198] A “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent, or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.

[0199] Subject” and “patient” refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.

[0200] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. As used herein "another" may mean at least a second or more.II. CompositionsCD47 Targeting AgentsA. Antibodies

[0201] In certain embodiments, compositions of the disclosure can be directed to cluster of differentiation 47 (CD47, aka integrin-associated protein (IAP)), i.e., comprising CD47targeting moieties. CD47 is an approximately 50 kDa glycosylated five transmembrane protein that is ubiquitously expressed by both hematopoietic cells such as T and B lymphocytes, monocytes, platelets and erythrocytes and non-hematopoietic cells. CD47 is involved in a range of cellular processes, including apoptosis, proliferation, adhesion, and migration. Furthermore, CD47 is known to play a key role in immune and angiogenic responses. CD47 has been found to be overexpressed in many different tumor cells. Because of this, anti-CD47 monoclonal antibodies have been proposed and studied as a therapeutic treatment for human cancers.

[0202] In some embodiments, a CD47 targeting agent comprises an anti-CD47 antibody or a fragment thereof. In some embodiments, a CD47 targeting agent neutralizes CD47. In some embodiments, a CD47 targeting agent comprises, but is not limited to, AO-176 (aka AO-104; Arch Oncology), CC-90002 (aka INB RX- 103; Cellgene), GenSci-059 (Changchun Genescience Pharmaceutical Co Ltd.), Lemzoparlimab (aka TJ011133, TJC4; LMab), Letaplimab (aka IBL188; Innovent), Ligufalimab (aka AK117; Akeso), Magrolimab (aka Hu5F9-G4; Gilead Sciences / Forty Seven), ZL-1201 (Zai Lab), IBL322 (Innovent), TG-1801 (Novimmune SA TG Therapeutics), B6H12 (Santa Cruz), BRIC126 (Santa Cruz), SRF231 (Surface Oncology), IMC-002 (ImmuneOncia Therapeutics, 3D Medicines), TQB2928 (Chia Tai Tianqing Pharmaceutical Group Co., Ltd.), PF-07257876 (Pfizer), HX009 (Hanxbio), STI- 6643 (Sorrento Therapeutics, Inc.) and / or MIAP410. The term “antibody” refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes chimeric, humanized, fully human, and bispecific antibodies. As used herein, the terms “antibody” or “immunoglobulin” are used interchangeably and refer to any of several classes of structurally related proteins that function as part of the immune response of an animal, including IgG, IgD, IgE, IgA, IgM, and related proteins, as well as polypeptides comprising antibody Complementarity Determining Region (CDR) domains that retain antigen-binding activity.

[0203] In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of AO-176 (aka AO-104; Arch Oncology). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of CC-90002 (aka INB RX- 103; Cellgene). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of GenSci-059 (Changchun Genescience Pharmaceutical Co Ltd.). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of Lemzoparlimab (aka TJ011133, TJC4; LMab). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of Letaplimab (aka IBL188; Innovent). In someembodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of Ligufalimab (aka AK117; Akeso). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of Magrolimab (aka Hu5F9- G4; Gilead Sciences / Forty Seven). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of ZL-1201 (Zai Lab). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of IBL322 (Innovent). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of TG-1801 (Novimmune SA TG Therapeutics). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of B6H12 (Santa Cruz). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of BRIC126 (Santa Cruz). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of SRF231 (Surface Oncology). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of IMC- 002 (ImmuneOncia Therapeutics, 3D Medicines). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of TQB2928 (Chia Tai Tianqing Pharmaceutical Group Co., Ltd.). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of PF-07257876 (Pfizer). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of HX009 (Hanxbio), In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of STL6643 (Sorrento Therapeutics, Inc.). In some embodiments, a CD47 targeting agent comprises, does not comprise, consists essentially of, or consists of MIAP410.

[0204] In some embodiments, a CD47 targeting agent comprises an Fc region derived from IgGl, IgG2, IgG2b, and / or IgG4. In some embodiments, an IgG2 comprising CD47 targeting agent comprises AO-176. In some embodiments, an IgG2b comprising CD47 targeting agent comprises BRIC126. In some embodiments, an IgG4 comprising CD47 targeting agent comprises Magrolimab, B6H12, BRIC126, SRF231, MIAP410, AO-176, CC-90002, GenSci- 059, Lemzoparlimab, Letaplimab, Ligufalimab, ZL-1201, IBL322, TG-1801, IMC-002, TQB2928, PF-07257876, HX009, and / or STI-6643.

[0205] The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody. An antigen may possess one or more epitopes that are capable of interacting with different antibodies.

[0206] The term “epitope” includes any region or portion of molecule capable eliciting an immune response by binding to an immunoglobulin or to a T-cell receptor. Epitope determinants may include chemically active surface groups such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen within a complex mixture.

[0207] The epitope regions of a given polypeptide can be identified using many different epitope mapping techniques are well known in the art, including: x-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, protein display arrays, see, e.g., Epitope Mapping Protocols, (Johan Rockberg and Johan Nilvebrant , Ed., 2018) Humana Press, New York, N.Y. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82:178-182 (1985); Geysen et al. Molec. Immunol. 23:709-715 (1986 See, e.g., Epitope Mapping Protocols, supra. Additionally, antigenic regions of proteins can also be predicted and identified using standard antigenicity and hydropathy plots.

[0208] An intact antibody is generally composed of two full-length heavy chains and two full-length light chains, but in some instances may include fewer chains, such as antibodies naturally occurring in camelids that may comprise only heavy chains. Antibodies as disclosed herein may be derived solely from a single source or may be “chimeric,” that is, different portions of the antibody may be derived from two different antibodies. For example, the variable or CDR regions may be derived from a rat or murine source, while the constant region is derived from a different animal source, such as a human. The antibodies or binding fragments may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term “antibody” includes derivatives, variants, fragments, and muteins thereof, examples of which are described below (Sela-Culang et al. Front Immunol. 2013; 4: 302; 2013)

[0209] The term “light chain” includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain has a molecular weight of around 25,000 Daltons and includes a variable region domain (abbreviated herein as VL), and a constant region domain (abbreviated herein as CL). There are two classifications of light chains, identified as kappa (K) and lambda ( ). The term “VL fragment” means a fragment of the light chain of a monoclonal antibody that includes all orpart of the light chain variable region, including CDRs. A VL fragment can further include light chain constant region sequences. The variable region domain of the light chain is at the amino-terminus of the polypeptide.

[0210] The term “heavy chain” includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain has a molecular weight of around 50,000 Daltons and includes a variable region domain (abbreviated herein as VH), and three constant region domains (abbreviated herein as CHI, CH2, and CH3). The term “VH fragment” means a fragment of the heavy chain of a monoclonal antibody that includes all or part of the heavy chain variable region, including CDRs. A VH fragment can further include heavy chain constant region sequences. The number of heavy chain constant region domains will depend on the isotype. The VH domain is at the aminoterminus of the polypeptide, and the CH domains are at the carboxy-terminus, with the CH3 being closest to the — COOH end. The isotype of an antibody can be IgM, IgD, IgG, IgA, or IgE and is defined by the heavy chains present of which there are five classifications: mu (p), delta (5), gamma (y), alpha (a), or epsilon (a) chains, respectively. IgG has several subtypes, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM subtypes include IgMl and IgM2. IgA subtypes include IgAl and IgA2.

[0211] Antibodies can be whole immunoglobulins of any isotype or classification, chimeric antibodies, or hybrid antibodies with specificity to two or more antigens. They may also be fragments (e.g., F(ab')2, Fab', Fab, Fv, and the like), including hybrid fragments. An immunoglobulin also includes natural, synthetic, or genetically engineered proteins that act like an antibody by binding to specific antigens to form a complex. The term antibody includes genetically engineered or otherwise modified forms of immunoglobulins, such as the following:

[0212] The term “monomer” means an antibody containing only one Ig unit. Monomers are the basic functional units of antibodies. The term “dimer” means an antibody containing two Ig units attached to one another via constant domains of the antibody heavy chains (the Fc, or fragment crystallizable, region). The complex may be stabilized by a joining (J) chain protein. The term “multimer” means an antibody containing more than two Ig units attached to one another via constant domains of the antibody heavy chains (the Fc region). The complex may be stabilized by a joining (J) chain protein.

[0213] The term “bivalent antibody” means an antibody that comprises two antigenbinding sites. The two binding sites may have the same antigen specificities or they may be bispecific, meaning the two antigen-binding sites have different antigen specificities.

[0214] Bispecific antibodies are a class of antibodies that have two paratopes with different binding sites for two or more distinct epitopes. In some embodiments, bispecific antibodies can be biparatopic, wherein a bispecific antibody may specifically recognize a different epitope from the same antigen. In some embodiments, bispecific antibodies can be constructed from a pair of different single domain antibodies termed “nanobodies”. Single domain antibodies are sourced and modified from cartilaginous fish and camelids. Nanobodies can be joined together by a linker using techniques typical to a person skilled in the art; such methods for selection and joining of nanobodies are described in PCT Publication No. WO2015044386A1, No. W02010037838A2, and Bever et al., Anal Chem. 86:7875-7882 (2014), each of which are specifically incorporated herein by reference in their entirety.

[0215] Bispecific antibodies can be constructed as: a whole IgG, Fab'2, Fab'PEG, a diabody, or alternatively as scFv. Diabodies and scFvs can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Bispecific antibodies may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992), each of which are specifically incorporated by reference in their entirety.

[0216] In certain aspects, the antigen-binding domain may be multispecific or hetero specific by multimerizing with VH and VL region pairs that bind a different antigen. For example, the antibody may bind to, or interact with, (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, or (c) at least one other component. Accordingly, aspects may include, but are not limited to, bispecific, trispecific, tetraspecific, and other multispecific antibodies or antigen-binding fragments thereof that are directed to epitopes and to other targets, such as Fc receptors on effector cells.

[0217] In some embodiments, multispecific antibodies can be used and directly linked via a short flexible polypeptide chain, using routine methods known in the art. One such example is diabodies that are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, and utilize a linker that is too short to allow for pairing between domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain creating two antigen binding sites. The linker functionality is applicable for embodiments of triabodies, tetrabodies, and higher order antibody multimers, (see, e.g., Hollinger et al., Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Polijak et al., Structure 2:1121-1123 (1994); Todorovska et al., J. Immunol. Methods 248:47-66 (2001)).

[0218] Bispecific diabodies, as opposed to bispecific whole antibodies, may also be advantageous because they can be readily constructed and expressed in E. coli. Diabodies (and other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94 / 13804) from libraries. If one arm of the diabody is kept constant, for instance, with a specificity directed against a protein, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Bispecific whole antibodies may be made by alternative engineering methods as described in Ridgeway et al., (Protein Eng., 9:616-621, 1996) and Krah et al., (N Biotechnol. 39:167-173, 2017), each of which is hereby incorporated by reference in their entirety.

[0219] Heteroconjugate antibodies are composed of two covalently linked monoclonal antibodies with different specificities. See, e.g., US Patent No. 6,010,902, incorporated herein by reference in its entirety.

[0220] The part of the Fv fragment of an antibody molecule that binds with high specificity to the epitope of the antigen is referred to herein as the “paratope.” The paratope consists of the amino acid residues that make contact with the epitope of an antigen to facilitate antigen recognition. Each of the two Fv fragments of an antibody is composed of the two variable domains, VH and VL, in dimerized configuration. The primary structure of each of the variable domains includes three hypervariable loops separated by, and flanked by, Framework Regions (FR). The hypervariable loops are the regions of highest primary sequences variability among the antibody molecules from any mammal. The term hypervariable loop is sometimes used interchangeably with the term “Complementarity Determining Region (CDR).” The length of the hypervariable loops (or CDRs) varies between antibody molecules. The framework regions of all antibody molecules from a given mammal have high primary sequence similarity / consensus. The consensus of framework regions can be used by one skilled in the art to identify both the framework regions and the hypervariable loops (or CDRs) which are interspersed among the framework regions. The hypervariable loops are given identifying names which distinguish their position within the polypeptide, and on which domain they occur. CDRs in the VE domain are identified as El, L2, and L3, with LI occurring at the most distal end and L3 occurring closest to the CL domain. The CDRs may also be given the names CDR-1, CDR-2, and CDR-3. The L3 (CDR-3) is generally the region of highest variability among all antibody molecules produced by a given organism. The CDRs are regions of the polypeptide chain arranged linearly in the primary structure, and separated from each other by Framework Regions. The amino terminal (N-terminal) end of the VL chain is named FR1. The region identified as FR2 occurs between LI and L2 hypervariable loops. FR3 occurs betweenL2 and L3 hypervariable loops, and the FR4 region is closest to the CL domain. This structure and nomenclature is repeated for the VH chain, which includes three CDRs identified as Hl, H2 and H3. The majority of amino acid residues in the variable domains, or Fv fragments (VH and VL), are part of the framework regions (approximately 85%). The three dimensional, or tertiary, structure of an antibody molecule is such that the framework regions are more internal to the molecule and provide the majority of the structure, with the CDRs on the external surface of the molecule.

[0221] Several methods have been developed and can be used by one skilled in the art to identify the exact amino acids that constitute each of these regions. This can be done using any of a number of multiple sequence alignment methods and algorithms, which identify the conserved amino acid residues that make up the framework regions, therefore identifying the CDRs that may vary in length but are located between framework regions. Three commonly used methods have been developed for identification of the CDRs of antibodies: Kabat (as described in T. T. Wu and E. A. Kabat, “AN ANALYSIS OF THE SEQUENCES OF THE VARIABLE REGIONS OF BENCE JONES PROTEINS AND MYELOMA LIGHT CHAINS AND THEIR IMPLICATIONS FOR ANTIBODY COMPLEMENTARITY,” J Exp Med, vol. 132, no. 2, pp. 211-250, Aug. 1970); Chothia (as described in C. Chothia et al., “Conformations of immunoglobulin hypervariable regions,” Nature, vol. 342, no. 6252, pp. 877-883, Dec. 1989); and IMGT (as described in M.-P. Lefranc et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Developmental & Comparative Immunology, vol. 27, no. 1, pp. 55-77, Jan. 2003). These methods each include unique numbering systems for the identification of the amino acid residues that constitute the variable regions. In most antibody molecules, the amino acid residues that actually contact the epitope of the antigen occur in the CDRs, although in some cases, residues within the framework regions contribute to antigen binding.

[0222] One skilled in the art can use any of several methods to determine the paratope of an antibody. These methods include: 1) Computational predictions of the tertiary structure of the antibody / epitope binding interactions based on the chemical nature of the amino acid sequence of the antibody variable region and composition of the epitope; 2) Hydrogendeuterium exchange and mass spectroscopy; 3) Polypeptide fragmentation and peptide mapping approaches in which one generates multiple overlapping peptide fragments from the full length of the polypeptide and evaluates the binding affinity of these peptides for the epitope; 4) Antibody Phage Display Library analysis in which the antibody Fab fragment encoding genes of the mammal are expressed by bacteriophage in such a way as to beincorporated into the coat of the phage. This population of Fab expressing phage are then allowed to interact with the antigen which has been immobilized or may be expressed in by a different exogenous expression system. Non-binding Fab fragments are washed away, thereby leaving only the specific binding Fab fragments attached to the antigen. The binding Fab fragments can be readily isolated and the genes which encode them determined. This approach can also be used for smaller regions of the Fab fragment including Fv fragments or specific VH and VL domains as appropriate.

[0223] In certain aspects, affinity matured antibodies are enhanced with one or more modifications in one or more CDRs thereof that result in an improvement in the affinity of the antibody for a target antigen as compared to a parent antibody that does not possess those alteration(s). Certain affinity matured antibodies will have nanomolar or picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art, e.g., Marks et al., Bio / Technology 10:779 (1992) describes affinity maturation by VH and VL domain shuffling, random mutagenesis of CDR and / or framework residues employed in phage display is described by Rajpal et al., PNAS. 24: 8466-8471 (2005) and Thie et al., Methods Mol Biol. 525:309-22 (2009) in conjugation with computation methods as demonstrated in Tiller et al., Front. Immunol. 8:986 (2017).

[0224] Chimeric immunoglobulins are the products of fused genes derived from different species; “humanized” chimeras generally have the framework region (FR) from human immunoglobulins and one or more CDRs are from a non-human source.

[0225] In certain aspects, portions of the heavy and / or light chain are identical or homologous to corresponding sequences from another particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived 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 biological activity. U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984). For methods relating to chimeric antibodies, see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851- 6855 (1985), each of which are specifically incorporated herein by reference in their entirety. CDR grafting is described, for example, in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are all hereby incorporated by reference for all purposes.

[0226] In some embodiments, minimizing the antibody polypeptide sequence from the non-human species optimizes chimeric antibody function and reduces immunogenicity. Specific amino acid residues from non-antigen recognizing regions of the non-human antibodyare modified to be homologous to corresponding residues in a human antibody or isotype. One example is the “CDR-grafted” antibody, in which an antibody comprises one or more CDRs from a particular species or belonging to a specific antibody class or subclass, while the remainder of the antibody chain(s) is identical or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass. For use in humans, the V region composed of CDR1, CDR2, and partial CDR3 for both the light and heavy chain variance region from a non-human immunoglobulin, are grafted with a human antibody framework region, replacing the naturally occurring antigen receptors of the human antibody with the non-human CDRs. In some instances, corresponding non-human residues replace framework region residues of the human immunoglobulin. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody to further refine performance. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1:105 (1998); Harris, Biochem. Soc. Transactions 23; 1035 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428 (1994); Verhoeyen et al., Science 239:1534-36 (1988).

[0227] Intrabodies are intracellularly localized immunoglobulins that bind to intracellular antigens as opposed to secreted antibodies, which bind antigens in the extracellular space.

[0228] Polyclonal antibody preparations typically include different antibodies against different determinants (epitopes). In order to produce polyclonal antibodies, a host, such as a rabbit or goat, is immunized with the antigen or antigen fragment, generally with an adjuvant and, if necessary, coupled to a carrier. Antibodies to the antigen are subsequently collected from the sera of the host. The polyclonal antibody can be affinity purified against the antigen rendering it monospecific.

[0229] Monoclonal antibodies or “mAb” refer to an antibody obtained from a population of homogeneous antibodies from an exclusive parental cell, e.g., the population is identical except for naturally occurring mutations that may be present in minor amounts. Each monoclonal antibody is directed against a single antigenic determinant.B. Fragments and Antigen-Binding Fragments a. Antigen-Binding Fragments

[0230] Certain aspects relate to antibody fragments, such as antibody fragments that bind to an antigen of interest, e.g., but not limited to, CD47. The term functional antibody fragment includes antigen-binding fragments of an antibody that retain the ability to specifically bind toan antigen. These fragments are constituted of various arrangements of the variable region heavy chain (VH) and / or light chain (VL); and in some embodiments, include constant region heavy chain 1 (CHI) and light chain (CL). In some embodiments, they lack the Fc region constituted of heavy chain 2 (CH2) and 3 (CH3) domains. Embodiments of antigen binding fragments and the modifications thereof may include: (i) the Fab fragment type constituted with the VL, VH, CL, and CHI domains; (ii) the Ed fragment type constituted with the VH and CHI domains; (iii) the Ev fragment type constituted with the VH and VL domains; (iv) the single domain fragment type, dAb, (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003) constituted with a single VH or VL domain; (v) isolated complementarity determining region (CDR) regions. Such terms are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, 2d ed., Wiley-Liss, Inc. New York, N.Y. (1990); Antibodies, 4:259-277 (2015). The citations in this paragraph are all incorporated by reference.

[0231] Antigen-binding fragments also include fragments of an antibody that retain exactly, at least, or at most 1, 2, or 3 complementarity determining regions (CDRs) from a light chain variable region. Eusions of CDR-containing sequences to an Ec region (or a CH2 or CH3 region thereof) are included within the scope of this definition including, for example, scEv fused, directly or indirectly, to an Ec region are included herein.

[0232] The term Fab fragment means a monovalent antigen-binding fragment of an antibody containing the VL, VH, CL and CHI domains. The term Fab' fragment means a monovalent antigen-binding fragment of a monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment includes the VL, VH, CL and CHI domains and all or part of the hinge region. The term F(ab')2 fragment means a bivalent antigen-binding fragment of a monoclonal antibody comprising two Fab' fragments linked by a disulfide bridge at the hinge region. An F(ab')2 fragment includes, for example, all or part of the two VH and VL domains, and can further include all or part of the two CL and CHI domains.

[0233] The term Fd fragment means a fragment of the heavy chain of a monoclonal antibody, which includes all or part of the VH, including the CDRs. An Fd fragment can further include CHI region sequences.

[0234] The term Fv fragment means a monovalent antigen-binding fragment of a monoclonal antibody, including all or part of the VL and VH, and absent of the CL and CHIdomains. The VL and VH include, for example, the CDRs. Single-chain antibodies (sFv or scFv) are Fv molecules in which the VL and VH regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding fragment. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are herein incorporated by reference. The term (scFv)2 means bivalent or bispecific sFv polypeptide chains that include oligomerization domains at their C-termini, separated from the sFv by a hinge region (Pack et al. 1992). The oligomerization domain comprises self-associating a- helices, e.g., leucine zippers, which can be further stabilized by additional disulfide bonds. (scFv)2 fragments are also known as “miniantibodies” or “minibodies.”

[0235] A single domain antibody is an antigen-binding fragment containing only a VH or the VL domain. In some instances, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens. b. Fragment Crystallizable Region, Fc

[0236] An Fc region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. The term “Fc polypeptide” as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are included. In certain embodiments, an Fc domain is fused to an antigen binding agent that does not comprise an scFv. In some embodiments, an Fc domain is fused to a “bait” protein, which may be a ligand (natural or modified) of a target antigenic protein of interest. c. Polypeptides with antibody CDRs & Scaffolding Domains that Display the CDRs

[0237] Antigen-binding peptide scaffolds, such as complementarity-determining regions (CDRs), are used to generate protein-binding molecules in accordance with the embodiments. Generally, a person skilled in the art can determine the type of protein scaffold on which to graft at least one of the CDRs. It is known that scaffolds, optimally, must meet a number of criteria such as: good phylogenetic conservation; known three-dimensional structure; small size; few or no post-transcriptional modifications; and / or be easy to produce, express, and purify. Skerra, J Mol Recognit, 13:167-87 (2000).

[0238] The protein scaffolds can be sourced from, but not limited to: fibronectin type III FN3 domain (known as “monobodies”), fibronectin type III domain 10, lipocalin, anticalin, Z- domain of protein A of Staphylococcus aureus, thioredoxin A or proteins with a repeated motif such as the “ankyrin repeat”, the “armadillo repeat”, the “leucine-rich repeat” and the “tetratricopeptide repeat”. Such proteins are described in US Patent Publication Nos. 2010 / 0285564, 2006 / 0058510, 2006 / 0088908, 2005 / 0106660, and PCT Publication No. W02006 / 056464, each of which are specifically incorporated herein by reference in their entirety. Scaffolds derived from toxins from scorpions, insects, plants, mollusks, etc., and the protein inhibitors of neuronal NO synthase (PIN) may also be used.C. Chemical Modification of Antibodies

[0239] In some aspects, also contemplated are glycosylation variants of antibodies, wherein the number and / or type of glycosylation site(s) has been altered compared to the amino acid sequences of the parent polypeptide. Glycosylation of the polypeptides can be altered, for example, by modifying one or more sites of glycosylation within the polypeptide sequence to increase the affinity of the polypeptide for antigen (U.S. Pat. Nos. 5,714,350 and 6,350,861). In certain embodiments, antibody protein variants comprise a greater or a lesser number of N- linked glycosylation sites than the native antibody. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate or alter this sequence will prevent addition of an N-linked carbohydrate chain present in the native polypeptide. For example, the glycosylation can be reduced by the deletion of an Asn or by substituting the Asn with a different amino acid. In other embodiments, one or more new N-linked glycosylation sites are created. Antibodies typically have an N-linked glycosylation site in the Fc region.

[0240] Additional antibody variants include cysteine variants, wherein one or more cysteine residues in the parent or native amino acid sequence are deleted from or substituted with another amino acid (e.g., serine). Cysteine variants are useful, inter alia, when antibodies must be refolded into a biologically active conformation. Cysteine variants may have fewer cysteine residues than the native antibody and typically have an even number to minimize interactions resulting from unpaired cysteines.

[0241] In some aspects, the polypeptides can be pegylated to increase biological half-life by reacting the polypeptide with polyethylene glycol (PEG) or a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to thepolypeptide. Polypeptide pegylation may be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). Methods for pegylating proteins are known in the art and can be applied to the polypeptides of the present disclosure to obtain PEGylated derivatives of antibodies. See, e.g., EP 0 154 316 and EP 0 401 384. In some aspects, the antibody is conjugated or otherwise linked to transthyretin (TTR) or a TTR variant. The TTR or TTR variant can be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly(n- vinyl pyrrolidone), polyethylene glycols, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide co-polymers, polyoxy ethylated polyols, and polyvinyl alcohols. As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins. a. Conjugation

[0242] Derivatives of the antibodies and antigen binding fragments that are described herein are also provided. The derivatized antibody or fragment thereof may comprise any molecule or substance that imparts a desired property to the antibody or fragment. The derivatized antibody can comprise, for example, a detectable (or labeling) moiety (e.g., a radioactive, colorimetric, antigenic, or enzymatic molecule, or a detectable bead), a molecule that binds to another molecule (e.g., biotin or streptavidin), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), or a molecule that increases the suitability of the antibody for a particular use (e.g., administration to a subject, such as a human subject, or other in vivo or in vitro uses).

[0243] Optionally, an antigen binding agent, such as an antibody or an immunological portion of an antibody, can be chemically conjugated to, or expressed as, a fusion protein with other proteins. In some aspects, polypeptides may be chemically modified by conjugating or fusing the polypeptide to serum protein, such as human serum albumin, to increase half-life of the resulting molecule. See, e.g., EP 0322094 and EP 0 486 525. In some aspects, the polypeptides may be conjugated to a diagnostic agent and used diagnostically, for example, to monitor the development or progression of a disease and determine the efficacy of a given treatment regimen. In some aspects, the polypeptides may also be conjugated to a therapeutic agent to provide a therapy in combination with the therapeutic effect of the polypeptide. Additional suitable conjugated molecules include ribonuclease (RNase), DNase I, an antisense nucleic acid, an inhibitory RNA molecule such as a siRNA molecule, an immuno stimulatory nucleic acid, aptamers, ribozymes, triplex forming molecules, and external guide sequences. The functional nucleic acid molecules may act as effectors, inhibitors, modulators, andstimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules may possess a de novo activity independent of any other molecules.

[0244] In some aspects, disclosed are antigen binding agents, such as antibodies and antibody-like molecules, that are linked to at least one agent to form an antigen targeting conjugate (e.g., an antibody conjugate). In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, an antibody may be covalently bound or complexed to one or more desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity, pore-forming activity, immunological activation activity, etc. Non-limiting examples of effector molecules include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides and the like. By contrast, a reporter molecule is defined as any moiety that may be detected using an assay. Non-limiting examples of reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands. b. Conjugate Types

[0245] Certain examples of antibody conjugates are those conjugates in which the antibody is linked to a detectable label. “Detectable labels” are compounds and / or elements that can be detected due to their specific functional properties, and / or chemical characteristics, the use of which allows the antibody to be detected, and / or further quantified if desired. Examples of detectable labels include, but not limited to, radioactive isotopes, fluorescers, semiconductor nanocrystals, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or haptens) and the like. Particular examples of labels are, but not limited to, horseradish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas red, luminol, NADPH and a- or P-galactosidase. Antibody conjugates include those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and / or to an enzyme to generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include, but are not limited to, urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary binding ligands can include biotin and / or avidin and streptavidin compounds. The uses of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference. Molecules containing azido groups may also be used toform covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter & Haley, 1983).

[0246] In some aspects, contemplated are immunoconjugates comprising an antibody or antigen-binding fragment thereof conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In this way, the agent of interest can be targeted directly to cells bearing cell surface antigen. The antibody and agent may be associated through non-covalent interactions such as through electrostatic forces, or by covalent bonds. Various linkers, known in the art, can be employed in order to form the immunoconjugate. In some embodiments, a linker is a cleavable linker and / or one that can be removed under reduced conditions. Additionally, in some embodiments, an immunoconjugate can be provided in the form of a fusion protein. In one aspect, an antibody may be conjugated to various therapeutic substances in order to target the cell surface antigen. Examples of conjugated agents include, but are not limited to, metal chelate complexes, drugs, toxins and other effector molecules, such as cytokines, lymphokines, chemokines, immunomodulators, radiosensitizers, asparaginase, carboranes, and radioactive halogens.

[0247] In antibody drug conjugates (ADC), an antibody (Ab) is conjugated to one or more drug moieties (D) through a linker (L). The ADC may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent, to form Ab-L, via a covalent bond, followed by reaction with a drug moiety D; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, followed by reaction with the nucleophilic group of an antibody. Antibody drug conjugates may also be produced by modification of the antibody to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. Alternatively, a fusion protein comprising the antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate. In yet another aspect, the antibody may be conjugated to a “receptor” (such as streptavidin) for utilization in tumor or cancer cell pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from thecirculation using a clearing agent and then administration of a “ligand” (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0248] Examples of antibody-drug conjugates known to a person skilled in the art are prodrugs useful for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos, Anticancer Res. 19:605-614 (1999); Niculescu-Duvaz and Springer, Adv. Drg. Del. Rev. 26:151-172 (1997); U.S. Pat. No. 4,975,278). In contrast, systematic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the target tumor cells (Baldwin et al., Lancet 1:603-5 (1986); Thorpe, (1985) “Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review,” In: Monoclonal Antibodies ‘84: Biological and Clinical Applications, A. Pincera et al., (eds.) pp. 475-506). Both polyclonal antibodies and monoclonal antibodies have been reported as useful in these strategies (Rowland et al., Cancer Immunol. Immunother. 21:183-87 (1986)).

[0249] In certain aspects, ADC include covalent or aggregative conjugates of antibodies, or antigen-binding fragments thereof, with other proteins or polypeptides, such as by expression of recombinant fusion proteins comprising heterologous polypeptides fused to the N-terminus or C-terminus of an antibody polypeptide. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide, e.g., the yeast alpha-factor leader, or a peptide such as an epitope tag (e.g., V5-His). Antibody-containing fusion proteins may comprise peptides added to facilitate purification or identification of the antibody (e.g., poly- His). An antibody polypeptide also can be linked to the FLAG® (Sigma- Aldrich, St. Louis, Mo.) peptide as described in Hopp et al., Bio / Technology 6:1204 (1988), and U.S. Pat. No. 5,011,912. Oligomers that contain one or more antibody polypeptides may be employed as antagonists. Oligomers may be in the form of covalently linked or non-covalently linked dimers, trimers, or higher oligomers. Oligomers comprising two or more antibody polypeptides are contemplated for use. Other oligomers include heterodimers, homotrimers, hetero trimers, homo tetramers, hetero tetramers, etc. In certain aspects, oligomers comprise multiple antibody polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the antibody polypeptides. Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting oligomerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of antibody polypeptides attached thereto, as described in more detail below.

[0250] In certain aspects, the linker is a cleavable linker. The cleavable linker may comprise a disulfide bond, ester bond, amide bond, thioether bond, carbamate, carbonate,triazole bond, hydrazone bond, and / or a protease sensitive protein sequence. Cleavage of the linker may be accomplished by any methods known in the art. In certain aspects, the linker is cleavable by changes in temperature, ionicity, or pH (e.g., acidic or reducing conditions). In certain aspects, the linker is cleaved within a discrete cellular compartment (e.g., lysosomes, endosomes, phagosomes, cytosol, and / or vacuoles). In certain aspects, the linker is cleavable in a phagosome. In certain aspects, the linker is photocleavable. In certain aspects, the linker is self-cleaving. In certain aspects, the linker is cleavable by an enzyme, e.g., a protease, amidase, carboxypeptidase, and / or asparaginyl endopeptidase.

[0251] In certain aspects a cleavable linker is necessary for proper function of an ADC. In certain aspects, the cleavable linker decreases the toxicity of an ADC. In certain aspects, the cleavable linker reduces the toxicity of LLO. In certain aspects, the cleavable linker decreases the hemolysis activity of LLO. In certain aspects, the cleavable linker limits the function of LLO to discrete cellular compartments (e.g., lysosomes, endosomes, phagosomes, cytosol, and / or vacuoles). c. Conjugation Methodology

[0252] Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and / or tetrachloro-3 -6 -diphenylglycouril-3 attached to the antibody (U.S. Patent Nos. 4,472,509 and 4,938,948, each incorporated herein by reference). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates may also be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis- diazonium derivatives (such as bos(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). In some aspects, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site, are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference). Site-specific attachmentof effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region has also been disclosed in the literature (O’Shannessy et al., 1987).

[0253] The term "click-chemistry reaction" is used as a chemical concept introduced by K. Barry Sharpless of the Scripps Research Institute to explain complementary chemical functional groups and a chemical reaction designed so that two molecules can rapidly and stably form a covalent bond. The click-chemistry reaction does not refer to a certain reaction, but refers to a concept of such a rapid and stable reaction. In any embodiment, the clickchemistry reaction must be modular, wide in scope, give high yields, generate only insignificant by-products, be stereospecific, physiologically stable, driven by a thermodynamic driving force (for example, greater than 84 kJ / mol), and / or have high atom economy.

[0254] The term "click-chemistry functional group" used in the present disclosure refers to a functional group that participates in a click-chemistry reaction. For example, a strained alkyne (for example, a cyclooctyne) corresponds to a click-chemistry functional group. In general, the click chemistry reaction requires at least two molecules, each of which contains click-chemistry functional groups complementary to each other. In this way, a pair of click-chemistry functional groups having reactivity with each other is often referred to as "partner clickchemistry functional groups.” In strain-promoted cycloaddition of a cyclooctyne with an azide, for example, the azide is a partner click-chemistry functional group for the cyclooctyne and other alkynes. Exemplary click-chemistry functional groups that may be used in the present disclosure include a terminal alkyne, an azide, a strained alkyne, a diene, a dienophile, a transcyclooctene, an alkene, a thiol, and / or a tetrazine, but the present disclosure is not limited thereto. Other click-chemistry functional groups are known to those skilled in the related art.Pore-Forming Agents

[0255] The term “pore-forming agent,” also “cytolysins” or “pore-forming cytolysins,” refers to a molecule or portion of a molecule capable of disrupting a cellular membrane through the formation of one or more transmembrane pores or channels in the lipid bilayer of a cell, including, but not limited to a cell membrane, vacuole, or vesicle, e.g., phagosomes, endosomes, and / or lysosomes. Pore-forming agents may have a variety of sources, including, but not limited to bacteria, fungi, plants, animals, and / or chemical synthesis. Pore-forming agents can be generated by methods known to those skilled in the art, e.g., proteolytic cleavage or expression of recombinant peptides. The ability of a candidate fragment to permeabilize the lipid bilayer of a cell can be assessed by methods known to those skilled in the art, e.g., by therelease of intracellular contents, such as ATP or radioactive label from pre-loaded cells or by the uptake of a dye, such as trypan blue which is excluded by intact cells.

[0256] Exemplary pore-forming-agents can include saponin compounds such as saponin- containing Immune Stimulating Complexes (ISCOMs) (see e.g., Cox and Coulter, 1997, Vaccine 15(3): 248-256 and U.S. Patent No. 6,352,697 ), phospholipases (see, e.g., Camilli et al., 1991, J. Exp. Med. 173: 751-754), pore-forming toxins (e.g., an a-toxin), natural cytolysins of gram-positive bacteria, such as Listeriolysin O (LLO, e.g., Mengaud et al., 1988, Infect. Immun. 56: 766-772 and Portnoy et al., 1992, Infect. Immun. 60: 2710-2717), streptolysin O (SLO, e.g., Palmer et al., 1998, Biochemistry 37(8): 2378-2383) and perfringolysin O (PFO, e.g., Rossjohn et al., Cell 89(5): 685-692). Pore-forming agents may further include proteins from the membrane attack complex / perforin (MACPF) family, cholesterol dependent cytolysin (CDC) family, and / or the Pleurotolysin B pore-forming family, e.g., but not limited to, perfringolysin O (PFO), alveolysin, streptolysin O (SEO), pneumolysin, ivanolysin, hemolysin, pyolysin, flavomodulin, tetanolysin O, MAC / perforin domain containing protein, complement component C8-alpha, complement component C6, complement component C9, complement component C7, complement component C8-beta, perforin-like protein 1, apextrin, hemopexin, photopexin a / b, SpoCl-CIC, toxin AvTX-60A, toxin PsTX-60B, MACPF domain-containing protein CADI, MACPF domain-containing protein NSE1, Macrophage expressed protein, Pleurotolysin, Erylysin, Ostreolysin, Cytolysin, Aegerolysin, intermedilysin (IEY), siulysin, aerolysin, ClyA, colicin, lysenin, CrylAa, Fragaceatoxin C, perforin, Tc holotoxin, a-haemolysin, SmhB, and / or Eisteriolysin O (FEO). Pore-forming agents may further include eucaryotic pore-forming agents such as defensin, magainin, melittin, complement, perforin, yeast killer toxin and histolysin. Pore-forming agents may further include synthetic pore-forming agents such as Pederson's crown ethers and / or valinomycin and other as described in Regen et al., Biochem. Biophys. Res. Commun. 159: 566-571, 1989, herein incorporated by reference. Where the target cell is phagosomal, acid activated poreforming agents may be advantageously used. For example, Eisteriolysin O exhibits greater pore-forming ability at mildly acidic pH (the pH conditions within the phagosome), thereby facilitating pore-forming activity in the target cell and delivery of vacuole (including phagosome and endosome) contents to the cytoplasm (see, e.g., Portnoy et al., Infect. Immun. 1992, 60: 2710-2717). In some embodiments, a pore-forming agent comprises, consists essentially of, or consists of FEO.

[0257] In one or more embodiments, the pore-forming agent is fused or otherwise linked to an antigen targeting agent, such as an antibody, wherein the fusion or linkage permits thedelivery of the pore-forming agent to a phagosome of a cell that engulfs a target cell. In another embodiment, the pore-forming agent is fused or otherwise liked to an antigen targeting agent, such as an antibody through a cleavable linker, wherein the cleavable linker permits the delivery and / or activation (e.g., through oligomerization) of the pore-forming agent to a phagosome of a cell that engulfs a target cell. In some embodiments, the conjugation between the antigen targeting agent and the pore-forming agent renders the pore-forming agent less cytotoxic (e.g., hemolysis levels, etc.), until and unless the linker is otherwise broken (e.g., through reducing conditions, a protease, etc.).III. Methods of Treatment

[0258] Embodiments of the present disclosure can concern methods for the use of compositions (e.g., ADCs, antigen binding agent conjugations, etc.) provided herein for treating or preventing a medical disease or disorder. In some embodiments, a method comprises treating and / or preventing a cancer through administration of one or more compositions provided herein. In some embodiments, methods include administering to the subject a therapeutically effective amount of an ADC, optionally as a co-therapy with one or more antibodies, thereby treating or preventing the disease in the subject, including reducing the risk of, reducing the severity of, and / or delaying the onset of the disease. In certain embodiments of the present disclosure, cancer or infection is treated by transfer of a composition comprising an ADC. In some embodiments, treatment with the ADC increases adaptive immune responses by promoting differentiation, activation, and / or recruitment of accessory immune cells to sites of malignancy, including at least primary tumor sites and / or metastasis.

[0259] Aspects of the current disclosure relate to methods for treating cancer. Cancers for which the present treatment methods are useful can include any malignant cell type, such as those found in a solid tumor or a hematological tumor. Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breastcancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0260] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acral lentiginous melanomas; nodular melanomas; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant;choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; B-cell lymphoma; low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.

[0261] In some embodiments, antigen targeting agent conjugate molecules described herein may be used for stimulating an immune response. The immune response stimulation may be done in vitro, in vivo, or ex vivo. In some embodiments, the CD47 targeting molecules described herein are for preventing relapse. In some embodiments, a method of treatment provided herein comprises administering a CD47 targeting molecule conjugated to a poreforming agent to a patient in need thereof (e.g., a cancer patient). In some embodiments, the CD47 targeting molecule is conjugated to a pore-forming agent through a cleavable linker. In some embodiments, the pore forming agent comprises Listeriolysin O (LLO). In some embodiments, methods of treatment comprising CD47-LLO further comprise administration of one or more additional therapeutic agents, such as but not limited to immune checkpoint inhibitors. In certain embodiments, combination treatments comprising CD47-LLO coupledwith an immune checkpoint inhibitor result in synergistic interactions for significantly improved treatment of a disease relative to either agent utilized as a monotherapy.

[0262] The therapy provided herein may comprise administration of a combination of therapeutic agents, such as a first cancer therapy and a second cancer therapy. The therapies may be administered in any suitable manner known in the art. For example, the first and second cancer treatment may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the first and second cancer treatments are administered in a separate composition. In some embodiments, the first and second cancer treatments are in the same composition. Embodiments of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed. Examples of therapies other than those of the present disclosure include surgery, chemotherapy, drug therapy, radiation, hormone therapy, immunotherapy (other than that of the present disclosure), or a combination thereof. In certain embodiments, the compositions comprising ADCs are administered in combination with one or more additional therapeutic agents. For example, the therapeutic agent may comprise T cells, an immunomodulatory agent, a monoclonal antibody, a chemotherapeutic agent, hormone(s), drugs of any kind, surgery, radiation, etc.

[0263] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0264] The treatments may include various “unit doses.” The term unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the composition of the present disclosure, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier,or vehicle, where appropriate. The specifications for the unit dosage forms of the present disclosure depend on the particular pharmacodynamics associated with the pharmaceutical composition in the particular subject. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose. A composition of the present disclosure can be provided in unit dosage form wherein each dosage unit, e.g., an injection, contains a predetermined amount of the composition, alone or in appropriate combination with other active agents.

[0265] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg / kg to 200 mg / kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg / kg, mg / kg, pg / day, or mg / day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and / or on multiple days, weeks, or months.

[0266] In certain embodiments, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM. In another embodiment, the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein). In other embodiments, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 pM or any range derivable therein. In certain embodiments, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case theblood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0267] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0268] It will be understood by those skilled in the art and made aware that dosage units of pg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of pg / ml or mM (blood levels), such as 4 pM to 100 pM. It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0269] In some embodiments, an ADC comprising a CD47 targeting molecule conjugated through a cleavable linker to a pore forming agent, such as but not limited to LLO, (CD-47 - ADC) is administered to an individual in need thereof. In some embodiments the individual is diagnosed with, has one or more symptoms of, or is suspected of having a cancer.

[0270] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is AO-176 (aka AO-104; Arch Oncology) (AO176-PFA). In some embodiments, AO-176 is conjugated through a cleavable linker to LLO (AO176-LLO) In some embodiments, treatment with AO176-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with AO176-LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0271] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is CC-90002 (aka INBRX-103; Cellgene) (CC90002-PFA). In some embodiments, CC-90002 is conjugated through a cleavable linker to LLO (CC90002- LLO) In some embodiments, treatment with CC90002-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with CC90002-LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0272] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is GenSci-059 (Changchun Genescience Pharmaceutical Co Ltd.) (GenSci059-PFA). In some embodiments, GenSci-059 is conjugated through a cleavable linker to LLO (GenSci059-LLO) In some embodiments, treatment with GenSci059-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with GenSci059-LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0273] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is Lemzoparlimab (aka TJ011133, TJC4; LMab) (IMab-PFA). In some embodiments, LMab is conjugated through a cleavable linker to LLO (IMab -LLO) In some embodiments, treatment with GenSci059-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with IMab -LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0274] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is Letaplimab (aka IBL188; Innovent) (Letaplimab-PFA). In some embodiments, Letaplimab is conjugated through a cleavable linker to LLO (Letaplimab- LLO) In some embodiments, treatment with Letaplimab-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with Letaplimab-LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0275] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is Ligufalimab (aka AK117; Akeso) (Ligufalimab-PFA). In some embodiments, Ligufalimab is conjugated through a cleavable linker to LLO (Ligufalimab-LLO) In some embodiments, treatment with Ligufalimab-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with Ligufalimab-LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0276] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is Magrolimab (aka Hu5F9-G4; Gilead Sciences / Forty Seven) (Magrolimab-PFA). In some embodiments, Magrolimab is conjugated through a cleavable linker to LLO (Magrolimab-LLO) In some embodiments, treatment with Magrolimab-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). Insome embodiments, treatment with Magrolimab-LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0277] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is ZL-1201 (Zai Lab) (ZL-1201-PFA). In some embodiments, ZL-1201 is conjugated through a cleavable linker to LLO (ZL-1201-LLO) In some embodiments, treatment with ZL-1201-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with ZL-1201- LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0278] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is IBL322 (Innovent) (IBI-322-PFA). In some embodiments, IBL322 is conjugated through a cleavable linker to LLO (IBI-322-LLO) In some embodiments, treatment with IBI-322-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with IBI-322-LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0279] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is TG-1801 (Novimmune SA TG Therapeutics) (TG-1801- PFA). In some embodiments, TG-1801 is conjugated through a cleavable linker to LLO (TG- 1801 -LLO) In some embodiments, treatment with TG-1801-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with TG-1801 -LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0280] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is B6H12 (Santa Cruz) (B6H12-PFA). In some embodiments, B6H12 is conjugated through a cleavable linker to LLO (B6H12-LLO) In some embodiments, treatment with B6H12-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with B6H12-LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0281] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is BRIC126 (Santa Cruz) (BRIC126-PFA). In some embodiments, BRIC126 is conjugated through a cleavable linker to LLO (BRIC126-LLO) In some embodiments, treatment with BRIC126-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatmentwith BRIC126-LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0282] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is SRF231 (Surface Oncology) (SRF231-PFA). In some embodiments, SRF231 is conjugated through a cleavable linker to LLO (SRF231-LLO) In some embodiments, treatment with SRF231-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with SRF231- LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0283] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is IMC-002 (ImmuneOncia Therapeutics, 3D Medicines) (IMC-002-PFA). In some embodiments, IMC-002 is conjugated through a cleavable linker to LLO (IMC-002-LLO) In some embodiments, treatment with IMC-002-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with IMC-002-LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0284] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is TQB2928 (Chia Tai Tianqing Pharmaceutical Group Co., Ltd.) (TQB2928-PFA). In some embodiments, TQB2928 is conjugated through a cleavable linker to LLO (TQB2928-LLO) In some embodiments, treatment with TQB2928-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with TQB2928-LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0285] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is PF-07257876 (Pfizer) (PF-07257876-PFA). In some embodiments, PF-07257876 is conjugated through a cleavable linker to LLO (PF-07257876- LLO) In some embodiments, treatment with PF-07257876-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with PF-07257876-LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0286] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is HX009 (Hanxbio) (HX009-PFA). In some embodiments, HX009 is conjugated through a cleavable linker to LLO (HX009-LLO) In some embodiments, treatment with HX009-PFA results in increased immune cell cytotoxicity against CD47 bearingcells (e.g., cancer cells). In some embodiments, treatment with HX009-LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0287] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is STI-6643 (Sorrento Therapeutics, Inc.) (STI-6643-PFA). In some embodiments, STI-6643 is conjugated through a cleavable linker to LLO (STI-6643- LLO) In some embodiments, treatment with STI-6643-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with STI-6643 -LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0288] In some embodiments, the CD47 targeting molecule conjugated to a pore forming agent through a cleavable linker is MIAP410 (MIAP410-PFA). In some embodiments, MIAP410 is conjugated through a cleavable linker to LLO (MIAP410-LLO) In some embodiments, treatment with MIAP410-PFA results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells). In some embodiments, treatment with MIAP410-LLO results in increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0289] In some embodiments, the CD47-ADC is administered to an individual in need thereof in combination with one or more monospecific, bispecific, and / or multi- specific antibodies. In some embodiments, combination administration to an individual in need thereof occurs in such a way as to have the CD47-ADC and antibodies be in proximity, and thus the CD-47-ADC and antibody are able to interact with the same or adjacent cells. In some embodiments, the two or more components are administered separately to an individual. In some embodiments, the two or more components are co-administered by any suitable route of administration, such as by co-infusion to the patient. In some embodiments, the two or more components may be used to activate immune cells in vitro or ex vivo.

[0290] In some embodiments, methods of using an ADC comprising a CD47 targeting molecule conjugated through a cleavable linker to a pore forming agent, such as but not limited to LLO, (CD47-ADC) used in conjunction with one or more monospecific, bispecific, and / or multi- specific antibodies results in synergistic effects. In some embodiments, combination of the CD-47- ADC with an immune checkpoint inhibitor results in a synergistic effect in antitumor immunity. In some embodiments, combination of the CD47-ADC with an immune checkpoint inhibitor results in a synergistic effect in anti-tumor immunity and increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0291] In some embodiments, an ADC comprising one or more LLO molecules and an antigen targeting molecule, such as but not limited to an antibody, conjugated through a cleavable linker (LLO-X) is administered to an individual in need thereof. In some embodiments, the LLO-X is administered to an individual in need thereof in combination with one or more monospecific, bispecific, and / or multi- specific antibodies. In some embodiments, combination administration to an individual in need thereof occurs in such a way as to have the LLO-X and antibodies be in proximity, and thus the LLO-X and antibody are able to interact with the same or adjacent cells. In some embodiments, the two or more components are administered separately to an individual. In some embodiments, the two or more components are co-administered by any suitable route of administration, such as by co-infusion to the patient. In some embodiments, the two or more components may be used to activate immune cells in vitro or ex vivo.

[0292] In some embodiments, methods of using an ADC comprising one or more LLO molecules and an antigen targeting molecule, such as but not limited to an antibody, conjugated through a cleavable linker (LLO-X) used in conjunction with one or more monospecific, bispecific, and / or multi- specific antibodies results in synergistic effects. In some embodiments, combination of the LLO-X with an immune checkpoint inhibitor results in a synergistic effect in anti-tumor immunity. In some embodiments, combination of the LLO-X with an immune checkpoint inhibitor results in a synergistic effect in anti-tumor immunity and increased immune cell cytotoxicity against CD47 bearing cells (e.g., cancer cells).

[0293] In some embodiments, the antigen targeting molecule conjugated to LLO-X is a CD19 antigen targeting molecule (CD19-LLO). In some embodiments, the antigen targeting molecule conjugated to LLO-X is Rituximab (RTX-LLO). In some embodiments, treatment with CD19-LLO results in increased immune cell cytotoxicity against CD 19 bearing cells (e.g., cancer cells). In some embodiments, treatment with RTX-LLO results in increased immune cell cytotoxicity against CD19 bearing cells (e.g., cancer cells). In some embodiments, the antigen targeting molecule conjugated to LLO-X is Tafasitamab (Tafasitamab-LLO). In some embodiments, treatment with Tafasitamab-LLO results in increased immune cell cytotoxicity against CD19 bearing cells (e.g., cancer cells).

[0294] In some embodiments, the antigen targeting molecule conjugated to LLO-X is a c- MET antigen targeting molecule (cMET-LLO). In some embodiments, the antigen targeting molecule conjugated to LLO-X is Amivantamab (Amivantamab-LLO). In some embodiments, treatment with cMET-LLO results in increased immune cell cytotoxicity against c-METbearing cells (e.g., cancer cells). In some embodiments, treatment with Amivantamab-LLO results in increased immune cell cytotoxicity against c-MET bearing cells (e.g., cancer cells).

[0295] In some embodiments, the antigen targeting molecule conjugated to LLO-X is an EGFR antigen targeting molecule (EGFR-LLO). In some embodiments, the antigen targeting molecule conjugated to LLO-X is Imgatuzumab (GA201-LLO). In some embodiments, treatment with EGFR-LLO results in increased immune cell cytotoxicity against EGFR bearing cells (e.g., cancer cells). In some embodiments, treatment with GA201-LLO results in increased immune cell cytotoxicity against EGFR bearing cells (e.g., cancer cells). In some embodiments, the antigen targeting molecule conjugated to LLO-X is Cetuximab (Cetuximab- LLO). In some embodiments, treatment with Cetuximab-LLO results in increased immune cell cytotoxicity against EGFR bearing cells (e.g., cancer cells).

[0296] In some embodiments, the antigen targeting molecule conjugated to LLO-X is an BCMA antigen targeting molecule (BCMA-LLO). In some embodiments, the antigen targeting molecule conjugated to LLO-X is Teclistamab (Teclistamab-LLO). In some embodiments, treatment with BCMA-LLO results in increased immune cell cytotoxicity against BCMA bearing cells (e.g., cancer cells). In some embodiments, treatment with Teclistamab-LLO results in increased immune cell cytotoxicity against BCMA bearing cells (e.g., cancer cells). In some embodiments, the antigen targeting molecule conjugated to LLO-X is Elranatamab (Elranatamab-LLO). In some embodiments, treatment with Elranatamab-LLO results in increased immune cell cytotoxicity against BCMA bearing cells (e.g., cancer cells).

[0297] In some embodiments, the antigen targeting molecule conjugated to LLO-X is an GPRC5D antigen targeting molecule (GPRC5D-LLO). In some embodiments, the antigen targeting molecule conjugated to LLO-X is Talquetamab (Talquetamab-LLO). In some embodiments, treatment with GPRC5D-LLO results in increased immune cell cytotoxicity against GPRC5D bearing cells (e.g., cancer cells). In some embodiments, treatment with Talquetamab-LLO results in increased immune cell cytotoxicity against GPRC5D bearing cells (e.g., cancer cells).

[0298] In some embodiments, the antigen targeting molecule conjugated to LLO-X is an HER2 antigen targeting molecule (HER2-LLO). In some embodiments, the antigen targeting molecule conjugated to LLO-X is Pertuzumab (Pertuzumab-LLO). In some embodiments, treatment with HER2-LLO results in increased immune cell cytotoxicity against HER2 bearing cells (e.g., cancer cells). In some embodiments, treatment with Pertuzumab-LLO results in increased immune cell cytotoxicity against HER2 bearing cells (e.g., cancer cells). In some embodiments, the antigen targeting molecule conjugated to LLO-X is Trastuzumab(Trastuzumab-LLO). In some embodiments, treatment with Trastuzumab-LLO results in increased immune cell cytotoxicity against HER2 bearing cells (e.g., cancer cells).

[0299] In some embodiments, the antigen targeting molecule conjugated to LLO-X is an CD30 antigen targeting molecule (CD30-LLO). In some embodiments, the antigen targeting molecule conjugated to LLO-X is Brentuximab (Brentuximab-LLO). In some embodiments, treatment with CD30-LLO results in increased immune cell cytotoxicity against CD30 bearing cells (e.g., cancer cells). In some embodiments, treatment with Brentuximab-LLO results in increased immune cell cytotoxicity against CD30 bearing cells (e.g., cancer cells).

[0300] In certain embodiments, cancers for which the present compositions and methods described herein are useful for treatment, prevention, and / or amelioration of symptoms include at least CD47, CD24, CD19, CD20, CD30, HER2, GPRC5D, EGER, EGER2, BCMA, and / or c-MET expressing cancers. In certain embodiments, cancers for which the present compositions and methods described herein are useful for treatment, prevention, and / or amelioration of symptoms include at least CD47 expressing cancers. In certain embodiments, cancers for which the present compositions and methods described herein are useful for treatment, prevention, and / or amelioration of symptoms include at least CD 19 expressing cancers. In certain embodiments, cancers for which the present compositions and methods described herein are useful for treatment, prevention, and / or amelioration of symptoms include at least CD20 expressing cancers. In certain embodiments, cancers for which the present compositions and methods described herein are useful for treatment, prevention, and / or amelioration of symptoms include at least EGFR expressing cancers. In certain embodiments, cancers for which the present compositions and methods described herein are useful for treatment, prevention, and / or amelioration of symptoms include at least BCMA expressing cancers. In certain embodiments, cancers for which the present compositions and methods described herein are useful for treatment, prevention, and / or amelioration of symptoms include at least c-MET expressing cancers. In certain embodiments, cancers for which the present compositions and methods described herein are useful for treatment, prevention, and / or amelioration of symptoms include at least CD70, and / or TROP2 expressing cancers.

[0301] In certain embodiments, compositions and methods described herein are utilized for treatment, prevention, and / or amelioration of symptoms associated with PDAC. In certain embodiments, compositions and methods described herein are utilized for treatment, prevention, and / or amelioration of symptoms associated with CRC. In certain embodiments, compositions and methods described herein are utilized for treatment, prevention, and / or amelioration of symptoms associated with ovarian cancer. In certain embodiments,compositions and methods described herein are utilized for treatment, prevention, and / or amelioration of symptoms associated with leukemias. In certain embodiments, compositions and methods described herein are utilized for treatment, prevention, and / or amelioration of symptoms associated with kidney cancer. In certain embodiments, compositions and methods described herein are utilized for treatment, prevention, and / or amelioration of symptoms associated with glioblastoma. In certain embodiments, compositions and methods described herein are utilized for treatment, prevention, and / or amelioration of symptoms associated with breast cancer. In certain embodiments, compositions and methods described herein are utilized for treatment, prevention, and / or amelioration of symptoms associated with RCC. In certain embodiments, compositions and methods described herein are utilized for treatment, prevention, and / or amelioration of symptoms associated with myeloma.

[0302] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget’s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreadingmelanoma; lentigo malignant melanoma; acral lentiginous melanomas; nodular melanomas; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi’s sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing’s sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin’s disease; Hodgkin’s; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin’s lymphomas; B-cell lymphoma; low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.

[0303] Particular embodiments concern methods of treatment of leukemia. Leukemia is a cancer of the blood or bone marrow and is characterized by an abnormal proliferation (production by multiplication) of blood cells, usually white blood cells (leukocytes). It is partof the broad group of diseases called hematological neoplasms. Leukemia is a broad term covering a spectrum of diseases. Leukemia is clinically and pathologically split into its acute and chronic forms.

[0304] Acute leukemia is characterized by the rapid proliferation of immature blood cells. This crowding makes the bone marrow unable to produce healthy blood cells. Acute forms of leukemia can occur in children and young adults. In fact, it is a more common cause of death for children in the U.S. than any other type of malignant disease. Immediate treatment is required in acute leukemia due to the rapid progression and accumulation of the malignant cells, which then spill over into the bloodstream and spread to other organs of the body. Central nervous system (CNS) involvement is uncommon, although the disease can occasionally cause cranial nerve palsies. Chronic leukemia is distinguished by the excessive buildup of relatively mature, but still abnormal, blood cells. Typically taking months to years to progress, the cells are produced at a much higher rate than normal cells, resulting in many abnormal white blood cells in the blood. Chronic leukemia mostly occurs in older people, but can theoretically occur in any age group. Whereas acute leukemia must be treated immediately, chronic forms are sometimes monitored for some time before treatment to ensure maximum effectiveness of therapy.

[0305] Furthermore, the diseases are classified into lymphocytic or lymphoblastic, which indicate that the cancerous change took place in a type of marrow cell that normally goes on to form lymphocytes, and myelogenous or myeloid, which indicate that the cancerous change took place in a type of marrow cell that normally goes on to form red cells, some types of white cells, and platelets (see lymphoid cells vs. myeloid cells).

[0306] Acute lymphocytic leukemia (also known as acute lymphoblastic leukemia, or ALL) is the most common type of leukemia in young children. This disease also affects adults, especially those aged 65 and older. Chronic lymphocytic leukemia (CLL) most often affects adults over the age of 55. It sometimes occurs in younger adults, but it almost never affects children. Acute myelogenous leukemia (also known as acute myeloid leukemia, or AML) occurs more commonly in adults than in children. This type of leukemia was previously called “acute nonlymphocytic leukemia.” Chronic myelogenous leukemia (CML) occurs mainly in adults. A very small number of children also develop this disease.

[0307] Lymphoma is a type of cancer that originates in lymphocytes (a type of white blood cell in the vertebrate immune system). There are many types of lymphoma. According to the U.S. National Institutes of Health, lymphomas account for about five percent of all cases of cancer in the United States, and Hodgkin's lymphoma in particular accounts for less than onepercent of all cases of cancer in the United States. Because the lymphatic system is part of the body's immune system, patients with a weakened immune system, such as from HIV infection or from certain drugs or medication, also have a higher incidence of lymphoma.

[0308] In certain embodiments of the disclosure, compositions comprising the ADCs are delivered to an individual in need thereof, such as an individual that has cancer or an infection. In at least some cases, the ADCs can enhance the individual’s immune system to attack the respective cancer or pathogenic cells. In some cases, the individual is provided with one or more doses of the compositions comprising ADC antibodies. In cases where the individual is provided with two or more doses of the ADC, the duration between the administrations should be sufficient to allow time for propagation in the individual, and in specific embodiments the duration between doses is 1, 2, 3, 4, 5, 6, 7, or more days.

[0309] Suitable modes of administration include intravenous, subcutaneous, intracavitary (for example by reservoir-access device), intraperitoneal, and direct injection into a tumor mass. A pharmaceutical composition of the present disclosure can be used alone or in combination with other well-established agents useful for treating cancer. Whether delivered alone or in combination with other agents, the pharmaceutical composition of the present disclosure can be delivered via various routes and to various sites in a mammalian, particularly human, body to achieve a particular effect. One skilled in the art will recognize that, although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. For example, intradermal delivery may be advantageously used over inhalation for the treatment of melanoma. Local or systemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, intraportal, intrahepatic, peritoneal, subcutaneous, or intradermal administration.

[0310] Desirably an effective amount or sufficient number of the ADCs is present in the composition and introduced into the subject such that long-term, specific, anti-tumor responses are established to reduce the size of a tumor or eliminate tumor growth or regrowth than would otherwise result in the absence of such treatment. Desirably, the amount of ADCs introduced into the subject causes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in tumor size when compared to otherwise same conditions wherein the ADCs are not present.

[0311] Accordingly, the amount of compositions comprising ADCs administered should take into account the route of administration and should be such that a sufficient number of thecompositions comprising ADCs will be introduced so as to achieve the desired therapeutic response. Furthermore, the amounts of each active agent included in the compositions described herein (e.g., the amount per certain body weight) can vary in different applications.

[0312] The values provided herein provide general guidance of the range of compositions comprising ADCs to be utilized by the practitioner upon optimizing the method of the present disclosure for practice of the present methods. The recitation herein of such ranges by no means precludes the use of a higher or lower amount of a component, as might be warranted in a particular application. For example, the actual dose and schedule can vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on interindividual differences in pharmacokinetics, drug disposition, and metabolism. One skilled in the art readily can make any necessary adjustments in accordance with the exigencies of the particular situation.A. Combination Therapies

[0313] In certain embodiments, the compositions and methods of the present embodiments involve a cancer therapy that is additional to the compositions comprising the ADCs. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, hormone therapy, immune cell therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

[0314] In some embodiments, the additional therapy is the administration of small molecule enzymatic inhibitor(s) or anti-metastatic agent(s). In some embodiments, the additional therapy is the administration of side-effect limiting agents (e.g., agents intended to lessen the occurrence and / or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is therapy targeting PBK / AKT / mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and / or chemopreventative agent(s). The additional therapy may be one or more of the chemotherapeutic agents known in the art.

[0315] An ADC therapy (in addition to the compositions of the disclosure) may be administered before, during, after, or in various combinations relative to an additional cancer therapy, such as immune checkpoint therapy. The administrations may be in intervals rangingfrom concurrently to minutes to days to weeks. In embodiments where the ADC therapy is provided to a patient separately from the composition(s) of the disclosure, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the ADC therapy and the disclosed compositions within about 12 to 24 or 72 h of each other and, more particularly, within about 6-12 h of each other. In some situations it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

[0316] Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy.1. Chemotherapy

[0317] A wide variety of chemotherapeutic agents may be used in accordance with the present embodiments. The term “chemotherapy” refers to the use of drugs to treat cancer. A “chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

[0318] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracilmustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PS Kpoly saccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine,famesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.2. Radiotherapy

[0319] Other factors that cause DNA damage and have been used extensively include what are commonly known as y-rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as micro waves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287), and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.3. Immunotherapy

[0320] The skilled artisan will understand that additional immunotherapies (outside of the disclosed ADC therapy) may be used in combination or in conjunction with methods of the embodiments.

[0321] In certain embodiments, additional immunotherapies comprise administration of one or more antibodies to a subject. In certain embodiments, the additional one or more antibodies that are administered to a subject may be the same one or more antibodies conjugated to an ADC. In some embodiments, the additional one or more antibodies that are administered to a subject may target one or more of the same antigens as the one or more antibodies conjugated to an ADC. In some embodiments, the additional one or more antibodies that are administered to a subject are administered prior to administration of ADC (e.g., CD47- LLO), for example but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days prior to administration of ADCs. In some embodiments, the additional one or more antibodies that are administered to a subject are administered after administration of ADCs (e.g., CD47-LLO), for example but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or longer than 28 days, after administration of ADCs. In some embodiments, the additional one or more antibodies that are administered to a subject are administered concurrently with administration of ADCs (e.g., CD47-LLO), for example but not limited to, 1, 2, 3, 4, 5, or more than 5 times.

[0322] In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab(RITUXAN®) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells other than those having knockdown or knockout of TGF-beta R2.

[0323] Antibody-drug conjugates have emerged as a breakthrough approach to the development of cancer therapeutics. Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) that are covalently linked to cell-killing drugs. This approach combines the high specificity of MAbs against their antigen targets with highly potent cytotoxic drugs, resulting in “armed” MAbs that deliver the payload (drug) to tumor cells with enriched levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in decreased toxicity and improved therapeutic index. The approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013 by FDA validated the approach. There are currently more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization are becoming more and more mature, the discovery and development of new ADCs are increasingly dependent on the identification and validation of new targets that are suitable to this approach and the generation of targeting MAbs. Two criteria for ADC targets are upregulated / high levels of expression in tumor cells and robust internalization.

[0324] In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiments. Common tumor markers include CD47, CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, and pl 55. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL- 12, GM-CSF, gamma- IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.

[0325] Examples of immunotherapies currently under investigation or in use are immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, andaromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, e.g., interferons of any kind, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-pl85 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed with the antibody therapies described herein.

[0326] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either turn up a signal e.g., co-stimulatory molecules) or turn down a signal. Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA). In particular, the immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.4. Surgery

[0327] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electro surgery, and microscopically-controlled surgery (Mohs’ surgery).

[0328] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.5. Other Agents

[0329] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.IV. Pharmaceutical Compositions

[0330] The present disclosure includes methods for treating disease and modulating immune responses in a subject in need thereof. The disclosure includes cells that may be in the form of a pharmaceutical composition that can be used to induce or modify an immune response.

[0331] Administration of the compositions according to the current disclosure will typically be via any common route. This includes, but is not limited to parenteral, orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. In certain embodiments, compositions disclosed herein are administered directly to the site of a tumor. In certain embodiments, compositions disclosed herein are administered directly to the site of a tumor before and / or after tumor resection.

[0332] Typically, compositions of the disclosure are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immune modifying. The quantity to be administered depends on the subject to be treated. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner.

[0333] The manner of application may be varied widely. Any of the conventional methods for administration of pharmaceutical compositions comprising peptide components areapplicable. The dosage of the pharmaceutical composition will depend on the route of administration and will vary according to the size and health of the subject.

[0334] In many instances, it will be desirable to have multiple administrations of at most about or at least about 3, 4, 5, 6, 7, 8, 9, 10 or more. The administrations may range from 2- day to 12-week intervals, more usually from one to two week intervals. The course of the administrations may be followed by assays for alloreactive immune responses and T cell activity.

[0335] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. The pharmaceutical compositions of the current disclosure are pharmaceutically acceptable compositions.

[0336] The compositions of the disclosure can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions and the preparations can also be emulsified.

[0337] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0338] Sterile injectable solutions are prepared by incorporating the active ingredients (i.e. cells of the disclosure) in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.

[0339] An effective amount of a composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed herein in association with its administration, i.e., the appropriateroute and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.V. Additional Therapies

[0340] The current methods and compositions of the disclosure may include one or more additional therapies known in the art and / or described herein. In some embodiments, the additional therapy or agent comprises an additional cancer treatment. Examples of such treatments are described herein.A. Immunotherapies

[0341] In some embodiments, the additional therapy or agent comprises a cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumour-associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines. Immunotherapies are known in the art, and some are described below.B. Inhibition of co-stimulatory molecules

[0342] In some embodiments, the immunotherapy comprises an inhibitor of a costimulatory molecule. In some embodiments, the inhibitor comprises an inhibitor of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.C. Dendritic cell therapy

[0343] Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.

[0344] One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte macrophage colony- stimulating factor (GM-CSF).

[0345] Dendritic cells can also be activated in vivo by making tumor cells express GM- CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[0346] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor- specific peptide / protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.

[0347] Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.D. CAR-T cell therapy

[0348] Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy.

[0349] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells. Scientists canremove T-cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells. Once the T cell has been engineered to become a CAR-T cell, it acts as a “living drug”. CAR-T cells create a link between an extracellular ligand recognition domain to an intracellular signaling molecule which in turn activates T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of molecule that is targeted.

[0350] Exemplary CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta). In some embodiments, the CAR-T therapy targets CD19.E. Cytokine therapy

[0351] Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.

[0352] Interferons are produced by the immune system. They are usually involved in antiviral response, but also have use for cancer. They fall in three groups: type I (IFNa and IFNP), type II (IFNy) and type III (IFN ).

[0353] Interleukins have an array of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.F. Adoptive T-cell therapy

[0354] Adoptive T cell therapy is a form of passive immunization by the transfusion of T- cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically they activate when the T-cell's surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumour death.

[0355] Multiple ways of producing and obtaining tumour targeted T-cells have been developed. T-cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the resultsreinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens.G. Checkpoint Inhibitors and Combination Treatment

[0356] In some embodiments, the additional therapy or agent comprises immune checkpoint inhibitors. Certain embodiments are further described below.1) PD-1, PDL1, and PDL2 inhibitors

[0357] PD -1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0358] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7- DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.

[0359] In some embodiments, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and / or PDL2. In another embodiment, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and / or B7-1. In another embodiment, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all incorporated herein by reference.

[0360] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD- 1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and Pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., animmunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX- 1106-04, MDX- 1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010 / 027827 and WO2011 / 066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0361] In some embodiments, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.

[0362] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or Pidilizumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or Pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or Pidilizumab. In another embodiment, the antibody competes for binding with and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.2) CTLA-4, B7-1, and B7-2

[0363] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD 152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co- stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits aninhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA- 4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some embodiments, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[0364] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0365] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. W02001 / 014424, W02000 / 037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.

[0366] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WOO 1 / 14424).

[0367] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another embodiment, the antibody competes for binding with and / or binds to the same epitope on PD-1, B7-1, or B7-2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.H. Oncolytic virus

[0368] In some embodiments, the additional therapy or agent comprises an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumour. Oncolytic viruses are thought not only to cause direct destruction of the tumour cells, but also to stimulate host anti-tumour immune responses for long-term immunotherapy.I. Polysaccharides

[0369] In some embodiments, the additional therapy or agent comprises polysaccharides. Certain compounds found in mushrooms, primarily polysaccharides, can up-regulate the immune system and may have anti-cancer properties. For example, beta- glucans such as lentinan have been shown in laboratory studies to stimulate macrophage, NK cells, T cells and immune system cytokines and have been investigated in clinical trials as immunologic adjuvants.J. Neoantigens

[0370] In some embodiments, the additional therapy or agent comprises neoantigen administration. Many tumors express mutations. These mutations potentially create new targetable antigens (neoantigens) for use in T cell immunotherapy. The presence of CD8+ T cells in cancer lesions, as identified using RNA sequencing data, is higher in tumors with a high mutational burden. The level of transcripts associated with cytolytic activity of natural killer cells and T cells positively correlates with mutational load in many human tumors.K. Chemotherapies

[0371] In some embodiments, the additional therapy or agent or agent comprises a chemotherapy. Suitable classes of chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin), (c) Natural Products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxanthrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., Interferon-a), and (d) Miscellaneous Agents, such asplatinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydiazine derivatives (e.g., procarbazine), and adrenocortical suppressants (e.g., taxol and mitotane). In some embodiments, cisplatin is a particularly suitable chemotherapeutic agent.

[0372] Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, with efficacious doses used in clinical applications including about 15 mg / m2to about 20 mg / m2for 5 days every three weeks for a total of three courses being contemplated in certain embodiments.

[0373] Other suitable chemotherapeutic agents include antimicrotubule agents, e.g., Paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”).

[0374] Nitrogen mustards are another suitable chemotherapeutic agent that may be useful as a cotherapy in certain the methods of the disclosure. A nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L- sarcolysin), and chlorambucil.

[0375] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluouracil; 5-FU) and floxuridine (fluorode- oxyuridine; FudR). 5-FU may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg / m2. Further, 5-FU dosing schedules may be for a variety of time periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.

[0376] Gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., “gemcitabine”), another suitable chemotherapeutic agent, is recommended for treatment of advanced and metastatic pancreatic cancer, and will therefore be useful in the present disclosure for these cancers as well, in some embodiments.

[0377] The amount of the chemotherapeutic agent delivered to the patient may be variable. In one suitable embodiment, the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered with compositions of the disclosure.L. Radiotherapy

[0378] In some embodiments, the additional therapy or agent or prior therapy comprises radiation, such as ionizing radiation. As used herein, “ionizing radiation” means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons). An exemplary and preferred ionizing radiation is an x-radiation. Means for delivering x-radiation to a target tissue or cell are well known in the art.

[0379] In some embodiments, the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some embodiments, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some embodiments, the amount of ionizing radiation is at least, at most, or exactly 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 40 Gy (or any derivable range therein). In some embodiments, the ionizing radiation is administered in at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 does (or any derivable range therein). When more than one dose is administered, the does may be about 1, 4, 8, 12, or 24 hours or 1, 2, 3, 4, 5, 6, 7, or 8 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range therein.

[0380] In some embodiments, the amount of IR may be presented as a total dose of IR, which is then administered in fractionated doses. For example, in some embodiments, the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each. In some embodiments, the total dose is 50-90 Gy, administered in 20-60 fractionated doses of 2-3 Gy each. In some embodiments, the total dose of IR is at least, at most, or about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, or 150 (or any derivable range therein). In some embodiments, the total dose is administered in fractionated doses of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein. In some embodiments, at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or 100 fractionated doses are administered (or any derivable rangetherein). In some embodiments, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) fractionated doses are administered per day. In some embodiments, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) fractionated doses are administered per week.M. Surgery

[0381] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electro surgery, and microscopically-controlled surgery (Mohs’ surgery).

[0382] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.N. Other Agents

[0383] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of ahyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.VI. Administration of Therapeutic Compositions

[0384] Methods of the disclosure include administration of a combination of therapeutic agents and / or administration of therapeutic agents, such as fecal matter and therapeutic regimens, such as steroid therapy or anti-integrin therapy, for example. The therapy may be administered in any suitable manner known in the art. For example, the therapies may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the therapies are in a separate composition. In some embodiments, the therapies are in the same composition.

[0385] Various combinations of the therapies may be employed, for example, one therapy designated “A” and another therapy designated “B”:

[0386] A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B

[0387] B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A

[0388] B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A

[0389] The therapies of the disclosure, such as the fecal matter from a healthy subject may be administered by the same route of administration or by different routes of administration. In some embodiments, the therapy is administered intracolonically, intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the microbial modulator is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

[0390] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg / kg to 200 mg / kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg / kg, mg / kg, pg / day, or mg / day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and / or on multiple days, weeks, or months.

[0391] In some embodiments, the therapeutically effective or sufficient amount of a therapeutic composition that is administered to a human will be in the range of about 0.01 to about 50 mg / kg of patient body weight whether by one or more administrations. In some embodiments, the therapeutic agent used is about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg administered daily, for example. In some embodiments, the therapeutic agent is administered at 15 mg / kg. However, other dosage regimens may be useful. In one embodiment, a therapeutic agent described herein is administered to a subject at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of 21-day cycles. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. The progress of this therapy is easily monitored by conventional techniques.

[0392] In certain embodiments, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM. In another embodiment, the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein). In other embodiments, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 pM or any range derivable therein. In certain embodiments, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0393] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0394] It will be understood by those skilled in the art and made aware that dosage units of pg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of pg / ml or mM (blood levels), such as 4 pM to 100 pM. It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.VII. Kits

[0395] Certain aspects of the disclosure also encompass kits for performing ...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A composition comprising, a CD47 targeting agent conjugated to a pore-forming agent.

2. The composition of claim 1, wherein the CD47 targeting agent comprises an antibody or a functional fragment thereof.

3. The composition of claim 2, wherein the antibody or functional fragment thereof comprises Magrolimab, B6H12, BRIC126, SRF231, MIAP410, AO-176, CC-90002, GenSci- 059, Lemzoparlimab, Letaplimab, Ligufalimab, ZL-1201, IBL322, TG-1801, IMC-002, TQB2928, PF-07257876, HX009, and / or STI-6643 .

4. The composition of claim 3, wherein the antibody or functional fragment thereof is humanized, optionally wherein the antibody or functional fragment thereof is an IgGl, IgG2, IgG2B, or IgG4 isotype.

5. The composition of claim 4, wherein the antigen targeting agent is capable of binding to an antigen expressed on a cancer cell.

6. The composition of claim 5, wherein the antigen targeting agent neutralizes the target antigen.

7. The composition of claim 6, wherein the cancer cell comprises a breast cancer, lung cancer, non-small cell lung cancer, brain cancer, glioblastoma, triple-negative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma cell.

8. The composition of claim 7, wherein the cancer overexpresses CD47.

9. The composition of any one of claims 1-8, wherein the pore forming agent comprises Listeriolysin O (LLO), melittin, proteins from the membrane attack complex / perforin (MACPF) family, cholesterol dependent cytolysin (CDC) family, and / or the Pleurotolysin B pore-forming family, e.g., but not limited to, perfringolysin O (PFO), alveolysin, streptolysin O (SLO), pneumolysin, ivanolysin, hemolysin, pyolysin, flavomodulin, tetanolysin O, MAC / perforin domain containing protein, complement component C8-alpha, complement component C6, complement component C9, complement component C7, complement component C8-beta, perforin-like protein 1, apextrin, hemopexin, photopexin a / b, SpoCl-CIC, toxin AvTX-60A, toxin PsTX-60B, MACPF domain-containing protein CADI, MACPF domain-containing protein NSL1, Macrophage expressed protein, Pleurotolysin, Erylysin, Ostreolysin, Cytolysin, Aegerolysin, intermedilysin (ILY), siulysin, aerolysin, ClyA, colicin, lysenin, CrylAa, Fragaceatoxin C, perforin, Tc holotoxin, a-haemolysin, and / or SmhB.

10. The composition of claims 9, wherein the pore forming agent comprises a bacterial peptide and / or bacterial toxin.

11. The composition of claim 9, wherein the pore forming agent is only active in an acidic environment with a pH of, of less than, or of about 6.5.

12. The composition of claim 11, wherein the pore forming agent is only active in an acidic environment with a pH of, less than, or of about 6.0 or 5.9, and / or is optimally active at a pH of or of about 5.5.

13. The composition of claim 9, wherein the pore forming agent is active in a phagosome.

14. The composition of claim 9, wherein the pore forming agent is not active in the cytoplasm of a cell.

15. The composition of claim 9, wherein the pore forming agent is not active at the cell membrane.

16. The composition of any one of claims 1-8, wherein the pore forming agent comprises, consists essentially of, or consists of LLO.

17. The composition of claim 9, wherein the CD47 antigen targeting agent and the poreforming agent are reversibly or irreversibly conjugated together.

18. The composition of claim 9, wherein the CD47 antigen targeting agent and the poreforming agent are conjugated through a cleavable linker.

19. The composition of claim 9, wherein the pore forming agent is active only once cleaved from the antigen targeting agent.

20. The composition of claim 9, wherein the CD47 antigen targeting agent and the poreforming agent are conjugated through click chemistry.

21. The composition of claim 20, wherein the click chemistry is copper-free click chemistry.

22. The composition of claim 19, wherein the click chemistry comprises a Polyethylene glycol (PEG), dibenzocyclooctyne (DBCO), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and / or a triazole linker.

23. The composition of claim 9, wherein the composition comprises at least 90% product showing a molar ration of 1 pore-forming agent molecule per CD47 antigen targeting agent molecule.

24. The composition of claim 9, wherein the pore-forming agent is conjugated to a primary amine on the CD47 antigen targeting agent.

25. The composition of claim 9, wherein the pore-forming agent is cleavable from the CD47 antigen targeting agent when exposed to reduced conditions.

26. A composition comprising, the CD47 antigen targeting agent conjugated to a poreforming agent of claim 9, wherein the composition comprises a pH greater than 7.0.

27. The composition of claim 9, further comprising a pharmaceutically acceptable excipient.

28. A method of making the composition of claim 9, the method comprising conjugating a CD47 antigen targeting agent to a pore-forming agent.

29. A method of treating a disease or disorder in a subject, the method comprising administering the composition of claim 27 to the subject.

30. A composition comprising, a CD47 antigen targeting agent conjugated to Listeriolysin O (LLO).

31. The composition of claim 30, wherein the CD47 antigen targeting agent comprises Magrolimab, B6H12, BRIC126, SRF231, MIAP410, AO-176, CC-90002, GenSci-059, Lemzoparlimab, Letaplimab, Ligufalimab, ZL-1201, IBI-322, TG-1801, IMC-002, TQB2928, PF-07257876, HX009, and / or STI-6643.

32. The composition of claim 31, wherein the CD47 antigen targeting agent is humanized.

33. The composition of claim 32, wherein the CD47 antigen targeting agent is capable of binding to a CD47 antigen expressed on a cancer cell.

34. The composition of claim 33, wherein the CD47 antigen targeting agent neutralizes the CD47 target antigen.

35. The composition of claim 34, wherein the cancer cell comprises a breast cancer, lung cancer, non-small cell lung cancer, brain cancer, glioblastoma, triple-negative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma cell.

36. The composition of claim 35, wherein the LLO permeabilizes phagolysosomes in an acidic environment with a pH of, of less than, or of about 6.5.

37. The composition of any one of claims 30-36, wherein the LLO permeabilizes phagolysosomes in an acidic environment with a pH of, of less than, or of about 6.0 or 5.9, and / or is optimally active at a pH of or of about 5.5.

38. The composition of claim 37, wherein the LLO is active in a phagosome.

39. The composition of claim 38, wherein the LLO is not active in the cytoplasm of a cell.

40. The composition of claim 39, wherein the LLO is not active at the cell membrane.

41. The composition of claim 40, wherein the CD47 antigen targeting agent and the LLO are conjugated together through a reversible or an irreversible linker.

42. The composition of claim 41, wherein the CD47 antigen targeting agent and the LLO are conjugated through a cleavable linker.

43. The composition of claim 42, wherein the LLO is active only once cleaved from the CD47 antigen targeting agent.

44. The composition of claim 43, wherein the CD47 antigen targeting agent and the LLO are conjugated through click chemistry.

45. The composition of claim 44, wherein the click chemistry is copper-free click chemistry.

46. The composition of claim 45, wherein the click chemistry comprises a Polyethylene glycol (PEG), dibenzocyclooctyne (DBCO), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and / or a triazole linker.

47. The composition of claim 46, wherein the composition comprises at least 90% product showing a molar ration of 1 LLO molecule per CD47 antigen targeting agent molecule.

48. The composition of claim 47, wherein the LLO is conjugated to a primary amine on the CD47 antigen targeting agent.

49. The composition of claim 48, wherein the LLO is cleavable from the CD47 antigen targeting agent when exposed to reduced conditions.

50. A composition comprising, the CD47 antigen targeting agent conjugated to the LLO of claim 49, wherein the composition comprises a pH of greater than 7.0.

51. The composition of claim 50, further comprising a pharmaceutically acceptable excipient.

52. A method of making the composition of claim 51, the method comprising conjugating a CD47 antigen targeting agent to LLO.

53. A method of treating a disease or disorder in a subject, the method comprising administering the composition of claim 51 to the subject.

54. A composition comprising, a Listeriolysin O (LLO) molecule conjugated to an antigen targeting agent, through a cleavable linker.

55. The composition of claim 54, wherein the antigen targeting agent comprises an antibody or functional fragment thereof.

56. The composition of claim 55, wherein the antibody or functional fragment thereof targets a CD47, CD24, PDL1, CD19, CD20, CD30, GPRC5D, EGFR, EGFR2, BCMA, c- MET, and / or HER2 antigen.

57. The composition of 56, wherein the antibody or fragment thereof is humanized, optionally wherein the antibody or functional fragment thereof is an IgGl, IgG2, IgG2B, or IgG4 isotype.

58. The composition of claim 57, wherein the antigen targeting agent is capable of binding to an antigen expressed on a cancer cell.

59. The composition of claim 58, wherein the antigen targeting agent neutralizes the target antigen.

60. The composition of claim 58, wherein the cancer cell comprises a breast cancer, lung cancer, non-small cell lung cancer, brain cancer, glioblastoma, triple-negative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma cell.

61. The composition of any one of claims 54-60, wherein the LLO is only active in an acidic environment with a pH of, of less than, or of about 6.5.

62. The composition of claim 60, wherein the LLO is only active in an acidic environment with a pH of, of less than, or of about 6.0 or 5.9, and / or is optimally active at a pH of or of about 5.5.

63. The composition of claim 62, wherein the LLO is active in a phagosome.

64. The composition of claim 63, wherein the LLO is not active in the cytoplasm of a cell.

65. The composition of claim 64, wherein the LLO is not active at the cell membrane.

66. The composition of claim 65, wherein the antigen targeting agent and the LLO are conjugated together.

67. The composition of claim 66, wherein the antigen targeting agent and the LLO are conjugated through a cleavable linker that can be cleaved upon exposure to reduced conditions.

68. The composition of claim 67, wherein the LLO is active only once cleaved from the antigen targeting agent.

69. The composition of claim 68, wherein the LLO molecule and the antigen targeting agent are conjugated through click chemistry.

70. The composition of claim 69, wherein the click chemistry is copper-free click chemistry.

71. The composition of claim 70, wherein the LLO is conjugated to a primary amine on the antigen targeting agent.

72. A composition comprising, the LLO molecule conjugated to an antigen targeting agent through a cleavable linker of claim 63, wherein the composition comprises a pH greater than 7.0.

73. The composition of claim 63, further comprising a pharmaceutically acceptable excipient.

74. A method of making the composition of claim 63, the method comprising conjugating an antigen targeting agent to LLO.

75. A method of treating a disease or disorder in a subject, the method comprising administering the composition of claim 73 to the subject.

76. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, and optionally a therapeutically effective amount of an immune checkpoint inhibitor.

77. The method of claim 76, wherein the pore-forming agent is or comprises LLO.

78. The method of claim 76 or 77, wherein the antigen targeting agent targets CD47.

79. The method of claim 78, wherein the patient has, has been diagnosed with, has one or more symptoms of, or is suspected of having a cancer.

80. The method of claim 79, wherein the cancer comprises breast cancer, lung cancer, nonsmall cell lung cancer, brain cancer, glioblastoma, triple-negative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma.

81. The method of claim 78, wherein administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, and a therapeutically effective amount of an immune checkpoint inhibitor comprises intratumoral, intraperitoneal, and or / intravenous delivery.

82. The method of claim 78, wherein the immune checkpoint inhibitor comprises PD-1 inhibitors (e.g., nivolumab, pembrolizumab, MED 10680, and / or pidilizumab), PDL2-inhibitors (e.g., AMP- 224, rHIgM12B7), PDL1 inhibitors (e.g., Durvalumab, atezolizumab, avelumab), CTLA-4 inhibitors, B7-1 inhibitors, and / or B7-2 inhibitors.

83. The method of claim 78, wherein the immune checkpoint inhibitor comprises a PD1- antigen targeting agent.

84. The method of claim 83, wherein the PDl-antigen targeting agent comprises nivolumab, pembrolizumab, MEDI0680, and / or pidilizumab.

85. The method of claim 78, wherein one or more additional therapies or therapeutic agents are co-administered before, after, or during the administration of a therapeutically effectiveamount of an antigen targeting agent conjugated to a pore-forming agent, and a therapeutically effective amount of an immune checkpoint inhibitor.

86. The method of claim 85, wherein the one or more additional therapies comprise tumor debulking, surgery, chemotherapy, radiotherapy, immunotherapy, and / or other agents.

87. The method of claim 78, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, the cancer accumulates intratumoral CD8+ T cells with TCRs specific to cancer antigens.

88. The method of claim 78, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, bone- marrow derived dendritic cells and / or bone-marrow derived macrophages display tumor- associated antigens.

89. The method of claim 88, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, bone- marrow derived dendritic cells and / or bone-marrow derived macrophages exhibit activation of stimulator of interferon genes (STING).

90. The method of claim 78, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent:APCs exhibit increased expression of genes encoding one or more of CD86, IL1, and IL 12, and / or lymphocytes exhibit increased expression of genes encoding CD40.

91. The method of claim 78, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, the antigen targeting agent conjugated to a pore-forming agent accumulates within one or more tumor.

92. The method of claim 91, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, the subject exhibits systemic antitumor immune memory.

93. The method of claim 92, wherein antitumor immune memory comprises: interferon-gamma production by isolated splenic CD8+ T cells upon contact with (i) one or more tumor cell and / or (ii) one or more tumor- associated antigen.

94. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent.

95. The method of claim 94 wherein the pore-forming agent is LLO.

96. The method of claim 94 or 95, wherein the antigen targeting agent targets CD47.

97. The method of claim 96, wherein the patient has, has been diagnosed with, has one or more symptoms of, or is suspected of having a cancer.

98. The method of claim 97, wherein the cancer comprises breast cancer, lung cancer, nonsmall cell lung cancer, brain cancer, glioblastoma, triple-negative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma.

99. The method of claim 96, wherein one or more additional therapies or therapeutic agents are co-administered before, after, or during the administration of a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent.

100. The method of claim 99, wherein the one or more additional therapies comprise tumor debulking, surgery, chemotherapy, radiotherapy, immunotherapy, and / or other agents.

101. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of a composition according to claims 1-8, 30-31, 54-60, 76-77, or 94-95.

102. The method of claim 101, wherein the patient has, has been diagnosed with, has one or more symptoms of, or is suspected of having a cancer.

103. The method of claim 102, wherein the cancer comprises breast cancer, lung cancer, non-small cell lung cancer, brain cancer, glioblastoma, triple-negative breast carcinoma, melanoma, leukemia, bladder tumor, pancreatic cancer, liver cancer, prostate cancer, adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, ovarian cancer, epithelial ovarian cancer, acute myeloid leukemia, myeloma, non-Hodgkin lymphoma, lymphoblastic lymphoma / acute lymphoblastic leukemia, multiple myeloma, head and neck squamous cell carcinoma, leiomyosarcoma, chronic myeloid leukemia, diffuse large cell B lymphoma, cholangiocarcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, and / or lymphoma.

104. The method of claim 103, wherein administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, comprises intratumoral, intraperitoneal, and or / intravenous delivery.

105. The method of claim 104, wherein one or more additional therapies or therapeutic agents are co-administered before, after, or during the administration of a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent.

106. The method of claim 105, wherein the one or more additional therapies comprise tumor debulking, surgery, chemotherapy, radiotherapy, immunotherapy, and / or other agents.

107. The method of claim 106, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, the tumor accumulates intratumoral CD8+ T cells with TCRs specific to tumor antigens.

108. The method of claim 107, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, bone-marrow derived dendritic cells and / or bone-marrow derived macrophages display tumor- associated antigens.

109. The method of claim 108, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, bone- marrow derived dendritic cells and / or bone-marrow derived macrophages exhibit activation of stimulator of interferon genes (STING).

110. The method of claim 109, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent,APCs exhibit increased expression of genes encoding one or more of CD86, IL1, and IL 12, and / or lymphocytes exhibit increased expression genes encoding CD40.

111. The method of claim 110, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, the antigen targeting agent conjugated to a pore-forming agent accumulates within one or more tumor.

112. The method of claim 111, wherein upon administering to a subject a therapeutically effective amount of an antigen targeting agent conjugated to a pore-forming agent, the subject exhibits systemic antitumor immune memory.

113. The method of claim 112, wherein antitumor immune memory comprises: interferon-gamma production by isolated splenic CD8+ T cells upon contact with (i) one or more tumor cell and / or (ii) one or more tumor- associated antigen.

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