Universal Receptor Immune Cell Therapy

Abstract

The present invention relates to methods for universal immune receptor cell-based therapy.

Description

[Technical field]

[0001] This application claims priority from AU2021 / 902320, filed July 28, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to methods for universal immune receptor cell-based therapy. [Background technology]

[0003] Despite the success of chimeric immune-based cell therapies, such as chimeric antigen receptor (CAR) T cells as cellular immunotherapy for hematological cancers, these therapies can have inherent complications that can occur, which can limit the therapeutic efficacy. The main toxicities can range from treatable B-cell aplasia to more severe cytokine release syndrome and neurotoxicity. Furthermore, traditional immune cell therapies have so far shown limited efficacy for treating solid tumors. This is due to many factors, including an immunosuppressive tumor microenvironment (TME), inefficient cell trafficking, and heterogeneity in antigen expression. In the context of both solid and hematological malignancies, relapse is common due to tumor escape. All of these are currently unmet needs in cellular immunotherapy that can be addressed by universal immune receptor expressing immune cells.

[0004] Universal immune receptors (UIRs) are composed of two separate components: (i) a standard intracellular signaling domain similar to conventional CARs with an extracellular adaptor protein (such as SpyCatcher), and (ii) a targeting antibody conjugated to an adaptor protein (such as SpyTAG) (WO2017 / 112784). The targeting antibody can then act as an immunological bridge to target tumor antigens and the extracellular adaptor on the SpyCatcher receptor to elicit an antigen-specific cellular response. By uncoupling the antigen recognition domain of the CAR from the intracellular signaling domain into a separate individuality element, it is then possible to target one or more different antigens with the same UIR to overcome immune escape or tumor heterogeneity, and to perform dose adjustment or withdrawal to modulate UIR immune cell function after administration, further increasing the safety of adoptive immunotherapy.

[0005] Improved universal immune receptor cell-based therapies are needed to more effectively and reliably treat diseases such as cancer while minimizing common problems encountered with current therapies such as CAR T. Summary of the Invention

[0006] The present invention provides a method for effectively treating subjects using the universal immune receptor system. The persistence of recombinant immune cells, such as CAR T cells, can be achieved by transient resting, which leads to epigenetic changes. In the present invention, this is achieved by periodic administration and interruption of administration of tagged binding agents.

[0007] In one aspect, the present invention provides a method of treating a disease in a subject that would benefit from immune cell therapy, comprising: i) administering to a subject an immune cell comprising a universal immune receptor, where the universal immune receptor may or may not be covalently linked to a molecule comprising a domain that binds an antigen associated with the disease; ii) administering the molecule to the subject at least twice within seven days following step i); iii) analyzing the subject for responsiveness to treatment at least about 21 days after step ii); iv) if the subject responds to treatment but disease is still detectable, repeating steps i) and ii).

[0008] In another aspect, there is provided a method of stimulating a universal immune receptor-mediated immune response against a tumor in a subject, comprising: i) administering to a subject an immune cell comprising a universal immune receptor, where the universal immune receptor may or may not be covalently linked to a molecule comprising a domain that binds an antigen associated with a tumor; ii) administering the molecule to the subject at least twice within seven days following step i); iii) analyzing the subject for responsiveness to treatment at least about 21 days after step ii); iv) if the subject responds to treatment but the tumor is still detectable, repeating steps i) and ii).

[0009] In one embodiment, the molecule does not bind to a universal immune receptor in step i).

[0010] In one embodiment, the molecule binds to a universal immune receptor in step i).

[0011] In one embodiment, in step ii), the molecule is administered twice, hi one embodiment, the molecule is administered on the third and sixth days after step i).

[0012] In one embodiment, in step ii), the molecule is administered three times, hi one embodiment, the molecule is administered on days 1, 4, and 6 after step i).

[0013] In certain embodiments, the subject is analyzed for responsiveness to the treatment about 21 to about 49 days after step ii). In certain embodiments, the subject is analyzed for responsiveness to the treatment about 21 days after step ii). In certain embodiments, the subject is analyzed for responsiveness to the treatment about 49 days after step ii).

[0014] In one embodiment, step ii) comprises administering the molecule to the subject at least once prior to step i) and at least twice within 7 days after step i).In another embodiment, step ii) comprises administering the molecule to the subject twice prior to step i) and at least twice within 7 days after step i).

[0015] In certain embodiments, step (ii) comprises one of the following dosing regimens: (a) dosing within a 24-48 hour time frame, and optionally repeating, within 7 days following administration of the immune cells of step i); (b) dosing every 24 hours within 7 days following administration of the immune cells of step i); (c) dosing intermittently, including pausing treatment for a given time frame, resuming treatment for a next time frame, and pausing treatment for a next time frame, within 7 days after administration of the immune cells in step i).

[0016] In any of (a), (b), or (c) above, the UIR cells administered in step i) may be unarmed or prearmed.

[0017] In another embodiment, the molecules are administered in different doses. In one example, the different doses are about 0.75 mg / m 2 , about 15mg / m 2 and about 75 mg / m 2 In another embodiment, the different dose is about 0.25 mg / m 2 , about 0.5mg / m 2 , about 0.75mg / m 2 , about 1mg / m 2, about 2.5mg / m 2 , about 5.0mg / m 2 , about 7.5mg / m 2 , about 10mg / m 2 , about 12.5mg / m 2 , about 15.0mg / m 2 , about 17.5mg / m 2 , about 20mg / m 2 , about 22.5mg / m 2 , about 25mg / m 2 , about 35mg / m 2 , about 45mg / m 2 , about 55mg / m 2 , about 65mg / m 2 , about 70mg / m 2 , about 75mg / m 2 , about 80mg / m 2 , about 85mg / m 2 , about 90mg / m 2 , about 95mg / m 2 In another embodiment, the dose is about 0.25 mg / m 2 ~2.0mg / m 2 , about 0.5mg / m 2 ~1.5mg / m 2 , or about 0.5 mg / m 2 ~1.0mg / m 2 In another embodiment, the dose is about 5 mg / m 2 ~25mg / m 2 , about 10mg / m 2 ~20mg / m 2 , or about 12 mg / m 2 ~17mg / m 2 In another embodiment, the dose is about 50 mg / m 2 ~100mg / m 2 , about 60mg / m 2 ~90mg / m 2 , or about 70 mg / m 2 ~80mg / m 2 may be also possible.

[0018] In another embodiment, the different doses of the molecule administered to the subject include about 0.25 mg, about 24 mg, or about 120 mg per about 1.6 body surface area (BSA). In another example, the different doses of the molecule administered to the subject include one of about 0.1 mg, about 0.15 mg, about 0.25 mg, about 0.35 mg, about 0.45 mg, about 0.6 mg, about 12 mg, about 16 mg, about 20 mg, about 24 mg, about 28 mg, about 32 mg, about 36 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, or more per about 1.6 BSA. In another embodiment, the different doses of the molecule administered to the subject include about 0.1 mg to 2.0 mg, about 0.2 mg to 1.5 mg, or about 0.2 mg to 0.75 mg. In another embodiment, the different doses of the molecule administered to the subject include about 4 mg to 36 mg, about 12 mg to 32 mg, or about 20 mg to 28 mg, hi another embodiment, the different doses of the molecule include about 60 mg to 160 mg, about 80 mg to 140 mg, or about 100 mg to 130 mg.

[0019] In one embodiment, step iv) comprises administering a universal immune receptor, which may or may not be covalently linked to a molecule that contains a domain that binds the same antigen as the molecule of step i), hi another embodiment, step iv) comprises administering a universal immune receptor, which may or may not be covalently linked to a molecule that contains a domain that binds a different antigen than the molecule of step i).

[0020] In one embodiment, the molecule comprises a domain that binds two or more antigens associated with a disease. In one embodiment, the molecule comprises a domain that binds two antigens associated with a disease, preferably cancer. In another embodiment, the molecule comprises a domain that binds three antigens associated with a disease, preferably cancer.

[0021] In another aspect, the invention provides a method of treating a disease in a subject that would benefit from immune cell therapy, comprising: i) administering to a subject an immune cell comprising a universal immune receptor, where the universal immune receptor may or may not be covalently linked to a molecule comprising a domain that binds an antigen associated with the disease; ii) administering the molecule to the subject at least every 2 or 3 days for about 14 days to about 28 days following step i); and iii) if the subject responds to treatment but disease is still detectable, repeating steps i) and ii).

[0022] In another aspect, there is provided a method of stimulating a universal immune receptor-mediated immune response against a tumor in a subject, comprising: i) administering to a subject an immune cell comprising a universal immune receptor, where the universal immune receptor may or may not be covalently linked to a molecule comprising a domain that binds an antigen associated with a tumor; ii) administering the molecule to the subject at least every 2 or 3 days for about 14 days to about 28 days following step i); and iii) if the subject responds to treatment but the tumor is still detectable, repeating steps i) and ii).

[0023] In certain embodiments of the above aspects, the molecule does not bind to a universal immune receptor in step i).

[0024] In certain embodiments of the above aspects, the molecule binds to a universal immune receptor in step i).

[0025] In certain embodiments of the above aspects, the molecule is administered every 3 days after step i).

[0026] In certain embodiments of the above aspects, the molecule is administered every 3 days for about 21 days following step i).

[0027] In certain embodiments of the above aspects, the subject is analyzed for responsiveness to treatment within 7 days, 5 days, 3 days, or 1 day of completing step ii).

[0028] In some embodiments, step i) comprises administering the molecule to the subject at least once prior to administration of the immune cells in step i) and every two or three days for about 14 to about 28 days after step i). In some embodiments, step ii) comprises administering the molecule to the subject twice prior to step i) and every two or three days for about 14 to about 28 days after step i).

[0029] In one embodiment, stimulating a universal immune receptor-mediated immune response against tumors comprises increasing cytokine levels in the subject, preferably increasing the levels of one or more or all of interferon gamma (IFN-γ), tumor necrosis factor (TNF), and interleukin-2 (IL-2).

[0030] In one embodiment, step iii) comprises administering a universal immune receptor, which may or may not be covalently linked to a molecule that contains a domain that binds the same antigen as the molecule of step i), hi another embodiment, step iii) comprises administering a universal immune receptor, which may or may not be covalently linked to a molecule that contains a domain that binds a different antigen than the molecule of step i).

[0031] In one embodiment, the molecule comprises a domain that binds two or more antigens associated with the disease. In one embodiment, the molecule comprises a domain that binds two antigens associated with the disease. In another embodiment, the molecule comprises a domain that binds three antigens associated with the disease.

[0032] In some embodiments, the treatment increases survival time in a subject. In some embodiments, survival time is increased compared to a subject not receiving the treatment. In some embodiments, survival time is increased by 3, 6, 9, 12, 24, 36, 48, 60, 72, 84, 96 months or more compared to a subject not receiving the treatment.

[0033] In some embodiments, the subject has been diagnosed with or is suspected of having a disease, such as cancer, an infectious disease, or an inflammatory disease. Thus, in some embodiments, the methods described herein include diagnosing the subject as having or being suspected of having a disease, such as cancer, an infectious disease, or an inflammatory disease.

[0034] In an embodiment, the method or use optionally further comprises administration of an additional therapeutic agent selected from the group consisting of chemotherapy, radiation therapy, surgery, bone marrow transplantation, drug therapy, cryoablation, or radiofrequency ablation.

[0035] In one embodiment, the universal immune receptor comprises a SpyCatcher or SpyTag extracellular binding domain bound to an extracellular hinge region, which in turn binds to a transmembrane domain, which in turn binds to an immune cell receptor intracellular signaling domain.

[0036] In some embodiments, the universal immune receptor intracellular signaling domain further comprises a costimulatory molecule.

[0037] In one embodiment, the SpyCatcher extracellular binding domain binds to the extracellular hinge domain. In an alternative embodiment, the SpyTag extracellular binding domain binds to the extracellular hinge domain.

[0038] In some embodiments, the molecule comprises a SpyCatcher or SpyTag and a domain.

[0039] In some embodiments, the domain is selected from the group consisting of an antibody, an antibody fragment, an scFv, a protein scaffold, a peptide, a ligand, an oligonucleotide, an aptamer, a tumor antigen, an autoantigen, a viral antigen, and any combination thereof, hi some embodiments, the domain is an antibody or an antibody fragment.

[0040] In one embodiment, the molecule comprises SpyTag. In an alternative embodiment, the molecule comprises SpyCatcher.

[0041] In an alternative embodiment, the above-mentioned SpyTyg is a SnoopTag and the above-mentioned SpyCatcher is a SnoopCatcher.

[0042] In some embodiments, the molecule is a polypeptide comprising a first domain that binds the extracellular binding domain and a second domain that binds an antigen associated with a disease, hi some embodiments, the molecule further comprises a third domain that is a labeling agent.

[0043] In one embodiment, the molecule is at about 0.25 mg / m 2 , about 0.5mg / m 2 , about 0.75mg / m 2 , about 1mg / m 2 , about 2.5mg / m 2 , about 5.0mg / m 2 , about 7.5mg / m 2 , about 10mg / m 2 , about 12.5mg / m 2 , about 15.0mg / m 2 , about 17.5mg / m 2 , about 20mg / m 2 , about 22.5mg / m 2 , about 25mg / m 2 , about 35mg / m 2 , about 45mg / m 2 , about 55mg / m 2 , about 65mg / m 2 , about 70mg / m 2 , about 75mg / m 2 , about 80mg / m 2 , about 85mg / m 2, about 90mg / m 2 , about 95mg / m 2 In another embodiment, the dose of the molecule administered to the subject is about 0.25 mg / m 2 ~2.0mg / m 2 , about 0.5mg / m 2 ~1.5mg / m 2 , or about 0.5 mg / m 2 ~1.0mg / m 2 In another embodiment, the dose of the molecule administered to the subject is about 5 mg / m 2 ~25mg / m 2 , about 10mg / m 2 ~20mg / m 2 , or about 12 mg / m 2 ~17mg / m 2 In another embodiment, the dose of the molecule administered to the subject is about 50 mg / m 2 ~100mg / m 2 , about 60mg / m 2 ~90mg / m 2 , or about 70 mg / m 2 ~80mg / m 2 Preferably, the dose of the molecule administered to the subject is about 0.75 mg / m 2 , about 15mg / m 2 , or about 75 mg / m 2 It is.

[0044] In certain embodiments, the molecule is administered to the subject at a dose of about 0.1 mg, about 0.15 mg, about 0.25 mg, about 0.35 mg, about 0.45 mg, about 0.6 mg, about 12 mg, about 16 mg, about 20 mg, about 24 mg, about 28 mg, about 32 mg, about 36 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, or more per about 1.6 body surface area (BSA). In another embodiment, the dose of the molecule administered to the subject is about 0.1 mg to 2.0 mg, about 0.2 mg to 1.5 mg, or about 0.2 mg to 0.75 mg. In another embodiment, the dose of the molecule administered to the subject is about 4 mg to 36 mg, about 12 mg to 32 mg, or about 20 mg to 28 mg. In another embodiment, the dose of the molecule administered to the subject is about 60 mg to 160 mg, about 80 mg to 140 mg, or about 100 mg to 130 mg. Preferably, the dose of the molecule administered to the subject is about 0.25 mg, about 24 mg, or about 120 mg. Those skilled in the art will understand how to calculate the equivalent dose for different BSAs.

[0045] In certain embodiments, the immune cells are T cells, NK cells, dendritic cells, myeloid cells, macrophages, stem cells, or combinations thereof.

[0046] In some embodiments, the T cell is a CD3+ T cell. In some embodiments, the T cell is a cytotoxic T cell, a gamma delta T cell, a T regulatory cell, or an iNKT cell.

[0047] In some embodiments, the methods described herein provide for enrichment of CD4+ and / or CD8+ T cells. In another embodiment, at least about 10% of the immune cells are CD8+ cells. In some embodiments, at least about 20% of the immune cells are CD8+ cells. In some embodiments, at least about 30% of the immune cells are CD8+ cells. In some embodiments, at least about 40% of the immune cells are CD8+ cells. In some embodiments, at least about 50% of the immune cells are CD8+ cells. In some embodiments, at least about 60% of the immune cells are CD8+ cells. In some embodiments, about 10% to about 60% of the immune cells are CD8+ cells. In some embodiments, about 10% to about 50% of the immune cells are CD8+ cells. In some embodiments, about 10% to about 40% of the immune cells are CD8+ cells. In some embodiments, about 10% to about 30% of the immune cells are CD8+ cells.

[0048] In another embodiment, the methods described herein provide for enrichment of CD45RO+CD45RA- T effector memory cells. In another embodiment, the methods described herein include enrichment of CD45RA+CD45RO- T central memory cells.

[0049] In embodiments where the invention provides for administration of immune cells comprising a universal immune receptor covalently linked to a molecule, the method expands CD8+ universal immune receptor cells in the spleen and / or tumor.

[0050] In certain embodiments, the cells are autologous. In alternative embodiments, the cells are allogeneic.

[0051] In certain embodiments, the disease is cancer, an infectious disease, or an inflammatory disease.

[0052] Examples of cancers that may be treated using the present invention include, but are not limited to, renal cell carcinoma, pancreatic cancer, head and neck cancer, prostate cancer, glioblastoma, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, melanoma, leukemia, acute myeloid leukemia (AML), or lymphoma.

[0053] In some embodiments, the subject is a mammal, hi some embodiments, the subject is a human.

[0054] The invention further provides for the use of immune cells comprising a universal immune receptor for the manufacture of a medicament for treating a disease in a subject that may benefit from immune cell therapy, the universal immune receptor may or may not be covalently linked to a molecule comprising a domain that binds an antigen associated with the disease, the molecule being administered to the subject at least twice within seven days following administration of the cells, the subject being analyzed for responsiveness to the treatment for at least 21 days following the seven days, and if the subject responds to the treatment but the disease is still detectable, the treatment is repeated.

[0055] Also provided are immune cells comprising a universal immune receptor for use in treating a disease in a subject that may benefit from immune cell therapy, the universal immune receptor may or may not be covalently linked to a molecule comprising a domain that binds an antigen associated with the disease, the molecule to be administered to the subject at least twice within seven days following administration of the cells, the subject to be analyzed for responsiveness to the treatment for at least 21 days following the seven days, and if the subject responds to the treatment but the disease is still detectable, the treatment is repeated.

[0056] Also provided is the use of immune cells comprising a universal immune receptor to manufacture a medicament for stimulating a universal immune receptor-mediated immune response against a tumor in a subject, where the universal immune receptor may or may not be covalently linked to a molecule comprising a domain that binds an antigen associated with the tumor, where the molecule would be administered to the subject at least twice within seven days following administration of the cells, where the subject would be analyzed for responsiveness to the treatment for at least 21 days following the seven days, where if the subject responds to the treatment but the tumor is still detectable, the treatment would be repeated.

[0057] Also provided is an immune cell comprising a universal immune receptor for use in stimulating a universal immune receptor-mediated immune response against a tumor in a subject, where the universal immune receptor may or may not be covalently linked to a molecule comprising a domain that binds an antigen associated with the tumor, where the molecule would be administered to the subject at least twice within seven days following administration of the cells, where the subject would be analyzed for responsiveness to treatment for at least 21 days following the seven days, where if the subject responds to treatment but the tumor is still detectable, the treatment would be repeated.

[0058] Also provided is the use of immune cells comprising a universal immune receptor for the manufacture of a medicament for treating a disease in a subject that may benefit from immune cell therapy, where the universal immune receptor may or may not be covalently linked to a molecule comprising a domain that binds an antigen associated with the disease, where the molecule is administered to the subject every 2 or 3 days for 14 to 28 days following administration of the cells, with the treatment being repeated if the subject responds to the treatment but disease is still detectable.

[0059] Also provided are immune cells comprising a universal immune receptor for use in treating a disease in a subject that may benefit from immune cell therapy, where the universal immune receptor may or may not be covalently linked to a molecule comprising a domain that binds an antigen associated with the disease, the molecule being administered to the subject every 2 or 3 days for 14 to 28 days following administration of the cells, with the treatment being repeated if the subject responds to the treatment but disease is still detectable.

[0060] Also provided is the use of immune cells comprising a universal immune receptor for the manufacture of a medicament for stimulating a universal immune receptor-mediated immune response against a tumor in a subject, where the universal immune receptor may or may not be covalently linked to a molecule comprising a domain that binds an antigen associated with the tumor, where the molecule is administered to the subject every 2 or 3 days for 14 to 28 days following administration of the cells, where the treatment is repeated if the subject responds to the treatment but the tumor is still detectable.

[0061] Also provided is an immune cell comprising a universal immune receptor for use in stimulating a universal immune receptor-mediated immune response against a tumor in a subject, where the universal immune receptor may or may not be covalently linked to a molecule comprising a domain that binds an antigen associated with the tumor, the molecule being administered to the subject every 2 or 3 days for 14 to 28 days following administration of the cells, with the treatment being repeated if the subject responds to the treatment but the tumor is still detectable.

[0062] In another aspect, the present invention provides a substantially purified and / or recombinant polypeptide comprising a sequence of amino acids provided as SEQ ID NO:5 or SEQ ID NO:6, or a sequence of amino acids that is at least 90% identical to one or both of SEQ ID NO:5 and SEQ ID NO:6, wherein the polypeptide is covalently linked to a protein comprising SpyCatcher and capable of binding the HER2 receptor on a cancer cell.

[0063] In another aspect, the present invention provides a substantially purified and / or recombinant polypeptide having an amino acid sequence comprising, or at least 90% identical to, the amino acid sequence provided as SEQ ID NO:7 and / or SEQ ID NO:10, or provided as SEQ ID NO:8 and / or SEQ ID NO:9, which is covalently linked to a protein comprising SpyCatcher and capable of binding the EGFRvIII receptor on a cancer cell.

[0064] In another aspect, the present invention provides a substantially purified and / or recombinant polypeptide comprising, or having an amino acid sequence that is at least 90% identical to, the sequence of amino acids provided as SEQ ID NO:11 and / or SEQ ID NO:14, or as provided as SEQ ID NO:12 and / or SEQ ID NO:13, wherein the polypeptide is covalently linked to a protein that comprises SpyCatcher and is capable of binding the IL-13Ra2 receptor on a cancer cell.

[0065] In another aspect, the present invention provides a substantially purified and / or recombinant polypeptide comprising a sequence of amino acids as provided as SEQ ID NO: 15 and / or SEQ ID NO: 16, or a sequence of amino acids which is at least 90% identical thereto, wherein the polypeptide is covalently linked to a protein comprising SpyCatcher and capable of binding the CD33 receptor on a cancer cell.

[0066] In another aspect, the present invention provides a substantially purified and / or recombinant polypeptide comprising a sequence of amino acids as provided as SEQ ID NO: 17 and / or SEQ ID NO: 18, or a sequence of amino acids which is at least 90% identical thereto, wherein the polypeptide is covalently bound to a protein comprising SpyCatcher and capable of binding a C-type lectin-like (CLL1) receptor on a cancer cell.

[0067] In another aspect, the present invention provides isolated and / or exogenous polynucleotides encoding the polypeptides of the present invention.

[0068] In a further aspect, the present invention provides a vector comprising a polynucleotide of the present invention.

[0069] In yet another aspect, the invention provides an isolated transgenic cell comprising a polynucleotide of the invention and / or a vector of the invention, hi one embodiment, the cell is a bacterial cell or a mammalian cell.

[0070] In yet another aspect, the invention provides pharmaceutical compositions comprising immune cells comprising a universal immune receptor, which may or may not be covalently linked to a molecule comprising a domain that binds an antigen associated with a disease, in one embodiment, the domain comprises one or more or all of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or a sequence of amino acids at least 90% identical thereto.

[0071] In another aspect, the invention provides a method for producing a polypeptide of the invention comprising culturing a cell of the invention and purifying the polypeptide from the cell or culture medium.

[0072] In one embodiment, the cells are cultured in the presence of an immune cell activator, preferably IL-2, an anti-CD3 antibody, or an anti-CD28 antibody. In another embodiment, the immune cell activator increases expression of Tim-3 and / or PD-1. In yet another embodiment, at least about 30% of the immune cells are Tim-3 and / or PD-1 positive.

[0073] Any embodiment herein shall apply mutatis mutandis to any other embodiment unless otherwise specified.

[0074] The present invention is not to be limited in scope by the specific embodiments described herein, which are for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the invention as described herein.

[0075] Throughout this specification, unless otherwise specified or the context requires otherwise, references to a single step, composition of matter, group of steps, or group of compositions of matter should be interpreted as encompassing one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0076] The invention will now be described by way of the following non-limiting examples and with reference to the accompanying drawings. [Brief description of the drawings]

[0077] [Figure 1] 1 is an example of a dosing schedule. [Diagram 2] 1 is an example of a dosing schedule. [Diagram 3] 1 is an example of a dosing schedule. [Figure 4] 1 is an example of a dosing schedule. [Diagram 5] 1 is an example of a dosing schedule. [Figure 6]The dosing regimen of the binders modulates functional UIR expression in vivo. (A) Expression of "unarmed" OmniCAR T cells (FLAG+ only) and "armed" OmniCAR T cells (FLAG+IgG+). (B) "Armed" OmniCAR receptor detected by IgG MFI in CD8+FLAG+ or CD4+FLAG+ CAR T cells armed with increasing concentrations of antibody binders. (C) Production of cytokines IFNγ, TNF, and IL-2 by OmniCAR T cells armed with increasing concentrations of binders co-cultured with MDA-MB231-HER2 tumors for 24 hours. (D) FACS plot showing % of armed OmniCAR receptor on T cells isolated from blood 1 or 7 days after adoptive transfer. (E) IgG+% of CD8+FLAG+ CAR T cells from blood 1 day after transfer for groups dosed with various doses of binders every 3-4 days. (F) Number of armed IgG+FLAG+OmniCAR T cells in blood 1 day after transfer for groups dosed with various doses of binder every 3-4 days. [Figure 7-1] Binder dosing regimens modulate T cell memory phenotype, expansion, and persistence in vivo. (A) Schematic of treatment regimens (low dose=1ug, high dose=25ug). Preconditioned tumor-bearing mice were treated with a single dose of 10-20 million unarmed or prearmed OmniCAR T cells according to treatment regimes 1-3, and further dosed with antibody binders. (B) Number of CD8+FLAG+ cells / uL of blood 1 day post-transfer. (C) MFI of IgG staining on OmniCAR T cells from blood 7 days post-transfer. (D) Number of total CD8+ T cells / uL of blood 7 days post-transfer for untransduced vs. unarmed OmniCAR groups. (E) Memory phenotype of CD45RO+CD45RA- (T effector memory) or CD45RA+CD45RO- (T central memory) populations. Data are presented as mean±SEM. [Figure 7-2] Same as above. [Figure 8]The dosing regimen of the binder modulates the antitumor efficacy in vivo. (A) Tumor growth curves of mice treated with pre-armed OmniCAR T cells and dosed with high doses of binders. (B) Spleens or (C) tumors were extracted at the end of treatment and CD8+FLAG+ OmniCAR T cells were counted. (D) TIM3+PD1+ population % of tumor-derived CD8+FLAG+ OmniCAR TILs at the end point. (E) Bioluminescence imaging to determine tumor burden over time in a mouse model of acute myeloid leukemia (AML). NSG mice were given 5 million KG-1 cells and animals were left untreated (control) or given pre-armed OmniCAR-T cells and 25ug of CD33 and CLL-1 binders on days 3, 6, and 9 after CAR-T transfer. Data are shown as mean ± SEM. [Figure 9] The dosing regimen and specific design of the binders modulates OmniCAR antigen-independent or antigen-dependent signaling. (A) TIM3 expression in unstimulated or stimulated (OKT3) T cells after 72 hours, subsetted by % CD8+FLAG+ (top) or % CD4+FLAG+ (bottom). (B) PD-1 expression (left) in unstimulated or stimulated (OKT3) T cells after 72 hours, subsetted by % CD8+FLAG+ (top) or % CD4+FLAG+ (bottom). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, data shown as mean ± SEM. [Figure 10-1]Metronomic dosing for sequential or simultaneous targeting of multiple tumor antigens. (A) Number of mixed tumor cultures of U251MG-HER2 (GFP) and U251MG-EGFRviii (mCherry) tumors. (B) Anti-HER2 armed OmniCAR T cells co-cultured with mixed tumor cultures. (C) Anti-EGFRviii armed OmniCAR T cells co-cultured with mixed tumor cultures. (D) Anti-EGFRviii armed OmniCAR T cells co-cultured with mixed tumor cultures with (HER2LT switching) or without (no switching) anti-HER2 binders 20 hours after co-culture. Data are shown as mean ± SD. (E) Histogram (top) of expression of the three binders on the same OmniCAR T cell sample (bottom). FACS plots binder expression in each double combination on the same OmniCAR T cell product. (F) Determination of the presence of HER2 and EGFRvIII antibody binders in the serum of mice. [Figure 10-2] Same as above. [Figure 11] Model of antigen-independent tonic signaling in OmniCARs compared to conventional CAR T cells to modulate memory and anti-tumor functional capabilities. (A) Schematic of metronomic dosing encompassing time, dose modulation, or combination strategies of multiple binders. (B) Model of antigen-independent modulation of tonic signaling and the interrelationship between memory phenotype and functional / anti-tumor capabilities.

[0078] Legend for sequence table SEQ ID NO:1 - SpyCatcher universal immune receptor amino acid sequence SEQ ID NO:2 - Nucleotide sequence encoding the SpyCatcher universal immune receptor SEQ ID NO:3 - Standard Her2-SpyTag binder with N-terminal signal SEQ ID NO: 4 - Short half-life Her2-SpyTag binder with N-terminal signal SEQ ID NO:5 - Standard Her2-SpyTag binder without N-terminal signal SEQ ID NO:6 - Short half-life Her2-SpyTag binder without N-terminal signal SEQ ID NO:7 - EGFRvIII heavy chain amino acid sequence SEQ ID NO:8 - EGFRvIII heavy chain with SpyTag amino acid sequence SEQ ID NO:9 - EGFRvIII light chain amino acid sequence SEQ ID NO:10 - EGFRvIII light chain with SpyTag amino acid sequence SEQ ID NO:11 - IL-13Ra2 heavy chain amino acid sequence SEQ ID NO:12 - IL-13Ra2 heavy chain with SpyTag amino acid sequence SEQ ID NO:13 - IL-13Ra2 light chain amino acid sequence SEQ ID NO:14 - IL-13Ra2 light chain with SpyTag amino acid sequence SEQ ID NO:15 - CD33 heavy chain amino acid sequence SEQ ID NO:16 - CD33 light chain amino acid sequence with SpyTag amino acid sequence SEQ ID NO:17 - CLL1 heavy chain amino acid sequence SEQ ID NO:18 - CLL1 light chain amino acid sequence with SpyTag amino acid sequence DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0079] General Techniques and Definitions Unless specifically defined otherwise, all technical and scientific terms used herein shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, cell-based immunotherapy, molecular genetics, protein chemistry, and biochemistry).

[0080] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Usubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current editions), Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J. E. Coligan et al. al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all updates until present), and other sources of information.

[0081] The term "and / or", e.g., "X and / or Y", is to be understood to mean either "X and Y" or "X or Y" and is to be interpreted as explicitly endorsing both meanings or either meaning.

[0082] As used herein, unless stated to the contrary, the term about refers to + / - 10%, more preferably + / - 5%, more preferably + / - 1% of the specified value.

[0083] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0084] As used herein, the term "subject" can be any animal. In one embodiment, the animal is a vertebrate. For example, the animal can be a mammal, a bird, a chordate, an amphibian, or a reptile. Exemplary subjects include, but are not limited to, humans, primates, livestock (e.g., sheep, cows, chickens, horses, donkeys, pigs), companion animals (e.g., dogs, cats), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), and captive wild animals (e.g., foxes, deer). In one embodiment, the mammal is a human. In some embodiments, the methods of the invention are for veterinary use.

[0085] The term "treat" or "treatment" as used herein refers to both direct treatment of a subject by a medical professional (e.g., by administering a therapeutic agent to the subject) or indirect treatment by at least one party (e.g., a doctor, nurse, pharmacist, or pharmaceutical sales representative) by (i) instructing the subject to self-treat according to the claimed methods (e.g., self-administering a drug), or (ii) providing any form of instructions instructing a third party to treat the subject according to the claimed methods. The meaning of the term "treat" or "treatment" also includes the prevention or reduction of the disease being treated, for example, by administering a therapeutic agent at an early enough stage to prevent or slow the progression of the disease.

[0086] As used herein, the term "subject responds to treatment but disease is still detectable" refers to a detectable reduction in disease (such as at least a 75% reduction, at least a 50% reduction, or at least a 25% reduction) (such as a reduction in tumor burden) but the disease is still present. Methods for detecting diseases that can be treated using the methods of the present invention are well known in the art and include imaging methods (such as PET, PET_SPECT, and MRI), cell detection, and pathogen detection techniques.

[0087] As used herein, "cytokine release syndrome" (CRS) refers to an acute systemic inflammatory syndrome characterized by fever and multiple organ dysfunction that is associated with chimeric antigen receptor cell therapy, therapeutic antibodies, and haploidentical allogeneic transplantation.

[0088] A polypeptide may be defined by the degree of identity (% identity) of its amino acid sequence to a reference amino acid sequence, or by the % identity to one reference amino acid sequence being greater than to another reference amino acid sequence. The % identity of a polypeptide to a reference amino acid sequence is typically determined by GAP analysis (Needleman and Wunsch, 1970; GCG program) with parameters of gap creation penalty=5 and gap extension penalty=0.3. The query sequence is at least 100 amino acids long, and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids long, and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over the entire length of the reference amino acid sequence.

[0089] It is understood that with respect to the defined polypeptides, higher identity percentage figures than those provided herein encompass preferred embodiments. Thus, it is preferred that the polypeptide comprises an amino acid sequence that is at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant designated SEQ ID NO, in terms of the minimum identity percentage figures, if applicable. In certain embodiments, for each of the ranges recited above, the identity percentage does not include 100%, i.e., the amino acid sequence is different from the designated SEQ ID NO.

[0090] As used herein, terms such as "combination therapy," "combined administration," or "co-administration" are meant to encompass the administration of selected therapeutic agents to a single subject, and are intended to include therapeutic regimens in which agents are administered by the same or different routes of administration or at the same or different times.

[0091] Universal Immune Receptor As used herein, a "universal immune receptor" or "UIR" is a chimeric antigen receptor system in which an immune cell recombinantly expresses a protein comprising an extracellular binding domain bound to an extracellular hinge region, which in turn binds to a transmembrane domain, which in turn binds to an immune cell receptor intracellular signaling domain. This system further comprises a soluble molecule comprising a first domain that binds the extracellular binding domain and a second domain that binds an antigen associated with a disease (such as a cancer antigen on the surface of a cancer cell). As used herein, the term "universal immune receptor" may refer to a molecule that is bound (also referred to as armed) or not bound (also referred to as unarmed) to the extracellular binding domain. A UIR for use in the present invention forms a covalent bond when the first domain binds the extracellular binding domain.

[0092] An example of a universal immune receptor for use in the present invention is the SpyTag / SpyCatcher system (WO2017 / 112784). The term "SpyTag / SpyCatcher system" encompasses versions of the system, such as version 1 (US9,547,003), version 2 (WO 2018 / 197854), and version 3 (WO2020 / 183198). As another example, a universal immune receptor for use in the present invention is the SnoopTag / SnoopCatcher system (Veggiani et al., 2016; WO2016 / 193746).

[0093] The term "chimeric antigen receptor" or alternatively "CAR" in the context of the present invention provides the cell with specificity for a target cell and intracellular signal generation when present in an immune cell, once the molecule is covalently linked to an extracellular binding domain that binds, for example, a cancer cell.

[0094] CAR (UIR) can be used to generate immune cells such as T cells, dendritic cells, or natural killer (NK) cells specific to selected targets. Suitable constructs for generating CAR are described in US5,843,728, US5,851,828, US5,912,170, US6,004,811, US6,284,240, US6,392,013, US6,410,014, US6,753,162, US8,211,422, and WO9215322. Alternative CAR constructs can be characterized as belonging to successive generations. First generation CARs typically consist of a single chain variable fragment of an antigen-specific antibody, e.g., comprising a VL linked to the VH of a particular antibody, linked by a flexible linker, e.g., the CD8a hinge domain and the CD8a transmembrane domain, to the transmembrane and intracellular signaling domains of either CD3C or FcRy or scFv-FcRy (see, e.g., US 7,741,465, US 5,912,172, and US 5,906,936). Second generation CARs incorporate the intracellular domain of one or more costimulatory molecules, such as CD28, CD28z, OX40 (CD134), or 4-1BB (CD137), within an endodomain, e.g., scFv-CD28 / OX40 / 4 BB-CD3 (see, e.g., US8,911,993, US8,916,381, US8,975,071, US9,101,584, US9,102,760, US9,102,761). Third generation CARs include combinations of costimulatory endodomains such as CD3C chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, or CD28 signaling domains, e.g., scFv-CD28-4BB-CD3C or scFv-CD28-OX40-CD3Q (see, e.g., US8,906,682, US8,399,645, US5,686,281, WO2014 / 134165, and WO2012 / 079000). In some embodiments, costimulation can be coordinated by expression of the CAR in antigen-specific T cells that are selected for activation and expansion following interaction with, for example, an antigen on a professional antigen-presenting cell using the costimulatory agent.For example, immune cells can be provided with additional engineered receptors to improve targeting of T cell attack and / or minimize side effects.

[0095] Those skilled in the art will recognize that an "antibody" generally comprises one or more polypeptide chains, e.g., V L A polypeptide comprising: H It will be appreciated that an antibody is considered to be a protein comprising at least one variable region consisting of a polypeptide comprising: H and V L The light chains from mammals are either kappa or lambda light chains, and the heavy chains from mammals are alpha, delta, epsilon, gamma, or mu. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or protein class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY). 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 ), or subclass. The term "antibody" also includes humanized antibodies, primatized antibodies, human antibodies, and chimeric antibodies.

[0096] The terms "full length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in a substantially intact form, as opposed to an antigen-binding fragment of an antibody. Specifically, whole antibodies include those having heavy and light chains, including the Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0097] The term "antibody fragment" as used herein includes antibody fragments that retain the ability to bind to a target antigen, e.g., Fab, Fab', F(ab')2, Fv, scFv fragments, other antigen-binding subsequences of antibodies, and can include those produced by modification of whole antibodies or synthesized de novo using recombinant DNA technology, and corresponding fragments obtained from antibodies other than IgG. These antibody fragments can be obtained by conventional procedures, such as proteolytic fragmentation procedures, as described in J. Goding, Monoclonal Antibodies: Principles and Practice, pp 98-118 (NY Academic Press 1983), and other techniques known to those skilled in the art. The fragments are screened for utility in the same manner as intact antibodies.

[0098] Suitable antibodies or antigen-binding fragments include, but are not limited to, IgG, IgA, IgM, IgE, monoclonal antibodies, Fab', rIgG (half antibodies), f(ab')2, nanobodies, chimeric antibodies, scFv, scFv multimers, single domain antibodies, or single domain fusion antibodies. In some embodiments, the antibody or antibody-like molecule is a monoclonal antibody or an antigen-binding fragment thereof. As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody exhibits a single binding specificity and affinity for a particular epitope.

[0099] In some embodiments, the molecule is a polypeptide, hi some embodiments, the molecule is a single polypeptide chain encoded by a single open reading frame.

[0100] In some embodiments, the molecule further comprises a third domain that is a labeling agent. In some embodiments, the labeling agent is a myc tag, a FLAG tag, a His tag, an HA tag, a fluorescent protein (e.g., green fluorescent protein (GFP)), a fluorophore (e.g., tetramethylrhodamine (TRITC), fluorescein isothiocyanate (FITC)), dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), histidine, biotin, streptavidin, avidin, horseradish peroxidase, palmitoylation, nitrosylation, alcalanine phosphatase, glucose oxidase, glutathione S-transferase (GST), maltose binding protein, radioisotopes, and any type of compound used for radioisotope labeling, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10 -tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

[0101] Molecules that contain a domain that binds an antigen associated with a disease can be produced by any means known in the art. In one embodiment, the molecule is produced and purified from a recombinant cell that expresses the molecule. In another embodiment, the molecule is synthetic.

[0102] Methods for preparing UIR-expressing cells Cell Source Prior to expansion and possible genetic or other modification, a cell population can be obtained from a subject that includes or consists of immune cells, such as T cells, dendritic cells, macrophages, natural killer (NK) cells, or combinations thereof. Immune cells can be obtained from a number of sources, such as peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors.

[0103] In certain embodiments of the present disclosure, immune cells, e.g., T cells, can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation. In a preferred embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, dendritic cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and, optionally, to place the cells in an appropriate buffer or medium for subsequent processing steps. In one embodiment, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the washing solution can lack calcium, lack magnesium, or lack many, but not all, divalent cations.

[0104] An initial activation step in the absence of calcium may lead to amplified activation. As will be readily understood by those skilled in the art, the washing step may be accomplished by methods known to those skilled in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) following the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0105] It is recognized that the methods of the present application can utilize culture medium conditions comprising 5% or less, e.g., 2% human AB serum, and can employ known culture medium conditions and compositions, e.g., those described in Smith et al. (2015).

[0106] In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or counterflow centrifugal elutriation.

[0107] The methods described herein can include, for example, the selection of a particular subpopulation of immune cells, e.g., T cells, that is a T regulatory cell depleted population. For example, a CD25+ depleted cell population can be obtained, for example, using negative selection techniques described herein. Preferably, the population of T regulatory depleted cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells.

[0108] In one embodiment, T regulation (T REG ) cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody or fragment thereof, or a CD25-binding ligand, IL-2. In one embodiment, the anti-CD25 antibody or fragment thereof, or the CD25-binding ligand, is conjugated to or otherwise coated on a substrate, e.g., a bead. In one embodiment, the anti-CD25 antibody or fragment thereof is conjugated to a substrate as described herein.

[0109] Without wishing to be bound by a particular theory, it is contemplated that reducing the levels of negative regulators of immune cells in a subject prior to apheresis or during the manufacture of a UIR-expressing cell product (e.g., reducing the levels of undesirable immune cells, e.g., T REG By reducing the number of T cells, the risk of recurrence in a subject can be reduced. reg Methods for depleting cells are known in the art. REG Methods for reducing cells include, but are not limited to, cyclophosphamide, anti-GITR antibodies (anti-GITR antibodies described herein), CD25-depletion, and combinations thereof.

[0110] In some embodiments, the method includes the step of: REGFor example, the production method may include contacting a sample, e.g., an apheresis sample, with an anti-GITR antibody and / or an anti-CD25 antibody (or fragment thereof, or a CD25-binding ligand) to, e.g., reduce the number of UIR-expressing cells (e.g., T cells, NK cells) prior to producing the UIR-expressing cell (e.g., T cell, NK cell) product. REG This involves depleting the cells.

[0111] Cells for stimulation can also be frozen after a washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more homogenous product by removing granulocytes and to some extent monocytes within the cell population. After a washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, and then the cells are frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as immediate uncontrolled freezing at -20°C or in liquid nitrogen, may also be used.

[0112] In certain embodiments, cryopreserved cells are thawed as described herein, washed, and allowed to rest at room temperature for 1 hour before being activated using the methods of the invention.

[0113] In the context of the present invention, collection of a blood sample or apheresis product from a subject at a time period before the expanded cells described herein may be needed is also contemplated. In this way, a source of expanded cells can be collected at any time needed, and desired cells, such as T cells, can be isolated and frozen for later use in immune cell therapy for any number of diseases or conditions that would benefit from immune cell therapy, such as those described herein. In one embodiment, a blood sample or apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or apheresis is taken from a generally healthy subject who is at risk of developing a disease but has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, T cells may be expanded, frozen, and used later. In certain embodiments, a sample is collected from a patient immediately after diagnosis of a particular disease described herein and prior to any treatment. In further embodiments, the cells are isolated from a blood sample or apheresis from the subject prior to any number of relevant therapeutic modalities, including, but not limited to, treatment with drugs such as natalizumab, efalizumab, antivirals, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation.

[0114] Methods for generating UIR-expressing cells In one embodiment, the method of the present invention includes the generation of a UIR-expressing cell by introducing a vector or nucleic acid encoding the UIR into a cell. Methods for introducing and expressing genes into cells are known in the art. In the context of an expression vector, the vector can be easily introduced into a host T cell, such as a mammalian, bacterial, yeast, or insect T cell, by any method in the art. For example, the expression vector can be transferred into the host T cell by physical, chemical, or biological means.

[0115] Physical methods for introducing a polynucleotide into a host T cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art (see, e.g., Sambrook Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press). A preferred method for introducing a polynucleotide into a host T cell is calcium phosphate transfection.

[0116] Biological methods for introducing a polynucleotide of interest into host T cells include the use of DNA and RNA vectors.Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, and adeno-associated virus, etc. (see, e.g., US5,350,674 and US5,585,362).

[0117] Chemical means for introducing polynucleotides into host T cells include macromolecular complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems, such as lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides by targeted nanoparticles or other suitable submicron-sized delivery systems.

[0118] An exemplary non-viral delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host T cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained as a suspension in lipid, contained in or complexed with a micelle, or otherwise associated with lipid. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be in a bilayer structure, as a micelle, or in a "collapsed" structure. They may also simply be interspersed in the solution, and in some cases may form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring lipids or synthetic lipids. For example, lipids include the naturally occurring lipid droplets in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma Aldrich, dicetyl phosphate ("DCP") can be obtained from K&K Laboratories, cholesterol ("Choi") can be obtained from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained, for example, from Avanti Polar Lipids, Inc. Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol."Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium.

[0119] Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components self-rearrange before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991). However, compositions that have structures in solution that differ from normal vesicular structures are also encompassed. For example, lipids may assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine nucleic acid complexes are also contemplated.

[0120] Regardless of the method used to introduce exogenous nucleic acid into host T cells or otherwise expose the cells to the inhibitors of the invention, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host T cells. Such assays include "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of the invention.

[0121] Methods for culturing and expanding immune cells Immune cells, such as T cells, may generally be activated and expanded using methods described, for example, in US6,352,694, US6,534,055, US6,905,680, US6,692,964, US5,858,358, US6,887,466, US6,905,681, US7,144,575, US7,067,318, US7,172,869, US7,232,566, US7,175,843, US5,883,223, US6,905,874, US6,797,514, US6,867,041, and US2006 / 0121005.

[0122] By expanding T cells by the methods disclosed herein, the cells can be expanded by about 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, 10,000x, 100,000x, 1,000,000x, 10,000,000x or more, and all integers or sub-integers therebetween. In one embodiment, the T cells are expanded in the range of about 20x to about 50x.

[0123] In one embodiment, the cells are cultured for about 7 to about 14 days, or about 7 to about 10 days.

[0124] Generally, a population of immune cells, e.g., T regulatory cell depleted cells, can be expanded by contacting the surface bound thereto with an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. Specifically, the T cell population can be stimulated as described herein, such as by contacting with an anti-CD3 antibody or an antigen-binding fragment thereof or an anti-CD2 antibody immobilized on the surface, or by contacting with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. For costimulation of accessory molecules on the surface of the T cells, a ligand that binds to the accessory molecule is used. For example, the population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate proliferation of the T cells. Anti-CD3 antibody and anti-CD28 antibody can be used to stimulate proliferation of either CD4+ T cells or CD8+ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), which may be used as well as others commonly known in the art (Berg et al., 1998; Haanen et al., 1999; Garland et al., 1999).

[0125] Suitable conditions for immune cell culture include an appropriate medium (e.g., Minimal Essential Medium or RPMI Media 1640, or X-vivo 15 (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF, and TNF-a, or any other additives for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Media may include RPMI1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in sufficient amounts for the growth and expansion of T cells. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells injected into subjects. Target T cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

[0126] Procedures for ex vivo expansion of hematopoietic stem and progenitor cells are described in US 5,199,942 and can be applied to the cells of the present invention. Other suitable methods are known in the art, and therefore the present invention is not limited to any particular method of ex vivo expansion of cells. Briefly, ex vivo culture and expansion of immune cells (e.g., T cells) includes (1) collecting mammalian-derived CD34+ hematopoietic stem and progenitor cells from peripheral blood collection or bone marrow explants, and (2) expanding such cells ex vivo. In addition to the cell growth factors described in US 5,199,942, other factors such as flt3-L, IL-1, IL-3, and c-kit ligand can be used for cell culture and expansion.

[0127] immune cells As used herein, the phrase "immune cell" refers to a cell that can affect or induce an immune response upon recognition of an antigen. In some embodiments, the immune cell is a T cell, a natural killer (NK) cell, a macrophage, a dendritic cell, or a stem cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. The cells may be autologous or allogeneic to the subject to which they are administered.

[0128] Examples of stem cells useful in the present invention include, but are not limited to, hematopoietic stem / progenitor cells and induced pluripotent stem cells.

[0129] As used herein, the phrase "cytotoxic activity" refers to the ability of immune cells, such as NK cells, to destroy living cells.

[0130] As used herein, the term "immune response" has its usual meaning in the art and includes both humoral and cellular immunity. An immune response may manifest as one or more of the following: development of anti-antigen antibodies, expansion of antigen-specific T cells, increase in tumor infiltrating lymphocytes (TILs), development of anti-tumor or anti-tumor antigen delayed-type hypersensitivity (DTH) responses, clearance of pathogens, inhibition of pathogen and / or tumor growth and / or spread, tumor reduction, reduction or elimination of metastasis, increase in time to recurrence, increase in pathogen or tumor-free survival, and increase in survival time. An immune response may be mediated by one or more of B cell activation, T cell activation, natural killer cell activation, activation of antigen-presenting cells (e.g., B cells, DCs, monocytes, and / or macrophages), cytokine production, chemokine production, specific cell surface marker expression, especially expression of costimulatory molecules. An immune response may be characterized as a humoral, cellular, Th1 or Th2 response, or a combination thereof. In certain embodiments, the immune response is an innate immune response.

[0131] T cells In some embodiments, the immune cell is a T cell, e.g., a UIR-T cell. T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on the cell surface. There are several subsets of T cells, each with different functions.

[0132] In one embodiment, the T cells are central memory (T CM ) T cells or comprising them. CM The cells patrol lymph nodes and provide central immune surveillance against known pathogens, but have not been described as performing primary tissue immune surveillance. In one embodiment, the T cells produced using the methods of the invention CM The cells are CD45RO+ CD62L+ T cells, preferably CD45RO+ CD62L hi Such cells may also be CCR7+.

[0133] In one embodiment, the T cells are central memory stem cells (T CM ) T cells or comprising them. SCM The cells are a rare subset of memory lymphocytes with stem cell-like self-renewal capacity and the pluripotent ability to reconstitute the full spectrum of memory and effector subsets. In one embodiment, T SCM The cells were CD27 + CD95 + Includes T cells.

[0134] As used herein, regulatory T cells (T REG ) or variants thereof refer to a population of T cells that are important in the maintenance of immune tolerance. Their main role is to shut down T cell-mediated immunity towards the end of an immune response and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus. REG Two major classes of cells have been described: Foxp3+ and Foxp3-.

[0135] Gamma delta (γδ) T cells are the prototype of "non-conventional" T cells and represent a relatively small subset of T cells in peripheral blood. They are defined by the expression of a heterodimeric T cell receptor (TCR) composed of a γ chain and a δ chain. This distinguishes them from CD4+ helper T cells and CD8+ cytotoxic T cells, which express the αβ TCR.

[0136] iNKT (invariant NKT) cells are classified as innate lymphocytes that rapidly secrete Th1 or Th2 cytokines when antigen binds to the TCR. Activated iNKT cells can regulate adaptive immune responses through recruitment, activation, or regulation of NK cell, DC, B cell, and T cell responses.

[0137] In one embodiment, the T cells are naive T cells. As used herein, the term "naive T cells" refers to a population of T cells that have matured and been released by the thymus, but have not yet encountered the corresponding antigen. In other words, naive T cells are in a stage between maturation and activation. Naive T cells are generally characterized by surface expression of L-selectin (CD62L) and CC chemokine receptor type 7 (CCR7), the absence of activation markers CD25, CD44, or CD69, and the absence of memory CD45RO isoforms. They also express a functional IL-7 receptor, consisting of the subunits IL-7 receptor-alpha, CD127, and the common-gamma chain, CD132.

[0138] A T cell that lacks a functional endogenous T cell receptor (TCR) may, for example, be engineered to not express any functional TCR on its surface, may be engineered to not express one or more subunits that comprise a functional TCR, or may be engineered to produce little to no functional TCR on its surface. Alternatively, the T cell may express a substantially impaired TCR, for example, by expression of a mutated or truncated form of one or more of the subunits of the TCR. The term "substantially impaired TCR" means that the TCR does not elicit a deleterious immune response in the host.

[0139] The T cells described herein can be engineered, for example, to not express a functional HLA on their surface. For example, the T cells described herein can be engineered to downregulate T cell surface expression HLA, e.g., HLA class I and / or HLA class II. In some embodiments, the T cells can lack a functional TCR and a functional HLA, e.g., HLA class I and / or HLA class II.

[0140] Modified T cells lacking expression of a functional TCR and / or HLA can be obtained by any suitable means, including knocking out or knocking down one or more subunits of the TCR or HLA. For example, the T cells can include knockdown of the TCR and / or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR) transcription activator-like effector nucleases (TALENs), or zinc finger endonucleases (ZFNs).

[0141] Natural killer cells In some embodiments, the immune cells are natural killer cells. Natural killer (NK) cells are CD56 CD3 large granular lymphocytes capable of killing infected and transformed cells and constitute an important cell subset of the innate immune system. Unlike cytotoxic CD8+ T lymphocytes, NK cells exert cytotoxicity against tumor cells without the need for prior sensitization and can also eradicate MHC-I-negative cells. In one embodiment, the NK cells are CD3-CD56+CD7+CD127-NKp46+T-bet+Eomes+. In one embodiment, the cytotoxic NK cells CD56 dim CD16+.

[0142] Dendritic cells In some embodiments, the immune cells are dendritic cells. Dendritic cells are a heterogeneous group of specialized antigen-presenting cells that originate in bone marrow from CD34+ stem cells and express major histocompatibility complex (MHC) class II molecules. Mature dendritic cells can prime, activate, and expand effector immune cells, such as T cells and NK cells. Dendritic cell therapy is known in the art (see, for example, Sabado et al., 2017). Briefly, dendritic cells can be isolated from a patient, exposed to disease-specific antigens, e.g., cancer-specific antigens, or genetically modified to express UIR or disease-specific antigens, and then infused back into the patient to prime, activate, and expand effector immune cells, e.g., T cells.

[0143] Bone marrow cells In some embodiments, the immune cells are myeloid cells. Granulocytes, monocytes, macrophages, and dendritic cells represent a subgroup of white blood cells collectively referred to as myeloid cells. They circulate through the blood and lymphatic system and are rapidly recruited to sites of tissue damage and infection via various chemokine receptors. Within tissues, they are activated for phagocytosis and secretion of inflammatory cytokines, thereby playing a major role in protective immunity. Bone marrow cell therapy is known in the art and may be useful in the treatment of cancer, infection, or disease. For example, myeloid cells are known to be abundant in the tumor stroma, and the presence of these cells may affect patient outcomes in many cancer types. Briefly, bone marrow cells may be isolated from a patient, exposed to disease-specific antigens, e.g., cancer-specific antigens, or genetically modified to express UIR or disease-specific antigens, and then infused back into the patient to prime, activate, and expand effector immune cells, e.g., T cells.

[0144] Macrophages Macrophages are specialized cells involved in the detection, phagocytosis, and destruction of bacteria and other harmful organisms. In addition, they can also initiate inflammation by presenting antigens to T cells and releasing molecules (known as cytokines) that activate other cells.

[0145] UIR-expressing cell therapy UIR cell therapy is a type of cell therapy in which immune cells (e.g., T cells) are genetically modified to express UIR, and the UIR-expressing cells (e.g., UIR-T cells) are infused into a recipient in need thereof. The infused cells can kill diseased cells expressing the target of UIR in the recipient. Unlike antibody therapy, UIR-modified immune cells (e.g., UIR-T cells) can replicate in vivo, resulting in long-term persistence that can result in sustained tumor control. In various embodiments, UIR cells are administered to a patient, and the UIR cells or their progeny persist in the patient for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, or 5 years after administration of the UIR cells to the patient.

[0146] In some embodiments, the UIR cells are pre-armed when administered to a subject. More specifically, the cells are exposed to molecules under conditions that covalently bind the UIR before administration to a subject, so that they can bind target cells (such as cancer cells) when administered.

[0147] In some embodiments, when administered to a subject, UIR cells are not pre-armed. More specifically, the cells are not exposed to molecules under conditions that covalently bind UIR before being administered to a subject. In this embodiment, the molecules are also administered to a subject so that the cells are armed in vivo for the cells to function.

[0148] In one embodiment, pre-armed UIR cells are administered followed by administration of the molecule every 3 days, for example, on days 1, 4, and 6, for about 21 days.

[0149] In one embodiment, pre-armed UIR cells are administered followed by administration of the molecule twice weekly, for example on days 3 and 6, followed by about 21 days of rest.

[0150] In one embodiment, disarmed UIR cells are administered followed by administration of the molecule twice weekly, for example on days 3 and 6, followed by about 21 days of rest.

[0151] In one embodiment, pre-armed UIR cells are administered followed by administration of the molecule three times a week, for example on days 1, 4, and 6, followed by about 21 days of rest.

[0152] In one embodiment, disarmed UIR cells are administered followed by administration of the molecule three times a week, for example on days 1, 4, and 6, followed by about 49 days of rest.

[0153] The present invention may be carried out in a number of cycles.For example, after the first cycle defined herein, the subject is analyzed to determine whether the subject has responded to treatment.If the subject responds to treatment but disease is still detectable, the subject may be subjected to a second cycle of treatment.Furthermore, after the second cycle, if the subject responds to treatment but disease is still detectable, the subject may be subjected to a third cycle of treatment.

[0154] In some embodiments, the subject is off-drug before determining whether they have responded to the treatment. In some embodiments, the subject is off-drug for about 21 days. In some embodiments, the subject is off-drug for about 28 days. In some embodiments, the subject is off-drug for about 35 days. In some embodiments, the subject is off-drug for about 42 days. In some embodiments, the subject is off-drug for about 49 days.

[0155] The present invention also includes a type of cell therapy in which immune cells (e.g., T cells) are modified to transiently express UIR, for example, by in vivo transcribed RNA, and the UIR-T cells are infused into a recipient in need thereof. The infused cells can kill tumor cells in the recipient. Thus, in various embodiments, the immune cells (e.g., UIR-T cells) administered to the patient are present for less than one month, for example, 3 weeks, 2 weeks, 1 week, after the UIR-T cells are administered to the patient. Without wishing to be bound by any particular theory, the anti-tumor immune response stimulated by the UIR-T cells may be an active or passive immune response, or alternatively, may result from a direct versus indirect immune response.

[0156] Where the present invention contemplates a method for stimulating a universal immune receptor-mediated immune response against a tumor in a subject, it is envisioned that the immune response stimulated by UIR-T cells, whether an active or passive immune response, or a direct versus indirect immune response, is sufficient to treat cancer in the subject.

[0157] As mentioned above, ex vivo procedures are well known in the art and are described above. Briefly, cells are isolated from a mammal (e.g., human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing UIR. The UIR-expressing cells (e.g., UIR-T cells) can be administered to a mammalian recipient to provide a therapeutic effect. The mammalian recipient can be a human, and the UIR-expressing cells can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic with respect to the recipient.

[0158] The UIR cells of the present invention may be administered as described herein either alone or as a pharmaceutical composition in combination with a diluent and / or in combination with other components such as IL-2 or other cytokines or cell populations. The immune cells may be administered either alone or as a pharmaceutical composition in combination with a diluent and / or in combination with other components such as IL-2, IL-15 or other cytokines or cell populations. Briefly, a pharmaceutical composition may include the immune cells described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions for use in the disclosed methods are, in some embodiments, formulated for intravenous administration.

[0159] The pharmaceutical composition comprising the cells described herein may be administered in a dose of 10 4 ~10 9 cells / kg body weight, e.g., 10 5 ~10 6 The cell composition may be administered at a dosage of cells / kg body weight (including all integer values ​​within these ranges). The cell composition may be administered multiple times at these dosages. The cells may be administered by using injection techniques commonly known in immunotherapy (see, for example, Rosenberg et al., 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by one of ordinary skill in the medical arts by monitoring the patient for symptoms of disease and adjusting treatment accordingly.

[0160] Pharmaceutical compositions containing the molecules (binding agents) described herein may be administered, for example, at a dosage of 0.5 mg to 5 mg per kg.

[0161] In certain embodiments, it may be desirable to administer activated immune cells to a subject, then subsequently redraw blood (or perform apheresis), activate and expand immune cells from the blood, and reinfuse these activated and expanded cells into the patient. This process can be performed multiple times every few weeks. In certain embodiments, immune cells can be activated from a blood draw of 10cc to 400cc. In certain embodiments, immune cells are activated from a blood draw of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc. Using this multiple blood draw / multiple reinfusion protocol can function to select for certain populations of immune cells.

[0162] Combination therapy Immune cells, such as UIR-T cells of the invention or immune cells produced by the methods of the invention, can be treated with a PRLR antagonist (e.g., an anti-PRLR antibody or a small molecule inhibitor of PRLR), an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or panitumumab] or a small molecule inhibitor of EGFR [e.g., gefitinib or erlotinib]), an antagonist of another EGFR family member, such as Her2 / ErbB2, ErbB3, or ErbB4 (e.g., an anti-ErbB2 [e.g., trastuzumab or T-DM1], anti-ErbB3, or anti-ErbB4 antibody, or a small molecule inhibitor of ErbB2, ErbB3, or ErbB4 activity), a cMET antagonist, or an anti-EGFR antagonist ... antagonists (e.g., anti-cMET antibodies), IGF1R antagonists (e.g., anti-IGF1R antibodies), B-raf inhibitors (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), PDGFR-alpha inhibitors (e.g., anti-PDGFR-alpha antibodies), PDGFR-beta inhibitors (e.g., anti-PDGFR-beta antibodies, or small molecule kinase inhibitors such as imatinib mesylate or sunitinib malate), PDGF ligand inhibitors (e.g., anti-PDGF-A, -B, -C, or -D antibodies, aptamers, siRNA, etc.), VEGF antagonists (e.g., VEGF-Trap such as aflibercept, e.g., US 7,087,See, e.g., 411 (also referred to herein as "VEGF inhibitor fusion proteins"), anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib)), DLL4 antagonists (e.g., anti-DLL4 antibodies disclosed in US 2009 / 0142354, such as REGN421), Ang2 antagonists (e.g., anti-Ang2 antibodies disclosed in US 2011 / 0027286, such as H1H685P), FOLH1 antagonists (e.g., anti-FOLH1 antibodies), STEAP1 or STEAP2 antagonists (e.g., anti-STEAP1 antibodies or anti-STEAP2 antibodies), TMPRSS2 antagonists (e.g., anti-TMPRSS2 antibodies), MSLN antagonists (e.g., For example, the therapeutically active agent may be co-formulated with and / or administered in combination with one or more additional therapeutically active ingredients selected from the group consisting of an anti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), a uroplakin antagonist (e.g., an anti-uroplakin [e.g., anti-UPK3A] antibody), a MUC16 antagonist (e.g., an anti-MUC16 antibody), a Tn antigen antagonist (e.g., an anti-Tn antibody), a CLEC12A antagonist (e.g., an anti-CLEC12A antibody), a TNFRSF17 antagonist (e.g., an anti-TNFRSF17 antibody), an LGR5 antagonist (e.g., an anti-LGR5 antibody), a monovalent CD20 antagonist (e.g., a monovalent anti-CD20 antibody such as rituximab), a PD-1 antibody, a PD-L1 antibody, a CD3 antibody, a CTLA-4 antibody, and the like. Other agents that may be beneficially administered in combination with the CAR-T cells of the invention include, for example, tamoxifen, aromatase inhibitors, and cytokine inhibitors (including small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or their respective receptors).

[0163] The present invention includes compositions and therapeutic formulations comprising any of the immune cells, such as UIR-T cells, described herein in combination with one or more chemotherapeutic agents. Exemplary chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide (Cytoxan™); alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, metholedopa, and uredopa; ethyleneimines and methylamelanamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenephosphoramide, and trimethylmelamine; chlorambucil, ... Nitrogen mustards such as nafazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide, melphalan, nobemitin, phenesterine, prednimustine, trophosphamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, and calicheamaycin. isin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubeni Antibiotics such as mexamex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluoracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;Androgens such as calcineurin, drostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamon; ol; nitracrine; pentostatin; phenameth; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g., paclitaxel (Taxol™, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (Taxotere™, Aventis Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamycin; capecitabine;and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included in this definition are antiestrogens, including, for example, tamoxifen, raloxifene, 4(5)-imidazole, 4-hydroxytamoxifen, trioxyphene, keoxyphene, LY 117018, onapristone, and toremifene (Fareston), and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, and pharmaceutically acceptable salts, acids, or derivatives of any of the above, that act to regulate or inhibit hormone action on tumors;

[0164] Administration of any of the disclosed therapeutic agents may be performed in any convenient manner, including injection, transfusion, or implantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intravenously (iv) injection, or intraperitoneally. In some embodiments, the disclosed compositions are administered by iv injection. The compositions may also be injected directly into a tumor, lymph node, or site of infection.

[0165] As will be appreciated by those skilled in the art, the above cells and / or molecules will be administered to a subject in a therapeutically effective amount. As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a therapeutic agent administered to alleviate to some extent or prevent the worsening of one or more symptoms of the disease or condition being treated. The result may be a reduction in signs, symptoms, or causes of a disease or prevention of progression, or any other desired change in a biological system. For example, an "effective amount" for therapeutic use is the amount of a therapeutic agent necessary to produce a clinically significant reduction in disease symptoms without undue adverse side effects.

[0166] The term "therapeutically effective amount" includes, for example, a prophylactically effective amount. An "effective amount" of a therapeutic agent is an amount effective to achieve a desired pharmacological effect or therapeutic improvement without undue adverse side effects. It is understood that an "effective amount" or "therapeutically effective amount" may vary from subject to subject due to variations in the subject's age, weight, general condition, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician, in the metabolism of any compound.

[0167] It is considered within the skill of the art to determine such therapeutically effective amounts by routine experimentation (including, but not limited to, dose escalation clinical trials). An appropriate "effective amount" in any individual case may be determined using techniques such as a dose escalation study.

[0168] When one or more therapeutic agents are used in combination, a "therapeutically effective amount" of each therapeutic agent can refer to the amount of that therapeutic agent that would be therapeutically effective when used by itself, or it may refer to a reduced amount that is therapeutically effective in combination with one or more additional therapeutic agents.

[0169] Treatment methods Immune cells, such as UIR-T cells, of the present invention or produced using the present invention are useful, inter alia, for the treatment, prevention, and / or amelioration of diseases or disorders. For example, the UIR-T cells of the present invention are useful for the treatment of cancer, infectious diseases, or inflammatory diseases. As another example, dendritic cells produced by the methods of the present invention can be used as dendritic cell vaccines (see, e.g., Datta et al., 2014) for treating, e.g., cancer, infectious diseases (such as bacterial or viral infections), or autoimmune diseases (such as diabetes). As a further example, NK cells, such as NK-UIR cells, can be used to treat cancer (see, e.g., Liu et al., 2021).

[0170] UIR cells may be used to treat primary and / or metastatic tumors occurring in the brain and meninges, oropharynx, lungs and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and adnexa, connective tissue, spleen, immune system, blood forming cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye. In certain embodiments, the UIR cells of the invention are used to treat one or more of the following cancers: renal cell carcinoma, pancreatic cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, melanoma, leukemia, or lymphoma.

[0171] In one embodiment, the UIR cells of the present invention are used to treat leukemia, such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, or chronic lymphocytic leukemia. In one embodiment, the leukemia is acute myeloid leukemia with a predominance of low CD33+ blasts.

[0172] In another embodiment, the UIR cell of the present invention is used to treat lymphoma, such as Hodgkin's lymphoma or non-Hodgkin's lymphoma. Types of non-Hodgkin's lymphoma include diffuse large B-cell lymphoma, anaplastic large cell lymphoma, Burkitt's lymphoma, lymphoblastic lymphoma, mantle cell lymphoma, or peripheral T-cell lymphoma. In one embodiment, the lymphoma is diffuse large B-cell lymphoma or non-Hodgkin's lymphoma with low levels of CD19 and / or CD20.

[0173] Examples of antigens that the armed universal immune receptor may bind include, but are not limited to, CD19, CD20, ROR1, CD22 carcinoembryonic antigen, alpha fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate specific antigen, melanoma associated antigen, mutated p53, mutated ras, HER2 / Neu, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, meothelin, GD3, HERV-K, IL-1 IRa, k chain, I chain, CSPG4, ERBB2, EGFRvIII, or VEGFR2.

[0174] In the context of the methods of treatment described herein, immune cells such as UIR cells may be administered as a monotherapy (i.e., as the only therapeutic agent) or in combination (combination therapy) with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0175] In one embodiment, a subject is at risk for developing cancer (e.g., cancer). A subject is at risk if the subject has a higher risk of developing cancer than a control population. The control population may include one or more subjects randomly selected (e.g., matched by age, sex, race, and / or ethnicity) from the general population who are cancer-free or have a family history of cancer. A subject may be considered at risk for cancer if a "risk factor" associated with cancer is found to be associated with the subject. Risk factors may include any activity, trait, event, or characteristic that is associated with a given disorder, for example, through statistical or epidemiological studies of a population of subjects. Thus, a subject may be classified as at risk for cancer even if the subject has not been specifically included in a study that identifies the underlying risk factor.

[0176] In one embodiment, the subject is at risk of developing cancer and the cells or compositions are administered prior to or after the onset of symptoms of the cancer. In one embodiment, the cells or compositions are administered prior to the onset of symptoms of the cancer. In one embodiment, the cells or compositions are administered after the onset of symptoms of the cancer. In one embodiment, the cells or compositions of the invention are administered in a dose that alleviates or reduces one or more of the symptoms of the cancer in the at-risk subject.

[0177] In some embodiments, the subject has been diagnosed with or is suspected of having a disease or disorder, such as cancer, an infectious disease, or an inflammatory disease. In some embodiments, the methods described herein include diagnosing the subject as having or being suspected of having a disease or disorder, such as cancer, an infectious disease, or an inflammatory disease.

[0178] As used herein, diagnosis refers to the determination that a subject or patient is in need of treatment with the immune cells of the present invention. The type of disease or disorder diagnosed according to the methods described herein can be any type known in the art or described herein.

[0179] In one embodiment, the step of identifying or diagnosing a subject in need of treatment with immune cells of the invention comprises determining that the subject has cancer; -Blood profiling, - cytological aspirate or tissue biopsy, - Imaging such as computed tomography (CT) scans, bone scans, magnetic resonance imaging (MRI), positron emission tomography (PET) scans, ultrasound and x-rays; -physical examination.

[0180] Examples of diseases that can be treated with NK cells include, but are not limited to, cancer (e.g., melanoma, prostate cancer, breast cancer, and liver cancer), as well as viral infections (e.g., infections with HSV, hepatitis viruses, human cytomegalovirus, influenza viruses, flaviviruses, and HIV-1), bacterial infections (e.g., infections with mycobacteria, listeria, and staphylococci), and protozoal infections (e.g., infections with malaria parasites), and fungal infections (e.g., infections with Aspergillus).

[0181] Examples of inflammatory diseases include, but are not limited to, antibiotic-resistant microbial infections, idiopathic pulmonary fibrosis, and Alzheimer's disease.

[0182] As will be clear to those skilled in the art, the "reduction" of cancer symptoms in a subject is compared to another subject that also suffers from cancer but is not treated by the methods described herein.This does not necessarily require a control comparison between two subjects.Rather, population data can be relied upon.For example, a population of subjects with cancer that are not treated by the methods described herein (optionally a population of subjects similar to the treated subjects, e.g., age, weight, race) is evaluated, and the average value is compared to the results of a subject or population of subjects that are treated by the methods described herein.

[0183] In one embodiment, the immune cells and methods of the invention are used to improve survival of subjects suffering from a disease or disorder, such as cancer, an infectious disease, or an inflammatory disease. When survival is contemplated, survival analysis can be performed using the Kaplan-Meier method. The Kaplan-Meier method can be used to estimate a survival function from survival data and measure the proportion of patients who survive a certain period of time after treatment. A Kaplan-Meier plot of a survival function is a series of horizontal steps of decreasing magnitude that, when a large enough sample is taken, approaches the true survival function for that population. The value of the survival function between successive distinct sampled observations ("clicks") is assumed to be constant. An important advantage of the Kaplan-Meier curve is that the method can take into account "censored" data loss from samples before the final outcome is observed (e.g., when a patient drops out of the study). In the plot, a small vertical check mark indicates loss where patient data was censored. If no truncation or censoring occurs, the Kaplan-Meier curve is equivalent to an empirical distribution.

[0184] In statistics, the log-rank test (also known as the Mantel-Cox test) is a hypothesis test that compares the survival distributions of two groups of patients. It is a non-parametric test and is suitable for use when the data are correctly censored. It is widely used in clinical trials to establish the efficacy of new drugs compared to a control group when the measurement is the time to event. The log-rank test statistic compares estimates of the hazard function of two groups at each observed event time. It is constructed by calculating the number of observed and expected events in one of the groups at each observed event time, and then adding these together to obtain an overall overview across all time points where there are events. The log-rank statistic can be derived as a score test of the Cox proportional hazards model that compares the two groups. It is therefore asymptotically equivalent to the likelihood ratio test statistic based on that model. EXAMPLES

[0185] Example 1 - Universal Immune Receptor A SpyCatcher universal immune receptor was produced, which has, in N-terminal to C-terminal order, a CD8a leader, SpyCatcher v003, a CD8a hinge, a CD8a transmembrane domain, a CD28z costimulatory domain, and a CD3z intracellular signaling domain (SEQ ID NO: 1), encoded by the polynucleotide sequence provided as SEQ ID NO:2.

[0186] SEQ ID NO:1: SpyCatcher universal immune receptor. The first underlined section is the CD8 leader, the second underlined section is SpyCatcher, the third underlined section is the CD8a hinge followed by the CD8a transmembrane domain, the fourth underlined section is the CD8a transmembrane domain followed by the CD28z co-stimulatory domain (excluding the C-terminal ID of this section), and the fifth underlined section is the CD3z intracellular signaling domain.

[0187] TIFF2024527963000001.tif39138

[0188] SEQ ID NO:2: Polynucleotide encoding the SpyCatcher universal immune receptor of SEQ ID NO:1.

[0189] In addition, two SpyTag binding molecules that bind Her2 were produced. One is referred to as a standard Her2 binder (SEQ ID NO:3), which contains an N-terminal signal peptide, an antibody variable domain that binds Her2, an antibody constant domain, a linker, and a SpyTag. The other is referred to as a short half-life binder, which has the antibody constant domain that confers a shorter half-life (SEQ ID NO:4). After expression and processing, the N-terminal signal peptide is removed, such that the administered molecule lacks this peptide (SEQ ID NOs:5 and 6, respectively).

[0190] SEQ ID NO:3: Canonical Her2 binding agent (heavy chain with SpyTag) with N-terminal signal sequence. The first underlined section is the N-terminal signal sequence followed by the antibody variable domain that binds Her2, the second underlined section is the antibody constant domain followed by a linker, and the third underlined section is the Spytag.

[0191] TIFF2024527963000002.tif58139

[0192] SEQ ID NO: 4: Short half-life Her2 variant with N-terminal signal sequence (heavy chain with SpyTag). The first underlined section is the N-terminal signal sequence followed by the antibody variable domain that binds Her2, the second underlined section is the short half-life antibody constant domain followed by a linker, and the third underlined section is the Spytag.

[0193] TIFF2024527963000003.tif58139

[0194] SEQ ID NO: 5: Standard Her2 binding agent (heavy chain with SpyTag) without the N-terminal signal sequence (anti-Her2-ST). EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSRGVPHIVMVDAYKRYK

[0195] SEQ ID NO: 6: Short half-life Her2 variant without N-terminal signal sequence (heavy chain with SpyTag).

[0196] TIFF2024527963000004.tif52138

[0197] The DNA sequence related to each binder was first de novo synthesized as a polynucleotide containing the SpyTag sequence, which was then cloned into a transfection-grade endotoxin-free plasmid (e.g., pAb20-hCL-1). This was then transiently transfected into a permissive cell line, such as TunaCHO, which was then cultured in DMEM / F12 for 14 days, after which the binder was purified using protein A purification, size exclusion chromatography, and ultra-performance liquid chromatography. Identity was confirmed using mass spectrometry. Additional binders have been developed and synthesized, and are disclosed herein by SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18.

[0198] Example 2 - Transfection and expansion of cells Either polymorphonuclear or enriched T cell preparations are activated with CD3 / CD28 beads for 24 hours and then transduced with lentiviruses encoding SpyCatcher constructs in the presence of retronectin or polybrene for an additional 24 hours, whereupon 50 IU / mL of recombinant IL-2 is added. CD3 / CD28 beads are removed after 7 days of continuous culture and T cells are cultured for an additional 7 days. Transduction efficiency is determined based on expression of SpyCatcher on the cell surface, and T cell numbers and phenotypes are determined using a Coulter Coulter and multiparameter flow cytometry.

[0199] Example 3 - Metronomic dosing of UIR-T cells To determine the effect of metronomic dosing on the persistence and activity of UIR-T cells, immunodeficient NOD-SCID-gamma (NSG) mice were administered 2–5 × 10 6 Either MCF7 or SKBR3 breast cancer cells are injected (via the mammary fat pad or subcutaneous injection) into the mouse. Tumor growth is measured every 2 days. 5-10 days after tumor injection or when tumors reach 20-30 mm 3 Once the mice reach 1Gy, precondition these mice with 0.5-1Gy total body irradiation. On the same day, pre-arm UIR T cells (1 x 10 in 200 µL PBS) together with anti-Her2-ST (anti-Her2 binder with SpyTag or short half-life version) at concentrations of 12.5 µg-50 µg per mouse every 3 days for 21 days. 7 Each patient will receive 100,000 IIR T cells (Figure 1) intravenously. Each dosing cycle will last for 21 days.

[0200] The effect of discontinuous dosing of ST binders on persistence and UIR-T activity was also evaluated in a 28-day dosing protocol, in which tumors with sizes between 20 and 30 mm 3When the IL-1 receptor agonist (IL-1 receptor agonist) is reached (day 0), pre-arm UIR T is administered and anti-Her2-ST (or a short half-life version) is administered twice during the first week (e.g., on days 3 and 6) at concentrations ranging from 12.5 µg to 50 µg per mouse, followed by a 21-day washout period (Figure 2).

[0201] The effect of in vivo arming and discontinuous dosing of ST binders on UIR-T persistence and cytotoxicity was also evaluated in an additional 28-day dosing protocol, in which tumors with sizes between 20 and 30 mm 3 When the IL-1 receptor agonist (IL-1 receptor agonist) is reached (day 0), unarmed UIR T is administered and anti-Her2-ST (or a short half-life version) is administered three times during the first week (e.g., on days 3 and 6) at concentrations ranging from 12.5 µg to 50 µg per mouse, followed by a 21-day washout period (Figure 3).

[0202] The effect of in vivo arming and discontinuous dosing of ST binders on UIR-T persistence and cytotoxicity was also evaluated in an additional 28-day dosing protocol, in which tumors with sizes between 20 and 30 mm 3 When the IL-1 receptor agonist (IL-1 receptor agonist) is reached (day 0), unarmed UIR T is administered and anti-Her2-ST (or a short half-life version) is administered three times during the first week (e.g., on days 1, 4, and 7) at concentrations ranging from 12.5 µg to 50 µg per mouse, followed by a 21-day washout period (Figure 4).

[0203] The effect of in vivo arming and discontinuous dosing of ST binders on UIR-T persistence and cytotoxicity was also evaluated in a longer 56-day dosing protocol, where tumors of size 20-30 mm 3 When the IL-1 receptor agonist (IL-1 receptor agonist) is reached (day 0), unarmed UIR T is administered and anti-Her2-ST (or a short half-life version) is administered three times during the first week (e.g., on days 1, 4, and 7) at concentrations ranging from 12.5 µg to 50 µg per mouse, followed by a 49-day washout period (Figure 5).

[0204] Example 4 - Antitumor efficacy and dosing of UIR-T cells Materials and Methods Cell lines and mouse models The human breast cancer cell line MDA-MB231 was procured from ATCC and used to inoculate NOD-SCID gamma (NSG) mice. PCR analysis on each cell line was performed periodically to ensure that the cell lines were mycoplasma negative. All tumor cell lines were cultured in DMEM (Gibco, Life Technologies, Grand Island, New York, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM glutamine, 0.1 mM non-essential amino acids, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were incubated at 37 °C, 5% CO 2 The cells were grown in a humidified incubator at 10%. Other cell lines included retroviral and lentiviral packaging lines, HEK293gp, PA317, and GP+E86, obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). GP+E86, PA317, HEK293T, and HEK293gp were maintained in the same medium as the tumor cell medium described above.

[0205] Third generation lentivirus production Third generation lentiviral envelope, packaging, and transfer plasmids were used, which were procured from Addgene. The transfer plasmid was a modified variant of the pULTRA plasmid. T175 flasks were pre-coated with poly-D-lysine (50 μg / mL) and 23 million HEK293T packaging cells were seeded after washing. After 24 hours, cells were transfected with a 1:1:1:1 molar ratio of plasmids using Lipofectamine 3000 reagent according to the manufacturer's instructions. Viral supernatants were collected on days 1, 2, and 3 post-transfection, filtered (0.45 μM), and concentrated with Lenti-X-Concentrater reagent from Takara Bio before freezing aliquots at -80°C.

[0206] Lentiviral transduction of human PBMCs Fresh donor human PBMCs were processed by diluting with PBS at a 1:1 ratio and then separated by Ficoll separation. White blood cells were separated from serum and red blood cells and collected, followed by further red blood cell lysis. After lysis, cells were activated with OKT3 (30 ng / mL), 600 IU of human IL-2, and complete RPMI (10% FCS, sodium pyruvate, glutamax, NEAA, HEPES, and penicillin / streptomycin) medium at a cell concentration of 1 million cells / mL for a minimum of 48 hours. After activation, activated T cells were re-seeded at 1 million T cells / mL with 600 IU of IL-2, followed by the addition of lentivirus at a functional titer of 1 MOI with LentiBoost Reagent (1:400). Cells were supplemented with medium and IL-2 and cultured for a minimum of 72 hours.

[0207] Flow cytometry The collected cells were first washed with FACS buffer and then stained with antibodies conjugated to fluorophores for 30 min on ice. The cells were washed at least twice with FACS buffer before being resuspended in FACS buffer and the beads (20 μL, approximately 20,000 beads) were counted. For intracellular or intranuclear staining, the stained cells were further fixed and permeabilized using BD Biosciences or eBioscience kits, respectively, according to the manufacturer's instructions. The fixed and permeabilized cells were then further stained with antibodies conjugated to fluorophores for 30 min at room temperature, followed by washing at least twice with perm / wash buffer and finally resuspending in perm / wash buffer with counting beads. The stained samples were acquired on a BD FACSymphony, FACSFortessa, or LSR (BD Biosciences). The number of targeted T cell populations was calculated using the number of samples / bead events of the targeted population times the number of input beads in the events.

[0208] Co-culture of CAR T cells with tumor cells For direct activation of human CAR T cells with OKT3 (1:1000), flat-bottom 96-well plates were pre-coated for at least 1 h at 37°C. For tumor co-culture, CAR T cells and tumor cells were co-cultured in flat-bottom 96-well plates with 50,000 tumor cells / well at various E:T ratios in 200 μL of supplemented RPMI medium. Cells were co-cultured for 24-72 h at 37°C and 5% CO2 as indicated.

[0209] Incucyte killing assay Tumor cells were first transduced to express target antigens linked to fluorescent markers, GFP for HER2 or mCherry for EGFRvIII. Tumors were seeded overnight at 5000 cells / well in 384-well plates, after which various ratios of CAR T cells were added to each well, using the Caspase 3 / 7 dye at the recommended concentration provided by the manufacturer. Plates were then incubated at 4°C for 24 hours at 37°C for 30 min at 5% CO. 2 The cells were then placed in an Incucyte machine, located inside a 37 °C incubator, and imaged every 1-4 h for a total of 24-72 h. Images were analyzed with the Incucyte analysis software.

[0210] Quantification of cytokine production The collected supernatants were analyzed by cytometric bead array (CBA) assay (BD Biosciences). 12.5 μL of supernatant was transferred to a V-bottom 96-well plate. Semi-serial dilutions of standards containing each cytokine tested were plated at a maximum concentration of 5000 pg / mL. Following the manufacturer's instructions, capture cocktails containing cytokine-specific beads at 0.25 μL beads / cytokine / well were made up to 12.5 μL / well (human) using BD bead buffer solution, respectively, and added to each well. The plates were then incubated at room temperature for up to 1 h. The same volume of beads for the PE detection antibody cocktail of mouse or human cytokines was made and added to each well. The plates were again incubated in the dark at room temperature for up to 1 h. Finally, the cells were washed at least twice with the provided wash buffer before being resuspended in 75–100 μL of wash buffer and analyzed with BD FacsVerse (Becton Dickinson) and data were processed with FCAP software (BD biosciences).

[0211] "Armoring" and "pre-arming" of OmniCAR T cells and dosing scheduling of antibody binding agents Disarmed OmniCAR T cells do not have the targeted antibody binder attached to the spyCatcher receptor CAR and cannot bind to tumor antigens, so they cannot activate or kill tumor cells. Antibody binders were added to the T cell medium (10 e6 OmniCAR T cells are "armed" by adding spyCatcher:spyTAG to OmniCAR T cells in 0.1% T cells / mL of spyCatcher and incubating for approximately 30 minutes at 37° C. in 5% CO2 to form functional spyCatcher:spyTAG receptors. "Pre-armed" OmniCAR T cells are CAR T cells that have been armed ex vivo or outside the body / mouse prior to adoptive transfer. Antibodies are washed out prior to adoptive transfer.

[0212] Adoptive transfer and treatment of tumor-bearing mice NSG mice were injected with 2-5x106 MDA-MB231 breast cancer cells transduced to express human HER2 antigen subcutaneously. After tumor establishment, mice were subsequently subjected to a preconditioning regimen of 0.5gy total body irradiation followed by adoptive transfer of OmniCAR T cells supplemented with 4 doses of 25,000IU IL-2 per mouse every 24 hours. Binding agents were delivered intratumorally, intravenously, or intraperitoneally based on a metronomic dosing schedule (Figure 11A).

[0213] Analysis of tumor-infiltrating immune subsets To investigate tumor-infiltrating CAR T cell phenotype, mice were euthanized and tumors or spleens were removed. Tumors were mechanically digested and enzymatically digested with DMEM supplemented with 1 mg / mL collagenase type IV (Sigma-Aldrich) and 0.02 mg / mL DNAse (Sigma-Aldrich) for 30 minutes at 37°C with constant shaking. The tumor single cell suspension was then filtered through a 70 μm filter before being aliquoted for flow cytometry staining. Spleens were also mechanically digested and filtered through a 70 μm filter before being stained.

[0214] result The dosing regimen of the binding agent regulates functional UIR expression in vivo. Using the protocol described above, we generated OmniCAR T cells using third generation lentiviruses and used anti-FLAG antibodies to detect the transduction efficiency and expression of non-antigen-specific OmniCAR receptors (Figure 6A). "Unarmed" OmniCAR T cells lack the antibody binder component and therefore cannot bind to tumor antigens to stimulate anti-tumor responses. Addition of anti-human IgG antibodies allows detection of full-length antibody binders when they spontaneously form complexes with OmniCAR receptors, resulting in "armed" OmniCAR T cells that are fully functional against their respective target antigens (Figure 6A).

[0215] Incubation with increasing concentrations of antibody binders against the human HER2 antigen increased detection by anti-IgG, corresponding to increased expression of the "armed" OmniCAR receptor in both CD8+ FLAG+ and CD4+ FLAG+ CAR T cells (Figure 6B). Correlating with increased expression of the armed receptor, OmniCAR T cells generated from three separate donors were activated when cocultured with the HER2-transduced breast cancer cell line MDA-MB231 (MDA-MB231-HER2) and secreted increasing concentrations of the functional cytokines IFNγ, TNF, and IL-2 (Figure 6C). OmniCAR T cells can also be armed after adoptive transfer into NSG mice. For this purpose, 10-20 million OmniCAR T cells were transferred into NSG mice and dosed every 3 days with increasing concentrations of antibody binders. Blood was collected on days 1 and 7 post-transfer, and "armed" FLAG+ CAR T cells were detected at both time points, with expression levels corresponding to increasing binder dosing concentrations (Figure 6D). At day 1 post-treatment, higher doses of binder resulted in increased detection of armed receptors (Figure 6E), as well as early expansion and numbers of armed OmniCAR T cells (Figure 6F). Similarly, armed receptor expression (MFI) at day 7 corresponded to increasing binder dosing (Figure 6D).

[0216] The dosing regimen of the binding agent modulates the memory phenotype, expansion, and persistence of T cells in vivo. To further explore the therapeutic efficacy of OmniCAR therapy, NSG mice were injected with human MDA-MB231-HER2 breast cancer cells. Once tumors were established, mice were treated with OmniCAR T cells and different doses and dosing regimens of anti-HER2 antibody binders (Figure 7A). Similar to previous observations, higher doses of antibody binders resulted in early expansion of CD8+ FLAG+ OmniCAR T cells in the mouse periphery (Figure 7B). Dosing of binders at >5ug / mouse resulted in greater expression of the "armed" OmniCAR receptor, consistent with dosing of binders in either tumor-bearing or non-tumor-bearing mice, or with longer intervals between doses of >1 week (Figure 7C).

[0217] By adjusting the dose and dosing strategy, the memory phenotype of OmniCAR T cells can be modulated in vivo, an effect observed in the background of tumor-bearing and non-tumor-bearing mice (Figure 7D). Low concentrations of binders resulted in a greater % of CD45RO+CD45RA-T effector memory subpopulation than pre-armed alone, while high concentrations of binders resulted in a greater % of CD45RA+CD45RO-T central memory subpopulation, which appears to be antigen-independent (Figure 7D). Indeed, much of the expansion observed at day 7 of peripherally transferred OmniCAR T cells in the high-dose treatment group was antigen-independent, indicating that the expansion of OmniCAR T cells is an intrinsic feature linked to modulation of signaling from the OmniCAR receptor (Figure 7E).

[0218] Dosing regimen of binding agents modulates antitumor efficacy in vivo Consistent with the expansion and function observations, mice treated with pre-armed OmniCAR T cells and high doses of antibody binders could mediate anti-tumor effects and reduce overall tumor size compared to untreated mice (Figure 8A). At the end of the study, spleens and tumors were isolated from the mice. As expected, treatment with high binder doses reduced engraftment in the spleen (Figure 8B). Similarly, this pattern translated to the number of tumor-infiltrating lymphocytes (TILs) detected within the tumor (Figure 8C). Higher dosages of binders could also drive increased antigen-specific signaling due to increased functional OmniCAR receptor expression, resulting in an increased % of TIM3+PD1+CD8+FLAG+CAR TILs (Figure 8D).

[0219] The effect of the proposed regimen was also tested in a model of acute myeloid leukemia (AML). Bioluminescence imaging was performed to determine the tumor burden over time in a mouse model of AML. NSG mice were given 5 million KG-1 cells, and the animals were left untreated (control) or given pre-armed OmniCAR-T cells and 25ug of CD33 and CLL-1 binding agent on days 3, 6, and 9 after CAR-T transfer. When compared to the control group, a significant effect on tumor growth was observed in mice treated with pre-armed OmniCAR-T cells and 25μg of CD33 and CLL-1 binding agent on days 3, 6, and 9 after CAR-T transfer (Figure 8E).

[0220] The dosing regimen or specific design of the binding agent modulates OmniCAR antigen-independent or antigen-dependent signaling. The unique ability of OmniCAR to drive superior expansion of CAR T cells after adoptive transfer compared to conventional CAR T or non-transduced T cells was previously shown to be antigen-independent (Figure 7D). To investigate the possible mechanisms involved, OmniCAR T cells were directly compared in vitro to non-transduced or conventional CAR T cells for levels of activation, proliferation, or expression of checkpoint T cell markers. After 72 h of over-coculture without stimulation in the presence of IL-2, both CD8 and CD4, unarmed or armed, OmniCAR T cells, respectively, upregulated expression of the activation marker TIM3 when compared to non-transduced or conventional CAR T cells (Figure 9A).

[0221] Importantly, both CD8 and CD4 armed OmniCAR T cells expressed higher levels of Tim3 compared to their unarmed counterparts (Figure 9A). Upon overstimulation with OKT3 (αCD3) for 72 hours, all CD8+ groups upregulated Tim3 to similar levels, but both unarmed and armed OmniCAR T cells had higher expression overall (Figure 9A). Interestingly, both unarmed and armed CD4+ OmniCAR T cells had significantly higher Tim3 expression compared to conventional CAR T cells, indicating higher levels of activation upon stimulation with OKT3 (Figure 9A). The second late checkpoint receptor, PD-1, was not highly expressed in vitro without stimulation, but was upregulated upon stimulation, but not by arming in the CD8+ fraction (Figure 9B). However, PD-1 expression was prominent in the CD4+ fraction, with unarmed and armed CD4+ OmniCAR T cells expressing higher levels of PD-1, and arming in unstimulated cells resulted in even higher PD-1 expression (Figure 9B). PD-1 remained elevated in stimulated CD4+ OmniCAR T cells, but armed OmniCAR T cells had reduced PD-1 compared to unarmed, potentially indicating reduced signaling from the armed OmniCAR receptor in CD4+ T cells (Figure 9B).

[0222] Taken together, unarmed or armed OmniCAR receptors have unique signaling effects on unstimulated or stimulated CD8 or CD4 fractions, respectively, highlighting their use to differentially regulate different fractions of the OmniCAR T cell product after adoptive transfer. Complex interactions between the CD8 and CD4 fractions can drive superior proliferation of transferred OmniCAR T cells.

[0223] Metronomic dosing for sequential or simultaneous targeting of multiple tumor antigens The advantage of the OmniCAR platform is that multiple tumor antigens can be targeted by sequential or simultaneous dosing of different antibody binders. The human glioblastoma cell line U251MG was modified to express either human HER2 or mutant EGFRvIII receptors separately. In mixed tumor cocultures, both U251MG-HER2 and U251MG-EGFRvIII had stable growth kinetics (Figure 10A). When OmniCAR T cells were armed against human HER2 antigen only, only HER2 expressing U251MG-HER2 cells were eliminated in mixed tumor cocultures (Figure 10B). Similarly, when OmniCAR T cells were armed against mutant EGFRvIII antigen only, only EGFRvII cells expressing U251MG-EGFRvIII were eliminated in mixed tumor cocultures (Figure 10C). Finally, to demonstrate antigen switching to different targets, EGFRvIII targeting OmniCAR T cells were co-cultured in a mixed tumor assay and 100 nM of HER2 binder was added 20 hours later (Figure 10D). Until the addition of the HER2 binder, only EGFRvIII-expressing tumors were eliminated, but CAR T cell killing was switched to HER2-expressing tumors after the addition of the HER2 binder, indicating rapid and efficient targeting of antigens sequentially (Figure 10D). To simultaneously target multiple tumor antigens, OmniCAR T cells can be armed with two or more antibody binders at once, with equivalent levels of expression and co-expression of three or more different binders (Figure 10E). Finally, the presence of HER2 and EGFRvIII antibody binders can be detected in the serum of mice two weeks after their administration (Figure 10F).

[0224] Chimeric antigen receptor (CAR) T-cell therapy has been widely successful in treating hematological malignancies and is in clinical trials to treat multiple solid tumors. However, there are several challenges related to unique toxicities and subsequent relapses that need to be addressed. Universal immune receptors are a rapidly emerging armoured form of adoptive immunotherapy that has the potential to address these challenges by increasing safety and reducing side effects (switching on / off CAR responses after infusion), as well as targeting multiple tumor antigens to overcome tumor escape mechanisms (such as antigen loss or antigen heterogeneity) that lead to tumor recurrence. In addition, universal CAR T cells may have the potential to have much greater versatility, lower cost, and off-the-shelf utility compared to conventional CAR therapies currently in the clinic. Thus, the metronomic dosing strategy highlighted in Figure 11A can endow OmniCAR T cells with the ability to regulate the expression of antigen-specific functional UIR expression in vitro and in vivo, as well as the memory differentiation, expansion, and persistence of T cells in vivo.

[0225] By adjusting the dosing regimen in time, through discontinuous, continuous, or periodic dosing, or by adjusting the dosing concentration (Figure 11A), it is possible to directly or indirectly modulate the strength of antigen-independent tonic signaling and antigen-dependent CAR signaling leading to the maintenance of optimal T cell memory subsets, T cell activation, and antitumor function (Figure 11B). Unique to the OmniCAR platform is the ability to modulate antigen-independent tonic signaling, which drives improved safety, antitumor activity, and T cell expansion at a higher level than traditional CAR T cells with a robust architecture and therefore fixed levels of tonic signaling (Figure 11B). Multiple antigens can also be targeted by the ability to rapidly and efficiently switch between antigen targets sequentially, or by arming with two or more antigen binders to simultaneously target multiple tumor antigens (Figures 10A-E). In conclusion, these findings support metronomic dosing as a strategy to synergistically combine with the flexibility of the OmniCAR UIR platform to fine-tune antitumor therapeutic responses following adoptive transfer.

[0226] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0227] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0228] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention and is not to be construed as an admission that any or all of such matters form part of the prior art or were common general knowledge in the art relevant to the present invention prior to the priority date of each claim of this application.

[0229] References Berg et al.(1998)Transplant Proc.30:3975-3977. Datta et al.(2014)Yale J Biol Med 87:491-518. Garland et al.(1999)J.Immunol Meth.227:53-63. Ghosh et al.(1991)Glycobiology 5:505-510. Haanen et al.(1999)J.Exp.Med.190:1319-1328. Lui et al.(2021)J Hemat Oncol 14:7. Rosenberg et al.(1988)New Eng.J.of Med 319:1676. Sabado et al.(2017)Cell Res 27:74-95. Smith et al.(2015)Clinical & Translational Immunology 4:e31. Veggiani et al.(2016)PNAS 113:1202-1207.

Claims

1. An immune cell comprising a universal immune receptor for use in treating a disease in a subject that may benefit from immune cell therapy or for use in stimulating a universal immune receptor-mediated immune response against a tumor in a subject, comprising: The universal immune receptor may or may not be covalently bound to a molecule containing a domain that binds an antigen associated with the disease. The molecule will be administered to the subject at least twice within 7 days following administration of the immune cells, and the subject will be analyzed for responsiveness to treatment for at least 21 days after the 7-day period, and if the subject responds to treatment but disease is still detectable, the treatment will be repeated. immune cells.

2. The immune cells described in claim 1, wherein the molecule is administered twice within 7 days after administration of the immune cells.

3. The immune cell of claim 2 , wherein the molecule is administered on days 3 and 6 following administration of the immune cell.

4. The immune cells of claim 1, wherein the subject is analyzed for responsiveness to the treatment 21 to 49 days after administration of the immune cells.

5. If the subject responds to treatment but disease is still detectable, the treatment is repeated, (i) administering a universal immune receptor, which may or may not be covalently linked to a molecule that contains a domain that binds the same antigen as the molecule used to administer the immune cells; or (ii) administering a universal immune receptor, which may or may not be covalently linked to a molecule that contains a domain that binds an antigen different from the molecule used to administer the immune cells; The immune cell of claim 1 , comprising:

6. An immune cell comprising a universal immune receptor for use in treating a disease in a subject that may benefit from immune cell therapy or for use in stimulating a universal immune receptor-mediated immune response against a tumor in a subject, comprising: The universal immune receptor may or may not be covalently bound to a molecule containing a domain that binds an antigen associated with the disease. the molecule is administered to the subject every two or three days for 14 to 28 days following administration of the immune cells, and if the subject responds to treatment but disease is still detectable, the treatment is repeated; immune cells.

7. If the subject responds to treatment but disease is still detectable, the treatment is repeated; administering a universal immune receptor, which may or may not be covalently linked to a molecule containing a domain that binds the same or a different antigen as the molecule used to administer the immune cells; or

8. The immune cell of claim 6, wherein the molecule comprises domains that bind two or more antigens associated with the disease, preferably two antigens associated with the disease. (i) the treatment increases the survival rate of the subject when compared to a subject not receiving the treatment; and / or (ii) the treatment optionally further comprises administration of an additional therapeutic agent selected from the group consisting of chemotherapy, radiation therapy, surgery, bone marrow transplantation, drug therapy, cryoablation, or radiofrequency ablation; The immune cell of claim 1.

10. 2. The immune cell of claim 1, wherein the universal immune receptor comprises a SpyCatcher or SpyTag extracellular binding domain linked to an extracellular hinge region, the extracellular binding domain in turn linked to a transmembrane domain, and the transmembrane domain in turn linked to an immune cell receptor intracellular signaling domain.

11. The immune cell of claim 10, wherein the universal immune receptor intracellular signaling domain further comprises a costimulatory molecule.

12. the SpyCatcher extracellular binding domain binds to the extracellular hinge domain; or the SpyTag extracellular binding domain binds to the extracellular hinge domain; The immune cell of claim 10.

13. The immune cell of claim 1 , wherein the molecule comprises SpyCatcher or SpyTag and the domain.

14. The molecule is about 0.25 mg / m ２ ~2.0 mg / m ２ at a dose of about 5 mg / m ２ ~25 mg / m ２ or about 50 mg / m ２ ~100 mg / m ２ The immune cells of claim 1 , administered at a dose of

15. The immune cell of claim 1 , wherein the immune cell is a T cell, a NK cell, a dendritic cell, a myeloid cell, a macrophage, a stem cell, or a combination thereof.

16. the cells are autologous, and / or the subject is a human; The immune cell of claim 1.

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