Methods for enhancing adoptive cell transfer immunotherapy

Abstract

The present invention relates to a method for enhancing adoptive cell transfer immunotherapy by administering a protein with IgG cysteine ​​protease or IgG endoglycosidase activity.

Description

[Technical Field]

[0001] The present invention relates to improvements in adoptive cell transfer immunotherapy. [Background technology]

[0002] Adoptive cell transfer immunotherapy is a newly established class of therapy for treating various diseases, including cancer, autoimmune conditions, and infectious diseases. Ideally, for the treatment of solid tumors or tumors of hematopoietic origin, patient T cells specific for tumor-associated antigens are expanded in vitro and then reinfused. The number, specificity, and efficacy of T cells are limiting factors in these treatments. One exemplary factor contributing to tumor escape is the downregulation of HLA, which is required for tumor antigen presentation.

[0003] Chimeric antigen receptor (CAR)-transfected cells are an emerging field of cell-based immunotherapy that harnesses the inherent cytolytic potential of a patient's own NK cells and T cells. These engineered autologous cells can be directed against B-cell malignancies or solid tumors such as colon or breast cancer through the introduction of a cell-surface-expressed chimeric antigen receptor (CAR), which receives its specificity from a tumor-specific scFv domain. CAR-expressing T cells combine the antitumor activity of cytotoxic T cells with the specificity and affinity of the scFv element derived from a tumor-associated antigen-specific antibody. Autologous T cells can be collected in sufficient numbers from a patient before being transfected in vitro with the selected CAR. Further expansion can yield large numbers of HLA-independent tumor-specific cytotoxic T cells.

[0004] The efficacy of CAR T cell (CAR-T) therapy may be limited by the persistence of CAR T cell activity and survival in patients after injection. However, the factors limiting CAR T cell survival and efficacy have not yet been fully explored and remain controversial. Although the humoral response to CAR T cells theoretically could trigger antibody-mediated effector mechanisms, little direct evidence has been found to suggest that antibodies are the limiting factor for CAR T cell success. Instead, tumor escape due to loss of target antigens such as CD19 (Majzner and Mackall, 2018, Cancer Discov., 8(10):1219-26), lack of CAR T cell polyfunctionality (Rossi et al., 2018, Blood, 132(8):804-814), or lack of tumor infiltration (Newick et al., 2017, Annu. Rev. Med. 68:139-152) are concerns regarding the success of CAR T cells. Repeated infusion failures are often attributed to T cell-mediated cytotoxicity, for example, caused by the presentation of mouse peptides by scFv in the HLA domain of CAR T cells (Turtle et al., 2016, J. Clin. Invest., 126(6):2123-2138). Consistent with this, tisagenlecleucel (Kymriah), an FDA-approved CAR T cell, is said to be unaffected by pre-existing and induced humoral immunity (Thudium-Muller, J. Clin. Oncology, 36(15), 2018).

[0005] Immunolifidase (IdeS), an immunoglobulin G-degrading cysteine ​​protease, is an IgG endopeptidase currently being developed as a rapid desensitization treatment for kidney transplantation. IdeS is highly specific and cleaves all subclasses of human IgG. IdeS may be useful for reducing competition for Fc receptors when administering antibody drug products (WO 2016 / 012285).

[0006] There is a need for improved methods for treating cancer, autoimmune conditions, infectious diseases, and conditions mediated by B cells that express harmful antibodies. Summary of the Invention

[0007] The present invention provides a method for improving the patient benefits of adoptive cell transfer immunotherapy, comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy. The inventors have demonstrated that the efficacy of adoptive cell transfer immunotherapy can be reduced by limiting the survival and persistence of activity of transferred cells, such as CAR-T cells, and have shown in the Examples that proteins having IgG cysteine ​​protease or IgG endoglycosidase activity can protect transferred cells. Specifically, the inventors have demonstrated that cell surface receptor-specific antibodies, including pre-existing antibodies and antibodies generated after administration of transferred cells, can shorten the potency of transferred cells, and that the therapeutic effect of transferred cells can benefit from removing the effector function of antibodies through conditioning of the recipient. Therefore, administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity can enhance the survival and activity of transferred cells, providing improved adoptive cell transfer immunotherapy treatment. The inventors have also shown that cell surface receptor-specific antibodies against receptor constructs, for example, on CAR-T cells and other cell-based therapeutics, can interfere with the interaction of the receptor with its target protein, and that proteins with IgG cysteine ​​protease or IgG endoglycosidase activity are effective in digesting antibodies and increasing binding of the receptor to its target.

[0008] As demonstrated in the Examples, treatment with the IgG cysteine ​​protease Immurifidase (IdeS) and the IgG endoglycosidase EndoS alleviates some of the limiting effector functions of antibodies and other cell-based therapeutics on CAR-T cells. Accordingly, the present invention provides methods for improving the patient benefit of adoptive cell transfer immunotherapy, comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with the adoptive cell transfer immunotherapy. In a preferred embodiment, the present invention provides a method for treating cancer, comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with the adoptive cell transfer immunotherapy.

[0009] One of the limiting factors of CAR-T therapy and other adoptive cell transfer immunotherapies may be naturally occurring pre-existing antibodies against the CAR construct, which affect its efficacy through attrition due to various antibody-mediated effector mechanisms, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent cellular cytotoxicity (ADCC), as well as osmotic stimulation or receptor activation-induced cell death. After injection, such antibodies may reduce the survival and persistence of CAR T cells in patients. Infusion of CAR-T cells induces elevated levels of antibodies against the chimeric receptor, which may prevent the CAR from interacting with its target cells during the first treatment course and even limit its expansion and persistence. Successful second treatment rounds may be even more challenging due to elevated levels and possibly higher affinity of anti-drug antibodies (ADAs). Even if CAR T cells are of autologous origin, the changes introduced by the chimeric receptor and the expression of viral antigens from the T cell transfection process make them vulnerable to the host's immune response. While some immunogenic moieties may be the junction regions between receptor components, the most prominent are scFv moieties, which in the early stages of CAR-T development were derived from tumor-specific murine IgG, for example. Although subsequent CAR constructs often use humanized IgG to reduce the number of foreign epitopes, these scFvs still contain neoepitopes in the antigen-binding domain. These foreign epitopes can serve as antigens for cellular host T cell responses and have been shown to limit CAR T cell survival (Harding et al., 2010, MAbs, 2(3):256-65; Meunier et al., 2019, Cell. & Mol. Immunology). Meanwhile, data from the Examples demonstrate that proteins with IgG cysteine ​​protease or IgG endoglycosidase activity can enhance the survival and activity of transfected cells and provide improved therapy through the inactivation of cell surface receptor-specific antibodies.

[0010] There are various possible mechanisms by which antibodies can affect the activity, expansion, and survival of transferred cells, such as CAR T cells. For example, CAR-specific antibodies may promote the destruction of CAR T cells by complement deposition, CDC, and / or ADCP. The examples demonstrate that proteins with IgG cysteine ​​protease or IgG endoglycosidase activity may be effective in mitigating these processes and thereby improving adoptive cell transfer immunotherapy. Furthermore, antibodies themselves may bind to receptors, triggering ADCC, exhaustion, or receptor activation-induced cell death. Proteins with IgG cysteine ​​protease or IgG endoglycosidase activity may also be useful in mitigating these processes.

[0011] In light of these developments, the present invention provides a method for improving the patient benefit of adoptive cell transfer immunotherapy, comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy. In certain embodiments, the protein is administered prior to adoptive cell transfer immunotherapy. The examples demonstrate that IdeS and EndoS are effective in inactivating pre-existing antibodies; for example, IdeS and EndoS were effective when used prior to the addition of a complement source or effector cells. The examples also demonstrate that antibodies present in the serum of healthy individuals and HLA-sensitized patients who have not received adoptive cell transfer immunotherapy can bind to receptor constructs and cells such as CAR T cells and mediate adverse effects such as ADCP, ADCC, and interference with target binding (such alloantibodies can be induced, for example, by pregnancy or blood transfusion), all of which can be reduced by IdeS treatment. In a preferred embodiment, the protein is administered after administration of adoptive cell transfer immunotherapy. The examples demonstrate that Ides and EndoS are effective in inactivating induced antibodies, as immune thrombocytopenia was reduced when Ides and EndoS were administered after anti-platelet specific antibodies. The examples also demonstrate that alloantibodies present in human serum can adversely affect adoptive cell transfer immunotherapy cells, such as CAR-T cells, and that the proteins of the present invention can reduce or prevent such adverse effects. Because adoptive cell transfer immunotherapy can induce such alloantibodies, the proteins of the present invention may be useful when administered after the administration of adoptive cell transfer immunotherapy.

[0012] The present invention also provides methods for improving the patient benefit of adoptive cell transfer immunotherapy, methods for prolonging the survival and / or enhancing the proliferation of cells administered as part of adoptive cell transfer, methods for conditioning or preparing a patient for adoptive cell transfer immunotherapy, and methods for lowering serum IgG levels or reducing complement and / or Fc receptor binding by serum IgG molecules in a patient undergoing or to undergo adoptive cell transfer immunotherapy, which methods comprise administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity. The effects shown in the examples for the polypeptides of the present invention will be of great benefit in such methods.

[0013] The present invention also provides a method for increasing the efficacy of adoptive cell transfer therapy or increasing binding between a cell surface receptor of an adoptive cell transfer therapy and its target, comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity before, after, or simultaneously with adoptive cell transfer immunotherapy. The examples demonstrate that the polypeptides of the present invention are effective in increasing such efficacy and binding. In a preferred such embodiment, the target is CD19, and preferably, the cell surface receptor is an anti-CD19 CAR.

[0014] In certain embodiments of any of the methods of the invention, proteins with IgG cysteine ​​protease or IgG endoglycosidase activity improve the patient benefit of adoptive cell transfer immunotherapy by removing IgG antibodies that inhibit the binding of cell surface receptors to their targets, or improve the treatment of cancer, autoimmune conditions, conditions mediated by harmful antibody-producing B cells, or infectious diseases. The antibodies may bind to cell surface receptors, particularly CARs, or to CAR adapter molecules, or to the target itself, and may sterically inhibit the binding of the receptor to its target. In certain embodiments, proteins of the invention increase the clearance of antibody fragments, particularly F(ab')2 fragments, that block the interaction between the target and the cell surface receptor. In a preferred embodiment, the target is CD19, and preferably the cell surface receptor is an anti-CD19 CAR.

[0015] In a preferred embodiment, the method of the invention uses an IgG cysteine ​​protease. In a particularly preferred embodiment, the IgG cysteine ​​protease is an IdeS or IdeZ polypeptide, most preferably an IdeS polypeptide, e.g., a polypeptide having a sequence at least 80% identical, e.g., at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 2, 4, or 5. The examples demonstrate that such polypeptides are effective in protecting cells.

[0016] In a further embodiment, the IgG endoglycosidase is an EndoS polypeptide, e.g., a polypeptide having a sequence at least 80% identical, e.g., at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 90. The examples demonstrate that such polypeptides are effective in protecting cells.

[0017] In a particularly preferred embodiment of the invention, the method comprises administering an IdeS polypeptide in combination with CAR-T therapy, preferably in the treatment of cancer.

[0018] In a particularly preferred embodiment, the methods of the invention comprise administering an IdeS polypeptide after adoptive cellular immunotherapy, e.g., administering an IdeS polypeptide to a patient who has previously undergone adoptive cellular immunotherapy. Such methods reduce the effects of ADA induced by cell therapy and improve cell survival. In a further particularly preferred embodiment, the IdeS polypeptide is administered to a patient who has previously undergone adoptive cellular immunotherapy and is scheduled to undergo additional adoptive cellular immunotherapy, such as with the same or a similar construct.

[0019] The present invention also provides compositions, particularly pharmaceutical compositions, comprising a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in the methods of the present invention.

[0020] Further embodiments of the present invention are provided in the numbered paragraphs below.

[0021] 1. A method for improving the patient benefit of adoptive cell transfer immunotherapy, comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy.

[0022] 2. A method of treating cancer comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy.

[0023] 3. A method of treating an autoimmune condition comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy.

[0024] 4. A method for treating a pathology mediated by harmful antibody-producing B cells comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy.

[0025] 5. A method for treating an infectious disease comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy.

[0026] 6. The method of any one of embodiments 1 to 5, wherein the protein having IgG cysteine ​​protease or IgG endoglycosidase activity is administered before the administration of adoptive cell transfer immunotherapy, or the protein having IgG cysteine ​​protease or IgG endoglycosidase activity is administered after the administration of adoptive cell transfer immunotherapy, or the protein having IgG cysteine ​​protease or IgG endoglycosidase activity is administered before the second or subsequent administration of adoptive cell transfer immunotherapy.

[0027] 7. A method of treating cancer, an autoimmune condition, a condition mediated by harmful antibody-producing B cells, or an infectious disease comprising administering a protein with IgG cysteine ​​protease or IgG endoglycosidase activity to a patient who has previously undergone and / or will undergo adoptive cell transfer immunotherapy.

[0028] 8. A method of treating cancer, an autoimmune condition, a condition mediated by harmful antibody-producing B cells, or an infectious disease, comprising administering adoptive cell transfer immunotherapy to a patient who has previously received and / or will receive a protein with IgG cysteine ​​protease or IgG endoglycosidase activity.

[0029] 9. The method of any one of embodiments 1-8, wherein adoptive cell transfer immunotherapy comprises administering T cells, natural killer cells, or dendritic cells expressing a chimeric antigen receptor or a T cell receptor.

[0030] 10. The method of any one of embodiments 1 to 9, which is a method of treating cancer, wherein the cancer is a hematological malignancy, such as leukemia (e.g., acute myeloid leukemia (AML), acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), acute mixed lineage leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia and large granular lymphocytic leukemia, myelodysplastic syndrome (MDS), myeloproliferative disorders (polycythemia vera, essential erythrocytosis), ... the cancer is selected from the list consisting of: thrombocytosis, primary myelofibrosis, and CML), lymphoma, multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS) and similar disorders, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, splenic marginal zone lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies.

[0031] 11. The method of any one of embodiments 1 to 10, which is a method of treating cancer, wherein the cancer is selected from the group consisting of solid cancer, e.g., bone cancer, breast cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, prostate cancer, rectal cancer, anal cancer, colon cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric tumors, bladder cancer, kidney or ureter cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, glioblastoma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, and squamous cell carcinoma.

[0032] 12. The method of any of embodiments 1-11, which is a method of treating an autoimmune condition, wherein the autoimmune condition is rheumatoid arthritis, systemic lupus erythematosus (lupus), inflammatory bowel disease (IBD), such as Crohn's disease or ulcerative colitis, multiple sclerosis (MS), type 1 diabetes, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, or vasculitis.

[0033] 13. The method of any one of embodiments 1-12, which is a method of treating an infectious disease, wherein the infectious disease is selected from the group consisting of human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), Epstein-Barr virus (EBV), human papillomavirus (HPV), Kaposi's sarcoma herpesvirus (KSHV), Lassa virus, cytomegalovirus (CMV), coronavirus such as COVID-19, Aspergillus fumigatus ( Aspergillus fumigatus ), and tuberculosis, preferably wherein the infection is a chronic infection.

[0034] 14. The method of any one of embodiments 1 to 13, which is a method for treating a condition mediated by B cells that produce harmful antibodies, wherein the condition is an autoimmune condition or a condition mediated by anti-drug antibodies.

[0035] 15. A method according to any one of embodiments 1 to 14, which increases the survival and / or proliferation of cells administered in adoptive cell transfer immunotherapy.

[0036] 16. The method of any one of embodiments 1 to 15, wherein the method reduces antibody-mediated complement fixation, complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), wasting, and / or receptor-activated cell death of cells administered in adoptive cell transfer immunotherapy.

[0037] 17. (i) Proteins with IgG cysteine ​​protease activity are isolated from Streptococcus pyogenes ( Streptococcus pyogenes ), optionally wherein the protein is IdeS or IdeZ; or (ii) Proteins with IgG endoglycosidase activity are isolated from Streptococcus pyogenes, Streptococcus pyogenes ( Streptococcus equi ), or Streptococcus zooepidemicus ( Streptococcus zooepidemicus ) or Yersinia pseudotuberculosis ( Corynebacterium pseudotuberculosis ), E. faecalis ( Enterococcus faecalis ), or Elizabethkingia meningoseptica ( Elizabethkingia meningoseptica 17. The method of any one of embodiments 1 to 16, wherein the protein is an IgG endoglycosidase derived from EndoS, CP40, EndoE, or EndoF2.

[0038] 18. (i) the protein having IgG cysteine ​​protease activity is a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2, 4, or 5, or a fragment or variant thereof having IgG cysteine ​​protease activity; or (ii) The method of embodiment 17, wherein the protein having IgG endoglycosidase activity is a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 90, or a fragment or variant thereof that has IgG endoglycosidase activity.

[0039] 19. (i) the protein having IgG cysteine ​​protease activity is a polypeptide having a sequence that is at least 80% identical, e.g., at least 85%, at least 90%, at least 95%, or at least 99% identical, to SEQ ID NO: 2, 4, or 5, or the IgG cysteine ​​protease comprises or consists of the sequence of any one of SEQ ID NOs: 6 to 25 and 55 to 69, optionally comprising an additional methionine at the N-terminus and / or a histidine tag at the C-terminus; or (ii) The method of embodiment 17, wherein the protein having IgG endoglycosidase activity is a polypeptide having a sequence that is at least 80% identical, e.g., at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 90.

[0040] 20. A protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in conditioning or preparing a patient for adoptive cell transfer immunotherapy.

[0041] 21. A protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving the patient benefit of adoptive cell transfer immunotherapy.

[0042] 22. A protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels or reducing complement or Fc receptor binding by serum IgG molecules in patients undergoing or to undergo adoptive cell transfer immunotherapy. [Brief explanation of the drawings]

[0043] [Figure 1A] Antibody-targeted C1q and C4d deposition on Daudi cells can be prevented by treatment with IdeS or EndoS. Daudi cells were incubated with RTX (or IgG1 isotype control) for approximately 2 hours with titrations of IdeS, followed by the addition of human serum complement for an additional 2 hours. Cells were aliquoted and stained with anti-human C1q, followed by final detection with SA-PE. Cells were analyzed in FL2 using an Accuri C6 flow cytometer. MFI values ​​are listed. [Figure 1B]Antibody-targeted C1q and C4d deposition on Daudi cells can be prevented by treatment with IdeS or EndoS. Daudi cells were incubated with RTX (or IgG1 isotype control) for approximately 2 hours with titrations of IdeS, followed by the addition of human serum complement for an additional 2 hours. Cells were aliquoted and stained with anti-human C4d, followed by final detection with SA-PE. Cells were analyzed in FL2 using an Accuri C6 flow cytometer. MFI values ​​are listed. [Figure 1C] Antibody-targeted C1q and C4d deposition on Daudi cells can be prevented by treatment with IdeS or EndoS. Daudi cells were incubated with RTX (or IgG1 isotype control) for approximately 2 hours with titrations of EndoS, followed by the addition of human serum complement for an additional 2 hours. Cells were aliquoted and stained with anti-human C4d, followed by final detection with SA-PE. Cells were analyzed in FL2 using an Accuri C6 flow cytometer. MFI values ​​are listed. [Figure 1D] Antibody-targeted C1q and C4d deposition on Daudi cells can be prevented by treatment with IdeS or EndoS. Daudi cells were incubated with RTX (or IgG1 isotype control) for approximately 2 hours with titrations of EndoS, followed by the addition of human serum complement for an additional 2 hours. Cells were aliquoted and stained with anti-human C4d, followed by final detection with SA-PE. Cells were analyzed in FL2 using an Accuri C6 flow cytometer. MFI values ​​are listed. [Figure 2]ADCC can be blocked or restored with IdeS or EndoS. CD20-positive Daudi cells were incubated with RTX (titrated from 0.2 μg / mL to 50 μg / mL) along with 50 μg / mL IdeS (squares), EndoS (triangles), or medium (diamonds). CDC was induced by adding baby rabbit serum as a complement source. Results were normalized to the cell viability without RTX, which was set at 100% after 90 minutes of incubation. Cell cytotoxicity was assessed using cell counting kit-8 (CCK8). A normal positive control group received only titrated RTX without therapeutic enzyme. This represents the maximum toxicity of RTX at the indicated concentrations. [Figure 3] Treatment of opsonized target cells with IdeS inhibits ADCP. Calcein-stained Daudi cells were opsonized with titrated RTX (from 7.5 μg / mL to 0.01 μg / mL) and incubated with 40 μg / mL IdeS (squares), EndoS (triangles), or no enzyme (medium alone, diamonds) prior to the addition of FarRed-stained THP1 effector cells. After 2 hours of incubation, cells were fixed and analyzed by flow cytometry in FL2 and FL4. The gated FL2-positive cells were designated as 100%. This population was further divided into FL2-single-positive and FL2-FL4-double-positive cells, which were then read for THP1-engulfed Daudi cells. [Figure 4] Anti-PLT antibodies cleaved with IdeS do not induce ITP in vitro. Thrombocytopenic anti-PLT IgG (250 μg / mouse) induced ITP in BALB / c mice after a single injection (squares). Singly cleaved anti-PLT IgG (triangles) only partially induced ITP at the same dose, whereas anti-PLT IgG fully cleaved to F(ab')2 and Fc did not affect normal platelet levels. Mice were intraperitoneally administered 0.25 mg / mouse of purified IgG in 200 μL PBS. Platelet counts were determined on day 1 using an automated cell counter, VetScan HM5. [Figure 5]Antibody-induced ITP can be blocked by in vivo IdeS treatment. BALB / c mice were primed for ITP by a single i.p. injection of purified intact rabbit anti-mouse platelet IgG (0.25 mg / mouse). One hour after ITP induction, treatment with three different doses of IdeS (0.2 μg / mouse, 2 μg / mouse, and 20 μg / mouse) was administered i.v. The "PBS only" (circle) treatment group did not receive IdeS and received only anti-PLT IgG as a positive control for ITP induction. Naive mice (diamonds) administered with carrier solution alone represent healthy controls. Platelet counts were determined on day 1 using an automated cell counter, VetScan HM5. [Figure 6] EndoS prevents ITP in mice even after anti-PLT antibody injection. BALB / c mice (n = 6 / group) were injected with 50 μg of anti-PLT IgG as a priming for ITP or with PBS only (circles) as a control for normal platelet levels. Thirty minutes later, they received a therapeutic ip injection of EndoS (10 μg / mouse, 30 μg / mouse, 90 μg / mouse). ITP-induced controls were injected with PBS (diamonds). Blood was collected 24 hours later and analyzed for platelet counts using an automated cell counter, VetScan HM5. [Figure 7-1] Identification of anti-CAR-specific antibodies—F(ab')2-specific polyclonal antibodies specifically bind to the CAR T cell receptor. Polyclonal rabbit anti-mouse F(ab')2 antibodies (10 μg / mL, 1 μg / mL) were evaluated for binding to primary CAR T cells, including (A) anti-CD19 CAR T cells and (B) anti-BCMA4 CAR T cells, as well as (C) mock-transfected T cells and (D) BCR-expressing Daudi cells as negative and positive controls, respectively. Bound anti-F(ab')2 antibodies were detected by flow cytometry analysis using biotinylated anti-rabbit Fc and SA-AF647. [Figure 7-2]Identification of anti-CAR-specific antibodies—F(ab')2-specific polyclonal antibodies specifically bind to the CAR T cell receptor. Binding of polyclonal rabbit anti-mouse F(ab')2 and anti-human F(ab')2 (10 μg / mL) was also assessed using the anti-CD19 CAR-Jurkat T cell line (E) (CARJ-ZP005, Creative Biolabs) by FACS analysis and expressed as MFI. [Figure 8A] Identification of HAMA and anti-CD19 CAR Jurkat T cell allospecific sera. (A) Normal human serum samples (BioIVT) were screened for human anti-mouse IgG antibodies (HAMA) using a validated sandwich ELISA kit (Biolegend). The threshold for HAMA-positive sera was >10 ng / mL. Sera from OneLambda were used as HLA class I positive and negative controls. [Figure 8B] Identification of HAMA and anti-CD19 CAR Jurkat T cell allospecific sera. (B) HAMA sera screened by ELISA were incubated with anti-CD19-CAR-Jurkat T cells. Jurkat wild-type cells served as a CAR-negative staining control. After incubation with a PE-conjugated goat anti-human Fc detection antibody, cells were analyzed by flow cytometry. Sera from OneLambda were used as HLA class I positive and negative controls. [Figure 8C] Identification of HAMA and anti-CD19 CAR Jurkat T cell allospecific sera. (C) HAMA-positive and -negative sera were selected and further tested for specific binding to anti-CD19 CAR-Jurkat T cells by flow cytometry analysis. These sera were further used to assay for IgG effector mechanisms with anti-CD19 CAR-Jurkat T cells. Sera from OneLambda were used as HLA class I positive and negative controls. [Figure 8D]Identification of HAMA and anti-CD19 CAR Jurkat T cell allospecific sera. (D) Sera from HLA-sensitized patients were screened for alloreactivity to anti-CD19-CAR-Jurkat T cells. Sera from OneLambda were used as HLA class I positive and negative controls. [Figure 9] CAR-specific antibody-induced ADCP can be blocked by immunofluorescence analysis. Polyclonal anti-F(ab')2 antibody opsonization of anti-CD19 CAR-Jurkat T cells for ADCP is blocked by immunofluorescence analysis. For flow cytometry-based ADCP analysis, target cells, including (A, B) anti-CD19 CAR-Jurkat, (D, E) wild-type Jurkat cells, and (C, F) CD20- and BCR-expressing Daudi cells, were stained with calcein AM and then incubated with the indicated concentrations of immunofluorescence-treated rabbit anti-mouse F(ab')2 or anti-human F(ab')2. The monocytic phagocytic effector cell line THP-1 was stained with CellTrace FarRed for 90 minutes before addition to the target cells. Phagocytosis was assessed by flow cytometry. The amount of double positive cells, reflecting phagocytosed target cells, is expressed as a percentage of target cells. [Figure 10] Immunolifidase prevents the induction of ADCP by anti-CD19 CAR-Jurkat T cells opsonized with allogeneic serum. Target cells, anti-CD19-CAR-Jurkat, were opsonized with serum from (A) a healthy donor and (B) a highly sensitized anti-HLA patient, with or without treatment with immunolifidase (10 μg / mL). After washing, target cells were incubated with FcγRI-expressing reporter cells at 37°C for 6 hours to induce ADCP. Luciferase activity, resulting from reporter cell activation, was measured using a luminescence reader, and the luminescence signal (RLU) is presented as the mean fold change in induction ± SD. [Figure 11]ADCC (V158) induced by anti-CD19 CAR-specific antibodies is blocked by immunofluorescence assay. Anti-CD19 CAR-Jurkat T cells were opsonized with the indicated concentrations of polyclonal rabbit (A) anti-mouse and (B) anti-human F(ab')2-specific antibodies, with or without immunofluorescence assay (20 μg / mL). ADCC induction was quantified using a luciferase reporter bioassay with high-affinity FcγRIIIa (V158)-transfected reporter cells (Promega, #G7015). (C) CAR-negative Jurkat wild-type cells were treated with rabbit anti-human IgG, F(ab')2, in the presence or absence of immunofluorescence assay. BCR- and CD20-positive Daudi cells represent the positive target cell control for ADCC induction by (D) rituximab and (E) anti-human F(ab')2-specific antibodies. Luminescent signals (RLU) from activated effector cells are presented as the mean fold change of induction (of duplicates) ±SD. [Figure 12] ADCC (F158) induced by anti-CD19 CAR-specific antibodies is blocked by immunofluorescence assay. Anti-CD19 CAR-Jurkat T cells were opsonized with the indicated concentrations of (A) polyclonal rabbit anti-mouse and (B) anti-human F(ab')2-specific antibodies, with or without immunofluorescence assay (20 μg / mL). ADCC induction was quantified using a luciferase reporter bioassay with low-affinity FcγRIIIa (F158)-transfected reporter cells (Promega, #G979A). (C) CAR-negative Jurkat wild-type cells were treated with rabbit anti-mouse IgG, F(ab')2, in the presence or absence of immunofluorescence assay. BCR- and CD20-positive Daudi cells represent the positive target cell control for ADCC induction by (D) rituximab and (E) anti-human F(ab')2-specific antibodies. Luminescent signals (RLU) from activated effector cells are presented as the mean fold change of induction (of duplicates) ±SD. [Figure 13]ADCC induction by HAMA-opsonized anti-CD19 CAR-Jurkat T cells can be blocked by immunofluorescence assay (IgA). A reporter cell line expressing the CD16 FcγRIIIa high-affinity (V158) allele was used to assay ADCC induction. A mouse mAb FMC63-based scFv-CD19-CAR Jurkat cell line was incubated with normal human serum samples previously tested by ELISA for HAMA levels against mouse IgG, with or without immunofluorescence assay. HAMA-positive (184, 187, 208, 250) and HAMA-negative (164) human sera were included in the ADCC assay. Luminescence signals (RLU) from activated effector cells are presented as the mean ± SD of (duplicate) induction values. [Figure 14] Immunofidase treatment of serum improves the association of target CD19 protein with anti-CD19 CAR T cells. Immunofidase treatment can increase CD19 protein binding to serum-exposed anti-CD19 CAR T cells. Anti-CD19 CAR-Jurkat T cells were incubated with HAMA-positive and -negative serum samples with or without immunofidase (10 μg / mL). IHAc (1 mM) was added to all samples to inactivate immunofidase during subsequent steps. The serum samples were incubated with anti-CD19 CAR-Jurkat T cells to allow binding of F(ab')2 to the anti-CD19 CAR for each available IgG. After washing, recombinant atto-647N-labeled human CD19-Fc protein (ATM9269, R&D Systems) was added to the cells, and the interaction of the anti-CD19 CAR with the CD19 target protein was evaluated by flow cytometry. Results are presented as median fluorescence intensity (FI). DETAILED DESCRIPTION OF THE INVENTION

[0044] A brief description of arrays SEQ ID NO: 1 is the complete sequence of IdeS, including the N-terminal methionine and signal sequence. Also available as NCBI reference sequence number WP_010922160.1.

[0045] SEQ ID NO: 2 is the mature sequence of IdeS, lacking the N-terminal methionine and signal sequence. Also available as Genbank accession number ADF13949.1.

[0046] SEQ ID NO: 3 is the full sequence of IdeZ, including the N-terminal methionine and signal sequence. Also available as NCBI reference sequence number WP_014622780.1.

[0047] SEQ ID NO: 4 is the mature sequence of IdeZ, lacking the N-terminal methionine and signal sequence.

[0048] SEQ ID NO: 5 is the sequence of hybrid IdeS / Z. The N-terminus is based on IdeZ, lacking the N-terminal methionine and signal sequence.

[0049] SEQ ID NOs: 6-25 are exemplary protease sequences for use in the methods of the present invention.

[0050] SEQ ID NO: 26 is the sequence of the IdeS polypeptide, which comprises the sequence of SEQ ID NO: 2 with an additional N-terminal methionine and histidine tag (internal reference pCART124).

[0051] SEQ ID NO: 27 is the sequence of the IdeZ polypeptide, which comprises the sequence of SEQ ID NO: 4 with an additional N-terminal methionine and histidine tag (internal reference pCART144).

[0052] SEQ ID NO: 28 is the sequence of the IdeS / Z polypeptide, which comprises the sequence of SEQ ID NO: 5 with an additional N-terminal methionine and histidine tag (internal reference pCART145).

[0053] SEQ ID NO:29 is the continuous sequence PLTPEQFRYNN corresponding to positions 63 to 73 of SEQ ID NO:3.

[0054] SEQ ID NO:30 is a contiguous sequence PPANFTQG corresponding to positions 58 to 65 of SEQ ID NO:1.

[0055] SEQ ID NO: 31 is the consecutive sequence DDYQRNATEAYAKEVPHQIT corresponding to positions 35 to 54 of SEQ ID NO: 3.

[0056] SEQ ID NO: 32 is the continuous sequence DSFSANQEIRYSEVTPYHVT corresponding to positions 30 to 49 of SEQ ID NO: 1.

[0057] SEQ ID NOs: 33 to 55 are nucleotide sequences encoding the above proteases.

[0058] SEQ ID NOs: 56-69 are exemplary protease sequences for use in the methods of the present invention.

[0059] SEQ ID NO: 70 is a contiguous NQTN sequence corresponding to positions 336 to 339 of SEQ ID NO: 1.

[0060] SEQ ID NO: 71 is the consecutive sequence DSFSANQEIR YSEVTPYHVT corresponding to positions 30 to 49 of SEQ ID NO: 1.

[0061] SEQ ID NOs: 72-86 are nucleotide sequences encoding the polypeptides disclosed herein.

[0062] SEQ ID NO: 87 is the consecutive sequence SFSANQEIRY SEVTPYHVT corresponding to positions 31 to 49 of SEQ ID NO:1.

[0063] SEQ ID NO: 88 is the sequence DYQRNATEAY AKEVPHQIT corresponding to positions 36 to 54 of the IdeZ polypeptide NCBI reference sequence number WP_014622780.1.

[0064] SEQ ID NO: 89 is the sequence DDYQRNATEA YAKEVPHQIT which may be present at the N-terminus of a polypeptide of the invention.

[0065] SEQ ID NO: 90 shows the amino acid sequence of mature endoglycosidase S (EndoS). The entire sequence, including the secretion signal, is available at Genbank accession number AAK00850.1.

[0066] Detailed Description of the Invention Methods for improving patient benefits from adoptive cell transfer immunotherapy The present invention provides a method for improving the patient benefits of adoptive cell transfer immunotherapy, comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy. The inventors have demonstrated that the efficacy of adoptive cell transfer immunotherapy can be reduced by limiting the survival and sustained activity of transferred cells, such as CAR-T cells, and have shown in the Examples that proteins having IgG cysteine ​​protease or IgG endoglycosidase activity can protect transferred cells. Specifically, the inventors have demonstrated that cell surface receptor-specific antibodies can shorten the potency of transferred cells, and that the therapeutic effect of transferred cells can benefit from removing the antibody effector function through recipient conditioning. Thus, administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity can enhance the survival and activity of transferred cells, providing improved therapy. The present inventors have also demonstrated that cell surface receptor-specific antibodies can interfere with the binding of receptors to their targets in adoptive cell transfer immunotherapy. Thus, the efficacy and effectiveness of adoptive cell transfer immunotherapy can be enhanced by administering proteins with IgG cysteine ​​protease or IgG endoglycosidase activity.

[0067] In certain embodiments, the present invention provides methods for prolonging survival and / or enhancing proliferation of cells administered as part of adoptive cell transfer, comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity before, after, or concomitantly with the adoptive cell transfer immunotherapy. In preferred embodiments, the method comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after a first adoptive cell transfer immunotherapy and before a second adoptive cell transfer immunotherapy.

[0068] In certain embodiments, the present invention provides methods of conditioning or preparing a patient for adoptive cell transfer immunotherapy comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0069] In certain embodiments, the present invention provides methods for lowering serum IgG levels or reducing complement or Fc receptor binding by serum IgG molecules in patients undergoing or to undergo adoptive cell transfer immunotherapy.

[0070] In certain embodiments, the present invention provides a method for increasing the efficacy of adoptive cell transfer therapy, the method comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity before, after, or concurrently with adoptive cell transfer immunotherapy. In certain embodiments, the present invention provides a method for increasing binding of a cell surface receptor in adoptive cell transfer therapy to its target, the method comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity before, after, or concurrently with adoptive cell transfer immunotherapy.

[0071] In certain embodiments, a method for improving patient benefits from adoptive cell transfer immunotherapy involves administering a protein with IgG cysteine ​​protease or IgG endoglycosidase activity followed by adoptive cell transfer immunotherapy. This method allows pre-existing anti-drug antibodies (ADAs) to be inactivated with the protein before administering the cells, allowing for better cell expansion and survival. ADAs can bind to any part of any cell therapy, including expressed CAR or TCRs or HLA antigens (particularly in allogeneic therapy). The examples demonstrate that IdeS and EndoS are effective in inactivating pre-existing antibodies; for example, IdeS and EndoS were effective when used before the addition of a complement source or effector cells. The examples also demonstrate that antibodies present in the serum of healthy individuals and HLA-sensitized patients who have not received adoptive cell transfer immunotherapy can bind to receptor constructs and cells, such as CAR T cells, and mediate adverse effects, such as ADCP, ADCC, and reduced target binding, all of which can be reduced by treatment with a protein of the invention. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in conditioning a patient for treatment with adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in a patient scheduled to receive adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in a patient who has previously donated blood for the development of adoptive cell transfer immunotherapy but has not yet received adoptive cell transfer immunotherapy. The present invention also provides an adoptive cell transfer immunotherapy composition for treating a patient who has previously been administered a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0072] In a preferred embodiment, a method for improving the patient benefit of adoptive cell transfer immunotherapy comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after administering adoptive cell transfer immunotherapy. Such a method makes it possible to inactivate pre-existing anti-drug antibodies (ADAs) and antibodies elicited by adoptive cell transfer immunotherapy, thereby improving the expansion and survival rate of transferred cells. The examples demonstrate that Ides and EndoS are effective in inactivating harmful polyclonal and induced antibodies, as immune thrombocytopenia was reduced when Ides and EndoS were administered after anti-platelet-specific antibodies. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving the patient benefit of previously administered adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating patients who have previously received adoptive cell transfer immunotherapy. The present invention also provides proteins having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in patients who have previously been treated with adoptive cell transfer immunotherapy, and adoptive cell transfer immunotherapy compositions for treating patients who are scheduled to receive a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0073] In a further preferred embodiment, a method for improving the patient benefit of adoptive cell transfer immunotherapy comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity both before and after multiple administrations of adoptive cell transfer immunotherapy. Such a method makes it possible to inactivate both pre-existing anti-drug antibodies (ADAs) and antibodies elicited by adoptive cell transfer immunotherapy, thereby improving the expansion and survival rate of transferred cells.

[0074] In a further preferred embodiment, a method for improving the patient benefit of adoptive cell transfer immunotherapy comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after administering a first adoptive cell transfer immunotherapy and before administering a second adoptive cell transfer immunotherapy. Thus, the protein is administered between two or more adoptive cell transfer immunotherapies. In such a method, any ADAs from previous injections are inactivated, allowing for better expansion and survival of the transferred cells. Preferably, in such an embodiment, the first and second, and any subsequent, adoptive cell transfer immunotherapies use the same or similar constructs and cells. In such an embodiment, the similar constructs or cells may have an ADA cross-reactive epitope. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating a patient who has already undergone a first adoptive cell transfer immunotherapy and is scheduled to undergo a second adoptive cell transfer immunotherapy. The present invention also provides proteins having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing IgG levels in patients undergoing multiple-dose adoptive cell transfer immunotherapy regimens. The present invention also provides proteins having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving the benefit to patients undergoing multiple-dose adoptive cell transfer immunotherapy regimens. The present invention also provides adoptive cell transfer immunotherapy compositions for treating patients who have previously received an adoptive cell transfer immunotherapy composition and who have previously received a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0075] In a further preferred embodiment, a method for improving the benefit of adoptive cell transfer immunotherapy for a patient comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after two or more administrations of adoptive cell transfer immunotherapy. Such a method can inactivate antibodies elicited by adoptive cell transfer immunotherapy, thereby improving the expansion and survival rate of transferred cells. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving the benefit of a patient who has already undergone two or more administrations of adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating a patient who has already undergone two or more administrations of adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in a patient who has already undergone two or more administrations of adoptive cell transfer immunotherapy.

[0076] In certain embodiments of the present invention, a method for improving patient benefit from adoptive cell transfer immunotherapy comprises administering multiple doses of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity and administering multiple doses of adoptive cell transfer immunotherapy. In certain embodiments, the multiple administrations of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity comprise administering the same enzyme. The repeated administrations of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity may be spaced apart by any suitable period, such as 1-7 days, 5-7 days, or 6-8 days. In certain embodiments, the multiple administrations of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity comprise administering different enzymes.

[0077] In a specific embodiment of the present invention, a method for improving the patient benefit of adoptive cell transfer immunotherapy comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity and simultaneously administering adoptive cell transfer immunotherapy. The examples demonstrate that because cell surface receptor-specific antibodies can interfere with the binding of adoptive cell transfer immunotherapy receptors to their targets, but treatment with a protein of the present invention can increase binding, co-administration of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity can increase the efficacy and effectiveness of adoptive cell transfer immunotherapy.

[0078] In preferred embodiments of the methods of the invention, administration of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity inactivates all or substantially all IgG molecules present in the patient's serum. In certain embodiments, the protein is administered in an amount sufficient to eliminate Fc receptor or complement binding by all or substantially all IgG molecules present in the patient's serum.

[0079] In methods of the present invention in which a protein having IgG cysteine ​​protease or IgG endoglycosidase activity is administered prior to adoptive cell transfer immunotherapy, the two administrations are preferably separated by a time interval sufficient to inactivate or eliminate Fc receptor and complement binding of all or substantially all IgG molecules present in the subject's serum. This interval may typically be at least 30 minutes and typically up to 21 days. Administration of the protein having IgG cysteine ​​protease or IgG endoglycosidase activity may occur simultaneously with lymphodepletion (e.g., on the same day) or 1 to 7 days before or after lymphodepletion. In certain embodiments, administration of the protein occurs between lymphodepletion and administration of cell therapy. "Substantially all" typically means that Fc receptor and complement binding by serum IgG is reduced to less than 30%, less than 20%, less than 15%, less than 10%, or less than 5% of the levels present prior to administration. For example, if the protein is a protease (such as IdeS), the interval would be the time required for the agent to cleave at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% of the serum IgG in the subject, as measured in any suitable assay in the subject.

[0080] In the methods of the present invention in which a protein having IgG cysteine ​​protease or IgG endoglycosidase activity is administered after adoptive cell transfer immunotherapy, the protein is preferably administered during the proliferation of the transferred cells, such as within 2 weeks, 1 week, 2 days, 1 day, or 5 hours of administering the adoptive cell transfer immunotherapy.

[0081] In a preferred embodiment, the method of the present invention is a method for reducing antibody-mediated complement deposition, CDC, ADCC, ADCP, wasting, or receptor-activated cell death of cells previously administered to a patient in adoptive cell transfer immunotherapy. In another preferred embodiment, the method of the present invention is a method for reducing antibody-mediated complement deposition, CDC, ADCC, ADCP, wasting, or receptor-activated cell death of cells subsequently administered to a patient in adoptive cell transfer immunotherapy. In a preferred embodiment, the present invention provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing antibody-mediated complement deposition, CDC, ADCC, ADCP, wasting, or receptor-activated cell death of cells previously administered to a patient in adoptive cell transfer immunotherapy. In another preferred embodiment, the present invention provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing antibody-mediated complement deposition, CDC, ADCC, ADCP, wasting, or receptor-activated cell death of cells subsequently administered to a patient in adoptive cell transfer immunotherapy. In a preferred embodiment, ADCC is mediated by FcγRIIIa(V158) or FcγRIIIa(F158) effector cells.

[0082] In certain embodiments, the methods of the present invention involve administering a protein with IgG cysteine ​​protease or IgG endoglycosidase activity to a patient with detectable levels of human anti-mouse antibodies (HAMA), which are immunoglobulins with specificity for mouse immunoglobulins. The administration may occur before, after, or simultaneously with adoptive cell transfer immunotherapy. The examples demonstrate that HAMA in patient serum can have deleterious effects on adoptive cell transfer immunotherapy cells, but these effects can be reduced or prevented by treatment with a protein of the present invention. HAMA can also be induced in normal individuals by contact with mouse antigens. It can be expected that patients receiving mouse mAb-based biologics will experience even higher frequencies and concentrations of HAMA, potentially even resulting in partial neutralization of these therapeutic agents.

[0083] In the methods of the present invention, the IgG cysteine ​​protease or IgG endoglycosidase may be co-administered with an immunosuppressant. In the methods of the present invention, the protease is preferably administered by intravenous infusion, but may be administered by any suitable route, including, for example, intradermal, subcutaneous, transdermal, intramuscular, intraarterial, intraperitoneal, intraarticular, intraosseous, intrathecal, intracerebroventricular, or other suitable administration route. The amount of the administered protease or endoglycosidase may be 0.01 mg / kg BW to 2 mg / kg BW, 0.05 to 1.5 mg / kg BW, 0.1 mg / kg BW to 1 mg / kg BW, preferably 0.15 mg / kg to 0.7 mg / kg BW, most preferably 0.2 mg / kg to 0.3 mg / kg BW, and particularly 0.25 mg / kg BW. The protein may be administered multiple times to the same subject, provided that the amount of anti-drug antibodies (ADA) in the subject's serum that can bind to the protein does not exceed a threshold determined by a clinician. The amount of ADA in the subject's serum that can bind to the protease can be determined by any suitable method, such as an agent-specific CAP FEIA (ImmunoCAP) test or titer assay.

[0084] How to Treat Cancer The present invention provides a method for treating cancer comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy. Cell protection as demonstrated in the Examples demonstrates that administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity can enhance the survival and activity of transferred cells, providing an improved cancer treatment.

[0085] In certain embodiments, a method of treating cancer comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity, followed by adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in conditioning a patient for cancer treatment by adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in a patient scheduled to receive adoptive cell transfer immunotherapy cancer treatment. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in a patient who has previously donated blood for the development of adoptive cell transfer immunotherapy but has not yet received adoptive cell transfer immunotherapy. The present invention also provides adoptive cell transfer immunotherapy compositions for treating cancer in patients who have previously received a protein with IgG cysteine ​​protease or IgG endoglycosidase activity.

[0086] In a preferred embodiment, a method for treating cancer comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after administering adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving a patient's benefit from previously administered adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating cancer in patients who have previously received adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in patients who have previously received adoptive cell transfer immunotherapy cancer treatment. The present invention also provides an adoptive cell transfer immunotherapy composition for treating cancer in a patient who will receive a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0087] In a further preferred embodiment, the method of treating cancer comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity both before and after multiple doses of adoptive cell transfer immunotherapy.

[0088] In a further preferred embodiment, the method of treating cancer comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after administration of a first adoptive cell transfer immunotherapy and before administration of a second adoptive cell transfer immunotherapy. Preferably, in such an embodiment, the first and second, and any subsequent, adoptive cell transfer immunotherapies use the same or similar constructs and cells. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating cancer in a patient who has already undergone a first adoptive cell transfer immunotherapy and is scheduled to undergo a second adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing IgG levels in a patient undergoing a regimen of multiple adoptive cell transfer immunotherapy. The present invention also provides proteins having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving the benefit to patients of multiple-dose cancer treatment regimens by adoptive cell transfer immunotherapy. The present invention also provides adoptive cell transfer immunotherapy compositions for treating cancer in patients who have previously received an adoptive cell transfer immunotherapy composition and who have previously received a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0089] In a further preferred embodiment, the method of treating cancer comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after two or more administrations of adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving the benefit of patients who have already received two or more administrations of adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating cancer in patients who have already received two or more administrations of adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in patients who have already received two or more administrations of cancer treatment with adoptive cell transfer immunotherapy.

[0090] In certain embodiments of the invention, a method of treating cancer comprises multiple administrations of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity and multiple administrations of adoptive cell transfer immunotherapy.

[0091] In certain embodiments of the invention, the method of treating cancer comprises the simultaneous administration of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity and adoptive cell transfer immunotherapy.

[0092] In the therapeutic methods of the present invention, a subject already suffering from cancer is administered an amount of protein sufficient to cure, alleviate, or partially halt the cancer or one or more of its symptoms, thereby undergoing adoptive cell transfer immunotherapy. Such therapeutic treatment may result in remission, stabilization, or reduction or elimination of metastasis of the cancer. An amount adequate to achieve this is defined as a "therapeutically effective amount." The subject may be identified by any suitable means as having cancer and being suitable for adoptive cell transfer immunotherapy.

[0093] Methods for Treating Autoimmune Conditions The present invention provides a method for treating autoimmune conditions comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy. The cell protection shown in the Examples demonstrates that administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity can enhance the survival and activity of transferred cells, providing an improved treatment for autoimmune conditions.

[0094] In a preferred embodiment, a method for treating an autoimmune condition comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity, followed by adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in conditioning a patient for treatment of an autoimmune condition with adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in a patient scheduled to receive treatment for an autoimmune condition with adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in a patient who has previously donated blood for the development of adoptive cell transfer immunotherapy but has not yet received adoptive cell transfer immunotherapy. The present invention also provides adoptive cell transfer immunotherapy compositions for treating autoimmune conditions in patients who have previously received a protein with IgG cysteine ​​protease or IgG endoglycosidase activity.

[0095] In a further preferred embodiment, the method for treating an autoimmune condition comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after administering adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving a patient's benefit from previously administered adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating an autoimmune condition in a patient who has previously undergone adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in a patient who has previously undergone adoptive cell transfer immunotherapy for an autoimmune condition. The present invention also provides an adoptive cell transfer immunotherapy composition for treating an autoimmune condition in a patient who will receive a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0096] In a further preferred embodiment, the method for treating an autoimmune condition comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity both before and after multiple doses of adoptive cell transfer immunotherapy.

[0097] In a further preferred embodiment, the method of treating an autoimmune condition comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after administration of a first adoptive cell transfer immunotherapy and before administration of a second adoptive cell transfer immunotherapy. Preferably, in such an embodiment, the first and second, and any subsequent, adoptive cell transfer immunotherapies use the same or similar constructs and cells. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating an autoimmune condition in a patient who has already undergone a first adoptive cell transfer immunotherapy and is scheduled to undergo a second adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing IgG levels in a patient undergoing a regimen of multiple adoptive cell transfer immunotherapy. The present invention also provides proteins having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving the benefit to patients of multiple-dose regimens for treating autoimmune conditions with adoptive cell transfer immunotherapy. The present invention also provides adoptive cell transfer immunotherapy compositions for treating autoimmune conditions in patients who have previously received an adoptive cell transfer immunotherapy composition and who have previously received a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0098] In a further preferred embodiment, the method of treating an autoimmune condition comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after two or more administrations of adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving the benefit of patients who have already undergone two or more administrations of adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating an autoimmune condition in patients who have already undergone two or more administrations of adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in patients who have already undergone two or more administrations of adoptive cell transfer immunotherapy for an autoimmune condition.

[0099] In certain embodiments of the invention, a method of treating an autoimmune condition comprises multiple administrations of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity and multiple administrations of adoptive cell transfer immunotherapy.

[0100] In the therapeutic methods of the present invention, a subject already suffering from an autoimmune condition is administered an amount of protein sufficient to cure, alleviate, or partially halt the autoimmune condition or one or more of its symptoms, thereby administering adoptive cell transfer immunotherapy. Such therapeutic treatment may result in remission, stabilization, reduced recurrence, or elimination of the autoimmune condition. An amount adequate to achieve this is defined as a "therapeutically effective amount." The subject may be identified by any suitable means as suffering from an autoimmune condition and suitable for adoptive cell transfer immunotherapy.

[0101] How to treat an infection The present invention provides a method for treating infectious diseases comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy. The protection of cells shown in the Examples demonstrates that administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity can enhance the survival and activity of transferred cells, providing an improved treatment for infectious diseases.

[0102] In certain embodiments, a method for treating an infectious disease comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity, followed by adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in conditioning a patient for treatment of an infectious disease with adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in a patient scheduled to receive treatment of an infectious disease with adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in a patient who has previously donated blood for the development of adoptive cell transfer immunotherapy but has not yet received adoptive cell transfer immunotherapy. The present invention also provides adoptive cell transfer immunotherapy compositions for treating infectious diseases in patients who have previously received a protein with IgG cysteine ​​protease or IgG endoglycosidase activity.

[0103] In a preferred embodiment, a method for treating an infectious disease comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after administering adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving a patient's benefit from previously administered adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating an infectious disease in a patient who has previously undergone adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in a patient who has previously undergone adoptive cell transfer immunotherapy treatment for an infectious disease. The present invention also provides an adoptive cell transfer immunotherapy composition for treating an infectious disease in a patient who will receive a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0104] In a further preferred embodiment, the method for treating an infectious disease comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity both before and after multiple doses of adoptive cell transfer immunotherapy.

[0105] In a further preferred embodiment, the method of treating an infectious disease comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after administration of a first adoptive cell transfer immunotherapy and before administration of a second adoptive cell transfer immunotherapy. Preferably, in such an embodiment, the first and second, and any subsequent, adoptive cell transfer immunotherapies use the same or similar constructs and cells. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating an infectious disease in a patient who has already undergone a first adoptive cell transfer immunotherapy and is scheduled to undergo a second adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing IgG levels in a patient undergoing a regimen of multiple adoptive cell transfer immunotherapy. The present invention also provides proteins having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving the benefit to patients of multiple-dose regimens for treating infectious diseases by adoptive cell transfer immunotherapy. The present invention also provides adoptive cell transfer immunotherapy compositions for treating infectious diseases in patients who have previously received an adoptive cell transfer immunotherapy composition and who have previously received a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0106] In a further preferred embodiment, the method for treating an infectious disease comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after two or more administrations of adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving the benefit of patients who have already received two or more administrations of adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating an infectious disease in patients who have already received two or more administrations of adoptive cell transfer immunotherapy. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing serum IgG levels in patients who have already received two or more administrations of adoptive cell transfer immunotherapy for an infectious disease.

[0107] In certain embodiments of the invention, a method for treating an infectious disease comprises multiple administrations of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity and multiple administrations of adoptive cell transfer immunotherapy.

[0108] In certain embodiments of the invention, the method of treating an infectious disease comprises the simultaneous administration of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity and adoptive cell transfer immunotherapy.

[0109] In the therapeutic methods of the present invention, a subject diagnosed with an infectious disease is administered an amount of protein sufficient to reduce or eliminate the virus from the patient and undergo adoptive cell transfer immunotherapy. Such therapeutic treatment may result in a reduction in viral load or a cure of the patient. An amount adequate to achieve this is defined as a "therapeutically effective amount." The subject may be identified by any suitable means as suffering from an infectious disease and suitable for adoptive cell transfer immunotherapy.

[0110] Methods for treating pathologies mediated by harmful antibody-producing B cells The present invention also provides a method for treating a condition mediated by B cells producing harmful antibodies, comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy. Cell protection, as demonstrated in the Examples, demonstrates that administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity can enhance the survival and activity of transferred cells, providing improved treatment for a condition mediated by B cells producing harmful antibodies. In certain embodiments, the harmful antibodies are pathogenic antibodies.

[0111] Examples of pathologies mediated by harmful antibody-producing B cells include autoimmune pathologies, in which B cells that produce antibodies against specific antigens mediate the disease process. Adoptive cell transfer immunotherapy can be used to eliminate problematic B cells. Similarly, B cells that produce anti-drug antibodies against primary therapy can be problematic and can be eliminated by adoptive cell transfer immunotherapy. In these cases, adoptive cell transfer immunotherapy can cause the problematic B cells to express the antigen they target.

[0112] In certain embodiments, a method of treating a condition comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity, followed by adoptive cell transfer immunotherapy.

[0113] In a preferred embodiment, the method of treating a condition comprises administering adoptive cell transfer immunotherapy followed by administration of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0114] In a further preferred embodiment, the method of treating a condition comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity both before and after multiple doses of adoptive cell transfer immunotherapy.

[0115] In a further preferred embodiment, the method of treating a condition comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after administration of a first adoptive cell transfer immunotherapy and before administration of a second adoptive cell transfer immunotherapy. Preferably, in such embodiments, the first and second, and any subsequent, adoptive cell transfer immunotherapies use the same or similar constructs and cells.

[0116] In a further preferred embodiment, the method of treating the condition comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after two or more administrations of adoptive cell transfer immunotherapy.

[0117] In certain embodiments of the invention, a method of treating a condition comprises multiple administrations of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity and multiple administrations of adoptive cell transfer immunotherapy.

[0118] In certain embodiments of the invention, the method of treating a condition comprises the simultaneous administration of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity and adoptive cell transfer immunotherapy.

[0119] In the therapeutic methods of the present invention, a subject already suffering from a condition mediated by harmful antibody-producing B cells is administered an amount of protein sufficient to cure, alleviate, or partially halt the autoimmune condition or one or more of its symptoms, thereby undergoing adoptive cell transfer immunotherapy. Such therapeutic treatment may result in remission, stabilization, reduced recurrence, or elimination of the condition. An amount adequate to accomplish this is defined as a "therapeutically effective amount." The subject may be identified by any suitable means as suffering from the condition and suitable for adoptive cell transfer immunotherapy.

[0120] Adoptive cell transfer immunotherapy The methods of the present invention enhance the benefits obtained from adoptive cell transfer (ACT) immunotherapy, thereby providing improved methods for treating cancer, autoimmune conditions, infectious diseases, and conditions mediated by harmful antibody-producing B cells. ACT immunotherapy is an established and powerful approach, particularly for treating cancer. ACT involves the passive transfer of ex vivo grown cells, most commonly immune-derived cells, into a host with the goal of transferring the immunological functions and characteristics of the graft.

[0121] The ACT may be autologous (e.g., isolated by leukapheresis, transduced, and selected immediately about four weeks prior to administration), as is common in adoptive T cell therapy, or it may be allogeneic, in which case the methods of the invention can improve the ACT by removing antibodies that recognize receptors and / or other antigens expressed on the allogeneic cells. Additionally, the ACT may be xenogeneic. In a preferred embodiment, the ACT is autologous.

[0122] ACT may also involve the transfer of autologous tumor-infiltrating lymphocytes (TILs), which can be used to treat patients with advanced solid tumors and hematological malignancies, such as melanoma.

[0123] ACT can also involve the transfer of "off-the-shelf" allogeneic lymphocytes isolated, prepared, and stored (e.g., frozen) from healthy donors, which can be used to treat patients with advanced solid tumors and hematologic malignancies, such as melanoma.

[0124] The adoptive cellular immunotherapy of the present invention may involve the administration of cells expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and may also include tumor-infiltrating lymphocytes (TILs). The population of cells expressing an antigen-recognizing CAR / TCR may include a population of activated T cells, natural killer (NK) cells, or dendritic cells. Dendritic cells can not only directly kill tumors but also present antigens. Dendritic cells can express, for example, 4-1BB or anti-CD19 CAR. The population of cells expressing a CAR / TCR may include a population of gene-edited cells.

[0125] ACT may use cell types such as T cells, natural killer (NK) cells, delta-gamma T cells, regulatory T cells, dendritic cells, and peripheral blood mononuclear cells. ACT may also use monocytes to induce differentiation into dendritic cells and / or macrophages after contact with tumor antigens.

[0126] According to a preferred embodiment of the present invention, the adoptive cell therapy can be CAR T cell therapy. CAR T cells can be engineered to target tumor antigens of interest by engineering a desired antigen-binding domain that specifically binds to the antigen on tumor cells. In a preferred embodiment, the cell therapy uses cells of hematopoietic origin. The examples demonstrate that the method of the present invention is particularly effective for cells of hematopoietic origin.

[0127] In a preferred embodiment, the CAR T cell therapy is selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CS-1, B cell maturation antigen (BCMA), MAGEA3, MAGEA3 / A6, KRAS, CLL1, MUC-1, HER2, EpCam, GD2, GPA7, PSCA, EGFR, EGFRvIII, ROR1, mesothelin, CD33 / IL3Ra, c-Met, CD37, PSMA, glycolipid F77, GD-2, gp100, NY-ESO-1 CAR T cells are used that target TCR, FR alpha, CD24, CD44, CD133, CD166, CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, or ULBP6, or a combination thereof (e.g., both CD33 and CD123). Preferred antigens are BCMA, CD19, CD20, and CD22.

[0128] Preferred adoptive cell transfer immunotherapies are CAR T-cell therapies (e.g., autologous and allogeneic cell therapies). CAR T-cell therapies for treating hematologic malignancies such as ALL, AML, NHL, DLBCL, and CLL are preferred. Examples of approved CAR T-cell therapies include, but are not limited to, KYMRIAH® (tisagenlecleucel) for treating NHL and DLBCL, and YESCARTA® (axicabtagene ciloleucel) for treating NHL.

[0129] According to certain aspects of the invention, the population of cells expressing a CAR / TCR or TIL may be autologous cells, allogeneic cells from another human donor, or xenogeneic cells from an animal of a different species.

[0130] According to certain aspects of the invention, populations of cells expressing CAR / TCR or TIL may be isolated by leukapheresis, transduced, and selected immediately or approximately four weeks prior to administration, as in the case of autologous stem cells, or may be isolated from healthy donors, prepared in advance, and then stored, e.g., as frozen preparations, for use with one or more patients, as in the case of so-called "off-the-shelf" allogeneic CAR-T stem cell therapies.

[0131] According to a particular embodiment of the present invention, the population of cells expressing a CAR / TCR may comprise a population of activated T cells, natural killer (NK) cells, or dendritic cells that express a CAR / TCR that recognizes an antigen. Dendritic cells not only directly kill tumors but also present antigens.

[0132] According to certain aspects of the invention, the antigen may be one that is expressed only on cancer cells or may be one that is preferentially expressed on cancer cells, such as a neo-antigen or a lineage-specific antigen (e.g., CD19 or CD20).

[0133] CAR T cells may comprise antigen-binding domains capable of targeting two or more different antigens (i.e., bispecific or bivalent, trispecific or trivalent, tetraspecific, etc.). Thus, CAR T cells may comprise a first antigen-binding domain that binds to a first antigen and a second antigen-binding domain that binds to a second antigen (e.g., a tandem CAR). For example, CAR T cells may comprise a CD19-binding domain and a CD22-binding domain, and thus can recognize and bind to both CD19 and CD22. Or, further, CAR T cells may comprise a CD19-binding domain and a CD20-binding domain, and thus can recognize and bind to both CD19 and CD20.

[0134] Alternatively, each cell in the cell population, or the entire cell population, may contain more than one different CAR T cell (e.g., construct), where each CAR T cell construct can recognize a different antigen. For example, a population of CAR T cells may target three antigens, such as HER2, IL13Rα2, and EphA2.

[0135] According to certain aspects of the present invention, populations of cells, whether autologous or allogeneic, can be engineered using gene editing techniques such as CRISPR / cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9), zinc finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs). These techniques are recognized and practiced in the art of genetic engineering and allow for the selective editing, disruption, or insertion of target sequences to modify the genome of cells of interest. Thus, isolated autologous or allogeneic cells for adoptive transfer as embodied in the present invention can be edited to delete or replace known genes or sequences. For example, T cell receptors (TCRs) can be deleted or replaced in allogeneic T cell populations before or after CAR-T transduction as a means of eliminating graft-versus-host disease in recipient patients.

[0136] According to certain aspects of the invention, a population of cells administered as adoptive cell transfer immunotherapy can comprise a population of T cells, NK cells, or dendritic cells expressing a CAR, wherein the CAR comprises an extracellular antibody or antibody fragment comprising a humanized anti-CD19 binding domain, a humanized anti-CD22 binding domain, a humanized anti-CD20 binding domain, or a humanized anti-BCMA binding domain, a transmembrane domain, and one or more cytoplasmic costimulatory signaling domains. The population of cells can comprise a population of cells expressing a CAR, wherein the CAR comprises two or more binding domains, such as a humanized anti-CD19 binding domain, a humanized anti-CD22 binding domain, a humanized anti-CD20 binding domain, and / or a humanized anti-BCMA binding domain, and an extracellular antibody or antibody fragment comprising a transmembrane domain and one or more cytoplasmic costimulatory signaling domains.

[0137] In certain embodiments of the present invention, the cell population administered as adoptive cell transfer immunotherapy expresses T cell receptors (TCRs). TCRs are antigen-specific molecules involved in recognizing antigenic peptides presented in association with products of the major histocompatibility complex (MHC) on the surface of antigen-presenting cells or any nucleated cell (e.g., all human cells in the body except red blood cells). In contrast, antibodies typically recognize soluble or cell-surface antigens and do not require MHC presentation. This system confers on T cells the potential to recognize, via the TCR, the entire array of intracellular antigens expressed by cells (including viral proteins), which are processed intracellularly into short peptides, bound to intracellular MHC molecules, and delivered to the surface as peptide-MHC complexes. This system allows the use of virtually any foreign protein (e.g., mutated cancer antigens or viral proteins) or aberrantly expressed proteins as targets for T cells.

[0138] In certain embodiments, particularly in methods for treating autoimmune conditions, adoptive cell transfer immunotherapy involves the administration of regulatory T cells. In certain such embodiments, the cells express a construct that targets an antigen in inflamed tissue. For example, in methods for treating multiple sclerosis, the cells target an antigen present in the brain, such as myelin oligodendrocyte glycoprotein (MOG), and in methods for treating Crohn's disease, the target antigen is commonly expressed in the intestine. Thus, the regulatory T cells target inflamed tissue and reduce inflammation.

[0139] In further embodiments, the methods use T cells to target B cell populations that produce harmful antibodies. In certain embodiments, the cells used are chimeric autoantibody receptor T (CAAR-T) cells. In such embodiments, the cells express a construct that presents an antigen recognized by problematic B cells, such as a drug antigen or an antigen of Table A, and the B cells are eliminated upon binding to the therapeutic cells. Such cells are also useful for treating autoimmune and other conditions caused by B cells that produce harmful antibodies.

[0140] According to certain aspects of the invention, the engineered CAR cells may be allogeneic from a healthy donor and may be further engineered to ablate or replace the endogenous TCR by gene editing techniques such as CRISPR / cas9, ZFN, or TALEN, where deletion of the endogenous TCR functions to eliminate CAR-driven graft-versus-host disease.

[0141] According to certain aspects of the invention, autologous cells (e.g., T cells, NK cells, or dendritic cells) may be collected from a subject. These cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. According to certain aspects of the invention, allogeneic or xenogeneic cells, typically isolated from healthy donors, may be used. When the T cells, NK cells, dendritic cells, or pluripotent stem cells are allogeneic or xenogeneic cells, any number of cell lines available in the art can be used.

[0142] 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. According to certain aspects of the invention, cells from an individual's circulating blood can be obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, B cells, monocytes, granulocytes, other nucleated white blood cells, red blood cells, and platelets.

[0143] Enrichment of a cell population by negative selection can be achieved using a combination of antibodies against surface markers specific to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD1 1b, CD16, HLA-DR, and CD8. According to certain embodiments of the present invention, it may be desirable to enrich or positively select a cell population. For example, positive enrichment for regulatory T cells can use positive selection for CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+.

[0144] The recovered cells may be engineered to express a CAR or TCR by any of a number of methods known in the art. Furthermore, the engineered cells may be expanded by any of a number of methods known in the art. As detailed above, the CAR or TCR may be bispecific, trispecific, or tetraspecific, the CAR or TCR may include a switch such as a goCAR or goTCR, or a safety switch CAR or TCR, and the CAR or TCR may express an immunomodulatory protein such as an armored CAR or TCR.

[0145] According to certain aspects of the invention, a blood sample or apheresis product may be collected from a subject at any time prior to when the expanded cells described herein may be needed. In this manner, a source of cells to be genetically engineered and expanded (or simply expanded in the case of TILs) may be collected at any time needed, and the desired cells, such as T cells, NK cells, dendritic cells, or TILs, may be isolated and frozen for later use in ACTs, such as those described herein.

[0146] According to certain aspects of the invention, the population of cells expressing a CAR / TCR may be administered to a subject in a split dose, where a first proportion of the total dose is administered on the first day of treatment, a second proportion of the total dose is administered on a subsequent day of treatment, and optionally a third proportion of the total dose is administered on an even later day of treatment.

[0147] An exemplary total dose is 10 3 ~10 11 cells / subject weight, e.g., 10 3 ~10 10 cells / subject weight, or 10 3 ~10 9 cells / subject weight, or 10 3 ~10 8 cells / subject weight, or 10 3 ~10 7 cells / subject weight, or 10 3 ~10 6 cells / subject weight, or 10 3 ~10 5 Further, an exemplary total dose is 10 4 ~10 11 cells / subject weight, e.g., 10 5 ~10 11 cells / subject weight, or 10 6 ~10 11 cells / subject weight, or 10 7 ~10 11 Includes cells / subject weight.

[0148] An exemplary total dose may be administered based on the patient's body surface area rather than body weight. Thus, the total dose may be administered based on the patient's body surface area rather than body weight. 3 ~10 13 cells / m 2 may include:

[0149] Exemplary doses may be based on a flat or constant dosing schedule, rather than on body weight or body surface area. Flat, constant dosing can avoid potential dose miscalculation. Furthermore, genotyping and phenotyping strategies, as well as therapeutic drug monitoring, can be used to calculate the appropriate dose. That is, dosing may be based on the patient's immune repertoire of immune suppressor cells (e.g., regulatory T cells, myeloid-derived suppressor cells) and / or disease burden. In this way, the total dose may be calculated based on a 10 3 ~10 13 It may contain whole cells.

[0150] According to certain aspects of the present invention, cells can be obtained from a subject immediately after treatment. In this regard, it has been observed that following certain cancer treatments, particularly treatment with drugs that damage the immune system, the quality of certain cells (e.g., T cells) obtained immediately after treatment, during the period when a subject would normally be recovering from treatment, can be optimal or improved for ex vivo expansion capacity. Similarly, after ex vivo manipulation using the methods described herein, these cells can be in a favorable state for enhanced engraftment and in vivo expansion. Thus, harvesting blood cells, including T cells, NK cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery period is contemplated within the context of the present invention.

[0151] According to certain aspects of the invention, the second administration may be a different population of cells expressing the same or a different CAR / TCR in the same or a different effective amount. The difference in the CAR / TCR may be in any aspect of the CAR / TCR, for example, a different binding or antigen recognition domain or costimulatory domain. The second administration may additionally or alternatively include secretory cells with IL-12 and may further include adjuvant immunotherapy with a small molecule inhibitor, such as an inhibitor of BTK, P13K, or IDO, either simultaneously or sequentially with the cell therapy infusion.

[0152] According to certain aspects of the invention, the methods may include administering one or more additional therapeutic agents in addition to adoptive cell transfer immunotherapy and the IgG cysteine ​​protease or endoglycosidase. Exemplary therapeutic agents include chemotherapeutic agents, anti-inflammatory agents, immunosuppressive agents, immunomodulatory agents, or combinations thereof.

[0153] Therapeutic agents may be administered according to any standard administration regimen known in the art. Exemplary chemotherapeutic agents include mitotic inhibitors such as taxanes, for example, docetaxel and paclitaxel, and vinca alkaloids, for example, vindesine, vincristine, vinblastine, and vinorelbine. Exemplary chemotherapeutic agents include topoisomerase inhibitors, such as topotecan.

[0154] Exemplary chemotherapeutic agents include growth factor inhibitors, tyrosine kinase inhibitors, histone deacetylase inhibitors, P38a MAP kinase inhibitors; inhibitors of angiogenesis, neovascularization, and / or other angiogenesis; colony-stimulating factors, erythropoietic agents, anti-allergic agents, immunosuppressive and / or immunomodulatory agents, viruses, viral proteins, immune checkpoint inhibitors, BCR inhibitors (e.g., BTK, P13K, etc.), immunometabolic agents (e.g., IDO, arginase, glutaminase inhibitors, etc.), etc. According to certain aspects of the invention, one or more therapeutic agents may comprise an anti-myeloma agent. Exemplary anti-myeloma agents include dexamethasone, melphalan, doxorubicin, bortezomib, lenalidomide, prednisone, carmustine, etoposide, cisplatin, vincristine, cyclophosphamide, and thalidomide, some of which are identified above as chemotherapeutic, anti-inflammatory, or immunosuppressive agents.

[0155] Cancer being treated The methods of the present invention can improve the treatment of any cancer that can be treated using adoptive cell transfer immunotherapy. In certain embodiments, the cancer is a hematological cancer, which is a cancer that originates in hematopoietic tissues, such as bone marrow or other cells of the immune system. Treatment of hematological cancers with adoptive cell transfer immunotherapy is well established and can benefit from the methods of the present invention. Hematological malignancies include leukemias (e.g., acute myeloid leukemia (AML), acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), acute mixed lineage leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, and large granular lymphocytic leukemia), myelodysplastic syndromes (MDS), myeloproliferative disorders (polycythemia vera, essential thrombocytosis, primary myelofibrosis, and CML), and other cancers that can be treated with adoptive cell transfer immunotherapy. ), lymphoma, multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS) and similar disorders, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, splenic marginal zone lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies.

[0156] In certain embodiments, the cancer being treated is B-cell lymphoma. In certain embodiments, the methods of the invention comprise multiple administrations of an IgG cysteine ​​protease or an IgG endoglycosidase, and the cancer being treated is B-cell lymphoma. In certain such embodiments, the method of treating cancer comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after two or more administrations of adoptive cell transfer immunotherapy, preferably including administration of CD19-expressing CAR-T cells. In certain such embodiments, the method also comprises immunosuppression. The methods of the invention may be particularly effective in treating tumors requiring immunosuppression.

[0157] In a further embodiment, the methods of the present invention are for use in the treatment of solid tumors. Treatment with adoptive cellular immunotherapy, which involves administering fewer cells, may increase the risk of the damaging effects of ADA. Additionally, treatment of solid tumors with adoptive cellular immunotherapy is less effective and may therefore benefit greatly from the methods of the present invention. "Solid cancer" includes, but is not limited to, bone cancer, breast cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, prostate cancer, rectal cancer, anal cancer, colon cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, glioblastoma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, and environmentally induced cancers including those induced by asbestos. Solid cancers currently being the subject of clinical trials using adoptive cellular immunotherapy include neuroblastoma, glioblastoma, CNS tumors (particularly recurrent or refractory HER2-positive), sarcoma (particularly HER2-positive), osteosarcoma (particularly metastatic HER2-positive), and liver tumors (particularly GPC3-positive pediatric tumors), and the methods of the present invention may be useful in treating these cancers.

[0158] In a preferred embodiment, the method of the invention is for treating cancer in a patient who is not B-cell impaired. In a preferred embodiment, the method of the invention is for treating cancer in a patient who is not immunosuppressed. Such patients may have more severe ADA.

[0159] Preferably, the method of the present invention is used to treat human patients. When the subject is human, the subject can be of any age. For example, the subject can be 60 years or older, 65 years or older, 70 years or older, 75 years or older, 80 years or older, 85 years or older, or 90 years or older. Alternatively, the subject can be 60 years or younger, 55 years or younger, 50 years or younger, 45 years or younger, 40 years or younger, 35 years or younger, 30 years or younger, 25 years or younger, or 20 years or younger. In the case of a human subject suffering from cancer, the subject can be newly diagnosed, relapsed and / or refractory, or in remission.

[0160] Autoimmune conditions treated The methods of the present invention can improve the treatment of any autoimmune condition that can be treated with adoptive cell transfer immunotherapy. In preferred embodiments, the autoimmune condition is selected from the list consisting of rheumatoid arthritis, systemic lupus erythematosus (lupus), inflammatory bowel disease (IBD), such as Crohn's disease or ulcerative colitis, multiple sclerosis (MS), type 1 diabetes, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, and vasculitis.

[0161] Table A below describes antigens associated with additional diseases that can be treated according to the present invention. The methods of the present invention can be used to treat any of these diseases or conditions.

[0162] [Table A-1]

[0163] [Table A-2]

[0164] [Table A-3]

[0165] Preferably, the method of the present invention is used to treat human patients. When the subject is human, the subject can be of any age. For example, the subject can be 60 years of age or older, 65 years of age or older, 70 years of age or older, 75 years of age or older, 80 years of age or older, 85 years of age or older, or 90 years of age or older. Alternatively, the subject can be 60 years of age or younger, 50 years of age or younger, 45 years of age or younger, 40 years of age or younger, 35 years of age or younger, 30 years of age or younger, 25 years of age or younger, or 20 years of age or younger. In the case of a human subject suffering from an autoimmune condition, the subject can be newly diagnosed, relapsed and / or refractory, or in remission.

[0166] Infections treated The methods of the present invention can improve the treatment of any infectious disease that can be treated using adoptive cell transfer immunotherapy. In a preferred embodiment, the infectious disease is selected from the list consisting of human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), Epstein-Barr virus (EBV), human papillomavirus (HPV), Kaposi's sarcoma herpesvirus (KSHV), Lassa virus, cytomegalovirus (CMV), coronaviruses such as COVID-19, Aspergillus fumigatus, and tuberculosis. Preferably, the infectious disease is a chronic infection. Preferably, the infectious disease is HIV.

[0167] In such embodiments, the transfected cells typically express a construct that recognizes an antigen on the target pathogen. Exemplary antigens include HIV gag protein, p55, p24, p18, envelope glycoprotein (env), gp160, gp120, gp41, reverse transcriptase (pol), p66, and p31; HAV core and surface antigens; HBV core and surface (S, M, and L) antigens; HCV core and surface (E2, NS2, NS3, NS4, and NS5) antigens; EBV EBVA, LMP2, EBNA1, and BZLF1; HPV E6 and E7; and CMV pp65, immediate early (IE) antigen, and IE1.

[0168] IgG cysteine ​​protease The present inventors have demonstrated that the use of IgG cysteine ​​proteases can protect cells and improve their survival, and may be useful in the treatment of cancer in combination with adoptive cell transfer immunotherapy. The IgG cysteine ​​proteases for use in the present invention are specific for IgG, the predominant class of antibody in mammalian serum.

[0169] In a preferred embodiment, the protease for use in the methods of the present invention is ImmuRifidase (IdeS) ( I mmunoglobulin G- d egrading e nzyme of SIdeS is an extracellular cysteine ​​protease produced by the human pathogen Streptococcus pyogenes (IgG-degrading enzyme of Streptococcus pyogenes). IdeS was originally isolated from serotype M1 group A streptococcus strains, but the ides gene has now been identified in all group A streptococcus strains tested. IdeS has an exceptionally high degree of substrate specificity, with IgG being its only identified substrate. IdeS catalyzes a single proteolytic cleavage in the lower hinge region of the heavy chains of all subclasses of human IgG. IdeS also catalyzes corresponding cleavage of the heavy chains of several subclasses of IgG in various animal species. IdeS efficiently cleaves IgG into Fc and F(ab')2 fragments via a two-step mechanism. In the first step, one (first) heavy chain of IgG is cleaved to generate a single-cleaved IgG (scIgG) molecule with a noncovalently bound Fc molecule. The scIgG molecule is effectively an intermediate product that retains the remaining (second) heavy chain of the original IgG molecule. In the second step of the mechanism, this second heavy chain is cleaved by IdeS to release the F(ab')2 fragment and the homodimeric Fc fragment, which are the products commonly observed under physiological conditions. Under reducing conditions, the F(ab')2 fragment can dissociate into two Fab fragments, and the homodimeric Fc fragment can dissociate into its constituent monomers. SEQ ID NO: 1 is the full sequence of IdeS, including the N-terminal methionine and signal sequence. It is also available as NCBI reference sequence number WP_010922160.1. SEQ ID NO: 2 is the mature sequence of IdeS, lacking the N-terminal methionine and signal sequence. It is also available as Genbank accession number ADF13949.1.

[0170] In another embodiment, the protease for use in the methods of the present invention is derived from Streptococcus equi ( Streptococcus equiIdeZ is an IgG cysteine ​​protease produced by the subspecies Zooepidemicus of the bacterium Bacillus subtilis. SEQ ID NO: 3 is the full sequence of IdeZ, including the N-terminal methionine and signal sequence. It is also available as NCBI reference sequence number WP_014622780.1. SEQ ID NO: 4 is the mature sequence of IdeZ, lacking the N-terminal methionine and signal sequence.

[0171] In another embodiment, the protease for use in the methods of the invention is a hybrid IdeS / Z, such as SEQ ID NO: 5. The N-terminus is based on IdeZ, lacking the N-terminal methionine and signal sequence.

[0172] In a preferred embodiment, a protease for use in the present invention may comprise or consist of SEQ ID NO: 2, 4, or 5. Proteases for use in the present invention may comprise an additional N-terminal methionine (M) residue and / or a C-terminal tag to aid in expression in and isolation from standard bacterial expression systems. Suitable tags include histidine tags, which may be directly linked to the C-terminus of a polypeptide or indirectly linked by any suitable linker sequence, e.g., three, four, or five glycine residues. Histidine tags typically consist of six histidine residues, but may be longer, typically up to seven, eight, nine, ten, or 20 amino acids, or may be shorter, e.g., five, four, three, two, or one amino acid.

[0173] In a further preferred embodiment, the protease for use in the present invention may comprise, consist essentially of, or consist of any one of the sequences of SEQ ID NOs: 6 to 25. These sequences represent IdeS and IdeZ polypeptides with increased protease activity and / or reduced immunogenicity. SEQ ID NOs: 6 to 25 may each optionally comprise an additional methionine at the N-terminus and / or a histidine tag at the C-terminus. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably linked to the C-terminus by a linker of 3 or 5 glycine residues.

[0174] In a further preferred embodiment, the protease for use in the present invention may comprise, consist essentially of, or consist of any one of the sequences set forth in SEQ ID NOs: 56-69. These sequences represent IdeS polypeptides with enhanced protease activity and / or reduced immunogenicity. Each of SEQ ID NOs: 56-69 may optionally comprise an additional methionine at the N-terminus and / or a histidine tag at the C-terminus. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably linked to the C-terminus by a linker of 3 or 5 glycine residues.

[0175] In a further preferred embodiment, the proteases for use in the present invention may comprise, consist essentially of, or consist of the sequence of any one of SEQ ID NOs: 6-25, optionally with up to three (e.g., one, two, or three) amino acid substitutions. SEQ ID NOs: 6-25 and variants thereof may each optionally include an additional methionine at the N-terminus and / or a histidine tag at the C-terminus.

[0176] In a further preferred embodiment, a protease for use in the present invention may comprise, consist essentially of, or consist of the sequence of any one of SEQ ID NOs: 56-69, optionally with up to three (e.g., one, two, or three) amino acid substitutions. SEQ ID NOs: 56-69 and variants thereof each may optionally include an additional methionine at the N-terminus and / or a histidine tag at the C-terminus.

[0177] Polypeptides of the present invention are typically at least 100 amino acids long, at least 150 amino acids long, at least 200 amino acids long, at least 250 amino acids long, at least 260 amino acids long, at least 270 amino acids long, at least 280 amino acids long, at least 290 amino acids long, at least 300 amino acids long, or at least 310 amino acids long. Polypeptides of the present invention are typically no longer than 400 amino acids long, no longer than 350 amino acids long, no longer than 340 amino acids long, no longer than 330 amino acids long, no longer than 320 amino acids long, or no longer than 315 amino acids long. It will be understood that any of the above lower limits can be combined with any of the above upper limits to provide a range for the length of a polypeptide of the present invention. For example, a polypeptide may be 100 to 400 amino acids long or 250 to 350 amino acids long. A polypeptide is preferably 290 to 320 amino acids long, and most preferably 300 to 315 amino acids long.

[0178] The primary structure (amino acid sequence) of the proteases of the present invention is based on the primary structure of IdeS, IdeZ, or IdeS / Z, specifically the amino acid sequence of SEQ ID NO: 2, 4, or 5, respectively. The sequences of the proteases of the present invention may include variants of the amino acid sequence of SEQ ID NO: 2, 4, or 5 that are at least 80% identical to the amino acid sequence of SEQ ID NO: 2, 4, or 5. The variant sequence may be at least 80%, at least 85%, preferably at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 2, 4, or 5. The variant may be identical to the sequence of SEQ ID NO: 2, 4, or 5 except that it contains one or more of the specified modifications identified in WO 2016 / 128558 or WO 2016 / 128559. Identity to the sequence of SEQ ID NO: 2, 4 or 5 may be measured over a region of at least 50, at least 100, at least 200, at least 300 or more contiguous amino acids of the sequence shown in SEQ ID NO: 2, 4 or 5, or more preferably over the full length of SEQ ID NO: 2, 4 or 5.

[0179] Proteases for use in the present invention may be IdeS, IdeZ, or IdeS / Z polypeptides comprising variants of the amino acid sequence of SEQ ID NO: 2, 4, or 5, in which modifications, such as amino acid additions, deletions, or substitutions, have been made relative to the sequence of SEQ ID NO: 2, 4, or 5. Such modifications are preferably conservative amino acid substitutions. In conservative substitutions, an amino acid is replaced with another amino acid of similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acid may have a similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acid being replaced. Alternatively, a conservative substitution may introduce another aromatic or aliphatic amino acid in place of an existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art.

[0180] IgG cysteine ​​protease activity can be assessed by any suitable method, for example, by incubating a polypeptide with a sample containing IgG and determining the presence of IgG cleavage products. Suitable methods are described in WO 2016 / 128559. Suitable assays include ELISA-based assays, such as those described in WO 2016 / 128559. In such assays, the wells of an assay plate are typically coated with an antibody target, such as bovine serum albumin (BSA). A sample of the polypeptide to be tested is then added to the well, followed by a sample of the target-specific antibody, in this example, an antibody specific for BSA. The polypeptide and antibody are allowed to interact under conditions suitable for IgG cysteine ​​protease activity. After a suitable interval, the assay plate is washed, and a detection antibody that specifically binds to the target-specific antibody is added under conditions suitable for binding to the target-specific antibody. The detection antibody binds to any intact target-specific antibody that is bound to the target in each well. After washing, the amount of detection antibody present in the well is proportional to the amount of target-specific antibody bound to that well. The detection antibody can be directly or indirectly conjugated to a label or another reporter system (such as an enzyme), so that the amount of detection antibody remaining in each well can be determined. The higher the potency of the test polypeptide present in the well, the less intact target-specific antibody remains, and therefore the less detection antibody is present. Typically, at least one well of a given assay plate contains IdeS instead of the test polypeptide, so that the potency of the test polypeptide can be directly compared to that of IdeS. IdeZ and IdeS / Z may also be included for comparison.

[0181] In other assays, the potency of a test polypeptide can be determined by directly visualizing and / or quantifying IgG fragments generated by cleavage of the IgG by the test polypeptide. This type of assay is also described in WO 2016 / 128559. In such assays, a sample of IgG is typically incubated with different concentrations of the test polypeptide (or one or more of IdeS, IdeZ, and IdeS / Z as controls) in a titration series. The products resulting from the incubation at each concentration are then separated using gel electrophoresis, for example, by SDS-PAGE. Whole IgG and fragments generated by cleavage of the IgG can then be identified by size and quantified by the intensity of staining with a suitable dye. The greater the amount of cleavage fragments, the greater the potency of the test polypeptide at a given concentration. Polypeptides of the present invention typically generate detectable amounts of cleavage fragments at lower concentrations (lower points in the titration series) than IdeZ and / or IdeS. This type of assay can also determine the amount of different fragments resulting from each cleavage event, and may therefore allow the identification of test polypeptides that are more effective at cleaving the first or second heavy chain of IgG molecules. Polypeptides of the invention may be more effective at cleaving the first chain of IgG molecules than the second chain, particularly when the IgG is of the IgG2 isotype. Polypeptides of the invention may be more effective at cleaving IgG1 than IgG2.

[0182] IgG endoglycosidase The present inventors have demonstrated that the use of IgG endoglycosidase can protect cells and improve their survival, and may be useful in the treatment of cancer in combination with adoptive cell transfer immunotherapy. The IgG endoglycosidase for use in the present invention is specific for IgG, the predominant class of antibody in mammalian serum.

[0183] The agent may be a protein with IgG endoglycosidase activity, preferably cleaving the glycan moiety at Asn-297 (Kabat numbering) in the Fc region of IgG. A preferred example of such a protein is EndoS ( Endo glycosidase of S EndoS is an endoglycosidase from Streptococcus pyogenes (Streptococcus pyogenes), which is shown to be effective in the Examples. EndoS hydrolyzes the β-1,4-di-N-acetylchitobiose core of the asparagine-linked glycan of normally glycosylated IgG (see Figure 18). The mature sequence of EndoS is provided as SEQ ID NO: 90. The protein may comprise or consist of the amino acid sequence of SEQ ID NO: 90, or may be a homolog thereof from another bacterium, e.g., Streptococcus pyogenes or Streptococcus zooepidemicus, or Mycobacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica. The inhibitor may be CP40, EndoE, or EndoF2.

[0184] Alternatively, the protein may be a variant of an EndoS protein comprising or consisting of any amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO: 90 and having IgG endoglycosidase activity. An EndoS protein variant may comprise or consist of an amino acid sequence having up to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more amino acid substitutions, insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 90, provided that the variant has IgG endoglycosidase activity. The amino acid substitutions are preferably conservative.

[0185] Alternatively, the agent may be a protein having IgG endoglycosidase activity comprising or consisting of a fragment of SEQ ID NO: 90, preferably the fragment having a length of 400-950 amino acids, 500-950 amino acids, 600-950 amino acids, 700-950 amino acids, or 800-950 amino acids. A preferred fragment consists of amino acids 1-409 of SEQ ID NO: 90, which corresponds to the enzymatically active α-domain of EndoS generated by cleavage with the streptococcal cysteine ​​proteinase SpeB. Fragments may be generated by deleting one or more amino acid residues of the amino acid sequence of SEQ ID NO: 90. Up to 1, 2, 3, 4, 5, 10, 20, 30, 40, 500, 100, 200, 300, 400, 500, or 550 residues, or more, may be deleted. The deleted residues may be adjacent to other residues.

[0186] Any fragment or variant of SEQ ID NO:2 preferably contains residues 191-199 of SEQ ID NO:90, i.e., Leu-191, Asp-192, Gly-193, Leu-194, Asp-195, Val-196, Asp-197, Val-198, and Glu-199 of SEQ ID NO:1. These amino acids constitute the complete chitinase family 18 active site, which ends with glutamic acid. Glutamic acid in the active site of chitinase is essential for enzymatic activity. Therefore, most preferably, a variant of SEQ ID NO:90 contains Glu-199 of SEQ ID NO:90. A variant of SEQ ID NO:2 may contain residues 191-199 of SEQ ID NO:90 with one or more conservative substitutions, provided that the variant contains Glu-199 of SEQ ID NO:90.

[0187] Assessment of IgG levels and timing of administration The appropriate timing of administration of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity and adoptive cell transfer immunotherapy in the methods of the invention can be determined, for example, using an assay to assess serum IgG levels. For example, the amount of time required for the protein to inactivate or eliminate Fc receptor and / or complement binding by substantially all IgG molecules present in the subject's serum may be measured. This can optionally be determined by testing serum samples taken from the individual and applying any suitable assay. Some exemplary suitable assays are described in the Examples.

[0188] Such assays can directly test for the presence of IgG molecules in serum samples that can bind to one or more Fc receptors, for example, in an ELISA. Alternatively, such assays can be indirect, in that they can test for the presence of one or more reaction products predicted to result from treating IgG with a protein with IgG cysteine ​​protease or IgG endoglycosidase activity. For example, if the agent is an enzyme that cleaves IgG proteins, serum samples can be assayed for the presence of intact IgG molecules or fragments resulting from cleavage. This can be accomplished by any suitable method, for example, by separating molecules and fragments based on molecular weight, for example, by mass spectrometry or SDS-PAGE, or by specifically detecting the molecules or fragments, for example, by ELISA. Alternatively, IgG can be detected by mixing a subject's serum with cells expressing FcgR and monitoring IgG binding by flow cytometry using a fluorescent dye-conjugated anti-human IgG.

[0189] Traditional methods for assessing the amount of IgG in samples, such as serum samples, in clinical settings rely on nephelometry and turbidimetry due to their speed, ease of use, and accuracy. In both nephelometry and turbidimetry, a light source is projected onto a liquid sample in a transparent container. Turbidimetry measures the decrease in light intensity, while nephelometry measures the scattering of light as it passes through the sample, which is proportional to the concentration of immunoglobulin in the solution. Both methods rely on the reaction of added anti-IgG antibodies with antigens in the sample to form antigen / antibody complexes (agglutination). Adding PEG can speed the reaction to its endpoint, increasing sensitivity and reducing the risk of false-negative results from samples containing excess antigen. In the case of IgG analysis, the F(ab')2 portion of IgG is cross-linked by anti-IgG antibodies, causing agglutination. However, these methods may not be suitable when some or all of the IgG present may not be intact. For example, if an IgG cysteine ​​protease (e.g., IdeS) is administered to a subject from whom a sample is collected, or if such a protease is administered to a sample, for example, in the methods of the present invention, cleavage fragments such as F(ab')2 and Fc fragments will be present. This does not affect the agglutination reactions of conventional nephelometric and turbidimetric assays as long as the F(ab')2 fragments are still present in the sample. Due to the shorter half-life of F(ab')2 fragments compared to intact IgG, agglutination decreases over time, but not proportionally to the amount of intact IgG present in the sample. Therefore, samples affected by the presence of IgG cysteine ​​proteases (e.g., IdeS) cannot be evaluated by conventional methods. The present inventors have developed a novel assay for IgG concentration that is compatible with samples affected by the presence of IgG cysteine ​​proteases (e.g., IdeS) and can be used in any clinical setting, including (but not limited to) in conjunction with other methods of the present invention.

[0190] The method can distinguish between intact IgG and IdeS-generated F(ab')2 fragments. This is achieved by utilizing antibodies that detect different fragments, i.e., anti-Fab and anti-Fc antibodies. The antibodies used in the assay must not be substrates for IgG cysteine ​​proteases (typically IdeS) that affect the sample. This prevents any active proteases that may be present in the sample from affecting the assay reagents. This can be achieved by testing IgG from different species or by using antibody fragments (i.e., Fab or F(ab')2 fragments) instead of whole antibodies. Typically, an anti-F(ab')2 agent is incubated with the sample as a capture reagent. The capture reagent is typically immobilized, for example, on the wells of an assay plate. The bound IgG is then detected by incubating with an anti-Fc agent as a detection reagent. Therefore, in contrast to nephelometry and turbidimetry, only IgGs that possess both Fab and Fc portions are detected. The detection reagent can typically be directly or indirectly conjugated to a moiety that facilitates detection, such as a fluorescent dye or an enzyme that reacts with a chromogenic substrate. The capture and detection reagents can be any other molecule that specifically recognizes the Fab or Fc portion of IgG, or can be used in the reverse order, i.e., capture with anti-Fc and detection with anti-Fab. The assay can be performed in any suitable format, such as conventional ELISA or Meso Scale Discovery format.

[0191] In some cases, such as when the IgG cysteine ​​protease is IdeS, the sample may contain intermediate fragments, such as scIgG, in which only one heavy chain is cleaved and the F(ab')2 remains attached to the other intact heavy chain. In such cases, the scIgG fragment may be erroneously identified as intact IgG by the assay. Therefore, the method may include a complementation step to assess the size of the fragments present in the sample. Because there are no disulfide bridges between the heavy chains below the hinge region, the Fc portion of the heavy chain in the scIgG fragment separates from the intact heavy chain under denaturing conditions as a protein of approximately 20-25 kDa. The different fragment sizes can be detected and quantified using any suitable method, such as SDS-PAGE. A specific embodiment of the method, including the optional complementation step, is described in Example 1 (see Efficacy Assessment). The method is particularly useful for assessing the efficacy of IdeS in clinical settings.

[0192] If the protein with IgG cysteine ​​protease or IgG endoglycosidase activity is an enzyme that cleaves glycan moieties on IgG, serum samples may be assayed for the presence of IgG molecules bearing either intact or cleaved glycans or the glycan fragments resulting from cleavage. This can be accomplished by any suitable method, for example, by separating the molecules and / or fragments based on molecular weight, for example, by mass spectrometry or SDS-PAGE, or by specifically detecting the molecules or fragments, for example, by ELISA.

[0193] In methods in which a protein having IgG cysteine ​​protease or IgG endoglycosidase activity is administered prior to adoptive cell transfer immunotherapy, the lower limit of the time interval between administration of the protein having IgG cysteine ​​protease or IgG endoglycosidase activity and adoptive cell transfer immunotherapy can be selected from at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, or at least 6 hours. The lower limit may be shorter than any of the above if Fc receptor binding by substantially all IgG molecules present in the subject's serum is determined to be sufficiently reduced or eliminated at an earlier time point. Preferably, the lower limit is 2 hours.

[0194] In methods in which a protein having IgG cysteine ​​protease or IgG endoglycosidase activity is administered prior to adoptive cell transfer immunotherapy, the upper limit of the time interval between administration of the protein having IgG cysteine ​​protease or IgG endoglycosidase activity and adoptive cell transfer immunotherapy may be selected independently of the lower limit and may be determined by the time required for endogenous production of IgG to begin to replace or completely replace the IgG molecules present in the subject's serum before the method was performed. This can be determined by testing serum samples taken from the individual and applying any suitable assay, such as those described above with respect to the lower limit. Newly synthesized IgG typically begins to reappear in the serum within 3-4 days and is completely replaced by about 3 weeks (21 days). Thus, the upper limit can be selected from: up to 21 days, up to 18 days, up to 14 days, up to 13 days, up to 12 days, up to 11 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, up to 24 hours, up to 18 hours, up to 12 hours, up to 10 hours, up to 8 hours, up to 7 hours, up to 6 hours, up to 5 hours, up to 4 hours, up to 3 hours, up to 2 hours, or up to 1 hour. Preferably, the upper limit is 48 hours.

[0195] The time interval between administration of the protein having IgG cysteine ​​protease or IgG endoglycosidase activity and adoptive cell transfer immunotherapy may be up to 24 hours, up to 12 hours, or up to 6 hours, such that both administration steps (a) and (b) can be performed on the same day or during the same visit to a treatment center, which is highly advantageous, especially when access to treatment centers may be limited.

[0196] In methods of administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after adoptive cell transfer immunotherapy to enhance the efficacy of immunotherapy, the timing of administration depends on the increase in IgG ADA. In certain embodiments, the protein is administered 4 to 8 days, e.g., 5 to 7 days, or 6 days, after adoptive cell transfer immunotherapy. This timing may be appropriate if the antibody response is a recall response. If the antibody response is a primary response, the protein may be administered more than one week after adoptive cell transfer immunotherapy, e.g., 10 days, 2 weeks, 3 weeks, or 4 weeks, or 10 days to 2 weeks, 10 days to 3 weeks, or 2 to 4 weeks.

[0197] Polypeptide production The polypeptides disclosed herein can be produced by any suitable means. For example, the polypeptides may be directly synthesized using standard techniques known in the art, such as Fmoc solid-phase chemistry, Boc solid-phase chemistry, or solution-phase peptide synthesis. Alternatively, the polypeptides may be produced by transforming cells, typically bacterial cells, with a nucleic acid molecule or vector encoding the polypeptide. Production of polypeptides by expression in bacterial host cells is described below and exemplified in WO 2016 / 128559.

[0198] Compositions and formulations comprising polypeptides The present invention also provides compositions comprising an IgG cysteine ​​protease or IgG endoglycosidase for use in the therapeutic methods of the invention. For example, the present invention provides compositions comprising one or more polypeptides of the invention and at least one pharmaceutically acceptable carrier or diluent. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the subject to which the composition is administered. Typically, the carrier and final composition are sterile and pyrogen-free.

[0199] Suitable compositions can be formulated using standard pharmaceutical formulation chemistry and methodology, all of which are readily available to those skilled in the art. For example, the agent may be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, reducing agents, etc., may be present in the excipient or vehicle. Suitable reducing agents include cysteine, thioglycerol, thioreducin, glutathione, etc. Excipients, vehicles, and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition and can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, thioglycerol, and ethanol. Pharmaceutically acceptable salts may be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and salts of organic acids such as acetates, propionates, malonates, benzoates, etc. A thorough discussion of pharmaceutically acceptable excipients, vehicles, and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0200] Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or multi-dose containers containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions may further comprise one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a composition for parenteral administration, the active ingredient is provided in a dry form (e.g., for powders or granules) for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water), after which the reconstituted composition is administered parenterally. The composition may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution may be formulated according to art-known methods and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations can be prepared using a non-toxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic mono- or diglycerides.

[0201] Other useful parenterally administrable compositions include those containing the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for controlled release or implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts. The compositions may be suitable for administration by any suitable route, including, for example, intradermal, subcutaneous, transdermal, intramuscular, intraarterial, intraperitoneal, intraarticular, intraosseous, or other suitable administration route. Preferred compositions are suitable for administration by intravenous infusion.

[0202] overview It is to be understood that the various applications of the disclosed products and methods can be tailored to the particular needs of the art, and that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0203] Furthermore, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes "polypeptides," and the like.

[0204] Unless specifically prohibited, the steps of the methods disclosed herein may be performed in any suitable order, and the order in which the steps are recited should not be considered limiting.

[0205] "Polypeptide" is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. Thus, the term "polypeptide" includes short peptide sequences and also includes longer polypeptides and proteins. As used herein, the term "amino acid" refers to natural and / or unnatural, i.e., synthetic, amino acids, including both D- and L-enantiomers, as well as amino acid analogs and peptidomimetics.

[0206] The terms "patient" and "subject" are used interchangeably and typically refer to a human. References to IgG typically refer to human IgG, unless otherwise specified.

[0207] The amino acid identity described above can be calculated using any suitable algorithm. For example, the PILEUP and BLAST algorithms can be used to calculate identity and align sequences (typically with their default settings, such as identifying equivalent or corresponding sequences), as described in Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value; when the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919), alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands (http: / / www.ncbi.nlm.nih.gov / ).

[0208] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in a comparison of a first sequence to a second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. Alternatively, the UWGCG package provides the BESTFIT program (e.g., used with its default settings) that can be used to calculate identity (Devereux et al. (1984) Nucleic Acids Research 12, 387-395).

[0209] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. [Example]

[0210] To investigate whether pre-existing or therapeutically induced antibodies have negative effects on CAR T cells and whether these effects can be mitigated through treatment with immurifidase or EndoS, the following experiments were performed. Using in vitro and in vitro models of IgG binding to cell surface receptors, a system was developed to mimic anti-drug antibody binding to CAR T cells. These models mimic the antibody-mediated effector mechanism of surface-bound antibodies on chimeric antigen receptors. The effects of treatment with immurifidase or EndoS were examined in the model systems.

[0211] Unless otherwise indicated, the methods used are standard biochemical and molecular biological techniques. Examples of suitable methodological textbooks include Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley and Sons, Inc.

[0212] Materials and Methods—Examples 1-4 Animals and cell lines Balb / c JBomTac, 6-week-old females, were purchased from Taconic and allowed to acclimate before use at age 9 weeks. Mice were housed and treated in accordance with ethical approval M72-13 (Lund). Daudi cells (ACC78) and THP1 monocytic cell line (AML; TIB-202) were cultured in complete D-MEM 10 medium (Glutamax D-MEM, 5% FCS and PEST).

[0213] Antibodies and Complement Rabbit anti-mouse platelet IgG (serum) (Code #CLA31440, Lot 6327, Cederland) was purified with protein G. For immune thrombocytopenia (ITP) experiments, purified IgG was treated with IdeS to generate scIgG and completely cleaved Fc / F(ab')2 fractions. The purity of the cleavage products was confirmed by SDS-page gel analysis. Anti-CD20 IgG (rituximab, N7022, 10 mg / mL) was purchased from Mabthera, and human IgG1 isotype negative control (#I5154) was purchased from Sigma. A cell counting kit, CCK-8, manufactured by Dojindo Laboratories (Japan) was used according to the manufacturer's instructions. Baby rabbit serum complement (CL3441; lot 6374; Cedarlane) was reconstituted with sterile water before use and diluted with medium to a final dilution of 1:10. Human blood was collected in serum-grade BD CAT tubes (#368815; spray-dried clot activator (silica, PVP, L-720)) and frozen at -20°C. Serum was tested for low toxicity to Daudi cells before use as a complement source. Mouse anti-human C4d (A213, Quidel) was biotinylated in-house and used at 1 μg / mL for cell staining. Polyclonal sheep anti-human C1q (MD-14-0162, Raybiotech) was fractionated into F(ab')2 fragments and purified using the FragIT kit (A2-FR2-025, Genovis) (final staining concentration, 75 μg / mL). Donkey anti-sheep IgG(H+L)-bio (Jackson, 713-065-003) was used as the detection antibody (2.5 μg / mL) with the fluorescent dye SA-PE (BD-Pharmingen, #554061). 7-AAD (Sigma, A9400) was used as a dead cell marker in the CDC assay. For the ADCP assay, THP1 cells were stained with FarRed DDAO-SE (Molecular Probes, C34553, lot: 33C1-1) at 1 mg / mL (approximately 2 mM) in DMSO, and Daudi cells were stained with calcein, AM (Invitrogen, C3099, lot 25257W); (1 mg / mL stock solution in DMSO).

[0214] enzyme His-tagged EndoS from Streptococcus pyogenes was expressed in E. coli and purified. Lipopolysaccharide (LPS) was removed using EndoTrap blue matrix. Purity was controlled on an SDS-page gel. Mass spectrometry analysis showed that an additional protein band after His-tag purification beyond the predicted 100 kD band originated from EndoS, not from host cell proteins. IdeS (IgG-degrading enzyme of Streptococcus pyogenesStreptococcus pyogenes IgG-degrading enzyme (S. pyogenes IgG-degrading enzyme) is a cysteine ​​endopeptidase secreted by the human pathogen S. pyogenes that highly specifically catalyzes a single proteolysis in the lower hinge of human immunoglobulin G (IgG) antibodies.

[0215] Example 1 - Antibody-mediated complement binding to target cells Using rituximab (RTX)-opsonized CD20-positive Daudi cells, a model system for ADA interactions in CAR-T therapy, in the sense that receptor-specific antibodies (RTX) bind to cell surface receptors (CD20) in a manner similar to ADA binding to CAR, we investigated the effects of pre-existing or therapeutically induced antibodies on cell therapy and the influence of IdeS and EndoS on such antibodies.

[0216] Complement deposition on Daudi cells Daudi cells (50 μL at 3 × 10E7 cells / mL) were treated with the anti-CD20 antibody RTX or human IgG1 negative control antibody at a final concentration of 2 μg / mL, along with a 1:10 step titration of IdeS or EndoS, starting at 100 μg / mL and decreasing to a final concentration of 0.01 μg / mL. V-shaped 96-well master plates containing a total volume of 150 μL / well were incubated for 110 min at 37°C. The plates were centrifuged, and a volume of 50 μL of supernatant was removed. 100 μL of complement was added in the form of human serum at a 1:5 dilution, resulting in a final serum dilution of 1:10. The plates were incubated for 120 min at 37°C. All dilutions and washing steps were performed in D-MEM supplemented with 0.5% BSA.

[0217] Flow cytometry staining for complement adhesion to cells C1q complement binding was assessed by incubation with sheep anti-C1q F(ab')2 prepared from polyclonal IgG (#MD-14-0162; RayBiotech) that had been previously digested into Fc and F(ab')2 fragments using resin-bound IdeS (FragIT kit, #A0-FR6; Genovis). The Fc fragment was removed using a protein A column. Polyclonal F(ab')2 was used at a final concentration of 75 μg / mL. Sheep anti-human C1q F(ab')2 was detected using biotinylated donkey anti-sheep IgG (H+L) (#713-065-003, Jackson ImmunoResearch). SA-PE was diluted in D-MEM (+0.5% BSA) and used as the fluorescent detection dye. C4d complement binding was assessed by incubation with biotinylated mouse anti-human C4d antibody (#A213; Quidel). SA-PE was diluted in D-MEM (0.5% BSA) and used as a fluorescent dye for detection. Finally, cells were transferred to FACS tubes and analyzed for mean fluorescence intensity (MFI) in FL2 using an Accuri C6 cytofluorometer.

[0218] result Complement deposition on IgG bound to target cells can initiate the classical complement pathway, which can lead to the formation of the membrane attack complex (MAC), causing cell death. Early steps in the complement cascade, promoted through antibody binding, can also signal and flag cells for complement-mediated phagocytosis via complement receptors. Thus, pre-existing and induced antibodies against CAR T cells may limit the persistence of CAR T in patients. Complement deposition on IgG-coated cells was examined using CD20-positive Daudi cells opsonized with RTX and then incubated with the indicated concentrations of IdeS or EndoS. After 2 hours, human serum was added as a complement source for an additional 2 hours. Cells were aliquoted and stained using anti-human C1q- or C4d-specific detection antibodies and SA-PE as a fluorochrome. Cells were analyzed for MFI by single-cell flow cytometry. IgG-induced C1q and C4d deposition can be blocked by IdeS and EndoS (see Figure 1), suggesting that IgG-primed CAR T cells are also protected from complement deposition by IdeS or EndoS treatment.

[0219] Example 2 - Complement-dependent cytotoxicity (CDC) Binding of antibodies, including pre-existing or induced CAR-specific antibodies, to receptor molecules can trigger full complement activation leading to the formation of cytotoxic MAC. To mimic this, Daudi cells were incubated with the CD20 receptor-specific antibody RTX and examined whether CDC could be blocked through treatment with IdeS or EndoS. Daudi cells (3 x 10E6) were incubated in a master plate with titrated concentrations of rituximab (RTX) (50 μg / mL in the well, serially diluted 1:2 down to 0.2 μg / mL) for 60 min at 37°C, along with IdeS or EndoS (50 μg / mL). 50 μL of the cell mix was transferred to an ELISA plate along with 50 μL of baby rabbit serum (1:5 dilution) as a complement source and incubated at 37°C for 45 minutes for CDC lysis. 10 μL of CCK8 was added to each 100 μL mix and incubated at 37°C for an additional 60 minutes in a CO2 incubator. HCl (50 μL, 0.1 M) was used as a stop solution. OD values ​​were taken at 450 nm. OD values ​​from wells without RTX were considered 100% viable.

[0220] result At an RTX concentration of 0.78 μg / mL, 70% of the cells in the medium group died, whereas in the presence of either IdeS or EndoS at this concentration, all Daudi cells survived (see Figure 2). The protective effect of EndoS decreased with increasing RTX concentration, resulting in a 30% survival rate at 50 μg / mL, a 60-fold higher RTX concentration than the 0.78 μg / mL RTX concentration that achieved the same survival rate without EndoS. Thus, even at high antibody concentrations (50 μg / mL), IdeS can block CDC, but EndoS also exhibits potent protective activity.

[0221] Example 3 - ADPC of RTX-opsonized Daudi cells by THP1 effector cells These experiments addressed the question of whether IdeS and EndoS could reduce FcγR-mediated phagocytosis (ADCP), one of the mechanisms that may limit CAR T cell persistence in the presence of pre-existing or induced CAR-specific antibodies.

[0222] cell culture THP1 (monocyte effector cells) and Daudi target cells were expanded in complete Glutamax D-MEM (5% FCS and PEST), washed and diluted in D-MEM 10 medium before the experiment.

[0223] Labeling of target cells Daudi cells were washed twice with PBS to remove proteins and then labeled. Cells were resuspended in PBS and stained with calcein (4 μL, from 1 mg / mL for 6 mL of cells) and incubated in the dark at RT. After 15 min, cells were washed twice with medium (0.5% BSA) and plated at 2 × 10 cells per mL in 3 mL of D-MEM 10 medium. 6 The cells were resuspended at 1000 cells / mL.

[0224] Labeling of effector cells THP1 cells were washed twice with PBS to remove proteins and resuspended in 5 mL of PBS. FarRed was added (5 μL per 5 mL of cells) and incubated in the dark at RT. After 20 min, cells were washed twice with medium and resuspended in D-MEM 10 at 3.6 × 10 6The cells were resuspended at 1000 cells / mL.

[0225] Target-effector cell incubation The titrated RTX and enzyme (IdeS or EndoS) were incubated for 60 minutes at 37°C. Daudi target cells (2 x 10E6 cells / mL, 50 μL) were incubated for 30 minutes for opsonization, followed by the addition of THP1 effector cells (3.6 x 10E6 cells / mL, 50 μL) and incubation for approximately 2 hours to allow ADCP of Daudi cells. Cells were fixed with 5% PFA for 3 minutes, washed, and transferred for FACS analysis. Cells were analyzed for MFI in FL2 (calcein) and FL4 (FarRed) using an Accuri C6 flow cytometer. Double-positive cells were considered ADCP-positive.

[0226] result The efficacy of IdeS and EndoS in protecting RTX-opsonized CD20+ Daudi cells from FcγR-mediated phagocytosis by monocytic THP1 effector cells was analyzed by flow cytometry. Briefly, calcein-labeled Daudi cells were opsonized with increasing concentrations of RTX, followed by the addition of a fixed concentration of IdeS or EndoS. After 30 min of incubation at 37°C, FarRed-labeled THP1-effector cells were added for approximately 2 h. Washed and fixed cells were then analyzed by flow cytometry. All calcein-positive cells in FL2 were gated and defined as 100% Daudi cells. All FL2-positive cells were then gated for FarRed positivity in FL4. These calcein / FarRed double-positive cells represent Daudi cells phagocytosed by THP1. Removal of the Fc portion by IdeS completely abolished the ability of THP1 cells to phagocytose opsonized Daudi cells. Furthermore, deglycosylation of RTX at N297 by EndoS also reduced phagocytosis in this model (see Figure 3). Thus, in ADCP, engulfment of antibody-opsonized cells by the monocytic cell line THP1 is completely blocked by IdeS treatment and reduced by EndoS treatment.

[0227] Example 4 - Platelet protection in an immune thrombocytopenia (ITP) model As demonstrated above using RTX and Daudi cells, IdeS and EndoS attenuate the effector function of cell surface receptor-specific antibodies. In the following experiments, we also investigated whether these enzymes protect antibody-sensitized endogenous cells from elimination in vivo using an ITP model. This demonstrates that harmful polyclonal antibodies against cell surface receptors can be inactivated through treatment with immunofidase or EndoS, which mimics the effects of these enzymes in the context of anti-CAR antibodies on CAR T cell persistence.

[0228] ITP in vivo EndoS treatment Nine-week-old female Balb / c mice were treated with a single i.p. injection of 200 μL of anti-platelet specific antibody (anti-PLT IgG) (50 μg IgG / mouse) purified by protein G from rabbit anti-mouse platelet serum (Cederland #CLA31440). JBomTac Mice were primed for immune thrombocytopenia (ITP). 30 minutes later, the indicated amount of EndoS (10 μg / mouse, 30 μg / mouse, or 90 μg / mouse) was injected i.p., respectively, with PBS serving as a negative control. In both cases, mice injected with the carrier solution (PBS) served as normal controls. Four hours after injection, mice were evaluated for hematoma or abnormal behavior. 24 hours later, blood samples were collected from the tail vein and placed in a Microvette CB300 (Sarstedt, Potassium-EDTA #16.444.100). Platelets in the blood samples were counted using a VetScan HM5 hematology analyzer.

[0229] Induction of ITP by IdeS-cleaved anti-PLT antibodies in vitro Anti-mouse PLT IgG was protein G purified from serum of a rabbit immunized with mouse platelets (code #CLA31440, Cederlane). IgG was processed to generate the IdeS cleavage product scIgG and the fully cleaved Fc / F(ab')2 fragment for in vivo experiments. Nine-week-old female Balb / c mice were treated with a single ip injection of 200 μL of either 250 μg / mouse anti-PLT IgG, scIgG, or Fc / F(ab')2 fragment. JBomTac Mice were primed for ITP. Some mice were injected with PBS to establish normal platelet control levels. 24 hours later, blood samples were collected from the tail vein and placed in a Microvette CB300 (Sarstedt, Potassium-EDTA #16.444.100). Platelets in the blood samples were counted using a VetScan HM5 hematology analyzer.

[0230] ITP in vivo IdeS treatment Nine-week-old female Balb / c mice were treated with a single ip injection of 200 μL of anti-PLT IgG (250 μg IgG / mouse) purified by protein G from rabbit anti-mouse platelet serum (Cederland #CLA31440). JBomTac Mice were primed for ITP. Anti-PLT IgG was injected i.p. 1 h later, IdeS (0 μg / mouse, 0.2 μg / mouse, 2 μg / mouse, 20 μg / mouse) was administered i.v. Control mice with normal platelet levels were injected with PBS alone. 24 h later, blood samples were collected from the tail vein and placed in a Microvette CB300 (Sarstedt, Potassium-EDTA #16.444.100). Platelets in the blood samples were counted using a VetScan HM5 hematology analyzer.

[0231] result The aim of this study was to evaluate the in vivo efficacy of IdeS cleavage products, scIgG or F(ab')2 IgG fragments, as mediators of effector function compared to intact anti-PLT IgG in vivo. Immune thrombocytopenia (ITP) was induced in BALB / c mice by a single i.p. injection of either rabbit anti-mouse platelet-purified intact IgG, rabbit scIgG, or F(ab')2 fragments at a dose of 0.25 mg / mouse. Mice were bled one day after induction of ITP, and blood was collected from the tail vein. Platelets were automatically counted using a VetScan HM5. Two naive mice administered PBS alone served as controls. These mice had a platelet count of 86 x 10 9 compared with mice injected with rabbit anti-mouse platelet-purified IgG, which was reduced to 657 × 10 platelets / L. 9 The mice injected with anti-PLT scIgG had normal platelet levels with platelets / L (see Figure 4). However, a slight decrease in platelets was observed. No induction of thrombocytopenia was observed in mice treated with purified rabbit anti-mouse platelet (F(ab')2 IgG fragment). From this experiment, we can conclude that protection from thrombocytopenia can be achieved through in vitro generation of scIgG or (F(ab')2) with IdeS. Similar protection can be predicted from other pre-existing or de novo antibodies targeting neoepitopes on chimeric antigen receptor T cells. Thrombocytopenia could be partially prevented when an scIgG preparation was injected and was completely abolished after cleavage of IgG with IdeS into Fc and F(ab')2 fragments (see Figure 4). To investigate whether IdeS has a therapeutic effect in vivo, mice were first injected with a platelet-reducing dose of anti-PLT rabbit IgG. One hour later, different doses of IdeS were injected. Blood was collected 24 hours later, and platelet counts were determined in the different groups. Two micrograms of IdeS per mouse was completely sufficient to restore platelets to normal levels in mice (see Figure 5). Therefore, we used an ITP mouse model to test the therapeutic effect of IdeS on harmful anti-platelet antibodies (anti-PLT) in vivo. Injection of intact polyclonal anti-PLT rabbit antibodies (250 μg) into mice induced a strong ITP phenotype accompanied by platelet depletion. Similar results were observed when EndoS was injected into anti-PLT IgG-sensitized mice. 50 μg of anti-PLT antibody was sufficient to reduce platelet counts from approximately 600 × 10E9 cells / L to 200 × 10E9 cells / L within 24 hours. 10 μg of EndoS per mouse was sufficient to protect mice from thrombocytopenia (see Figure 6). These experiments demonstrate that IdeS or EndoS can be used to block or restore pathogenic antibody-mediated effector functions of opsonized cells. Based on these data, these enzymes are predicted to also improve CAR T / NK cell survival and efficacy through inactivation of pre-existing and treatment-induced ADAs. In summary, we demonstrated that the tested antibody-mediated effector functions (C1q and C4d deposition, CDC, ADCP) that mimic the ADA response to CAR-T cells can be blocked through the use of IdeS and EndoS.

[0232] Example 5 Experiments were conducted to determine whether antibodies against cognate epitopes on single-chain variable fragment (scFv) chimeric antigen receptors or CAR-T cells have deleterious effects. Therefore, CAR- and allospecific antibodies from different sources were tested for binding and Fc-mediated antibody effector functions, i.e., antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC), of CAR-T cells. Furthermore, it was confirmed that immunofluorescence treatment could protect CAR-T cells from these deleterious IgG effector mechanisms.

[0233] Materials and Methods cell CAR-T cell line, anti-CD19scFv(FMC63)-h(28ζ) based on clone E6-1 CAR-Jurkat T cells (CARJ-ZP005, Creative biolabs, Shirley, NY, USA); Jurkat wild type (wt) (clone E6-1) (#EP-CL-0129, ElabScience, Houston, TX, USA); primary human CAR T cells, anti-BCMA4 CAR T cells (BCMA-4-TM8-4-1BB-CD3 zeta CAR-T cells) (PM-CAR1037, ProMab Biotechnologies, Richmond, CA, USA); anti-CD19 CAR T cells (CD19-scFv-Flag-TM--CD28-CD3 zeta CAR-T cells) (PM-CAR1007, ProMab Mock-scFv control T cells (PM-CAR1000, ProMab Biotechnologies); anti-CD19-scFv was derived from the murine CD19-specific monoclonal antibody FMC63. THP-1 monocytic cell line (ACC16, DSMZ, Braunschweig, Germany) and IgM BCR and CD20-positive Daudi cells (ACC78, DSMZ). All cells were cultured in complete RPMI 10 medium (RPMI, 10% FCS, and PEST). Anti-CD19 CAR-Jurkat T cells were cultured under puromycin (1 mg / mL) selection.

[0234] Antibodies and human serum CAR scFv cross-reactive anti-F(ab')2 specific antibodies included affinity-purified rabbit anti-human IgG, F(ab')2 fragment specific (#309-005-006, Jackson ImmunoResearch, West Grove, PA, USA), affinity-purified rabbit anti-mouse IgG, F(ab')2 fragment specific (#315-005-006, Jackson ImmunoResearch), goat anti-human IgG-Fc-PE (LS-AB2, OneLambda), and biotinylated goat anti-rabbit Fc (#111-066-046, Jackson ImmunoResearch). Detection of biotinylated goat anti-rabbit Fc was achieved by using streptavidin-Alexa Fluor 647 (SA-AF647) (#016-600-084, Jackson ImmunoResearch). Human serum samples (n = 119, both males and females) were purchased from BioIVT (BioIVT, Westbury, NY, USA) and used for human anti-mouse antibody (HAMA) quantification and allospecific antibody screening. Anonymous clinical trial samples from HLA-hypersensitized patients (n = 8) and phase 1 healthy volunteers (n = 11) were screened for HAMA and HLA alloreactivity to anti-CD19 CAR-Jurkat T cells. HLA alloreactive control sera, including FlowPRA HLA class I positive control serum (FL1-PC, OneLambda, West Hills, CA, USA), FlowPRA HLA class II positive control serum (FL1-PC, OneLambda), and HLA negative control serum (LS-NC, LABScreen, OneLambda), were purchased from OneLambda or were research samples donated by healthy donors.

[0235] ELISA-based HAMA detection The bridging ELISA kit "LEGEND MAX Human Anti-Mouse Ig (HAMA)" (catalog number 438307, lot number B329842, BioLegend, San Diego, CA, USA) was used to detect HAMA in human serum. Briefly, a plate precoated with mouse IgG was washed and incubated with undiluted human serum samples (BioIVT), HAMA quality control, standard curve samples, and mouse IgG conjugate. The contents were discarded, and the plate was washed with wash buffer. Substrate solution was added, and the plate was incubated in the dark at room temperature (RT) for 15 minutes. The reaction was terminated by adding stop solution. Absorbance was measured at 450 nm and 570 nm within 30 minutes using a SpectraMax i3x spectrophotometer (SpectraMax i3x, Molecular Devices, San Jose, CA, USA). OD results were analyzed using a four-parameter logistic curve-fitting algorithm in Graph Pad Prism 9 (GraphPad Software, San Diego, CA, USA). Serum samples with HAMA >10 ng / mL were considered HAMA positive.

[0236] Screening of CAR-specific human sera by flow cytometry Anti-CD19 CAR-Jurkat or wild-type Jurkat T cells (1 x 10E5 cells / well) were washed with PBS and centrifuged at 300 g for 5 minutes. The cell pellet was resuspended in 50 μL of human serum sample selected from ELISA-based HAMA detection. The serum-incubated cells were then washed, stained with goat anti-human Fc-PE antibody (OneLambda), and analyzed by flow cytometry (CytoFLEX flow cytometer, #C02945, Beckman Coulter) at FL2 for mean fluorescence intensity (MFI) levels.

[0237] Screening of CAR-T Jurkat alloresponding human sera by flow cytometry Anti-CD19 CAR-Jurkat T cells (1 x 10E5) were washed with PBS and centrifuged at 300g for 5 minutes. The cell pellet was resuspended in 50 μL of the indicated human serum sample. FlowPRA Class I positive control serum (OneLambda), FlowPRA Class II positive control serum (OneLambda), and HLA negative control serum (OneLambda) were used as controls. Bound alloantibodies were detected using PE-conjugated goat anti-human IgG (LS-AB2, OneLambda). After the washing step, the cells were resuspended in PBS and analyzed for MFI values ​​using a flow cytometer (CytoFLEX).

[0238] Polyclonal IgG binding to primary CAR-T Primary human CAR T cells, including anti-BCMA4 CAR T cells (PM-CAR1037, ProMab Biotechnologies), anti-CD19 CAR T cells (PM-CAR1007, ProMab Biotechnologies), and mock scFv control cells (PM-CAR1000, ProMab Biotechnologies), were thawed, washed, and incubated with rabbit anti-mouse IgG, F(ab)2 specific (#315-005-005, Jackson ImmunoResearch) for 30 minutes. BCR-expressing Daudi cells were stained as a F(ab')2 positive control. The cells were then washed and sequentially incubated with biotinylated goat anti-rabbit Fc antibody and SA-AF647 (#016-600-084, Jackson ImmunoResearch). A similar approach was used to evaluate the binding of polyclonal anti-mouse and anti-human IgG antibodies to anti-CD19 CAR-Jurkat T cells.

[0239] Antibody-dependent cellular phagocytosis (ADCP) using FcγRI-expressing effector cells Anti-CD19 CAR-Jurkat T cells were incubated with serum and tested for ADCP induction using the FcγRI Reporter Bioassay Kit (#CS1781C01, Promega, Madison, WI, USA). Briefly, human serum samples (BioIVT and anonymous serum from the 06-study), HLA class I positive control serum (FL1-PC, OneLambda), and HLA negative control serum (#LS-NC, OneLambda) were incubated with or without 10 μg / mL immunofidase at 37°C for 30 minutes. Anti-CD19 CAR-Jurkat target T cells (CARJ-ZP005, Creative Biolabs) (7500 cells / well) were centrifuged, washed once with D-PBS, and resuspended in the provided assay buffer (4% low IgG serum in RPMI-1640). Target cells were incubated with immunofluorescence-treated or untreated antibody and serum for 1 hour at 37°C. After this, effector cells (75,000 cells / well) expressing FcγRI (Promega) were added to the opsonized target cells and incubated for 6 hours at 37°C. Finally, cells were incubated with Bio-Glo™ Luciferase Assay Reagent (Promega) for 10 minutes at ambient temperature, after which luciferase activity was measured using a luminescence reader (SpectraMax i3x) with an integration time of 0.5 seconds per well. Data were analyzed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). The background of the plate containing assay buffer was calculated as the average of three replicate experiments, while the no-antibody control containing cells alone in the presence or absence of immunofluorescence was calculated as the average of two replicate experiments. Fold induction (FoI) was calculated as follows: FoI=RLU (induction-background) / RLU (no antibody control-background).

[0240] Antibody-dependent cellular cytotoxicity (ADCC) using effector cells expressing high-affinity FcgRIIIa (V158) and low-affinity FcgRIIIa (F158) Antibodies (rituximab, MabThera, Roche, Basel, Switzerland), rabbit anti-human IgG, F(ab')2 specific (JacksonImmunoResearch), rabbit anti-mouse IgG, F(ab')2 specific (JacksonImmunoResearch), and human serum (BioIVT and anonymized serum from an HLA-highly sensitized patient) were incubated with or without 20 μg / mL of immunofidase at 37°C for 30 min and then stored at 4°C overnight. Target cells (7500 cells / well) including anti-CD19 CAR-Jurkat (CARJ-ZP005, Creative Biolabs), Jurkat wild-type (EP-CL-0129, Elabscience, Houston, TX, USA), and Daudi (ACC78, DSMZ, Braunschweig, Germany) cells were centrifuged and resuspended in D-PBS (GIBCO Life Technologies, Grand Rapids, TX, USA). The cells were washed once with RPMI-1640 (Promega, Madison, NY, USA) and resuspended in assay buffer (4% low IgG serum in RPMI-1640 (Promega, Madison, WI, USA)) and incubated with immunofluorescence-treated or non-treated antibody and serum for 1 hour at 37°C. After this, 75,000 effector cells expressing low affinity (#G979A, Promega) or high affinity (#G701A, Promega) FcγRIIIa were added to the opsonized target cells and incubated for 6 hours at 37°C. Finally, the cells were incubated with Bio-Glo™ Luciferase Assay Reagent (Promega) for 10 minutes at ambient temperature, after which luciferase activity was measured using a luminescence reader (SpectraMax i3x) with an integration time of 0.5 seconds per well. Raw data were exported and analyzed using GraphPad Prism 9.0 (GraphPad The assay was analyzed using the ELISA software. The background of the plate containing assay buffer was calculated as the average of three replicates, while the no-antibody control containing cells alone in the presence / absence of immunofidase was calculated as the average of two replicates. An HLA-negative control representing a serum sample from a healthy donor lacking quantitative anti-HLA class I and class II antibodies was also included.Fold induction (FoI) was calculated as follows: FoI=RLU (induction-background) / RLU (no antibody control-background).

[0241] Flow cytometry-based ADCP assay ADCP target cells (anti-CD19 CAR-Jurkat T cells, Jurkat wild-type T cells, and Daudi cells) were stained with calcein-AM (C3099, Invitrogen, Carlsbad, CA, USA) before incubation with the indicated concentrations of immunofidase-treated (±10 μg / mL) rabbit anti-human IgG, F(ab')2-specific (#309-005-006, Jackson ImmunoResearch) or anti-mouse IgG, F(ab')2-specific (#315-005-006, Jackson ImmunoResearch) antibodies. Alternatively, target cell pellets were resuspended undiluted with 25 μL of immunofidase-treated (±10 μg / mL) human serum samples for opsonization with HAMA or allogeneic IgG. The monocytic effector cell line, THP-1, was stained with CellTrace FarRed DDAO-SE (C34553, Molecular Probes, Eugene, OR, USA) and then added to the target cells and incubated at 37°C for 90 minutes. Phagocytosis was assessed by flow cytometry (CytoFLEX flow cytometer, #C02945, Beckman Coulter). The amount of FL1 and FL4 double-positive cells, reflecting target cells engulfed by THP1 cells, was expressed as a percentage of target cells.

[0242] CD19-protein binding blocking experiment Rabbit anti-mouse IgG, F(ab')2-specific antibody (Jackson ImmunoResearch Laboratories) and human serum (BioIVT) were incubated with or without 10 μg / mL immunofidase at 37°C for 60 min. IHAc (1 mM) (I4386, Sigma-Aldrich, St. Louis, MO, USA) was added to all samples for 30 min to inactivate the immunofidase during the subsequent incubation step. Anti-CD19 CAR-Jurkat and Jurkat wild-type T cells were incubated with the prepared serum and IgG samples for 60 min at RT. Recombinant human CD19-Fc chimeric protein, atto 647N conjugate (ATM9269, R&D systems, Minneapolis, MN, USA) was added to the cells and incubated for 45 min before analysis by flow cytometry (CytoFLEX flow cytometer, #C02945, Beckman Coulter).

[0243] result F(ab') 2 The specific polyclonal antibody specifically binds to the CAR T cell receptor Primary human T cells transfected with anti-CD19 or BCMA-specific chimeric antigen receptors to generate autologous CAR T cells were used to identify CAR-specific antibodies. Even in the case of autologous CAR T cell therapy, the antigen-specific scFv domain of the CAR, if derived from a mouse monoclonal antibody, contains epitopes that are foreign to the recipient. To investigate the effect of CAR-specific antibodies on CAR T cells, CAR-specific antibodies from different sources were tested for binding. A polyclonal rabbit anti-mouse F(ab')2-specific antibody showed cross-reactivity with anti-CD19 CAR T cells (Figure 7A) and anti-BCMA-CAR T cells (Figure 7B), whereas mock-transfected T cells (Figure 7C) remained unstained. B cell receptor-expressing Daudi cells (Figure 7D), previously shown to cross-react with polyclonal rabbit anti-mouse F(ab')2 detection reagent, were used for F(ab')2 positive control staining. Staining of the human T cell line Jurkat transduced with an anti-human CD19 CAR, whose receptor scFv is based on the mouse mAb FMC63, demonstrated strong binding of the rabbit anti-mouse F(ab')2 antibody. Even with the rabbit anti-human F(ab')2 antibody, some cross-reactivity was observed. These data confirm that antibodies can bind to a variety of different receptor constructs directed against different targets. Because such binding can have negative effects on adoptive cell transfer immunotherapies such as CAR T cell therapy, we further investigated the anti-CAR T cell antibody-mediated effector function and the extent to which it could be blocked by treatment with immunofidase.

[0244] Identification of HAMA and CD19-CAR Jurkat T cell allospecific sera One group of potentially CAR-specific antibodies is human anti-mouse antibodies (HAMA). HAMA levels in human serum samples can be quantified by sandwich ELISA using assay plates coated with mouse IgG. Human sera were screened for HAMA using a validated HAMA ELISA kit (Figure 8A). A selection of identified HAMA-positive and -negative samples was tested for binding to anti-CD19 CAR-Jurkat T cells. Jurkat wild-type T cells were included to distinguish HAMA-specific IgG binding from HLA alloreactivity. ELISA demonstrated that HAMA-positive sera also specifically bound to the anti-CD19 CAR T cell receptor (Figure 8B). Sera screened for HAMA by ELISA are shown (Figure 8C). These samples were then further screened for IgG binding by flow cytometry. This allowed us to select HAMA or anti-CD19 CAR T cell alloreactive sera to further elucidate the effects of these different IgG groups on anti-CD19 CAR-Jurkat T cells. HAMA is thought to be induced in normal individuals by contact with mouse antigens. It can be predicted that the frequency and concentration of HAMA will be even higher in patients receiving mouse mAb-based biologics, potentially even resulting in partial neutralization of these therapeutics. Compared to healthy individuals, higher levels of alloantibodies against anti-CD19 CAR-Jurkat T cells can be detected in the serum of HLA-sensitized transplant patients (Figure 8D). Five of eight anti-HLA-highly sensitized patients screened were positive for anti-CD19 CAR-Jurkat T cells, and one patient was anti-HLA-highly sensitized. Two of the screened individuals did not have alloantibodies against anti-CD19 CAR-Jurkat cells. These data demonstrate that antibodies capable of binding to receptor constructs are detectable in human patients, including healthy individuals, and are elevated in patients who have undergone treatments that can enhance HLA sensitization, such as transplantation. Alloantibodies are induced not only during organ transplantation but also due to pregnancy and blood transfusions. Infusion of allogeneic cell therapy into patients may further induce alloantibodies, potentially compromising allogeneic CAR T cell processing.

[0245] Polyclonal anti-F(ab') for ADCP 2 Antibody opsonization of anti-CD19 CAR T cells is blocked by immurifidase treatment, and immurifidase blocks ADCP induction by CD19-CAR Jurkat T cells opsonized with allogeneic serum. Polyclonal anti-F(ab')2 antibodies specific for the CD19 scFv CAR domain and HLA-specific antibodies against allogeneic anti-CD19 CAR-Jurkat T cells were tested for induction of antibody-mediated cellular phagocytosis (ADCP) and its blockage through treatment with the IgG-cleaving enzyme immurifidase (see Figures 9 and 10). A flow cytometry-based ADCP model was used with calcein-stained target cells and CellTrace FarRed-labeled monocytic THP1 effector cells. Cell acquisition by flow cytometry allowed for the identification of single- and double-positive cells, i.e., phagocytosed cells. Anti-CD19 CAR-Jurkat target cells were opsonized with either a rabbit anti-mouse F(ab')2 antibody (Figure 9A) or a cross-reactive anti-human F(ab')2 antibody (Figure 9B). In both cases, the addition of immurifidase (10 μg / mL) blocked ADCP of target cells. There was no uptake of Jurkat wild-type cells, indicating that phagocytosis was anti-CD19 CAR-dependent (Figures 9D, E). BCR-positive Daudi target cells were used as a positive control for F(ab')2 antibody-mediated ADCP (Figures 9C, F). These data demonstrate that receptor-specific antibodies can induce ADCP against T cells, which is predicted to have a negative effect on cell therapy. The data also demonstrate that treatment with immunofidase is effective in blocking ADCP. Furthermore, opsonization and induction of ADCP by human sera sensitized to allogeneic Jurkat CAR T cells was tested using a bioluminescent FcγRI reporter assay (#CS1781C01, Promega). Sera from a normal individual (Figure 10A) and an anti-HLA highly sensitized patient (Figure 10B) were able to opsonize anti-CD19 CAR-Jurkat T cells and induce ADCP. The induction of ADCP by serum IgG against allogeneic cells could be reduced through treatment with immurifidase. Thus, these data confirm that antibodies in the serum of both normal individuals and sensitized patients induce ADCP against adoptively transferred immunotherapy cells, which can be reduced by immurifidase treatment.

[0246] ADCC(V158) induced by FcγRIIIa allele(V158) and allele(F158) anti-CD19 CAR-specific antibodies is blocked by immunofluorescence treatment. Antibody-dependent cellular cytotoxicity (ADCC) is triggered by IgG-opsonized target cells engaging FcγRIIIa (CD16a) on effector cells. Two major CD16a alleles exist in the general population: a high-affinity variant with valine at position 158 (V158) and a low-affinity variant with phenylalanine at position 158 (F158). These alleles were introduced into effector cells for an ADCC bioluminescence assay (Promega). Anti-CD19 CAR-Jurkat T cells were incubated with either a rabbit anti-mouse (Figure 11A) or anti-human (Figure 11B) F(ab')2 antibody, with or without immunofluorescence (20 μg / mL) treatment. Opsonized target cells were incubated with high-affinity FcγRIIIa (V158) effector cells. Target cells opsonized with anti-mouse F(ab')2 antibody strongly induced ADCC. Immunofidase-treated samples completely abolished ADCC induction (Fig. 11A). Polyclonal anti-human F(ab')2 induced a weaker stimulatory effect but was still able to induce ADCC at 100 μg / mL (Fig. 11B). No effect was observed with negative control Jurkat wild-type cells (Fig. 11C). Daudi cells opsonized with rituximab (Fig. 11D) or anti-human F(ab')2 (Fig. 11E) were used as positive controls. Furthermore, immunofidase treatment of opsonized Daudi target cells blocked ADCC induction (Fig. 11D, E). Similar results were observed using a low-affinity FcγRIIIa ADCC bioluminescence reporter assay (Figure 12). Anti-CD19 CAR-Jurkat T cells treated with anti-mouse F(ab')2 induced ADCC signaling, which could be blocked by treatment with immurifidase (Figure 12A). On the other hand, polyclonal anti-human F(ab')2 antibodies failed to induce ADCC signaling, even at the highest antibody concentration (100 μg / mL) (Figure 12B). Daudi cells incubated with rituximab (Figure 12D) or anti-human F(ab')2 (Figure 12E) induced low-affinity FcγRIIIa ADCC, an effect that was blocked by treatment with immurifidase. These data further demonstrate that receptor-specific antibodies can mediate detrimental effects on adoptive cell transfer immunotherapy cells, particularly ADCC, which is predicted to negatively impact cell therapy. The data also demonstrate that treatment with immunofidase is effective in blocking ADCC.

[0247] ADCC induction by HAMA-opsonized anti-CD19 CAR-Jurkat T cells can be blocked by immunofluorescence treatment ELISA-HAMA negative (164) and positive (184, 187, 208, 250) sera, treated or not with 20 μg / mL of immurifidase, were incubated with anti-CD19 CAR-Jurkat T cells, after which the opsonized target cells were added to effector cells transfected with the FcγRIIIa high-affinity allele (Promega). The bioluminescence signal from the two HAMA-positive sera (184, 250) was reduced in the presence of immurifidase (Figure 13). This suggests that a portion of the mouse IgG-binding HAMA antibodies in human serum bind to the anti-CD19 CAR in a manner that can induce CD16 FcγRIIIa activation on effector cells. Thus, these data further demonstrate that antibodies in human serum mediate detrimental effects on adoptively transferred immunotherapy cells, which can be reduced by immurifidase treatment.

[0248] Binding of CD19 protein to serum-exposed anti-CD19 CAR T cells can be improved by immunofidase treatment For CAR T-cell therapy to be successful, specific interaction of the chimeric antigen receptor with target proteins is a necessary step. Binding of serum IgG to anti-CD19 CARs may interfere with cognate binding, such as with CD19 on target cells. HAMA-positive and -negative serum samples identified by ELISA (Figure 14) were tested for interference with binding of recombinant atto-647N-labeled human CD19 protein to anti-CD19 CAR-Jurkat T cells. Digestion of serum IgG with immunofidase increased the median FI signal from anti-CD19 CAR-Jurkat T cells in most samples, suggesting increased binding of atto-647N-labeled CD19 protein. This suggests that the interaction of adoptive cell immunotherapies, such as anti-CD19 CAR T cells, with target proteins can be enhanced simply by removing the IgG-Fc portion through immunofidase treatment, thereby reducing steric hindrance. Blocking the F(ab')2 fragment may result in a shorter half-life than the intact IgG equivalent, making the in vitro effect of immunofidase even stronger in vivo.

Claims

1. A pharmaceutical composition for enhancing the survival and activity of transferred cells, administered in combination with adoptive cell transfer immunotherapy, the pharmaceutical composition comprising a protein having IgG cysteine ​​protease or IgG endoglycosidase activity, wherein: (i) the protein having IgG cysteine ​​protease activity is an IgG cysteine ​​protease derived from streptococci, such as Streptococcus pyogenes, and optionally the protein is IdeS or IdeZ; or (ii) the protein having IgG endoglycosidase activity is derived from streptococci such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica IgG endoglycosidase from Saccharomyces meningoseptica, optionally wherein the protein is EndoS, CP40, EndoE, or EndoF2; Pharmaceutical compositions.

2. A pharmaceutical composition for treating cancer administered in combination with adoptive cell transfer immunotherapy, the pharmaceutical composition comprising a protein having IgG cysteine ​​protease or IgG endoglycosidase activity, wherein: (i) the protein having IgG cysteine ​​protease activity is an IgG cysteine ​​protease derived from streptococci, such as Streptococcus pyogenes, and optionally the protein is IdeS or IdeZ; or (ii) the protein having IgG endoglycosidase activity is derived from streptococci such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica IgG endoglycosidase from Saccharomyces meningoseptica, optionally wherein the protein is EndoS, CP40, EndoE, or EndoF2; Pharmaceutical compositions.

3. A pharmaceutical composition for treating an autoimmune condition administered in combination with adoptive cell transfer immunotherapy, the pharmaceutical composition comprising a protein having IgG cysteine ​​protease or IgG endoglycosidase activity, wherein: (i) the protein having IgG cysteine ​​protease activity is an IgG cysteine ​​protease derived from streptococci, such as Streptococcus pyogenes, and optionally the protein is IdeS or IdeZ; or (ii) the protein having IgG endoglycosidase activity is derived from streptococci such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica IgG endoglycosidase from Saccharomyces meningoseptica, optionally wherein the protein is EndoS, CP40, EndoE, or EndoF2; Pharmaceutical compositions.

4. A pharmaceutical composition for treating a condition mediated by harmful antibody-producing B cells administered in combination with adoptive cell transfer immunotherapy, the pharmaceutical composition comprising a protein having IgG cysteine ​​protease or IgG endoglycosidase activity, wherein: (i) the protein having IgG cysteine ​​protease activity is an IgG cysteine ​​protease derived from streptococci, such as Streptococcus pyogenes, and optionally the protein is IdeS or IdeZ; or (ii) the protein having IgG endoglycosidase activity is derived from streptococci such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica IgG endoglycosidase from Saccharomyces meningoseptica, optionally wherein the protein is EndoS, CP40, EndoE, or EndoF2; Pharmaceutical compositions.

5. A pharmaceutical composition for treating an infectious disease administered in combination with adoptive cell transfer immunotherapy, the pharmaceutical composition comprising a protein having IgG cysteine ​​protease or IgG endoglycosidase activity, wherein: (i) the protein having IgG cysteine ​​protease activity is an IgG cysteine ​​protease derived from streptococci, such as Streptococcus pyogenes, and optionally the protein is IdeS or IdeZ; or (ii) the protein having IgG endoglycosidase activity is derived from streptococci such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica IgG endoglycosidase from Saccharomyces meningoseptica, optionally wherein the protein is EndoS, CP40, EndoE, or EndoF2; Pharmaceutical compositions.

6. A pharmaceutical composition described in any one of claims 1 to 5, comprising a protein having the activity of the IgG cysteine ​​protease or IgG endoglycosidase, which is administered before the adoptive cell transfer immunotherapy is administered, after the adoptive cell transfer immunotherapy is administered, or simultaneously with the adoptive cell transfer immunotherapy.

7. A pharmaceutical composition for treating cancer, an autoimmune condition, a condition mediated by harmful antibody-producing B cells, or an infectious disease, the pharmaceutical composition comprising a protein having IgG cysteine ​​protease or IgG endoglycosidase activity, administered to a patient who has previously undergone and / or will undergo adoptive cell transfer immunotherapy, wherein: (i) the protein having IgG cysteine ​​protease activity is an IgG cysteine ​​protease derived from streptococci, such as Streptococcus pyogenes, and optionally the protein is IdeS or IdeZ; or (ii) the protein having IgG endoglycosidase activity is derived from streptococci such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica IgG endoglycosidase from Saccharomyces meningoseptica, optionally wherein the protein is EndoS, CP40, EndoE, or EndoF2; Pharmaceutical compositions.

8. A pharmaceutical composition for treating cancer, an autoimmune condition, a condition mediated by harmful antibody-producing B cells, or an infectious disease, the pharmaceutical composition comprising cells used in adoptive cell transfer immunotherapy, administered to a patient who has previously received and / or will receive a protein having IgG cysteine ​​protease or IgG endoglycosidase activity, wherein: (i) the protein having IgG cysteine ​​protease activity is an IgG cysteine ​​protease derived from streptococci, such as Streptococcus pyogenes, and optionally the protein is IdeS or IdeZ; or (ii) the protein having IgG endoglycosidase activity is derived from streptococci such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica IgG endoglycosidase from Saccharomyces meningoseptica, optionally wherein the protein is EndoS, CP40, EndoE, or EndoF2; Pharmaceutical compositions.

9. A pharmaceutical composition described in any one of claims 1 to 8, comprising cells used in adoptive cell transfer immunotherapy, which comprises administering T cells, natural killer cells, or dendritic cells expressing a chimeric antigen receptor or a T cell receptor.

10. A pharmaceutical composition described in any one of claims 1 to 9, comprising cells used in adoptive cell transfer immunotherapy, which increase the survival rate and / or proliferation of administered cells.

11. A pharmaceutical composition according to any one of claims 1 to 10, comprising cells used in adoptive cell transfer immunotherapy, which reduce antibody-mediated complement deposition, complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), wasting and / or receptor-activated cell death of administered cells.

12. (iii) the protein having IgG cysteine ​​protease activity is a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2, 4, or 5, or a fragment or variant thereof having IgG cysteine ​​protease activity; or (iv) The pharmaceutical composition of claim 1, wherein the protein having IgG endoglycosidase activity is a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 90, or a fragment or variant thereof having IgG endoglycosidase activity.

13. (iii) the protein having IgG cysteine ​​protease activity is a polypeptide having a sequence that is at least 80% identical, e.g., at least 85%, at least 90%, at least 95%, or at least 99% identical, to SEQ ID NO: 2, 4, or 5, or the IgG cysteine ​​protease comprises or consists of the sequence of any one of SEQ ID NOs: 6-25 and 55-69, optionally comprising an additional methionine at the N-terminus and / or a histidine tag at the C-terminus; or (iv) The pharmaceutical composition of claim 1, wherein the protein having IgG endoglycosidase activity is a polypeptide having a sequence that is at least 80% identical, such as at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

90.

14. 1. A pharmaceutical composition for use in conditioning or preparing a patient for adoptive cell transfer immunotherapy, for use in enhancing the survival and activity of transferred cells, or for use in reducing serum IgG levels or reducing complement or Fc receptor binding by serum IgG molecules in a patient undergoing or to be undergoing adoptive cell transfer immunotherapy, the pharmaceutical composition comprising a protein having the activity of an IgG cysteine ​​protease or an IgG endoglycosidase, wherein: (i) the protein having IgG cysteine ​​protease activity is an IgG cysteine ​​protease derived from streptococci, such as Streptococcus pyogenes, and optionally the protein is IdeS or IdeZ; or (ii) the protein having IgG endoglycosidase activity is derived from streptococci such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica IgG endoglycosidase from Saccharomyces meningoseptica, optionally wherein the protein is EndoS, CP40, EndoE, or EndoF2; Pharmaceutical compositions.

15. A protein having IgG cysteine ​​protease or IgG endoglycosidase activity is formulated in a first pharmaceutical composition, and cells used in the adoptive cell transfer immunotherapy are formulated in a second pharmaceutical composition, wherein: (i) the protein having IgG cysteine ​​protease activity is an IgG cysteine ​​protease derived from streptococci, such as Streptococcus pyogenes, and optionally the protein is IdeS or IdeZ; or (ii) the protein having IgG endoglycosidase activity is derived from streptococci such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica IgG endoglycosidase from Saccharomyces meningoseptica, optionally wherein the protein is EndoS, CP40, EndoE, or EndoF2; The pharmaceutical composition according to any one of claims 1 to 14. (i) a protein having IgG cysteine ​​protease or IgG endoglycosidase activity is synthesized using Fmoc solid-phase chemistry, Boc solid-phase chemistry, or solution-phase peptide synthesis; or (ii) the protein having IgG cysteine ​​protease or IgG endoglycosidase activity is synthesized by transforming a bacterial cell with a nucleic acid molecule or vector encoding the protein having IgG cysteine ​​protease or IgG endoglycosidase activity, wherein: (i) the protein having IgG cysteine ​​protease activity is an IgG cysteine ​​protease derived from streptococci, such as Streptococcus pyogenes, and optionally the protein is IdeS or IdeZ; or (ii) the protein having IgG endoglycosidase activity is derived from streptococci such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica IgG endoglycosidase from Saccharomyces meningoseptica, optionally wherein the protein is EndoS, CP40, EndoE, or EndoF2; The pharmaceutical composition according to any one of claims 1 to 15.

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