Methods for enhancing adoptive cell transfer immunotherapy

Abstract

The present invention relates to a method for enhancing adoptive cell transfer immunotherapy targeted to immunoglobulin light chains 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 that targets immunoglobulin light chains. [Background technology]

[0002] Adoptive cell transfer immunotherapy is a newly established class of therapy for treating various diseases, including cancer. For the treatment of solid tumors or tumors of hematopoietic origin, ideally the patient's T cells specific for tumor-associated antigens are expanded in vitro and then reinjected. The number, specificity, activity, and efficacy of T cells are limiting factors of these treatments. One exemplary factor of tumor escape is the downregulation of HLA, which is necessary for tumor antigen presentation.

[0003] Chimeric antigen receptor (CAR)-transfected cells are an emerging field of cell-based immunotherapy that exploits the inherent cytolytic potential of the patient's own NK 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) that receives its specificity from a tumor-specific scFv domain. Chimeric antigen receptor (CAR)-expressing T cells combine the antitumor activity of cytotoxic T cells with the specificity and affinity of scFv elements from tumor-associated antigen-specific antibodies. Autologous T cells can be collected in sufficient numbers from a patient before being transfected in vitro with the CAR of choice. Further expansion results in large numbers of HLA-independent tumor-specific cytotoxic T cells.

[0004] Adoptive cell transfer immunotherapy targeting immunoglobulin light chains is under development to treat lymphoid malignancies (Ranganathan et al., Clin Cancer Res, 2021 and Vera et al., Blood 2006;108). B lymphocytes express surface monoclonal immunoglobulins with either kappa or lambda light chains, as do many lymphoid malignancies. Therefore, adoptive cell transfer immunotherapy targeting immunoglobulin light chains expressed by lymphoid malignancies is expected to have anticancer activity. Since non-cancerous cells expressing other immunoglobulin light chain series are not affected by adoptive cell transfer immunotherapy, such adoptive cell transfer immunotherapy is also expected to have minimal adverse effects on the patient's immunity.

[0005] The efficacy of CAR T cell (CAR-T) therapy may be limited by the persistence of CAR T cell survival and activity in patients after injection. However, the factors limiting CAR T cell survival and efficacy have not yet been fully explored and remain controversial. Although, theoretically, humoral responses to CAR T cells may trigger antibody-mediated effector mechanisms, little direct evidence has been found to show that the limiting factor of CAR T cell success is caused by antibodies. Instead, tumor escape due to loss of target antigens such as CD19 (Majzner and Mackall, 2018, Cancer Discov., 8(10):1219-26), lack of polyfunctionality of CAR T cells (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 for the success of CAR T cells. Repeated infusion failures are often due to T cell-mediated cytotoxicity caused, for example, by the presentation of mouse peptides by scFvs in the HLA of CAR T cells (Turtle et al.,-2016, J. Clin. Invest.,126(6):2123-2138). In line with this, tisagenlecleucel (Kymriah), an FDA-approved CAR T cell, is said to be immune to pre-existing and induced humoral immunity (Thudium-Muller, J. Clin. Oncology,36(15),2018).

[0006] Immunoglobulin G-degrading cysteine ​​protease, ImmuRifidase (IdeS), is an IgG endopeptidase currently being developed as a rapid desensitization treatment in kidney transplantation. ImmuRifidase is highly specific and cleaves all subclasses of human IgG. ImmuRifidase may be useful to reduce competition for Fc receptors when administering antibody drug products (WO 2016 / 012285).

[0007] Improved methods for treating cancer are needed. Summary of the Invention

[0008] The present invention provides a method for improving the patient benefit of adoptive cell transfer immunotherapy targeting immunoglobulin light chains, comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with the immunoglobulin light chain-targeting adoptive cell transfer immunotherapy. The inventors have revealed that the efficacy of immunoglobulin light chain-targeting adoptive cell transfer immunotherapy can be reduced by stimulation of cells by immunoglobulins in plasma, leading to cell exhaustion and reduced function. In addition, the limited survival time and sustained activity of transferred cells such as CAR-T cells can limit the efficacy of immunoglobulin light chain-targeting adoptive cell transfer immunotherapy. The inventors have shown in the examples that a protein having IgG cysteine ​​protease or IgG endoglycosidase activity can remove and / or inactivate soluble immunoglobulins, reduce off-tumor cell stimulation, and thus improve anti-cancer activity. Furthermore, the efficacy of adoptive cell transfer immunotherapy targeting immunoglobulin light chains may be reduced by cell surface receptors binding to soluble immunoglobulins, which may block the interaction of adoptive cell transfer immunotherapy with target tumor cells. Proteins with IgG cysteine ​​protease or IgG endoglycosidase activity may be effective in digesting soluble antibodies and increasing receptor binding to targets on tumors. The inventors also demonstrated in the Examples that proteins with IgG cysteine ​​protease or IgG endoglycosidase activity may protect transferred cells. Specifically, the inventors revealed that cell surface receptor-specific antibodies, including pre-existing antibodies and antibodies generated after administration of transferred cells, may shorten the potential of transferred cells, and the therapeutic effect of transferred cells may benefit from removing the effector function of antibodies through conditioning of the recipient. Soluble antibodies bound by adoptive cell transfer immunotherapy targeting immunoglobulin light chains may also have similar adverse effects on transferred cells. Thus, administration of proteins with IgG cysteine ​​protease or IgG endoglycosidase activity can enhance the survival and activity of transferred cells, providing an improved adoptive cell transfer immunotherapy treatment.The inventors have also demonstrated that cell surface receptor-specific antibodies against, for example, receptor constructs 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 the antibodies and increasing binding of the receptor to its target.

[0009] As demonstrated in the Examples, treatment with the IgG cysteine ​​protease ImmuRifidase (IdeS) can reduce or prevent cytokine production by CAR-T cells targeting immunoglobulin light chains in the presence of soluble immunoglobulin. As further demonstrated in the Examples, treatment with the IgG cysteine ​​protease ImmuRifidase (IdeS) and the IgG endoglycosidase EndoS relieves some of the limiting effector functions of antibodies and other cell-based therapeutics on CAR-T cells. Thus, the present invention provides a method for improving the patient benefit of adoptive cell transfer immunotherapy targeting immunoglobulin light chains, comprising administering a protein having the activity of an IgG cysteine ​​protease or an IgG endoglycosidase in combination with the immunoglobulin light chain-targeting adoptive cell transfer immunotherapy. In a preferred embodiment, the present invention provides a method of treating cancer, particularly B cell neoplasms, comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy targeted to immunoglobulin light chains.

[0010] One of the limiting factors of CAR-T therapy and other adoptive cell transfer immunotherapies targeting immunoglobulin light chains may be that anti-kappa and anti-lambda CAR constructs bind to immunoglobulins in plasma. This may stimulate CAR-T cells outside the tumor, resulting in cell exhaustion and reduced function. As a result, the presence of soluble immunoglobulins in the patient may cause CAR-T to be exhausted faster and maximal tumor cell cytotoxicity may not be achieved. Furthermore, soluble immunoglobulins may block the interaction between CAR-T therapy and its target on tumor cells. According to the present invention, IgG cysteine ​​proteases and IgG endoglycosidases can be used to reduce off-tumor stimulation and binding of CAR-T cells by plasma immunoglobulins. When plasma immunoglobulins are cleaved, their stability and half-life are reduced, and the adverse effects on CAR-T cells are reduced even if plasma immunoglobulins are not completely destroyed (e.g., antibody fragments remain). Also, cleaving immunoglobulins can prevent cross-linking between CAR and FcgR, which could otherwise lead to off-tumor activation and exhaustion.

[0011] One further limiting factor 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 various antibody-mediated effector mechanisms such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), attrition due to osmotic stimulation or receptor activation-induced cell death. After injection, such antibodies may reduce the survival and persistence of activity of CAR T cells in patients. Infusion of CAR-T cells induces elevated levels of antibodies against the chimeric receptor, which may prevent the interaction of the CAR with its target cells during the first course of treatment and even limit its expansion and persistence. Successful second rounds of treatment may be even more difficult due to elevated levels and possibly higher affinity of anti-drug antibodies (ADA). Even if the CAR T cells are cells 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. Some immunogenic moieties may be junction regions between receptor components, but most notably the scFv moieties, which in the early stages of CAR-T development were taken from, for example, tumor-specific mouse IgG. 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 be 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). On the other hand, the data in 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 inactivation of cell surface receptor-specific antibodies.Soluble antibodies bound by adoptive cell transfer immunotherapy targeting immunoglobulin light chains can also activate similar antibody-mediated effector mechanisms against the transferred cells, and these adverse effects can be reduced using proteins with IgG cysteine ​​protease or IgG endoglycosidase activity according to the present invention.

[0012] There are various possible mechanisms by which the activity, expansion, and survival of transferred cells, such as CAR T cells, can be affected by antibodies. For example, CAR-specific antibodies can promote the destruction of CAR T cells by complement deposition CDC and / or ADCP. Soluble antibodies bound to cell surface receptors can also promote the destruction of CAR-T cells by the same mechanisms. The examples demonstrate that proteins with IgG cysteine ​​protease or IgG endoglycosidase activity can be effective in mitigating these processes, thereby improving adoptive cell transfer immunotherapy. Antibodies themselves can also bind to receptors, causing 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.

[0013] In light of these developments, the present invention provides a method for improving the patient benefit of immunoglobulin light chain targeted adoptive cell transfer immunotherapy, comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with immunoglobulin light chain targeted adoptive cell transfer immunotherapy. Preferably, the immunotherapy is a cancer therapy, more preferably a therapy for B cell neoplasms. In other embodiments, the immunotherapy is a therapy for an antibody-mediated autoimmune disease. Optionally, the disease is selected from the group consisting of juvenile arthritis (particularly juvenile idiopathic arthritis), rheumatoid arthritis, generalized myxedematous lichen (sclerosing myxedema), Graves' disease, IgA-driven bullous dermatosis, IgG4-driven bullous pemphigoid, Sjogren's syndrome, and lupus mastitis.

[0014] In certain embodiments, the protein is administered prior to adoptive cell transfer immunotherapy. Pre-administration of the protein can remove and / or inactivate immunoglobulins from plasma, maximizing the anti-cancer activity of the cells when administered. The examples also 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 been administered adoptive cell transfer immunotherapy can bind to cells such as receptor constructs and CAR T cells and mediate deleterious 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 certain embodiments, the protein is administered after administering adoptive cell transfer immunotherapy. The examples demonstrate that Ides and EndoS are effective in inactivating induced antibodies, since immune thrombocytopenia was reduced when Ides and EndoS were administered after antiplatelet 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.Since adoptive cell transfer immunotherapy can induce such alloantibodies, the proteins of the present invention may be useful when administered after administration of adoptive cell transfer immunotherapy.

[0015] The present invention also provides a method for improving the benefit of adoptive cell transfer immunotherapy to a patient, for increasing the survival time and / or proliferation of cells administered as part of adoptive cell transfer, for conditioning or preparing a patient for adoptive cell transfer immunotherapy, for lowering plasma IgG levels or reducing complement and / or Fc receptor binding by plasma IgG molecules in a patient undergoing or to undergo adoptive cell transfer immunotherapy, comprising 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. Preferably, the immunotherapy is a cancer therapy, more preferably a therapy for B-cell neoplasms.

[0016] The present invention also provides a method for increasing the efficacy of adoptive cell transfer therapy or increasing the binding between the cell surface receptor of adoptive cell transfer therapy and its target, comprising administering a protein having the activity of IgG cysteine ​​protease or IgG endoglycosidase before, after, or simultaneously with an adoptive cell transfer immunotherapy targeting immunoglobulin light chain. 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 a kappa light chain or a lambda light chain, and preferably the cell surface receptor is an anti-kappa or anti-lambda CAR.

[0017] In certain embodiments of any of the methods of the invention, the protein having the activity of an IgG cysteine ​​protease or an IgG endoglycosidase improves the benefit to the patient of adoptive cell transfer immunotherapy or improves the treatment of cancer by removing and / or inactivating immunoglobulins in the plasma that bind and cause cell wasting by adoptive cell transfer therapy. Also, in certain embodiments of any of the methods of the invention, the protein having the activity of an IgG cysteine ​​protease or an IgG endoglycosidase improves the benefit to the patient of adoptive cell transfer immunotherapy or improves the treatment of cancer by removing IgG antibodies that inhibit the binding of the cell surface receptor of adoptive cell transfer therapy to its target. The antibodies may bind to the cell surface receptor, in particular the CAR, or may bind to the CAR adaptor molecule, or may bind to the target itself, and may sterically inhibit the binding of the receptor to its target.

[0018] 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 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, 5, or 91. The examples demonstrate that such polypeptides are effective in protecting cells and removing and / or inactivating circulating immunoglobulins.

[0019] In a further embodiment, the IgG endoglycosidase is an EndoS polypeptide, e.g., 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. The examples demonstrate that such polypeptides are effective in protecting cells and removing and / or inactivating circulating immunoglobulins.

[0020] In a particularly preferred embodiment of the invention, the method comprises administering an IdeS polypeptide in combination with CAR-T therapy targeting immunoglobulin light chains in the treatment of B cell neoplasms.

[0021] In a particularly preferred embodiment, the method of the invention comprises administering an IdeS polypeptide prior to adoptive cellular immunotherapy, for example administering an IdeS polypeptide to a patient who is to undergo adoptive cellular immunotherapy that targets immunoglobulin light chains. Such a method reduces immunoglobulins in the plasma that may otherwise deplete and reduce the efficacy of the cellular therapy or block the interaction between the cellular therapy and its target on the tumor cells.

[0022] 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.

[0023] In a preferred embodiment of any aspect of the invention, the adoptive cellular immunotherapy does not express a protein having IgG cysteine ​​protease or IgG endoglycosidase activity. In a preferred embodiment of any aspect of the invention, the protein having IgG cysteine ​​protease or IgG endoglycosidase activity is administered to the patient in the form of an isolated protein or a composition comprising the isolated protein. This measure is advantageous because the protein having IgG cysteine ​​protease or IgG endoglycosidase activity is not directly bound to the transferred cells and is therefore less likely to cleave the immunoglobulin light chains expressed by the tumor cells. The adoptive cellular immunotherapy and the protein having IgG cysteine ​​protease or IgG endoglycosidase activity can be administered separately in separate compositions, even if administered simultaneously. Such an approach is expected to be particularly effective, for example, because plasma immunoglobulins can be systemically removed or inactivated to maximize the activity of the administered adoptive cellular immunotherapy.

[0024] In certain embodiments of the invention, proteins with IgG cysteine ​​protease or IgG endoglycosidase activity are used to inactivate soluble immunoglobulins targeted by adoptive cellular immunotherapy, but not to inactivate anti-drug antibodies to the adoptive cellular immunotherapy, hi certain embodiments, patients treated according to the invention do not have anti-drug antibodies to the adoptive cellular immunotherapy, or are not predicted or suspected to have such antibodies.

[0025] Further embodiments of the present invention are provided in the following numbered paragraphs. 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 said adoptive cell transfer immunotherapy targeting immunoglobulin light chains. 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 targeting immunoglobulin light chains. 3. The method of embodiment 1 or 2, wherein the protein having IgG cysteine ​​protease or IgG endoglycosidase activity is administered before administering adoptive cell transfer immunotherapy, or the protein having IgG cysteine ​​protease or IgG endoglycosidase activity is administered after administering adoptive cell transfer immunotherapy, or the protein having IgG cysteine ​​protease or IgG endoglycosidase activity is administered before administering a second or subsequent adoptive cell transfer immunotherapy. 4. A method of treating cancer comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity to a patient who has previously undergone and / or will undergo adoptive cell transfer immunotherapy targeted to immunoglobulin light chains. 5. A method of treating cancer comprising administering immunoglobulin light chain-targeted 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. 6. A method of treating an antibody-mediated autoimmune disease comprising administering immunoglobulin light chain-targeted adoptive cell transfer immunotherapy to a patient who has previously received and / or will receive a protein having IgG cysteine ​​protease or IgG endoglycosidase activity; Optionally, the antibody-mediated autoimmune disease is selected from the group consisting of juvenile arthritis (particularly juvenile idiopathic arthritis), rheumatoid arthritis, generalized myxedematous lichen (sclerosing myxedema), Graves' disease, IgA-driven bullous dermatosis, IgG4-driven bullous pemphigoid, Sjogren's syndrome, and lupus mastitis. 7. The method of any one of embodiments 1-6, 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. 8. The method of embodiment 7, wherein the chimeric antigen receptor or T cell receptor comprises a binding domain, such as an scFv, that specifically binds to an immunoglobulin light chain, such as a human kappa immunoglobulin light chain or a human lambda immunoglobulin light chain. 9. The method of any one of embodiments 1 to 8, which is a method for treating cancer, wherein the cancer is a B cell neoplasm, e.g., a B cell lymphoma or a B cell leukemia. 10. The method of embodiment 9, wherein the cancer is selected from the group consisting of precursor B-cell acute lymphoblastic leukemia / lymphoblastic lymphoma (LBL), B-cell acute lymphoblastic leukemia; B-cell chronic lymphocytic leukemia (CLL); small lymphocytic lymphoma; B-cell prolymphocytic leukemia; lymphoplasmacytic lymphoma / immunocytoma; mantle cell lymphoma; follicular lymphoma; extranodal marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) type; nodal marginal zone B-cell lymphoma; splenic marginal zone lymphoma; hairy cell leukemia; plasmacytoma / plasma cell myeloma; diffuse large B-cell lymphoma (such as primary mediastinal B-cell lymphoma), Burkitt lymphoma, Burkitt-like lymphoma; primary central nervous system (CNS) lymphoma, and primary intraocular lymphoma. 11. A method according to any one of embodiments 1 to 10, which increases the activity of cells administered in adoptive cell transfer immunotherapy. 12. A method according to any one of embodiments 1 to 11, which increases the viability and / or proliferation of cells administered in adoptive cell transfer immunotherapy. 13. The method of any of embodiments 1-12, which 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. 14. (i) the protein having IgG cysteine ​​protease activity is an IgG cysteine ​​protease from a streptococcus, such as Streptococcus pyogenes, and optionally the protein is IdeS or IdeZ; or (ii) The method according to any one of embodiments 1 to 13, wherein the protein having IgG endoglycosidase activity is an IgG endoglycosidase from a streptococcus, such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica, and optionally the protein is EndoS, CP40, EndoE, or EndoF2. 15. (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, 5, or 91, or a fragment or variant thereof having IgG cysteine ​​protease activity; or (ii) The method of embodiment 14, 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. 16. (i) a 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, 5, or 91, 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 (ii) The method of embodiment 14, 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. 17. A protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in conditioning or preparing a patient for immunoglobulin light chain targeted adoptive cell transfer immunotherapy. 18. A protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving the patient benefit of adoptive cell transfer immunotherapy targeted to immunoglobulin light chains. 19. A protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing plasma IgG levels or decreasing complement or Fc receptor binding by plasma IgG molecules in patients undergoing or to undergo adoptive cell transfer immunotherapy targeted to immunoglobulin light chains. [Brief description of the drawings]

[0026] [Figure 1] Antibody-targeted deposition of C1q and C4d on Daudi cells can be avoided by treatment with IdeS or EndoS. Daudi cells were incubated with RTX (or IgG1 isotype control) for approximately 2 hours with titrations of IdeS (A+B) or EndoS (C+D), followed by addition of human serum complement for another 2 hours. Cells were aliquoted and stained with anti-human C1q (A+C) or C4d (B+D), followed by final detection with SA-PE. Cells were analyzed in FL2 using an Accuri C6 flow cytometer. MFI values ​​are listed. [Diagram 2]ADCC can be blocked or reduced with IdeS or EndoS. CD20-positive Daudi cells were incubated with RTX (titrated from 0.2 μg / mL to 50 μg / mL) together with 50 μg / mL IdeS (squares), EndoS (triangles), or medium (diamonds). CDC was induced through the addition of baby rabbit serum as a complement source. Results were normalized as cell viability without RTX, which was set as 100% after 90 min incubation. Cell cytotoxicity was assessed using cell counting kit-8 (CCK8). Normal positive control groups were administered only titrated RTX without therapeutic enzyme. This represents the maximum toxicity of RTX at the indicated concentrations. [Diagram 3] IdeS treatment of opsonized target cells blocks ADCP. Calcein-stained Daudi cells were opsonized with titrated RTX (from 7.5 μg / mL to 0.01 μg / mL) and incubated with either 40 μg / mL IdeS (squares), EndoS (triangles), or no enzyme (medium only, diamonds) prior to the addition of FarRed-stained THP1 effector cells. After 2 h of incubation, cells were fixed and analyzed by flow cytometer in FL2 and FL4. Gated FL2-positive cells were taken as 100%. This population was further divided into FL2 single-positive and FL2-FL4 double-positive cells, which were read for THP1 phagocytosed 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 purified IgG at 0.25 mg / mouse in 200 μL PBS. Platelet counts were determined on day 1 using an automated cell counter VetScan HM5. [Diagram 5]Antibody-induced ITP can be blocked by IdeS treatment in vivo. BALB / c mice were primed for ITP by a single ip injection of purified intact rabbit anti-mouse platelet IgG (0.25 mg / mouse). One hour after induction of ITP, treatment with IdeS was administered iv at three different doses (0.2 μg / mouse, 2 μg / mouse, 20 μg / mouse). The "PBS only" (circles) treatment group did not receive IdeS but only anti-PLT-IgG as a positive control for ITP induction. Naïve mice that received only carrier solution (diamonds) 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 priming for ITP or with PBS only (circles) as a control for normal levels of platelets. Thirty minutes later, they were injected with a therapeutic ip injection with EndoS (10 μg / mouse, 30 μg / mouse, 90 μg / mouse). ITP induction 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]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. Similarly, binding of polyclonal rabbit anti-mouse F(ab')2 and anti-human F(ab')2 (10 μg / mL) was also assessed using anti-CD19 CAR-Jurkat T cell line (E) (CARJ-ZP005, Creative biolabs) by FACS analysis and expressed as MFI. [Figure 8] 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 >10ng / mL. (B) ELISA screened HAMA sera were incubated with anti-CD19-CAR-Jurkat T cells. Jurkat wild type cells served as CAR negative staining control. After incubation with PE-conjugated goat anti-human Fc detection antibody, cells were analyzed by flow cytometry. (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 mechanism with anti-CD19 CAR-Jurkat T cells. (D) Sera from HLA-sensitized patients were screened for alloreactivity against anti-CD19-CAR-Jurkat T cells. Sera from OneLambda were used as HLA class I positive and negative controls. [Figure 9]ADCP induced by CAR-specific antibodies can be blocked by immurifidase treatment - polyclonal anti-F(ab')2 antibody opsonization of anti-CD19 CAR-Jurkat T cells for ADCP is blocked by immurifidase treatment. For flow cytometry-based ADCP analysis, target cells including (A, B) anti-CD19 CAR-Jurkat, (D, E) Jurkat wild-type cells, and (C, F) CD20- and BCR-expressing Daudi cells were stained with calcein AM and then incubated with the indicated concentrations of immurifidase-treated rabbit (A, C, D) anti-mouse F(ab')2 or (B, E, F) anti-human F(ab')2. The monocytic phagocytic effector cell line THP-1 was stained with CellTrace FarRed before addition to target cells for 90 min. 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] ImmuRifidase blocks 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 immuRifidase (10 μg / mL). After washing, target cells were incubated with FcγRI-expressing reporter cells for 6 h at 37°C to induce ADCP. Luciferase activity, a result of reporter cell activation, was measured using a luminescence reader, and luminescence signals (RLU) are presented as the mean fold change of induction ± SD. [Figure 11]ADCC (V158) induced by anti-CD19 CAR-specific antibodies is blocked by immunofluorescence treatment. 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 (20 μg / mL). Induction of ADCC was quantified using a luciferase reporter bioassay of 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 / absence of immunofluorescence. 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 treatment. 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 (20 μg / mL). Induction of ADCC was quantified using a luciferase reporter bioassay of 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 / absence of immunofluorescence. 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 enzyme treatment. A reporter cell line expressing the CD16 FcγRIIIa high affinity (V158) allele was used to assay for ADCC induction. Mouse mAb FMC63-based scFv-CD19-CAR Jurkat cell lines were incubated with normal human serum samples previously tested by ELISA for HAMA levels against mouse IgG in the presence / absence of immunofluorescence enzyme, and 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] ImmuRifidase treatment of serum improves the association of target CD19-protein with anti-CD19 CAR T cells - CD19-protein binding to serum-exposed anti-CD19 CAR T cells can be increased by immuRifidase treatment. Anti-CD19 CAR-Jurkat T cells were incubated with HAMA positive and negative serum samples with or without immuRifidase (10 μg / mL). IHAc (1 mM) was added to all samples to inactivate immuRifidase during the next step. Serum samples were incubated with anti-CD19 CAR-Jurkat T cells to bind F(ab')2 to anti-CD19 CAR for each possible IgG. After washing, recombinant atto-647N-labeled human CD19-Fc protein (ATM9269, R&D systems) was added to the cells and the interaction of anti-CD19 CAR with CD19-target protein was evaluated by flow cytometry. Results are presented as median fluorescence intensity (FI). [Figure 15]Absence of IFNγ production in in vitro co-culture of CAR-T cells targeting immunoglobulin light chains with soluble immunoglobulin. T cells from two healthy human donors (BC170909 and BC170803) were transduced with either CD19.CAR, kappa.CD28, or non-transduced (NTD). NTD and CD19.CAR served as negative controls, and would not predict IFNγ production from either cell type when plated with soluble immunoglobulin. They were then plated in serum with or without IdeS, either without soluble immunoglobulin (denoted TCM) or varying soluble immunoglobulin concentrations ranging from 10%, 50%, and 90% soluble immunoglobulin in serum. Supernatants were then harvested from the co-cultures 24 hours after plating, and IFNγ concentrations were measured by ELISA. NTD and CD19.CAR did not produce IFNγ in either plating condition, as expected. Kappa.CD28 (denoted as K28 in the graph) produced increased amounts of IFNγ in the presence of soluble immunoglobulin. However, the ability of kappa.CD28 to produce IFNγ is abolished when IdeS is added to the co-culture, as shown on the right side of the graph. In each group of bars, the bars represent NTD, CD19, and K28 from left to right. [Figure 16]Suppression of IFNγ production seen in kappa.CAR. T cells from another healthy donor were transduced with CD19.CAR, kappa.CD28, a CAR construct targeting lambda light chain (lambda.CD28), and NTD. Kappa.CD28 continued to show IFNγ production when plated under increasing concentrations of soluble immunoglobulin, and IFNγ production was reduced in the presence of IdeS. The lambda.CD28 CAR construct showed no significant difference with or without the IdeS molecule. This is likely due to the polyclonal nature of the soluble immunoglobulin serum used for co-culture, and the ratio of lambda light chains being below the threshold required to activate lambda.CD28 CAR T cells. In Figure 16A, in each group of bars, the bars show NTD 1E6, NTD 2E6, and NTD 3E6 from left to right. In Figure 16B, in each group of bars, the bars represent from left to right CD19.CD28z 1E6, CD19.CD28z 2E6, CD19.CD28z 3E6. In Figure 16C, in each group of bars, the bars represent from left to right kappa.CD28 1E6, kappa.CD28 2E6, kappa.CD28 3E6. In Figure 16D, in each group of bars, the bars represent from left to right lambda.CD28 1E6, lambda.CD28 2E6, lambda.CD28 3E6.

[0027] A brief description of the sequence SEQ ID NO: 1 is the complete sequence of IdeS, including the N-terminal methionine and signal sequence. Also available as NCBI reference sequence WP_010922160.1.

[0028] SEQ ID NO:2 is the mature sequence of IdeS, lacking the N-terminal methionine and signal sequence. Also available as Genbank Accession No. ADF13949.1.

[0029] SEQ ID NO: 3 is the complete sequence of IdeZ, including the N-terminal methionine and signal sequence. Also available as NCBI reference sequence WP_014622780.1.

[0030] SEQ ID NO:4 is the mature sequence of IdeZ, lacking the N-terminal methionine and signal sequence.

[0031] 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.

[0032] SEQ ID NOs:6-25 are exemplary protease sequences for use in the methods of the present invention.

[0033] 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).

[0034] 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).

[0035] 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).

[0036] SEQ ID NO:29 is the consecutive sequence PLTPEQFRYNN corresponding to positions 63 to 73 of SEQ ID NO:3.

[0037] SEQ ID NO:30 is a consecutive sequence PPANFTQG corresponding to positions 58 to 65 of SEQ ID NO:1.

[0038] SEQ ID NO:31 is the consecutive sequence DDYQRNATEAYAKEVPHQIT corresponding to positions 35 to 54 of SEQ ID NO:3.

[0039] SEQ ID NO:32 is the consecutive sequence DSFSANQEIRYSEVTPYHVT corresponding to positions 30 to 49 of SEQ ID NO:1.

[0040] SEQ ID NOs: 33 to 55 are nucleotide sequences encoding the above proteases.

[0041] SEQ ID NOs:56-69 are exemplary protease sequences for use in the methods of the present invention.

[0042] SEQ ID NO:70 is a contiguous sequence NQTN corresponding to positions 336 to 339 of SEQ ID NO:1.

[0043] SEQ ID NO:71 is the consecutive sequence DSFSANQEIR YSEVTPYHVT corresponding to positions 30 to 49 of SEQ ID NO:1.

[0044] SEQ ID NOs:72-86 are nucleotide sequences encoding the polypeptides disclosed herein.

[0045] SEQ ID NO:87 is the consecutive sequence SFSANQEIRY SEVTPYHVT corresponding to positions 31 to 49 of SEQ ID NO:1.

[0046] SEQ ID NO: 88 is the sequence DYQRNATEAY AKEVPHQIT, corresponding to positions 36 to 54 of the IdeZ polypeptide NCBI reference sequence WP_014622780.1.

[0047] SEQ ID NO: 89 is the sequence DDYQRNATEA YAKEVPHQIT which may be present at the N-terminus of a polypeptide of the invention.

[0048] SEQ ID NO: 90 shows the amino acid sequence of mature endoglycosidase S (EndoS). The entire sequence including the secretion signal is available in Genbank under the accession number AAK00850.1.

[0049] SEQ ID NO:91 represents a polypeptide having IgG cysteine ​​protease activity and that is more efficient at cleaving human IgG than IdeZ.

[0050] SEQ ID NO:92 is related to SEQ ID NO:91 and is identical to SEQ ID NO:91 except that the first 20 amino acids at the N-terminus of SEQ ID NO:91 are deleted.

[0051] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Methods for improving patient benefits from adoptive cell transfer immunotherapy 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 the immunoglobulin light chain-targeting adoptive cell transfer immunotherapy. The inventors have found that immunoglobulins in plasma can be bound by the immunoglobulin light chain-targeting transferred cells, which can cause exhaustion and block the interaction between the transferred cells and their targets on the tumor. The inventors have demonstrated that a protein having IgG cysteine ​​protease or IgG endoglycosidase activity can cleave immunoglobulins in plasma, which helps to maintain the anti-cancer activity of the transferred therapeutic cells. Thus, the activity of the immunoglobulin light chain-targeting adoptive cell transfer immunotherapy can be increased or maintained by administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0052] The inventors have also 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 the inventors have shown in the Examples that proteins with IgG cysteine ​​protease or IgG endoglycosidase activity can protect the transferred cells. Specifically, the inventors have demonstrated that cell surface receptor-specific antibodies can shorten the potential of transferred cells, and the therapeutic effect of transferred cells can benefit from removing the effector function of the antibody through conditioning of the recipient. Similarly, soluble antibodies bound by adoptive cell transfer immunotherapy targeting immunoglobulin light chains can also shorten the capacity of transferred cells. Thus, the administration of proteins with IgG cysteine ​​protease or IgG endoglycosidase activity can enhance the survival and activity of transferred cells, providing improved treatment. The inventors have also demonstrated that cell surface receptor-specific antibodies can interfere with the binding of the receptor of adoptive cell transfer immunotherapy to its target. Therefore, the efficacy and effectiveness of adoptive cell transfer immunotherapy can be increased by administering proteins with IgG cysteine ​​protease or IgG endoglycosidase activity.

[0053] In certain embodiments, the invention provides methods of maintaining or increasing the activity of cells administered as part of adoptive cell transfer targeting immunoglobulin light chains, comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity before, after, or concomitantly with the adoptive cell transfer immunotherapy. In certain embodiments, the invention provides methods of extending the survival and / or enhancing the 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.

[0054] In certain embodiments, the invention provides methods of conditioning or preparing a patient for immunoglobulin light chain targeted adoptive cell transfer immunotherapy comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0055] In certain embodiments, the invention provides methods of lowering plasma IgG levels or decreasing complement or Fc receptor binding by plasma IgG molecules in patients undergoing or to undergo adoptive cell transfer immunotherapy targeted to immunoglobulin light chains.

[0056] In certain embodiments, the present invention provides a method for increasing the efficacy of adoptive cell transfer therapy targeting immunoglobulin light chains, comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity before, after, or simultaneously with the adoptive cell transfer immunotherapy targeting immunoglobulin light chains.In certain embodiments, the present invention provides a method for increasing binding of a cell surface receptor of an adoptive cell transfer therapy to its target, comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity before, after, or simultaneously with the adoptive cell transfer immunotherapy.

[0057] In certain embodiments, a method for improving the patient benefit of adoptive cell transfer immunotherapy targeting immunoglobulin light chains includes administering a protein with IgG cysteine ​​protease or IgG endoglycosidase activity followed by adoptive cell transfer immunotherapy. Such a method allows for the removal and / or inactivation of immunoglobulins present in the plasma and allows for the protein to inactivate pre-existing anti-drug antibodies (ADAs) before administering the cells, allowing for better cell activity, expansion, and survival. ADAs can be bound to any part of any cell therapy, including expressed CAR or TCR or HLA antigens (especially in allogeneic therapy). The examples demonstrate that proteins such as IdeS are effective in removing antibodies that stimulate or block CAR-T cells targeting immunoglobulin light chains. The examples also 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 been treated with adoptive cell transfer immunotherapy can bind to cells such as receptor constructs and CAR T cells and mediate adverse effects such as ADCP, ADCC, and reduced target binding, all of which can be reduced by treatment with the proteins of the invention. The invention also provides proteins having IgG cysteine ​​protease or IgG endoglycosidase activity for use in conditioning patients for treatment with immunoglobulin light chain-targeted adoptive cell transfer immunotherapy. The invention also provides proteins having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing plasma IgG levels in patients who will be treated with immunoglobulin light chain-targeted adoptive cell transfer immunotherapy.The present invention also provides proteins having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing plasma IgG levels in patients who have previously donated blood for the development of immunoglobulin light chain targeted adoptive cell transfer immunotherapy and have not yet received immunoglobulin light chain targeted adoptive cell transfer immunotherapy.The present invention also provides immunoglobulin light chain targeted adoptive cell transfer immunotherapy compositions for treating patients who have previously been administered a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0058] In a preferred embodiment, the method for improving the patient benefit of adoptive cell transfer immunotherapy comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity prior to administering adoptive cell transfer immunotherapy. By such a method, soluble plasma immunoglobulins are cleaved so that they are unable to stimulate, deplete or block immunoglobulin light chain specific cells. Thus, the activity of the transferred cells is improved. The invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving the patient benefit of adoptive cell transfer immunotherapy that the patient is scheduled to undergo. The invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating a patient scheduled to undergo adoptive cell transfer immunotherapy. The invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing plasma IgG levels in a patient scheduled to undergo adoptive cell transfer immunotherapy. The present invention also provides adoptive cell transfer immunotherapy compositions for treating patients who have previously been administered a protein with IgG cysteine ​​protease or IgG endoglycosidase activity.

[0059] In a particular embodiment, the 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 allows inactivating existing anti-drug antibodies (ADA) and antibodies elicited by adoptive cell transfer immunotherapy. Such a method also allows inactivating soluble antibodies to which the transferred cells may otherwise bind. Thus, the expansion and survival rate of the transferred cells is improved. 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 antiplatelet specific antibodies. The 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 invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in the treatment of a patient who has previously undergone adoptive cell transfer immunotherapy.The invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in the reduction of plasma IgG levels in a patient who has previously undergone treatment with adoptive cell transfer immunotherapy.The invention also provides an adoptive cell transfer immunotherapy composition for treating a patient who will receive a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0060] In a further 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 (ADA) and antibodies elicited by adoptive cell transfer immunotherapy. Such a method also allows inactivation of soluble antibodies that may otherwise bind to the transferred cells. Thus, the expansion and survival rate of the transferred cells is improved.

[0061] In further embodiments, the method of 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 during two or more adoptive cell transfer immunotherapy treatments. In such a method, any ADA from previous injections is inactivated, allowing the transferred cells to better expand and survive. In such a method, soluble antibodies that may otherwise bind the transferred cells can also be inactivated. Also, soluble antibodies that may consume or block the transferred cells are removed or inactivated. Preferably, in such an embodiment, the first and second, and any subsequent adoptive cell transfer immunotherapy, use the same or similar constructs and cells. In such an embodiment, the similar constructs or cells may have ADA cross-reactive epitopes. The present invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in the treatment of a patient who has already undergone a first adoptive cell transfer immunotherapy and is 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 the reduction of IgG levels in a patient undergoing a regimen of multiple 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 a patient undergoing a regimen of multiple adoptive cell transfer immunotherapy treatment. The present invention also provides an adoptive cell transfer immunotherapy composition for treating a patient who has previously been administered an adoptive cell transfer immunotherapy composition and who has previously been administered a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

[0062] In a further 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 sessions of adoptive cell transfer immunotherapy. Such a method can inactivate antibodies elicited by adoptive cell transfer immunotherapy. Such a method can also inactivate soluble antibodies to which the transferred cells may otherwise bind. Also, soluble antibodies that may consume or block the transferred cells are removed or inactivated. Thus, the expansion and survival of the transferred cells is improved. The 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 been administered two or more sessions of adoptive cell transfer immunotherapy. The invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating a patient who has already been administered two or more sessions 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 treatments with adoptive cell transfer immunotherapy.

[0063] In certain embodiments of the invention, the method of improving the patient benefit of 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 doses of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity comprise administering the same enzyme. The repeated doses 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 doses of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity comprise administering different enzymes.

[0064] In a particular embodiment of the invention, the method of 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 co-administration of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity can increase the efficacy and effectiveness of adoptive cell transfer immunotherapy, since cell surface receptor-specific antibodies can interfere with the binding of the adoptive cell transfer immunotherapy receptor to its target, but binding can be increased by treatment with a protein of the invention.

[0065] Administration of a protein with IgG cysteine ​​protease or IgG endoglycosidase activity reduces the stability, half-life, and Fc effector function of plasma IgG immunoglobulins, thereby reducing the adverse effects on immunotherapy cells. Also, cleavage of IgG immunoglobulins can inhibit cross-linking between immunotherapy cells and FcgR, which may otherwise lead to off-tumor activation and exhaustion. In a preferred embodiment of the method of the present invention, administration of a protein with IgG cysteine ​​protease or IgG endoglycosidase activity cleaves all or substantially all IgG molecules present in the patient's plasma.

[0066] In a preferred embodiment of the method 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 plasma, hi a specific embodiment, 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 plasma.

[0067] In certain embodiments of the method of the invention, antibodies in the blood are removed or inactivated by administration of a protein having IgG cysteine ​​protease or IgG endoglycosidase activity. In certain embodiments, antibodies in lymph are also inactivated. In certain embodiments, antibodies in interstitial fluid are also inactivated. Thus, removal or inactivation of antibodies from plasma according to the invention may include removal or inactivation of antibodies from lymph and / or interstitial fluid. Removal or inactivation of antibodies in lymph and / or interstitial fluid delays replenishment of plasma antibodies, thereby reducing the adverse effect of plasma antibodies on the imported cells for a longer period.

[0068] In the method of the invention where a protein having IgG cysteine ​​protease or IgG endoglycosidase activity is administered prior to administering adoptive cell transfer immunotherapy, the two administrations and administrations are preferably separated by a time interval sufficient to cleave all or substantially all IgG molecules present in the plasma of the subject. The 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 be performed simultaneously with lymphodepletion (e.g., on the same day) or may be performed 1-7 days before or after lymphodepletion. In certain embodiments, administration of the protein is performed between lymphodepletion and administration of cell therapy. By "substantially all" it is typically meant that Fc receptor and complement binding by plasma 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 plasma IgG in the subject, as measured by any suitable assay in the subject.

[0069] In the methods of the 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, within 1 week, within 2 days, within 1 day, or within 5 hours of administration of the adoptive cell transfer immunotherapy.

[0070] In a preferred embodiment, the method of the invention is a method of reducing antibody-mediated complement deposition, CDC, ADCC, ADCP, exhaustion, 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 invention is a method of reducing antibody-mediated complement deposition, CDC, ADCC, ADCP, exhaustion, or receptor-activated cell death of cells subsequently administered to a patient in adoptive cell transfer immunotherapy. In a preferred embodiment, the invention provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing antibody-mediated complement deposition, CDC, ADCC, ADCP, exhaustion, or receptor-activated cell death of cells previously administered to a patient in adoptive cell transfer immunotherapy. In another preferred embodiment, the invention provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing antibody-mediated complement deposition, CDC, ADCC, ADCP, exhaustion, 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.

[0071] In a particular embodiment, the method of the present invention comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity to a patient with detectable levels of human anti-mouse antibodies (HAMA), an immunoglobulin with specificity for mouse immunoglobulins. The administration may be before, after, or at the same time as 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 the protein of the present invention. HAMA can also be induced in normal individuals by contact with mouse antigens. It can be expected that the frequency and concentration of HAMA will be even higher in patients receiving mouse mAb-based biologics, potentially even causing partial neutralization of these therapeutics.

[0072] In the methods of the invention, the IgG cysteine ​​protease or IgG endoglycosidase may be co-administered with an immunosuppressant. In the methods of the 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 route of administration. The amount of protease or endoglycosidase administered 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, especially 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 plasma that can bind to the protein does not exceed a threshold determined by the clinician. The amount of ADA in the subject's plasma that can bind to the protease can be determined by any suitable method, such as an agent-specific CAP FEIA (ImmunoCAP) test or a titer assay.

[0073] How to Treat Cancer The present invention provides a method of treating cancer comprising administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy targeting immunoglobulin light chains. The reduction in cytokine production and stimulation in the presence of soluble immunoglobulins shown in the examples indicates that administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity can reduce attrition and increase activity of transferred cells, providing improved cancer therapy. Also, the protection of cells shown in the examples indicates that administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity can increase viability and activity of transferred cells, providing improved cancer therapy.

[0074] In a preferred embodiment, the method of treating cancer comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity, followed by immunoglobulin light chain targeted adoptive cell transfer immunotherapy. The invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in conditioning a patient for cancer treatment with immunoglobulin light chain targeted adoptive cell transfer immunotherapy. The invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing plasma IgG levels in a patient who will be treated for cancer with immunoglobulin light chain targeted adoptive cell transfer immunotherapy. The invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing plasma IgG levels in a patient who has previously donated blood for the development of immunoglobulin light chain targeted adoptive cell transfer immunotherapy, but has not yet been treated with adoptive cell transfer immunotherapy. The present invention also provides adoptive cell transfer immunotherapy compositions targeting immunoglobulin light chains for treating cancer in patients who have previously been administered a protein with IgG cysteine ​​protease or IgG endoglycosidase activity.

[0075] In certain embodiments, the method of treating cancer comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after administering adoptive cell transfer immunotherapy. The invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving the benefit to a patient of previously administered adoptive cell transfer immunotherapy. The invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating cancer in a patient who has previously received adoptive cell transfer immunotherapy. The invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing plasma IgG levels in a patient who has previously received cancer treatment by adoptive cell transfer immunotherapy. The 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.

[0076] In a further embodiment, the method of treating cancer comprises administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity both before and after multiple administrations of adoptive cell transfer immunotherapy.

[0077] In further embodiments, 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 embodiments, the first and second and any subsequent adoptive cell transfer immunotherapy use the same or similar constructs and cells. The 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 to undergo a second adoptive cell transfer immunotherapy. The 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 invention also provides proteins having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving the benefit to patients of multiple cancer treatment regimens by adoptive cell transfer immunotherapy.The 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.

[0078] In a further 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 invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in improving the benefit of patients who have already been administered adoptive cell transfer immunotherapy two or more times. The invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in treating cancer in patients who have already been administered adoptive cell transfer immunotherapy two or more times. The invention also provides a protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in reducing plasma IgG levels in patients who have already been administered cancer treatment by adoptive cell transfer immunotherapy two or more times.

[0079] 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.

[0080] 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.

[0081] In the therapeutic method 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, and undergoes adoptive cell transfer immunotherapy. Such therapeutic treatment may result in remission, stabilization, 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 as suffering from cancer and suitable for adoptive cell transfer immunotherapy by any suitable means.

[0082] Adoptive cell transfer immunotherapy The methods of the present invention increase the benefits obtained from adoptive cell transfer (ACT) immunotherapy, thereby providing an improved method of treating cancer and other diseases such as antibody-mediated autoimmune diseases. ACT immunotherapy is a well-established and powerful approach, particularly for treating cancer. ACT involves the passive transfer of ex vivo grown cells, most commonly immune-derived cells, into the host with the goal of transferring the immunological functions and characteristics of the graft.

[0083] The ACT immunotherapy used according to the present invention targets the immunoglobulin light chain. The immunoglobulin light chain may be a kappa or lambda light chain. Preferably, the immunoglobulin light chain is a human immunoglobulin light chain. The ACT immunotherapy binds to the immunoglobulin light chain by expressing a receptor construct, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR), that includes a binding domain, such as an scFv, that specifically binds to the immunoglobulin light chain. An exemplary antibody that targets the kappa light chain of human immunoglobulin is produced by the CRL-1758 (ATCC) hybridoma. An exemplary antibody that targets the lambda light chain of human immunoglobulin is produced by the HP6054 (ATCC) hybridoma. Other antibodies that target the kappa or lambda light chain are also readily available. An scFv or alternative construct that binds to the kappa or lambda light chain of human immunoglobulin can be readily made using the variable regions of such antibodies.

[0084] The ACT may be autologous (e.g., isolated by leukapheresis, transduced, and selected immediately about 4 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 may 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.

[0085] ACT may also involve the transfer of autologous tumor-infiltrating lymphocytes (TILs), which can be used to treat patients with advanced solid tumors, such as melanoma, and hematological malignancies.

[0086] ACT may also involve the transfer of "off-the-shelf" allogeneic lymphocytes isolated, prepared, and stored (e.g., frozen) from healthy donors that can be used to treat patients with advanced solid tumors, such as melanoma, and hematological malignancies.

[0087] The adoptive cellular immunotherapy of the present invention may include administration of cells expressing chimeric antigen receptors (CARs) or T cell receptors (TCRs), and may include tumor infiltrating lymphocytes (TILs). The population of cells expressing CAR / TCRs that recognize antigens may include a population of activated T cells or 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, anti-kappa or lambda CARs. The population of cells expressing CAR / TCRs may include a population of gene-edited cells.

[0088] 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.

[0089] 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 kappa or lambda light chains by engineering a desired antigen binding domain that specifically binds to kappa or lambda light chains expressed 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.

[0090] In a preferred embodiment, the adoptive cell therapy, preferably CAR T cell therapy, uses cells that target kappa or lambda light chains. Exemplary CAR-T cells are described in Ranganathan et al., Clin Cancer Res, 2021 and Vera et al., Blood 2006;108. An exemplary antibody that targets the kappa light chain of human immunoglobulin is produced by the CRL-1758 (ATCC) hybridoma. An exemplary antibody that targets the kappa light chain of human immunoglobulin is produced by the HP6054 (ATCC) hybridoma. Other antibodies that target the kappa or lambda light chain are also readily available. scFv or alternative constructs that bind to the kappa or lambda light chain of human immunoglobulin can be readily generated using the variable regions of such antibodies, for example, as described in Ranganathan et al., Clin Cancer Res, 2021 and Vera et al., Blood 2006;108. A CAR-T construct for use in the present invention may comprise a human IgG1 CH2-CH3 region and a hinge and zeta chain of the TCR / CD3 complex, and optionally a CD28 domain. A CAR-T construct for use in the present invention may comprise a human CD8a hinge, and transmembrane domain, and a CD28 costimulatory endodomain, and an intracytoplasmic CD3z chain of the TCR / CD3 complex.

[0091] Preferred adoptive cell transfer immunotherapy is CAR T cell therapy (e.g., autologous cell therapy and allogeneic cell therapy). CAR T cell therapy is preferred for treating hematological malignancies such as ALL, AML, NHL, DLBCL, and CLL. Examples of approved CAR T cell therapy include, but are not limited to, KYMRIAH® (tisagenlecleucel) for treating NHL and DLBCL, and YESCARTA® (axicabtageneciloleucel) for treating NHL.

[0092] According to certain aspects of the invention, the population of cells expressing CAR / TCR or TIL may be autologous cells, allogeneic cells from another human donor, or xenogeneic cells from an animal of a different species.

[0093] 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 4 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., in frozen preparations, for one or more patients, as in the case of so-called "off-the-shelf" allogeneic CAR-T stem cell therapies.

[0094] According to a particular embodiment of the present invention, the population of cells expressing CAR / TCR may comprise a population of activated T cells or natural killer (NK) cells or dendritic cells expressing CAR / TCR that recognizes an antigen. Dendritic cells can not only directly kill tumors but also present antigens.

[0095] 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., tandem CAR). For example, CAR T cells may comprise an immunoglobulin light chain binding domain and a CD19 or CD22 binding domain, and thus can recognize and bind both immunoglobulin light chain and CD19 or CD22. Or even, CAR T cells may comprise an immunoglobulin light chain binding domain and a CD20 binding domain, and thus can recognize and bind both immunoglobulin light chain and CD20.

[0096] 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 can target three antigens.

[0097] According to certain aspects of the present invention, a population 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 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 to eliminate graft-versus-host disease in the recipient patient.

[0098] According to certain aspects of the invention, the population of cells administered as adoptive cell transfer immunotherapy may comprise a population of T cells, NK cells, or dendritic cells expressing a CAR, the CAR comprising an extracellular antibody or antibody fragment comprising a humanized anti-kappa or lambda light chain binding domain, a transmembrane domain, and one or more cytoplasmic costimulatory signaling domains.

[0099] In a particular embodiment 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 the recognition of 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 antigen presentation by MHC. This system endows T cells with the potential to recognize, via the TCR, the entire set 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.

[0100] 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.

[0101] According to certain aspects of the invention, autologous cells (e.g., T cells or 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, splenic tissue, and tumors. According to certain aspects of the invention, allogeneic or xenogeneic cells, typically isolated from healthy donors, may be used. When T cells, NK cells, dendritic cells, or pluripotent stem cells are allogeneic or xenogeneic cells, any number of cell lines available in the art may be used.

[0102] The 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 the circulating blood of an individual 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.

[0103] 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 aspects of the 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+.

[0104] 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 immune-modulating protein, such as an armored CAR or TCR.

[0105] 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. Thus, a source of cells to be engineered and expanded (or simply expanded in the case of TILs) may be collected at any time needed, and 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.

[0106] According to certain aspects of the invention, a population of cells expressing a CAR / TCR may be administered to a subject by dose splitting, 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.

[0107] 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.

[0108] Exemplary total doses 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.

[0109] 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 immune repertoire and / or disease burden of the patient's immune suppressor cells (e.g., regulatory T cells, myeloid-derived suppressor cells). Thus, the total dose may be determined based on a dose of 10 3 ~10 13 The sample may include whole cells.

[0110] 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 after certain cancer treatments, particularly treatments 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, it is contemplated in the context of the present invention to collect blood cells, including T cells, NK cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery period.

[0111] According to certain aspects of the invention, the second administration may be a different population of cells expressing the same or different CAR / TCR in the same or different effective amount. The difference in the CAR / TCR may be in any aspect of the CAR / TCR, such as, for example, different binding or antigen recognition domains or co-stimulatory domains. The second administration may additionally or alternatively include secretory cells with IL-12 and may further include adjuvant immunotherapy with small molecule inhibitors such as inhibitors of BTK, P13K, IDO, either simultaneously or sequentially with the cell therapy infusion.

[0112] According to certain aspects of the invention, the method may include administration of 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.

[0113] The therapeutic agent may be administered according to any standard administration regimen known in the art.Exemplary chemotherapeutic agents include mitotic inhibitors such as taxanes, e.g., docetaxel and paclitaxel, and vinca alkaloids, e.g., vindesine, vincristine, vinblastine, and vinorelbine.Exemplary chemotherapeutic agents include topoisomerase inhibitors, e.g., topotecan.

[0114] Exemplary chemotherapeutic agents include growth factor inhibitors, tyrosine kinase inhibitors, histone deacetylase inhibitors, P38a MAP kinase inhibitors; angiogenesis, neovascularization, and / or other angiogenesis inhibitors; 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.), and the like. According to certain aspects of the invention, the one or more therapeutic agents may include an antimyeloma agent. Exemplary antimyeloma agents include dexamethasone, melphalan, doxorubicin, bortezomib, lenalidomide, prednisone, carmustine, etoposide, cisplatin, vincristine, cyclophosphamide, and thalidomide, some of which are set forth above as chemotherapeutic, anti-inflammatory, or immunosuppressive agents.

[0115] Cancer being treated The method of the present invention can improve the treatment of any cancer that can be treated with immunoglobulin light chain-targeted adoptive cell transfer immunotherapy. The method of the present invention is particularly useful in methods of treating B cell neoplasms. Since B lymphocytes express surface monoclonal immunoglobulins with either kappa or lambda light chains, B cell neoplasms are generally expected to be amenable to immunoglobulin light chain-targeted adoptive cell transfer immunotherapy. Examples of B cell neoplasms that may be treatable with immunoglobulin light chain-targeted adoptive cell transfer immunotherapy and are treated in preferred embodiments of the present invention include precursor B cell acute lymphoblastic leukemia / lymphoblastic lymphoma (LBL), B cell acute lymphoblastic leukemia; B cell chronic lymphocytic leukemia (CLL); small lymphocytic lymphoma; B cell prolymphocytic leukemia; lymphoplasmacytic lymphoma / immunocytoma. ;mantle cell lymphoma;follicular lymphoma;extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) type;nodal marginal zone B-cell lymphoma;splenic marginal zone lymphoma;hairy cell leukemia;plasmacytoma / plasma cell myeloma;diffuse large B-cell lymphoma (such as primary mediastinal B-cell lymphoma), Burkitt lymphoma, Burkitt-like lymphoma;primary central nervous system (CNS) lymphoma, and primary intraocular lymphoma. In certain embodiments, the cancer to be treated is a B-cell lymphoma. In certain embodiments, the cancer to be treated is a B-cell leukemia. In a preferred embodiment, the cancer to be treated is B-cell non-Hodgkin's lymphoma (B-NHL), in particular diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), or aggressive follicular lymphoma (FL), which are established to express surface immunoglobulins clonally restricted to either kappa or lambda light chains.

[0116] In certain embodiments, the cancer to be treated is a B-cell lymphoma. In certain embodiments, the method of the present invention comprises administering multiple doses of IgG cysteine ​​protease or IgG endoglycosidase, and the cancer to be treated is a B-cell lymphoma. In certain such embodiments, the method of treating cancer comprises administering a protein having the activity of IgG cysteine ​​protease or IgG endoglycosidase after two or more doses of adoptive cell transfer immunotherapy, preferably including administration of CAR-T cells targeting immunoglobulin light chains, particularly kappa light chains or lambda light chains. In certain such embodiments, the method also comprises immunosuppression. The method of the present invention may be particularly effective in treating tumors that require immunosuppression.

[0117] In certain embodiments, the cancer being treated expresses kappa or lambda light chains on its surface, hi certain embodiments, the patient being treated has been determined to have a B cell neoplasm that expresses kappa or lambda light chains on its surface.

[0118] Patient anti-cancer responses against solid tumors are driven not only by cancerous cells but also by the tumor microenvironment. This microenvironment is created by non-malignant cells such as fibroblasts, T cells, and B lymphocytes and can be tolerogenic. T cells in particular have not only tumor lytic functions but also subgroups that can express a regulatory suppressor phenotype that reduces the immune system's natural anti-tumor response. The same dual role has been observed for B cells. For example, Breg cells (e.g., those that produce IL10 in the tumor mass) can suppress endogenous anti-tumor responses.

[0119] Using light chain-specific CAR T cells to remove Breg cells from the tumor environment may be advantageous in the treatment of cancer (Leong and Bryant, Transl Lung Cancer Res. 2021 Jun;10(6):2830-2841). However, removing most B cells, for example with rituximab, may make already weakened cancer patients more susceptible to infections. In such cases, it is particularly advantageous to treat patients with CAR T cells, e.g., kappa or lambda light chain-specific CAR T cells, using the methods of the invention to remove most of the B cell mass in the tumor while sparing the respective light chain B cell populations, protecting the patient from completely removed humoral responsiveness.

[0120] Preferably, the method of the present invention is used to treat human patients. When the subject is a human, the subject may be of any age. For example, the subject may 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 may 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 may be newly diagnosed, relapsed and / or refractory, or in remission.

[0121] Other diseases being treated The method of the present invention is suitable for improving the treatment of any disease that can be treated with adoptive cell transfer immunotherapy targeting immunoglobulin light chains. Some autoimmune diseases are characterized by the presence of monoclonal or oligoclonal immunoglobulins with either kappa or lambda light chains. Targeting and eliminating B cells that produce kappa or lambda Ig without completely suppressing the humoral arm of the immune system may be beneficial for these patients.

[0122] Examples of suitable diseases include Ig light chain biased autoimmune diseases such as juvenile arthritis (particularly juvenile idiopathic arthritis), rheumatoid arthritis, generalized myxedematous lichen (sclerosing myxedema), Graves' disease, IgA-driven bullous dermatosis, IgG4-driven bullous pemphigoid, Sjogren's syndrome, and lupus mastitis.

[0123] Juvenile arthritis can be treated using the methods of the invention, as lambda light chain levels were significantly elevated in these patients (Low et al.; Scand J Immunol. 2007 Jan; 65(1):76-83). Furthermore, k:λ light chain bias of citrullinated protein antibodies (ACPA) has been observed in rheumatoid arthritis (Slot et al., PLoS One. 2021 Mar 30; 16(3):e0247847.).

[0124] Systemic scleromyxedema is characterized by monoclonal gammopathy (commonly IgGλ) and systemic manifestations including neurological, rheumatic, cardiac, pulmonary, gastrointestinal, hematological, and ocular manifestations. Apart from the main group of patients expressing IgGλ, there are also subgroups of patients expressing clonal kappa or lambda IgA, or IgM kappa.

[0125] Graves' disease is caused by thyroid-stimulating autoantibodies activating thyrotropin receptors, which activate target organs. Increased signaling can lead to thyroid hyperplasia, increased thyroid hormone secretion, and clinical thyrotoxicosis, which can lead to life-threatening thyroid storms. Many patients have kappa or lambda oligoclonal IgG1 antibodies (Chazenbalk et al.; J Clin Invest. 2002 Jul;110(2):209-17).

[0126] Bullous dermatosis and bullous pemphigoid are subtypes of autoimmune bullous skin diseases. Skin blistering is thought to be driven by antibodies against skin matrix components. Anti-basement membrane zone antibodies predominate in bullous pemphigoid, which show a bias towards kappa light chains in IgG4. Patients presenting with linear IgA bullous dermatosis may have a predominance of either kappa or lambda light chain IgA (Flotte and Baird; J Immunol. 1986 Jan; 136(2):491-6).

[0127] IgA lambda bias has also been observed in Sjogren's syndrome. (Jasani; J Pathol. 1988 Jan; 154(1):1-5.)

[0128] In some cases of lupus mastitis, patients were observed to have a plasma cell infiltrate restricted to kappa light chains, but did not display the immunophenotype of plasma cell neoplasms (Yan et al. Surgical and Experimental Pathology volume 3, Article number: 24 (2020)). Such patients may benefit from treatment with the methods of the present invention.

[0129] 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 plasma.

[0130] In a preferred embodiment, the protease for use in the method 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. IdeS was originally isolated from a group A Streptococcus pyogenes serotype M1 strain, 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 cleavages in the heavy chains of several subclasses of IgG in various animals. 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 truncated IgG (scIgG) molecule with a non-covalently bound Fc molecule. The scIgG molecule is in fact 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. Also available as NCBI reference sequence WP_010922160.1. 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.

[0131] In another embodiment, the protease for use in the methods of the invention is IdeZ, an IgG cysteine ​​protease produced by Streptococcus equi subspecies zooepidemicus, a bacterium found primarily in horses. SEQ ID NO:3 is the full sequence of IdeZ, including the N-terminal methionine and signal sequence. Also available as NCBI reference sequence WP_014622780.1. SEQ ID NO:4 is the mature sequence of IdeZ, lacking the N-terminal methionine and signal sequence.

[0132] 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.

[0133] In a preferred embodiment, a protease for use in the present invention may comprise or consist of SEQ ID NO: 2, 4 or 5. A protease for use in the present invention may comprise an additional methionine (M) residue at the N-terminus and / or a tag at the C-terminus 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, for example, three, four or five glycine residues. A histidine tag typically consists of six histidine residues, but may be typically up to 7, up to 8, up to 9, up to 10 or up to 20 amino acids longer, for example 5, 4, 3, 2 or 1 amino acid shorter.

[0134] 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. Each of SEQ ID NOs: 6 to 25 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 6 histidine residues. The histidine tag is preferably linked to the C-terminus by a linker of 3x glycine residues or 5x glycine residues.

[0135] 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 SEQ ID NOs: 56-69. These sequences represent IdeS polypeptides with increased 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 6 histidine residues. The histidine tag is preferably linked to the C-terminus by a linker of 3x glycine residues or 5x glycine residues.

[0136] In a further preferred embodiment, the protease for use in the present invention may comprise, consist essentially of, or consist of the sequence of any one of SEQ ID NOs: 6 to 25, optionally with up to three (e.g., one, two, or three) amino acid substitutions. Each of SEQ ID NOs: 6 to 25 and variants thereof may optionally include an additional methionine at the N-terminus and / or a histidine tag at the C-terminus.

[0137] In a further preferred embodiment, the 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. Each of SEQ ID NOs: 56-69 and variants thereof may optionally include an additional methionine at the N-terminus and / or a histidine tag at the C-terminus.

[0138] In a further preferred embodiment, a protease for use in the present invention may comprise, consist essentially of, or consist of the sequence of SEQ ID NO:91, SEQ ID NO:92, or a variant of SEQ ID NO:91 or SEQ ID NO:92 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modification(s) relative to SEQ ID NO:91 or SEQ ID NO:92, respectively, provided that the sequence retains (a) an asparagine (N) at a position corresponding to position 10-95 of SEQ ID NO:3, (b) an aspartic acid (D) at a position corresponding to position 99 of SEQ ID NO:3, and (c) an asparagine (N) at a position corresponding to position 226 of SEQ ID NO:3, provided that the polypeptide is at least as effective at cleaving human IgG as a polypeptide consisting of the amino acid sequence of SEQ ID NO:91 or 92, respectively, when measured in the same assay. Preferably, at least one of the modifications, or all of the modifications, does not result in the same amino acid as the amino acid present at the corresponding position in the polypeptide sequence of SEQ ID NO:4.

[0139] Polypeptides of the 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 invention are typically no more than 400 amino acids long, no more than 350 amino acids long, no more than 340 amino acids long, no more than 330 amino acids long, no more than 320 amino acids long, or no more 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 invention. For example, a polypeptide may be 100-400 amino acids long or 250-350 amino acids long. A polypeptide is preferably 290-320 amino acids long, most preferably 300-315 amino acids long.

[0140] The primary structure (amino acid sequence) of the protease 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 sequence of the protease of the present invention may include a variant of the amino acid sequence of SEQ ID NO: 2, 4, or 5 that is 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 entire length of SEQ ID NO:4 or 5.

[0141] The protease for use in the present invention may be an IdeS, IdeZ, or IdeS / Z polypeptide comprising a variant of the amino acid sequence of SEQ ID NO: 2, 4, or 5, which has been modified, for example, by addition, deletion, or substitution of amino acids, 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, basic, acidic, neutral, or charge as 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.

[0142] IgG cysteine ​​protease activity can be assessed by any suitable method, for example, by incubating the 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 that was 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.

[0143] In other assays, the potency of a test polypeptide can be determined by directly visualizing and / or quantifying fragments of IgG resulting from cleavage of 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. Total IgG and fragments resulting from cleavage of 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. The 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 an IgG molecule. Polypeptides of the invention may be more effective at cleaving the first chain of an IgG molecule 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.

[0144] 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 plasma.

[0145] The agent may be a protein that has 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, 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 FIG. 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 homologue thereof from another bacterium, for example Streptococcus pyogenes or Streptococcus zooepidemicus, or Mycobacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica. The agent may be CP40, EndoE, or EndoF2.

[0146] 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. A variant of an EndoS protein 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.

[0147] Alternatively, the agent may be a protein comprising or consisting of a fragment of SEQ ID NO: 90 and having IgG endoglycosidase activity, preferably the fragment has 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, 50, 100, 200, 300, 400, 500 or 550 residues or more may be deleted. The deleted residues may be adjacent to other residues.

[0148] Any fragment or variant of SEQ ID NO:2 preferably comprises 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. The glutamic acid in the active site of chitinase is essential for enzymatic activity. Thus, most preferably, the variant of SEQ ID NO:90 contains Glu-199 of SEQ ID NO:90. The 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.

[0149] Assessment of IgG levels and timing of administration The appropriate timing of administration of the protein having IgG cysteine ​​protease or IgG endoglycosidase activity and adoptive cell transfer immunotherapy in the method of the present invention can be determined, for example, using an assay to evaluate plasma or 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 serum or plasma of the subject may be measured. This can be determined, optionally, by testing serum or plasma samples taken from the individual and applying any suitable assay. Some exemplary suitable assays are described in the Examples.

[0150] Such assays can directly test for the presence of IgG molecules in serum or plasma 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 that has IgG cysteine ​​protease or IgG endoglycosidase activity. For example, if the agent is an enzyme that cleaves IgG protein, serum or plasma 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 the 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 serum or plasma from a subject with cells expressing FcgR and monitoring IgG binding by flow cytometry using a fluorochrome-conjugated anti-human IgG.

[0151] Traditional methods for assessing the amount of IgG in a sample, such as a serum or plasma sample, in a clinical setting rely on nephelometry and turbidimetry for 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 principles are based on the reaction of added anti-IgG antibodies with antigens in the sample to form antigen / antibody complexes (agglutination). Addition of PEG allows the reaction to proceed more quickly to the end point, 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 an agglutination reaction. However, such methods may not be suitable when some or all of the IgG present may not be intact. For example, when an IgG cysteine ​​protease (such as IdeS) is administered to a subject from whom a sample was taken, for example in the method of the present invention, or when such a protease is administered to a sample, cleavage fragments such as F(ab')2 and Fc fragments will be present. This does not affect the agglutination reaction of conventional nephelometric and turbidimetric methods, as long as the F(ab')2 fragments are still present in the sample. Due to the short half-life of the F(ab')2 fragments compared to intact IgG, agglutination decreases over time, but not in proportion to the amount of intact IgG present in the sample. Thus, samples affected by the presence of IgG cysteine ​​proteases (such as IdeS) cannot be evaluated by conventional methods. The inventors have developed a novel assay for IgG concentration that is compatible with samples affected by the presence of IgG cysteine ​​proteases (such as IdeS) and can be used in any clinical situation, including (but not limited to) in combination with other methods of the present invention.

[0152] The method can distinguish between intact IgG and F(ab')2 fragments generated by IdeS. This was 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 avoids 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 agent. The capture agent 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 agent. Thus, in contrast to nephelometry and turbidimetry, only IgGs that carry 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, and can be used in the reverse order, i.e., capture with anti-Fc and detect with anti-Fab. The assay can be performed in any suitable format, such as a conventional ELISA or Meso Scale Discovery format.

[0153] In some cases, such as when the IgG cysteine ​​protease is IdeS, the sample may contain intermediate fragments such as scIgG where only one heavy chain has been 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. Thus, the method may include a complementation step to evaluate the size of the fragments present in the sample. Since there are no disulfide bridges between the heavy chains below the hinge region, the Fc portion of the heavy chain in the scIgG fragment is separated from the intact heavy chain as a protein of about 20-25 kDa under denaturing conditions. 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 evaluating the efficacy of IdeS in a clinical setting.

[0154] If the protein having IgG cysteine ​​protease or IgG endoglycosidase activity is an enzyme that cleaves glycan moieties on IgG, serum or plasma samples may be assayed for the presence of IgG molecules bearing either normal or cleaved glycans or for 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.

[0155] In the method of administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity 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 it is determined that Fc receptor binding by substantially all IgG molecules present in the serum or plasma of the subject is sufficiently reduced or eliminated at an earlier time point. Preferably, the lower limit is 2 hours.

[0156] 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 serum or plasma of the subject prior to carrying out the method. This may be determined by testing a sample of serum or plasma 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 plasma within 3-4 days, with all replacement being completed 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.

[0157] The time interval between administration of the protein having IgG cysteine ​​protease or IgG endoglycosidase activity and the 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 a treatment center may be limited.

[0158] In the method of administering a protein having IgG cysteine ​​protease or IgG endoglycosidase activity after adoptive cell transfer immunotherapy to enhance the efficacy of the immunotherapy, the timing of administration depends on the increase in IgG ADA. In a particular embodiment, the protein is administered 4-8 days, for example, 5-7 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, for example, 10 days, 2 weeks, 3 weeks, or 4 weeks, or 10 days to 2 weeks, 10 days to 3 weeks, 2-4 weeks.

[0159] Safety switch A concern when administering any type of adoptive cell transfer immunotherapy is the possibility of serious cytokine release syndrome (CSR) or other complications. Therefore, it is desirable from a safety perspective to be able to temporarily halt the lysis of target cells by adoptive cell transfer immunotherapy. Suitable means for doing this are known in the art. For example, depletion of CAR T cells can be achieved by designing a CAR construct that also expresses a suicide gene, such as inducible caspase 9 (iCasp9), herpes simplex virus tyrosine kinase (HSV-TK), or human thymidylate kinase (TMPK).

[0160] In the case of B cell receptor (BCR)-specific CAR T cells, the BCR serves as a triggering target for kappa or light chain CAR T cells. Transient removal of the BCR from the cell surface reduces the activity of effector CAR T cells.

[0161] Removal of the CAR can be accomplished in a number of ways. For example, cells may be engineered to contain a CAR spacer that connects the extracellular ligand binding domain(s) and the intracellular signaling domain. Advantageously, these spacers may be susceptible to cleavage by proteases. This is useful because CAR T cells of any given scFv specificity that contain these spacers may become temporarily blinded when exposed to proteases, which may result in the elimination of all CAR T cells in the patient.

[0162] Preferably, the spacer may comprise the constant region of an IgG, such as IgG1 or IgG4. In these embodiments, the spacer may be cleaved by an IgG cleaving enzyme, such as an immunofidase. These CAR spacers may comprise a CH2 or CH2-CH3 domain. The IgG may have a wild-type sequence or may be mutated. For example, the spacer may be mutated to reduce FcR-mediated recognition of cells in vivo compared to CAR T cells that do not contain a mutated spacer.

[0163] Suitable spacers are known in the art. For example, Jonnalagadda et al. (Mol Ther. 2015 Apr; 23(4): 757-768) describe CAR Ts that contain IgG4 Fc spacers with mutations that reduce FcR binding. Similarly, Savoldo et al. (J Clin Invest. 2011 May; 121(5): 1822-6) describe CAR Ts that contain a spacer region from human IgG1-CH2CH3 domains cloned in frame between scFv and signaling domains (see also Hudecek et al. (Cancer Immunol Res. 2015 Feb; 3(2): 125-35)).

[0164] It may also be performed using other IgG cleaving enzymes, such as cysteine ​​proteases or gingipains, such as those cloned from Bdellovibrio bacteriovorus.

[0165] Alternatively, the IgG cysteine ​​protease is administered prior to administering adoptive cell transfer immunotherapy. In these embodiments, it is preferred to allow sufficient time (preferably more than 3 days, e.g. more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, or more than 10 days) between administering the IgG cysteine ​​protease and initiating adoptive cell transfer immunotherapy to allow at least some or all of the protease to be cleared from the patient's blood. Suitable time frames are known to those skilled in the art.

[0166] Polypeptide production The polypeptides disclosed herein can be produced by any suitable means. For example, the polypeptides can 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 can be produced by transforming a cell, typically a bacterial cell, with a nucleic acid molecule or vector encoding the polypeptide. The production of polypeptides by expression in bacterial host cells is described below and exemplified in WO2016 / 128559.

[0167] Compositions and formulations comprising polypeptides The present invention also provides compositions comprising IgG cysteine ​​proteases or IgG endoglycosidases for use in the therapeutic methods of the present invention. For example, the present invention provides compositions comprising one or more polypeptides of the present invention and at least one pharma- ceutically 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.

[0168] The formulation of suitable compositions can be carried out 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 pharma-ceutically acceptable excipients or vehicles. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, reducing agents, and the like, may be present in the excipients or vehicles. Suitable reducing agents include cysteine, thioglycerol, thioreducin, glutathione, and the like. 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. These may include pharma- ceutically acceptable salts, e.g., 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 pharma- ceutically acceptable excipients, vehicles, and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0169] 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 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, stabilizing agents, 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), and the reconstituted composition is then administered parenterally. The compositions 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 known art 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 may 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.

[0170] Other parenterally administrable compositions that are useful include those that contain 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 pharma- ceutically 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 routes. Preferred compositions are suitable for administration by intravenous infusion.

[0171] overview It is to be understood that the various applications of the disclosed products and methods can be tailored according to the particular needs of the art, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0172] Moreover, as used in the 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.

[0173] 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.

[0174] "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, but 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- or L-optical isomers, as well as amino acid analogs and peptidomimetics.

[0175] The terms "patient" and "subject" are used interchangeably and typically refer to a human. References to IgG, unless otherwise specified, typically refer to human IgG.

[0176] The above-mentioned amino acid identity can be calculated using any suitable algorithm. For example, PILEUP and BLAST algorithms can be used to calculate identity and align sequences (typically with their default settings to identify equivalent or corresponding sequences, etc.) 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 the query sequence that match or meet some positive threshold score T when aligned with words of the same length in database sequences. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as long 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 negatively 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), alignment (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. http: / / www.ncbi.nlm.nih.gov / ).

[0177] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, for example, 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).

[0178] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0179] Working Example The experiments in Examples 1-5 were performed 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. A system was developed to mimic anti-drug antibody binding to CAR T cells by using in vitro and in vitro models of IgG binding to cell surface receptors. These models mimic the antibody-mediated effector mechanism of surface-bound antibodies on chimeric antigen receptors. The effect of treatment with ImmuRifidase or EndoS was investigated in the model systems.

[0180] Unless otherwise indicated, the methods used are standard biochemical and molecular biology 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.

[0181] 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 prior to use at the age of 9 weeks. Mice were housed and treated in accordance with ethical approval M72-13 (Lund).

[0182] 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).

[0183] 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.

[0184] Anti-CD20 IgG (rituximab, N7022, 10 mg / mL) was purchased from Mabthera and human IgG1 isotype negative control (#I5154) was purchased from Sigma.

[0185] A cell counting kit, CCK-8, manufactured by Dojindo Laboratories (Japan) was used according to the manufacturer's instructions.

[0186] Baby rabbit serum complement (CL3441; lot 6374; Cedarlane) was reconstituted with sterile water before use and diluted in medium to a final dilution of 1:10.

[0187] Human blood was collected in BD CAT tubes for serum (#368815; spray dried clot activator (silica, PVP, L-720)) and frozen at -20°C. Serum was tested for low toxicity towards Daudi cells before use as a complement source.

[0188] Mouse anti-human C4d (A213, Quidel) was biotinylated in-house and used at 1 μg / mL for cell staining.

[0189] Polyclonal sheep anti-human C1q (MD-14-0162, Raybiotech) was fractionated to F(ab')2 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 detection antibody (2.5 μg / mL) together with the fluorescent dye SA-PE (BD-Pharmingen, #554061).

[0190] 7-AAD (Sigma, A9400) was used as a dead cell marker in the CDC assay.

[0191] 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).

[0192] 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 SDS-page gels. Additional protein bands after His-tag purification beyond the predicted 100 kD band were shown by mass spectrometry to be of EndoS origin and not host cell proteins.

[0193] IdeS (IgG-degrading enzyme of Streptococcus pyogenes) is a cysteine ​​endopeptidase secreted by the human pathogen Streptococcus pyogenes that highly specifically catalyzes a single proteolysis in the lower hinge of human immunoglobulin G (IgG) antibodies.

[0194] 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 a receptor-specific antibody (RTX) binds to a cell surface receptor (CD20) in a manner similar to ADA binding to CAR, we investigated the effect of pre-existing or therapeutically induced antibodies on cell therapy and the influence of IdeS and EndoS on such antibodies.

[0195] Complement deposition on Daudi cells Daudi cells (50 μL at 3×10E7 cells / mL) were treated with anti-CD20 antibody RTX or human IgG1 negative control antibody at a final concentration of 2 μg / mL together with IdeS or EndoS in a 1:10 step titration starting at 100 μg / mL and going down 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 dilution of 1:5, resulting in a final serum dilution of 1:10. The plates were incubated for 120 min at 37° C. All dilution and washing steps were performed in D-MEM supplemented with 0.5% BSA.

[0196] Flow cytometric staining for complement adhesion to cells C1q complement binding was assessed through incubation with sheep anti-C1q F(ab')2 generated from polyclonal IgG (#MD-14-0162; RayBiotech) that had been previously cleaved into Fc and F(ab')2 fragments using resin-bound IdeS (FragIT kit, #A0-FR6; Genovis). The Fc fragment was removed with 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 detection fluorochrome.

[0197] C4d complement binding was assessed through incubation with biotinylated mouse anti-human C4d antibody (#A213; Quidel). SA-PE was diluted in D-MEM (0,5% BSA) and used as a detection fluorescent dye. Finally, cells were transferred to FACS tubes and analyzed for mean fluorescence intensity (MFI) in FL2 using an Accuri C6 cytofluorometer.

[0198] 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) and cell death. Early steps in the complement cascade, promoted through antibody binding, can also 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.

[0199] 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 another 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.

[0200] 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, which is predicted to be true for light chain-specific CAR-T cells.

[0201] 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 the 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.

[0202] Daudi cells (3x10E6) were incubated in a master plate for 60 min at 37°C with titrated concentrations of rituximab (RTX) (50 μg / mL in well, 1:2 serial dilutions down to 0.2 μg / mL) and with IdeS or EndoS (50 μg / mL).

[0203] 50 μL of cell mix was transferred to ELISA plate with 50 μL of baby rabbit serum (1:5 dilution) as a complement source and incubated at 37°C for 45 min for CDC lysis. 10 μL of CCK8 was added to each 100 μL mix and incubated at 37°C for another 60 min 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 as 100% viability.

[0204] result At an RTX concentration of 0.78 μg / mL, 70% of the cells in the medium group die, whereas in the presence of IdeS or EndoS at this concentration, all Daudi cells survive (see Figure 2). The protective effect of EndoS decreases with increasing RTX concentration, resulting in a 30% viability at 50 μg / mL, which is a 60-fold higher RTX concentration than the same viability at 0.78 μg / mL without EndoS. Thus, even at high antibody concentrations (50 μg / mL), IdeS is able to block CDC, whereas EndoS also shows strong protective activity. This is expected to be true for light chain-specific CAR-T cells as well.

[0205] 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.

[0206] 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 prior to the experiment.

[0207] 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 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 in 3 mL of D-MEM 10 medium. 6 Resuspended at 100 cells / mL.

[0208] 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 at RT in the dark. After 20 min, cells were washed twice with medium and resuspended in D-MEM 10 at 3.6 × 10 6 Resuspended at 100 cells / mL.

[0209] Target-effector cell incubation Titrated RTX and enzyme (IdeS or EndoS) were incubated for 60 min at 37°C. Daudi target cells (50 μL at 2×10E6 cells / mL) were incubated for 30 min to opsonize, after which THP1 effector cells (50 μL at 3.6×10E6 cells / mL) were added and incubated for approximately 2 h to allow ADCP of Daudi cells. Cells were fixed with 5% PFA for 3 min, washed, and transferred for FACS analysis. Cells were analyzed using an Accuri C6 flow cytometer for MFI in FL2 (calcein) and FL4 (FarRed). Double positive cells were considered ADCP positive.

[0210] result The efficacy of IdeS and EndoS in protecting RTX-opsonized CD20-positive 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. The 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 RarRed positivity in FL4. These calcein / FarRed double-positive cells are 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).

[0211] 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, which is predicted to also be true for light chain-specific CAR-T cells. Example 4 - Platelet protection in immune thrombocytopenia (ITP) model As demonstrated above using RTX and Daudi cells, IdeS and EndoS reduce the effector function of cell surface receptor-specific antibodies. In the following experiments, using a thrombocytopenic thrombocytopenic (ITP) model, we also investigated whether these enzymes protect antibody-sensitized endogenous cells from elimination in vivo. This demonstrates that harmful polyclonal antibodies against cell surface receptors can be inactivated through treatment with immunofluorescence enzymes or EndoS, which mimic the effects of these enzymes in the context of anti-CAR antibodies on CAR T cell persistence.

[0212] ITP in vivo EndoS treatment Nine-week-old female Balb / c mice were immunized with antiplatelet-specific antibodies (anti-PLT IgG) (50 μg IgG / mouse) purified by protein G from rabbit anti-mouse platelet serum (Cederland #CLA31440) in a single ip injection of 200 μL. JBomTac Mice were primed for immune thrombocytopenia (ITP). After 30 min, the indicated amount of EndoS (10 μg / mouse, 30 μg / mouse, 90 μg / mouse) was injected ip, respectively with PBS as a negative control. Mice injected with carrier solution (PBS) in both cases were used as normal controls. Mice were evaluated for hematoma or abnormal behavior 4 hours after injection. After 24 hours, blood samples were taken from the tail vein and placed in a Microvette CB300 (Sarstedt, Potassium-EDTA #16.444.100). Platelets in the blood samples were counted with a hematology analyzer VetScan HM5.

[0213] Induction of ITP by IdeS-cleaved anti-PLT antibodies in vitro Anti-mouse PLT IgG was protein G purified from serum of rabbits 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.

[0214] Nine-week-old female Balb / c mice were immunized with either anti-PLT IgG, scIgG, or Fc / F(ab')2 fragments at 250 μg / mouse in a single ip injection of 200 μL. JBomTac Mice were primed for ITP. Some mice were injected with PBS to establish normal control levels of platelets. After 24 hours, blood samples were taken 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 hematology analyzer, VetScan HM5.

[0215] ITP in vivo IdeS treatment Nine-week-old female Balb / c mice were cultured using 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 ip 1 h before IdeS (0 μg / mouse, 0.2 μg / mouse, 2 μg / mouse, 20 μg / mouse) was administered iv. Control mice with normal platelet levels were injected with PBS only. After 24 h, blood samples were taken from the tail vein and placed in a Microvette CB300 (Sarstedt, Potassium-EDTA #16.444.100). Platelets in the blood samples were counted with a hematology analyzer VetScan HM5.

[0216] 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 in comparison with intact anti-PLT IgG in vivo.

[0217] Immune thrombocytopenia (ITP) was induced in BALB / c mice by a single ip 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 counted automatically on a VetScan HM5. Two naive mice that received PBS only served as control mice. These mice had a platelet count of 86 × 10 9 compared with mice injected with rabbit anti-mouse platelet-purified IgG, which was reduced to 657 × 10 platelets / L. 9The mice injected with anti-PLT scIgG had normal platelet levels with 0.01% platelets / L (see Figure 4). However, a slight decrease in platelets was observed in mice injected with anti-PLT scIgG. No induction of thrombocytopenia was observed in mice treated with purified rabbit anti-mouse platelet (F(ab')2 IgG fragment).

[0218] From this experiment, it can be concluded that a protective effect against thrombocytopenia can be achieved through in vitro generation of scIgG or (F(ab')2) by IdeS. Similar protective effects can be predicted from other pre-existing or de novo antibodies targeting neoepitopes on chimeric antigen receptor T cells, including light chain-specific CAR-T cells.

[0219] Thrombocytopenia could be partially prevented when an scIgG preparation was injected and was completely abolished after cleavage of IgG into Fc and F(ab')2 fragments with IdeS (see Figure 4).

[0220] To investigate whether IdeS also has a therapeutic effect in vivo, mice were first injected with a platelet-reducing dose of anti-PLT rabbit IgG. After 1 h, different doses of IdeS were injected. After 24 h, blood was collected and platelet counts in the different groups were determined. 2 μg of IdeS per mouse was completely sufficient to restore platelets to normal levels in mice (see Figure 5). Therefore, the therapeutic effect of IdeS against harmful anti-platelet antibodies (anti-PLT) was tested in vivo using an ITP mouse model. Injection of mice with intact polyclonal anti-PLT rabbit antibodies (250 μg) induces a strong ITP phenotype accompanied by platelet depletion.

[0221] Similar results were seen when EndoS was injected into anti-PTL 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).

[0222] 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, it is predicted that these enzymes will also improve CAR T / NK cell survival and efficacy through inactivation of pre-existing and treatment-induced ADA.

[0223] In summary, it was shown that the antibody-mediated effector functions tested (C1q and C4d deposition, CDC, ADCP) that mimic the ADA response against CAR-T cells can be blocked through the use of IdeS and EndoS.

[0224] Example 5 Experiments were performed to determine whether antibodies against single chain variable fragment (scFv) chimeric antigen receptors or cognate epitopes on 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 determined that immunofluorescence enzyme treatment could protect CAR-T cells from these deleterious IgG effector mechanisms.

[0225] 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). The anti-CD19-scFv was derived from the mouse CD19-specific monoclonal antibody FMC63.

[0226] THP-1 monocytic cell line (ACC16, DSMZ, Braunschweig, Germany). 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.

[0227] Antibodies and human serum CAR scFv cross-reactive anti-F(ab')2 specific antibodies include affinity purified rabbit anti-human IgG, F(ab')2 fragment specific (#309-005-006, JacksonImmunoResearch, West Grove, PA, USA), affinity purified rabbit anti-mouse IgG, F(ab')2 fragment specific (#315-005-006, JacksonImmunoResearch), goat anti-human IgG-Fc-PE (LS-AB2, OneLambda), and biotinylated goat anti-rabbit Fc (#111-066-046, JacksonImmunoResearch). Detection of biotinylated goat anti-rabbit Fc was achieved by using streptavidin-Alexa Fluor 647 (SA-AF647) (#016-600-084, JacksonImmunoResearch).

[0228] 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 highly sensitized patients (n=8) and phase 1 healthy volunteers (n=11) were screened for HAMA and HLA alloreactivity to anti-CD19 CAR-Jurkat T cells. Control sera for HLA alloreactivity, 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 from healthy donors.

[0229] ELISA-based HAMA detection Bridging ELISA kit "LEGEND MAX Human Anti-Mouse Ig (HAMA)" (Cat. No. 438307, Lot No. B329842, BioLegend, San Diego, CA, USA) was used to detect HAMA in human serum. Briefly, mouse IgG pre-coated plates were washed and incubated with undiluted human serum samples (BioIVT), HAMA quality control, standard curve samples, and mouse IgG conjugate. Contents were discarded and plates were washed with wash buffer. Substrate solution was added and plates were incubated at room temperature (RT) for 15 min in the dark. Reactions were terminated by adding stop solution. Absorbance was measured at 450 nm and 570 nm within 30 min using a SpectraMax i3x spectrophotometer (SpectraMax i3x, Molecular Devices, San Jose, CA, USA). OD results were analyzed with Graph Pad Prism 9 (GraphPad Software, San Diego, CA, USA) using a four-parameter logistic curve fitting algorithm. Serum samples with HAMA >10 ng / mL were considered HAMA positive.

[0230] Screening of CAR-specific human sera by flow cytometry Anti-CD19 CAR-Jurkat or Jurkat wild-type T cells (1×10E5 / well) were washed with PBS and centrifuged at 300g for 5 min. 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.

[0231] Screening of CAR-T Jurkat alloreactive human sera by flow cytometry Anti-CD19 CAR-Jurkat T cells (1x10E5) were washed with PBS and centrifuged at 300g for 5 min. The cell pellet was resuspended in 50 μL of the indicated human serum sample. As controls, FlowPRA class I positive control serum (OneLambda), FlowPRA class II positive control serum (OneLambda), and HLA negative control serum (OneLambda) were used. Bound alloantibodies were detected using PE-conjugated goat anti-human IgG (LS-AB2, OneLambda). After washing steps, the cells were resuspended in PBS and analyzed for MFI values ​​using a flow cytometer (CytoFLEX).

[0232] 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, JacksonImmunoResearch) for 30 minutes. Daudi cells expressing BCR were stained as a F(ab')2 positive control. Cells were then washed and incubated sequentially with biotinylated goat anti-rabbit Fc antibody and SA-AF647 (#016-600-084, JacksonImmunoResearch). 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.

[0233] 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 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 immu-fidase for 30 min at 37°C. 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 the immunofluorescence-treated / untreated antibodies and serum for 1 h at 37°C. After this, effector cells (75000 cells / well) expressing FcγRI (Promega) were added to the opsonized target cells and incubated for 6 h at 37°C. Finally, cells were incubated with Bio-Glo™ Luciferase Assay Reagent (Promega) for 10 min at ambient temperature, after which luciferase activity was measured using a luminescence reader (SpectraMax i3x) with an integration time set at 0.5 s / 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 replicates, while the no-antibody control containing only cells in the presence / absence of immunofluorescence was calculated as the average of two replicates. Fold induction (FoI) was calculated as follows: FoI=RLU (induction−background) / RLU (no antibody control−background).

[0234] 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 anonymous serum from HLA-highly sensitized patients) 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, MD, USA) for 1 h. The cells were washed once with 100 μl of IgG-treated or untreated antibody and serum (Promega, Madison, NY, USA), resuspended in assay buffer (4% low IgG serum in RPMI-1640 (Promega, Madison, WI, USA)) and incubated with immunofluorescence-treated or untreated antibody and serum for 1 h 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 h at 37°C. Finally, the cells were incubated with Bio-Glo™ Luciferase Assay Reagent (Promega) for 10 min at ambient temperature, after which luciferase activity was measured using a luminescence reader (SpectraMax i3x) with an integration time of 0.5 s / well. Raw data was 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, whereas the no antibody control containing only cells in the presence / absence of immunofidase was calculated as the average of two replicates. An HLA negative control was also included, representing a serum sample of a healthy donor with no quantitative anti-HLA class I or class II antibodies.Fold induction (FoI) was calculated as follows: FoI=RLU (induction−background) / RLU (no antibody control−background).

[0235] 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, JacksonImmunoResearch) or anti-mouse IgG, F(ab')2 specific (#315-005-006, JacksonImmunoResearch) antibodies. Alternatively, target cell pellets were resuspended undiluted with immunofidase-treated (±10 μg / mL) human serum samples (25 μL) 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 min. Phagocytosis was assessed by flow cytometry (CytoFLEX flow cytometer, #C02945, Beckman Coulter). The amount of FL1 and FL4 double positive cells, reflecting the target cells phagocytosed by THP1 cells, was expressed as a percentage of target cells.

[0236] CD19-protein binding blocking experiment Rabbit anti-mouse IgG, F(ab')2 specific antibody (JacksonImmunoResearch Laboratories) and human serum (BioIVT) were incubated with or without 10 μg / mL ImmuRifidase for 60 min at 37°C. IHAc (1 mM) (I4386, Sigma-Aldrich, St. Louis, MO, USA) was added to all samples for 30 min to inactivate ImmuRifidase during the subsequent incubation steps. 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).

[0237] result F(ab')2-specific polyclonal antibodies specifically bind 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, for example, originates from a mouse monoclonal antibody, and 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 to anti-CD19 CAR T cells (Figure 7A) and anti-BCMA-CAR T cells (Figure 7B), while 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. Some cross-reactivity was observed using rabbit anti-human F(ab')2. These data confirm that antibodies can bind a variety of different receptor constructs against different targets. Since such binding can have a negative effect on adoptive cell transfer immunotherapy such as CAR T cell therapy, we further used the antibody to investigate anti-CAR T cell antibody-mediated effector function and the extent to which it can be blocked by treatment with immuRifidase.

[0238] Identification of HAMA and CD19-CAR Jurkat T cell allospecific sera One group of antibodies that are potentially CAR-specific are human anti-mouse antibodies (HAMA). HAMA levels in human serum samples can be quantified by sandwich ELISA using mouse IgG-coated assay plates. Human sera were screened for HAMA using a validated HAMA ELISA kit (Figure 8A). A selection of identified HAMA-positive and -negative samples were 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 It could be demonstrated that HAMA-positive sera also specifically bind 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 by flow cytometry for IgG binding. This allowed the selection of HAMA or anti-CD19 CAR T cell alloreactive sera to further elucidate the effect 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. In patients receiving mouse mAb-based biologics, it can be expected that the frequency and concentration of HAMA will be even higher, possibly even causing 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 was anti-HLA highly sensitized. Two of the screened individuals did not have alloantibodies against anti-CD19 CAR Jurkat cells.

[0239] These data demonstrate that antibodies capable of binding to the receptor construct are detectable in human patients, including healthy individuals, and are increased in patients who have undergone treatments that can enhance HLA sensitization, such as transplantation.

[0240] 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. Opsonization of anti-CD19 CAR T cells with polyclonal anti-F(ab')2 antibodies for ADCP is blocked by immuRifidase treatment, and immuRifidase blocks ADCP induction by CD19-CAR Jurkat T cells opsonized with allogeneic serum.

[0241] 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 blocking through treatment with the IgG-cleaving enzyme immu-fidase (see Figures 9 and 10). A flow cytometry-based ADCP model was used with target cells stained with calcein and monocytic THP1 effector cells labeled with CellTrace FarRed. Acquisition of cells by flow cytometry allowed 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 immu-fidase (10 μg / mL) blocked ADCP of the target cells. There was no uptake of Jurkat wild-type cells, indicating that phagocytosis was anti-CD19 CAR-dependent (Fig. 9D,E). BCR-positive Daudi target cells were used as a positive control for F(ab')2 antibody-mediated ADCP (Fig. 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 against allogeneic Jurkat CAR T cells was tested using a bioluminescent FcγRI reporter assay (#CS1781C01, Promega, #CS1781C01). Sera from normal individuals (Fig. 10A) and anti-HLA highly sensitized patients (Fig. 10B) were able to opsonize anti-CD19 CAR-Jurkat T cells and induce ADCP. 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.

[0242] ADCC(V158) induced by FcγRIIIa allele(V158) and allele(F158) anti-CD19 CAR-specific antibodies is blocked by immunofluorescence enzyme treatment. Antibody-dependent cellular cytotoxicity (ADCC) is triggered through engagement of FcγRIIIa (CD16a) on effector cells by IgG-opsonized target cells. There are two major CD16a alleles 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 rabbit anti-mouse (Figure 11A) or anti-human (Figure 11B) F(ab')2 antibodies with or without immunofluorescence (20 μg / mL) treatment. Opsonized target cells were incubated with high-affinity FcγRIIIa (V158) effector cells. ADCC was strongly induced by target cells opsonized with anti-mouse F(ab')2 antibodies. In the samples treated with immurifidase, the induction of ADCC was completely abolished (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 in the 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. Immurifidase treatment of opsonized Daudi target cells also prevented the induction of ADCC (Fig. 11D, E).

[0243] Similar results were seen 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 could not 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 immurifidase treatment.

[0244] 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.

[0245] ADCC induction by HAMA-opsonized anti-CD19 CAR-Jurkat T cells can be blocked by immunofluorescence (IHC) 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 (Promega) transfected with FcγRIIIa high affinity alleles. 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 binds to the anti-CD19 CAR in a manner that can trigger CD16 FcγRIIIa on effector cells. Thus, these data further demonstrate that antibodies in human serum mediate deleterious effects on adoptively transferred immunotherapy cells that can be reduced by immurifidase treatment.

[0246] Binding of CD19 protein to serum-exposed anti-CD19 CAR T cells can be improved by immunofluorescence treatment For CAR T cell therapy to be successful, specific interaction of its chimeric antigen receptor with target proteins is a necessary step. Binding of serum IgG to anti-CD19 CAR may interfere with cognate association with CD19 on target cells. HAMA positive and negative serum samples (Figure 14) identified by ELISA 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 immuRifidase resulted in an increase in 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 immunotherapy, such as anti-CD19 CAR T cells, with target proteins can be increased simply by removing the IgG-Fc portion by immuRifidase treatment, thereby reducing steric hindrance. By blocking the F(ab')2 fragment, which has a shorter half-life than the intact IgG equivalent, the in vitro effect of the immunofidase may be even stronger in vivo.

[0247] Example 6 - Loss of cytokine production from CAR-T cells cultured with soluble immunoglobulin in the presence of IdeS

[0248] Introduction In this study, we investigated whether CAR-T cell constructs targeting immunoglobulin light chains (such as those disclosed in Ranganathan et al., Clin Cancer Res, 2021 and Vera et al., Blood 2006;108) exhibit baseline production of the cytokines IFNg and IL-2 in the presence of immunoglobulin. We also investigated whether the addition of IdeS to the suspension would cleave the soluble immunoglobulin and reduce or eliminate cytokine production.

[0249] method In the first experiment, T cells were isolated from peripheral blood mononuclear cells obtained from two healthy human donors. T cells were transduced with a CAR construct targeting CD19 (CD19.CAR) or a CAR construct targeting kappa light chain (kappa.28). A population of non-transduced cells (NTD) was used as a negative control. The cells were then plated in four different culture conditions, each condition having an increasing concentration of soluble immunoglobulin in it. IdeS was then added to another group of similarly plated CAR-T cells to observe the variation in cytokine production.

[0250] In the second experiment, the first experiment was repeated using a second light chain-targeting construct, a lambda light chain-targeting CAR (lambda.28). T cells obtained from healthy human donors were used and were either transduced with CD19.CAR, kappa.28, lambda.28, or not transduced (NTD). As in the previous experiment, cells were plated in the same increasing concentrations of soluble immunoglobulin, and with or without IdeS.

[0251] result As shown in Figure 1, NTD cells and CD19.CAR did not produce any interferon gamma (IFNγ) regardless of the concentration of soluble immunoglobulin present in the serum, confirming that the assay was working since NTD cells and CD19.CAR do not recognize soluble immunoglobulin.

[0252] Kappa.28 T cells produced IFNγ in the presence of soluble immunoglobulin, indicating baseline cytokine production. Furthermore, more IFNγ was produced in serum containing higher concentrations of soluble immunoglobulin. However, addition of IdeS to similarly plated kappa.28 cells suppressed IFNγ production. These results were reproducible in both donors. These data demonstrate that light chain-specific CAR-T cells can be stimulated off-tumor by soluble immunoglobulin and that this stimulation can be suppressed by IdeS. Thus, IdeS administration is predicted to be useful in maintaining CAR-T cell activity and reducing attrition.

[0253] Similar results were observed in the second experiment, as shown in Figure 2. These data confirm that light chain-specific CAR-T cells can be stimulated off-tumor by soluble immunoglobulins, and that this stimulation can be suppressed by IdeS. Therefore, IdeS administration is predicted to be useful for maintaining the activity of CAR-T cells and reducing attrition. The lambda.28 construct did not produce minimal IFNγ, which may be due to the fact that the soluble human immunoglobulin serum used is polyclonal and has a kappa:lambda ratio of 2:1 as naturally occurring in the human body. Therefore, the amount of lambda light chain in the soluble immunoglobulin serum used may not have been sufficient to elicit cytokine production.

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Claims

1. A protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy targeting immunoglobulin light chains for use in improving the patient benefits of said immunotherapy.

2. A protein having IgG cysteine ​​protease or IgG endoglycosidase activity in combination with adoptive cell transfer immunotherapy targeting immunoglobulin light chains for use in the treatment of cancer.

3. The protein of claim 1, wherein the protein having IgG cysteine ​​protease or IgG endoglycosidase activity is administered before the adoptive cell transfer immunotherapy is administered, or the protein having IgG cysteine ​​protease or IgG endoglycosidase activity is administered after the adoptive cell transfer immunotherapy is administered.

4. A protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in the treatment of cancer, wherein the treatment of cancer comprises administering the protein to a patient who has previously undergone and / or will undergo adoptive cell transfer immunotherapy targeted to immunoglobulin light chains.

5. Adoptive cell transfer immunotherapy targeted to immunoglobulin light chains for use in the treatment of cancer, wherein said treatment of cancer comprises administering said immunotherapy to a patient who has previously received and / or will receive a protein having IgG cysteine ​​protease or IgG endoglycosidase activity.

6. 1. An immunoglobulin light chain targeted adoptive cell transfer immunotherapy for use in the treatment of an antibody-mediated autoimmune disease, wherein said treatment of said antibody-mediated autoimmune disease comprises administering said immunotherapy to a patient who has previously received and / or will receive a protein having IgG cysteine ​​protease or IgG endoglycosidase activity; Optionally, the antibody-mediated autoimmune disease is selected from the group consisting of juvenile arthritis (particularly juvenile idiopathic arthritis), rheumatoid arthritis, generalized myxedematous lichen (sclerosing myxedema), Graves' disease, IgA-driven bullous dermatosis, IgG4-driven bullous pemphigoid, Sjogren's syndrome, and lupus mastitis.

7. 7. The protein of any one of claims 1 to 4 or the immunotherapy of claim 5 or 6, wherein the adoptive cell transfer immunotherapy targeting the immunoglobulin light chain comprises administering T cells, natural killer cells, or dendritic cells expressing a chimeric antigen receptor or a T cell receptor.

8. 8. The protein or immunotherapy of claim 7, wherein the chimeric antigen receptor or T cell receptor comprises a binding domain, such as an scFv, that specifically binds to an immunoglobulin light chain, such as a human kappa immunoglobulin light chain or a human lambda immunoglobulin light chain.

9. A protein according to any one of claims 1 to 4 or an immunotherapy according to claim 5 or 6, which increases the activity, viability and / or proliferation of cells administered in said adoptive cell transfer immunotherapy.

10. 7. The protein of any one of claims 1 to 4 or the immunotherapy of claim 5 or 6, which reduces antibody-mediated complement deposition, complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), depletion, receptor-activated cell death, receptor blocking, and / or immunoglobulin cross-linking of cells administered in the adoptive cell transfer immunotherapy.

11. A protein described in any one of claims 1 to 4 or an immunotherapy described in claim 5 or 6, wherein the target disease is cancer, and the cancer is a B-cell neoplasm such as B-cell lymphoma or B-cell leukemia.

12. (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 and optionally, the protein is EndoS, CP40, EndoE, or EndoF. ２ The protein according to any one of claims 1 to 4 or the immunotherapy according to claim 5 or 6,

13. (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, 5, or 91, or a fragment or variant thereof having IgG cysteine ​​protease activity; or (ii) The protein or immunotherapy of claim 12, 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.

14. (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, 5, or 91, 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 (ii) The protein or immunotherapy of claim 13, 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.

15. 1. A protein having IgG cysteine ​​protease or IgG endoglycosidase activity for use in conditioning or preparing a patient for immunoglobulin light chain targeted adoptive cell transfer immunotherapy, for use in improving the benefit to a patient of immunoglobulin light chain targeted adoptive cell transfer immunotherapy, or for use in reducing plasma IgG levels or reducing complement or Fc receptor binding by plasma IgG molecules in a patient undergoing or to undergo immunoglobulin light chain targeted adoptive cell transfer immunotherapy.

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