Compositions and methods for enhancing chimeric antigen receptor (CAR) T cell therapy

Abstract

The present disclosure relates to improving CAR-T expansion and / or survival using CD3 antibodies alone or in combination with other costimulatory molecules, such as anti-IL-6 receptor monoclonal antibodies, anti-CD28 monoclonal antibodies, IL-17 monoclonal antibodies, or specific inhibitors of the phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), or mammalian target of rapamycin (mTOR) signaling pathways.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority of U.S. Provisional Patent Application No. 63 / 040,101, filed on June 17, 2020, and U.S. Provisional Patent Application No. 63 / 058,783, filed on July 30, 2020, the contents of which are hereby incorporated by reference in their entirety.

[0002] Reference to Sequence Listing This application was filed electronically via EFS - Web and includes an electronically submitted sequence listing in.txt format. The.txt file was created on June 16, 2021, and contains a sequence listing titled "TIZI_027_001WO_SeqList_ST25.txt" with a size of 33 KB. The sequence listing contained in this.txt file is part of this specification and is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates to the use of CD3 antibodies alone or in combination with other costimulatory molecules such as anti - IL - 6 receptor monoclonal antibodies, anti - CD28 monoclonal antibodies, or specific inhibitors of the phosphatidylinositol 3 - kinase (PI3K), protein kinase B (AKT), or mammalian target of rapamycin (mTOR) signaling pathways for improving cell therapy, CAR - T expansion, and / or survival.

Background Art

[0004] Chimeric antigen receptor T cells (CAR-Ts) specifically enhance T cell function while leveraging the expansion and killing power of cytotoxic T cells, thereby circumventing common problems in T cell therapy, such as dependence on human leukocyte antigen (HLA) interactions for activating T cell effector function. HLA is frequently downregulated in cancer cells, promoting immune system evasion, which makes it more difficult for engineered T cells to activate effector responses. By expressing CARs and binding to tumor-specific surface proteins, T cells can reduce their dependence on HLA-mediated activation and induce effector responses. A basic CAR is a single-chain variable region antibody fragment (scFV) consisting of an antigen-binding domain, an extracellular domain, a transmembrane domain, and an intracellular signaling domain. The intracellular domain is typically the CD3ζ chain, which is typically associated with the T cell receptor (TCR) complex. CAR-T cells combine the T cell kinetics with the antigen specificity of the antibody, allowing them to bind to tumor antigens without antigen treatment and independently of HLA-mediated antigen presentation.

[0005] CAR-T cell therapy has been successful in treating B-cell leukemia, most notably acute B-cell lymphoblastic leukemia (B-cell ALL) treated with anti-CD19 CAR-T cells.

[0006] Despite encouraging clinical outcomes, relapse rates after CAR-T therapy occur, limiting the usefulness of this promising treatment approach. Therefore, it is crucial to address the current limitations of CAR-T cell therapy. This disclosure provides compositions, methods, and embodiments that meet this unmet need. [Overview of the Initiative]

[0007] In one embodiment, the present disclosure provides a method for improving cell expansion and / or viability, comprising the step of contacting cells with a composition comprising an anti-CD3 antibody. In some embodiments, the cells are engineered cells. In some embodiments, the contact is ex vivo, in vivo, or both.

[0008] In some embodiments, the cells are lymphocytes. In some embodiments, the lymphocytes are B cells or T cells. In some embodiments, the T cells are CAR-T cells. In some embodiments, the cells are stem cells. In some embodiments, the stem cells are human embryonic stem cells, tissue-specific stem cells, neural stem cells, mesenchymal stem cells, hematopoietic stem cells, induced pluripotent stem cells, epidermal stem cells, epithelial stem cells, and / or neural stem cells.

[0009] In one embodiment, the present disclosure provides a method for augmenting cell therapy in a subject, comprising the step of administering a composition comprising an anti-CD3 antibody to a subject in need thereof. In some embodiments, the cell therapy is CAR-T cell therapy. In some embodiments, the cell therapy is stem cell therapy. In some embodiments, the stem cells are human embryonic stem cells, tissue-specific stem cells, neural stem cells, mesenchymal stem cells, hematopoietic stem cells, induced pluripotent stem cells, epidermal stem cells, epithelial stem cells, and / or neural stem cells.

[0010] In some embodiments, compositions containing an anti-CD3 antibody improve the clinical outcomes of cell therapy. In some embodiments, the anti-CD3 antibody is a monoclonal antibody, a bispecific antibody, or a tripspecific antibody.

[0011] In some embodiments, the bispecific antibody has specificity for CD3 and IL-6R, CD28, or TNF.

[0012] In some embodiments, the triplicate antibody has specificity for i) CD3, IL6R, and CD28; ii) CD3, IL6R, and TNF; iii) CD3, CD28, and TNF; or iv) IL-6, IL-17.

[0013] In some embodiments, the composition comprising an anti-CD3 antibody further comprises one or more co-stimulators. In some embodiments, the co-stimulators are a CD28 antibody, an IL-6R antibody, a PI3K inhibitor, an Akt inhibitor, or an mTor inhibitor. In some embodiments, the CD28 antibody is a monoclonal antibody, a bispecific antibody, or a tripspecific antibody.

[0014] In some embodiments, the bispecific antibody has specificity for CD28 and IL-6R or TNF.

[0015] In some embodiments, the triplicate antibody has specificity for CD28, IL-6R, and TNF.

[0016] In some embodiments, the IL-6R antibody is a monoclonal antibody, a bispecific antibody, or a triplicate antibody. In some embodiments, the bispecific antibody has specificity for IL-6R and CD28 or TNF. In some embodiments, the triplicate antibody has specificity for IL-6R, CD28, and TNF.

[0017] In some embodiments, the CD3 antibody and / or CD28 antibody are coated onto macroporous beads. In some embodiments, the CD3 antibody is administered via nasal, oral, subcutaneous, intravenous, or inhalation.

[0018] In some embodiments, the CD3 antibody includes a heavy chain complementarity determination region 1 (CDRH1) containing the amino acid sequence GYGMH (SEQ ID NO: 42), a heavy chain complementarity determination region 2 (CDRH2) containing the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO: 43), a heavy chain complementarity determination region 3 (CDRH3) containing the amino acid sequence QMGYWHFDL (SEQ ID NO: 44), a light chain complementarity determination region 1 (CDRL1) containing the amino acid sequence RASQSVSSYLA (SEQ ID NO: 45), a light chain complementarity determination region 2 (CDRL2) containing the amino acid sequence DASNRAT (SEQ ID NO: 46), and a light chain complementarity determination region 3 (CDRL3) containing the amino acid sequence QQRSNWPPLT (SEQ ID NO: 47).

[0019] In some embodiments, the CD3 antibody includes a variable heavy chain amino acid sequence containing the amino acid sequence of SEQ ID NO: 48 and a variable light chain amino acid sequence containing the amino acid sequence of SEQ ID NO: 49. In some embodiments, the CD3 antibody includes a heavy chain amino acid sequence containing the amino acid sequence of SEQ ID NO: 50 and a light chain amino acid sequence containing the amino acid sequence of SEQ ID NO: 51.

[0020] In some embodiments, the IL-6R antibody includes a VH CDR1 region containing the amino acid sequence of SEQ ID NO: 15, a VH CDR2 region containing the amino acid sequence of SEQ ID NO: 37, a VH CDR3 region containing the amino acid sequence of SEQ ID NO: 35, a VL CDR1 region containing the amino acid sequence of SEQ ID NO: 24, a VL CDR2 region containing the amino acid sequence of SEQ ID NO: 25, and a VL CDR3 region containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, the IL-6R antibody is tocilizumab or sarilumab.

[0021] In some embodiments, the anti-CD3 antibody is administered before, after, both before and after, and / or concurrently with the administration of the cell therapy composition to the target. In some embodiments, the anti-CD3 antibody is administered 24 to 48 hours before the administration of the cell therapy composition to the target. In some embodiments, the anti-CD3 antibody is administered 14 to 21 days after the administration of the cell therapy composition.

[0022] In some embodiments, administration of an anti-CD3 antibody prior to administration of a cell therapy composition results in lymphocyte depletion and / or immunosuppression.

[0023] In some embodiments, the anti-CD3 antibody is formulated in a pharmaceutical composition comprising the anti-CD3 antibody and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition contains a unit dose of 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, or 4 mg of the anti-CD3 antibody. [Brief explanation of the drawing]

[0024] [Figure 1]A diagram showing the method of the present disclosure. Specifically, it is to manipulate CAR-T cells to kill tumor cells in cancer patients. The process of ex vivo and in vivo expansion of CAR-T cells can be improved by co-stimulation with anti-CD3 / anti-CD28 and small molecule inhibitors of the PI-3K / Akt / mTOR axis. Anti-IL-6 receptor mAb can also enhance the survival of CAR-T cells.

[0025] [Figure 2] A plot showing the pharmacokinetic profile of foralumab administered subcutaneously or intravenously in a mouse model.

[0026] [Figure 3] A table showing the pharmacokinetic profile of foralumab administered intravenously using different dosing regimens in human subjects.

[0027] [Figure 4] A table showing the apparent Cmax and AUC values of foralumab administered intravenously using different dosing regimens in human subjects.

[0028] [Figure 5] A set of plots showing the time-course levels of foralumab in plasma after intravenous administration in human subjects.

[0029] [Figure 6] A set of plots showing the time-course levels of foralumab in plasma and CD3 regulation after intravenous administration in human subjects.

[0030] [Figure 7] A set of plots showing the levels of CD3 regulation on CD4+ and CD8+ T cells over time after intravenous administration of foralumab at different dosages in human subjects.

[0031] [Figure 8]This plot shows the level of CD3 regulation on CD4+ and CD8+ T cells over time after intravenous administration of foralumab at different doses in human subjects.

[0032] [Figure 9] This table shows the mean TCR-CD3 regulation on CD45+ lymphocytes at different time points after intravenous administration of foralumab at different dosages in human subjects.

[0033] [Figure 10] This table shows the mean peak cytokine concentrations after intravenous administration of foralumab at different dosages in human subjects.

[0034] [Figure 11] This table shows a summary of all serious adverse events in human subjects after intravenous administration of foralumab.

[0035] [Figure 12] This table lists the reported infusion-related reactions (IRRs) in individual human subjects after intravenous administration of foralumab.

[0036] [Figure 13] This table lists the reported infusion-related reactions (IRRs) in individual human subjects after intravenous administration of foralumab.

[0037] [Figure 14] This table summarizes the abnormal liver function test results in human subjects after intravenous administration of foralumab.

[0038] [Figure 15A] This figure shows an example embodiment of a clinical dosing plan for the administration of foralmab to augment CAR-T cell therapy. [Figure 15B] This figure shows an example embodiment of a clinical dosing plan for the administration of foralmab to augment CAR-T cell therapy. [Figure 15C]This figure shows an example embodiment of a clinical dosing plan for the administration of foralmab to augment CAR-T cell therapy. [Modes for carrying out the invention]

[0039] This disclosure provides compositions and methods for improving cell therapies such as chimeric antigen receptor T cell therapy (CAR-T cell therapy), which represents a promising novel therapeutic option for patients with various hematological and solid tumors, and possibly patients with autoimmune diseases. However, poor CAR-T expansion and survival in the complex manufacturing process and ex vivo process are costly. Improved CAR-T therapy can be achieved through more efficient production processes by providing ancillary therapies to optimize the ex vivo expansion state and / or improve the clinical benefits of CAR-T cells. Accordingly, this disclosure provides compositions and in vivo treatment plans for increasing the ex vivo expansion of CAR-T cells. Specifically, the compositions and methods of this disclosure utilize an anti-CD3 monoclonal antibody either alone or in combination with other co-stimulating molecules such as an anti-IL-6 receptor (IL-6R) monoclonal antibody, an anti-CD28 monoclonal antibody, or a specific inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway. More specifically, intravenous or subcutaneous administration of foralumab, a fully human anti-CD3 monoclonal antibody, can be used as a lymphocyte depletion agent to improve CAR-T cell survival. The use of foralumab for enhancement of lymphocyte depletion to replace other lymphocyte depletion agents such as cyclophosphamide / fludarabine (cy / flu) can be administered alone or in combination with other co-stimulating molecules such as anti-IL-6, anti-IL-17, or other anti-inflammatory agents at different stages before and after CAR-T therapy.

[0040] Anti-CD3 antibody Antibodies specific to the CD3 epsilon chain (CD3ε) and their antigen-binding fragments are referred to herein as “anti-CD3 antibodies” or “CD3 antibodies,” and compositions are referred to herein as “anti-CD3 antibody compositions.” Any anti-CD3 antibodies known in the art are suitable for use in this disclosure. Anti-CD3 antibodies are monoclonal antibodies.

[0041] An anti-CD3 antibody can be any antibody specific to CD3. Anti-CD3 antibodies can be polyclonal, monoclonal, recombinant, e.g., chimeric, deimmunized or humanized, fully human, non-human, e.g., mouse, single-chain, or single-domain antibodies. In some embodiments, the antibody may have effector function and be capable of immobilizing complement. In some embodiments, the antibody may have or may not have reduced ability to bind to the Fc receptor. For example, an anti-CD3 antibody may be an isotype or subtype, fragment, or other variant that does not support binding to the Fc receptor, for example, having a mutagenic or deficient Fc receptor binding region. The antibody may be conjugated to a toxin or contrast agent.

[0042] OKT3 (muromonab / Orthoclone OKT3(TM), Ortho Biotech, Raritan, NJ; U.S. Patent No. 4,361,549); hOKT3(1(Herold et al., NEJM 346(22):1692-1698(2002)); HuM291(Nuvion(TM), Protein Design Labs, Fremont, Calif.);gOKT3-5(Alegre et al., J. Immunol. 148(11):3461-8 (1992);1F4(Tanaka et al., J. Immunol. 142:2791-2795 (1989));G4.18(Nicholls et al., Transplantation 55:459-468 (1993));145-2C11(Davignon et al., Numerous anti-CD3 antibodies are known, including, but not limited to, those described in J. Immunol. 141(6):1848-54 (1988); and Frenken et al., Transplantation 51(4):881-7 (1991); U.S. Patents 6,491,9116, 6,406,696, and 6,143,297.

[0043] Methods for producing such antibodies are also known. Full-length CD3 protein or CD3 antigen peptide fragments can be used as immunogens, or they can be used to identify anti-CD3 antibodies produced using other immunogens, such as cells, membrane preparations, etc., as described in U.S. Patents No. 4,361,549 and 4,654,210, using E-rosette-positive purified normal human peripheral T cells. Anti-CD3 antibodies can bind to epitopes on any domain or region of CD3.

[0044] Chimeric, humanized, deimmunized, or fully human antibodies are desirable for repeated administration, including therapeutic treatments in human subjects.

[0045] Chimeric antibodies contain portions of two different antibodies, typically from two different species. Generally, such antibodies contain a human constant region and a variable region derived from another species, e.g., a mouse variable region. For example, mouse / human chimeric antibodies exhibiting binding characteristics of the parental mouse antibody and effector functions associated with the human constant region have been reported. See, for example, U.S. Patent No. 4,816,567 by Cabilly et al.; U.S. Patent No. 4,978,745 by Shoemaker et al.; U.S. Patent No. 4,975,369 by Beavers et al.; and U.S. Patent No. 4,816,397 by Boss et al., all of which are incorporated herein by reference. Generally, these chimeric antibodies are constructed by preparing a genomic gene library from DNA extracted from existing mouse hybridomas (Nishimura et al., Cancer Research, 47:999 (1987)). The library is then screened for variable region genes derived from both the heavy and light chains that exhibit the correct antibody fragment rearrangement pattern. Alternatively, a cDNA library may be extracted from hybridomas and prepared from screened RNA, or the variable region may be obtained by polymerase chain reaction. The cloned variable region gene is then ligated into an expression vector containing a cloned cassette of a suitable heavy-chain or light-chain human constant region gene. The chimeric gene can then be expressed in a select cell line, such as a mouse myeloma cell. Such chimeric antibodies are used in human therapy.

[0046] Humanized antibodies are well known in the art. Typically, "humanization" results in antibodies that are less immunogenic but retain all the antigen-binding properties of the original molecule. To retain all the antigen-binding properties of the original antibody, the structure of its binding site must be precisely reproduced in the "humanized" version. This can be achieved by (a) transplanting the entire non-human variable domain into the human constant region to obtain a chimeric antibody (Morrison et al., Proc. Natl. Acad. Sci., USA 81:6801 (1984); Morrison and Oi, Adv. Immunol. 44:65 (1988)) (preserving ligand-binding properties but also retaining the immunogenicity of the non-human variable domain); (b) transplanting only the non-human CDR into the constant region with or without the retention of the human framework and definitive framework residues (Jones et al. Nature, 321:522 (1986); Verhoeyen et al., Science 239:1539 (1988)); or (c) transplanting the entire non-human variable domain (to preserve ligand-binding properties) but "covering" it with a human-like surface through precise substitution of exposed residues (to reduce antigenicity) (Padlan, Molec. Immunol. 28:489) (1991)) This may be achievable by transplanting the binding site of a non-human antibody into a human framework.

[0047] Humanization by CDR transplantation typically involves transplanting only the CDR into a human framework with human fragments and constant regions. Theoretically, this should substantially eliminate immunogenicity (unless allotype or idiotype differences exist). However, it has also been reported that some framework residues of the original antibody need to be conserved (Riechmann et al., Nature 332:323 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10,029 (1989)). Framework residues that need to be conserved can be identified by computer modeling. Alternatively, definitive framework residues may be identified by comparing them with known antibody binding site structures (Padlan, Molec. Immun. 31(3):169-217 (1994)). The compositions and methods of the present disclosure also include a partially humanized antibody in which six heavy and light chain CDRs and a limited number of structural amino acids of a mouse monoclonal antibody are transplanted into a CDR-depleted human IgG scaffold by recombinant technology (Jones et al., Nature 321:522-525 (1986)).

[0048] Deimmunizing antibodies are produced by replacing immunogenic epitopes in the mouse variable domain with benign amino acid sequences to obtain a deimmunizing variable domain. This deimmunizing variable domain is then gene-bound to the human IgG constant domain to obtain a deimmunizing antibody (Biovation, Aberdeen, Scotland).

[0049] Anti-CD3 antibodies can also be single-chain antibodies. Single-chain antibodies (scFVs) can be manipulated (see, for example, Colcher et al., Ann. NY Acad. Sci. 880:263-80 (1999); and Reiter, Clin. Cancer Res. 2:245-52 (1996)). Single-chain antibodies can be dimerized or polymerized to obtain polyvalent antibodies with specificity for different epitopes of the same target CD3 protein. In some embodiments, the antibody is monovalent, as described, for example, in Abbs et al., Ther. Immunol. 1(6):325-31 (1994), which is incorporated herein by reference.

[0050] An exemplary anti-CD3 antibody includes a heavy chain complementarity determination region 1 (CDRH1) containing the amino acid sequence GYGMH (SEQ ID NO: 42), a heavy chain complementarity determination region 2 (CDRH2) containing the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO: 43), a heavy chain complementarity determination region 3 (CDRH3) containing the amino acid sequence QMGYWHFDL (SEQ ID NO: 44), a light chain complementarity determination region 1 (CDRL1) containing the amino acid sequence RASQSVSSYLA (SEQ ID NO: 45), a light chain complementarity determination region 2 (CDRL2) containing the amino acid sequence DASNRAT (SEQ ID NO: 46), and a light chain complementarity determination region 3 (CDRL3) containing the amino acid sequence QQRSNWPPLT (SEQ ID NO: 47).

[0051] In some embodiments, the anti-CD3 antibody is [ka] Variable heavy chain amino acid sequence including (SEQ ID NO: 48) and [ka] Contains a variable light chain amino acid sequence including (SEQ ID NO: 49).

[0052] Preferably, the anti-CD3 antibody is [ka] (SEQ ID NO: 50) and heavy chain amino acid sequence [ka] It contains a light chain amino acid sequence including (SEQ ID NO: 51). This anti-CD3 antibody is referred to herein as NI-0401, foralmab, or 28F11-AE (see, for example, Dean Y, Depis F, Kosco-Vilbois M. “Combination therapies in the context of anti-CD3 antibodies for the treatment of autoimmune diseases.” Swiss Med Wkly. (2012) (its contents are incorporated herein by reference in their entirety)).

[0053] In some embodiments, the anti-CD3 antibody is a fully human antibody or a humanized antibody. In some embodiments, the anti-CD3 antibody preparation comprises a full-length anti-CD3 antibody. In alternative embodiments, the anti-CD3 antibody preparation comprises an antibody fragment that specifically binds to CD3. In some embodiments, the anti-CD3 antibody preparation comprises a combination of a full-length anti-CD3 antibody that specifically binds to CD3 and an antigen-binding fragment.

[0054] In some embodiments, the antibody or antigen-binding fragment that binds to CD3 is a monoclonal antibody, a domain antibody, a single-chain antibody, a Fab fragment, an F(ab')2 fragment, an scFv, scAb, a dAb, a single-domain heavy-chain antibody, or a single-domain light-chain antibody. In some embodiments, such an antibody or antigen-binding fragment that binds to CD3 is a mouse, other rodent, chimeric, humanized, or fully human monoclonal antibody.

[0055] Optionally, the anti-CD3 antibody or its antigen-binding fragment used in the formulations of this disclosure contains at least one amino acid mutation. Typically, the mutation is located in the constant region. The mutation results in an antibody with altered effector function. The effector function of an antibody is altered by changing, i.e., enhancing or reducing, the antibody's affinity for effector molecules such as Fc receptors or complement components. For example, the mutation results in an antibody capable of reducing cytokine release from T cells. For example, the mutation is located in the heavy chain, at amino acid residues 234, 235, 265, or 297, or a combination thereof. Preferably, the mutation results in an alanine residue at any of positions 234, 235, 265, or 297, or a glutamate residue at position 235, or a combination thereof.

[0056] Preferably, the anti-CD3 antibodies provided herein contain one or more mutations that interfere with the heavy chain constant region-mediated release of one or more cytokines in vivo.

[0057] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment used in the formulations of this disclosure is a fully human antibody. The fully human CD3 antibodies used herein include, for example, L234A and L235E mutations in the Fc region such that cytokine release upon exposure to the anti-CD3 antibody is significantly reduced or eliminated. The L234A and L235E mutations in the Fc region of the anti-CD3 antibodies provided herein reduce or eliminate cytokine release when the anti-CD3 antibody is exposed to human leukocytes, while the mutations described below maintain significant cytokine release. For example, a significant reduction in cytokine release is defined by comparing the cytokine release upon exposure to an anti-CD3 antibody having the L234A and L235E mutations in the Fc region with the cytokine release level upon exposure to another anti-CD3 antibody having one or more of the mutations described below. Other mutations in the Fc region include, for example, L234A and L235A, L235E, N297A, D265A, or combinations thereof.

[0058] The term "cytokine" refers to all human cytokines known in the art, including but not limited to IL-2, IFN-gamma, TNF-α, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, that bind to extracellular receptors expressed on the cell surface and thereby regulate cellular functions.

[0059] The anti-CD3 formulation contains an anti-CD3 antibody in a unit dose ranging from approximately 0.01 mg to approximately 25 mg; or from 0.01 mg to approximately 10 mg. For example, the unit dose is approximately 0.01, 0.02, 0.03, 0.04, 0.50, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9, 9.5, 10 mg or more. Preferably, the unit dose is 0.05 mg, 0.1 mg, 0.5 mg, 1.0 mg, 2.5 mg, 5.0 mg, or 10 mg.

[0060] The anti-CD3 antibody preparation comprises one or more salts (buffered salts), one or more polyols, and one or more excipients. The preparations of this disclosure may also contain buffers or preservatives. The anti-CD3 antibody preparation is buffered in a solution with a pH in the range of about 4 to 8; about 4 to 7; about 4 to 6; about 5 to 6; or about 5.5 to 6.5. Preferably, the pH is 5.5.

[0061] Examples of salts include those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, boric acid, formic acid, malonic acid, succinic acid, etc. Such salts can also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium, or calcium salts. Examples of buffers include phosphates, citrates, acetates, and 2-(N-morpholino)ethanesulfonic acid (MES).

[0062] The formulations of this disclosure may include a buffer system. As used in this application, the terms “buffer” or “buffer system” typically mean a compound that, in combination with at least one other compound, provides a buffer system in solution that exhibits buffering ability, i.e., the ability to moderately neutralize either an acid or a base (alkali) with a relatively small change or no change in the original pH.

[0063] Buffers include borate buffers, phosphate buffers, calcium buffers, and combinations and mixtures thereof. Borate buffers include, for example, boric acid and its salts, such as sodium borate or potassium borate. Borate buffers also include compounds such as potassium tetraborate or potassium metaborate that produce boric acid or its salts in solution.

[0064] A phosphate buffer system comprises one or more monobasic phosphates, dibasic phosphates, etc. Particularly useful phosphate buffers are those selected from alkali metal and / or alkaline earth metal phosphates. Examples of suitable phosphate buffers include one or more of dibasic sodium phosphate (Na2HPO4), monobasic sodium phosphate (NaH2PO4), and monobasic potassium phosphate (KH2PO4). Phosphate buffer components are frequently used in amounts of 0.01% or ~0.5% (w / v), calculated as phosphate ions.

[0065] Other known buffer compounds can be optionally added according to the CD3 formulation, such as citrate, sodium bicarbonate, and TRIS. Other components in the solution can also affect the buffering capacity while having other functions. For example, EDTA, often used as a compounding agent, can have a noteworthy effect on the buffering capacity of the solution.

[0066] Preferred salts for use in the formulations of this disclosure include sodium chloride, sodium acetate, sodium acetate trihydrate, and sodium citrate.

[0067] The salt concentrations in the formulations according to this disclosure are between approximately 10 mM and 500 mM, between approximately 25 mM and 250 mM, and between approximately 25 nM and 150 mM.

[0068] The concentration of sodium acetate trihydrate is in the range of approximately 10 mM to 100 mM. For example, sodium acetate trihydrate is approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM. Preferably, the sodium acetate trihydrate is 25 mM.

[0069] Sodium chloride at a concentration in the range of approximately 50 mM to 500 mM. For example, sodium chloride concentrations of approximately 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 mM. Preferably, the sodium chloride concentration is approximately 125 mM.

[0070] The sodium citrate concentration is in the range of approximately 10 mM to 100 mM. For example, the sodium citrate concentration is approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM. Preferably, the sodium citrate concentration is in the range of approximately 25 to 50 mM.

[0071] In some embodiments, the salts are sodium acetate trihydrate at concentrations ranging from about 25 mm to 100 mm and sodium chloride at concentrations ranging from about 150 mm to 500 mm.

[0072] Preferably, the formulation contains about 25 mM sodium acetate trihydrate and about 150 mM sodium chloride.

[0073] The formulation contains one or more polyols as fillers and / or stabilizing excipients. The polyols include, for example, trehalose, mannitol, maltose, lactose, sucrose, sorbitol, or glycerol. The polyols are present in concentrations ranging from about 0.1% to 50% or 5% to 25%. For example, the polyols may be present in concentrations of about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%.

[0074] In some embodiments, the polyol is trehalose in a concentration ranging from about 1% to 50% or 5% to 25%. For example, the trehalose concentration may be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%. Preferably, the trehalose concentration is about 10% or about 20%. Most preferably, the trehalose concentration is about 20%.

[0075] In some embodiments, the polyol is sorbitol in a concentration ranging from about 1% to about 10%. In some embodiments, the polyol is glycerol in a concentration ranging from about 1% to about 10%.

[0076] In some embodiments, the polyol is mannitol in a concentration ranging from about 0.1% to about 10%. In some embodiments, the polyol is maltose in a concentration ranging from about 1% to about 10%.

[0077] The formulation comprises one or more excipients and / or surfactants to inhibit or otherwise reduce antibody aggregation. Suitable excipients for reducing antibody aggregation include, for non-limiting illustrative purposes, surfactants, e.g., polysorbate 20 or polysorbate 80. In some embodiments, polysorbate 20 or polysorbate 80 is present in concentrations ranging from about 0.01 to 1% or about 0.01 to 0.05%. For example, polysorbate 20 or polysorbate 80 is present in concentrations of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0%.

[0078] Preferably, the surfactant is polysorbate 80 in a concentration of about 0.01 to 0.05%. More preferably, the polysorbate 80 is 0.02%.

[0079] The formulation comprises one or more excipients for reducing antibody oxidation. Suitable excipients for reducing antibody oxidation include, for non-limiting illustrative purposes, antioxidants. Antioxidants include, for example, methionine, D-arginine, BHT, or ascorbic acid. Antioxidants are present in concentrations ranging from about 0.01% to 1%; 0.1% to 1%; or 0.1% to 0.5%. In some embodiments, the antioxidant is methionine. In some embodiments, methionine is present in concentrations ranging from about 0.01% to 1%; 0.1% to 1%; or 0.1% to 0.5%. For example, methionine is present at concentrations of approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0%. Preferably, the methionine concentration is approximately 0.1%.

[0080] The formulation contains one or more chelating agents, such as ethylenediaminetetraacetic acid (EDTA). The chelating agent is present in concentrations within the range of 0.01% to 1%, 0.1% to 1%, or 0.1% to 0.5%. For example, the chelating agent is present in concentrations of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%. Preferably, the chelating agent is EDTA at a concentration of about 0.1%.

[0081] In some embodiments, the formulation includes one or more excipients to increase stability. In some embodiments, the excipient for increasing stability is human serum albumin. In some embodiments, the human serum albumin is present in a range of about 1 mg to about 5 mg.

[0082] In some embodiments, the formulation comprises magnesium stearate (Mg stearate), an amino acid, or both magnesium stearate and an amino acid. Suitable amino acids include, for example, leucine, arginine, histidine, or a combination thereof.

[0083] In some embodiments, one or more additional excipients are low-moisture microcrystalline cellulose such as Avicel, polyethylene glycol (PEG), or starch.

[0084] Further examples of pharmaceutically acceptable carriers and excipients useful in the formulations of this disclosure include binders, fillers, disintegrants, lubricants, antimicrobial agents, antioxidants, and coatings, e.g., binders: corn starch, potato starch, other starches, gelatin, natural and synthetic gums, e.g., acacia, xanthan gum, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethylcellulose, cellulose acetate, carboxymethylcellulose calcium, carboxymethylcellulose sodium), polyvinylpyrrolidone (e.g., povidone, crospovidone, copovidone, etc.), methylcellulose, Methocel, pregelatinized starch (e.g., Colorcon, Ltd.)Sold by STARCH 1500 (registered trademark) and STARCH 1500 LM (registered trademark), hydroxypropyl methylcellulose, microcrystalline cellulose (FMC Corporation, Marcus Hook, PA, USA), Emdex, Plasdone, or mixtures thereof; Fillers: Talc, Calcium Carboxylate (e.g., granules or powder), Dibasic Calcium Phosphate, Tribasic Calcium Phosphate, Calcium Sulfate (e.g., granules or powder), Microcrystalline Cellulose, Powdered Cellulose, Dextrose, Kaolin, Mannitol, Silicic Acid, Sorbitol, Starch, Pregelatinized Starch, Dextrose, Fructose, Honey, Anhydrous Lactose, Lactose Monohydrate, Lactose and Aspartame, Lactose and Cellulose, Lactose and Microcrystalline Cellulose, Maltodextrin, Maltose, Mannitol, Microcrystalline Cellulose and Guar Gum, Molasses, Sucrose, or mixtures thereof; Disintegrants: Agar, Alginic Acid, Calcium Carboxylate, Microcrystalline Cellulose, Croscarmellose Sodium, Crospovidone, Potassium polaritrate, sodium starch glycolate (e.g., Explotab), potato or tapioca starch, other starches, pregelatinized starch, clay, other algins, other celluloses, gums (such as gellan), low-substituted hydroxypropylcellulose, polyplasdone, or mixtures thereof, lubricants: calcium stearate, magnesium stearate, mineral oil, diesel fuel, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, complitol, stearic acid, sodium lauryl sulfate, sodium stearyl fumarate (e.g., Pruv), vegetable fatty acid lubricants, talc, hydrogenated vegetable oils (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, silica gel (AEROSIL) 200, WR Grace Co., Baltimore, MD USA), synthetic silica for solidified aerosols (Deaussa Co., Piano, TX USA), exothermic silicon dioxide (CAB-O-SIL, Cabot Co.), Boston, MA USA), or mixtures thereof, anti-caking agents: calcium silicate, magnesium silicate, silicon dioxide, colloidal silicon dioxide, talc, or mixtures thereof, antimicrobial agents: benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, butylparaben, cetylpyridinium chloride, cresol, chlorobutanol, dehydroacetic acid, ethylparaben, methylparaben, phenol, phenylethyl alcohol, phenoxyethanol, phenylmercury acetate, phenylmercury nitrate, potassium sorbate, propylparaben, sodium benzoate, sodium dehydroacetate, sodium propionate, sorbic acid, thimerosol, thymo, or mixtures thereof, acid Anti-corrosion agents include, but are not limited to, ascorbic acid, BHA, BHT, EDTA, or mixtures thereof, and coating agents include sodium carboxymethylcellulose, cellulose acetate phthalate, ethylcellulose, gelatin, pharmaceutical glazing agents, hydroxypropylcellulose, hydroxypropyl methylcellulose (hypromellose), hydroxypropyl methylcellulose phthalate, methylcellulose, polyethylene glycol, polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide, carnauba wax, microcrystalline wax, gellan gum, maltodextrin, methacrylate, microcrystalline cellulose, and carrageenan or mixtures thereof.

[0085] The formulations also include Pluronic®, poloxamer (e.g., Lutrol® and Poloxamer 188), ascorbic acid, glutathione, protease inhibitors (e.g., soy trypsin inhibitors, organic acids), pH lowering agents, creams and lotions (e.g., maltodextrin and carrageenan); chewable tablet materials (e.g., dextrose, fructose, lactose monohydrate, lactose and aspartame, lactose and cellulose, maltodextrin, maltose, mannitol, microcrystalline cellulose and guar gum, sorbitol crystals); parenteral preparations (e.g., mannitol and povidone); plasticizers (e.g., dibutyl sebacate, coating materials, polyvinyl acetate phthalate); powdered lubricants (e.g., glyceryl behenate); soft gelatin capsules (e.g., sorbitol special solution); coating spheres (e.g., sugar spheres); spheroidizing agents (behenate). Glyceryl acid and microcrystalline cellulose, etc.); Suspension / gelling agents (carrageenan, gellan gum, mannitol, microcrystalline cellulose, povidone, sodium starch glycolate, xanthan gum, etc.); Sweeteners (aspartame, aspartame and lactose, dextrose, fructose, honey, maltodextrin, maltose, mannitol, molasses, sorbitol crystals, sorbitol special solution, sucrose, etc.); Wet granulating agents (calcium carboxylate, lactose anhydride, lactose monohydrate, maltodextrin, mannitol, microcrystalline cellulose, povidone, starch, etc.), caramel, sodium carboxymethylcellulose, cherry cream flavor and cherry flavor, citrate anhydride, citric acid, powdered sugar, D&C Red No. 33, D&C Yellow #10 Aluminum Lake, Disodium Edetate, 15% Ethyl Alcohol, FD&C Yellow No. 6 Aluminum Lake, FD&C Blue #1 Aluminum Lake, FD&C Blue No. 1, FD&C Blue No. 2 Aluminum Lake, FD&C Green No. 3, FD&C Red No. 40, FD&C Yellow No. 6 Aluminum Lake, FD&C Yellow No. 6, FD&C Yellow No.10. This may include, but is not limited to, other excipients and their categories, including glycerol palmitostearate, glyceryl monostearate, indigo carmine, lecithin, mannitol, methyl and propylparabens, monoammonium glycyrrhizinate, natural and artificial orange flavors, pharmaceutical glazing agents, poloxamer 188, polydextrose, polysorbate 20, polysorbate 80, polyvidone, pregelatinized corn starch, pregelatinized starch, red iron oxide, sodium saccharin, sodium carboxymethyl ether, sodium chloride, sodium citrate, sodium phosphate, strawberry flavor, synthetic black iron oxide, synthetic red iron oxide, titanium dioxide, and white wax.

[0086] CD3 antibodies formulated for enteral, parenteral, or nasal administration. For example, CD3 antibodies are formulated for nasal, oral, inhalation, subcutaneous, or intravenous administration.

[0087] For enteral administration, i.e., oral administration, the formulation may be in capsule or tablet form. Parenteral administration includes intravenous, subcutaneous, intramuscular, and intra-articular administration, and may be in liquid or lyophilized powder form in a sealed vial or other container. The preferred oral dose range is 0.1 mg to 5 mg daily. For example, doses of 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, or 5.0 mg are administered daily. The dose is administered once or twice daily.

[0088] For nasal administration, the formulation may be an aerosol in a sealed vial or other suitable container. The preferred nasal dose range is 0.05 mg to 1 mg daily. For example, doses of 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1.0 mg may be administered daily. The dose should be divided evenly among the nostrils. The dose may be administered once or twice daily.

[0089] In some embodiments, the anti-CD3 antibody preparation is a subcutaneous preparation. In some embodiments, the subcutaneous anti-CD3 antibody preparation is contained in a sealed vial or other container. The preferred subcutaneous dose range is 0.2 mg to 5 mg daily. For example, doses of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, or 5.0 mg are administered daily. The dose is administered once or twice daily. A preferred preparation for subcutaneous administration is a preferred dose of anti-CD3 antibody in 125 mM sodium chloride containing 0.02% polysorbate 80 in 25 mM sodium acetate buffer at pH 5.5.

[0090] In some embodiments, the anti-CD3 antibody preparation is an inhalation preparation. For inhalation administration, the preparation may be an aerosol in a sealed vial or other suitable container. Administration by inhalation may be in the form of an inhaler or nebulizer. Nebulizers and / or inhalers are portable. Optionally, nebulizers and / or inhalers may be of different sizes to suit children and / or adults.

[0091] The preferred inhalation dose range is 0.1 mg to 5 mg daily. For example, doses of 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, or 5.0 mg are administered daily. The dose is administered once or twice daily.

[0092] The particles in the particulate formulation have diameters ranging from approximately 1 mm to approximately 5 mm, for example, less than 5 mm in diameter, less than 4 mm in diameter, less than 3 mm in diameter, less than 2 mm in diameter, and approximately 1 mm in diameter.

[0093] The particles of a particle formulation containing an anti-CD3 antibody or its antigen-binding fragment have an average diameter of about 0.1 mm to about 50 mm. The particles of a particle formulation containing an anti-CD3 antibody or its antigen-binding fragment have an average diameter of about 1 mm to about 10 mm, for example, an average diameter of less than 10 mm, an average diameter of less than 9 mm, an average diameter of less than 8 mm, an average diameter of less than 7 mm, an average diameter of less than 6 mm, an average diameter of less than 5 mm, an average diameter of less than 4 mm, an average diameter of less than 3 mm, and an average diameter of about 2 mm. In some embodiments, the particles have an average diameter of about 2 mm to 5 mm. In some embodiments, the particles have an average diameter of 2 mm to 5 mm, where each particle has a diameter of less than about 50 mm.

[0094] In some embodiments, the CD3 antibody is available in sustained-release and controlled-release formulations. Methods for producing sustained-release and controlled-release formulations are known in the art and include, for example, the use of macroporous beads.

[0095] In some embodiments, the anti-CD3 antibody preparation comprises a full-length anti-CD3 antibody. In some embodiments, the anti-CD3 antibody preparation comprises an antibody fragment that specifically binds to CD3. In some embodiments, the anti-CD3 antibody preparation comprises a combination of a full-length anti-CD3 antibody that specifically binds to CD3 and an antigen-binding fragment. IL-6 / IL-6 receptor antibody

[0096] Examples of IL-6 / IL-6 receptor (IL-6R) antibodies useful in the compositions and methods of this disclosure include, for example, Actemra® (tocilizumab) or Kevzera® (sarilumab) and anti-IL-6 mAbs.

[0097] Other IL-6R antibodies include the 39B9 VL1 antibody, the 39B9 VL5 antibody, the 12A antibody, and the 5C antibody, the contents of which are described in their entirety in International Patent Application Publication No. 2009 / 140348, which is incorporated herein by reference in its entirety. These antibodies exhibit specificity for human IL-6R and / or both IL-6R and IL-6Rc, and they have been shown to inhibit the functional activity of IL-6Rc in vitro (i.e., binding to gp130 and inducing the signaling cascade).

[0098] In some embodiments, the anti-6Rc antibody includes light chain and heavy chain sequences. In some embodiments, the light chain sequence of the anti-6R antibody is SEQ ID NO: 53. In some embodiments, the heavy chain sequence of the anti-6Rc antibody is SEQ ID NO: 52. In some embodiments, the anti-6Rc antibody includes SEQ ID NO: 53 and SEQ ID NO: 52.

[0099] The 39B9 VL1 and 39B9 VL5 antibodies share a common heavy chain variable region (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1. The 39B9 VL1 antibody contains a light chain variable region (SEQ ID NO: 4) encoded by the nucleic acid sequence shown in SEQ ID NO: 3. The 39B9 VL5 antibody contains a light chain variable region (SEQ ID NO: 6) encoded by the nucleic acid sequence shown in SEQ ID NO: 5. The 12A antibody contains a heavy chain variable region (SEQ ID NO: 8) encoded by the nucleic acid sequence shown in SEQ ID NO: 7. The 12A antibody contains a light chain variable region (SEQ ID NO: 10) encoded by the nucleic acid sequence shown in SEQ ID NO: 9. The 5C antibody contains a heavy chain variable region (SEQ ID NO: 12) encoded by the nucleic acid sequence shown in SEQ ID NO: 11. The 5C antibody contains a light chain variable region (SEQ ID NO: 14) encoded by the nucleic acid sequence shown in SEQ ID NO: 13. [Table 1]

[0100] The huIL-6R antibody of this disclosure further includes, for example, the heavy chain complementarity-determining regions (VH CDRs) shown in Table 2, the light chain complementarity-determining regions (VL CDRs) shown in Table 3, and combinations thereof. [Table 2] [Table 3]

[0101] The huIL-6R antibodies of this disclosure work to modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with the functional activity of IL-6Rc. The functional activity of IL-6Rc includes, for example, intracellular signaling via activation of the JAK / STAT pathway and the MAPK cascade, acute phase protein production, antibody production, and cell differentiation and / or proliferation. For example, the huIL-6R antibodies completely or partially inhibit the functional activity of IL-6Rc by partially or completely modulating, block, inhibit, reduce, antagonize, neutralize, or otherwise interfering with the binding of IL-6Rc to the signaling receptor component gp130.

[0102] A huIL-6R antibody is considered to completely modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with IL-6Rc functional activity when the level of IL-6Rc functional activity in the presence of the huIL-6R antibody is reduced by at least 95%, e.g., 96%, 97%, 98%, 99%, or 100%, compared to the level of IL-6Rc functional activity in the absence of binding to the huIL-6R antibody described herein. A huIL-6R antibody is considered to partially modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with IL-6Rc functional activity when the level of IL-6Rc functional activity in the presence of the huIL-6R antibody is reduced to less than 95% of the level of IL-6Rc functional activity in the absence of binding to the huIL-6R antibody described herein, for example, by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90%. huIL-6R antibody variant

[0103] Variants of huIL-6R antibodies are prepared using any of the various techniques recognized in the art. For example, a variant huIL-6R antibody may contain an antibody having one or more amino acid modifications, such as amino acid substitutions at positions within the antibody sequence.

[0104] Preferred positions for amino acid substitutions are indicated in Table 4 below as residues highlighted in bold and underlined. These bold / underlined amino acid residues can be substituted with any amino acid residue. In preferred embodiments, the bold / underlined amino acid residues are substituted with the amino acid residues shown in Table 4 below. In these embodiments, the antibody includes (i) a common amino acid sequence QQSXSYPLT (SEQ ID NO: 31) in the light chain complementarity determination region 3 (CDR3) (wherein X is N or Q); (ii) a common amino acid sequence GIIPX1FX2TTKYAQX3FQG (SEQ ID NO: 32) in the heavy chain complementarity determination region 2 (CDR2) (wherein X1 is L or A, X2 is D or E, and X3 is Q or K); (iii) a common amino acid sequence DRDILTDYYPXGGMDV (SEQ ID NO: 33) in the heavy chain complementarity determination region 3 (CDR3) (wherein X is M or L); and (iv) a common amino acid sequence TAVXYCAR (SEQ ID NO: 34) in the framework region 3 (FRW3) (wherein X is F or Y).

[0105] The NI-1201-wild-type (NI-1201-WT) antibodies listed in Table 4 contain the amino acid sequence QQSNSYPLT (SEQ ID NO: 26) in the light chain CDR3 region, the amino acid sequence GIIPLFDTTKYAQQFQG (SEQ ID NO: 16) in the heavy chain CDR2 region, the amino acid sequence DRDILTDYYPMGGMDV (SEQ ID NO: 35) in the heavy chain CDR3 region, and the amino acid sequence TAVFYCAR (SEQ ID NO: 36) in the FRW3 region.

[0106] The NI-1201-A antibodies listed in Table 4 contain the amino acid sequence QQSNSYPLT (SEQ ID NO: 26) in the light chain CDR3 region, the amino acid sequence GIIPLFDTTKYAQKFQG (SEQ ID NO: 37) in the heavy chain CDR2 region, the amino acid sequence DRDILTDYYPMGGMDV (SEQ ID NO: 35) in the heavy chain CDR3 region, and the amino acid sequence TAVYYCAR (SEQ ID NO: 38) in the FRW3 region.

[0107] The NI-1201-B antibodies listed in Table 4 contain the amino acid sequence QQSNSYPLT (SEQ ID NO: 26) in the light chain CDR3 region, the amino acid sequence GIIPLFDTTKYAQKFQG (SEQ ID NO: 37) in the heavy chain CDR2 region, the amino acid sequence DRDILTDYYPLGGMDV (SEQ ID NO: 39) in the heavy chain CDR3 region, and the amino acid sequence TAVYYCAR (SEQ ID NO: 38) in the FRW3 region.

[0108] The NI-1201-C antibodies listed in Table 4 contain the amino acid sequence QQSNSYPLT (SEQ ID NO: 26) in the light chain CDR3 region, the amino acid sequence GIIPAFETTKYAQKFQG (SEQ ID NO: 40) in the heavy chain CDR2 region, the amino acid sequence DRDILTDYYPLGGMDV (SEQ ID NO: 39) in the heavy chain CDR3 region, and the amino acid sequence TAVYYCAR (SEQ ID NO: 38) in the FRW3 region.

[0109] The NI-1201-D antibodies listed in Table 4 contain the amino acid sequence QQSQSYPLT (SEQ ID NO: 41) in the light chain CDR3 region, the amino acid sequence GIIPAFETTKYAQKFQG (SEQ ID NO: 40) in the heavy chain CDR2 region, the amino acid sequence DRDILTDYYPLGGMDV (SEQ ID NO: 39) in the heavy chain CDR3 region, and the amino acid sequence TAVYYCAR (SEQ ID NO: 38) in the FRW3 region.

[0110] The NI-1201-E antibodies listed in Table 4 contain the amino acid sequence QQSQSYPLT (SEQ ID NO: 41) in the light chain CDR3 region, the amino acid sequence GIIPLFDTTKYAQKFQG (SEQ ID NO: 37) in the heavy chain CDR2 region, the amino acid sequence DRDILTDYYPLGGMDV (SEQ ID NO: 39) in the heavy chain CDR3 region, and the amino acid sequence TAVYYCAR (SEQ ID NO: 38) in the FRW3 region.

[0111] The NI-1201-F antibodies listed in Table 4 contain the amino acid sequence QQSNSYPLT (SEQ ID NO: 26) in the light chain CDR3 region, the amino acid sequence GIIPAFDTTKYAQKFQG (SEQ ID NO: 42) in the heavy chain CDR2 region, the amino acid sequence DRDILTDYYPLGGMDV (SEQ ID NO: 39) in the heavy chain CDR3 region, and the amino acid sequence TAVYYCAR (SEQ ID NO: 38) in the FRW3 region.

[0112] The NI-1201-G antibodies listed in Table 4 contain the amino acid sequence QQSQSYPLT (SEQ ID NO: 41) in the light chain CDR3 region, the amino acid sequence GIIPAFDTTKYAQKFQG (SEQ ID NO: 42) in the heavy chain CDR2 region, the amino acid sequence DRDILTDYYPLGGMDV (SEQ ID NO: 39) in the heavy chain CDR3 region, and the amino acid sequence TAVYYCAR (SEQ ID NO: 38) in the FRW3 region. [Table 4]

[0113] CD28 antibody

[0114] A CD28-specific antibody and its antigen-binding fragment are referred to herein as an anti-CD28 antibody, and the composition is referred herein as an "anti-IL6 / IL6 receptor antibody composition." Any anti-CD28 receptor antibody known in the art is suitable for use in this disclosure. The anti-CD28 receptor antibody is a monoclonal antibody.

[0115] CD28 (surface antigen classification 28) is one of the proteins expressed on T cells that delivers co-stimulatory signals necessary for T cell activation and survival. In addition to T cell receptor (TCR) stimulation, CD28-mediated T cell stimulation can deliver potent signals for the production of various interleukins (particularly IL-6).

[0116] CD28 is a receptor for the CD80(B7.1) and CD86(B7.2) proteins. When activated by Toll-like receptor ligands, CD80 expression is upregulated in antigen-presenting cells (APCs). CD86 expression on antigen-presenting cells is constitutive (expression is independent of environmental factors). Phosphatidylinositol 3-kinase (PI3K) inhibitors

[0117] PI3K inhibitors are members of a unique and conserved family of intracellular lipid kinases that phosphorylate the 3'-OH group on phosphatidylinositol or phosphoinositides. PI3K inhibitors are key signaling enzymes that relay signals from cell surface receptors to downstream effectors. The PI3K family comprises 15 kinases with distinct substrate specificities, expression patterns, and regulatory mechanisms. Class I PI3K inhibitors are typically activated by tyrosine kinases or G protein-binding receptors to generate PIP3, which then binds to downstream effectors such as those in the Akt / PDK1 pathway, mTOR, Tec family kinases, and Rho family GTPases.

[0118] The PI3K signaling pathway is known to be one of the most highly mutated in human cancers. PI3K signaling is also a key factor in disease states, including hematological malignancies, non-Hodgkin lymphomas (e.g., diffuse large B-cell lymphoma), allergic contact dermatitis, rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, chronic obstructive pulmonary disease, psoriasis, multiple sclerosis, asthma, diabetic complications and associated disorders, and cardiovascular inflammatory complications such as acute coronary syndrome. PI3K-delta and PI3K-gamma isoforms are preferentially expressed in normal and malignant leukocytes.

[0119] Downstream mediators of the PI3K signaling pathway include Akt and the mammalian target of rapamycin (mTOR). One important function of Akt is to enhance mTOR activation through phosphorylation of TSC2 and other mechanisms. mTOR is a serine-threonine kinase associated with the PI3K family of lipid kinases and is involved in a wide range of biological processes, including cell growth, cell proliferation, cell migration, and survival.

[0120] PI3K inhibitors can be small molecules. As used herein, “small molecule” means a composition having a molecular weight in the range of less than about 5 kD to 50 daltons, for example, less than about 4 kD, less than about 3.5 kD, less than about 3 kD, less than about 2.5 kD, less than about 2 kD, less than about 1.5 kD, less than about 1 kD, less than 750 daltons, less than 500 daltons, less than about 450 daltons, less than about 400 daltons, less than about 350 daltons, less than 300 daltons, less than 250 daltons, less than about 200 daltons, less than about 150 daltons, or less than 100 daltons. Small molecules can be, for example, nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids, or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened using any of the assays of this disclosure.

[0121] PI3K inhibitors are antibodies or fragments thereof that inhibit PI3K activity.

[0122] PI3K inhibitors, including PI3K-delta inhibitors, PI3K-gamma inhibitors, and PI3K-delta / gamma inhibitors, are well known in the art.

[0123] Exemplary PI3K inhibitors include, for example, Wartmannin, LY294002, Hisviscon C, Idelalisib, Copanlisib, Duvelisib, Alpelisib, Taselicib, Perifosin, Idelalisib, Buparisib, Umbralisib, PX-866, Dactricib, CUDC-907, Voxtalisib, CUDC-907, ME-401, IPI-549, SF1126, RP6530, INK1117, Pictilisib, XL147, Paromide 529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, RP6503, PI-103, GNE-477, and AEZS-136. Protein kinase B (AKT) inhibitors

[0124] Protein kinase B (PKB), also known as Akt, is a serine / threonine-specific protein kinase that plays a key role in several cellular processes, including glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration.

[0125] Akt1 is involved in cell survival pathways by inhibiting the apoptotic process. Akt1 can also induce protein synthesis pathways and is therefore a key signaling protein in cellular pathways that lead to skeletal muscle hypertrophy and general tissue growth. Mouse models with complete deletion of Akt1 exhibit stunted growth and increased spontaneous apoptosis in tissues such as the testes and thymus. Because it can block apoptosis and thereby promote cell survival, Akt1 is involved as a major factor in many types of cancer. Akt (now also called Akt1) was originally identified as AKT8, an oncogene in retroviral transformations.

[0126] Akt2 is a crucial signaling molecule in the insulin signaling pathway. It is necessary to induce glucose transport. In mice where Akt1 is null but Akt2 is normal, glucose homeostasis is not disrupted, but the animals are small, which is consistent with the role of Akt1 in growth. In contrast, mice lacking Akt2 but with normal Akt1 exhibit mild growth deficiency and show a diabetic phenotype (insulin resistance), which is also consistent with the idea that Akt2 is more specific to the insulin receptor signaling pathway.

[0127] Akt inhibitors can be small molecules. As used herein, “small molecule” means a composition having a molecular weight in the range of less than about 5 kD to 50 daltons, for example, less than about 4 kD, less than about 3.5 kD, less than about 3 kD, less than about 2.5 kD, less than about 2 kD, less than about 1.5 kD, less than about 1 kD, less than 750 daltons, less than 500 daltons, less than about 450 daltons, less than about 400 daltons, less than 350 daltons, less than 300 daltons, less than 250 daltons, less than about 200 daltons, less than about 150 daltons, or less than 100 daltons. Small molecules can be, for example, nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids, or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened using any of the assays of this disclosure.

[0128] Akt inhibitors are antibodies or fragments thereof that inhibit Akt activity.

[0129] Akt inhibitors are well known in the art. Exemplary Akt inhibitors include, for example, VQD-002, perifosine, miltefosine, MK-2206, AZD5363, and ipatasertib. Mammalian target of rapamycin (mTOR) inhibitors

[0130] mTOR inhibitors are a type of drug that inhibits the mammalian target of rapamycin (mTOR) and are serine / threonine-specific protein kinases belonging to the phosphatidylinositol 3-kinase (PI3K)-related kinase (PIKK) family. mTOR regulates cellular metabolism, growth, and proliferation by forming two protein complexes, mTORC1 and mTORC2, and by signaling through them. The most established mTOR inhibitors are the so-called rapalogs (rapamycin and its analogues), which have shown tumor responses in clinical trials for various tumor types.

[0131] Growth factors, amino acids, ATP, and oxygen levels appear to regulate mTOR signaling. Several downstream pathways that control cell cycle progression, translation, initiation, transcriptional stress response, protein stability, and cell survival signal via mTOR.

[0132] The serine / threonine kinase mTOR is a downstream effector of the PI3K / AKT pathway and forms two distinct polyprotein complexes, mTORC1 and mTORC2. These two complexes have a separate network of protein partners, feedback loops, substrates, and regulators. mTORC1 consists of mTOR and two positive regulatory subunits, raptor and mammalian LST8 (mLST8), as well as two negative regulators, proline-rich AKT substrate 40 (PRAS40) and DEPTOR. mTORC2 consists of mTOR, mLST8, mSin1, protor, rictor, and DEPTOR.

[0133] mTOR inhibitors can be small molecules. As used herein, “small molecule” means a composition having a molecular weight in the range of less than about 5 kD to 50 daltons, for example, less than about 4 kD, less than about 3.5 kD, less than about 3 kD, less than about 2.5 kD, less than about 2 kD, less than about 1.5 kD, less than about 1 kD, less than 750 daltons, less than 500 daltons, less than about 450 daltons, less than about 400 daltons, less than about 350 daltons, less than 300 daltons, less than 250 daltons, less than about 200 daltons, less than about 150 daltons, or less than 100 daltons. Small molecules can be, for example, nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids, or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened using any of the assays of this disclosure.

[0134] mTor inhibitors are antibodies or fragments thereof that inhibit mTor activity.

[0135] mTOR inhibitors are well known in the art. First-generation mTOR inhibitors included rapamycin, sirolimus, temsirolimus (CCI-779), everolimus (RAD001), and ridafololimus (AP-23573).

[0136] Second-generation mTOR inhibitors are known as ATP-competitive mTOR kinase inhibitors. mTORC1 / mTORC2 dual inhibitors are designed to compete with ATP at the catalytic site of mTOR. They inhibit all kinase-dependent functions of mTORC1 and mTORC2, and therefore, unlike rapalogs that target only mTORC1, they block the feedback activation of PI3K / AKT signaling. These types of inhibitors have been developed, and some of them are being tested in clinical trials. Similar to rapalogs, they reduce protein translation, attenuate cell cycle progression, and inhibit angiogenesis in many cancer cell lines, and in human cancers.

[0137] The close interaction of mTOR with the PI3K pathway has also led to the development of mTOR / PI3K dual inhibitors. Compared to drugs that inhibit either mTORC1 or PI3K, these drugs have the advantage of inhibiting all catalytic isoforms of mTORC1, mTORC2, and PI3K. Targeting both kinases simultaneously reduces the upregulation of PI3K, which typically occurs with mTORC1 inhibition. Methods to enhance ex vivo expansion of CAR-T cells

[0138] Typically, CAR-T cells are stimulated ex vivo for their expansion to obtain a sufficient number of cells before in vivo injection. This ex vivo process is important for having a sufficient number of CAR-Ts at a relatively early differentiation stage. After ex vivo clonal expansion, the CAR-Ts are injected back into the patient's circulation, where they undergo a "transport" process toward their site of action. Transport to the site of action can be mediated through the overexpression of chemokines or molecules that direct the CAR-Ts toward their target site. While treatment with CAR-Ts has shown promising results, they have also been shown to induce toxicity in patients through macrophage activation syndrome (MAS), cytokine release syndrome (CRS), tumor lysis syndrome (TLS), and autoimmune toxicity.

[0139] Macrophage activation syndrome is a condition characterized by increased T cell expansion and elevated macrophage activation levels in vivo.

[0140] Cytokine release syndrome, or "cytokine storm," is a severe immune response in which the body releases too many cytokines into the bloodstream too rapidly. While cytokines play a vital role in a normal immune response, having large amounts of them suddenly released into the body is harmful. In vivo T-cell clonal expansion and response to antigens lead to a "cytokine storm." The presence of high levels of cytokines results in an increased immune response (e.g., B cells, NK cells, macrophages, PMNs), inflammation, and tissue damage. In particular, IL-6 is commonly elevated during a cytokine storm and, at high levels, can lead to transsignaling using soluble IL-6 receptors.

[0141] Tumor lysis syndrome involves massive tumor cell lysis, releasing a large amount of tumor cell contents into the systemic circulation. This often leads to hyperkalemia, hyperuricemia, hypophosphatemia, and hypocalcemia.

[0142] When CAR-T receptors attack the correct antigenic target, but the tissue is non-malignant, autoimmune toxicity, or "on-target, off-tumor toxicity," occurs. The risk of autoimmune toxicity increases after treatment with checkpoint inhibitors. Potential solutions to mitigate this toxicity include treatment with corticosteroids and treatment with IL-6 signaling-specific inhibitors.

[0143] Current T cell expansion technologies only partially replicate the in vivo microenvironment found in human lymph nodes. Typically, in CART-T cell production, T cells are activated under close cell-to-cell contact via antigen-presenting cells (APCs), such as dendritic cells (DCs), which present the peptide major histocompatibility complex (MHC) to the T cells and various other co-stimulatory signals. These similar quarters enable efficient autocrine / parasecretory signaling among expanding T cells, which secrete IL-2 and other cytokines to aid their own growth. In addition, lymphoid tissue is composed of extracellular matrix (ECM) components such as collagen, which provide signals to upregulate proliferation, cytokine production, and pro-survivability pathways. Various solutions have been proposed to make the T cell expansion process more physiological. One strategy is to use feeder cell cultures modified to produce activation signals similar to those of DCs. While this has the theoretical ability to mimic many components of lymph nodes, it is difficult to reproduce on a large scale due to the complexity and inherent variability of using cell lines in a fully GMP-compliant manner. Others have proposed biomaterial-based solutions to circumvent this problem, including lipid-coated microrods, 3D scaffolds via either Matrigel or 3D-printed lattices, oval beads, and polydimethylsiloxane (PDMS) beads conjugated with mAbs that replicate the cell membrane, respectively, with broad boundary contact surfaces, 3D structures, or soft-surface T cells, which is a normal experience in vivo. While these have been shown to yield superior expansion compared to traditional microbeads, there is no method that can demonstrate preferential expansion of functional memory and CD4 T cell populations. Generally, T cells in earlier differentiated states, such as memory cells, have been shown to yield superior antitumor efficacy, likely due to their high ability to replicate, zoospore, and engraft, leading to long-term endurance responses. Similarly, CD4 T cells are equally important to antitumor efficacy due to their cytokine-releasing properties and their ability to resist exhaustion.

[0144] In some embodiments, CAR-T cells are stimulated ex vivo for their expansion prior to injection in cancer patients. The process for efficient CAR-T cell expansion requires activation of the TCR complex and simultaneous stimulation with anti-CD3 / anti-CD28. Simultaneous stimulation with anti-CD3 is important because it can bind to the CD3 subunit and activate the TCR complex without requiring antigenic peptides derived from antigen-presenting cells. Similarly, anti-CD28 can bind to CD28 and stimulate T cells without requiring CD80 or CD86 derived from antigen-presenting cells. Therefore, simultaneous stimulation with anti-CD3 and / or anti-CD28 is useful for CAR-T in vivo expansion.

[0145] For ex vivo expansion, it is necessary to provide a surface to which CAR-T cells can bind, which can be achieved by using either a plate or bead-conjugated anti-CD3 to promote the binding of CD3 components of T cell receptors on CAR-T cells, which is necessary to create a surface that mimics an immune synapse. Similarly, CD28 is an essential co-stimulatory molecule required to bring about naive T cell proliferation. Therefore, anti-CD28 antibodies can be added to immobilized anti-CD3, i.e., plate or bead-conjugated anti-CD3, or alternatively, to a solution.

[0146] Key considerations include easy industrialization and the ability to integrate with scalable systems such as bioreactors. In T cell expansion culture, the use of porous microcarriers functionalized with anti-CD3 and anti-CD28 mAbs has been demonstrated. While microcarriers have historically been used through the bioprocess industry for adherent cultures of stem cells and Chinese hamster ovary (CHO) cells, they have not been used for suspension cells such as T cells. The macroporous structure within the beads allows T cells to grow within and along the surface, resulting in sufficient cell-to-cell contact for enhanced autocrine and parasecrine signaling. Furthermore, the beads are composed of gelatin, which is a collagen derivative and therefore possesses adhesion domains also present in lymph nodes. Finally, the 3D surface of the carrier provides a larger contact surface for T cells, which can mimic the large contact surface range that occurs between T cells and DCs. Traditional beads coated with anti-CD3 / anti-CD28 have not only resulted in superior CAR-T cell expansion but have also been shown to consistently produce higher frequencies of memory and CD4 T cells (CCR7+CD62L+) across multiple donors. Therefore, the antitumor activity of CAR-T cells can be enhanced by using anti-CD3 / anti-CD28 coated microbeads in conjunction with specific inhibitors of the PI3K-AKT-mTOR pathway and IL-6 signaling. How to enhance CAR-T cell therapy

[0147] This disclosure provides dosing protocols for administering antibodies to enhance CAR-T therapy. Example dosing protocols can be seen in Figures 15A-C. The antibody may be, for example, an antibody or fragment thereof that targets a T cell surface protein. In some embodiments, the antibody that binds to the T cell surface protein may be a protein that activates T cells. In some embodiments, the antibody binds to CD3 (i.e., an anti-CD3 antibody). In some embodiments, the antibody may be any of the antibodies or fragments thereof described herein. In some embodiments, the antibody is foralmab.

[0148] The anti-CD3 antibody used in the methods for augmenting CAR-T cell therapy described herein may be administered in any manner that achieves the desired outcome of augmenting CAR-T therapy. Augmentation of CAR-T cell therapy may include, but is not limited to, achieving lymphocyte depletion, improving safety and tolerability, improving pharmacokinetics, and / or improving the cytokine profile, reducing host-versus-graft disease, or reducing graft-versus-host disease. In some embodiments, the anti-CD3 antibody is administered intravenously. In some embodiments, the anti-CD3 antibody is administered by subcutaneous injection. In some embodiments, the anti-CD3 antibody is administered orally. In some embodiments, the anti-CD3 antibody is administered intranasally.

[0149] The anti-CD3 antibody used in the methods for augmenting CAR-T cell therapy described herein may be administered in any dose that achieves the desired outcome of augmenting CAR-T therapy (e.g., achieving lymphocyte depletion, improved safety and tolerability, improved pharmacokinetics, and / or improved cytokine profile). In some embodiments, the anti-CD3 antibody dose is approximately 0.5 mg / day, approximately 1.0 mg / day, approximately 1.5 mg / day, approximately 2.0 mg / day, approximately 2.5 mg / day, approximately 3.0 mg / day, approximately 3.5 mg / day, approximately 4 mg / day, approximately 5 mg / day, approximately 5.5 mg / day, approximately 6 mg / day, approximately 6.5 mg / day, approximately 7 mg / day, approximately 7.5 mg / day, approximately 8 mg / day, approximately 8.5 mg / day, approximately 9 mg / day, approximately 9.5 mg / day, or approximately 10 mg / day.

[0150] In one embodiment, the method described herein includes a step of enhancing CAR-T therapy, which includes administering an antibody to a subject prior to the administration of a CAR-T cell composition, which may be referred to herein as “pre-medication” or “first cycle.” In some embodiments, pre-medication induces lymphocyte depletion and / or immunosuppression in the subject. Lymphocyte depletion and immunosuppression can enhance CAR-T therapy by promoting the survival of engineered CAR-T cells in vivo and reducing the serious toxic effects observed in CAR-T therapy. In some embodiments, pre-medication reduces host-versus-graft disease. In some embodiments, pre-medication improves the safety and tolerability of treatment with CAR-T cells. In some embodiments, pre-medication improves the pharmacokinetics of CAR-T therapy. In some embodiments, pre-medication improves the cytokine profile associated with CAR-T therapy. In some embodiments, pre-medication is performed approximately 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, or 120 hours before administration of the CAR-T cell composition. In some embodiments, pre-medication is performed once or multiple times before administration of the CAR-T cell composition. In some embodiments, pre-medication is repeated until lymphocyte depletion and / or immunosuppression are observed.

[0151] In one embodiment, the method described herein includes the step of administering a CAR-T cell composition to a target after pre-medication. The CAR-T cell composition can be any CAR-T cell composition. The CAR-T cell composition can be derived, for example, from autologous cells or allogeneic T cells. The CAR-T cell composition can be directed to any target. The target can be, for example, a tumor-associated antigen or a pathogen antigen. The CAR-T cell composition can be administered by intravenous injection. The response to the administration of the CAR-T cell composition can be monitored sequentially or at specific time points. In some embodiments, an anti-CD3 antibody is administered co-administered with the CAR-T cell composition. In some embodiments, the response to the administration of the CAR-T composition is monitored 7, 10, 12, 13, 14, 21, or 28 days after administration of the CAR-T cell composition. It is understood in the art that the specific measurement of the response to an administered CAR-T cell composition is determined by the type of CAR-T cell composition, CAR-T cell therapy indicators, the needs of the subject receiving the CAR-T cell composition, and the certified healthcare professional responsible for treating the subject. The measured response may include, for example, an assessment of toxicity or adverse events in the subject. The measured response may include, for example, an assessment of disease progression in the subject. The measured response may include, for example, an assessment of the presence of pathogens in the subject. In some embodiments, the administration of the CAR-T cell composition to the subject is repeated once or multiple times. In some embodiments, the CAR-T cell composition is repeatedly administered to the subject until a predetermined measured response is reached.

[0152] In one embodiment, the method described herein includes the step of administering an anti-CD3 antibody after administration of a CAR-T cell composition. Enhancement of CAR-T therapy by administering an antibody to a subject after administration of a CAR-T cell composition may be referred to as “post-medication” or “second cycle.” In some embodiments, post-medication induces lymphocyte depletion and / or immunosuppression in the subject. Lymphocyte depletion and immunosuppression can enhance CAR-T therapy by promoting the survival of engineered CAR-T cells in vivo and reducing the severe toxic effects observed in CAR-T therapy. In some embodiments, post-medication improves the safety and tolerability of the treatment using CAR-T cells. In some embodiments, post-medication improves the pharmacokinetics of CAR-T therapy. In some embodiments, post-medication improves the cytokine profile associated with CAR-T therapy. In some embodiments, post-medication is part of a treatment plan that includes the co-administration of an anti-drug antibody, e.g., an antibody or small molecule drug that depletes the CAR-T cell composition. In some embodiments, post-medication is administered 7, 14, 21, or 28 days after administration of the CAR-T cell composition. In some embodiments, post-medication is administered once or multiple times after administration of the CAR-T cell composition. In some embodiments, post-medication is repeated until lymphocyte depletion and / or immunosuppression are observed.

[0153] In some embodiments, the antibody administered before or after the CAR-T cell composition is an anti-CD3 antibody. In some embodiments, the antibody administered before or after the CAR-T cell composition is an antibody described herein. In some embodiments, the antibody administered before or after the CAR-T cell composition is forallumab. In some embodiments, the antibody administered before or after the CAR-T cell composition is delivered intravenously. In some embodiments, the antibody administered pre- or post-dose is delivered by subcutaneous injection. In some embodiments, the antibody administered before or after the CAR-T cell composition is delivered as a pharmaceutical composition comprising forallumab and a pharmaceutically acceptable carrier. The pharmaceutical composition may be a pharmaceutical composition described herein. In some embodiments, the pharmaceutical composition comprises a unit dose of antibody. In some embodiments, the unit dose is 1, 2, 3, or 4 mg of antibody. In some embodiments, the pharmaceutical composition is formulated for delayed and extended release using a carrier. In some embodiments, the carrier is nanoparticles.

[0154] Antibodies or pharmaceutical compositions administered as pre-medication or post-medication can be combined with one or more additional agents. In some embodiments, the antibody or pharmaceutical composition is administered in combination with a steroid. In some embodiments, the antibody or pharmaceutical composition is administered in combination with a PI3K inhibitor. In some embodiments, the antibody or pharmaceutical composition is administered in combination with an ATK inhibitor. In some embodiments, the antibody or pharmaceutical composition is administered in combination with an mTOR inhibitor. In some embodiments, the antibody or pharmaceutical composition is administered together with an anti-IL6R antibody. Ways to enhance cell therapy

[0155] In one embodiment, the present disclosure provides a method for augmenting cell therapy. Cell therapy can be any therapy that includes the step of transplanting (i.e., administering) cells for treatment of a subject in need thereof. In some embodiments, cell therapy includes allogeneic cell therapy. In some embodiments, cell therapy includes autologous cell therapy. In some embodiments, cell therapy includes xenocellular cell therapy.

[0156] The cell therapies envisioned herein may include any cell type appropriate for the treatment of the target requiring them. In some embodiments, the cell therapy includes stem cells. In some embodiments, the stem cells are human embryonic stem cells. In some embodiments, the stem cells are tissue-specific stem cells. In some embodiments, the stem cells are neural stem cells. In some embodiments, the stem cells are mesenchymal stem cells. In some embodiments, the stem cells are hematopoietic stem cells. In some embodiments, the stem cells are induced pluripotent stem cells. In some embodiments, the stem cells are epidermal stem cells. In some embodiments, the stem cells are epithelial stem cells. In some embodiments, the stem cells are neural stem cells. In some embodiments, the cell therapy includes differentiated or mature cells. In some embodiments, the cell therapy includes immune cells. In some embodiments, the immune cells are lymphocytes. In some embodiments, the immune cells are T lymphocytes. In some embodiments, the immune cells are B cells. In some embodiments, the immune cells are regulatory T cells. In some embodiments, the immune cells are CD4+ T cells. In some embodiments, the immune cells are CD8+ T cells. In some embodiments, the immune cells are hyper T cells. In some embodiments, the immune cells are cytotoxic T cells. In some embodiments, the immune cells are natural killer cells. In some embodiments, the immune cells are NKT cells.

[0157] In some embodiments, cell therapy includes engineered cells. Engineered cells can be autologous, allogeneic, or artificial cells containing exogenous polynucleotides. For example, engineered cells may contain exogenous or foreign polynucleotides delivered to the cells by transfection or transduction. Transfection or transduction can be achieved using any method known in the art for delivering exogenous polynucleotides to cells. For example, plasmid polynucleotides can be delivered using, for example, electroporation or lipid-based transfection reagents (e.g., lipofectamine). Engineered cells may be infected or transduced using a viral vector that delivers exogenous polynucleotides. Transduction can be achieved by delivering exogenous polynucleotides to cells using a viral vector such as a lentivirus, adenovirus, or adeno-associated virus. Engineered cells may be genetically modified stably or transiently. Methods for manipulating polynucleotides and vectors for use in the delivery of exogenous polynucleotides to cells are generally known in the art (e.g., Sambrook. Molecular cloning: a Laboratory manual. Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY 2012).

[0158] Exogenous polynucleotides may contain transcriptional regulatory elements (e.g., promoters or enhancers). Exogenous polynucleotides may encode products expressed by engineered cells. Transcriptional regulatory elements may be operably ligated to, i.e., to control the expression of, the product encoded by the exogenous polynucleotide. Exogenous polynucleotides may contain non-coding RNA products, such as interfering RNA or guide RNA for targeted nuclease systems. Exogenous polynucleotides may contain products encoding polypeptides.

[0159] Manipulated cells can express polypeptides encoded by exogenous polynucleotides. Polypeptide expression can be inducible, repressive, or constitutive. Polypeptides can be, without limitation, artificial receptors, chimeric antigen receptors, manipulated T cell receptors, fusion proteins, factors that promote the survival of manipulated cells, suicide genes, cytokines, or any polypeptide that enhances the safety or efficacy of cell therapy.

[0160] Manipulated cells may have one or more modifications to endogenous genes. Modifications to endogenous genes can be achieved using any gene editing method known in the art. For example, common gene editing methods include targeted nuclease systems such as CRISPR / Cas, TALEN, and zinc finger nucleases. The modifications may be, for example, targeted mutations (e.g., deletions) to a specific gene or a transcriptional regulatory element operably linked to a specific gene. The specific gene may be an immune-related gene, such as a gene encoding a polypeptide in the major histocompatibility complex (e.g., beta-2-microglobulin or human leukocyte antigen). The gene may have a functional role in the safety and efficacy of cell therapy. For example, the functional role may be the regulation of host-versus-graft or graft-versus-host disease.

[0161] This disclosure provides dosing protocols for administering antibodies to enhance cell therapy. Example dosing protocols can be seen in Figures 15A-C. The antibody may be, for example, an antibody or fragment thereof that targets a T cell surface protein. In some embodiments, the antibody that binds to the T cell surface protein may be a protein that activates T cells. In some embodiments, the antibody binds to CD3 (i.e., an anti-CD3 antibody). In some embodiments, the antibody may be any of the antibodies or fragments thereof described herein. In some embodiments, the antibody is foralmab.

[0162] The anti-CD3 antibody used in the methods for augmenting cell therapy described herein may be administered in any manner that achieves the desired outcome of augmenting cell therapy. Augmentation of cell therapy may include, but is not limited to, achieving lymphocyte depletion, improving safety and tolerability, improving pharmacokinetics, and / or improving the cytokine profile, reducing host-versus-graft disease, or reducing graft-versus-host disease. In some embodiments, the anti-CD3 antibody is administered intravenously. In some embodiments, the anti-CD3 antibody is administered by subcutaneous injection. In some embodiments, the anti-CD3 antibody is administered orally. In some embodiments, the anti-CD3 antibody is administered intranasally.

[0163] The anti-CD3 antibody used in the methods for augmenting cell therapy described herein may be administered in any dose that achieves the desired outcome of augmenting cell therapy (e.g., reduction of graft-versus-host disease, reduction of host-versus-graft disease, achievement of lymphocyte depletion, improvement of safety and tolerability, improvement of pharmacokinetics, and / or improvement of cytokine profile). In some embodiments, the anti-CD3 antibody dose is approximately 0.5 mg / day, approximately 1.0 mg / day, approximately 1.5 mg / day, approximately 2.0 mg / day, approximately 2.5 mg / day, approximately 3.0 mg / day, approximately 3.5 mg / day, approximately 4 mg / day, approximately 5 mg / day, approximately 5.5 mg / day, approximately 6 mg / day, approximately 6.5 mg / day, approximately 7 mg / day, approximately 7.5 mg / day, approximately 8 mg / day, approximately 8.5 mg / day, approximately 9 mg / day, approximately 9.5 mg / day, or approximately 10 mg / day.

[0164] In one embodiment, the method described herein includes a step of enhancing cell therapy, which includes administering an antibody to a subject prior to the administration of a cell therapy composition, a step that may be referred to herein as “pre-dosing” or “first cycle.” In some embodiments, pre-dosing induces lymphocyte depletion and / or immunosuppression in the subject. Lymphocyte depletion and immunosuppression can enhance cell therapy by promoting the survival of transplanted cells in vivo and reducing the serious toxic effects observed in cell therapy. In some embodiments, pre-dosing improves the safety and tolerability of the treatment using transplanted cells. In some embodiments, pre-dosing improves the pharmacokinetics of cell therapy. In some embodiments, pre-dosing improves the cytokine profile associated with cell therapy. In some embodiments, pre-dosing is performed about 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, or 120 hours before administration of the cell therapy composition. In some embodiments, premedication is performed once or multiple times before administration of the cell therapy composition. In some embodiments, premedication is repeated until lymphocyte depletion and / or immunosuppression are observed.

[0165] In one embodiment, the method described herein includes the step of administering a cell therapy composition to a subject after pre-dosing. The cell therapy composition can be any cell therapy composition. In some embodiments, an anti-CD3 antibody is administered co-administered with the cell therapy composition. In some embodiments, the response to the administration of the cell therapy composition is monitored 7, 10, 12, 13, 14, 21, or 28 days after administration. It is understood in the art that the specific measurement of the response to the administered cell therapy composition is determined by the type of cell therapy composition, the cell therapy composition index, the needs of the subject receiving the cell therapy composition, and the certified healthcare professional responsible for treating the subject. The measured response may include, for example, an assessment of toxicity or adverse events in the subject. The measured response may include, for example, an assessment of disease progression in the subject. The measured response may include, for example, an assessment of the presence of a pathogen in the subject. In some embodiments, the administration of the cell therapy composition to the subject is repeated once or multiple times. In some embodiments, the cell therapy composition is repeatedly administered to the subject until a predetermined measured response is reached.

[0166] In one embodiment, the method described herein includes the step of administering an anti-CD3 antibody after administration of a cell therapy composition. Enhancement of cell therapy by administering an antibody to a subject after administration of a cell therapy composition may be referred to as “post-dosing” or “second cycle.” In some embodiments, post-dosing induces lymphocyte depletion and / or immunosuppression in the subject. Lymphocyte depletion and immunosuppression can enhance cell therapy by promoting the survival of in vivo administered or transplanted cells and reducing the serious toxic effects observed in cell therapy. In some embodiments, post-dosing improves the safety and tolerability of the treatment using cell therapy. In some embodiments, post-dosing improves the pharmacokinetics of cell therapy. In some embodiments, post-dosing improves the cytokine profile associated with cell therapy. In some embodiments, post-dosing is part of a treatment plan that includes the co-administration of an anti-drug antibody, e.g., an antibody or small molecule drug that depletes, maintains, or promotes the persistence of the cell therapy composition. In some embodiments, post-medication is administered 7, 14, 21, or 28 days after administration of the cell therapy composition. In some embodiments, post-medication is administered once or multiple times after administration of the cell therapy composition. In some embodiments, post-medication is repeated until lymphocyte depletion and / or immunosuppression are observed.

[0167] In some embodiments, the antibody administered before or after the cell therapy composition is an anti-CD3 antibody. In some embodiments, the antibody administered before or after the CAR-T cell composition is an antibody described herein. In some embodiments, the antibody administered before or after the CAR-T cell composition is forallumab. In some embodiments, the antibody administered before or after the cell therapy composition is delivered intravenously. In some embodiments, the antibody administered in pre- or post-medication is delivered by subcutaneous injection. In some embodiments, the antibody administered before or after the cell therapy composition is delivered as a pharmaceutical composition comprising forallumab and a pharmaceutically acceptable carrier. The pharmaceutical composition may be a pharmaceutical composition described herein. In some embodiments, the pharmaceutical composition contains a unit dose of antibody. In some embodiments, the unit dose is 1, 2, 3, or 4 mg of antibody. In some embodiments, the pharmaceutical composition is formulated for delayed and extended release using a carrier. In some embodiments, the carrier is nanoparticles.

[0168] Antibodies or pharmaceutical compositions administered as pre-medication or post-medication can be combined with one or more additional agents. In some embodiments, the antibody or pharmaceutical composition is administered in combination with a steroid. In some embodiments, the antibody or pharmaceutical composition is administered in combination with a PI3K inhibitor. In some embodiments, the antibody or pharmaceutical composition is administered in combination with an ATK inhibitor. In some embodiments, the antibody or pharmaceutical composition is administered in combination with an mTOR inhibitor. In some embodiments, the antibody or pharmaceutical composition is administered together with an anti-IL6R antibody. Pharmaceutical composition

[0169] CD3 antibodies, IL-6Rc antibodies, CD28 antibodies, PI3K inhibitors, Akt inhibitors, and mTor inhibitors (also referred to herein as “active compounds”), as well as their derivatives, fragments, analogs, and homologs, are incorporated into pharmaceutical compositions suitable for administration (also referred to herein as “therapeutic compositions”).

[0170] When it is desired that active compounds be administered together and via the same route of administration, the active compounds may be formulated in the same therapeutic composition. Alternatively, the active compounds may be formulated in different therapeutic compositions. This is useful when it is desired that the active compounds be administered separately and / or via different routes of administration.

[0171] Principles and considerations involved in the preparation of such pharmaceutical compositions, as well as guidance in the selection of components, are provided, for example, in Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0172] When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable region sequence of the antibody, a peptide molecule that retains the ability to bind to the target protein sequence can be designed. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology (see, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The active compounds described herein, and their pharmaceutically acceptable salts, are used in pharmaceutical preparations in combination with pharmaceutically acceptable carriers or diluents. Suitable pharmaceutically acceptable carriers include inert solid packing or diluents and sterile aqueous or organic solutions. The active compounds are present in such compositions in amounts sufficient to yield the desired dosage within the range described herein.

[0173] Formulations intended for ex vivo use and in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filtration membrane.

[0174] The pharmaceutical compositions of this disclosure may be administered orally, nasally, dermatologically, pulmonaryly, by inhalation, orally, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, subarachnoidally, and parenterally. Those skilled in the art will recognize the advantages of certain routes of administration.

[0175] The active compound may be formulated to include, but not limited to, a carrier or diluent such as water, saline solution, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and non-volatile oils may also be used.

[0176] Systemic administration may also be by mucosal or percutaneous means. For mucosal or percutaneous administration, penetrating nematocysts suitable for the barrier to be traversed are used in the formulation. Such penetrating nematocysts are commonly known in the art and, for example, for mucosal administration, include surfactants, bile salts, and fusidic acid derivatives. Mucosal administration can be achieved through the use of nasal sprays or suppositories. For percutaneous administration, the active compound is formulated in the form of ointments, blister packs, gels, or creams, as is commonly known in the art.

[0177] Compositions suitable for injectable use include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® EL (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy injection is possible. It must be stable under manufacturing and storage conditions and must be protected from contamination by microorganisms such as bacteria and fungi. Carriers can be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Correct fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the particle size required in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonizing agents in the composition, such as sugars and polyhydric alcohols, such as mannitol, sorbitol, and sodium chloride. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0178] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent, along with one or a combination of the components listed above, and, if necessary, by subsequent filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required components different from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and lyophilization, which produce the active ingredient powder and any additional desired components derived from its solution that has already been sterile filtered.

[0179] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents, e.g., water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid (EDTA); buffers, e.g., acetates, citrates, or phosphates; and agents for adjusting osmotic pressure, e.g., sodium chloride or dextrose. pH can be adjusted using an acid or base, e.g., hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in ampoules, disposable syringes, or multi-dose vials made from glass or plastic.

[0180] Generally, dispersions are prepared by incorporating the active compound into a basic dispersion medium and a sterile vehicle containing the required components, different from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and lyophilization, which yield the active ingredient powder and any additional desired components derived from its already sterile filtered solution.

[0181] Oral compositions generally contain an inert diluent or a pharmaceutically acceptable food-grade carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier for use as an oral rinse, where the compound in the fluid carrier is applied orally, used to rinse the mouth, and then spat out or swallowed. pharmaceutically inconsistent binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar properties: binders, e.g., microcrystalline cellulose, gum tragacanth, or gelatin; excipients, e.g., starch, or lactose; disintegrants, e.g., alginic acid, Primogel, or corn starch; lubricants, e.g., magnesium stearate or Sterotes; lubricants, e.g., colloidal silicon dioxide; sweeteners, e.g., sucrose or saccharin; or flavoring agents, e.g., peppermint, methyl salicylate, or orange flavor.

[0182] The composition, formulation, or pharmaceutically acceptable composition contains excipients, such as stabilizers, preservatives, phospholipids, and / or other components, to improve stability and shelf life, and, in the case of dactinomycin nanoparticles, to improve uniform particle size. Exemplary excipients include, but are not limited to, trehalose (1-20%), surfactants, sodium chloride (50-150 mM), EDTA or EGTA (0.1-1 mM), and buffers, such as sodium citrate buffer (10-50 mM).

[0183] For administration by inhalation, the active compound is delivered in the form of an aerosol spray from a compressed container or a suitable spraying agent, such as a dispenser or nebulizer containing a gas, such as carbon dioxide.

[0184] Inhalation administration may be in the form of an inhaler or nebulizer. Nebulizers and / or inhalers are portable. Optionally, nebulizers and / or inhalers may be of different sizes to suit children and / or adults.

[0185] In some embodiments, vials containing the stabilization and formulation solutions of the active compounds described herein are inserted into an inhaler and / or nebulizer. In some embodiments, vials containing the stabilization and formulation solutions of the active compounds described herein are inserted into the bottom of the inhaler and / or nebulizer. In some embodiments, the pharmaceutical composition is dispersed as a fine aerosol through the opening.

[0186] The compounds can also be prepared in the form of suppositories for rectal delivery (e.g., using conventional suppository bases, e.g., cocoa butter and other glycerides) or retained enemas.

[0187] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable solid hydrophobic polymer matrices containing antibodies, where the matrix is ​​in the form of a formed article, e.g., a film or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Patent No. 3,773,919), L-glutamic acid and ethyl-L-glutamic acid copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., LUPRON DEPOT® (injectable microparticles composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers, e.g., ethylene-vinyl acetate and lactic acid-glycolic acid, allow for molecular release over 100 days, while certain hydrogels release proteins for shorter periods. definition

[0188] Unless otherwise defined, scientific and technical terms used in connection with this disclosure will have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms include plurals, and plural terms include singulars. Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, and protein and oligo or polynucleotide chemistry and hybridization described herein are well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzyme reactions and purification techniques are performed according to the manufacturer's specifications, or as commonly achieved in the art, or as described herein. The aforementioned techniques and methods are generally performed according to conventional methods well known in the art, as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature, laboratory methods, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, as well as for patient care.

[0189] As used in accordance with this disclosure, the following terms shall have the meanings set forth below unless otherwise indicated.

[0190] As used herein, the term “antibody” refers to a molecule containing an immunoglobulin molecule and an immunoglobulin (Ig) molecule with an immunoactive portion, i.e., an antigen-binding site that specifically binds to (immunely reacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single-stranded, Fab, Fab', and F(ab')2 fragments, as well as Fab expression libraries. “Specifically binds” or “immunely reacts” means that the antibody reacts with one or more antigenic determinants of a desired antigen and does not react (i.e., bind) with other polypeptides, or binds with other polypeptides with a much lower affinity (Kd > 10⁶).

[0191] The basic antibody structural unit is known to include a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids, primarily involved in antigen recognition. The carboxyl-terminal portion of each chain defines a constant region, primarily involved in effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, defining antibody isotypes as IgM, IgD, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, accompanied by a heavy chain containing a "D" region of approximately 10 or more amino acids. For general information, see Fundamental Immunology Ch. 7 (Paul, W., ea., 2nd ed. Raven Press, NY (1989)). The variable regions of each light / heavy chain pair form antibody binding sites.

[0192] As used herein, the terms “monoclonal antibody” (MAb) or “monoclonal antibody composition” refer to a group of antibody molecules containing only one molecular species of antibody molecule, each consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity-determining region (CDR) of a monoclonal antibody is identical across all molecules in the group. MAbs contain antigen-binding sites that have the ability to immunely react with specific epitopes of an antigen, characterized by their unique binding affinity to the antigen.

[0193] Generally, antibody molecules obtained from humans belong to one of the classes IgG, IgM, IgA, IgE, and IgD, where the properties of the heavy chain present in the molecule differ from one another. A particular class similarly has subclasses, such as IgG1, IgG2, etc. Furthermore, in humans, the light chain can be either a copper chain or a lambda chain.

[0194] As used herein, the term “epitope” includes any protein determinant that has the ability to specifically bind to an immunoglobulin, scFv, or T cell receptor. Epitope determinants typically consist of chemically active surface molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural features as well as specific charge characteristics. An antibody is said to bind specifically to an antigen when its dissociation constant is ≤1 μM; preferably ≤100 nM, most preferably ≤10 nM.

[0195] As used herein, the terms “immunological binding,” “immunological binding properties,” and “specific binding” refer to a type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction can be expressed in terms of the dissociation constant (Kd) of the interaction, where a smaller Kd indicates higher affinity. The immunological binding properties of a selected polypeptide are quantified using methods well known in the art. One such method requires measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that equally affect the rate in both directions. Thus, both the “on-rate constant” (Kon) and the “off-rate constant” (Koff) can be determined by calculating the concentrations and actual rates of binding and dissociation (see Nature 361:186-87 (1993)). The Koff / Kon ratio allows for the dissociation of all parameters unrelated to affinity and is equal to the dissociation constant Kd. (Generally, see Davies et al. (1990) Annual Rev Biochem 59:439-473). The antibody of this disclosure is said to bind specifically to the CD3 epitope when the equilibrium binding constant (Kd) is about 1 μM, preferably about 100 nM, more preferably about 10 nM, and most preferably about 100 pM to about 1 pM, as measured by an assay such as a radioligand binding assay or similar assay known to those skilled in the art.

[0196] Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, amino acids with aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; amino acids with aliphatic-hydroxyl side chains are serine and threonine; amino acids with amide-containing side chains are asparagine and glutamine; amino acids with aromatic side chains are phenylalanine, tyrosine, and tryptophan; amino acids with basic side chains are lysine, arginine, and histidine; and amino acids with sulfur-containing side chains are cysteine ​​and methionine. Preferred conservative amino acid substitutions include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0197] As discussed herein, minor changes in the amino acid sequence of antibody or immunoglobulin molecules are intended to be covered by this disclosure, provided that the changes in amino acid sequence are maintained by at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid substitutions are intended. Conservative substitutions occur in which their side chains are within a family of amino acids to which they relate. Amino acids encoded by genes are generally divided into families: (1) acidic amino acids, such as aspartic acid and glutamic acid; (2) basic amino acids, such as lysine, arginine, and histidine; (3) nonpolar amino acids, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids, such as glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include (i) the aliphatic-hydroxy family, serine and threonine; (ii) the amide-containing family, asparagine and glutamine; (iii) the aliphatic family, alanine, valine, leucine, and isoleucine; and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine.

[0198] The term "pharmaceutical" as used herein refers to a compound, a mixture of compounds, a biological polymer, or an extract made from a biological material.

[0199] The term "patient" includes both human and veterinary subjects.

[0200] This disclosure also includes Fv, Fab, Fab', and F(ab')2 anti-CD3 antibody fragments, single-chain anti-CD3 antibodies, bispecific anti-CD3 antibodies, heteroconjugate anti-CD3 antibodies, triplicate antibodies, immunoconjugates, and their fragments.

[0201] A bispecific antibody is an antibody that has binding specificity to at least two different antigens. In this application, one of the binding specificities is to CD3. The second binding target is any other antigen, and advantageously, a cell surface protein or receptor or receptor subunit.

[0202] All publications and patent documents referenced herein are incorporated herein by reference to the extent that each such publication or document is specifically and individually indicated to be incorporated herein by reference. No reference to publications and patent documents is intended to constitute an acknowledgment that any of them is relevant prior art, nor does it constitute an acknowledgment of any of them in accordance with their content or date. This disclosure is provided herein for illustrative purposes only, and those skilled in the art will understand that this disclosure can be made in various embodiments, and that the foregoing description and the following examples are for illustrative purposes only and do not limit the scope of the following claims. [Examples]

[0203] Example 1: Bioavailability study of foralumab administered by subcutaneous delivery in a mouse model.

[0204] The purpose of this study was to compare the pharmacokinetic (PK) profiles of foralumab administered intravenously and subcutaneously in a mouse model.

[0205] The results of this study demonstrate the feasibility of administering foralumab via subcutaneous injection, representing a possible human therapeutic route. The intravenous administration route is used as a control because it has been validated in previous preclinical repeated dose-toxicity studies and early clinical studies on foralumab administration.

[0206] research design

[0207] The mouse model used in this study was a human CD3 epsilon transgenic mouse model possessing a humanized CD3 epsilon chain of the CD3 coreceptor within the functional mouse immune system. This model was used to determine the in vivo efficacy of human-specific immunotherapy targeting the human CD3 epsilon chain. A total of 132 mice (66 males and 66 females) were used. The groups were administered either intravenously (IV) or subcutaneously (SC). Study groups 1, 2, and 3 consisted of 18 males and 18 females, and were divided into three groups at each time point. Intravenous (IV) group 4 had 3 males and 3 females, and subcutaneous placebo group 5 had 9 males and 9 females. At each time point, 3 males and 3 females were used as described below (Table 1.1).

[0208] [Table 1.1]

[0209] Medication preparations

[0210] Foralmab (NI-0401) was formulated in 125 mM sodium chloride with 25 mM sodium acetate buffer, 0.02% polysorbate 80, and pH 5.5. The vehicle control (placebo) used was 125 mM sodium chloride with 25 mM sodium acetate buffer, 0.02% polysorbate 80, and pH 5.5.

[0211] Dose level and volume

[0212] A single dose of 0.3 mg / kg was selected, as it has already been shown to induce up to a 70% reduction in T cells in peripheral blood and an 80% regulation of the human CD3 epsilon molecule at the T cell membrane in LCD3 transgenic mice. The dose volume was 2.5 mL / kg, administered manually as a bolus.

[0213] Administration of test substance

[0214] A single dose of foralumab or placebo was administered subcutaneously to the outside of the abdominal wall or intravenously via the posterior orbital sinus.

[0215] blood sample

[0216] Blood samples were collected from mice by intracardiac puncture under local anesthesia at the indicated time points after administration. The samples were collected in Plasma Separator Tubes (BD), and the plasma was separated by centrifugation. Aliquots of 40-50 μL of plasma were frozen and stored at -50°C.

[0217] Results and Conclusions

[0218] Subcutaneous administration of foralmab achieves efficient delivery into the bloodstream and is pharmacologically active (Figure 2, Table 1.2). Blood levels of subcutaneously delivered foralmab peaked 6–24 hours after delivery (Table 1.3). The bioavailability of subcutaneously delivered foralmab at 0.3 mg / kg is approximately 55% (Table 1.2). Increasing the dose of foralmab up to 0.6 mg / kg via subcutaneous delivery increases bioavailability to 92% (Table 1.2). Due to a 50% reduction in Cmax achieved via subcutaneous delivery compared to intravenous administration, infusion-related reactions are likely to be reduced. Taken together, subcutaneous delivery of foralmab is an effective method for administering foralmab and related antibodies. [Table 1.2] [Table 1.3] Example 2: Pharmacokinetic, therapeutic, and safety studies of foralumab (NI-0401) administered intravenously.

[0219] The primary objective of these studies was to evaluate the safety and tolerability of foralumab in human subjects. The studies included evaluation of the pharmacokinetic profile, immunogenicity, and pharmacokinetic effects of foralumab delivered intravenously over a 5-day period.

[0220] Test methods, dosages, and administration methods

[0221] Foralmab human monoclonal antibody was supplied in 3 mL vials, each containing 2 mL of foralmab at a concentration of 2.0 mg / mL. Each vial contained 4.0 mg of foralmab. The dose of foralmab was administered by intravenous infusion over 2 hours. Foralmab dosing schedules were prepared for eight different cohorts, according to Table 2.1. [Table 2.1]

[0222] The pharmacokinetic profile was determined by measuring foralmab plasma levels at the indicated time after administration. Foralmab plasma levels were measured using a ligand-binding assay. A specific anti-foralmab antibody was used as the capture reagent and fluorescently labeled anti-human IgG1. The sensitivity of the assay was 20 ng / mL (lower limit of detection).

[0223] Pharmacokinetic results

[0224] Plasma levels of foralumab were measured using a ligand-binding assay. A specific anti-foralumab antibody was used as the capture reagent, and fluorescently labeled anti-human IgG1 was used. The sensitivity of the assay was 20 ng / mL (lower limit of detection).

[0225] Regarding the study design shown in Table 2.1, the Cmax and AUC0-t parameters on day 5 of the study were 584.4 ng / mL (range 28.43-1860.1 ng / mL) and 28570 ng.h / mL (range 1453-106494 ng.h / mL), respectively, across the dose range of 0.73-3.73 mg.

[0226] Representative sample collections of pharmacokinetic profiles for three individuals from another study are shown in Figure 3. These subjects were treated with either a five-dose dose of 1.0 mg (approximately + / - 500 μg / m2), a two-dose dose of 2.0 mg (approximately + / - 1000 μg / m2), or a single dose of 10.0 mg (approximately + / - 5000 μg / m2). For these three subjects, the concentration profiles were sufficient to estimate the apparent Cmax (1 hour after infusion) and AUC0-6, both of which showed a clear increase with administration (Figure 4). The apparent Cmax values ​​for the 1.0 mg, 2.0 mg, and 10.0 mg doses were 110 ng / mL, 350 ng / mL, and 2800 ng / mL, respectively. The AUC values ​​for the 1.0 mg, 2.0 mg, and 10.0 mg doses were 440 ng.-hours / mL, 1700 ng.-hours / mL, and 11800 ng.-hours / mL, respectively.

[0227] Figure 5 shows a graph summarizing the foralmab PK data obtained after a single 2-hour infusion of 10.0 mg (subjects 001-0001 in Figure 4). Following the intravenous infusion of 10.0 mg of the study drug over 2 hours, the plasma concentration of foralmab increased rapidly during the infusion period, as expected. Post-infusion, the concentration values ​​declined in essentially a single exponential manner. The initial rapid decline in plasma concentration is attributed to the binding of the drug to its target on circulating T cells and drug distribution. The estimated plasma endpoint half-life after a single dose of 10.0 mg of foralmab was approximately 13 hours, although the endpoint half-life may be considerably longer using more sensitive assays (theoretically, it would take approximately 78 hours to be removed from systemic circulation, which is 6 times the endpoint half-life). The measured half-life of foralmab is shorter than predicted for IgG1 molecule (typically about 3 weeks) due to the rapid uptake of the drug by target cells.

[0228] Exposure based on AUC0~t during days 1-5 of the study indicated the presence of foralumab accumulation in plasma. One subject (030-0003 in Figure 4) exposed to five doses of 1.0 mg foralumab, corresponding to approximately 21 μg / kg / administered (+ / - 500 μg / m2 / administered), had increasing plasma drug concentrations throughout the treatment period (Figure 6). When accumulation occurred, the PK and PD profiles in this subject correlated, both peaking at the end of the treatment period, as shown in the graph below (Figure 6). This may be due to depletion of the target, which has a reduced effect on the frequency of administration and / or mAb properties.

[0229] These findings as a whole suggest that intravenous administration of foralumab at doses of 1.0 mg (i.e., 21 μg / kg or 500 μg / m2) and above may have resulted in drug accumulation over a 5-day administration period. However, the drug is expected to be rapidly eliminated, approximately 3–4 days after the final dose.

[0230] Pharmacodynamic results

[0231] For all pharmacodynamic analyses, there was no distinction in predictive pharmacology based on the foralumab dose. All dose levels of foralumab had effects on the TCR-CD3 complex and cell populations over the observed time course.

[0232] TCR-CD3 complex regulation was measured in CD8+ or CD4+ T cells after the start of treatment at the indicated time points (Figure 7). Maximum TCR-CD3 complex regulation was observed in all treatment groups at the end of the treatment period on day 5 of the study. The mean regulation across all treatment groups on day 5 of the study was 81.1%, with the highest mean TCR-CD3 complex regulation at this point observed in treatment cohort 8 (94%) (Figure 8). TCR-CD3 regulation gradually decreased towards the end of the treatment period (Figure 8). All patients in all treatment cohorts (for which CD3 regulation data was available) achieved CD3 regulation above 50% (Figure 8). CD3 regulation across all treatment groups remained above 50% for a mean duration of 8.7 days and above 30% for a mean duration of 12.9 days.

[0233] Figure 9 summarizes the kinetic profiles of TCR-CD3 regulation for each cohort (dose 500–1500 μg / m2) of three patients. The profiles showed a dose-dependent effect, peaking on day 5 and then gradually decreasing until day 21 (week 3). Three maximum dose treatment groups have been tested to date, and the 2–3 mg / day range exhibited the same profile with an average TCR-CD3 regulation of approximately 50% on day 10, indicating that these dose treatment groups reached a plateau (Figure 9).

[0234] Circulating leukocyte and subpopulation counts were performed during and after foralumab administration. A transient increase in CD45+ leukocyte counts was observed in all foralumab-treated cohorts (overall mean increase of 68.7%) 6 hours after administration on day 1 of the study, followed by a return to near-baseline or below-baseline levels in most cohorts. Up to week 3, CD45+ leukocyte counts remained below baseline in all cohorts except for cohorts 1 and 4. In all treatment groups, there was a rapid and almost complete elimination of circulating CD45+ lymphocytes, CD3+ T cells, CD3+CD4+ T cells (helper T cells), and CD3+CD8+ T cells (cytotoxic T cells) within 24 hours of the first infusion, followed by a return to near-baseline levels by week 3. These results strongly suggest that foralumab can induce lymphocyte depletion in humans. Recovery in Cohort 1 was more rapid than in all other cohorts, except for CD8+ cell counts, but there was no apparent dose-response in reduction or recovery. There was also a rapid decrease in CD3-CD19+ (B cell) and CD3-CD16+CD56+ (natural killer cell) counts in all treatment groups 6 hours after administration on day 1 of the study, accompanied by variable (dose-independent) recovery in these cell counts on day 3 of the study and above baseline values ​​at week 3 in the majority of cases.

[0235] Cytokine levels were assessed in subjects treated with foralumab (Figure 10). Substantial variation in cytokine release after foralumab treatment was observed among subjects, and this did not appear to be dose-dependent. In patients with a marked increase in pro-inflammatory cytokines, this was accompanied by symptoms suggestive of an infusion-related reaction (IRR), although most symptoms were mild and short-lived. Most patients had little to no evidence of pro-inflammatory cytokine release on subsequent treatment days.

[0236] safety results

[0237] Adverse events (AEs) were evaluated in human subjects who received foralumab intravenously (Figure 11). Of the 24 subjects, 19 patients (79%) experienced a total of 94 AEs, of which 3 (3%) were serious adverse events (SAEs). 58 AEs (62%) were of mild severity, 32 (34%) were of moderate severity, and 4 (4.3%) were serious. Of the 91 non-serious AEs, 68 (75%) were considered to have a reasonable possibility of being drug-related by the researchers. AEs that occurred during the 5-day treatment period were reported during or within 24 hours of foralumab infusion and were therefore primarily defined as infusion-related reactions (IRRs) (61%) (Figures 12 and 13). The most common internal risk factors (IRRs) were chills (9 events in 6 patients), fever (8 events in 7 patients), headache (8 events in 6 patients), hypotension (5 events in 3 patients), and elevated ALT (3 events in 3 patients).

[0238] One IRR was reported as a SAE, and a patient in cohort 5 had a transient elevation of ALT on day 2 of the study, which led to discontinuation of the study drug treatment. Visual examination showed a clear dose response in reporting treatment-related AEs, with higher dose cohorts reporting more drug-related AEs, and all hematological and lymphatic disorders reported by the two highest dose cohorts.

[0239] Three SAEs were reported, two of which were considered unrelated to the study drug. One patient experienced a relapse of Crohn's disease eight days after completing the five-day treatment, resulting in an extended hospital stay. A second patient had multiple fractures of the humerus with displaced fragments after a fall in the street one and a half months after the end of the study treatment. One SAE was considered related to the study drug, a transient increase in ALT on day two of the study, leading to discontinuation of the study drug treatment. Few AEs were reported after the five-day treatment period during the study (18 events). There were no deaths or AEs leading to discontinuation of the study.

[0240] Hematological and biochemical analyses in subjects receiving foralumab generally showed that mean hemoglobin, hematocrit, and red blood cell counts remained stable over time across all treatment cohorts, and there was no substantial change over time in mean platelet counts across treatment cohorts. Up to day 5 of the study, there was a decrease from baseline in mean total white blood cell counts of -2.98 10E9 / L across all treatment cohorts, without evidence of dose-response. White blood cell counts recovered by week 12. Mean neutrophil and monocyte counts showed a similar pattern, with a mean decrease on day 5 of the study and recovery by weeks 12 and 3, respectively.

[0241] Liver function was assessed in patients who received foralumab, and several transient, non-critical liver laboratory abnormalities were detected (Figure 14). Five patients (15%) had isolated, transient elevations of ALP above the upper limit of the normal range, beginning at 2, 3, and 4 weeks in two cases, and at 2, 3, and 4 weeks in the other cases, respectively. One patient had pre-existing abnormal ALP levels. One patient had a transient, isolated elevation of AST / ALT 3.5 times above the upper limit of the normal range on 5 days, which normalized at 2 weeks. Six patients (18%) had liver laboratory abnormalities, indicating mild, transient cholesterol-induced liver injury. Elevated serum bilirubin was not associated with these abnormalities, except in one patient. Of these six patients, three had pre-existing liver laboratory abnormalities of similar magnitude. Elevated liver enzymes mainly occurred on 5 days and returned to baseline within one week. One patient had mild jaundice (elevated bilirubin on day 2, which resolved on day 4), and another patient had hepatomegaly (a second elevation in liver enzymes recurred at week 4 in this patient, who also had pre-existing liver test abnormalities). No other signs of liver failure were present in these cases, and no contributing factors were identified.

[0242] conclusion

[0243] Based on the safety data, it can be concluded that these studies did not reach dose-limited toxic doses. However, the safety profile of foralumab was extended from a daily dose of 500 mg / m2 (approximately 1.00 mg) to 1750 mg / m2 (approximately 3.5 mg) with steroid premedication. This study demonstrated that foralumab is pharmacologically active. Foralumab affected the TCR-CD3 complex and T cell subsets, which reflected the expected pharmacology of the drug and its targets. After foralumab treatment, lymphocyte counts decreased to below the normal range in all patients. There was no apparent effect of foralumab doses on persistent lymphocyte depletion. Mean lymphocyte counts returned to near pre-treatment levels by week 4. Lymphocyte depletion was a predicted effect of anti-CD3 antibody administration. Other Embodiments

[0244] This disclosure is described in conjunction with its detailed description, but the foregoing description is intended to describe, and not limit, the scope of this disclosure as defined by the attached claims. Other aspects, advantages, and modifications are within the scope of the following claims:

Claims

1. 1. A composition comprising a bispecific antibody having specificity for CD3 and IL6R for use in a method for improving cell expansion and / or survival, comprising: The method includes contacting a cell in vivo with the composition; The bispecific antibody comprises: (a) a heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence GYGMH (SEQ ID NO: 42), a heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO: 43), a heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence QMGYWHFDL (SEQ ID NO: 44), a light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSYLA (SEQ ID NO: 45), a light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence DASNRAT (SEQ ID NO: 46), and a light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQRSNWPPLT (SEQ ID NO: 47), which has specificity for CD3; (b) a heavy chain CDR1 region comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain CDR2 region comprising the amino acid sequence of SEQ ID NO: 37, a heavy chain CDR3 region comprising the amino acid sequence of SEQ ID NO: 35, a light chain CDR1 region comprising the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 region comprising the amino acid sequence of SEQ ID NO: 25, and a light chain CDR3 region comprising the amino acid sequence of SEQ ID NO: 26, which have specificity for IL-6R; A composition comprising:

2. 1. A method for improving cell expansion and / or survival, comprising: The method comprises contacting cells ex vivo with a composition comprising a bispecific antibody having specificity for CD3 and IL6R; The bispecific antibody comprises: (a) a heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence GYGMH (SEQ ID NO: 42), a heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO: 43), a heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence QMGYWHFDL (SEQ ID NO: 44), a light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSYLA (SEQ ID NO: 45), a light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence DASNRAT (SEQ ID NO: 46), and a light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQRSNWPPLT (SEQ ID NO: 47), which has specificity for CD3; (b) a heavy chain CDR1 region comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain CDR2 region comprising the amino acid sequence of SEQ ID NO: 37, a heavy chain CDR3 region comprising the amino acid sequence of SEQ ID NO: 35, a light chain CDR1 region comprising the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 region comprising the amino acid sequence of SEQ ID NO: 25, and a light chain CDR3 region comprising the amino acid sequence of SEQ ID NO: 26, which have specificity for IL-6R; A method comprising:

3. The composition of claim 1 , wherein the cell is an engineered cell.

4. the cells are lymphocytes, optionally said lymphocytes are B cells or T cells; 2. The composition of claim 1, further optionally wherein the T cells are CAR-T cells.

5. the cells are stem cells, 2. The composition of claim 1, wherein optionally the stem cells are human embryonic stem cells, tissue-specific stem cells, neural stem cells, mesenchymal stem cells, hematopoietic stem cells, induced pluripotent stem cells, epidermal stem cells, epithelial stem cells, and / or neural stem cells.

6. 1. A composition comprising a bispecific antibody having specificity for CD3 and IL6R for use in a method of enhancing cell therapy, comprising: The method includes administering the composition to a subject in need thereof; the cell therapy is CAR-T cell therapy or stem cell therapy; The bispecific antibody comprises: (a) a heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence GYGMH (SEQ ID NO: 42), a heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO: 43), a heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence QMGYWHFDL (SEQ ID NO: 44), a light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSYLA (SEQ ID NO: 45), a light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence DASNRAT (SEQ ID NO: 46), and a light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQRSNWPPLT (SEQ ID NO: 47), which has specificity for CD3; (b) a heavy chain CDR1 region comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain CDR2 region comprising the amino acid sequence of SEQ ID NO: 37, a heavy chain CDR3 region comprising the amino acid sequence of SEQ ID NO: 35, a light chain CDR1 region comprising the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 region comprising the amino acid sequence of SEQ ID NO: 25, and a light chain CDR3 region comprising the amino acid sequence of SEQ ID NO: 26, which have specificity for IL-6R; A composition comprising:

7. The composition of claim 6, wherein the stem cells are human embryonic stem cells, tissue-specific stem cells, neural stem cells, mesenchymal stem cells, hematopoietic stem cells, induced pluripotent stem cells, epidermal stem cells, epithelial stem cells, and / or neural stem cells.

8. The composition of any one of claims 1 and 3-7, further comprising one or more costimulatory agents.

9. the one or more costimulatory agents are an anti-CD28 antibody, an anti-IL-6R antibody, a PI3K inhibitor, an Akt inhibitor, or an mTor inhibitor; Depending on the situation, (a) the CD28 antibody is a monoclonal antibody, a bispecific antibody, or a trispecific antibody, optionally wherein the bispecific antibody has specificity for CD28 and IL-6R or TNF, and the trispecific antibody has specificity for CD28, IL-6R, and TNF; or (b) the anti-IL-6R antibody is a monoclonal antibody, a bispecific antibody, or a trispecific antibody, optionally wherein the bispecific antibody has specificity for IL-6R and CD28 or TNF, or the trispecific antibody has specificity for IL-6R, CD28, and TNF; The composition of claim 8.

10. The composition of any one of claims 1 and 3 to 9, wherein the bispecific antibody is coated onto macroporous beads.

11. The composition of any one of claims 1 and 3 to 10, wherein the composition antibody is administered nasally, orally, subcutaneously, intravenously or by inhalation.

12. the bispecific antibody (a) a variable heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 48 and a variable light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 49; or (b) a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 50 and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 51; The composition of any one of claims 1 and 3 to 11, comprising:

13. 12. The composition of claim 11, wherein the bispecific antibody comprises tocilizumab or sarilumab.

14. administered before, after, both before and after, and / or simultaneously with administration of a cell therapy cell composition to a subject; 14. The composition of any one of claims 6 to 13, optionally administered 24 to 48 hours prior to administration of a cell therapy composition to a subject, or 14 to 21 days after administration of a cell therapy composition.

15. comprising the bispecific antibody and a pharmaceutically acceptable carrier, 15. The composition of any one of claims 1 and 3-14, optionally comprising the bispecific antibody in a unit dose of 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, or 4.0 mg.

16. The method of claim 2, wherein the cell is an engineered cell.

17. the cells are lymphocytes, optionally said lymphocytes are B cells or T cells; 3. The method of claim 2, further optionally wherein the T cells are CAR-T cells.

18. the cells are stem cells, 3. The method of claim 2, wherein optionally the stem cells are human embryonic stem cells, tissue-specific stem cells, neural stem cells, mesenchymal stem cells, hematopoietic stem cells, induced pluripotent stem cells, epidermal stem cells, epithelial stem cells, and / or neural stem cells.

19. 19. The method of any one of claims 2 and 16-18, further comprising one or more costimulatory agents.

20. the one or more costimulatory agents are an anti-CD28 antibody, an anti-IL-6R antibody, a PI3K inhibitor, an Akt inhibitor, or an mTor inhibitor; Depending on the situation, (a) the CD28 antibody is a monoclonal antibody, a bispecific antibody, or a trispecific antibody, optionally wherein the bispecific antibody has specificity for CD28 and IL-6R or TNF, or wherein the trispecific antibody has specificity for CD28, IL-6R, and TNF; or (b) the anti-IL-6R antibody is a monoclonal antibody, a bispecific antibody, or a trispecific antibody, optionally wherein the bispecific antibody has specificity for IL-6R and CD28 or TNF, or the trispecific antibody has specificity for IL-6R, CD28, and TNF; 20. The method of claim 19.

21. The method of any one of claims 2 and 16 to 20, wherein the bispecific antibody is coated onto macroporous beads.

22. 22. The method of any one of claims 2 and 16-21, wherein the composition is administered nasally, orally, subcutaneously, intravenously or by inhalation.

23. the bispecific antibody (a) a variable heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 48 and a variable light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 49; or (b) a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 50 and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 51; 23. The method of any one of claims 2 and 16 to 22, comprising:

24. 23. The method of claim 22, wherein the bispecific antibody comprises tocilizumab or sarilumab.

25. the composition comprises the bispecific antibody and a pharmaceutically acceptable carrier; 25. The method of any one of claims 2 and 16-24, optionally wherein the composition comprises the bispecific antibody in a unit dose of 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, or 4.0 mg.

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