Methods for reducing bispecific T cell engager or chimeric antigen receptor T cell-mediated cytokine release syndrome using interleukins

Abstract

The present disclosure provides various methods, including a method of reducing the severity of bispecific T cell engager (BiTE) or chimeric antigen receptor T cell (CAR-T) induced cytokine release syndrome (CRS), comprising administering to a patient in need thereof an amount of a composition comprising interleukin 10 (IL-10) or an IL-10 agent, interleukin 4 (IL-4) or an IL-4 agent, or a combination thereof. In one embodiment, the composition comprises human or viral IL-10, muteins, variants, fusion proteins, and fragments thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 312,697, filed February 22, 2022, the disclosure of which is incorporated by reference in its entirety herein.

[0002] Use of Electronic Sequence Listing The contents of the electronic sequence listing (039451-00100-Sequence-Listing.xml; size: 37,997 bytes; and creation date: February 22, 2023) are incorporated by reference herein in their entirety. [Background technology]

[0003] background Cytokine release syndrome (CRS) is a dosing and treatment-related toxicity resulting from bispecific T cell engager (BiTE) (Hosseini, 2020) activation of T cells and application of chimeric antigen receptor T cells (CAR-T) to patients (Maude, 2014; Norelli, 2018). CRS is primarily defined by the uncontrolled induction of high levels of interleukin-6 (IL-6) and interleukin-1β (IL-1b) (Liu, 2018), tumor necrosis factor α (TNFα) (Chen, 2021), and interferon gamma (IFNγ) (Shimabukuro-Vornhagen, 2018) in the serum of treated patients.

[0004] To date, the molecular circuitry responsible for the induction of CRS is unknown. Based on our findings, it has been determined that the induction of CRS in BiTE and CAR-T patients is likely due to T cell receptor (TCR):BiTE or CAR-T:tumor-associated antigen (TAA) clustering-mediated activation of CD4+ T cells. This activation subsequently leads to the secretion of IL-2 (Brandl, 2007). The secretion of IL-2 then drives monocyte secretion of proinflammatory cytokines associated with CRS (Bosco, 2000; Musso, 1992; Strieter, 1989). Summary of the Invention [Means for solving the problem]

[0005] The inventors have found that treatment of monocytes with IL-10 or IL-4, IL-12, IL-15, IL-7 or any combination of the foregoing, or extended half-life versions of any of these, or any diakine including IL-10, IL-4, IL-12, IL-15, IL-7, or IL-2, binds the cognate cytokine receptors on monocytes and directly inhibits the induction of pro-inflammatory cytokines by IL-2.

[0006] In one aspect, the application relates to a method of treating monocytes with either IL-10 or IL-4, IL-12, IL-15, IL-7, extended half-life versions thereof, combinations of IL-10 and IL-4, IL-10 and IL-2, IL-10 and IL-7, IL-10 and IL-12, IL-10 and IL-15, or a fusion protein or diakine comprising at least two cytokines, where at least one of the at least two cytokines is IL-10 or IL-4, IL-12, IL-15, or IL-7, to reduce CRS associated with BiTE or CAR-T therapy. In one embodiment, the monocytes are treated with IL-10, or IL-4, IL-12, IL-15, IL-7 or a half-life extended version thereof, or a diakine in which at least one of the at least two cytokines is IL-10, IL-4, IL-12, IL-15, or IL-7. In another embodiment, the patient is treated with IL-10 or a half-life extended version thereof and a BiTE or CAR-T. In yet another embodiment, the patient is treated with IL-4 or a half-life extended version thereof and a BiTE or CAR-T. In another embodiment, the patient is treated with a diakine comprising IL-10 and any one of IFN-α, IL-2, IL-4, IL-7, IL-12, IL-15, IL-21, or IL-27 in an amount sufficient to reduce CRS. In yet another embodiment, the patient is treated with a diakine comprising IL-4 and any one of IFN-α, IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, or IL-27 in an amount sufficient to reduce CRS.

[0007] In another aspect, the present application relates to a method of reducing BiTE or CAR-T associated CRS, comprising administering to a subject a BiTE or CAR-T treatment modality in combination with IL-10, IL-4, or any combination thereof, or a diakine comprising at least one of IL-10, IL-4, IL-2. In one embodiment, the method comprises administering to a patient in need thereof the BiTE or CAR-T treatment before, after, or simultaneously with IL-10 or IL-4, IL-12, IL-15, or IL-7, or a half-life extended version thereof, or a diakine comprising IL-10 or IL-4 in combination with IFN-α, IL-2, IL-7, IL-12, IL-15, IL-21, or IL-27.

[0008] In yet another aspect, the application relates to a method of inhibiting induction of pro-inflammatory cytokines in a patient undergoing BiTE or CAR-T therapy, comprising administering to the patient undergoing said therapy a dose of IL-10 or IL-4 or extended half-life versions thereof, or diakines including IL-10 or IL-4 in combination with IFN-α, IL-2, IL-7, IL-12, IL-15, IL-21 or IL-27, in an amount sufficient to suppress CRS caused by said pro-inflammatory cytokines.

[0009] The above simplified summary of representative aspects serves to provide a basic understanding of the present disclosure. This summary is not an extensive overview of all contemplated aspects, and is not intended to identify key or critical elements of all aspects, nor to delineate the scope of any or all aspects of the present disclosure. Its sole purpose is to present one or more aspects in a simplified form as a prelude to the more detailed description of the present disclosure that follows. To accomplish the foregoing, one or more aspects of the present disclosure include the features described and illustratively pointed out in the claims. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a graph measuring levels of IL1β, IFNγ, TNFα, IFNa2α, IL-12, and IL-6 in PBMCs in response to increasing levels of IL-2 exposure.

[0011] [Diagram 2] FIG. 2 is a graph measuring the levels of IFNγ, IL-6, and TNFα induction from PBMC in response to 1 μg / mL anti-CD3 in the presence of diakine, IL-10, and IL-2.

[0012] [Diagram 3] FIG. 3. Mouse serum cytokines resulting from diakine exposure in vivo.

[0013] [Figure 4] FIG. 4. Non-human primate serum cytokine levels resulting from diakine exposure in vivo.

[0014] [Diagram 5] FIG. 5 is a graph measuring the cytotoxicity of CD8+ T cells exposed to diakine and BiTE.

[0015] [Figure 6] FIG. 6 is a graph measuring IFNγ and TNFα levels in CD8+ T cells exposed to various concentrations of BiTE.

[0016] [Figure 7] FIG. 7 is a schematic diagram of the proposed IL-2-mediated CRS circuit in response to BiTE or CAR-T therapy.

[0017] [Figure 8]FIG. 8. Diakine combinations including IL-10 and IL-2 targeting EGFR in combination with a CD3×CD19 BiTE enhance CD8+ T cell-induced tumor cell cytolysis.

[0018] [Figure 9] FIG. 9. Combination of diakine (DK210 EGFR) with CD3×CD19 BiTE exhibits enhanced cytolytic effector molecules and regulated CRS.

[0019] [Figure 10-1] FIG. 10. Intracellular FACS analysis of diakine (DK210 EGFR) and CD3×CD19 BiTE treatment of PBMC cultures containing RajiGFP+ tumor cells. [Figure 10-2] FIG. 10. Intracellular FACS analysis of diakine (DK210 EGFR) and CD3×CD19 BiTE treatment of PBMC cultures containing RajiGFP+ tumor cells. [Figure 10-3] FIG. 10. Intracellular FACS analysis of diakine (DK210 EGFR) and CD3×CD19 BiTE treatment of PBMC cultures containing RajiGFP+ tumor cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Detailed Description Exemplary aspects are described herein in the context of using IL-10, IL-4, IL-12, IL-15, IL-7, or half-life-extended versions thereof, or diakines including at least one of IL-10 or IL-4 in combination with IFN-α, IL-2, IL-7, IL-12, IL-15, or IL-27 in a method of suppressing, inhibiting, reducing, or preventing CRS associated with BiTE or CAR-T therapeutic modalities. Those skilled in the art will understand that the following description is merely illustrative and is not intended to be limiting in any way. Other aspects will be readily suggested to those skilled in the art who have the benefit of this disclosure. Reference is made in detail to the implementation of the exemplary aspects as illustrated in the accompanying drawings. The same reference designations are used to the extent possible throughout the drawings and the following detailed description to refer to the same or similar items.

[0021] Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the various described embodiments, the preferred materials and methods are described herein.

[0022] Unless otherwise indicated, the embodiments described herein use conventional methods and techniques of molecular biology, biochemistry, pharmacology, chemistry, and immunology well known to those skilled in the art.See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); Handbook of Experimental Immunology, Vol. I-IV (DM Weir and CC Blackwell, eds., Blackwell Scientific Publications); ALLehninger, Biochemistry (Worth Publishers, Inc., latest edition).Many of the general techniques for designing and producing diakines have been previously described in US Patent Publication No. 20220017587. This includes types of IL-10 variants (including but not limited to human, mouse, CMV and / or EBV forms of IL-10), as well as assays for testing IL-10 variants, diakines, and other known assay methods.

[0023] The following terms are used to describe the various embodiments discussed herein and are intended to be defined as set forth below.

[0024] As used herein in describing various embodiments, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0025] The term "about" refers to deviations between 0.0001-5% from a given number or range of numbers. In one embodiment, the term "about" refers to deviations between 1-10% from a given number or range of numbers. In one embodiment, the term "about" refers to deviations up to 25% from a given number or range of numbers. In a more specific embodiment, the term "about" refers to differences of 1-25% in terms of nucleotide sequence homology or amino acid sequence homology when compared to a wild-type sequence.

[0026] The term "agent" when referring to various interleukins (ILs), such as "IL-10 agent" or "IL-4 agent," is intended to be broadly interpreted and includes, for example, human and non-human forms (including homologs), variants (including muteins), fragments, and fusion proteins of interleukin polypeptides, as well as polypeptides having, for example, leader sequences (e.g., signal peptides), and modified versions of the foregoing. The present disclosure also contemplates nucleic acid molecules encoding the foregoing, vectors, etc., that contain the nucleic acid molecules, and cells (e.g., transformed and host cells) that express the interleukin agents. The terms "variant," "analog," and "mutein" refer to biologically active derivatives of a reference molecule that retain a desired activity (such as, for example, anti-inflammatory activity). In general, the terms "variant," "variants," "analog," and "mutein," when referring to a polypeptide, refer to a compound having a naturally occurring polypeptide sequence and structure that has one or more amino acid additions, substitutions (which may be conservative in nature), and / or deletions relative to the naturally occurring molecule. For example, the terms "IL-10 variant," "variant IL-10," "IL-10 variant molecule," and grammatical variations and plurals thereof are all intended to be equivalent terms that refer to variant forms of IL-10 amino acid (or nucleic acid) sequences that differ from the wild-type IL-10 form by anywhere from 1-25% in sequence identity or homology. Thus, for example, an EBV IL-10 variant molecule differs from wild-type EBV IL-10 by having one or more amino acid (or nucleotide sequence encoding those amino acids) additions, substitutions, and / or deletions.

[0027] The term "fusion protein" refers to the combination or conjugation of two or more proteins or polypeptides, resulting in a novel arrangement of proteins that do not normally occur in nature. The fusion protein is the result of the covalent linkage of the two or more proteins or polypeptides. The two or more proteins that make up the fusion protein can be arranged in any configuration, from amino terminus ("NH2") to carboxy terminus ("COOH").

[0028] The terms "homolog," "homology," "homologous," or "substantially homologous" refer to the percent identity between at least two polynucleotide or at least two polypeptide sequences. Sequences are homologous to one another if they exhibit at least about 50%, preferably at least about 75%, more preferably at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules.

[0029] The term "sequence identity" refers to exact nucleotide-by-nucleotide or amino acid-by-amino acid correspondence. Sequence identity can range from 100% sequence identity to 50% sequence identity. Percent sequence identity can be determined using a variety of methods, including but not limited to direct comparison of sequence information between two molecules (a reference sequence and a sequence with unknown percent identity to the reference sequence) by aligning the sequences, counting the number of exact matches between the two aligned sequences, dividing by the length of the reference sequence, and multiplying the result by 100. Readily available computer programs can be used to assist in identifying percent identity.

[0030] The terms "subject," "individual," or "patient" are used interchangeably herein and refer to vertebrates, preferably mammals, including, but not limited to, murines, rodents, simians, humans, farm animals, sport animals, and certain pets.

[0031] The term "administering" includes routes of administration that enable the active ingredients of the present application to perform their intended function.

[0032] A "therapeutically effective amount" or "effective amount," as it relates to administering, for example, an IL variant, fusion protein, dual cytokine fusion protein, or diakine thereof described herein, refers to an amount sufficient to promote a certain biological activity, such as suppression of myeloid cell function, enhanced Kupffer cell activity, and / or CD8 + T cells or enhanced CD8 + These may include the lack of any effect on T cell activity, as well as blocking the mast cell upregulation or preventing degranulation of Fc receptors, or promoting or enhancing the effect of combination therapy (e.g., CAR-T therapy), or suppressing the induction of cytokines from monocytes or macrophages.Therefore, "effective amount" improves or prevents the symptoms or signs of a medical condition.Effective amount also means an amount sufficient to enable or facilitate diagnosis.

[0033] The terms "treat", "treating" or "treatment" refer to a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the underlying cause of the disease or condition itself, rather than only the symptoms. The treatment can be any reduction from its original level, and can be, but is not limited to, the complete elimination of the disease, condition, or symptoms of the disease or condition.

[0034] The term "half-life extended" as used in this application refers to a protein that includes one or more additional moieties (e.g., protein or PEGylation) and that extends and / or enhances circulation time in a subject by anywhere from 1-100 fold over the protein in the absence of the additional moieties. As it relates to ILs, in one preferred embodiment, the half-life extended IL refers to IL-10 or IL-4, IL-7, IL-12, or IL-15 conjugated to an scFv, whereby the circulation time is extended by anywhere from 1-10 fold over the IL in the absence of the scFv. In another embodiment, the half-life extended version of an IL is represented by Formula I: NH2-(Y)-(X 1 )-(X 2 )-(Y)-COOH, The configuration of "Y" is any monomer derived from either a homodimeric or heterodimeric cytokine; "X 1 " is a VL or VH region obtained from the first monoclonal antibody; "X 2 " is a VH or VL region obtained from the first monoclonal antibody, Here, X 1 If VL, then X 2 is VH or X 1 If VH, then X 2 is VL, and the VH and VL together form an scFv.

[0035] The term "diakine" or "DK" as used in this application refers to a dual cytokine fusion protein that includes two monomers of a dimeric cytokine (which can be either a homodimer (e.g., IL-10 or an IL-10 variant) or a heterodimer (e.g., IL-12 or an IL-12 variant)) fused to a monomeric cytokine (e.g., IL-2, IL-4, IL-7, IL-15, IL-21, IL-28, IL-29) or together with a dimeric cytokine (e.g., IL-12 or IL-10), whereby both the dimeric and monomeric cytokines are fused onto a half-life extended antigen targeting domain. Representative diakines are described in detail in U.S. Patent No. 11,292,822 (IL-10-based diakines) and co-pending U.S. Patent Application No. 18 / 065,504 (double dimeric cytokine-based diakines), both of which are incorporated herein by reference in their entirety. In one embodiment, the diakine, which may be used in a method in combination with BiTE or CAR-T, is represented by Formula II: NH2-(Y)-(X 1 )-(Z n )-(X 2 )-(Y)-COOH (Formula II) is represented by where "Y" is any monomer derived from either a homodimeric or heterodimeric cytokine; "X 1 " is a VL or VH region obtained from the first monoclonal antibody; "X 2 " is a VH or VL region obtained from the first monoclonal antibody, Here, X 1 If VL, then X 2 is VH or X 1 If VH, then X 2is a VL, said VH and VL together forming an scFv; "Z" is a second cytokine, wherein said second cytokine is any monomeric cytokine or another dimeric cytokine; "n" is an integer selected from 0 to 2. In one embodiment, the dimeric cytokine may include IFN-α, IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, and IL-27. In another embodiment, the monomeric cytokine may include IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-21, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferon-α, -β, -γ, TGF-β, or tumor necrosis factor-α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13. The VH and VL of the diakine are scFvs and may be derived from any monoclonal antibody, but are preferably derived from an antibody capable of targeting a specific antigen. Monoclonal antibodies from which the scFv (as applicable to Formula I and Formula II) may be derived are selected from: EGFR; CD52; CD14; various immune checkpoint targets (such as, but not limited to, PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4); CD19; CD20; CD22; CD47; GD-2; VEGFR1; VEGFR2; HER2; PDGFR; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, CD14, FAPα; 5T4; Trop2; EDB-FN; TGFβ Trap; MAdCAM, β7 integrin subunit; α4β7 integrin; α4 integrin SR-A1;SR-A3;SR-A4;SR-A5;SR-A6;SR-B;dSR-C1;SR-D1;SR-E1;SR-F1;SR-F2;SR-G;SR-H1;SR-H2;SR-I1;SR-J1;CD123;CD33;BCMA;PSA;PSMA;CEA;GPC3;BCMA;DLL3;MUC17;CLDN 18;gpA33;HIV or Ebola.In one embodiment, the scFv (if it is of Formula I or Formula II) is a grafted scFv, in which the VH and VL framework regions are derived from a first antibody (e.g., an anti-Ebola antibody) and the CDRs are derived from a second antibody (e.g., an antibody targeting EGFR; CD52; CD14; various immune checkpoint targets (e.g., but not limited to, PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4); CD19; CD20; CD22; CD47; GD-2; VEGFR1; VEGFR2; HER2; PDGFR; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, CD14, FAPα; 5T4; Trop2; EDB-FN; TGFβ Trap; MAdCAM, β7 integrin subunit; α4β7 integrin; α4 integrin SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; SR-J1; CD123; CD33; BCMA; PSA; PSMA; CEA; GPC3; BCMA; DLL3; MUC17; CLDN 18; gpA33; HIV or Ebola).In one preferred embodiment, the scFv is derived from an anti-Ebola antibody, where the VH and VL framework regions of the anti-Ebola antibody are selected from the group consisting of EGFR; CD52; CD14; various immune checkpoint targets (such as, but not limited to, PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4); CD19; CD20; CD22; CD47; GD-2; VEGFR1; VEGFR2; HER2; PDGFR; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, CD14, FAPα; 5T4; Trop2; EDB-FN; TGFβ Trap; MAdCAM, β7 integrin subunit; α4β7 integrin; α4 integrin Antibodies specific for SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; SR-J1; CD123; CD33; BCMA; PSA; PSMA; CEA; GPC3; BCMA; DLL3; MUC17; CLDN 18; gpA33; more preferably substituted or grafted with six CDR regions derived from anti-EGFR, anti-MAdCAM, anti-VEGFR1, anti-VEGFR2, anti-PDGFR, or anti-CD14, anti-CD19, anti-CD20, or anti-CD22.

[0036] In another embodiment, the diakine (which can be used in the method of combining with BiTE or CAR-T) is the diakine described in U.S. Patent No. 11,292,822 (IL-10-based diakine) or co-pending U.S. Patent Application No. 18 / 065,504 (double dimer cytokine-based diakine), both of which are incorporated by reference in their entirety.In yet another embodiment, the diakine (which can be used in the method of combining with BiTE or CAR-T) is the diakine described in Formula III: NH2-(IL-10)-(X 1 )-(Z n )-(X 2 )-(IL-10)-COOH (Formula III); wherein "IL-10" is a monomer; "X 1 " is a VL or VH region derived from the first monoclonal antibody; "X 2 " is a VH or VL region derived from the first monoclonal antibody, Here, X 1 If VL, then X 2 is VH or X 1 If VH, then X 2 is VL; wherein the first monoclonal antibody is an anti-Ebola antibody or an scFv framework region derived therefrom; wherein the VL and VH derived from said anti-Ebola antibody comprise three light chain CDRs and three heavy chain CDRs grafted with three light chain CDRs and three heavy chain CDRs derived from a second monoclonal antibody; "Z" is a cytokine other than IL-10; "n" is an integer 1; where the following variables in Table 1 apply to Formula III: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0037] In another embodiment, the diakine, which may be used in a method in combination with BiTE or CAR-T, is a diakine described in Tables 2a-2d and 3a-3d in co-pending U.S. patent application Ser. No. 18 / 065,504, which is incorporated by reference in its entirety.

[0038] In another aspect, the protein molecule or nucleic acid molecule encoding the dual cytokine fusion protein or DK can be formulated as a pharmaceutical composition comprising a therapeutically effective amount of the dual cytokine fusion protein and a pharmaceutical carrier and / or a pharma- ceutical acceptable excipient. The pharmaceutical composition can be formulated with commonly used buffers, excipients, preservatives, stabilizers. The pharmaceutical composition comprising the dual cytokine fusion protein is mixed with a pharma- ceutical acceptable carrier or excipient. A variety of pharmaceutical carriers are known in the art and can be used in the pharmaceutical composition. For example, the carrier can be any compatible, non-toxic substance suitable for delivering the dual cytokine fusion protein composition of the present application to a patient. Examples of suitable carriers include saline, Ringer's solution, dextrose solution, and Hank's solution. The carrier may also include any poloxamer generally known to those skilled in the art, including but not limited to those having molecular weights of 2900 (L64), 3400 (P65), 4200 (P84), 4600 (P85), 11,400 (F88), 4950 (P103), 5900 (P104), 6500 (P105), 14,600 (F108), 5750 (P123), and 12,600 (F127). The carrier may also include an emulsifier, including but not limited to polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80, to name a few. Non-aqueous carriers, such as fixed oils and ethyl oleate, may also be used. The carrier may also include additives, such as substances that enhance isotonicity and chemical stability, such as buffers and preservatives. See, e.g., Remington's Pharmaceutical Sciences and the United States Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984). Formulations of therapeutic and diagnostic agents may be prepared by mixing with a physiologically acceptable carrier, excipient, or stabilizer, for example, in the form of a lyophilized powder, a slurry, an aqueous solution or a suspension.

[0039] The pharmaceutical composition is formulated for administration to a patient in a therapeutically effective amount sufficient to provide the desired therapeutic result. Preferably, such amount has minimal negative side effects. In one embodiment, the amount of the dual cytokine fusion protein administered is sufficient to treat or prevent an inflammatory disease or condition. In another embodiment, the amount of the dual cytokine fusion protein administered is sufficient to treat or prevent an immune disease or disorder. In yet another embodiment, the amount of the diakine or dual cytokine fusion protein administered is sufficient to treat or prevent CRS mediated by BiTE or CAR-T therapy. The amount administered may vary between patients and should be determined by considering the subject's or patient's disease or condition, the patient's overall health, the method of administration, the severity of side effects, etc.

[0040] The effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method, route and dose of administration, and the severity of side effects.The appropriate dose to be administered to a patient is typically determined by a clinician using parameters or factors known or suspected in the art that affect or are expected to affect treatment.Generally, the dose is started at a somewhat lower amount than the optimal dose, which is then increased in small increments until the desired or optimal effect is achieved relative to any negative side effects.Important diagnostic measures include, for example, measures of symptoms of inflammation or the level of inflammatory cytokines produced.

[0041] The method for determining the dosage of the presently described dual cytokine fusion protein is substantially similar to that described in US Pat. No. 10,858,412. Generally, the presently described dual cytokine fusion protein has a dosage in the range of 0.01 mg / kg to 1 mg / kg, preferably 0.025 mg / kg to 0.5 mg / kg. The dual cytokine fusion protein may be administered daily, three times a week, twice a week, weekly, bimonthly, or monthly. The effective amount of the therapeutic agent affects the level of inhibition of CRS caused by BiTE or CAR-T therapy. In yet another embodiment, the diakine is administered at a concentration of 0.1 ng / mL to 200 ng / mL, preferably 10 ng / mL to 100 ng / mL. Generally, the addition of diakine is lower than the dose required for BiTE or CAR-T modalities.

[0042] The compositions of the present application can be administered orally or injected into the body. Formulations for oral use can include compounds to further protect IL or DK molecules from proteases in the digestive tract. Injections are usually intramuscular, subcutaneous, intradermal or intravenous. Alternatively, intraarticular injection or other routes can be used in appropriate circumstances. Dual cytokine fusion proteins administered parenterally are preferably formulated in a unit dose injectable form (solution, suspension, emulsion) in association with a pharmaceutical carrier and / or a pharma-ceutically acceptable excipient. In other embodiments, the compositions of the present application can be introduced into the patient's body by an implantable or injectable drug delivery system.

[0043] Those skilled in the art will recognize that adoptive cell therapy (e.g., adoptive T cell therapy) is well known and is carried out according to procedures previously described. See, e.g., U.S. Patent No. 4,690,915. These methods may include autologous transfer (i.e., derived from the patient) or allogenic transfer (i.e., derived from another subject other than the patient to be treated).

[0044] The CAR-T or TCR-T cells are administered by methods known and conventionally practiced by those familiar with adoptive cell therapy. In one embodiment, the administration methods include, but are not limited to, bolus injection, intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, subtenon injection, retrobulbar injection, periocular injection, or posterior juxtascleral delivery. In some embodiments, they are administered parenterally, intrapulmonary, and intranasally, or intralesional or intratumoral administration. Parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In certain embodiments, the recombinantly engineered CAR-T or TCR-T is administered as a single bolus, multiple boluses, or continuous infusion. Commonly known CAR-T therapies include idecabtagene vicleucel, lisocabtagene maraleucel, brexucabtagene autoleucel, tisagenlecleucel, or axicabtagene ciloleucel.

[0045] In one embodiment, the diakine and the CAR-T are administered in separate subsequent periods, e.g., where the diakine (e.g., DK2 10 vegfr2 or DK2 10EGFR or any of those referenced in Table 1 above) is administered prior to administration of recombinantly engineered CAR-T cells. In other embodiments, the diakine and the CAR-T are administered simultaneously. In other embodiments, the diakine is administered 1-3 days prior to the CAR-T treatment and then administered simultaneously with the CAR-T and / or 1-7 days after CAR-T administration. The diakine can be administered once a day or week, or 2-3 times a week, in combination with or with the CAR-T. In another aspect, the diakine is utilized in the expansion and / or thawing procedure of CAR-T cells prior to administration. When reconstituting CAR-T cells from cryopreserved stocks, the CAR-T is typically quiesced in the presence of cytokines beneficial to the CAR-T (e.g., low dose IL-2). In one aspect, the CAR-T cells can be stimulated in the presence of diakine or expanded from cryopreserved stocks. In one aspect, the CAR-T is expanded or stimulated in the presence of 0.001 to 300 ng / mL of diakine, more preferably 0.01 to 200 ng / mL of diakine.

[0046] Similarly, the diakine and the BiTE can be administered at separate subsequent times, where the diakine (e.g., DK2 10 CD20 or DK2 10 EGFR or DK2 10 HER2 or DK2 10The diakine is administered 1-3 days prior to administration of the BiTE (e.g., CD3xCD19 BiTE). In other embodiments, the diakine is administered 1-3 days prior to the BiTE and then administered simultaneously with the BiTE and / or 1-7 days after BiTE administration. The diakine can be administered once a day or week, or 2-3 times a week, in combination with or with the BiTE. BiTEs generally follow the format of a bispecific antibody that fuses anti-CD3 and anti-TAA together. In one embodiment, the method combines IL-10 or IL-4, or half-life extended versions thereof, or IL-10 or IL-4 and diakines including IFN-α, IL-2, IL-7, IL-12, IL-15, IL-21 or IL-27 with a BiTE (e.g., one with bispecificity for CD3 and CD33, BCMA, CD19, CD20, CD22, PSMA, EGFR, DLL3, MUC17, CLDN18, CEA, HER2, HER3, EpCAM, gpA33, GPC3, GD2 5T4, VEGFR2, PDGFR, PDL1, or PD1, to name a few). In a preferred embodiment, the BiTE is anti-CD3 and anti-CD19, anti-CD20, anti-HER2, anti-HER3, anti-PSMA, or anti-BCMA.

[0047] CRS is generally understood to be associated with a concomitant increase in serum IL-6, IL-1b, TNFα, IFNα / γ, IL-12, and IL-23. Although these cytokines are generally associated with CRS, it is unknown whether one, two, or all of these cytokines work together to result in the toxic side effects associated with CRS. We believe that a potential underlying cause of CRS is the fact that many patients with severe CRS induced by BiTE or CAR-T treatment develop vascular leak syndrome. Vascular or capillary leak syndrome is also observed as a primarily dose-limiting, and often fatal, toxicity associated with high-dose IL-2 treatment. Furthermore, we found that treatment of human peripheral blood mononuclear cells (PBMCs) or human whole blood cells with increasing concentrations of IL-2 results in the induction of a series of pro-inflammatory cytokines reminiscent of CRS. Figure 1.

[0048] Without being bound to any particular theory, the inventors believe that there is a similarity between the cytokines induced by treatment with IL-2 alone, and IL-10 (e.g., DK2 10 We believe that the apparent blockade of induction of these secondary cytokines by diakines, including EGFR (EGFR) or IL-4, IL-12, IL-15, IL-7, or any combination of these, suppresses or blocks IL-2-mediated CRS. Addition of tumor cells expressing specific tumor-associated antigens (TAA) to PBMCs with a titration range of anti-TAA:anti-CD3 BiTEs has been shown by others to result in induction of a range of pro-inflammatory cytokines (Fu, 2019). BiTE stimulation also appears to result in an initial induction of TNFα, IL-2, and IL-4, followed by transient induction of other cytokines (Brandl, 2007). Diakines (DK2 10 The levels of IFNγ, IL-6, and TNFα induction from PBMCs in response to exposure to anti-CD3 in the presence of EGFR, IL-10, and IL-2 were significantly higher in DK2 mice than in controls. 10These results suggest that the presence of EGFR, unlike increased concentrations of IL-2, prevents the induction of significant secondary proinflammatory cytokines associated with CRS. 10 Fusion with the high affinity EBV IL-10, also known as EGFR (internally designated DV07, SEQ ID NO:5), prevents IL-2-mediated induction of secondary cytokines both from PBMCs and from anti-CD3 stimulated PBMCs. Figure 2. Linking IL-10 to IL-2 prevents induction of DK2 in mice. 10 We further investigated whether application of EGFR prevents the induction of peripheral cytokines induced by IL-2 alone. 10 Treatment of non-human primates with (EGFR) similarly does not result in significant induction of peripheral plasma cytokines.

[0049] In addition, diakines (e.g., DK2 10 Pre-exposure of CD8+ T cells to EGFR (eg, EGFR) dramatically enhances the cells' subsequent ability to engage BiTEs and result in tumor cell cytolysis (Figure 5). Furthermore, these cells appear to secrete similar levels of IFNγ, but lower levels of TNFα, compared to cells stimulated with BiTEs alone (Figure 6). Thus, we propose that the molecular circuitry responsible for BiTE- or CAR-T-mediated CRS is a cascade of pro-inflammatory cytokine release that is first triggered by CD4+ T cell engagement with BiTEs or CAR-T, and subsequently induces monocytes / macrophages to induce IL-2 secretion and undergo further secretion of pro-inflammatory cytokines (Figure 7). EXAMPLES

[0050] Example 1: Diakine enhances BiTE-mediated tumor cell cytolysis DK2 10 (EGFR) has previously been shown to stimulate CD8+ T cells for subsequent tumor cell lysis in vitro and in vivo. See U.S. Patent No. 11,292,822, in which DK210 (EGFR), which is a representative example of a diakine, has been shown to both enhance BiTE-mediated tumor cell cytolysis and suppress BiTE-mediated CRS.

[0051] First, DK2 10 (EGFR) has been shown to enhance BiTE-mediated tumor cell cytotoxicity. In this in vitro model, DK2 10 The combined antitumor efficacy of (EGFR) and CD19 BiTEs has been evaluated upon multiple exposure to target tumor cells.

[0052] CD8+ T cells are isolated from fresh donor leukopaks via magnetic bead isolation according to the manufacturer's suggested protocol (Miltenyi). The isolated CD8+ T cells were collected at a concentration of 2.5×10 6 Cells were plated at 100 cells / well and incubated with various concentrations (0 or 100 ng / mL) of DK2 in AIMV for 2 days. 10 (EGFR) and various concentrations of DK2 10 After 2 days of exposure to (EGFR), CD8+ T cells were harvested, counted, washed, and finally incubated with the corresponding concentration of DK2. 10 The CD8+ cells (effectors) and Raji-GFP cells (targets) are then combined at a 10:1 effector to target ratio. The effector and target cell mixtures (which express CD19 BiTE alone, DK2 ... 10 (EGFR) alone or CD19 BiTE and DK2 10(EGFR) combinations) were monitored over a 5-day period using the IncuCyte® S3 Live-Cell Analysis System (Essen Bioscience / Sartorius). Additionally, additional plates were seeded under the same conditions as described above and used for subsequent successive rounds of cytotoxicity assays. Every 3 days, medium was aspirated from the wells and the appropriate concentration of DK2 was added. 10 Fresh medium containing either (EGFR), CD19 BiTE, or a combination of the two is added to the wells. After 5 days of exposure, cells are harvested, counted, washed, and re-exposed to conditions similar to those described above. The percentage of (GFP) loss is measured as an index of cytotoxicity.

[0053] DK2 10 CD3xCD19 BiTE when combined with (EGFR) enhances tumor cell cytotoxicity. See Figures 5 and 8. CD3XCD19 BiTE and DK2 10 (EGFR) are tested both alone and in combination using CD8+ T cells from normal healthy human donors. Figure 8. Effector cells were subjected to multiple exposures (five consecutive cell lysis) to target tumor cells (Raji-GFP) and cytotoxicity was measured via loss of GFP. DK2 10 In vitro treatment with BiTEs in combination with (EGFR) inhibited DK2 10 T cell activation at EGFR induces CD8+ T cell anti-Raji GFP+ When assessing response, it is suggested to enhance the response to BITE.

[0054] Example 2: Diakine reduces BiTE-mediated CRS One of the current clinical challenges with BiTE is the marked induction of CRS (Zhou, 2021). 10(EGFR) treatment (see Figures 1, 3, and 4) appears to prevent IL-2-mediated CRS, and therefore 100 ng / mL DK2 10 PBMCs with or without (EGFR), Raji GFP+ DK2 cells, using in vitro culture with 0.1ng / mL CD19 BiTE 10 To determine whether (EGFR) can suppress BiTE-mediated CRS.

[0055] PBMCs are isolated from leukocyte packs collected from healthy donors using Ficoll density gradient method. Equal volumes of HBSS and donor samples are transferred individually to conical tubes. Ficoll is slowly added to create a bottom layer, and the samples are then centrifuged at 400×g for 30 min at 25° C. PBMCs are harvested from the top layer and then washed twice using Aim V medium (300×g for 8 min). The isolated PBMCs were collected at 2×10 6 DK2 at various concentrations (0 or 100 ng / mL) in AIMV was incubated with 100 mL of 100 mL of 100% 100% 15 ... 10 The Raji tumor cells were counted, washed, and transfected with AIMV into DK2 cells and incubated for 2 days (37° C., 5% CO2). 10 Cells were resuspended in various concentrations of CD19 BiTE (0, or 0.1 ng / mL final) with or without (EGFR) (100 ng / mL) and stimulated for 2 days. PBMCs (assuming 10% CD8+ T cells (effector)) and Raji cells (target) were combined at a 10:1 effector to target ratio. After 24 hours of incubation, supernatants were harvested and cytokine secretion was measured via multiplexed capture assay (MSD) and ELISA.

[0056] As illustrated in FIG. 9, the data show that DK2 10These results suggest that the presence of (EGFR) significantly promotes tumor cell lysis (induction of IFN-γ, granzyme B, and perforin) while limiting the induction of BiTE-mediated CRS at non-functional concentrations of BiTE (0.1 ng / mL). Data shown are taken 1 to 24 hours after exposure. Longitudinal data show a reduction in CRS over time in these conditions. FIG. 10.

[0057] DK2 10 PBMC + Raji affected by (EGFR) GFP+ To better understand the cell types present in the cultures, we assessed 48-h cultures by intracellular fluorescence-activated cell sorting (FACS). 10 These results suggest that the presence of EGFR polarizes both CD4+ and CD8+ T cells to express primarily IFN-γ, while reducing TNF-α and IL-2 production. MHC II positive cells, designated as antigen presenting cells (APCs), also showed reduced TNF-α, IL-6 and IL-1β production, which is consistent with the DK2 10 These results suggest that (EGFR) mediates the multifaceted cell type regulation of CRS associated with BiTE-mediated T cell activation. Figure 10. [ka]

Claims

1. 1. A composition for reducing the severity of bispecific T cell engager (BiTE) or chimeric antigen receptor T cell (CAR-T)-induced cytokine release syndrome (CRS), the composition comprising: (i) a certain amount of Formula II NH 2 -(Y)-(X 1 )-(Z n )-(X 2 )-(Y)-COOH (Formula II) A dual cytokine fusion protein of where "Y" is a monomer of IL10 comprising the sequence of SEQ ID NO: 5 or a variant thereof; "X 1 " is a VL or VH region derived from the first monoclonal antibody; "X2" is a VH or VL region derived from said first monoclonal antibody; wherein when X 1 is VL, X 2 is VH, or when X 1 is VH, X 2 is VL; the first monoclonal antibody is an anti-Ebola antibody; the VL and VH derived from the anti-Ebola antibody comprise three light chain CDRs and three heavy chain CDRs grafted with three light chain CDRs and three heavy chain CDRs derived from an anti-EGFR monoclonal antibody; "Z" is IL-2; a dual cytokine fusion protein, wherein "n" is the integer 1; (ii) an amount of a bispecific antibody or a CAR-T, wherein the bispecific antibody comprises specificity for CD3 and a tumor-associated antigen; wherein administering reduces the induction of the pro-inflammatory cytokines IL-1β, IL-6, TNFα, IL-12, IFNα, IFNγ, IL-23, or any combination thereof.

2. 10. The composition of claim 1, wherein the dual cytokine fusion protein further comprises an scFv targeting domain that targets a receptor different from the bispecific antibody or CAR-T.

3. 10. The composition of claim 1, wherein the bispecific antibody or CAR-T therapy targets a hematological or solid tumor.

4. The bispecific antibody or CAR-T may be selected from the group consisting of TNFRSF17, IL3RA, SDC1, CD5, CD19, CD20, CD22, CD23, CD33, CD38, CD44, CD70, CD133, CD174, CD274, CD276, CEACAM6, GFRA1, ITGB6, MS4A1, TNFRSF8, NCAM1, ULBP1, ULBP2, IL1RAP, CEACAM5 (CEA), MET, EGFR, EGFRvIII, ENPP1, and DLL3. , CLDN18, BCAM, PSMA, MUC17, HER2, HER3, FGFR4, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Mucl, PDCD1, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, SLAMF7, GD2, 5T4, PSCA, GPNMB, CSPG4, PDGFR, PDL1, PD1, or TEM1.

5. 2. The composition of claim 1, wherein the CAR-T therapy is idecbutagen biculeucel, lisocabtagene malareucel, brexcabtagene outrucel, tisagen luculeucel, or axicabtagene ciloreucel.

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