Dual cytokine fusions including IL-10 and adoptive cell therapy or bispecific T cell engagers to treat cancer

Abstract

The present application relates to a method of treating cancer or tumors comprising administering a dual cytokine fusion protein composition, pharmaceutical composition thereof, and / or formulation thereof comprising a single chain variable fragment scaffold system and an IL-10 or IL-10 variant molecule fused to a second cytokine, in combination with adoptive cell therapy or bispecific T cell engager, wherein the second cytokine is linked at the hinge region of the scFv. The present disclosure relates to the field of biotechnology, and more specifically to the use of novel dual cytokine fusion proteins called Diakines comprising interleukin-10 ("IL-10") and interleukin-2 ("IL-2") in combination with conventional adoptive cell therapy ("ACT") or bispecific T cell engager ("BiTE") therapy to treat cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 265,521, filed December 16, 2021, the contents of which are incorporated by reference herein in their entirety.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (039451-00085-Sequence-Listing.xml; size: 107,499 bytes; and creation date: December 16, 2022) are incorporated herein by reference in their entirety.

[0003] FIELD OF THEINVENTION The present disclosure relates to the field of biotechnology, and more specifically, to the use of novel dual cytokine fusion proteins, called Diakines, comprising interleukin-10 ("IL-10") and interleukin-2 ("IL-2"), in combination with conventional adoptive cellular therapy ("ACT") or bispecific T cell engager ("BiTE") therapy to treat cancer. [Background technology]

[0004] Introduction IL-10, commonly termed an inhibitor of cytokine synthesis (Malefyt, Interleukin 10 inhibits cytokine synthesis by human monocytes: An autoregulatory role of IL-10 produced by monocytes, 1991), has been shown to suppress inflammatory responses (Fedorak, 2000) and, more recently, to inhibit CD8 T cells that induce interferon-γ ("IFNγ")-dependent antitumor immune responses. +IL-10 is a pleiotropic cytokine known for both activating and suppressing T cells (Mumm, 2011). IL-10 is a non-covalent homodimeric cytokine with structural similarity to IFNγ. IL-10 binds to the IL-10 receptor, which consists of two subunits, IL10 receptor 1 (IL10R1) and IL-10 receptor 2 (IL10R2) (Moore, 2001). The IL-10 receptor complex is expressed on the surface of most hematopoietic cells, with the highest expression in macrophages and T cells. IL-10 has been reported to be both an immunosuppressive (Schreiber, 2000) and immune stimulatory (Mumm, 2011) cytokine, but clinical evaluation of IL-10 treatment of Crohn's patients has resulted in an inverse dose response (Fedorak, 2000; Schreiber, 2000), while treatment of cancer patients with PEGylated IL-10 has resulted in a potent, dose-titrable antitumor response (Naing, 2018). PEGylated IL-10 antitumor responses require endogenous CD8+ T cells and IFNγ (Mumm, 2011). Treatment of tumor-bearing animals with PEGylated IL-10 results in an increase in intratumoral CD8+ T cells and an increase in IFNγ per cell. Most recently, however, cancer patients treated with PEGylated IL-10 have resulted in evidence of immune stimulation but not an increase in antitumor responses (Spigel, 2020).

[0005] Interleukin-2 ("IL-2") is a four-helix bundle pleiotropic cytokine known to induce antitumor immune responses (Jiang, 2016), but also acts as a cytotoxic antagonist to natural killer ("NK") cells and CD4 +It also exhibits high toxicity due to unregulated activation and secretion of IFNγ by T cells and the expansion of T regulatory cells (Chinen, 2016). For this reason, many groups have attempted to mutate IL-2 to reduce its binding to high affinity receptors, resulting in reduced IL-2 toxicity (Chen, 2018). These muteins have not met with substantial clinical success (Bentebibe, 2019), suggesting that other mechanisms need to be employed to reduce the lethal toxicity potential of IL-2.

[0006] IL-10 inhibits NK and CD4 + It has been reported that IL-2, which drives the production of IFNγ secreted by both CD8 T cells and IL-2-induced CD8 T cells, is suppressed (Scott, 2006). + It has also been reported to act as a cofactor for T cell proliferation (Groux, 1998). Thus, it is not known whether IL-2 and IL-10 coactivate cells of the immune system or counteract each other.

[0007] Surprisingly, it was discovered that Epstein-Barr Virus ("EBV") IL-10 variants having one or more amino acid substitutions in the key IL-10 receptor binding domain region (at amino acid positions 31, 75, or both of the mature EBV IL-10 amino acid sequence of SEQ ID NO:3) altered the ability of EBV IL-10 to bind to and activate the IL-10 receptor. These modifications included the ability to increase the affinity of EBV IL-10 for the IL-10 receptor. The inventors discovered that EBV IL-10 variant molecules act as IL-10 receptor agonists, capable of treating immune diseases, inflammatory diseases or conditions, and treating cancer. The high affinity EBV IL-10 variant (designated "DV07" and containing two amino acid substitutions at positions 31 and 75) when incorporated as a monomer into a single chain variable domain (scFv) scaffold system containing non-immunogenic variable heavy ("VH") and variable light ("VL") domains resulted in a half-life extended molecule that folded properly and remained functionally active. See U.S. Patent Nos. 10,858,412; 10975,133; 10,981,965; 10,975,134; and 10,981,966, each of which is incorporated herein by reference in its entirety, and see also FIG. 1 for a representative schematic. The EBV IL-10 variant incorporated into the scaffold system was shown to be capable of targeting inflammatory cells (e.g., monocytes / macrophages / dendritic cells) and immune cells (e.g., CD8 +In one iteration, the scFv scaffold system utilized VH and VL regions originally from a human anti-Ebola antibody. The single cytokine fusion protein was designated DeboDV07. Id. The inventors also found that when six complementarity determining regions ("CDRs") from the VH and VL regions of an scFv originally from a human anti-Ebola antibody were grafted with six CDRs from antibodies that recognize different tumor associated antigens ("TAA"), e.g., anti-EGFR, anti-VEGFR1, anti-VEGFR2, and anti-HER2 antibodies (i.e., replacing the six CDRs of one antibody with the six CDRs from a second antibody), IL-10 functionality was not destroyed. See U.S. Patent Nos. 10,858,412; 10,975,133; 10,981,965; 10,975,134; and 10,981,966.

[0008] The inventors have improved DeboDV07 by incorporating a second cytokine into a single cytokine fusion protein (described above; see also co-pending US Application No. 17 / 199,239, filed March 11, 2021, which is incorporated herein by reference in its entirety). Specifically, the second cytokine is incorporated into the linker region between the VH and VL regions of the scFv. See, for example, FIG. 2, supra (representative schematic of a dual cytokine fusion protein). The dual cytokine fusion protein, when grafted with CDRs from different TAA-targeting monoclonal antibodies, can deliver both IL-10 and another cytokine (such as, but not limited to, IL-2) to a specific TAA. In this application, the inventors describe the novel use of dual cytokine fusion proteins known as Diakines™ ("DK") in support of adoptive cell therapy ("ACT"), including but not limited to other known immunotherapy modalities such as chimeric antigen receptor T cells ("CAR-T"), engineered T cell receptor T cells ("TCR-T"), natural killer ("NK") cells and / or tumor infiltrating lymphocytes ("TIL"), and bispecific T cell engagers ("BiTEs"). As used in this application, the term "diakine" or "diakines" is a generic term referring to a novel class of dual cytokine fusion proteins linked together in a targeted, half-life extending scFv.

[0009] Regarding CAR-T cell therapy, this therapy has shown great success in hematological malignancies, but there are limited comparable examples when using these CAR-T cells in solid tissue tumors (Ma, 2019; Castellarin, 2018; Wagner, 2020).

[0010] Challenges with current approaches appear to share similarities with most immune stimulatory therapies. Specifically, challenges include off-target toxicity (Bonifant, 2016; Bianca Santomasso, 2019), CAR-T cell persistence (Jafarzadeh, 2020; Christopher DeRenzo, 2019), and the ability to infiltrate the tumor microenvironment ("TME") (Rodriguez-Garcia, 2020; Zou, 2019). Both IL-2 (Groux, 1998; Ross, 2018) and IL-10 (Berman, 1996; Chan, 2015; Naing A., 2018; Naing A., Safety, Antitumor Activity, and Immune Activation of Pegylated Recombinant Human Interleukin-10 (AM0010) in Patients With Advanced Solid Tumors, 2016; Emmerich, 2012; Mumm J., 2011) specifically enhance antitumor T cell function. However, while PEGylated IL-10 has been reported to enhance CAR-T function (McCauley, 2018), the use of IL-2 in combination with CAR-T cell therapy is challenging, mainly due to IL-2-associated toxicity (Tang, 2018). Thus, in one aspect, the present application provides: (1) a method for controlling IL-2-mediated toxicity, comprising administering to the patient a therapeutically effective amount of DK2 10 This problem is overcome by using diakines that contain both IL2 and IL10, (2) IL12 and IL10, (3) IL7 and IL10, or (4) IL15 and IL-10, referred to as DK2, to name a few, by concentrating coupled IL-2 and IL10 in the tumor vasculature via targeting TAAs, e.g., against EGFR2, HER2, or VEGFR2 (Smith, 2010). 10Both tumor-specific activation of CAR-T and direct suppression of cytokine release syndrome and IL-2 toxicity by DV07 drive TME activation of CAR-T cells, allowing activation, infiltration and persistence while limiting toxicity. The same premise also seems promising for ACT therapy and BiTE. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Pat. No. 10,858,412 [Patent Document 2] U.S. Pat. No. 10,975,133 [Patent Document 3] U.S. Pat. No. 10,981,965 [Patent Document 4] U.S. Pat. No. 10,975,134 [Patent Document 5] U.S. Pat. No. 10,981,966 [Patent Document 6] U.S. Pat. No. 10,858,412 [Patent Document 7] U.S. Pat. No. 10,975,133 [Patent Document 8] U.S. Pat. No. 10,981,965 [Patent Document 9] U.S. Pat. No. 10,975,134 [Patent Document 10] U.S. Pat. No. 10,981,966 [Non-patent literature]

[0012] [Non-Patent Document 1] Malefyt, Interleukin 10 inhbits cytokine synthesis by human monocytes: An autoregulatory role of IL-10 produced by monocytes, 1991 [Non-Patent Document 2] Naing A. , Safety, Antitumor Activity, and Immune Activation of Pegylated Recombinant Human Interleukin-10 (AM0010) in Patients With Advanced Solid Tumors, 2016 Summary of the Invention [Means for solving the problem]

[0013] Summary of Various Aspects of the Invention The present disclosure relates generally to methods of using a dual cytokine fusion protein, termed Diakine, in combination with conventional immunotherapy.

[0014] Thus, in a first aspect, the present disclosure relates to a method of using a diakin comprising IL-10 or various IL-10 variants, a half-life extension targeting domain, and a second cytokine in combination with conventional immunotherapy, including, but not limited to, engineered immune cells (e.g., CAR-T cells, TCR-T cells, TIL or NK cells) or BiTEs. In certain embodiments, the method uses a diakin comprising IL-10, such as, but not limited to, human, murine, cytomegalovirus ("CMV") or EBV IL-10 forms, or an IL-10 variant molecule thereof, where the IL-10 variant has one or more amino acid substitutions that affect the IL-10 receptor binding domain. In certain embodiments, the method uses a diakin comprising IL-10, IL-12 or IL-27, or a variant thereof. Each of the aforementioned diakin types, including IL-10, IL-12 or IL-27, also includes a second cytokine, which is a different cytokine from the first cytokine, and acts in tandem with IL-10, IL-12 or IL-27, or a variant thereof, such that the first and second cytokines are coupled and have an additive or synergistic effect when targeted together to a specific antigen by the antigen targeting domain of the diakin that extends the half-life. These second cytokines include, among others, 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, IFN-α, IFN-β, IFN-γ, TGF-β, or TNF-α, TNF-β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13, preferably IL-2. The antigen targeting domain of the diakin comprises a targeting domain selected from an antibody, an antibody fragment (e.g., scFv, antigen-binding fragment), or an antigen-binding portion that directs the diakin to the target antigen recognized by the variable heavy (VH) and variable light (VL) regions of the antibody, antibody fragment, or antigen-binding portion thereof. In certain embodiments, the antigen targeting domain is an scFv.In certain aspects, the scFv has specificity for a tumor-associated antigen (TAA), and the TAA is selected from various antigenic targets found on the surface of solid or hematological tumors. In one aspect, the antigen targeting domain is an scFv that comprises three CDRs in the VH region and three CDRs in the VL region of the scFv. In another aspect, the scFv retains the original VH and VL framework regions, but may be grafted with three CDRs in the VH and three CDRs in the VL from another antibody. In other embodiments, the engineered cell comprises a recombinant antigen receptor, such as, but not limited to, a CAR, a T cell receptor ("TCR") or a functional non-TCR, preferably a CAR that specifically targets a tumor-associated antigen (TAA). In certain embodiments, the engineered cell is a T cell.

[0015] In yet another aspect, the disclosure provides a method of using a diaquine of formula (I) in combination with a TAA that targets an engineered immune cell or a BiTE, wherein formula (I) is NH2-(IL10)-(X 1 )-(Z n )-(X 2 )-(IL10)-COOH (Formula I); (In the formula, "IL10" is a monomer of IL-10, wherein the IL-10 is human, mouse, CMV or EBV IL-10, or a variant thereof, more preferably, the IL10 is a monomer comprising a sequence selected from SEQ ID NO: 1, 3, 7, 9 or 10; "X 1 " is a VL or VH region obtained from a first monoclonal antibody; "X 2 " is a VH or VL region obtained from a first monoclonal antibody; 1 If VL, then X 2 is VH or X 1 If VH, then X 2is a VL, preferably the VH and VL regions are scFvs obtained from a human anti-Ebola antibody, the VH and VL regions being grafted with six CDRs (three from the VH and three from the VL) from a second antibody; "Z" is any cytokine other than IL-10, preferably 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, IFN-α, IFN-β, IFN-γ, TGF-β, or TNF-α, TNF-β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13; "n" is an integer selected from 1 to 2. and; The second antibody is targeted to VEGFR2, CD3, CD4, CD5, CD7, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD44, CD52, CD56, CD70, CD79B, CD117, CD123, CD138, CD147, IL-22R1, B-cell mutant antigen (BCMA), C-type lectin-like molecule 1 (CLL01), CD5, CD147, latent membrane protein 1 (LMP-1), signal transduction phosphokinase (SEQ ID NO: 1), and IL-22R2. serotype 1, serotype 2, serotype 3, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11, serotype 12, serotype 13, serotype 14, serotype 15, serotype 16, serotype 17, serotype 18, serotype 19, serotype 20, serotype 21, serotype 22, serotype 23, serotype 24, serotype 25, serotype 26, serotype 27, serotype 28, serotype 29, serotype 30, serotype 31, serotype 32, serotype 33, serotype 34, serotype 35, serotype 36, serotype 37, serotype 38, serotype 39, serotype 40, serotype 41, serotype 42, serotype 43, serotype 44, serotype 45, serotype 46, serotype 47, serotype 48, serotype 49, serotype 49, serotype 49, serotype 49, serotype 49, serotype 49, serotype 41, serotype 42, serotype 43, serotype 44, serotype 45, serotype 46, serotype 47, serotype 48, serotype 49, serotype 49, serotype 49, It concerns the method.

[0016] In yet another aspect, the disclosure relates to methods of using the IL-10 diakines of formula (II) in combination with TAAs that target immune cells or BiTEs. NH2-(IL10)-(L)-(X 1 )-(L)-(Z n )-(L)-(X 2)-(L)-(IL10)-COOH (formula II); (In the formula, "IL-10" is a monomeric sequence selected from SEQ ID NOs: 1, 3, 7, 9 or 10; "L" is an optional linker, more preferably the linker is selected from SEQ ID NOs: 39, 40 or 41; "X 1 " is a VL or VH region obtained from a first monoclonal antibody; "X 2 " is a VH or VL region obtained from a first monoclonal antibody; 1 If VL, then X 2 is VH or X 1 If VH, then X 2 is a VL, preferably the VH and VL regions are scFvs obtained from a human anti-Ebola antibody, and the VH and VL regions are grafted with six CDRs (three from the VH and three from the VL) from a second antibody, the second antibody being one of the following CDRs: VEGFR2, CD3, CD4, CD5, CD7, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD44, CD52, CD56, CD70, CD79B, CD117, CD1 23, CD138, CD147, IL-22R1, BCMA, CLL01, CD5, CD147, lLMP-1, SLAMF7, NY-ESO-1, TACI, CS-1, CXCR4, NKG2D, B7-H3, EGFR, PD-1, P DL-1, HER2, HER3, EpCAM, PSCA, MUC1, Lewis Y, GPC3, AXL, Claudin 18.2, GD2, CTLA-4, CEA, PDGFR, MESO, PSCA, PSMA, BCMA, or PSA; "Z" stands for 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, I a cytokine selected from FN-α, IFN-β, IFN-γ, TGF-β, or TNF-α, TNF-β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13, preferably IL-2; "n" is an integer selected from 1 to 2.

[0017] In another aspect, the present disclosure provides a method of treating cancer, comprising administering an effective amount of Diakine, preferably DK2, in combination with an engineered immune cell, preferably CAR-T therapy or BiTE. 10 , DK7 10 , DK12 10 , DK15 10 DK21 10 , DK27 10 , DKIFNa 10 to a subject in need thereof, 10 , DK7 10 , DK12 10 , DK15 10 DK21 10 , DK27 10 or DKIFNa 10 But VEGFR2, CD3, CD4, CD5, CD7, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD44, CD52, CD56, CD70, CD79B , CD117, CD123, CD138, CD147, IL-22R1, BCMA, CLL01, CD5, CD147, lLMP-1, SLAMF7, NY-ESO-1, TACI, CS-1, CXCR4, NK The present invention relates to methods comprising an anti-Ebola scaffold system (including a VH / VL pair or scFv) grafted with CDRs from an antibody having specificity for G2D, B7-H3, EGFR, PD-1, PDL-1, HER2, HER3, EpCAM, PSCA, MUC1, Lewis Y, GPC3, AXL, claudin 18.2, GD2, CTLA-4, CEA, PDGFR, MESO, PSCA, PSMA, BCMA, or PSA.

[0018] In another aspect, the present disclosure relates to methods of using the IL-12 diakin of formula (III) in combination with engineered immune cells or BiTEs that target TAA.

[0019] NH2-(R 1 )-(X 1 )-(Zn )-(X 2 )-(R 2 )-COOH (formula IIIa);

[0020] NH2-(R 2 )-(X 1 )-(Z n )-(X 2 )-(R 1 )-COOH (formula IIIb); (In the formula, "R 1 " is an alpha subunit from any multi-subunit first cytokine, preferably either the IL-12-alpha subunit (p35) or the IL-27 alpha subunit (p28), more preferably the subunit of SEQ ID NO: 45 or 47; "R 2 " is a beta subunit from any multi-subunit first cytokine, preferably either the IL-12-beta subunit (p40) or the IL-27 beta subunit (EBI3), more preferably the subunit of SEQ ID NO: 46 or 48; Here, R 1 is the alpha subunit of the first cytokine, R 2 is the beta subunit of the first cytokine; or R 1 If p35, then R 2 is p40; or R 1 If p28, then R 2 is EBI3; or R 1 is SEQ ID NO: 45 or 47, then R 2 is SEQ ID NO: 46 or 48; or R 1 is SEQ ID NO: 46 or 48, then R 2 is SEQ ID NO: 45 or 47; "X 1 " is a VL or VH region obtained from a first monoclonal antibody; "X 2 " is a VH or VL region obtained from a first monoclonal antibody; 1 If VL, then X2 is VH or X 1 If VH, then X 2 is VL; "Z" is any cytokine that enhances the biological function of a multisubunit cytokine, preferably IFN-2a, IL-28, IL-29; "n" is an integer selected from 1 to 2, The first monoclonal antibody is selected from the group consisting of VEGFR2, CD3, CD4, CD5, CD7, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD44, CD52, CD56, CD70, CD79B, CD117, CD123, CD138, CD147, IL-22R1, BCMA, CLL01, CD5, CD147, lLMP-1, SLAMF7, NY-ESO-1, an anti-Ebola antibody grafted with CDRs from a second antibody having specificity for TACI, CS-1, CXCR4, NKG2D, B7-H3, EGFR, PD-1, PDL-1, HER2, HER3, EpCAM, PSCA, MUC1, Lewis Y, GPC3, AXL, claudin 18.2, GD2, CTLA-4, CEA, PDGFR, MESO, PSCA, PSMA, BCMA, or PSA).

[0021] In yet another aspect, the disclosure provides a method of using a diakin having two multisubunit cytokines of formula (IV), such as IL12, IL-27, or IL-10, in combination with an engineered immune cell or BiTE that targets a TAA, wherein the diakin has formula (IV): NH2-(R 1 )-(L a )-(X 1 )-(L a )-(W 1 )-(L b )-(W 2 )-(L a )-(X 2 )-(L a )-(R 2 )-COOH (formula IV); (In the formula, "R 1" is the alpha subunit of a first cytokine, e.g., IL-12 or IL-27, or a first monomer of a homodimeric cytokine, e.g., IL-10, where R 1 is preferably p40; "R 2 " is a beta alpha subunit of a first cytokine, e.g., IL-12 or IL-27, or a second monomer of a homodimeric cytokine, e.g., IL-10, where R 2 is preferably p35; "L a " is an optional linker; preferably, SEQ ID NO: 43-44; "L b " is an optional linker; preferably GGGSGGG or SEQ ID NO:43; "X 1 " is a VL or VH region obtained from a first monoclonal antibody; "X 2 " is a VH or VL region obtained from a first monoclonal antibody; 1 If VL, then X 2 is VH or X 1 If VH, then X 2 is VL; "W 1 " is a first cytokine, e.g., the alpha subunit of IL-12 or IL-27, or a first monomer of a homodimeric cytokine, e.g., IL-10, preferably a first monomer of IL-10; "W 2 " is a first cytokine, e.g., a beta alpha subunit of IL-12 or IL-27, or a second monomer of a homodimeric cytokine, e.g., IL-10, preferably a second monomer of IL-10; The first monoclonal antibody is directed against VEGFR2, CD3, CD4, CD5, CD7, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD44, CD52, CD56, CD70, CD79B, CD117, CD123, CD138, CD147, IL-22R1, BCMA, CLL01, CD5, CD147, lLMP-1, SLAMF7, NY- grafted with CDRs from antibodies with specificity for ESO-1, TACI, CS-1, CXCR4, NKG2D, B7-H3, EGFR, PD-1, PDL-1, HER2, HER3, EpCAM, PSCA, MUC1, Lewis Y, GPC3, AXL, claudin 18.2, GD2, CTLA-4, CEA, PDGFR, MESO, PSCA, PSMA, BCMA, or PSA) This relates to a method.

[0022] The above simplified summary of representative embodiments serves to provide a basic understanding of the present disclosure. This summary is not a detailed overview of all contemplated embodiments, nor is it intended to identify key or critical elements of all embodiments, nor is it intended to delineate the scope of any or all embodiments of the present disclosure. Its sole purpose is to present one or more embodiments 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 embodiments of the present disclosure include the features recited in the claims and the exemplified examples pointed out therein. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 is a schematic diagram of a single embodiment of a first generation IL-10 fusion protein, which is a cytokine fusion protein previously described in US Pat. No. 10,858,412.

[0024] [Diagram 2]FIG. 2 is a schematic diagram of a diakinin embodied in the present disclosure, in which a dual cytokine fusion protein comprises an IL-10 monomer (or an IL10 variant) linked at its terminus, and a second cytokine is incorporated into the linker between the VH and VL of the scFv.

[0025] [Diagram 3] FIG. 3 is a schematic diagram of a diakinin embodied in the present disclosure, where the dual cytokine fusion protein contains two multisubunit cytokines, one linked at the terminus (e.g., IL-12 or IL-27) and the other fused between the linker region of an scFv (e.g., two IL-10 monomers, or an IL-10 variant thereof).

[0026] [Figure 4] FIG. 4 is a schematic diagram of a proposed mechanism utilizing DK210 grafted with CDRs targeting, for example, VEGFR2.

[0027] [Diagram 5] Figures 5A-5F are grafts demonstrating cytokine induction of IL-1β, IFN-γ, TNF-α, IL-12p70, IFNα2a and IL-6 in naive PBMCs from healthy donors in response to Diakine (DK210), IL10 or IL2.

[0028] [Figure 6] 6A-6D are grafts demonstrating cytokine induction of IL-4, IL-17, IL-8 and GM-CSF in naive PBMCs from healthy donors in response to Diakine (DK210), IL-10 or IL-2.

[0029] [Figure 7] FIG. 7. Assessment of granzyme B levels in CD8+ T cells in response to increasing concentrations of diaquine in response to anti-CD3 stimulation at 24, 48 and 72 hours.

[0030] [Figure 8] FIG. 8 is an assessment of IFN-γ levels in CD8+ T cells in response to increasing concentrations of Diaquine in response to anti-CD3 stimulation at 24, 48 and 72 hours.

[0031] [Figure 9] FIG. 9 is an assessment of TNF-α levels in CD8+ T cells in response to increasing concentrations of Diaquine in response to anti-CD3 stimulation at 24, 48 and 72 hours.

[0032] [Figure 10] Figures 10A and 10B show the effect of combining Diakine (DK210CD19) with CAR T cells (CD20 CAR T) in reducing target tumor cells (Raji), where CD8+ T cells are primed in the presence of Diakine for 1 day. Figure 10A shows an effector to target ratio of 3:1, and Figure 10B shows an effector to target ratio of 1:3.

[0033] [Figure 11] Figures 11A-11F show the effect of combining Diakin (DK210egfr) with BiTEs (0.01 ng / mL CD3xCD19 BiTEs) in reducing target tumor cells (Raji), where CD8+ T cells are primed in the presence of Diakin for 2 days. Figures 11A-11D provide cytokine secretion levels of TNF-alpha, IFN-gamma, Granzyme B and Perforin of CD8+ T cells in the presence of DK210 and CD19 BiTEs. Figures 11E and 11F provide cytolysis profiles of CD8+ T cells when combined with DK210 and CD19 BiTEs.

[0034] [Figure 12]Figures 12A-12F show the effect of combining Diakine (DK210CD19) with BiTEs (0.1 ng / mL CD3xCD20 BiTEs) in reducing target tumor cells (Raji), where CD8+ T cells are primed in the presence of Diakine for 3 days. Figures 12A-12D provide cytokine secretion levels of TNF-alpha, IFN-gamma, Granzyme B and Perforin of CD8+ T cells in the presence of DK210 and CD20 BiTEs. Figures 12E and 12F provide cytolysis profiles of CD8+ T cells when combined with DK210 and CD20 BiTEs.

[0035] [Figure 13] Figure 13 shows the effect of combining Diakine (DK710) with CD19 BiTE. The data provides a comparison of control (i.e., no DK710, no CD19 BiTE), DK710 alone, CD19 BiTE alone, and the combination of DK710 and CD19 BiTE. The data shows that the combination of DK710 and CD19 BiTE has enhanced cell lysis relative to CD19 BiTE.

[0036] [Figure 14] Figure 14 shows the effect of combining Diakine (DK1210) with CD19 BiTE. The data provides a comparison of control (i.e., no DK1210, no CD19 BiTE), DK1210 alone, CD19 BiTE alone, and the combination of DK1210 and CD19 BiTE. The data shows that the combination of DK1210 and CD19 BiTE has enhanced cell lysis relative to CD19 BiTE. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description Exemplary embodiments include a class of dual cytokine fusion proteins referred to herein as diakines, whereby the diakines comprise IL-10, IL-12 or IL-27, and a method of treating cancer comprising administering IL-10 and IL-2 (DK27) in combination with engineered immune cells or ACTs (e.g., CAR T, TCT T, TIL or NK) or BiTEs that target tumor-associated antigens (TAA). 10 ), or IL-10 and IL-7 (DK7 10 ), IL-10 and IL-12 (DK12 10 ), IL-10 and IL-15 (DK15 10 ), IL-10 and IL-21 (DK21 10 ), IL-10 and IFN-gamma (DKIFNa 10 ), or IL-10 and IL-27 (DK27 10 The present invention is described in the context of a method comprising administering a diaquinone containing a diaquinone derivative to a patient having a pulmonary circulation. Those skilled in the art will appreciate that the following description is merely illustrative and is not intended to be limiting in any way. Other embodiments will readily suggest themselves to those skilled in the art, having the benefit of this disclosure. Reference will now be made in detail to the practice of the exemplary embodiments as set forth in the accompanying disclosure and drawings. The same reference labels will be used, to the extent possible, throughout the drawings and the following description to refer to the same or similar items.

[0038] 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.

[0039] Unless otherwise indicated, the embodiments described herein employ conventional methods and techniques of molecular biology, biochemistry, pharmacology, chemistry and immunology well known to those skilled in the art. Many of the general techniques for designing and producing IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, IFN-alpha, or IL-27 variants, including but not limited to assays for testing human, mouse, CMV and / or EBV forms of IL-10, and IL-10 variants, aglycosylated or deglycosylated forms of each of the aforementioned cytokines and their variants, are accomplished by known methods readily available in the art and detailed. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Handbook of Experimental Immunology, Vols. I-IV (DM Weir and CC Blackwell eds., Blackwell Scientific Publications); AL Lehninger, Biochemistry (Worth Publishers, Inc., current addition). N-terminal aldehyde based PEGylation chemistries are also well known in the art. definition

[0040] The following terms are used to describe the various embodiments discussed herein and are intended to be defined as set forth below.

[0041] As used herein, in describing various embodiments, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0042] The term "about" refers to a deviation of 0.0001-5% of the indicated number or range of numbers. In one embodiment, the term "about" refers to a deviation of 1-10% of the indicated number or range of numbers. In one embodiment, the term "about" refers to a deviation of up to 25% of the indicated number or range of numbers. In a more specific embodiment, the term "about" refers to a difference of 1-25% in terms of nucleotide or amino acid sequence homology when compared to the wild-type sequence.

[0043] The term "interleukin-10" or "IL-10" refers to a protein comprising two identical subunits non-covalently linked to form a homodimer, where IL-10 is an intercalated dimer of two six-helix bundles (helices A-F). As used herein, unless otherwise indicated, "interleukin-10" and "IL-10" refer to human IL-10 ("hIL-10"; Genbank Accession No. NP_000563; or U.S. Patent No. 6,217,857) protein (SEQ ID NO: 1) or nucleic acid (SEQ ID NO: 2); murine IL-10 ("mIL-10"; Genbank Accession No.: M37897; or U.S. Patent No. 6,217,857) protein (SEQ ID NO: 7) or nucleic acid (SEQ ID NO: 8); or viral IL-10 ("vIL-10"). Viral IL-10 homologs may be derived from EBV or CMV (Genbank accession numbers NC_007605 and DQ367962, respectively). The term EBV-IL10 refers to the EBV homolog of the IL-10 protein (SEQ ID NO: 3) or nucleic acid (SEQ ID NO: 4). The term CMV-IL10 refers to the CMV homolog of the IL-10 protein (SEQ ID NO: 5) or nucleic acid (SEQ ID NO: 6). The term "monomeric" IL-10 or "monomer of" IL-10, as used herein, refers to individual subunits of IL-10 or variant IL-10 that, when non-covalently connected, form homodimers of IL-10 or variant IL-10. The terms "wild type," "wt," and "native" are used interchangeably herein to refer to the sequence of a protein (e.g., IL-10, CMV-IL10, or EBV IL-10) that is naturally and commonly found in the species of origin of the particular IL-10 in question. For example, the term "wild-type" or "native" EBV IL-10 would therefore correspond to the amino acid sequence most commonly found in nature.

[0044] The term "interleukin-12" or "IL-12" refers to a protein comprising alpha (p35) and beta (p40) subunits that are non-covalently linked to form a heterodimer. As used herein, unless otherwise indicated, "interleukin-12" and "IL-12" refer to human, mouse, or variant forms thereof. For example, the term "wild type" or "native" would therefore correspond to the amino acid sequences most commonly found in nature for the alpha and beta subunits.

[0045] The term "interleukin-27" or "IL-27" refers to a protein comprising alpha (p28) and beta (EBI3) subunits that are non-covalently linked to form a heterodimer. As used herein, unless otherwise indicated, "interleukin-27" and "IL-27" refer to human, mouse, or variant forms thereof. For example, the term "wild type" or "native" would therefore correspond to the amino acid sequences most commonly found in nature for the alpha and beta subunits.

[0046] The terms "variant", "analog" and "mutein" refer to a biologically active derivative of a reference molecule that retains a desired activity, such as, for example, anti-inflammatory activity. In general, the terms "variant", "variants", "analogs" and "muteins", when referring to a polypeptide, refer to one or more compounds having a native polypeptide sequence, as well as structures having one or more amino acid additions, substitutions (which may be conservative in nature), and / or deletions relative to the native molecule. Thus, the terms "IL-10 variant", "variant IL-10", "IL-10 variant molecule", and grammatical variations and plurals thereof are all intended to be equivalent to terms referring to an IL-10 amino acid (or nucleic acid) sequence that differs from wild-type IL-10 by any of 1-25% of 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 amino acid-encoding nucleotide sequence) additions, substitutions and / or deletions. Thus, in one form, an EBV IL-10 variant differs from the wild-type sequence of SEQ ID NO:3 by having about a 1%-25% difference in sequence homology amounting to a total of about 1-42 amino acid differences. In one embodiment, the IL-10 variant is an EBV IL-10 that contains an A75I amino acid mutation (internally designated "DV06"; SEQ ID NO:14), or both the V31L and A75I amino acid mutations (internally designated "DV07"; SEQ ID NO:16). The terms "IL-12 variant," "variant IL-12," "IL-12 variant molecule," "IL-27 variant," "variant IL-27," "IL-27 variant molecule," and grammatical variations and plurals thereof, are all intended to be equivalent terms that refer to an IL-12 or IL-27 amino acid (or nucleic acid) sequence that differs from wild-type IL-12 or IL-27. Amino acid sequence differences for IL-12 or IL-27 may be additions, deletions or substitutions within the alpha, beta, or both subunits that are anywhere from 1-25% sequence identity or homology.These variant forms include modifications to the protein to its deglycosylated (deglycosylated or aglycosylated) form.

[0047] The term "fusion protein" refers to the combination or conjugation of two or more proteins or polypeptides resulting in a novel arrangement of proteins not normally found in nature. A fusion protein is the result of the covalent linkage of two or more proteins or polypeptides. The two or more proteins making up the fusion protein may be arranged in any configuration from amino terminus ("NH2") to carboxy terminus ("COOH"). Thus, for example, the carboxy terminus of one protein may be covalently linked to either the carboxy terminus or amino terminus of another protein. An exemplary fusion protein may include combining a monomeric IL-10 or monomeric variant IL-10 molecule with one or more antibody variable domains (i.e., VH and / or VL) or single chain variable region ("scFv").

[0048] The terms "homolog," "homology," "homologous," or "substantially homologous" refer to the percent identity between at least two polynucleotide sequences 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 molecule.

[0049] The term "sequence identity" refers to an 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 an unknown percent identity to the reference sequence) by aligning the sequences, counting the exact number of 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 aid in identifying percent identity.

[0050] The terms "subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, including, but not limited to, murines, rodents, apes, humans, farm animals, sport animals, and certain pets.

[0051] The term "administering" includes routes of administration that allow the active ingredients of the present application to perform their intended function.

[0052] A "therapeutically effective amount" or "effective amount" refers to, for example, an EBV IL-10 variant, fusion protein, dual cytokine fusion protein, or DK2 thereof as described herein. 10 , DK7 10 , DK12 10 , DK15 10 DK21 10 , DK27 10 Or DKIFNa 10 When referring to administering a TAA to a subject, particularly in the context of combining with an engineered immune cell or ACT or BiTE that targets the TAA, it refers to an amount sufficient to promote a certain biological activity. These may include, for example, suppression of myeloid cell function, enhancement of Kupffer cell activity, and / or enhancement of CD8 + T cells or enhanced CD8 +It may include the lack of any effect on T cell activity, as well as blocking the upregulation of Fc receptors on mast cells, or preventing degranulation, or promoting or enhancing the effect of combination therapy (e.g., CAR-T therapy, or BiTE).Therefore, "effective amount" treats, ameliorates, or prevents the symptoms or signs of a medical condition.Effective amount also means an amount sufficient to enable or facilitate diagnosis.

[0053] The term "treat" or "treatment" refers 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 just the symptoms. Treatment can be any reduction from native levels, and can be, but is not limited to, the complete elimination of the disease, condition, or symptoms of the disease or condition.

[0054] The term "diakines" or "DKs" as used herein refers to a general class of dual cytokine fusion proteins that include IL-10 (monomer of IL-10), IL-12 or IL-27, or variants thereof, fused together with another cytokine on a half-life extending antigen targeting domain. Specifically, diakines take the form of Formula I, II, IIIa, IIIb, IV.

[0055] "DK2 10 " refers to a diakin comprising IL-10 or an IL-10 variant (e.g., SEQ ID NO: 10), IL-2 (e.g., SEQ ID NO: 9) linked between the VH and VL regions of the scFv, as depicted diagrammatically in FIG. 2, where the diakin can be targeted or non-targeted. Targeted DK2 10 can be made into a targeting molecule by using an scFv that binds to a specific antigen, e.g., a TAA, or by grafting onto an scFv scaffold CDRs from an antibody that targets a TAA. These molecules are 10 (target name). For example, DK2 targeted by EGFR 10 "DK2 10 (egfr)" or "DK2 10egfr". In one embodiment, DK2 10 egfr is SEQ ID NO: 19. In another example, DK2 is targeted to HER2. 10 "DK2 10 (her2)" or "DK2 10 her2." In one embodiment, DK2 10 her2 is SEQ ID NO: 21, 23 or 25.

[0056] "DK7 10 The term "DK7" refers to diakines, including IL-7 or IL-7 variants (e.g., aglycosylated or deglycosylated forms), and IL-10 and IL-10 variants (e.g., SEQ ID NO: 10, or aglycosylated or deglycosylated forms thereof) linked between the VH and VL regions of the scFv, as depicted diagrammatically in FIG. 2, where the diakines can be targeted or non-targeted. Targeted DK7 10 can be made into a targeting molecule by using an scFv that binds to a specific antigen, e.g., a TAA, or by grafting onto an scFv scaffold CDRs from an antibody that targets a TAA. These molecules include DK7 10 (target name). For example, DK7 targeted to EGFR 10 "DK7 10 (egfr)" or "DK7 10 egfr." In one embodiment, DK7 10 egfr is SEQ ID NO: 36. In another example, (a) DK7 targeted to HER2 10 "DK7 10 (her2)" or "DK12 10 her2” and (b) DK7 targeted to CD20 10 "DK7 10 (CD20)" or "DK12 10 In one embodiment, DK7 10 her2 is SEQ ID NO: 37; DK7 10 CD20 is sequence number 38.

[0057] "DK12 10 " refers to diakines, including IL-12 or IL-12 variants (e.g., aglycosylated or deglycosylated forms), and IL-10 and IL-10 variants (e.g., SEQ ID NO: 10, or aglycosylated or deglycosylated forms thereof) linked between the VH and VL regions of the scFv, as depicted diagrammatically in FIG. 3, where the diakines can be targeted or non-targeted. Targeted DK12 10 can be made into a targeting molecule by using an scFv that binds to a specific antigen, e.g., a TAA, or by grafting onto an scFv scaffold CDRs from an antibody that targets a TAA. These molecules are 10 (target name). For example, DK12 targeted to EGFR 10 "DK12 10 (egfr)" or "DK12 10 egfr." In one embodiment, DK12 10 egfr is SEQ ID NO: 26 to 32. In another example, (a) DK12 targeted to HER2 10 "DK12 10 (her2)" or "DK12 10 her2” and (b) DK12 targeted to CD20. 10 "DK12 10 (CD20)" or "DK12 10 In one embodiment, DK12 10 CD20 is sequence number 34 or 35.

[0058] "DK15 10 " refers to diakines, including IL-15 or IL-15 variants (e.g., aglycosylated or deglycosylated forms), and IL-10 and IL-10 variants (e.g., SEQ ID NO: 10, or aglycosylated or deglycosylated forms thereof) linked between the VH and VL regions of the scFv, as depicted diagrammatically in FIG. 2, where the diakines can be targeted or non-targeted. Targeted DK15 10can be made into a targeting molecule by using an scFv that binds to a specific antigen, e.g., a TAA, or by grafting onto an scFv scaffold CDRs from an antibody that targets a TAA. These molecules include DK15 10 (target name). For example, DK15 targeted to EGFR 10 "DK15 10 (egfr)" or "DK15 10 egfr" is displayed.

[0059] "DK21 10 " refers to diakines, including IL-21 or IL-21 variants (e.g., aglycosylated or deglycosylated forms), and IL-10 and IL-10 variants (e.g., SEQ ID NO: 10, or aglycosylated or deglycosylated forms thereof) linked between the VH and VL regions of the scFv, as depicted diagrammatically in FIG. 2, where the diakines can be targeted or non-targeted. Targeted DK21 10 can be made into a targeting molecule by using an scFv that binds to a specific antigen, e.g., a TAA, or by grafting onto an scFv scaffold CDRs from an antibody that targets a TAA. These molecules include DK21 10 (target name). For example, DK21 targeted to EGFR 10 "DK21 10 (egfr)" or "DK21 10 egfr" is displayed.

[0060] "DK27 10 " refers to diakines, including IL-27 or IL-27 variants (e.g., aglycosylated or deglycosylated forms), and IL-10 and IL-10 variants (e.g., SEQ ID NO: 10, or aglycosylated or deglycosylated forms thereof) linked between the VH and VL regions of the scFv, as depicted diagrammatically in FIG. 2, where the diakines can be targeted or non-targeted. Targeted DK27 10may be made into a targeting molecule by using an scFv that binds to a specific antigen, e.g., a TAA, or by grafting onto an scFv scaffold CDRs from an antibody that targets a TAA. These molecules include DK27 10 (target name). For example, DK27 targeted to EGFR 10 "DK27 10 (egfr)" or "DK27 10 egfr" is displayed.

[0061] "DKIFNa 10 " refers to diakines, including IFNa or IFNa variants (e.g., aglycosylated or deglycosylated forms), and IL-10 and IL-10 variants (e.g., SEQ ID NO: 10, or aglycosylated or deglycosylated forms thereof) linked between the VH and VL regions of the scFv, as depicted diagrammatically in FIG. 2, where the diakines can be targeted or non-targeted. Targeted DKIFNa 10 may be made into a targeting molecule by using an scFv that binds to a specific antigen, e.g., a TAA, or by grafting onto an scFv scaffold CDRs from an antibody that targets a TAA. These molecules include DKIFNa 10 (target name). For example, DKIFNa, which targets EGFR, 10 "DKIFNa 10 (egfr)" or "DKIFNa 10 egfr" is displayed.

[0062] "DK12 IFNa The term "DK12" refers to diakines, including IFNa or IFNa variants (e.g., aglycosylated or deglycosylated forms), and IL-12 and IL-12 variants (e.g., aglycosylated or deglycosylated forms thereof) linked between the VH and VL regions of scFv, as depicted diagrammatically in FIG. 2, where the diakines can be targeted or non-targeted. Targeted DK12 IFNacan be made into a targeting molecule by using an scFv that binds to a specific antigen, e.g., a TAA, or by grafting onto an scFv scaffold CDRs from an antibody that targets a TAA. These molecules are IFNa (target name). For example, DK12 targeted to EGFR IFNa "DK12 IFNa (egfr)" or "DK12 IFNa egfr" is displayed.

[0063] The term "tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as referred to herein.

[0064] Tumors can be benign or malignant. Benign tumors are characterized as not undergoing metastasis. Malignant cells are cancer cells and can undergo metastasis. Tumors on which the methods of the present application can be performed include, but are not limited to, adenoma, angio-sarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hamartoma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and teratoma. Tumors include acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, bronchial adenocarcinoma, capillary, carcinoid, carcinoma, carcinosarcoma, cavernous, cholangiocarcinoma, chondrosarcoma, choriod plexus papilloma / carcinoma, clear cell carcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal, epithelioid, Ewing's sarcoma, fibrolamellar type, focal nodular hyperplasia, gastrinoma, germ cell tumor, glioblastoma, glucagonoma, hemangiblastoma, hemangioendothelioma, hemangioma, hepatocellular adenoma, hepatocellular adenomatosis, hepatocellular carcinoma, insulinoma, intraepithelial neoplasia neoplasia), interepithelial squamous cell neoplasm, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, lentigo maligna melanoma, malignant melanoma, malignant mesothelioma, medulloblastoma, medulloepithelioma, melanoma, meningeal, mesothelial, metastatic carcinoma, mucoepidermoid carcinoma, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, oat cell carcinoma, oligodendroglia, osteosarcoma, papillary serous adenocarcinoma of the pancreas, pineal cell, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous cell carcinoma, squamous cell carcinoma, submesothelial, superficial spreading melanoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipoma, well-differentiated carcinoma, and Wilms' tumor.

[0065] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, carcinoma of the peritoneum, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer. Structure of dual cytokine fusion proteins

[0066] The present disclosure relates to the use of diakines, previously described in US Patent Application No. 17 / 199,239 (filed March 11, 2021, incorporated herein by reference in its entirety), to increase or enhance the function of known immunotherapies, such as engineered immune cell or BiTE therapies targeting TAA. Briefly, the diakines used in the methods of the present application, which are an improvement over previous versions of IL-10 fusion proteins previously described in US Patent Nos. 10,858,412 and 10,975,133 (incorporated herein by reference in their entirety), comprise two monomers of IL-10 or an IL-10 variant molecule, and a second cytokine molecule linked to the hinge region of an scFv. The diakines are constructed in the first generation IL-10 fusion molecules (FIG. 1) described in US Patent Nos. 10,858,412 and 10,975,133. Briefly, the first generation IL-10 fusion protein is constructed on a VH and VL scFv scaffold featuring two monomers of IL-10 at each end (i.e., a first IL-10 monomer at the amino terminus and a second IL-10 monomer at the carboxy terminus). The scaffold system includes an scFv derived from a human monoclonal anti-Ebola antibody with six complementarity determining regions ("CDRs") with CDRs 1-3 of VH and CDRs 1-3 of VL. The VH and VL regions can target the IL-10 fusion protein to a specific antigen, which is achieved by replacing the six CDR regions of the VH and VL pair (three CDRs of VH and three CDRs of VL) with the six CDR regions from the VH and VL of a receptor or antigen targeting antibody, or an antigen-binding fragment thereof. The ability to replace and optimize the six CDR and framework regions of an scFv, as well as grafting CDRs onto the scFv scaffold described herein, is well known and performed by one of skill in the art.

[0067] In a first aspect, the present application relates to a dual cytokine fusion protein, termed Diakine, which comprises IL-10 or an IL-10 variant and at least one other cytokine, whereby the dual cytokine fusion protein has a combined or synergistic functionality when compared to the IL-10 fusion protein previously described in US Pat. No. 10,858,412. Figure 2 is a representative diagram of Diakine containing IL-10. In particular, Diakine utilizes a scaffold system made of scFv, which comprises a VH and a VL, whereby two monomers of IL-10 terminate the dual cytokine fusion protein at the amino and carboxy termini. The second cytokine is conjugated to the monomer of IL-10 (or an IL-10 variant) by fusing between the VH and VL regions of the scFv, which is the hinge region of the scFv. Diakine is capable of forming a functional protein complex, whereby monomers of IL-10 (or an IL-10 variant) homodimerize into a functional IL-10 molecule, and the VH and VL regions pair together to form an scFv complex that allows antigen binding and recognition.

[0068] In certain embodiments, the IL-10 containing diakin or dual cytokine fusion protein is represented by Formula I NH2-(IL-10)-(X 1 )-(Z n )-(X 2 )-(IL-10)-COOH (In the formula, "IL10" is a monomer of IL-10, wherein the IL-10 is human, mouse, CMV or EBV IL-10, or a variant thereof, more preferably, the IL10 is a monomer comprising a sequence selected from SEQ ID NO: 1, 3, 7, 9 or 10; "X1" is a VL or VH region obtained from a first monoclonal antibody; "X2" is a VH or VL region obtained from a first monoclonal antibody; where if X1 is a VL then X2 is a VH, or if X1 is a VH then X2 is a VL, and preferably the VH and VL regions are scFvs obtained from a human anti-Ebola antibody, and the VH and VL regions are grafted with six CDRs (three from the VH and three from the VL) from a second antibody; "Z" is any cytokine other than IL-10, preferably 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, IFN-α, IFN-β, IFN-γ, TGF-β, or TNF-α, TNF-β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13; "n" is an integer selected from 1 to 2. It is a structure having the following structure. In a preferred embodiment, IL-10 is a high affinity variant designated DV07, which contains substitutions at amino acid positions 31 and 75 of SEQ ID NO: 10. In another embodiment, the VH and VL regions are scFvs derived from any monoclonal antibody capable of binding to a TAA found on the surface of a solid or hematological tumor. In another aspect, the scFv is derived from an antibody against VEGFR2. In one aspect, the scFv is derived from a human monoclonal anti-Ebola antibody, where the six CDRs (three in VH and three in VL) of the anti-Ebola antibody are optionally replaced with six CDRs from any monoclonal antibody capable of binding to a TAA. In another aspect, the scFv comprises VH and VL framework regions derived from a human monoclonal anti-Ebola antibody grafted with CDRs having specificity for a TAA associated with a solid or hematological tumor. In one aspect, the VH and VL regions of the scFv are grafted with six CDRs (three from VH and three from VL) from an anti-VEGFR2 antibody. In yet another preferred embodiment, the second cytokine is IL-2, more preferably SEQ ID NO:9.

[0069] In another embodiment, the diakin or dual cytokine fusion protein comprising IL-10 is represented by Formula II NH2-(IL10)-(L)-(X 1 )-(L)-(Z n )-(L)-(X 2 )-(L)-(IL10)-COOH (In the formula, "IL-10" refers to IL-10 monomer, such as, but not limited to, human, mouse, CMV, EBV IL-10, or a variant thereof; "L" is a linker, preferably a linker of SEQ ID NO: 39, 40 or 41; "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; where X 1If VL, then X 2 is VH or X 1 If VH, then X 2 is VL, and X 1 and X 2 together are scFvs; "Z" is a second cytokine, where the second cytokine is a cytokine other than IL-10; "n" is an integer selected from 1 to 2. It is a structure having the following structure. In a preferred embodiment, IL-10 is a high affinity variant designated DV07, which contains substitutions at amino acid positions 31 and 75 of SEQ ID NO: 10. In another embodiment, the VH and VL regions are scFvs derived from any monoclonal antibody capable of binding to a TAA found on the surface of a solid or hematological tumor. In another embodiment, the scFv is derived from an antibody against VEGFR2. In one embodiment, the scFv is derived from a human monoclonal anti-Ebola antibody, where the six CDRs (three of the VH and three of the VL) of the anti-Ebola antibody are optionally replaced with six CDRs from any monoclonal antibody capable of binding to a TAA. In another embodiment, the scFv comprises VH and VL framework regions derived from a human monoclonal anti-Ebola antibody grafted with CDRs having specificity for a TAA associated with a solid or hematological tumor. In one aspect, the VH and VL regions of the scFv are grafted with six CDRs (three from VH and three from VL) from an anti-VEGFR2 antibody. In yet another preferred embodiment, the second cytokine is IL-2, more preferably SEQ ID NO:9.

[0070] In one embodiment, the IL-10 monomer comprises any form of IL-10, including human (SEQ ID NO: 1), CMV (SEQ ID NO: 5), EBV (SEQ ID NO: 3) or mouse (SEQ ID NO: 7). In another embodiment, the IL-10 monomer is a modified or variant form of EBV IL-10 (SEQ ID NO: 3), including that described in U.S. Pat. No. 10,858,412. In a preferred embodiment, the EBV IL-10 comprises two substitutions in SEQ ID NO: 3 at amino acid positions 31 and 75 ("DV07"). In yet another embodiment, the IL-10 monomer is of the sequence of SEQ ID NO: 1, 3, 7 or 10. The first and second monomers of the IL-10 or IL-10 variant molecule are located at the termini of the fusion protein, respectively (i.e., the first monomer at the amino terminus and the second monomer at the carboxy terminus), as depicted in FIG. 2.

[0071] In another aspect, the present disclosure relates to methods of using the IL-12 diakin of formula (III) in combination with engineered immune cells or BiTEs that target TAA.

[0072] NH2-(R 1 )-(X 1 )-(Z n )-(X 2 )-(R 2 )-COOH (formula IIIa);

[0073] NH2-(R 2 )-(X 1 )-(Z n )-(X 2 )-(R 1 )-COOH (formula IIIb); (In the formula, "R 1 " is an alpha subunit from any multi-subunit first cytokine, preferably either the IL-12-alpha subunit (p35) or the IL-27 alpha subunit (p28), more preferably the subunit of SEQ ID NO: 45 or 47; "R 2" is a beta subunit from any multi-subunit first cytokine, preferably either the IL-12-beta subunit (p40) or the IL-27 beta subunit (EBI3), more preferably the subunit of SEQ ID NO: 46 or 48; Here, R 1 is the alpha subunit of the first cytokine, R 2 is the beta subunit of the first cytokine; or R 1 If p35, then R 2 is p40; or R 1 If p28, then R 2 is EBI3; or R 1 is SEQ ID NO: 45 or 47, then R 2 is SEQ ID NO: 46 or 48; or R 1 is SEQ ID NO: 46 or 48, then R 2 is SEQ ID NO: 45 or 47; "X 1 " is a VL or VH region obtained from a first monoclonal antibody; "X 2 " is a VH or VL region obtained from a first monoclonal antibody; 1 If VL, then X 2 is VH or X 1 If VH, then X 2 is VL; "Z" is any cytokine that enhances the biological function of a multisubunit cytokine, preferably IFN-2a, IL-28, IL-29; "n" is an integer selected from 1 to 2, The first monoclonal antibody is selected from the group consisting of VEGFR2, CD3, CD4, CD5, CD7, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD44, CD52, CD56, CD70, CD79B, CD117, CD123, CD138, CD147, IL-22R1, BCMA, CLL01, CD5, CD147, lLMP-1, SLAMF7, NY-ESO-1, an anti-Ebola antibody grafted with CDRs from a second antibody having specificity for TACI, CS-1, CXCR4, NKG2D, B7-H3, EGFR, PD-1, PDL-1, HER2, HER3, EpCAM, PSCA, MUC1, Lewis Y, GPC3, AXL, claudin 18.2, GD2, CTLA-4, CEA, PDGFR, MESO, PSCA, PSMA, BCMA, or PSA).

[0074] In yet another aspect, the disclosure provides a method of using a diakin having two multisubunit cytokines of formula (IV), such as IL12, IL-27, or IL-10, in combination with an engineered immune cell or BiTE that targets a TAA, wherein the diakin has formula (IV): NH2-(R 1 )-(L a )-(X 1 )-(L a )-(W 1 )-(L b )-(W 2 )-(L a )-(X 2 )-(L a )-(R 2 )-COOH (formula IV); (In the formula, "R 1 " is the alpha subunit of a first cytokine, e.g., IL-12 or IL-27, or a first monomer of a homodimeric cytokine, e.g., IL-10, where R 1 is preferably p40; "R 2" is a beta alpha subunit of a first cytokine, e.g., IL-12 or IL-27, or a second monomer of a homodimeric cytokine, e.g., IL-10, where R 2 is preferably p35; "L a " is an optional linker; preferably SEQ ID NO: 43 or 44; "L b " is an optional linker; preferably SEQ ID NO: GGGSGGG or SEQ ID NO: 42; "X 1 " is a VL or VH region obtained from a first monoclonal antibody; "X 2 " is a VH or VL region obtained from a first monoclonal antibody; 1 If VL, then X 2 is VH or X 1 If VH, then X 2 is VL; "W 1 " is a first cytokine, e.g., the alpha subunit of IL-12 or IL-27, or a first monomer of a homodimeric cytokine, e.g., IL-10, preferably a first monomer of IL-10; "W 2 " is a first cytokine, e.g., a beta alpha subunit of IL-12 or IL-27, or a second monomer of a homodimeric cytokine, e.g., IL-10, preferably a second monomer of IL-10; The first monoclonal antibody is directed against VEGFR2, CD3, CD4, CD5, CD7, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD44, CD52, CD56, CD70, CD79B, CD117, CD123, CD138, CD147, IL-22R1, BCMA, CLL01, CD5, CD147, lLMP-1, SLAMF7, NY- grafted with CDRs from antibodies with specificity for ESO-1, TACI, CS-1, CXCR4, NKG2D, B7-H3, EGFR, PD-1, PDL-1, HER2, HER3, EpCAM, PSCA, MUC1, Lewis Y, GPC3, AXL, claudin 18.2, GD2, CTLA-4, CEA, PDGFR, MESO, PSCA, PSMA, BCMA, or PSA) This relates to a method.

[0075] The VH and VL regions are derived from an antibody, antibody fragment, or antigen-binding fragment thereof. Antigen-binding fragments include, but are not limited to, scFv, Fab, F(ab')2, V-NAR, diabody, or nanobody. Preferably, the VH and VL are derived from a single chain variable fragment ("scFv"). In one embodiment, the scFv is obtained from a human monoclonal anti-Ebola antibody. In another embodiment, the scFv comprises a framework region from an anti-Ebola antibody and six CDRs (three VH and three VL) from a monoclonal antibody specific for any TAA expressed on the surface of a solid or hematological tumor. The scFv antibodies or graftable CDRs include VEGFR2, CD3, CD4, CD5, CD7, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD44, CD52, CD56, CD70, CD79B, CD117, CD123, CD138, CD147, IL-22R1, BCMA, CLL01, CD5, CD147, ILMP-1, SLAMF7, NY-ES The antibody is derived from a monoclonal antibody selected from O-1, TACI, CS-1, CXCR4, NKG2D, B7-H3, EGFR, PD-1, PDL-1, HER2, HER3, EpCAM, PSCA, MUC1, Lewis Y, GPC3, AXL, claudin 18.2, GD2, CTLA-4, CEA, PDGFR, MESO, PSCA, PSMA, BCMA or PSA specific antibodies, or multi-targeting forms thereof.

[0076] In another embodiment, the diakins comprising IL-10, IL-12 or IL-27 comprise VH and VL pairs from a single antibody. The VH and VL pairs act as scaffolds to which the monomers of IL-10 or its variants can attach, so that the monomers of IL-10 or its variants can homodimerize into a functional IL-10 molecule. Thus, those skilled in the art will recognize that the VH and VL scaffolds used in the fusion protein can be selected based on the desired physical characteristics required for proper homodimerization of the IL-10 monomer or IL-10 monomer variant and / or the desire to maintain the VH and VL targeting ability. Similarly, those skilled in the art will also understand that the six CDRs in the VH and VL pair (three CDRs from VH and three CDRs from VL) can also be replaced with six CDRs from other antibodies to obtain a specific targeting fusion protein. In one embodiment, three CDRs from the VH and three CDRs from the VL (i.e., VH and VL pairs) of any monoclonal antibody may be grafted onto a scaffold system comprising SEQ ID NO: 12 or 15. The scaffold system described in SEQ ID NO: 12 or 15, when produced as a diakin, also comprises a second cytokine linked within the hinge region of the VH and VL portions of the molecule. If the dual cytokine fusion protein is not intended to target any specific antigen, it is also envisioned that the VH and VL pairs may be selected as scaffolds that do not target any particular antigen (or are antigens of low abundance in vivo), e.g., VH and VL pairs from anti-HIV and / or anti-Ebola antibodies. Thus, in an embodiment, the IL-10 fusion protein of the present application may comprise a VH and VL pair from a human anti-Ebola antibody, more preferably the VH and VL sequences found in SEQ ID NO: 12 or 15. The fusion protein may comprise a range of one to four variable regions. In another embodiment, the variable regions can be from the same antibody or from at least two different antibodies.

[0077] In another aspect, the target specificity of the antibody variable chain, or VH and VL pair, or the six CDRs of the VH and VL pair, may include, but is not limited to, targeting proteins, cell receptors and / or tumor-associated antigens. In another embodiment, the CDR regions from any VH and VL pair may be grafted onto the scaffold system described above, such a scaffold preferably comprising a system called Debo (schematically represented in FIG. 1), whereby an IL-10 monomer is linked to an scFv comprising the VH and VL regions of a human anti-Ebola antibody, and a second cytokine is linked to the hinge region of the scFv (schematically represented in FIG. 2). More preferably, grafting onto the Debo scaffold system occurs on a scaffold comprising the sequence of SEQ ID NO: 12 or 15. In yet another embodiment, the variable region, or the six CDRs of the VH and VL pair, or the VH and VL pair, are obtained from variable regions from antibodies targeting antigens associated with various diseases (e.g., cancer), or those that are not typically or rarely found in the serum of healthy subjects, such as antibodies against EGFR, PDGFR, VEGFR1, VEGFR2, Her2Neu, FGFR, GPC3, or other tumor-associated antigens, MAdCAM, ICAM, VCAM, CD14, or other inflammation-associated cell surface proteins, HIV and / or Ebola. Thus, in one embodiment, the variable region is obtained or derived from, for example, an anti-EGFR, anti-MAdCAM, anti-HIV (Chan et al, J. Virol, 2018, 92(18):e006411-19), anti-ICAM, anti-VCAM, anti-CD14, or anti-Ebola (mAbs described in US Patent Publication No. 2018 / 0180614, particularly Tables 2, 3 and 4, which are incorporated by reference herein in their entirety) antibody. In another embodiment, the variable region is obtained or derived from an antibody that can concentrate the concentration of cytokines, e.g., IL-10 and IL-2, to a specific target region such that IL-10 and IL-2 can induce their biological effects.Such antibodies may include those that target receptors or antigens that are overexpressed or upregulated in a particular diseased region, or that are specifically expressed in a particular affected region. For example, the variable regions may target epidermal growth factor receptor (EGFR); CD52; CD14; various immune checkpoint targets, including but not limited to PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4; CD20; CD47; GD-2; VEGFR1; VEGFR2; HER2; PDGFR; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TGFβ, to name a few. 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; and SR-J1 specific antibodies may be obtained. Other variable regions include CD3, CD4, CD5, CD7, CD19, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD56, CD70, CD79B, CD117, CD123, CD138, CD147, B-cell mutated antigen (BCMA), C-type lectin-like molecule 1 (CLL01), latent membrane protein 1 (LMP-1), signaling lymphoid activation molecule F7 (SLAMF7) , NY-ESO-1, transmembrane activator and CAML interactor (TACI), CS-1, CXCR4, NKG2D, B7-H3, EGFR, HER3, EpCAM, mesothelin, PSCA, MUC1, Lewis Y, GPC3, AXL, claudin 18.2, GD2, CTLA-4, CEA, PDGFR, mesothelin (MESO), PSCA, PSA specific antibodies. Monomers of IL-10 (e.g., human, CMV or EBV) or variant IL-10 molecules (as described herein) are conjugated to either the amino or carboxy terminus of the variable region (VH or VL) such that the monomeric IL-10 or variant IL-10 molecules are capable of dimerizing with one another.In a preferred embodiment, a monomer of IL-10 (or a variant IL-10) is fused to a VH and VL pair according to Formula I or II, where the IL-10 monomer is the EBV IL-10, DV05, DV06, or DV07 form of IL-10.

[0078] Diakine, dual cytokine fusion protein or dual cytokine fusion protein complex may also have antigen targeting function. Diakine or dual cytokine fusion protein or dual cytokine fusion protein complex comprises VH and VL pair that can be associated together to form antigen binding site or ABS. In some configurations, IL-10 monomer or its IL-10 variant monomer is covalently linked to the end that contains the antigen binding site. The variable region may be further modified (e.g., by addition, subtraction, or substitution) by changing one or more amino acids that reduce antigenicity in the subject. Other modifications to the variable region may include substitution, deletion, or addition of amino acids found outside the six CDR regions of the VH and VL regions and serve to increase the stability and expression of the VH and VL regions of the scFv. For example, modifications can include those in which the CDR regions are derived from the VH and VL regions of an anti-EGFR or anti-VEGFR1 or anti-VEGFR2 antibody and the regions outside the CDRs are optimized to stabilize the scFv and / or to increase expression, which can be used based on the linkage of a second cytokine between the VH and VL regions of the scFv. To demonstrate that these types of modifications are within the skill of the art, similar modifications to the CDR and regions outside the CDRs were made to DK2, which includes DV07 and targets human HER2. 10 Morphology (i.e., DK2 10 Her2) molecules, such as those set forth in SEQ ID NOs: 52-54, or 55, more preferably SEQ ID NO: 21 (variant 4) or 23 (variant 5). These and other modifications have been made to DK2, including DV07, which targets human VEGFR1 or VEGFR2A. 10These modifications may also be made to any form of molecule and one of skill in the art would be able to determine other modifications to stabilize the scFv and / or optimize the sequence for expression purposes.

[0079] The VH and VL pairs form a scaffold onto which the CDR regions obtained for multiple antibodies can be grafted or grafted. Such antibody CDR regions include known and above-described antibodies. The CDR regions in the above-described VH and VL scaffolds include the following numbered amino acid positions available for CDR grafting / insertion: [Table A] In a preferred embodiment, the dual cytokine fusion protein comprising IL-10 has a VH and VL pair derived from an anti-Ebola antibody (e.g., as set forth in SEQ ID NO: 19), whereby the six CDR regions from the anti-Ebola antibody have been removed, and a specific targeting antibody, such as, but not limited to, 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. The present invention includes a previously described scaffold IL-10 fusion protein grafted with the VH and VL pairs of 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; and SR-J1. In an embodiment, the six anti-Ebola CDR regions are replaced with six CDR regions from an anti-EGFR, anti-MAdCAM, anti-VEGFR1, anti-VEGFR2, anti-PDGFR, or anti-CD14, anti-CD19, anti-CD20, anti-CD22, more preferably, an anti-VEGFR2 antibody.

[0080] In yet another aspect, the second cytokine is fused between the VH and VL of the scFv as depicted in Figure 2. The second cytokine is conjugated, fused, or linked between the VH or VL regions of the scFv such that the second cytokine retains its functional properties. In one embodiment, the second cytokine is different from the IL-10 monomer. In another embodiment, the second cytokine is IL-10. In one embodiment, the second cytokine is IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-17, IL-21, IL-26, IL-27, IL-28a, IL28b, IL-29, TSLP, 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, or variants or muteins thereof, including but not limited to high, medium, and low receptor affinity variants. In a preferred embodiment, the second cytokine in the diakin with IL-10 is IL-2. In a more preferred embodiment, the diakin or dual cytokine fusion protein is DK2. 10in the form of, wherein the IL-10 monomer is an IL-10 variant molecule linked to a scaffold system (i.e., Debo) comprising VH and VL framework regions from DV07; a human anti-Ebola antibody; and anti-EGFR, anti-HER2, anti-CD14, anti-VEGFR1, anti-VEGFR2, anti-MAdCAM, or anti-PDGFR, anti-Cd19, anti-Cd20, anti-CD22, anti-CD3, anti-CD4, anti-CD5, anti-CD6, anti-CD7, anti-CD8, anti-CD9, anti-CD10, anti-CD11, anti-CD12, anti-CD13, anti-CD14, anti-CD15, anti-CD16, anti-CD17, anti-CD18, anti-CD19, anti-CD ...19, anti-CD20, anti-CD22, anti-CD3, anti-CD19, anti-CD20, anti-CD22, anti-CD3, anti-CD19, anti-CD20, anti-CD22, anti-CD3, anti-CD19, anti-CD20, anti-CD19, anti-CD22, anti-CD3, anti-CD19, anti-CD20, anti-CD22, anti-CD3, anti-CD19, anti-CD19, anti-CD20, anti-CD22, anti-CD3, anti-CD19, anti-CD20, anti-CD19 CD4, anti-CD5, anti-CD7, anti-CD25, anti-CD30, anti-CD33, anti-CD34, anti-CD38, anti-CD40, anti-CD52, anti-CD56, anti-CD70, anti-CD79B, anti-CD117, anti-CD123, anti-CD138, anti-CD147, anti-B cell mutant antigen (BCMA), anti-C-type lectin-like molecule 1 (CLL01), anti-CD5, anti-CD147, anti-latent membrane protein 1 (LMP-1), anti The grafted CDRs from an antibody selected from signaling lymphocyte activation molecule F7 (SLAMF7), anti-NY-ESO-1, anti-transmembrane activating factor and CAML interactor (TACI), anti-CS-1, anti-CXCR4, anti-NKG2D, anti-B7-H3, anti-PD-1, anti-PDL-1, anti-HER3, anti-EpCAM, anti-mesothelin, anti-PSCA, anti-MUC1, anti-Lewis Y, anti-GPC3, anti-AXL, anti-claudin 18.2, anti-GD2, anti-CTLA-4, anti-CEA, anti-mesothelin (MESO), anti-PSCA, or anti-PSA, and a second cytokine selected from IFNα, IL2, IL7, IL15, IL21, IL28, IL29, or high, medium or low affinity variants thereof, linked to the hinge region of the VH and VL pair. In a most preferred embodiment, the diakin or dual cytokine fusion protein is selected from DK2, including DV07. 10 It is a fusion protein of the form grafted with six CDRs from an anti-VEGFR2 antibody.

[0081] In another embodiment, the diakin is a combination of a first cytokine (or a high affinity variant thereof), a second cytokine (or a high affinity variant thereof), and a targeting scFv (or CDRs derived from a monoclonal antibody and grafted onto a framework region derived from an anti-Ebola antibody), as shown in Table 1 below. [Table 1-1] [Table 1-2]

[0082] In other embodiments, methods of using potential diakines, or combinations of at least one diakin and TAA-targeted engineered immune cells, ACT, or BiTEs, include those listed in Tables 2a-2e) below. [Table 2a] [Table 2b] [Table 2c-1] [Table 2c-2] [Table 2d-1] [Table 2d-2] [Table 2e-1] [Table 2e-2]

[0083] In yet another aspect, the diakin or dual cytokine fusion protein comprising IL-10, IL-12 or IL-27 incorporates a linker. Those skilled in the art know that linkers or spacers can be used to achieve the correct spatial arrangement of the various fusion protein moieties and thus select the appropriate linker for use in forming the dual cytokine fusion protein comprising IL-10. In a more preferred embodiment, the linker or spacer can be a random amino acid sequence (e.g., SEQ ID NOs: 39-44) or a constant region of an antibody. The constant region can be derived from, but is not limited to, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD or IgE. In one embodiment, the linker or spacer is a constant heavy ("CH") region 1, CH2, or CH3. In another aspect, the linker or spacer can further comprise at least two interchain disulfide bonds.

[0084] In another aspect, the present disclosure relates to nucleic acid molecules encoding diakines or dual cytokine fusion proteins comprising IL-10, IL-12 or IL-27 and a second cytokine. These nucleic acid molecules are described in US Patent Application Serial No. 17 / 110,104. Polynucleotide sequences encoding diakines or dual cytokine fusion proteins comprising IL-10 and a second cytokine may also include modifications that do not change the functional properties of the described dual cytokine fusion proteins. Such modifications use conventional recombinant DNA techniques and methods. For example, specific amino acid sequence additions or substitutions may be introduced into the IL-10 sequence at the nucleic acid (DNA) level using site-directed mutagenesis using synthetic oligonucleotides, which is also well known in the art. In a preferred embodiment, nucleic acid molecules encoding dual cytokine fusion proteins comprising IL-10 and a second cytokine may include insertions, deletions or substitutions (e.g., denaturing the code) that do not change the functionality of the IL-10 variant molecule. Nucleotide sequences encoding IL-10, IL-12, IL-27 variants and / or the entire fusion proteins described herein may differ from the amino acid sequences due to the degeneracy of the genetic code and may be 70-99%, preferably 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the aforementioned sequences. Thus, embodiments of the present disclosure include nucleic acid sequences that encode the proteins of SEQ ID NOs: 35, 46-58 or 59, but that differ by 70-99% due to the degeneracy of the genetic code.

[0085] The nucleotide sequences encoding the diakin or dual cytokine fusion proteins described herein may further include well-known sequences, for example, useful for the expression, production or secretion of the protein. Such sequences may include, for example, a leader sequence, a signal peptide, and / or a translation initiation site / sequence (e.g., Kozak consensus sequence). The nucleotide sequences described herein may also include one of several restriction enzyme sites, allowing insertion into various expression systems / vectors.

[0086] In another aspect, the nucleotide sequence encoding the dual cytokine fusion protein may be used directly in gene therapy. In one embodiment, the variant IL-10, IL-12 or IL-27 molecule or fusion protein of the present application can be delivered by any method known in the art, including direct administration of mutant IL-10, IL-12 or IL-27 protein and gene therapy using a vector encoding mutant IL-10 protein. Gene therapy may be achieved using plasmid DNA or a viral vector, such as an adeno-associated viral vector, an adenoviral vector, a retroviral vector, etc. In some embodiments, the viral vectors of the present application are administered as viral particles, and they are also administered as plasmids (e.g., as "naked" DNA).

[0087] Other methods for delivery of nucleotide sequences include those already known in the art. These would include delivery of nucleotide sequences encoding IL-10 or IL-10 variant molecules, such as, but not limited to, DNA, RNA, siRNA, mRNA, oligonucleotides, or variants thereof, by cell penetrating peptides, hydrophobic moieties, electrostatic complexes, liposomes, ligands, liposomal nanoparticles, lipoproteins (preferably HDL or LDL), folate-targeted liposomes, antibodies (e.g., folate receptor, transferrin receptor), targeting peptides, or by aptamers. Nucleotide sequences encoding IL-10, IL-12, or IL-27 variant molecules may be delivered to a subject by direct injection, infusion, patch, bandage, mist or aerosol, or by thin film delivery. The nucleotides (or proteins) may be to any area where targeted delivery of cytokine stimulation is desired. These would include, for example, the lungs, GI tract, skin, liver, brain by intracranial injection, deep metastatic tumor lesions via ultrasound-guided injection.

[0088] In another aspect, the present disclosure relates to a method for preparing and purifying diakin or dual cytokine fusion proteins comprising IL-10, IL-12 or IL-27.For example, the nucleic acid sequence encoding the diakin or dual cytokine fusion proteins described herein may be used to recombinantly produce the fusion proteins.For example, using conventional molecular biology and protein expression techniques, the diakin or dual cytokine fusion proteins described herein may be expressed and purified from mammalian cell systems.These systems include well-known eukaryotic cell expression vector systems and host cells.A variety of suitable expression vectors may be used and are well known to those skilled in the art, which can be used for the expression and introduction of variant IL-10, IL-12 or IL-27 molecules and fusion proteins. These vectors include, for example, pUC-type vectors, pBR-type vectors, pBI-type vectors, pGA-type, pBinl9, pBI121, pGreen series, pCAMBRIA series, pPZP series, pPCV001, pGA482, pCLD04541, pBIBAC series, pYLTAC series, pSB11, pSB1, pGPTV series, and viral vectors, etc. Well-known host cell systems include, but are not limited to, expression in CHO cells.

[0089] The expression vector carrying the diakin or dual cytokine fusion protein may also contain other vector components necessary for the functionality of the vector. For example, the vector may contain signal sequences, tag sequences, protease specific sequences, selection markers, and other sequence regulatory sequences, such as promoters, necessary for proper replication and expression of the dual cytokine fusion protein. The specific promoters utilized in the vector are not particularly limited, so long as they are capable of driving the expression of the dual cytokine fusion protein in a variety of host cell types. Similarly, the type of tag promoter is not particularly limited, so long as the tag sequence makes the purification of the expressed variant IL-10 molecule easier or more straightforward. These include, for example, 6-histidine, GST, MBP, HAT, HN, S, TF, Trx, Nus, biotin, FLAG, myc, RCFP, GFP, and the like, which can be used. Protease recognition sequences can be used, for example, but are not limited to, recognition sequences for factor Xa, thrombin, HRV, 3C protease, and the like. The selection marker is not particularly limited, so long as it can detect the transformed rice plant cell, for example, a neomycin resistance gene, a kanamycin resistance gene, or a hygromycin resistance gene, and any selection marker can be used.

[0090] The diakin or dual cytokine fusion proteins described herein may also contain additional amino acid sequences that aid in the recovery or purification of the fusion protein during the manufacturing process. These may include various sequence modifications or affinity tags, such as, but not limited to, protein A, albumin binding protein, alkaline phosphatase, FLAG epitope, galactose binding protein, histidine tag, and any other tag known in the art. See, for example, Kimple et al. (Curr. Protoc. Protein Sci., 2013, 73:Unit 9.9, Table 9.91, which is incorporated herein by reference in its entirety). In one aspect, the affinity tag is a histidine tag with an amino acid sequence of 6 histidines. The histidine tag may be removed from the final product or may be left intact. In another embodiment, the affinity tag incorporated into the fusion protein (e.g., into the VH region of the fusion protein described herein) is a protein A modification. One of skill in the art will understand that any of the dual cytokine fusion protein sequences described herein may be modified to incorporate Protein A modifications by inserting point amino acid substitutions within the antibody framework regions as described in the art.

[0091] In another aspect, the proteins and nucleic acid molecules encoding the dual cytokine fusion proteins may be formulated as pharmaceutical compositions comprising a therapeutically effective amount of the dual cytokine fusion proteins and pharmaceutical carriers and / or pharma- ceutical acceptable excipients. The pharmaceutical compositions may be formulated with commonly used buffers, excipients, preservatives, and stabilizers. The pharmaceutical compositions comprising the dual cytokine fusion proteins are mixed with pharma- ceutical acceptable carriers or excipients. A variety of pharmaceutical carriers are known in the art and may be used in the pharmaceutical compositions. For example, the carrier may be any compatible, non-toxic substance suitable for delivering the dual cytokine fusion protein compositions of the present application to a patient. Examples of suitable carriers include common saline, Ringer's solution, dextrose solution, and Hank's solution. Carriers may also include any poloxamer commonly known to those of skill 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). Carriers may also include emulsifiers, 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. Carriers may also contain additives, such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives, see, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984). Formulations of therapeutic and diagnostic agents may be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers, for example in the form of a lyophilized powder, a slurry, an aqueous solution or a suspension.

[0092] 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 diakin or dual cytokine fusion protein administered is sufficient to treat or prevent cancer. The amount administered may vary between patients and needs to 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.

[0093] The effective amount for a particular patient may vary depending on factors such as the condition to be treated, the overall health of the patient, the method and dosage of the route of administration, and the severity of side effects.The appropriate dosage to be administered to a patient is typically determined by a clinician using parameters or factors that are known or suspected in the art to affect treatment, or that are predicted to affect treatment.Generally, dosage is started at an amount that is somewhat less than the optimal dosage, which is then increased in small increments until desired or optimal effect is achieved compared to any negative side effects.Important diagnostic measures include, for example, symptoms of inflammation, or the level of inflammatory cytokines produced.

[0094] The method for determining the dosage of the dual cytokine fusion protein described herein is substantially similar to that described in U.S. Patent No. 10,858,412. In general, the dual cytokine fusion protein described herein has a dosage ranging from 0.5 micrograms / kilogram to 100 micrograms / kilogram. The dual cytokine fusion protein may be administered daily, three times a week, twice a week, weekly, bimonthly, or monthly. An effective amount of a therapeutic agent affects the level of inflammation or a disease or condition by alleviating symptoms. For example, the effect may include a level of effect of at least 10%; at least 20%; at least about 30%; at least 40%; at least 50%; or higher, such that the disease or condition is reduced or completely treated.

[0095] The compositions of the present application can be administered orally or injected into the body. Formulations for oral use can also include compounds that further protect the variant IL-10 molecule from proteases in the gastrointestinal tract. Injections are usually intramuscular, subcutaneous, intradermal or intravenous. Alternatively, intraarticular injection or other routes can be used in appropriate situations. Parenterally administered dual cytokine fusion proteins are preferably formulated into unit dosage injection forms (solutions, suspensions, emulsions) in association with pharmaceutical carriers and / or pharma-ceutically acceptable excipients. In other embodiments, the compositions of the present application can be introduced into the patient's body by implantable or injectable drug delivery systems. Testing for Diakine or Dual Cytokine Fusion Proteins

[0096] A number of screening assays are known and available to those skilled in the art to test for the desired biological functions, which in one embodiment include, but are not limited to, reducing anti-inflammatory responses, reducing T cell stimulation, enhancing T cell function, enhancing Kupffer cell function, and reducing mast cell degranulation.

[0097] For example, it is known that IL-10 exposure primes T cells to generate and secrete more IFNγ upon T cell receptor stimulation. At the same time, IL-10 exposure prevents the secretion of TNFα, IL-6 and other pro-inflammatory cytokines secreted by monocytes / macrophages in response to LPS. IL-10 inhibits FoxP3 + CD4 + T reg In one embodiment, dual cytokine fusion proteins that lack T cell effects, including both stimulatory and inhibitory responses, but maximize monocyte / macrophage suppression, are positively selected. In one embodiment, screening for dual cytokine fusion proteins with increased anti-inflammatory effects is positively selected for the treatment of autoimmune disease, anti-inflammatory disease, or both. In yet another embodiment, dual cytokine fusion proteins that maximize T cell biology, including both stimulatory and inhibitory responses, and also have enhanced Kupffer cell capture are selected for development for the treatment of cancer. Various assay and screening methods for dual cytokine fusion proteins have been previously described in co-pending U.S. Patent No. 10,858,412, which is incorporated herein by reference in its entirety. See U.S. Patent Application No. 16 / 811,718, pages 39-42. Recombinantly Engineered Cells or ACTs

[0098] In one aspect, the methods disclosed herein include a combination of diakinin and ACT therapy, for example, but not limited to, recombinantly engineered cells include recombinant antigen receptors in the form of chimeric antigen receptors (CARs), TCRs or functional non-TCRs, TILs, NK cells, preferably CAR-Ts that target TAA associated with solid tumors. In some embodiments, the antigen receptor comprises an extracellular antigen recognition domain that specifically binds to the TAA antigen, and an intracellular signaling domain.

[0099] A typical recombinantly expressed CAR or TCR contains a transmembrane domain and an intracellular domain as an extracellular antigen recognition domain ("EARD").

[0100] EARD can be specific for protein, polypeptide or carbohydrate. EARD targeting TAA is particularly useful for treating cancer. For example, EARD can target TAA, such as but not limited to EGFR, VEGFR1, VEGFR2, EGP-2, EGP-4, OEPHa2, ErbB2, 3 or 4, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, carcinoembryonic antigen (CEA), prostate specific antigen (PSA), PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, and MAGE. A3, CD3, CD4, CD5, CD7, CD23, CD24, CD30, CD33, CD34, CD38, CD40, CD44, CD52, CD56, CD70, CD79B, CD117, C D123, CD138, CD147, BCMA, CLL01, LMP-1, SLAMF7, NY-ESO-1, TCAI, CS-1, CCR4, ROR1, tEGFR, MUC1, MUC16, P It can target SCA, NKG2D ligand, MART-1, gp100, tumor fetal antigen, ROR1, TAG72, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1 cell adhesion molecule, MAGE-AL mesothelin, CE7, Wilms tumor 1 (WT-1), or cyclin. Those skilled in the art will recognize that the recombinantly engineered cells that contain EARD depend on the tumor or cancer that is treated. Therefore, those skilled in the art can select the appropriate CAR-T or TCR-T with appropriate EARD to target cancer.

[0101] The transmembrane domain of the CAR or TCR is one that is normally associated with an artificial hydrophobic region, or EARD, or a commonly known transmembrane domain, including, but not limited to, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a domain from the alpha, beta, or zeta chain of the TCR.

[0102] Those skilled in the art will also recognize that the intracellular domain of a CAR-T or TCR-T cell generally comprises one or more of the following intracellular signaling domains: ITAM (e.g., CD3-zeta "CD3ζ"), costimulatory domain I ("CM1") (e.g., CD28, CD134, CD137 / 4-1BB or ICOS), and / or costimulatory domain II ("CMII") (e.g., CD134 or CD137 / 4-1BB).

[0103] The recombinantly engineered cells can be T cells, including CD4+ or CD8+ T cells. In other aspects, the T cells are either autologous or allogeneic, preferably autologous. In some embodiments, the ratio of CD4+ cells to CD8+ cells is about 1:5 to about 5:1. In some embodiments, the ratio of CD4+ cells to CD8+ cells is about 1:2 to about 2:1. In some embodiments, the dose of cells includes about 0.2×106 cells / kg to about 6×106 cells / kg of subject's body weight, about 0.5×106 cells / kg to about 3×106 cells / kg, about 0.75×106 cells / kg to about 2.5×106 cells / kg, or about 1×106 cells / kg to about 2×106 cells / kg, inclusive. Bispecific Monoclonal Antibodies

[0104] In one embodiment, the method disclosed herein includes the combination of Dikaine and bispecific monoclonal antibodies (BSMabs). BSMabs are generally known in the art and include, but are not limited to, bispecific T cell engagers (BiTEs), tandem single chain variable fragments (taFvs), diabodies (Dbs), single chain diabodies (scDbs), triple bodies or tetravalent antibodies or fragments thereof, dual affinity retargeting antibodies (DARTs), or Trident technology. Representative examples of BSMabs include BiTEs, which broadly describe bispecific monoclonal antibodies that can engage two different antigenic determinants or targets. Traditionally, BiTEs are composed of two scFv molecules, where the first scFv can recognize polyclonal immune cells (e.g., CD8+ or CD4+ T cells or NK cells) and the second scFv can recognize tumor antigen targets or TAA. This is generally achieved by having a first scFv that recognizes CD3 on the surface of the T cell (or NK cell), and a second scFv that recognizes a TAA, for example CD20.

[0105] In one aspect, the BiTE comprises an scFv with specificity for CD3, while a second scFv can have specificity for a variety of TAAs found on the surface of both hematological and solid tumors. In one embodiment, the first scFv specific for CD3 is capable of binding to EGFR, VEGFR1, VEGFR2, EGP-2, EGP-4, OEPHa2, ErbB2, 3 or 4, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, carcinoembryonic antigen (CEA), prostate specific antigen (PSA), PSMA, Her2 / neu, HER3, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, and MAGE. A3, CD3, CD4, CD5, CD7, CD23, CD24, CD30, CD33, CD34, CD38, CD40, CD44, CD52, CD56, CD70, CD79B, CD117, CD123, CD138, CD147, BC MA, CLL01, LMP-1, SLAMF7, NY-ESO-1, TCAI, CS-1, CCR4, ROR1, tEGFR, MUC1, MUC16, PSCA, NKG2D ligand, B7-H3, PD-1, PD-L1, EpCAM, P It may be combined with a second scFv specific for SCA, GPC3, AXL, claudin 18.2, CTLA-4, CEA, PSA, MART-1, gp100, oncofetal antigen, ROR1, TAG72, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1 cell adhesion molecule, MAGE-AL mesothelin, CE7, Wilms tumor 1 (WT-1), or cyclin. In one embodiment, the BSMab (more specifically, BiTE) has one specificity for a TAA, while Dikaine has specificity for a different TAA. For example, BiTE can be BSMab with specificity for CD3 and CD20, while Dikaine is DK2 with VH and VL scFv specific for CD19. 10 It is the form. Methods of treatment and / or prevention using Diakine in combination with ACT or BiTE

[0106] Without being bound to any particular theory, the inventor believes that targeting both IL-10 and IL-2 to tumor vasculature, for example by directing diakines to VEGFR2, allows activation, infiltration and persistence while limiting toxicity through both tumor-specific activation of CAR-T cells and direct suppression of cytokine release syndrome and IL-2 toxicity by DV07 (high affinity form of IL-10).The inventor also believes that targeting IL-10 alone to tumor vasculature using a single cytokine version of the fusion protein in Dvegfr2DV07, as described in US Patent No. 10,858,412 (see also Figure 1 for a representative example of structure), is also an effective tumor-specific activation of CAR-T cells. In another embodiment, based on a similar theory, DK2 contains a combination of IL-10 and IL-7, or IL-12 and IL-10, or IL-10 and IL-15, or IL-10 and IFN-alpha, or IL-10 and IL-21, or IL-10 and IL-27. 10 They believe that a form of Diakine could prime the immune system to enhance or strengthen the function of traditional ACT or BiTE therapies.

[0107] Thus, in one aspect, the present disclosure relates to a method of treating and / or preventing a malignant tumor disease or condition, or cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a diakin or dual cytokine fusion protein comprising IL-10, IL-12, IL-27, and a second cytokine, in combination with an ACT, e.g., a CAR-T cell or a TCR-T cell, or a BSMab, e.g., a BiTE. Such a dual cytokine fusion protein preferably comprises a monomer of DV07, as described above, and grafted with CDRs from any antibody-targeted tumor associated antigen ("TAA"), DK2. 10 DK7 10 , DK12 10 , DK15 10 , DKIFNa 10 , DK21 10or DK27 10 In a preferred embodiment, the dual cytokine fusion protein is in the form of DK2, DV07, 10 The dosage form is a diaquinone. Those skilled in the art will appreciate that the dosage of the diaquinone or dual cytokine fusion protein described herein may be adjusted as needed or depending on the desired outcome. In one embodiment, the diaquinone is administered to a patient in need thereof at a dose of approximately 0.001-0.25 mg / kg, preferably at a dose range of 0.01-0.2 mg / kg. In another embodiment, the dose administered to a patient in need thereof is sufficient to achieve a serum or plasma concentration in the range of 0.0005-250 ng / mL, preferably 0.001-200 ng / mL.

[0108] Those skilled in the art will recognize that adoptive cell therapy (e.g., adoptive T cell therapy) is carried out according to well-known and previously described procedures. See, for example, U.S. Patent No. 4,690,915. These methods can include autologous transfer (i.e., derived from the patient) or allogeneic transfer (i.e., derived from another subject other than the patient being treated).

[0109] CAR-T or TCR-T cells are administered by known methods and routinely performed by those familiar with adoptive cell therapy. In one embodiment, 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, peribulbar injection or posterior juxtascleral delivery. In some embodiments, they are administered parenterally, intrapulmonary, and intranasally, or intralesional or intratumoral administration. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In certain embodiments, recombinantly engineered CAR-T or TCR-T are administered as a single bolus, multiple boluses or continuous infusion.

[0110] In one embodiment, the dual cytokine fusion protein and the CAR-T are administered at separate subsequent time periods, where, for example, diakinin (e.g., DK2 10 vegfr2) is administered prior to administration of recombinantly engineered CAR-T cells. In other embodiments, the dual cytokine fusion protein and CAR-T administered are administered simultaneously. In other embodiments, diaquine is administered 1-3 days prior to CAR-T therapy and then administered simultaneously with CAR-T and / or 1-7 days after CAR-T administration. Diaquine may be administered once a day or once a week, or 2-3 times a week, in combination with or in conjunction with CAR-T. In another aspect, diaquine is utilized in the expansion and / or thawing procedure of CAR-T cells prior to administration. When reconstituting CAR-T cells from cryopreserved stocks, CAR-Ts are typically rested in the presence of CAR-T beneficial cytokines (e.g., low dose IL-2). In one aspect, CAR-T cells may be primed or expanded from cryopreserved stocks in the presence of diaquine. In one embodiment, CAR-Ts are expanded or primed in the presence of 0.001 to 300 ng / mL of diaquine, more preferably 0.01 to 200 ng / mL of diaquine.

[0111] Similarly, the diaquinone and the BiTE are administered at separate subsequent times, where the diaquinone (e.g., DK2 10 In another embodiment, the diaquinone is administered 1-3 days prior to administration of the BiTE (e.g., CD3xCD19 BiTE). In another embodiment, the diaquinone is administered 1-3 days prior to administration of the BiTE, and then administered simultaneously with the BiTE and / or 1-7 days after BiTE administration. The diaquinone may be administered once daily or weekly, or 2-3 times weekly, in combination with or in conjunction with the BiTE.

[0112] In other embodiments, the present disclosure also contemplates methods of co-administration or co-treatment with a third therapeutic agent, such as a cytokine, steroid, chemotherapeutic agent, antibiotic, anti-inflammatory agent, or radiation therapy, as are known in the art. These include other therapeutic agents, such as, but not limited to, one or more of the following: chemotherapeutic agents, interferon-β, such as IFNβ-1α and IFN-β-1β; protein stimulating myelin basic protein; corticosteroids; IL-1 inhibitors; TNF inhibitors; anti-TNFα antibodies, anti-IL-6 antibodies, IL-1br-Ig fusions, anti-IL-23 antibodies, antibodies against CD40 ligand and CD80; antagonists of IL-12 and IL-23, such as antagonists of the p40 subunit of IL-12 and IL-23 (e.g., inhibitory antibodies against the p40 subunit); IL-22 antagonists; small molecule inhibitors, such as methotrexate. These treatments may include combination treatments with methotrexate, leflunomide, sirolimus (rapamycin), and analogs thereof, such as CCI-779; Cox-2 and cPLA2 inhibitors; NSAIDs; p38 inhibitors; TPL-2; Mk-2; NFkβ inhibitors; RAGE or soluble RAGE; P-selectin or PSGL-1 inhibitors (e.g., small molecule inhibitors, antibodies thereto, e.g., antibodies to P-selectin); estrogen receptor beta (ERB) agonists or ERB-NFkβ antagonists.

[0113] In addition, useful combination treatments for administration with the dual cytokine fusion protein may include, for example, TNF inhibitors, including chimeric, humanized, effectively human, human or in vitro-generated antibodies that bind to TNF, or antigen-binding fragments thereof; soluble fragments of TNF receptors, such as p55 or p75 human TNF receptors, or derivatives thereof, such as 75kd TNFR-IgG (75kD TNF receptor-IgG fusion protein, ENBREL™), p55kD TNF receptor-IgG fusion protein; and TNF enzyme antagonists, such as TNFα-converting enzyme (TACE) inhibitors. Other combination treatments with anti-inflammatory agents / drugs include, but are not limited to, standard nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase 2 inhibitors. The NSAIDs may include aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac and / or tolmetin. The cyclooxygenase 2 inhibitor used in the compositions according to the present application may be, for example, celecoxib or rofecoxib.

[0114] Additional therapeutic agents that may be co-administered and / or co-formulated with the dual cytokine fusion protein include interferon-β, e.g., IFNβ-1α and IFNβ-1β; COPAXONE®; corticosteroids; IL-1 inhibitors; TNF antagonists (e.g., soluble fragments of TNF receptors, e.g., p55 or p75 human TNF receptors, or derivatives thereof, e.g., 75 kd TNFR-IgG; antibodies against CD40 ligand and CD80; and I antagonists of IL-12 and / or IL-23, such as antagonists of the p40 subunit of IL-12 and IL-23 (e.g., inhibitory antibodies that bind to the p40 subunit of IL-12 and IL-23); methotrexate, leflunomide, and sirolimus (rapamycin), or analogs thereof, such as one or more of CCI-779. Other therapeutic agents may include Imfimzi or Atezolizumb.

[0115] Representative chemotherapeutic agents that may be co-administered with the dual cytokine fusion proteins described herein include, but are not limited to, the following non-exhaustive list: alkylating agents, such as thiotepa and cyclosphosphamide (CYTOXAN™); alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, metuledopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide, and trimethylolomelamime; nitrogen mustards, such as chiorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novoenbiquine, phenesterine, prednisolone, phenethylamine ... nimustine, trofosfamide, uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, Xorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate;Purine analogues, for example, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;pyrimidine analogues, for example, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU;androgens, for example, calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone;antiadrenal, for example, aminoglutethimide, mitotane, trilostane;folic acid supplements, for example, floric acid;aceglatone;aldophosphamide glycosides;aminolevulinic acid;amsacrine;bestrabulil;bisantrene;edatrexate;defofamine;demecolcine;diaziquone;eflo Lunithine (elfornithine); Elliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK (registered trademark); Razoxane; Sizofiran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2',2''-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside (Ara-C); Cyclophosphamide; Thiotepa; Taxoids, such as paclitaxel (TAXOL (registered trademark) Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (Taxotere™, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; Xeloda® Roche, Switzerland; ibandronate; CPT11;Topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxyphene, keoxyphene, LY117018, onapristone and toremifene (Fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. EXAMPLES

[0116] Example 1 Diakine and CAR-T combination Various effector to target (E:T) ratios of CAR-T to tumor cells will be examined to determine whether combining Diakine with CAR-T enhances the function of CAR-T cytotoxicity. Briefly, CAR-T cells (Promab) were treated with 0, 10, or 100 ng / mL of Diakine (DK2 10 The cells are primed for 1-3 days in the presence of CD19. Raji cells stably transfected with constitutively expressing GFP are used as a model tumor cell line to examine CAR-T function. After priming, CAR-T cells are mixed at an E:T ratio of 3:1 or 1:3, infected with Raji-GFP cells, and monitored in an IncuCyte S3 system (Sartorius). The cytolytic efficacy of CAR-T cells is measured by loss of GFP over a period of approximately 48 hours.

[0117] The percentage of tumor cell growth (i.e., loss of GFP is a measure of target cells being lysed) is measured over time. After 24 hours, the combination of CD20 CAR-T cells and Raji cells with Diakine (10 ng / mL or 100 ng / mL) at a 3:1 effector-to-target cell ratio showed approximately 6.5-fold reduction in tumor cell growth when compared to CAR-T cells alone. See FIG. 9A. Similarly, at 24 hours, the combination of CD20 CAR-T cells and Raji cells with Diakine (10 ng / mL or 100 ng / mL) at a 1:3 effector-to-target cell ratio showed approximately 2-fold reduction in tumor cell growth when compared to CAR-T cells alone. See FIG. 10B. These data demonstrate that CAR-T has enhanced and more potent cytotoxic function when Diakine is used to prime CAR-T cells prior to administration. Example 2 Treatment of solid tumors with Diakine in combination with CAR-T

[0118] In this method of treating cancer, the subject is administered an effective amount of diakinin (e.g., DK2 10 vegfr2), which can target both IL-10 and IL-2 to the tumor microenvironment that overexpresses TAA, e.g., VEGFR2. Then, using standard protocols for adoptive cell therapy, CAR-T cells that express EARD targeting overexpressed antigens, e.g., her2 / neu or PSA, in tumor cells are administered. Before administration to the patient, the CAR-T cells are expanded, optionally in the presence of diaquine. In preparing the engineered cells, the CAR is selected so that its EARD specifically binds to an antigen epitope specific to the tumor to be treated. Example 3 Combination of Diakine and BiTE

[0119] The rationale for determining whether diaquine would be beneficial to current BiTE therapy was initiated by understanding how diaquine behaves when exposed to PBMCs. Using a model system for determining T cell responses (see columns 29-30 of U.S. Patent No. 10,858,412), PBMCs were exposed to various concentrations of diaquine (0 ng / mL to 10 ug / mL of DK2 10 We then exposed PBMCs to IL-1β, IFN-γ, TNF-α, IL-12p70, IFNα2a, and IL6, respectively, and to Figures 6A-6D for IL-4, IL-17, IL-8, and GM-CSF, respectively. These data support the conclusion that combining IL-10 (DV07) and IL-2 together, DK2 10 We demonstrate that diakinin, constituted as a mAb, dramatically suppresses the induction of cytokines associated with IL-2 alone, giving way to a molecule capable of enhancing cytotoxic function without a similar surge in proinflammatory cytokines.

[0120] Next, Diakin (DK2 10 CD8+ T cell responses to anti-CD3 / anti-CD28 priming were evaluated. Briefly, CD8+ T cells are magnetically selected and activated with anti-CD3 / anti-CD28 for 3 days. After activation, CD8+ T cells are rested and primed for 24, 48 and 72 hours in the presence of Diakine. After the resting and priming period (i.e., 24, 48 or 72 hours), the levels of granzyme B, IFN-γ and TNF-α are evaluated as a result of 4, 20, 48 and 72 hour anti-CD3 stimulation periods. DK2 at around 1 ng / mL 10 Priming of CD8+ T cells with a form of diakinin resulted in increased levels of granzyme B (Fig. 7) with a concomitant increase in levels of IFN-γ (Fig. 8), whereas TNF-α levels (Fig. 9) were suppressed. Taken together, these data suggest that cytotoxic T cells are able to activate DK2 10We demonstrate that when primed with this form of diakinin, induction of inflammatory cytokines is relatively suppressed and, upon stimulation, CD8+ T cells are potently activated against targets.

[0121] Using the rationale presented above, commercially available BiTEs, anti-CD3 x anti-CD19 and anti-CD3 x anti-CD20, were used in combination with Diakine DK2, respectively. 10 egfr and DK2 10 Briefly, freshly isolated CD8+ T cells from healthy donors were assessed for their cytolytic capacity against Raji cells stably transfected with constitutively expressing GFP. Raji cells have also been previously determined to express EGFR, CD19 and CD20 on the cell surface (as demonstrated by FACS, data not shown). CD8+ T cells were cultured in 0–100 ng / mL diclofenac (DK2 10 egfr and DK2 10 The Raji cells are primed for 1-2 days in the presence of CD19. The efficacy of the BiTE to lyse Raji cells is measured and monitored in an IncuCyte S3 system (Sartorius) by the disappearance of GFP over a period of approximately 48 hours. Concurrently, analyte levels of IFN-γ, TNF-α, granzyme B and performin, cytokines, proteases and proteins important for cytotoxic function, are assessed.

[0122] 11E-11F show the results of the 48-hour period of DK2 10 Figures 12E-12F provide an assessment of the combination of egfr-form diakinin with the lowest functional CD3xCD19 BiTE concentration (0.01 ng / mL) over a 48 hour period. 10 These data provide an assessment of the combination of the CD19 form of Diakin with the lowest functional CD3xCD20 BiTE concentration (0.1ng / mL). 10 egfr or DK2 10Combining CD19 with BiTEs, CD3xCD19 or CD3xCD20, respectively, suggests dramatically enhancing the safety profile (i.e., reduced inflammatory cytokine induction) or enhancing cytolytic function (see Figures 11A-D and 12A-12D).

[0123] IL-7 and IL-10 in combination with BiTE (DK7 10 ) or IL12 and IL-10 (DK12 10 Additional diakines, including , were also evaluated using the same assay described above. See Figures 13 and 14. These data suggest that combining different types of diakines in combination with BiTEs is effective in enhancing cytolytic function. Example 4 Treatment of tumors with Diakine in combination with BiTE

[0124] In this method of treating cancer, the subject is administered an effective amount of DK2 10 A form of Dikaine, capable of targeting both IL-10 and IL-2, is administered to the hematologic tumor or tumor microenvironment that overexpresses the TAA, 1-3 days prior to administration of the BiTE. Dikaine is then co-administered with the BiTE to the patient in need thereof, using standard protocols associated with BiTE administration.

[0125] This specification uses examples to disclose aspects of the present disclosure, including preferred embodiments, and to enable any person skilled in the art to practice the aspects, including making and using any device or system, and performing any incorporated methods. The patentable scope of these aspects is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements that have minor differences from the literal language of the claims. Aspects from the various embodiments described, and other known equivalents to each such aspect, may be mixed and matched by those skilled in the art to construct additional embodiments and techniques in accordance with the principles of the present application. [ka] [ka] [ka] [ka] [ka]

Claims

1. 1. A combination for treating a patient with cancer and / or tumor, said combination comprising a targeted diakin and a bispecific T cell engager (TCE), The targeted diakin comprises a first single chain variable fragment (scFv) having specificity for IL10, IL2, and a first tumor-associated antigen (TAA), wherein the TAA is selected from the group consisting of CD4, CD5, CD7, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD52, CD56, CD70, CD79B, CD117, CD123, CD138, CD147, B-cell mutated antigen (BCMA), C-type lectin-like molecule 1 (CLL01), latent membrane protein 1 (LTP1), and the like. LMP-1), signaling lymphocyte activation molecule F7 (SLAMF7), NY-ESO-1, transmembrane activator and CAML interactor (TACI), CS-1, CXCR4, NKG2D, B7-H3, EGFR, PD-1, PDL-1, HER2, HER3, EpCAM, mesothelin, PSCA, MUC1, Lewis Y, GPC3, AXL, claudin 18.2, GD2, CTLA-4, CEA, PDGFR, VEGFR2, mesothelin (MESO), PSCA, or PSA; the TCE comprises at least a first antigen-binding specificity for CD3 and a second antigen-binding specificity for a TAA that is different from the TAA-binding specificity of the Diakin scFv; Combination.

2. The combination of claim 1, wherein the cancer is a blood cancer.

3. The combination of claim 2, wherein the hematological cancer is lymphoma or leukemia.

4. 3. The combination of claim 2, wherein the hematological cancer is B-cell acute lymphocytic leukemia (B-ALL), multiple myeloma (MM), B-cell lymphoma, chronic lymphocytic leukemia (CLL), T-cell acute lymphoblastic leukemia, or non-Hodgkin's lymphoma.

5. The combination of claim 1, wherein the cancer is a solid cancer or tumor.

6. 6. The combination of claim 5, wherein the solid cancer is neuroblastoma, small cell lung cancer, melanoma, ovarian cancer, renal cell carcinoma, colon cancer, lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia and lymphoma.

7. 2. The combination of claim 1, wherein the IL10 is human IL10, EBV IL10 or CMV IL10, or a variant and / or mutein thereof.

8. 2. The combination of claim 1, wherein the diaquinone comprises IL10 of SEQ ID NO: 1, 3, 5 or 7.

9. The combination of claim 1, wherein the IL2 is human.

10. 2. The combination of claim 1, wherein the IL2 is wild-type IL2, or a variant or mutein thereof.

11. 2. The combination of claim 1, wherein said Diaquine and said BiTE are administered to said patient at separate times.

12. 2. The combination of claim 1, wherein the Diaquine is administered to the patient 1 to 3 days before the administration of the BiTE.

13. 2. The combination of claim 1, wherein said Diaquine and said BiTE are administered to said patient simultaneously.

14. 2. The combination of claim 1, wherein the diaquinone is administered to the patient 1 to 3 days before the administration of the BiTE, and then the diaquinone is readministered to the patient 1 to 3 days after the administration of the BiTE.

15. The combination according to claim 1, characterized in that said Diaquine is administered at a dosage concentration ranging from 0.001 to 200 mg / kg.

16. 2. The combination of claim 1, wherein said Diaquine is administered at a dose that achieves a serum or plasma concentration of about 0.0001 to 200 ng / mL.

17. 2. The combination of claim 1, wherein said diaquine is administered subcutaneously or intravenously.

18. A composition for treating a patient having cancer and / or a tumor, the composition comprising a targeted diakin and administered in combination with a bispecific T cell engager (TCE); The targeted diakin comprises a first single chain variable fragment (scFv) having specificity for IL10, IL2, and a first tumor-associated antigen (TAA), wherein the TAA is selected from the group consisting of CD4, CD5, CD7, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD52, CD56, CD70, CD79B, CD117, CD123, CD138, CD147, B-cell mutated antigen (BCMA), C-type lectin-like molecule 1 (CLL01), latent membrane protein 1 (LTP1), and the like. LMP-1), signaling lymphocyte activation molecule F7 (SLAMF7), NY-ESO-1, transmembrane activator and CAML interactor (TACI), CS-1, CXCR4, NKG2D, B7-H3, EGFR, PD-1, PDL-1, HER2, HER3, EpCAM, mesothelin, PSCA, MUC1, Lewis Y, GPC3, AXL, claudin 18.2, GD2, CTLA-4, CEA, PDGFR, VEGFR2, mesothelin (MESO), PSCA, or PSA; the TCE comprises at least a first antigen-binding specificity for CD3 and a second antigen-binding specificity for a TAA that is different from the TAA-binding specificity of the Diakin scFv; composition.

19. A composition for treating a patient having cancer and / or a tumor, the composition comprising a bispecific T cell engager (TCE) administered in combination with a targeted diakinin; The targeted diakin comprises a first single chain variable fragment (scFv) having specificity for IL10, IL2, and a first tumor-associated antigen (TAA), wherein the TAA is selected from the group consisting of CD4, CD5, CD7, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD52, CD56, CD70, CD79B, CD117, CD123, CD138, CD147, B-cell mutated antigen (BCMA), C-type lectin-like molecule 1 (CLL01), latent membrane protein 1 (LTP1), and the like. LMP-1), signaling lymphocyte activation molecule F7 (SLAMF7), NY-ESO-1, transmembrane activator and CAML interactor (TACI), CS-1, CXCR4, NKG2D, B7-H3, EGFR, PD-1, PDL-1, HER2, HER3, EpCAM, mesothelin, PSCA, MUC1, Lewis Y, GPC3, AXL, claudin 18.2, GD2, CTLA-4, CEA, PDGFR, VEGFR2, mesothelin (MESO), PSCA, or PSA; the TCE comprises at least a first antigen-binding specificity for CD3 and a second antigen-binding specificity for a TAA that is different from the TAA-binding specificity of the Diakin scFv; composition.

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