WO2023115033A2 - Use of dual cytokine fusion proteins comprising il-10 and adoptive cell therapies or bispecific t-cell engagers to treat cancer - Google Patents

Use of dual cytokine fusion proteins comprising il-10 and adoptive cell therapies or bispecific t-cell engagers to treat cancer Download PDF

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WO2023115033A2
WO2023115033A2 PCT/US2022/081862 US2022081862W WO2023115033A2 WO 2023115033 A2 WO2023115033 A2 WO 2023115033A2 US 2022081862 W US2022081862 W US 2022081862W WO 2023115033 A2 WO2023115033 A2 WO 2023115033A2
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diakine
cell
cancer
antibody
car
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WO2023115033A3 (en
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John Mumm
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Deka Biosciences Inc
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Deka Biosciences Inc
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Priority to AU2022413694A priority patent/AU2022413694A1/en
Priority to MX2024007404A priority patent/MX2024007404A/es
Priority to CN202280091741.8A priority patent/CN118695868A/zh
Priority to CA3240977A priority patent/CA3240977A1/en
Priority to JP2024536179A priority patent/JP2024546999A/ja
Priority to IL313588A priority patent/IL313588A/en
Priority to EP22908772.1A priority patent/EP4447996A4/en
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Definitions

  • the present disclosure relates to the field of biotechnology, and more specifically, to the use of a novel dual cytokine fusion protein, called Diakines, comprising Interleukin-10 (“IL-10”) and lnterleukin-2 (“IL-2”) in combination with conventional Adoptive Cell Therapies (“ACTs”) or Bispecific T-cell Engagers (“BiTE”) therapies to treat cancer.
  • Diakines comprising Interleukin-10 (“IL-10”) and lnterleukin-2 (“IL-2”) in combination with conventional Adoptive Cell Therapies (“ACTs”) or Bispecific T-cell Engagers (“BiTE”) therapies to treat cancer.
  • IL-10 originally named cytokine synthesis inhibitory factor (Malefyt,
  • Interleukin 10 inhbits cytokine synthesis by human monocytes An autoreglatory role of IL-10 produced by monocytes, 1991 ), is a pleiotropic cytokine known to both suppress inflammatory response (Fedorak, 2000), and more recently activate CD8 + T cells to induce Interferon y (“IFNy”) dependent anti-tumor immune responses (Mumm J. , 2011 ).
  • IFNy Interferon y dependent anti-tumor immune responses
  • IL-10 is a non-covalent homo-dimeric cytokine with structural similarities to IFNy.
  • IL-10 binds to the IL-10 receptor, which consists of two subunits of the IL10 receptor 1 (IL10R1) and two subunits of the IL-10 receptor 2 (IL10R2) (Moore, 2001 ).
  • the IL-10 receptor complex is expressed on the surface of most hematopoietic cells and most highly expressed on macrophages and T-cells. While IL-10 has been reported to be both an immunosuppressive (Schreiber, 2000) and an immunostimulatory cytokine (Mumm, 2011 ), clinical evaluation of IL-10 treatment of Crohn’s patients resulted in an inverse dose response (Fedorak, 2000; Schreiber, 2000), whereas treatment of cancer patients with PEGylated IL-10 resulted in dose titratable potent anti-tumor responses (Naing, 2018). PEGylated IL-10 anti-tumor response requires endogenous CD8+ T cells and IFNy (Mumm, 2011 ).
  • IL-2 lnterleukin-2
  • NK natural killer
  • IL-10 has been reported to suppress IL-2 driven IFNy production secreted by both NK and CD4 + T cells (Scott, 2006), but it has also been reported to act as a cofactor for IL-2 induced CD8 + T cell proliferation (Groux, 1998). It is therefore not known whether IL-2 and IL-10 will co-activate cells of the immune system or cancel each other out.
  • Epstein-Barr virus (“EBV”) IL-10 variants with one or more amino acid substitutions (at amino acid position 31 , 75, or at both positions of the mature EBV IL-10 amino acid sequence of SEQ ID No. 3) in key IL-10 receptor binding domain regions, 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.
  • EBV IL- 10 variant molecules act as IL-10 receptor agonists capable of treating immune diseases, inflammatory diseases or conditions, and in treating cancer.
  • the high affinity EBV IL-10 variant (which comprises two amino acid substitutions at positions 31 and 75 termed “DV07”), when incorporated as monomers into a single chain variable region (scFv) scaffolding system comprising non-immunogenic variable heavy (“VH”) and variable light (“VL”) regions, resulted in a half-life extended molecule that properly folded and remained functionally active.
  • VH variable heavy
  • VL variable light
  • the EBV IL-10 variants incorporated into the scaffolding system showed enhanced IL-10 function on both inflammatory cells (e.g., monocytes/macrophages/dendritic cells) and immune cells (e.g., CD8 + T-cells).
  • inflammatory cells e.g., monocytes/macrophages/dendritic cells
  • immune cells e.g., CD8 + T-cells.
  • the scFv scaffolding system utilized VH and VL regions originally obtained from a human anti-ebola antibody.
  • the single cytokine fusion protein was termed DeboDV07.
  • the inventor also found that IL-10 functionality was not disrupted when the 6 complementarity-determining regions (“CDRs”) from the VH and VL regions of the scFv originally obtained from the human anti-ebola antibody were engrafted (i.e.
  • TAA tumor associated antigen
  • the inventor improved DeboDV07 by incorporating a second cytokine into the single cytokine fusion protein (described above; see, also co-pending U.S. Application 17/199,239, filed March 11 , 2021 , incorporated by reference in its entirety).
  • a second cytokine is incorporated in the linker region between the VH and VL region of a scFv. See, e.g., Id. at FIG. 2 (a representative schematic diagram of the dual cytokine fusion protein).
  • the dual cytokine fusion protein is capable of delivering both IL-10 and another cytokine (such as but not limited to IL-2) to specific TAAs when engrafted with CDRs from various TAA targeting monoclonal antibodies.
  • DK DiakinesTM
  • ACT Adoptive Cell Therapies
  • CAR-T Chimeric Antigen Receptor T-cell
  • TCR-T engineered T-Cell Receptor T-cell
  • NK Natural Killer
  • TILs Tumor-Inflitrating Lymphocytes
  • BiTE Bispecific T-cell Engagers
  • the term “diakine” or “diakines” is a generic term that refers to a novel class of dual cytokine fusion proteins linked to together on a targeted and half-life extending scFv.
  • the therapies have exhibited significant success in hematological malignancies, but there have been limited equivalent examples when using these CAR-T cells in solid tissue tumors (Ma, 2019; Castellarin, 2018; Wagner, 2020).
  • the challenges with the current approach appear to share similarities with most immune stimulatory therapies.
  • the 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).
  • the present application overcomes this issue by using, in one aspect, a diakine comprising both (1 ) IL2 and IL10, termed DK2 10 , that controls IL-2 mediated toxicity, (2) IL12 and IL10, (3) IL7 and IL10, or (4) IL15 and IL-10.
  • DK2 10 a diakine comprising both (1 ) IL2 and IL10, termed DK2 10 , that controls IL-2 mediated toxicity, (2) IL12 and IL10, (3) IL7 and IL10, or (4) IL15 and IL-10.
  • DK2 10 By enriching the coupled IL-2 and IL10 into the tumor vasculature via targeting to a TAA, such as EGFR2, HER2, or VEGFR2 (Smith, 2010), to name a few, DK2 10 will drive TME activation of CAR-T cells, enabling activation, infiltration and persistence while limiting toxicity both through tumor specific activation of the CAR-T and directly suppressing cytokine release syndrome and IL-2 toxicity by DV07. The same premise is also believed to be promising for ACT therapies and BiTEs.
  • TAA such as EGFR2, HER2, or VEGFR2
  • the present disclosure generally relates to a method of using a dual cytokine fusion protein, termed a diakine, in combination with conventional immunotherapies.
  • the present disclosure relates to a method of using a diakine, comprising IL-10 or various IL-10 variants, a half-life extending targeting domain, and a second cytokine, in combination with conventional immunotherapies, including but not limited to, engineered immune cells (such as CAR-T cells, TCR-T cells, TILs, or NK cells) or BiTEs.
  • the method uses a diakine comprising an IL-10, such as but not limited to human, mouse, cytomegalovirus, (“CMV”), or EBV IL-10 forms or IL-10 variant molecules thereof, wherein the IL-10 variant has one or more amino acid substitution(s) that impact the IL-10 receptor binding domains.
  • CMV cytomegalovirus
  • EBV IL-10 forms or IL-10 variant molecules thereof, wherein the IL-10 variant has one or more amino acid substitution(s) that impact the IL-10 receptor binding domains.
  • the method uses a diakine comprising an IL-10, IL-12 or IL-27 or variants thereof.
  • a diakine comprising an IL-10, IL-12 or IL-27 or variants thereof.
  • Each of the aforementioned diakine varieties - comprising IL-10 IL-12, or IL-27 - will also include a second cytokine, which is a cytokine that is different from the first cytokine and works in tandem with the IL-10, IL- 12 or IL-27 or variants thereof, such that there is an additive or synergistic effect when the first and second cytokines are coupled and targeted together to a specific antigen by the half-life extending antigen targeting domain of the diakine.
  • These second cytokines include, amongst 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-a, IFN-
  • the antigen targeting domain of the diakine comprises a targeting domain selected from an antibody, antibody fragment (e.g., scFv, antigen binding fragment), or antigen binding portion that directs the diakine to a target antigen recognized by the variable heavy (VH) and variable light (VL) chain regions of the antibody, antibody fragment, or antigen binding portion thereof.
  • the antigen targeting domain is a scFv.
  • the scFv has specificity for a tumor associated antigen (TAA), the TAA being selected from a variety of antigenic targets found on the surface of solid or hematological tumors.
  • TAA tumor associated antigen
  • the antigen targeting domain is a scFv comprising 3 CDRs in the VH and 3 CDRs in the VL region of the scFv.
  • the scFv may be engrafted with 3 CDRs in the VH and 3 CDRs in the VL from another antibody, but retaining the original VH and VL framework regions.
  • the engineered cell includes 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).
  • TAA tumor associated antigen
  • the engineered cell is a T-cell.
  • the present disclosure relates to a method of using a diakine of formula (I) in combination with a TAA targeting engineered immune cell or a BiTE, wherein formula (I) is: NH 2 -(IL10)-(X 1 )-(Zn)-(X 2 )-(IL10)-COOH (Formula I); wherein
  • 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 a IL10 is monomer comprising a sequence selected from SEQ ID Nos: 1 , 3, 7, 9, or 10,
  • X 1 is a VL orVH region obtained from a first monoclonal antibody
  • X 2 is a VH orVL region obtained from the first monoclonal antibody
  • the VH and VL regions are a scFv obtained from a human anti-ebola antibody, and the VH and VL regions are engrafted with 6 CDRs (3 from the VH and 3 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-a, IFN-p, IFN-y, TGF-p, or TNF-a, TNF-p, basic FGF, EGF, PDGF, IL-4, IL-11 , or IL-13; and
  • n is an integer selected from 1 -2, wherein the second antibody is 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 maturation antigen (BCMA), C-type lectin-like molecule-1 (CLL01 ), CD5, CD147, latent membrane protein 1 (LMP-1 ), signaling lymphocytic 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, PSCA, MUC1 , Lewis-Y, GPC3, AXL, Claudin18.2, GD2, CTLA-4, CEA,
  • the present disclosure relates to a method of using an IL-10 diakine of formula (II) in combination with a TAA targeting immune cell or a BiTE
  • IL-10 is a monomer sequence selected from SEQ ID Nos: 1 , 3, 7, 9, or 10; “L” is any linker, more preferably the linker is selected from SEQ ID No: 39, 40, or 41 ;
  • X 1 is a VL or VH region obtained from a first monoclonal antibody
  • X 2 is a VH orVL region obtained from the first monoclonal antibody
  • the VH and VL regions are a scFv obtained from a human anti-ebola antibody, and the VH and VL regions are engrafted with 6 CDRs (3 from the VH and 3 from the VL) from a second antibody
  • the second antibody is 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
  • Z is a cytokine selected from 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-a, IFN-
  • n is an integer selected from 1 -2.
  • the present disclosure relates to a method of treating cancer comprising administering to a subject in need thereof, an effective amount of the diakine, preferably a DK2 10 , DK7 10 , DK12 10 , DK15 10 DK21 10 , DK27 10 , DKIFNa 10 in combination with an engineered immune cells, preferably CAR-T therapy, or a BiTE, wherein the DK2 10 , DK7 10 , DK12 10 , DK15 10 DK21 10 , DK27 10 , or DKIFNa 10 includes and anti-ebola scaffolding system (which comprises a VHA/L pair or scFv) engrafted with CDRs from an antibody with specificity for VEGFR2, CD3, CD4, CD5, CD7, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD44, CD52, CD56, CD70, CD79B, CD117, CD123, CD
  • an engineered immune cells
  • R 1 is an alpha subunit from any multi-subunit first cytokine, preferably either IL-12-alpha subunit (p35) or IL-27 alpha subunit (p28), more preferably a subunit of SEQ ID No: 45 or 47
  • R 2 is a beta subunit from any multi-subunit first cytokine, preferably either IL- 12-beta subunit (p40) or IL-27 beta subunit (EBI3), more preferably a subunit of SEQ ID No: 46 or 48; wherein when R 1 is an alpha subunit of the first cytokine, R 2 is a beta subunit of the first cytokine; or when R 1 is p35, R 2 is p40; or when R 1 is p28, R 2 is EBI3; or when R 1 is SEQ ID No: 45 or 47, R 2 is SEQ ID No: 46 or 48; or when R 1 is SEQ ID No: 46 or 48, 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 the first monoclonal antibody; wherein when X 1 is a VL, X 2 is a VH or when X 1 is a VH, X 2 is a VL;
  • Z is any cytokine that enhances the biological function of the multi-subunit cytokine, preferably IFNa-2a, IL-28, IL-29, and
  • n is an integer selected from 1 -2, wherein the first monoclonal antibody is an anti-ebola antibody that is engrafted with CDRs from a second antibody with specificity for 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-ESO-1 , TACI, CS-1 , CXCR4, NKG2D, B7-H3, EGFR, PD-1 , PDL-1 , HER2, HER3, EpCAM, PSCA, MUC1 , Lewis-Y, GPC3, AXL, Claudin18.2, GD2, CTLA-4, CEA, PDGFR, MESO, PSCA, PSMA, BCMA, or PSA.
  • the present disclosure relates to a to a method of using a diakine having two multi-subunit cytokines, such as IL12, IL-27, or IL-10, of formula (IV) in combination with a TAA targeting engineered immune cell or a BiTE, said diakine being Formula (IV):
  • R 1 is an alpha subunit of a first cytokine, such as IL-12 or IL-27 or a first monomer of a homodimeric cytokine, such as IL-10, wherein R 1 is preferably p40;
  • R 2 is a beta alpha subunit of the first cytokine, such as IL-12 or IL-27 or a second monomer of the homodimeric cytokine, such as IL-10, wherein R 2 is preferably p35;
  • “La” is any linker; preferably SEQ ID No: 43-44;
  • Lb is any 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 orVL region obtained from the first monoclonal antibody; wherein when X 1 is a VL, X 2 is a VH or when X 1 is a VH, X 2 is a VL;
  • W 1 is an alpha subunit of a first cytokine, such as IL-12 or IL-27 or a first monomer of a homodimeric cytokine, such as IL-10, preferably a first monomer of IL-10;
  • W 2 is a beta alpha subunit of the first cytokine, such as IL-12 or IL-27 or a second monomer of the homodimeric cytokine, such as IL-10, preferably a second monomer of IL-10, wherein the first monoclonal antibody is engrafted with CDRs from an antibody with specificity for 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-ESO-1 , TACI, CS-1 , CXCR4, NKG2D, B7-H3, EGFR, PD-1 , PDL-1 , HER2, HER3, EpCAM, PSCA, MUC1 , Lewis-Y,
  • 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 U.S. Patent 10,858,412.
  • FIG. 2 is a schematic diagram of a diakine embodied in the present disclosure, wherein the dual cytokine fusion protein comprises terminally linked IL-10 monomers (or IL10 variants), where a second cytokine is incorporated into the linker between the VH and VL of a scFv.
  • FIG. 3 is a schematic diagram of a diakine embodied in the present disclosure, wherein the dual cytokine fusion protein comprises two multi-subunit cytokines, wherein one is terminally linked (e.g., IL-12 or IL-27) and another is fused between the linker region of the scFv (e.g., two IL-10 monomers or IL-10 variants thereof).
  • the dual cytokine fusion protein comprises two multi-subunit cytokines, wherein one is terminally linked (e.g., IL-12 or IL-27) and another is fused between the linker region of the scFv (e.g., two IL-10 monomers or IL-10 variants thereof).
  • FIG. 4 is a schematic drawing of the proposed mechanism of utilizing a DK2 10 engrafted with CDRs targeting, for example, VEGFR2.
  • FIG. 5A-5F are grafts demonstrating the cytokine induction of IL-1 p, IFN- y, TNF-a, IL-12p70, IFNa2a, and IL-6 in naive PBMC from healthy donors in response to a diakine (DK2 10 ), IL10, or IL2.
  • DK2 10 diakine
  • FIG 6A-6D are grafts demonstrating the cytokine induction of IL-4, IL-17, IL-8, and GM-CSF in naive PBMC from healthy donors in response to a diakine (DK2 10 ), IL-10, or IL-2.
  • FIG. 7 is an evaluation of granzyme B levels in CD8+ T cells in response to escalating concentration of diakine in response to anti-CD3 stimulation at 24, 48, and 72 hours.
  • FIG. 8 is an evaluation of IFN-y levels in CD8+ T cells in response to escalating concentration of diakine in response to anti-CD3 stimulation at 24, 48, and 72 hours.
  • FIG. 9 is an evaluation of TNF-a levels in CD8+ T cells in response to escalating concentration of diakine in response to anti-CD3 stimulation at 24, 48, and 72 hours.
  • FIG. 10A & 10B shows the effects of combining a diakine (DK2 10 CD19) with a CAR T cell (CD20 CAR T) on reducing target tumor cells (Raji), wherein the CD8+ T cells are primed in the presence of a diakine for 1 day.
  • FIG. 10A is a 3:1 effector to target ratio and
  • FIG 10B is a 1 :3 effector to target ratio.
  • FIG. 11 A-11 F shows the effects of combining a diakine (DK2 10 egfr) with a BiTE (CD3xCD19 BiTE at 0.01 ng/mL)) on reducing target tumor cells (Raji), wherein the CD8+ T cells are primed in the presence of a diakine for 2 day.
  • FIG. 11A-11 D provides the cytokine secretion levels of TNF-alpha, IFN-gamma, granzyme B, and perforin of the CD8+ T cells in the presence of DK2 10 and CD19 BiTE.
  • FIG. 11 E and 11 F provide the cytolytic profiles of CD8+ T cells when combined with DK2 10 and CD19 BiTE.
  • FIG. 12A-12F shows the effects of combining a diakine (DK2 10 CD19) with a BiTE (CD3xCD20 BiTE at 0.1 ng/mL) on reducing target tumor cells (Raji), wherein the CD8+ T cells are primed in the presence of a diakine for 3 day.
  • FIG. 12A- 12D provides the cytokine secretion levels of TNF-alpha, IFN-gamma, granzyme B, and perforin of the CD8+ T cells in the presence of DK2 10 and CD20 BiTE.
  • FIG. 12E and 12F provide the cytolytic profiles of CD8+ T cells when combined with DK2 10 and CD20 BiTE.
  • FIG. 13 shows the effect of combining a diakine (DK7 10 ) with a CD19 BiTE.
  • the data provides a comparison of control (i.e., no DK7 10 , no CD19 BiTE), DK7 10 alone, CD19 BiTE alone, and the combination of DK7 10 and CD19 BiTE.
  • the data shows that the combination of DK7 10 and CD19 BiTE has enhanced cytolysis over CD19 BiTE.
  • FIG. 14 shows the effect of combining a diakine (DK12 10 ) with a CD19 BiTE.
  • the data provides a comparison of control (i.e., no DK12 10 , no CD19 BiTE), DK12 10 alone, CD19 BiTE alone, and the combination of DK12 10 and CD19 BiTE.
  • the data shows that the combination of DK12 10 and CD19 BiTE has enhanced cytolysis over CD19 BiTE.
  • diakines a class of dual cytokine fusion proteins termed diakines, whereby the diakines comprises IL-10, IL- 12, or IL-27, and methods of treating cancer comprising administering a diakine comprising IL-10 and IL-2 (DK2 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 ) in combination with a tumor associated antigen (TAA) targeting engineered immune cells or ACT (such as a CAR T, TCT T, TIL or NK ) or a BiTE.
  • TAA tumor associated antigen
  • the embodiments described herein employ conventional methods and techniques of molecular biology, biochemistry, pharmacology, chemistry, and immunology, well known to a person skilled in the art. Many of the general techniques for designing and fabricating the IL-2, IL-7, IL-10, IL- 12, IL-15, IL-21 , IFN-alpha, or IL-27 variants, including but not limited to human, mouse, CMV and/or EBV forms of IL-10, as well as the assays for testing the IL-10 variants, aglycosylated or deglycosylated forms of each of the aforementioned cytokines and variants thereof, which are achieved by known methods readily available and detailed in the art.
  • the term “about”, refers to a deviance of between 0.0001 -5% from the indicated number or range of numbers. In one embodiment, the term “about”, refers to a deviance of between 1 -10% from the indicated number or range of numbers. In one embodiment, the term “about”, refers to a deviance of up to 25% from 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 sequence homology or amino acid sequence homology when compared to a wild-type sequence.
  • interleukin-10 refers to a protein comprising two identical subunits non-covalently joined to form a homodimer, where IL-10 is an intercalated dimer of two six helix bundle (helix A-F).
  • IL-10 refers to human IL-10 (“hlL-10”; Genbank Accession No. NP_000563; or U.S. Pat. No. 6,217,857) protein (SEQ ID No: 1 ) or nucleic acid (SEQ ID No: 2); mouse IL-10 (“mlL-10”; Genbank Accession No: M37897; or U.S. Pat. No.
  • EBV- IL10 refers to the EBV homolog of IL-10 protein (SEQ ID No: 3) or the nucleic acid (SEQ ID No: 4).
  • CMV-IL10 refers to the CMV homolog of IL-10 protein (SEQ ID No: 5) or the nucleic acid (SEQ ID No: 6).
  • IL-10 refers to the individual subunits of IL-10 or variant IL-10 that, when non-covalently joined, form a homodimer of IL-10 or variant IL-10.
  • wild-type refers to the sequence of the protein (e.g. IL-10, CMV-IL10 or EBV IL- 10) as commonly found in nature in the species of origin of the specific IL-10 in question.
  • wild-type or “native” EBV IL-10 would thus correspond to an amino acid sequence that is most commonly found in nature.
  • interleukin-12 refers to a protein comprising an alpha (p35) and beta (p40) subunit, non-covalently joined to form a heterodimer.
  • interleukin-12 and IL-12 refers to human, mouse, or variant forms thereof.
  • wild-type or “native” would thus correspond to an amino acid sequence that is most commonly found in nature for the alpha and beta subunits.
  • interleukin-27 refers to a protein comprising an alpha (p28) and beta (EBI3) subunit, non-covalently joined to form a heterodimer.
  • interleukin-27 and IL-27 refers to human; mouse, or variant forms thereof.
  • wild-type or “native” would thus correspond to an amino acid sequence that is most commonly found in nature for the alpha and beta subunits.
  • variant refers to biologically active derivatives of the reference molecule, that retain a desired activity, such as, for example, anti-inflammatory activity.
  • desired activity such as, for example, anti-inflammatory activity.
  • variant refers to a compound or compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (which may be conservative in nature), and/or deletions, relative to the native molecule.
  • an EBV IL-10 variant molecule is one that differs from wild-type EBV IL-10 by having one or more amino acid (or nucleotide sequence encoding the amino acid) additions, substitutions and/or deletions.
  • an EBV IL-10 variant is one that differs from the wild type sequence of SEQ ID No.:3 by having about 1 % to 25% difference in sequence homology, which amounts to about 1 -42 amino acid difference.
  • an IL-10 variant is an EBV IL-10 comprising a A75I amino acid mutation (internally denoted as “DV06”; SEQ ID No: 14), or both V31 L and a A75I amino acid mutations (internally denoted as “DV07”; SEQ ID No: 16).
  • IL-12 variant refers to an IL-12 or IL-27 amino acid (or nucleic acid) sequence that differs from wild-type IL-12 or IL-27.
  • the difference in amino acid sequence for IL-12 or IL-27 may be additions, deletions, or substitutions within the alpha, beta, or both subunits such that there is anywhere from 1 -25% in sequence identity or homology.
  • variant forms include modifications to the glycosylation (deglycosylated or aglycosylated) forms thereof to the protein.
  • fusion protein refers to a combination or conjugation of two or more proteins or polypeptides that results in a novel arrangement of proteins that do not normally exist naturally.
  • the fusion protein is a result of covalent linkages of the two or more proteins or polypeptides.
  • the two or more proteins that make up the fusion protein may be arranged in any configuration from amino-terminal end (“NH2”) to carboxy-terminal end (“COOH”).
  • NH2 amino-terminal end
  • COOH carboxy-terminal end
  • the carboxy-terminal end of one protein may be covalently linked to either the carboxy terminal end or the amino terminal end of another protein.
  • Exemplary fusion proteins may include combining a monomeric IL-10 or a monomeric variant IL-10 molecule with one or more antibody variable domains (i.e., VH and/or VL) or single chain variable region (“scFv”).
  • sequence identity refers to an exact nucleotide-by-nucleotide or amino acid-by-amino acid correspondence. The sequence identity may range from 100% sequence identity to 50% sequence identity.
  • a percent sequence identity can be determined using a variety of methods including but not limited to a direct comparison of the sequence information between two molecules (the reference sequence and a sequence with 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 the identification of percent identity.
  • subject refers to a vertebrate, preferably a mammal.
  • Mammals include, but are not limited to, murine, rodent, simian, human, farm animals, sport animals, and certain pets.
  • administering includes routes of administration which allow the active ingredient of the application to perform their intended function.
  • an “effective amount” will treat, ameliorate or prevent a symptom or sign of the medical condition. Effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • treat 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.
  • the treatment can be any reduction from native levels and can be, but is not limited to, the complete ablation of the disease, condition, or the symptoms of the disease or condition.
  • diakine refers to a general class of dual cytokine fusion protein comprising IL-10 (monomers of IL-10), IL-12, or IL-27 or variants thereof fused together with another cytokine onto a half-life extending antigen targeting domain.
  • a diakine takes the form of Formula I, II, Illa, lllb, IV.
  • DK2 10 refers to a diakine, which is schematically represented in FIG. 2, comprising an IL-10 or IL-10 variant (e.g., SEQ ID No. 10), an IL-2 (e.g., SEQ ID No: 9) that is linked between the VH and VL regions of a scFv, where the diakine may be targeted or untargeted.
  • Targeted DK2 10 may be made into a targeting molecule by using a scFv that binds to a specific antigen, such as a TAA, or engrafting onto a scFv scaffolding CDRs from an antibody that target the TAA. These molecules will be denoted as DK2 10 (target name).
  • DK2 10 that is targeted to EGFR will be denoted as “DK2 10 (egfr)” or “DK2 10 egfr.”
  • DK2 10 egfr is SEQ ID No: 19.
  • DK2 10 that is targeted to HER2 will be denoted as “DK2 10 (her2)” or “DK2 10 her2.”
  • DK2 10 her2 is SEQ ID No: 21 , 23, or 25.
  • DK7 10 refers to a diakine, which is schematically represented in FIG. 2, comprising an IL-7 or an IL-7 variant (such as an aglycosylated or deglycosylated form) and an IL-10 and IL-10 variant (e.g., SEQ ID No: 10, or aglycosylated or deglycosylated forms thereof) that is linked between the VH and VL regions of a scFv, where the diakine may be targeted or untargeted.
  • an IL-7 or an IL-7 variant such as an aglycosylated or deglycosylated form
  • an IL-10 and IL-10 variant e.g., SEQ ID No: 10
  • SEQ ID No: 10 aglycosylated or deglycosylated forms thereof
  • DK7 10 that is targeted to: (a) HER2 will be denoted as “DK7 10 (her2)” or “DK12 10 her2”; and (b) CD20 will be denoted “DK7 10 (CD20)” or “DK12 10 CD20”
  • DK7 10 her2 is SEQ ID No: 37
  • DK7 10 CD20 is SEQ ID No: 38.
  • DK12 10 refers to a diakine, which is schematically represented in FIG.
  • Targeted DK12 10 may be made into a targeting molecule by using a scFv that binds to a specific antigen, such as a TAA or engrafting onto a scFv scaffolding CDRs from an antibody that target the TAA. These molecules will be denoted as DK12 10 (target name).
  • DK12 10 that is targeted to EGFR will be denoted as “DK12 10 (egfr)” or “DK12 10 egfr.”
  • DK12 10 egfr is SEQ ID No: 26-32.
  • DK12 10 that is targeted to: (a) HER2 will be denoted as “DK12 10 (her2)” or “DK12 10 her2”; and (b) CD20 will be denoted “DK12 10 (CD20)” or “DK12 10 CD20”
  • DK12 10 CD20 is SEQ ID No: 34 or 35.
  • DK15 10 refers to a diakine, which is schematically represented in FIG. 2, comprising an IL-15 or an IL-15 variant (such as an aglycosylated or deglycosylated form) and an IL-10 and IL-10 variant (e.g., SEQ ID No: 10, or aglycosylated or deglycosylated forms thereof) that is linked between the VH and VL regions of a scFv, where the diakine may be targeted or untargeted.
  • an IL-15 or an IL-15 variant such as an aglycosylated or deglycosylated form
  • an IL-10 and IL-10 variant e.g., SEQ ID No: 10
  • SEQ ID No: 10 aglycosylated or deglycosylated forms thereof
  • Targeted DK15 10 may be made into a targeting molecule by using a scFv that binds to a specific antigen, such as a TAA or engrafting onto a scFv scaffolding CDRs from an antibody that target the TAA.
  • DK15 10 target name
  • DK15 10 that is targeted to EGFR will be denoted as “DK15 10 (egfr)” or “DK15 10 egfr.”
  • DK21 10 refers to a diakine, which is schematically represented in FIG. 2, comprising an IL-21 or an IL-21 variant (such as an aglycosylated or deglycosylated form) and an IL-10 and IL-10 variant (e.g., SEQ ID No: 10, or aglycosylated or deglycosylated forms thereof) that is linked between the VH and VL regions of a scFv, where the diakine may be targeted or untargeted.
  • an IL-21 or an IL-21 variant such as an aglycosylated or deglycosylated form
  • an IL-10 and IL-10 variant e.g., SEQ ID No: 10
  • SEQ ID No: 10 aglycosylated or deglycosylated forms thereof
  • Targeted DK21 10 may be made into a targeting molecule by using a scFv that binds to a specific antigen, such as a TAA or engrafting onto a scFv scaffolding CDRs from an antibody that target the TAA.
  • DK21 10 target name
  • DK21 10 target name
  • DK21 10 egfr
  • DK21 10 egfr DK21 10 egfr
  • DK27 10 refers to a diakine, which is schematically represented in FIG.
  • Targeted DK27 10 may be made into a targeting molecule by using a scFv that binds to a specific antigen, such as a TAA or engrafting onto a scFv scaffolding CDRs from an antibody that target the TAA. These molecules will be denoted as DK27 10 (target name). For example, DK27 10 that is targeted to EGFR will be denoted as “DK27 10 (egfr)” or “DK27 10 egfr.”
  • DKIFNa 10 refers to a diakine, which is schematically represented in FIG. 2, comprising an IFNa or an IFNa variant (such as an aglycosylated or deglycosylated form) and an IL-10 and IL-10 variant (e.g., SEQ ID No: 10, or aglycosylated or deglycosylated forms thereof) that is linked between the VH and VL regions of a scFv, where the diakine may be targeted or untargeted.
  • IFNa or an IFNa variant such as an aglycosylated or deglycosylated form
  • IL-10 and IL-10 variant e.g., SEQ ID No: 10
  • SEQ ID No: 10 aglycosylated or deglycosylated forms thereof
  • Targeted DKIFNa 10 may be made into a targeting molecule by using a scFv that binds to a specific antigen, such as a TAA or engrafting onto a scFv scaffolding CDRs from an antibody that target the TAA.
  • DKIFNa 10 target name
  • DKIFNa 10 that is targeted to EGFR will be denoted as “DKIFNa 10 (egfr)” or “DKIFNa 10 egfr.”
  • DK12 IFNa refers to a diakine, which is schematically represented in FIG. 2, comprising an IFNa or an IFNa variant (such as an aglycosylated or deglycosylated form) and an IL-12 and IL-12 variant (e.g., aglycosylated or deglycosylated forms thereof) that is linked between the VH and VL regions of a scFv, where the diakine may be targeted or untargeted.
  • IFNa or an IFNa variant such as an aglycosylated or deglycosylated form
  • IL-12 and IL-12 variant e.g., aglycosylated or deglycosylated forms thereof
  • Targeted DK12 IFNa may be made into a targeting molecule by using a scFv that binds to a specific antigen, such as a TAA or engrafting onto a scFv scaffolding CDRs from an antibody that target the TAA.
  • DK12 IFNa target name
  • DK12 IFNa that is targeted to EGFR will be denoted as “DK12 IFNa (egfr)” or “DK12 IFNa egfr.”
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • a tumor can be benign or malignant.
  • a benign tumor is characterized as not undergoing metastasis.
  • a malignant cell is a cancer cell and can undergo metastasis.
  • Tumors on which the methods of the present application can be performed include, without limitation, adenoma, angio-sarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hamartoma, hemangioendothelioma, hemangiosarcoma, hematoma, hepato-blastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and teratoma.
  • the tumor can be chosen from acral lentiginous melanoma, actinic keratoses, adenocarcinoma, adenoid cycstic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors, bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinomas, capillary, carcinoids, carcinoma, carcinosarcoma, cavernous, cholangio-carcinoma, chondosarcoma, choriod plexus papilloma/carcinoma, clear cell carcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal, epitheloid, Ewing's sarcoma, fibrolamellar, focal nodular hyperplasia, gas
  • cancer refers to or describe the 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 and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.
  • the present disclosure relates to the use of a diakine, which was previously described in U.S. Application 17/199,239 (filed March 11 , 2021 , and incorporated by reference in its entirety), to augment or enhance the function of known immunotherapies, such as TAA targeting engineered immune cells or BiTE therapy.
  • the diakine used in the method of the present application which is an improvement on an earlier version of an IL-10 fusion protein previously described in U.S. Patents 10,858,412 and 10,975,133 (incorporated by reference it its entirety), comprises two monomers of IL-10 or IL-10 variant molecules and a second cytokine molecule linked in the hinge region of a scFv.
  • the diakine is built on the first- generation IL-10 fusion molecule (FIG. 1 ) which is described in U.S. Patents 10,858,412 and 10,975,133.
  • the first-generation IL-10 fusion protein is constructed on a VH and VL scFv scaffolding featuring two monomers of IL-10 on each end (i.e., a first IL-10 monomer on the amino terminal end and a second IL-10 monomer on the carboxy terminal end).
  • the scaffolding system comprises a scFv which was obtained from a human monoclonal anti-ebola antibody, with 6 complementarity-determining regions (“CDRs”) having CDRs 1 -3 in the VH and CDRs 1 -3 in the VL.
  • the VH and VL regions are capable of targeting the IL-10 fusion protein to a specific antigen, which is accomplished by substituting the 6 CDR regions of the VH and VL pair (3 CDRs in the VH and 3 CDRs in the VL) with 6 CDR regions from a VH and VL of a receptor or antigen targeting antibody, or antigen binding fragment thereof.
  • the ability to substitute and optimize the 6 CDR and framework regions of the scFv and to engraft CDRs into the scFv scaffolding described herein, is well known and practiced by those of skill in the art.
  • the present application relates to a dual cytokine fusion protein, termed a diakine, comprising 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 U.S. Patent 10,858,412.
  • FIG. 2 is a representative diagram of the diakine comprising IL-10.
  • the diakine utilizes a scaffolding system made up of a scFv that comprises a VH and VL whereby two monomers of IL-10 terminate the dual cytokine fusion protein at the amino and carboxy terminal ends.
  • the second cytokine is conjugated to the monomers of IL-10 (or IL-10 variants) by being fused between the VH and VL regions of the scFv, which is the hinge region of the scFv.
  • the diakine is capable of forming a functional protein complex whereby the monomers of IL-10 (or IL-10 variants) homodimerize into a functional IL-10 molecule and the VH and VL regions form a pair that associate together to form a scFv complex that permits antigen binding and recognition.
  • the diakine or dual cytokine fusion protein comprising IL-10 is a structure having formula I
  • 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 a IL10 is monomer comprising a sequence selected from SEQ ID Nos: 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 the first monoclonal antibody
  • the VH and VL regions are a scFv obtained from a human anti-ebola antibody, and the VH and VL regions are engrafted with 6 CDRs (3 from the VH and 3 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-a, IFN-
  • n is an integer selected from 1 -2
  • the IL-10 is the high affinity variant termed DV07 that comprises substitutions at amino acid positions 31 and 75 of SEQ ID No: 10.
  • the VH and VL regions are a scFv obtained from any monoclonal antibody that is capable of binding to a TAA found on the surface of a solid or hematological tumor.
  • the scFv is obtained from an antibody directed to VEGFR2.
  • the scFv is obtained from a human monoclonal anti-Ebola antibody, wherein the 6 CDRs (3 in the VH and 3 in the VL) of the anti-Ebola antibody are optionally replaced with 6 CDRs from any monoclonal antibody that is capable of binding to a TAA.
  • the scFv comprises the VH and VL framework regions from a human monoclonal anti-Ebola antibody that have been engrafted with CDRs having specificity for a TAA associated with solid or hematologic tumors.
  • the VH and VL regions of the scFv are engrafted with 6 CDRs (3 from the VH and 3 from the VL) from an anti-VEGFR2, and antibody.
  • the second cytokine is IL-2, more preferably SEQ ID No: 9.
  • the diakine or dual cytokine fusion protein comprising IL-10 is a structure having formula II
  • IL-10 is an IL-10 monomer, such as but not limited to a human, mouse, CMV, EBV IL-10, or variants 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 a first monoclonal antibody
  • X 2 is a VH orVL region obtained from the first monoclonal antibody; wherein when X 1 is a VL, X 2 is a VH or when X 1 is a VH, X 2 is a VL, and X 1 and X 2 together for a scFv; “Z” is a second cytokine, wherein the second cytokine is a cytokine other than IL-10; and
  • n is an integer selected from 1 -2.
  • the IL-10 is the high affinity variant termed DV07 that comprises substitutions at amino acid positions 31 and 75 of SEQ ID No: 10.
  • the VH and VL regions are a scFv obtained from any monoclonal antibody that is capable of binding to a TAA found on the surface of a solid or hematological tumor.
  • the scFv is obtained from an antibody directed to VEGFR2
  • the scFv is obtained from a human monoclonal anti-Ebola antibody, wherein the 6 CDRs (3 in the VH and 3 in the VL) of the anti-Ebola antibody are optionally replaced with 6 CDRs from any monoclonal antibody that is capable of binding to a TAA.
  • the scFv comprises the VH and VL framework regions from a human monoclonal anti-Ebola antibody that have been engrafted with CDRs having specificity for a TAA associated with solid or hematologic tumors.
  • the VH and VL regions of the scFv are engrafted with 6 CDRs (3 from the VH and 3 from the VL) from an anti-VEGFR2, ant antibody.
  • the second cytokine is IL-2, more preferably SEQ ID No: 9.
  • the IL-10 monomer includes 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).
  • the IL-10 monomer is a modified or variant form of EBV IL-10 (SEQ ID NO.: 3), including those that are described in U.S. Patent 10,858,412.
  • the EBV IL-10 comprises two substitutions in SEQ ID No. 3 at amino acid position 31 and 75 (“DV07”).
  • the IL-10 monomer is a sequence of SEQ ID No: 1 , 3, 7, or 10.
  • the first and second monomers of IL-10 or IL-10 variant molecule are each located at the terminal ends of the fusion protein (i.e. , the first monomer at the amino terminal end and the second monomer at the carboxy terminal end) as represented by FIG 2.
  • the present disclosure relates to a method of using an IL-12 diakine of formula (III) in combination with a TAA targeting engineered immune cell or a BiTE
  • R 1 is an alpha subunit from any multi-subunit first cytokine, preferably either IL-12-alpha subunit (p35) or IL-27 alpha subunit (p28), more preferably a subunit of SEQ ID No: 45 or 47;
  • R 2 is a beta subunit from any multi-subunit first cytokine, preferably either IL- 12-beta subunit (p40) or IL-27 beta subunit (EBI3), more preferably a subunit of SEQ ID No: 46 or 48; wherein when R 1 is an alpha subunit of the first cytokine, R 2 is a beta subunit of the first cytokine; or when R 1 is p35, R 2 is p40; or when R 1 is p28, R 2 is EBI3; or when R 1 is SEQ ID No: 45 or 47, R 2 is SEQ ID No: 46 or 48; or when R 1 is SEQ ID No: 46 or 48, 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 the first monoclonal antibody; wherein when X 1 is a VL, X 2 is a VH or when X 1 is a VH, X 2 is a VL;
  • Z is any cytokine that enhances the biological function of the multi-subunit cytokine, preferably IFNa-2a, IL-28, IL-29, and
  • n is an integer selected from 1 -2, wherein the first monoclonal antibody is an anti-ebola antibody that is engrafted with CDRs from a second antibody with specificity for 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-ESO-1 , TACI, CS-1 , CXCR4, NKG2D, B7-H3, EGFR, PD-1 , PDL-1 , HER2, HER3, EpCAM, PSCA, MUC1 , Lewis-Y, GPC3, AXL, Claudin18.2, GD2, CTLA-4, CEA, PDGFR, MESO, PSCA, PSMA, BCMA, or PSA.
  • the present disclosure relates to a to a method of using a diakine having two multi-subunit cytokines, such as IL12, IL-27, or IL-10, of formula (IV) in combination with a TAA targeting engineered immune cell or a BiTE, said diakine being Formula (IV): NH2-(R 1 )-(La)-(X 1 )-(La)-(W 1 )-(Lb)-(W 2 )-(La)-(X 2 )-(L a )-(R 2 )-COOH (Formula IV); wherein
  • R 1 is an alpha subunit of a first cytokine, such as IL-12 or IL-27 or a first monomer of a homodimeric cytokine, such as IL-10, wherein R 1 is preferably p40;
  • R 2 is a beta alpha subunit of the first cytokine, such as IL-12 or IL-27 or a second monomer of the homodimeric cytokine, such as IL-10, wherein R 2 is preferably p35;
  • “La” is any linker; preferably SEQ ID No.: 43 or 44;
  • Lb is any 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 orVL region obtained from the first monoclonal antibody; wherein when X 1 is a VL, X 2 is a VH or when X 1 is a VH, X 2 is a VL;
  • W 1 is an alpha subunit of a first cytokine, such as IL-12 or IL-27 or a first monomer of a homodimeric cytokine, such as IL-10, preferably a first monomer of IL-10;
  • W 2 is a beta alpha subunit of the first cytokine, such as IL-12 or IL-27 or a second monomer of the homodimeric cytokine, such as IL-10, preferably a second monomer of IL-10, wherein the first monoclonal antibody is engrafted with CDRs from an antibody with specificity for 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-ESO-1 , TACI, CS-1 , CXCR4, NKG2D, B7-H3, EGFR, PD-1 , PDL-1 , HER2, HER3, EpCAM, PSCA, MUC1 , Lewis-Y,
  • the VH and VL regions are from an antibody, antibody fragment, or antigen binding fragment thereof.
  • the antigen binding fragment includes, but is not limited to, a scFv, Fab, F(ab’)2, V-NAR, diabody, or nanobody.
  • the VH and VL are from a single chain variable fragment (“scFv”).
  • the scFv is obtained from a human monoclonal anti-Ebola antibody.
  • the scFv comprises the framework region from an anti-Ebola antibody and 6 CDRs (3 VH and 3 VL) from a monoclonal antibody specific for any TAA that is expressed on the surface of a solid or hematological tumor.
  • the scFv antibody or the engraftable CDRs are obtained from a monoclonal antibody selected from an antibody that is specific for 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-ESO-1 , TACI, CS-1 , CXCR4, NKG2D, B7-H3, EGFR, PD-1 , PDL-1 , HER2, HER3, EpCAM, PSCA, MUC1 , Lewis-Y, GPC3, AXL, Claudin18.2, GD2, CTLA-4, CEA, PDGFR, MESO, PSCA, PSMA, BCMA, or PSA, or multitargeting forms thereof.
  • the diakine comprising IL-10, IL-12, or IL-27 includes a VH and VL pair from a single antibody.
  • the VH and VL pair act as a scaffolding onto which monomers of IL-10 or variants thereof may be attached such that the monomers of IL-10 or variants thereof may be able to homodimerize into a functioning IL-10 molecule.
  • the VH and VL scaffolding used in the fusion protein may be selected based on the desired physical attributes needed for proper homodimerization of the IL-10 monomers or IL-10 monomer variants and/or the desire to maintain VH and VL targeting ability.
  • the 6 CDRs within the VH and VL pair (3 CDRs from the VH and 3 CDRs from VL) may also be substituted with 6 CDRs from other antibodies to obtain a specifically targeted fusion protein.
  • 3 CDRs from a VH and 3 CDRs from a VL (i.e. , a VH and VL pair) of any monoclonal antibody may be engrafted into a scaffolding system comprising SEQ No: 12, or 15.
  • the scaffolding system described in SEQ ID No: 12 or 15, when fabricated as a diakine, will also include a second cytokine linked within the hinge region of the VH and VL portions of the molecule.
  • the dual cytokine fusion protein is not intended to target any specific antigen, a VH and VL pair may be selected as the scaffolding that does not target any particular antigen (or is an antigen in low abundance in vivo), such as the VH and VL pair from an anti-HIV and/or anti-Ebola antibody.
  • the IL-10 fusion protein of the present application may include a VH and VL pair from a human anti-Ebola antibody, more preferably the VH and VL sequence found in SEQ ID No: 12 or 15.
  • the fusion protein may comprise a range of 1 -4 variable regions.
  • the variable regions may be from the same antibody or from at least two different antibodies.
  • the target specificity of the antibody variable chains or VH and VL pair or the 6 CDRs of the VH and VL pair may include, but not limited to those targeting proteins, cellular receptors, and/or tumor associated antigens.
  • the CDR regions from any VH and VL pair may be engrafted into the scaffolding system described above, such scaffolding preferably includes a system termed Debo (schematically represented by FIG. 1 ), whereby IL-10 monomers are linked to a scFv comprising VH and VL regions of a human anti-Ebola antibody and the second cytokine is linked in the hinge region of the scFv (schematically represented by FIG. 2).
  • variable regions or VH and VL pair or the 6 CDRs of the VH and VL pair are obtained from antibodies that target antigens associated with various diseases (e.g., cancer) or those that are not typically found or rarely found in the serum of a healthy subject, for example variable regions from antibodies directed to 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.
  • variable regions are obtained or derived from 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 (US Published Application 2018/0180614, incorporated by reference in its entirety, especially mAbs described in Tables 2, 3, and 4) antibodies, for example.
  • the variable regions are obtained or derived from antibodies capable of enriching the concentration of cytokines, such as IL-10 and IL-2, to a specific target area so as to enable IL-10 and IL-2 to elicit its biological effect.
  • variable regions might be obtained from antibodies specific for epidermal growth factor receptor (EGFR); CD52; CD14; various immune check point targets, such as 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, FAPa; 5T4; Trop2; EDB-FN; TGF0 Trap; MAdCAM, 07 integrin subunit; a407 integrin; a4 integrin SR-A1 ; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1 ; SR- D
  • variable regions might include those that are obtained from antibodies specific for CD3, CD4, CD5, CD7, CD19, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD56, CD70, CD79B, CD117, CD123, CD138, CD147, B cell Maturation Antigen (BCMA), -type lectin like molecule-1 (CLL01 ), latent membrane protein 1 (LMP-1 ), signaling lymphatic 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, Claudin18.2, GD2, CTLA- 4, CEA, PDGFR, mesothelin (MESO), PSCA, PSA.
  • BCMA B cell Maturation Antigen
  • CLL01 latent membrane
  • a monomer of IL-10 e.g., human, CMV, or EBV
  • variant IL-10 molecule is conjugated to either the amino terminal end or the carboxy terminal end of a variable region (VH or VL), such that the monomer IL-10 or variant IL-10 molecule is able to dimerize with one another.
  • the monomers of IL-10 or variant IL-10) are fused to the VH and VL pair in accordance to formula I or II, wherein the IL-10 monomer is an EBV IL-10, DV05, DV06, or DV07 form of IL-10.
  • the diakine, dual cytokine fusion protein or dual cytokine fusion protein complex may also have an antigen targeting functionality.
  • the diakine or dual cytokine fusion protein or dual cytokine fusion protein complex will comprise a VH and VL pair that is able to associate together to form an antigen binding site or ABS.
  • the IL-10 monomers or IL-10 variant monomers thereof will be covalently linked to the end comprising the antigen binding site.
  • the variable regions may be further modified (e.g., by addition, subtraction, or substitution) by altering one or more amino acids that reduce antigenicity in a subject.
  • modifications to the variable region may include amino acids substitutions, deletions, or additions that are found outside of the 6 CDR regions of the VH and VL regions and serve to increase stability and expression of the VH and VL regions of the scFv.
  • the modifications may include modifications wherein the CDR regions are obtained from the VH and VL regions of an anti-EGFR or anti-VEGFR1 or anti-VEGFR2 antibody and the regions outside of the CDRs are optimized to stabilize the scFv and/or optimized to increase expression, which may be used as a basis for linking the second cytokine between the VH and VL regions of the scFv.
  • the VH and VL pair form a scaffolding onto which CDR regions obtained for a plurality of antibodies may be grafted or engrafted.
  • Such antibody CDR regions include those antibodies known and described above.
  • the CDR regions in the above described VH and VL scaffolding will include the following number of amino acid positions available for CDR engraftment/insertion:
  • the dual cytokine fusion protein comprising IL-10 will include the previously described scaffolding IL-10 fusion protein where the VH and VL pair is derived from an anti-ebola antibody (such as those described in SEQ ID No: 19) whereby the 6 CDR regions from the anti-ebola antibody are removed and engrafted with a VH and VL pair of a specific targeting antibody, such as but not limited to EGFR; CD52; CD14; various immune check point 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, FAPa; 5T4; Trop2; EDB-FN; TGF0 Trap; MAdCam, 07 integrin subunit;
  • the 6 anti-ebola CDR regions are substituted with 6 CDR regions from 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.
  • the second cytokine is fused between the VH and VL of a scFv, as depicted in FIG 2.
  • the second cytokine is conjugated, fused or linked between the VH or VL region of the scFv such that the second cytokine retains its functional properties.
  • the second cytokine is different from the IL- 10 monomer.
  • the second cytokine is IL-10.
  • 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, interferons-a, -
  • the second cytokine in the diakine comprising IL-10 is an IL-2.
  • the diakine or dual cytokine fusion protein is in DK2 10 form, where the IL-10 monomer is DV07; the IL-10 variant molecule linked to a scaffolding system comprising the VH and VL framework regions from a human anti-Ebola antibody (i.e.
  • CDRs obtained from an antibody selected from an 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-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 maturation antigen (BCMA), anti-C-type lectin-like molecule-1 (CLL01 ), anti-CD5, anti-CD147, anti-latent membrane protein 1 (LMP-1 ), anti-signaling lymphocytic activation molecule F7 (SLAMF7), anti-NY-ESO-1
  • the diakine is a combination of a first cytokine (or high affinity variants thereof), a second cytokine (or high affinity variants thereof), and a targeting scFv (or CDRs obtained from monoclonal antibodies and engrafted into a framework region obtained from an anti-ebola antibody) as set forth in the Table 1 below:
  • a possible diakine or a method of using a combination of at least one of the diakines and a TAA targeted engineered immune cells, ACT, or BiTEs combinations include one of the following listed in Table 2a-2e).
  • the diakine or dual cytokine fusion protein comprising IL-10, IL-12 or IL-27 incorporates linkers.
  • linkers or spacers are used to achieve proper spatial configuration of the various fusion protein parts and therefore may select the appropriate linker to use in the formation of the dual cytokine fusion protein comprising IL-10.
  • the linker or spacer may be a random amino acid sequence (such as SEQ ID No: 39-44) or a constant region of an antibody.
  • the constant region can be derived from, but not limited to lgG1 , lgG2, lgG3, lgG4, IgA, IgM, IgD, or IgE.
  • the linker or spacer is a constant heavy (“CH”) region 1 , CH2, or CH3.
  • the linker or spacer may further comprise at least two interchain disulfide bonds.
  • the present disclosure relates to nucleic acid molecules that encode for the diakine or dual cytokine fusion protein comprising IL-10, IL-12, or IL-27 and a second cytokine.
  • These nucleic acid molecules are described in U.S. Application No. 17/110, 104.
  • the polynucleotide sequences that encode for the diakine or dual cytokine fusion protein comprising IL-10 and a second cytokine may also include modifications that do not alter the functional properties of the described dual cytokine fusion protein. Such modifications will employ conventional recombinant DNA techniques and methods.
  • the addition or substitution of specific amino acid sequences may be introduced into an IL-10 sequence at the nucleic acid (DNA) level using site-directed mutagenesis methods employing synthetic oligonucleotides, which methods are also well known in the art.
  • the nucleic acid molecules encoding the dual cytokine fusion protein comprising IL-10 and a second cytokine may include insertions, deletions, or substitutions (e.g., degenerate code) that do not alter the functionality of the IL-10 variant molecule.
  • nucleotide sequences encoding the IL-10, IL-12, IL-27 variant and/or the overall 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. Accordingly, an embodiment of the present disclosure includes a nucleic acid sequence that encodes a protein of SEQ ID Nos: 35, 46-58, or 59 but differing by 70-99% due to the degeneracy of the genetic code.
  • the nucleotide sequences encoding the diakine or dual cytokine fusion proteins described herein may further comprise well known sequences that aid in, for example, the expression, production, or secretion of the proteins. Such sequences may include, for example a leader sequence, signal peptide, and/or translation initiation sites/sequence (e.g. Kozak consensus sequence).
  • sequences described herein may also include one of more restriction enzyme sites that allow for insertion into various expression systems/vectors.
  • the nucleotide sequences encoding the dual cytokine fusion protein may be used directly in gene therapy.
  • the variant IL-10, IL-12, or IL-27 molecules or fusion protein of the present application can be delivered by any method know in the art, including direct administration of the mutant IL-10, IL-12, or IL-27 proteins and gene therapy with a vector encoding the mutant IL- 10 protein.
  • Gene therapy may be accomplished using plasmid DNA or a viral vector, such as an adeno-associated virus vector, an adenovirus vector, a retroviral vector, etc.
  • the viral vectors of the application are administered as virus particles, and in others they are administered as plasmids (e.g. as “naked” DNA).
  • nucleotide sequences include those which are already known in the art. These would include the delivery of the nucleotide sequences, such as but not limited to DNA, RNA, siRNA, mRNA, oligonucleotides, or variants thereof, encoding the IL-10 or IL-10 variant molecules by a cell penetrating peptide, a hydrophobic moiety, an electrostatic complex, a liposome, a ligand, a liposomal nanoparticle, a lipoprotein (preferably HDL or LDL), a folate targeted liposome, an antibody (such as Folate receptor, transferrin receptor), a targeting peptide, or by an aptamer.
  • the nucleotide sequences encoding IL-10, IL-12 or IL-27 variant molecules may be delivered to a subject by direct injection, infusion, patches, bandages, mist or aerosol, or by thin film delivery.
  • the nucleotide (or the protein) may be directed to any region that is desired for targeted delivery of a cytokine stimulus. These would include, for example, the lung, the Gl tract, the skin, liver, brain though intracranial injection, deep seated metastatic tumor lesions via ultrasound guided injections.
  • the present disclosure relates to methods of preparing and purifying the diakine or dual cytokine fusion protein comprising IL-10, IL-12, or IL- 27.
  • nucleic acid sequences that encode the diakine or dual cytokine fusion protein described herein may be used to recombinantly produce the fusion proteins.
  • the diakine or dual cytokine fusion protein 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 a person skilled in the art, which can be used for expression and introduction of the 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, pPCVOOl , pGA482, pCLD04541 , pBIBAC series, pYLTAC series, pSB11 , pSB1 , pGPTV series, and viral vectors and the like can be used.
  • Well known host cell systems include but not limited to expression in CHO cells.
  • the expression vectors harboring the diakine or dual cytokine fusion protein may also include other vector componentry required for vector functionality.
  • the vector may include signal sequences, tag sequences, protease identification sequences, selection markers and other sequences regulatory sequences, such as promoters, required for proper replication and expression of the dual cytokine fusion protein.
  • the particular promoters utilized in the vector are not particularly limited as long as they can drive the expression of the dual cytokine fusion protein in a variety of host cell types.
  • the type of Tag promoters are not be limited as long as the Tag sequence makes for simplier or easier purification of expressed variant IL-10 molecule easier.
  • protease can be used.
  • Protease recognition sequences are not particularly limited, for instance, recognition sequences such as Factor Xa, Thrombin, HRV, 3C protease can be used.
  • Selected markers are not particularly limited as long as these can detect transformed rice plant cells, for example, neomycin-resistant genes, kanamycin- resistant genes, hygromycin-resistant genes and the like can be used.
  • the diakine or dual cytokine fusion protein described above may also include additional amino acid sequences that aid in the recovery or purification of the fusion proteins during the manufacturing process.
  • additional amino acid sequences that aid in the recovery or purification of the fusion proteins during the manufacturing process.
  • These may include various sequence modifications or affinity tags, such as but not limited to protein A, album inbinding protein, alkaline phosphatase, FLAG epitope, galactose-binding protein, histidine tags, and any other tags that are well known in the art. See, e.g., Kimple et al (Curr. Protoc. Protein Sci. , 2013, 73: Unit 9.9, Table 9.91 , incorporated by reference in its entirety).
  • the affinity tag is an histidine tag having an amino acid sequence of 6 histidines.
  • the histidine tag may be removed or left intact from the final product.
  • the affinity tag is a protein A modification that is incorporated into the fusion protein (e.g., into the VH region of the fusion proteins described herein).
  • a person of skill in the art will understand that any dual cytokine fusion protein sequence described herein can be modified to incorporate a protein A modification by inserting amino acid point substitutions within the antibody framework regions as described in the art.
  • the protein and nucleic acid molecules encoding dual cytokine fusion protein may be formulated as a pharmaceutical composition comprising a therapeutically effective amount of the dual cytokine fusion protein and a pharmaceutical carrier and/or pharmaceutically acceptable excipients.
  • the pharmaceutical composition may be formulated with commonly used buffers, excipients, preservatives, stabilizers.
  • the pharmaceutical compositions comprising the dual cytokine fusion protein is mixed with a pharmaceutically acceptable carrier or excipient.
  • Various pharmaceutical carriers are known in the art and may be used in the pharmaceutical composition.
  • the carrier can be any compatible, nontoxic substance suitable for delivering the dual cytokine fusion protein compositions of the application to a patient.
  • Carriers may also include any poloxamers generally 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.
  • the carrier may also include additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives, see, e.g., Remington's Pharmaceutical Sciences and U.S. 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 in the form of lyophilized powders, slurries, aqueous solutions or suspensions, for example.
  • the pharmaceutical composition will be 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.
  • the amount of dual cytokine fusion protein administered will be sufficient to treat or prevent inflammatory diseases or condition.
  • the amount of dual cytokine fusion protein administered will be sufficient to treat or prevent immune diseases or disorders.
  • the amount of diakine or dual cytokine fusion protein administered will be sufficient to treat or prevent cancer.
  • the amount administered may vary from patient to patient and will need to be determined by considering the subject’s or patient’s disease or condition, the overall health of the patient, method of administration, the seventy of side-effects, and the like.
  • An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the seventy of side effects.
  • the appropriate dose administered to a patient is typically determined by a clinician using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects.
  • Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced.
  • the method for determining the dosing of the presently described dual cytokine fusion protein will be substantially similar to that described in U.S. Patent 10,858,412. Generally, the presently described dual cytokine fusion protein will have a dosing in the range of 0.5 microgram/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 therapeutic will impact the level of inflammation or disease or condition by relieving the symptom.
  • the impact might include a level of impact that is at least 10%; at least 20%; at least about 30%; at least 40%; at least 50%; or more such that the disease or condition is alleviated or fully treated.
  • compositions of the application can be administered orally or injected into the body.
  • Formulations for oral use can also include compounds to further protect the variant IL-10 molecules from proteases in the gastrointestinal tract. Injections are usually intramuscular, subcutaneous, intradermal or intravenous. Alternatively, intraarticular injection or other routes could be used in appropriate circumstances.
  • Parenterally administered dual cytokine fusion protein are preferably formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutical carrier and/or pharmaceutically acceptable excipients.
  • compositions of the application may be introduced into a patient's body by implantable or injectable drug delivery system.
  • the desired biological function includes, but are not limited to, reduced anti-inflammatory response, reduce T-cell stimulation, enhanced T-cell function, enhanced Kupffer cell functionality and reduced mast cell degranulation.
  • IL-10 exposure primes T cells to generate and secrete more IFNy upon T cell receptor stimulation. Simultaneously, IL-10 exposure prevents the secretion of TNFa, IL-6 and other pro-inflammatory cytokines secreted from monocytes/macrophages in response to LPS. IL-10 also suppresses FoxP3 + CD4 + Treg proliferation.
  • the dual cytokine fusion protein that maximize monocyte/macrophage suppression but lack T cell effects, including both stimulatory and suppressive responses will be positively selected.
  • screening for dual cytokine fusion proteins that possess increased antiinflammatory effects will be positively selected for the treatment of autoimmune, anti- inflammatory disease or both.
  • dual cytokine fusion proteins that maximize T cell biology, including both stimulatory and suppressive responses, and also possesses enhanced Kupffer cell scavenging, will be selected to develop for the treatment of cancer.
  • Various assays and methods of screening the dual cytokine fusion proteins are previously described in co-pending U.S. Patent 10,858,412, which is incorporated by reference in its entirety. See, U.S. Application 16/811 ,718 Specification at pages 39-42.
  • the methods disclosed herein will include the combination of the diakine with an ACT therapy, such as but not limited to the recombinantly engineered cell includes a recombinant antigen receptor in the form of a chimeric antigen receptor (CAR), a TCR, or a functional non-TCR, TILs, NK cells, preferably a CAR-T that targets a TAA associated with a solid tumor.
  • the antigen receptor comprises an extracellular antigen-recognition domain that specifically binds to a TAA antigen and an intracellular signaling domain.
  • a typical recombinantly expressed CAR or TCR will comprise as extracellular antigen-recognition domain (“EARD”), a transmembrane domain, and an intracellular domain.
  • EARD extracellular antigen-recognition domain
  • the EARD may be specific for proteins, polypeptides, or carbohydrates. Specifically useful for treating cancers are EARDs that target TAAs.
  • the EARD may target TAAs, 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, anti-folate 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, CD123, CD138,
  • the recombinantly engineered cell comprising the EARD will be dictated by the tumor or cancer to be treated. Accordingly, those of skill in the art will be capable of selected the appropriate CAR-T or TCR-T with the appropriate EARD to target the cancer.
  • Transmembrane domains of the CAR or the TCR are artificial hydrophobic regions or those commonly associated with the EARD or generally well- known transmembrane domains. These include, but are not limited to domains from CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154, or alpha, beta or zeta chain of the TCR.
  • the intracellular domains of the CAR-T or TCR-T cells will comprise one or more of the following intracellular signaling domains: an ITAM (e.g., CD3-zeta “CD3 ”), a costimulatory domain I (“CM1”) (e.g., CD28, CD134, CD137/4-1 BB, or lCOS), and/or a costimulatory domain II (“CMII”) (e.g., CD134 or CD137/4-1 BB).
  • ITAM e.g., CD3-zeta “CD3 ”
  • CM1 costimulatory domain I
  • CMII costimulatory domain II
  • the recombinantly engineered cell may be T-cells, including CD4+ or CD8+ T cells.
  • the T-cells are either autologous or allogenic cells, preferably autologous.
  • the ratio of CD4+ to CD8+ cells is between about 1 :5 and about 5:1. In some embodiments, the ratio of CD4+ to CD8+ cells is between about 1 :2 and about 2:1.
  • the dose of cells comprises between about 0.2x106 cells/kg body weight of the subject and about 6x106 cells/kg, about 0.5x106 cells/kg body weight of the subject and about 3x106 cells/kg, between about 0.75x106 cells/kg and about 2.5x106 cells/kg or between about 1 X106 cells/kg and about 2x106 cells/kg, each inclusive.
  • the methods disclosed herein will include the combination of a Dikaine with a bispecific monoclonal antibody (BSMab).
  • BSMab are generally known in the art and include, without limitation bispecific T cell Engagers (BiTEs), tandem single chain variable fragments (taFvs), diabodies (Dbs), single chain diabodies (scDbs), triple bodies or trivalent antibodies or fragments thereof, dualaffinity retargeting antibodies (DARTs), or Trident technology.
  • BiTEs bispecific T cell Engagers
  • taFvs tandem single chain variable fragments
  • Dbs diabodies
  • scDbs single chain diabodies
  • Trident technology dualaffinity retargeting antibodies
  • a representative example of a BSMab includes BiTEs, which are broadly described as being a bispecific monoclonal antibody capable of engaging two different antigenic determinants or targets.
  • BiTEs are composed of two scFv molecules wherein the first scFv is capable of recognizing polyclonal immune cells (e.g., CD8+or CD4+ T-cells or NK cells) and the second scFv is capable of recognizing a tumor antigenic target or TAA.
  • This is generally accomplished by having the first scFv recognizing a CD3 on the surface of a T cell (or NK cells) and the second scFv recognizing a TAA, such as CD20.
  • the BiTE will include a scFv having specificity for CD3, while the second scFv may have specificity for a variety of TAAs that are found on the surface of both hematological and solid tumors.
  • the first scFv specific for for CD3 may be combined with a second scFv specific for 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
  • the BSMab (more specifically the BiTE) will have one specificity to a TAA, while the diakine will have specificity for a different TAA.
  • the BiTE may be a BSMab having specificity for CD3 and CD20, while the Dikaine will be in DK2 10 form having a VH and VL scFv specific for CD19.
  • the inventor believes that targeting both IL-10 and IL-2 to the tumor vasculature, such as by directing a diakine to VEGFR2, will enable activation, infiltration and persistence while limiting toxicity both through tumor specific activation of the CAR-T cells and direct suppression of cytokine release syndrome and IL-2 toxicity by DV07 (the high affinity form of IL-10).
  • the inventor also believes that targeting IL-10 alone to the tumor vasculature using a single cytokine version of the fusion protein in Dvegfr2DV07, as described in U.S.
  • Patent 10,858,412 (see also, FIG.1 as a representative examples of the structure) will also be effective tumor specific activation of the CAR-T cells.
  • diakines comprising the 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.
  • the inventor believes that diakines in DK2 10 form are capable of priming the immune system to enhance or potentiate the function of conventional ACT or BiTE therapies.
  • the present disclosure relates to methods of treating and/or preventing malignant diseases or conditions or cancer comprising administering to a subject in need thereof a therapeutically effective amount of a diakine or a dual cytokine fusion protein comprising IL-10, IL-12, IL-27 and a second cytokine in combination with ACT, such as CAR-T cells or TCR-T cells, or a BSMab, such as a BiTE.
  • ACT such as CAR-T cells or TCR-T cells
  • BSMab such as a BiTE.
  • Such a dual cytokine fusion protein may preferably be in DK2 10 DK7 10 , DK12 10 , DK15 10 , DKIFNa 10 , DK21 10 , or DK27 10 form, with monomers of DV07 and engrafted with CDRs from any antibody targeting a tumor associated antigen (“TAA”).
  • TAA tumor associated antigen
  • the dual cytokine fusion protein is in DK2 10 form comprising DV07.
  • the diakine is administered to a patient in need thereof at a dose of approximately 0.001 to 0.25 mg/kg, preferably in the dose range of 0.01 to 0.2mg/kg.
  • the dose administered to the patient in need thereof is sufficient to achieve a serum or plasma concentration of 0.0005 to 250 ng/mL, preferably in the range of 0.001 to 200 ng/mL.
  • adoptive cell therapy such as adoptive T-cell therapy
  • adoptive cell therapy is well known and practiced according to procedures previously described. See, e.g., U.S. Pat. No. 4,690,915. These methods may include autologous transfer (i.e., derived from the patient) or allogenic transfer (i.e. derived from another subject other than the patient to be treated).
  • autologous transfer i.e., derived from the patient
  • allogenic transfer i.e. derived from another subject other than the patient to be treated.
  • the CAR-T or TCR-T cells are administered by methods known and conventionally practiced by those familiar with adaptive cell therapy.
  • the administration method includes, but is not limited to bolus infusion, intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • they are administered by parenteral, intrapulmonary, and intranasal, or intralesional or intrtumoral administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the recombinantly engineered CAR-T or TCR-T is administered as a single bolus administration, multiple bolus or continuous infusion.
  • the dual cytokine fusion protein and the CAR-T are administered in separate subsequent time periods, wherein, for example, the diakine (such as DK2 10 vegfr2) is administered prior to the administration of a recombinantly engineered CAR-T cell.
  • the diakine such as DK2 10 vegfr2
  • the diakine is administered 1 -3 days before the CAR-T therapy and then simultaneously administered along with the CAR-T, and/or 1 -7 days following CAR-T administration.
  • the diakine may be administered once a day or week, or 2-3 times a week in combination or conjunction with the CAR-T.
  • the diakine is utilized in the expansion and/or thawing procedure of the CAR-T cells prior to administration.
  • the CAR- T Upon reconstituting CAR-T cells from cryopreserved stock, the CAR- T are typically rested in the presence of CAR-T beneficial cytokines (e.g., low dose IL- 2).
  • the CAR-T cells may be primed or expanded from cryopreserved stocks in the presence of a diakine.
  • the CAR-T is expanded or primed in the presence of 0.001 to 300 ng/mL of a diakine, more preferably 0.01 to 200 ng/mL of a diakine.
  • the diakine and the BiTE are administered in separate subsequent time periods, wherein the diakine (e.g., DK2 10 CD20) is administered 1 -3 days before administering the BiTE (e.g., CD3xCD19 BiTE).
  • the diakine is administered 1 -3 days before the BiTE and then simultaneously administered along with the BiTE, and/or 1 -7 days following BiTE administration.
  • the diakine may be administered once a day or week, or 2-3 times a week in combination or conjunction with the BiTE.
  • the present disclosure also contemplates methods of co-administration or treatment with a third therapeutic agent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, anti-inflammatory agents, or radiation, are well known in the art.
  • a third therapeutic agent e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, anti-inflammatory agents, or radiation
  • chemotherapeutics interferon-[3, for example, IFN
  • the combination treatment useful for administration with the dual cytokine fusion protein may include TNF inhibitors include, e.g., chimeric, humanized, effectively human, human or in vitro generated antibodies, or antigenbinding fragments thereof, that bind to TNF; soluble fragments of a TNF receptor, e.g., p55 or p75 human TNF receptor or derivatives thereof, e.g., 75 kd TNFR-IgG (75 kD TNF receptor-IgG fusion protein, ENBRELTM), p55 kD TNF receptor-IgG fusion protein; and TNF enzyme antagonists, e.g., TNFa converting enzyme (TACE) inhibitors.
  • TNF inhibitors include, e.g., chimeric, humanized, effectively human, human or in vitro generated antibodies, or antigenbinding fragments thereof, that bind to TNF; soluble fragments of a TNF receptor, e.g., p55 or p75 human TNF receptor
  • NSAID non-steroidal anti-inflammatory drugs
  • cyclo-oxygenase-2 inhibitors may include aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac, and/or tolmetin.
  • the cyclo- oxygenase-2 inhibitor employed in compositions according to the application could, for example, be celecoxib or rofecoxib.
  • Additional therapeutic agents that can be co-administered and/or coformulated with the dual cytokine fusion protein include one or more of: interferon-[3, for example, IFN [3-1 a and IFN [3-1 [3; COPAXONE®; corticosteroids; IL-1 inhibitors; TNF antagonists (e.g., a soluble fragment of a TNF receptor, e.g., p55 or p75 human TNF receptor or derivatives thereof, e.g., 75 kdTNFR-IgG; antibodies to CD40 ligand and CD80; and antagonists of IL-12 and/or IL-23, e.g., antagonists of a 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 a sirolimus (rapamycin) or an analog thereof, e.g.
  • Representative chemotherapeutic agents that may be co-administered with the dual cytokine fusion protein described herein may include for following non- exhaustive list: include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime nitrogen mustards such as chiorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin,
  • paclitaxel TAXOL® Bristol-Myers Squibb Oncology, Princeton, N.J.
  • doxetaxel TexotereTM, 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 inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, 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.
  • CAR-T cells are primed 1 -3 days in the presence of diakine (DK2 10 CD19) at 0, 10, or 100 ng/mL.
  • DK2 10 CD19 diakine
  • Raji cells which were stably transfected with a constititively expressing GFP, are used as the model tumor cell line to examine CAR-T function.
  • CAR-T cell cytolytic effectiveness is measured by the disappearance of GFP over a period of approximately 48 hours.
  • the percentage of tumor cell growth i.e., disappearance of GFP is a measure that the target cells are being cytolyzed is measured over time.
  • EXAMPLE 2 Treatment of Solid Tumor with Diakine in combination with a CAR- T
  • the subject is administered an effective amount of a diakine, (e.g., DK2 10 vegfr2), which is capable of targeting both IL-10 and IL-2 to the tumor microenvironment, which overexpresses a TAA, such as VEGFR2.
  • a diakine e.g., DK2 10 vegfr2
  • a CAR-T cell expressing a EARD targeting an over-expressed antigens on tumor cells such as her2/neu or PSA
  • the CAR-T cells Prior to administering to the patient, the CAR-T cells are expanded, optionally in the presence of diakine.
  • the CAR is chosen such that its EARD specifically binds to the antigenic epitope specific for the tumor to be treated.
  • CD8+ T cell response to diakine (DK2 10 ) priming was assessed. Briefly, CD8+ T cells are magnetically sorted and activated with anti-CD3/anti-CD28 for 3 days. Following activation, the CD8+ T cells are allowed to rest and prime in the presence of a diakine for 24, 48, and 72 hours. Following the rest and priming period (i.e. , 24, 48, or 72 hours), levels of Granzyme B, IFN-y, and TNF-a are evaluated as a result of an anti-CD3 stimulation period for 4, 20, 48, and 72 hours.
  • DK2 10 diakine priming
  • CD8+ T cells are primed for a period of 1 -2 days in the presence of 0 to 100 ng/mL of diakine (DK2 10 egfr and DK2 10 CD19).
  • BiTE effectiveness to cytolyze Raji cells is measured and monitored on an IncuCyte S3 system (Sartorius) through the disappearance of GFP over a period of approximately 48 hours.
  • IFN-y, TNF-a, granzyme B, and performin which are the cytokines, proteases, and proteins important for cytotoxic function, are assessed.
  • FIG. 11 E-11 F provide the assessment of combining a diakine in DK2 10 egfrform with the lowest functional CD3xCD19 BiTE concentration (0.01 ng/mL) at a 48-hour time point.
  • FIG. 12E-12F provide the assessment of combining a diakine in DK2 10 CD19 form with the lowest functional CD3xCD20 BiTE concentration (0.1 ng/mL) at a 48-hour time point.
  • the subject is administered an effective amount of a diakine in DK2 10 form, which is capable of targeting both IL-10 and IL-2 to a hematological tumor or a tumor microenvironment, which overexpresses the TAA 1 -3 days prior to the administration of a BiTE.
  • a Dikaine is then simultaneously administered to the patient in need thereof along with the BiTE.
  • IL-4 suppresses IL-1 , TNF-a and PGE2 production by human peritoneal macrophages. Immunology.
  • Interleukin 10 inhbits cytokine synthesis by human monocytes An autoreglatory role of IL-10 produced by monocytes. JEM.
  • VEGFR2 and VEGFR-3 are Localized Primarily to the Vasculature in Human Primary Solid Cancers. Human Cancer Biology.
  • Th2 cytokines and asthma lnterleukin-4 its role in the pathogenesis of asthma, and targeting it for asthma treatment with interleukin- 4 receptor antagonists. Respiratory Research.

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KR1020247023470A KR20240161974A (ko) 2021-12-16 2022-12-16 암을 치료하기 위한 il-10을 포함하는 이중 시토카인 융합체 및 입양 세포 요법 또는 이중특이적 t-세포 연관체
AU2022413694A AU2022413694A1 (en) 2021-12-16 2022-12-16 Dual cytokine fusions comprising il-10 and adoptive cell therapies or bispecific t-cell engagers to treat cancer
MX2024007404A MX2024007404A (es) 2021-12-16 2022-12-16 Fusiones de citocinas dobles que comprenden interleucina 10 (il-10) y terapias con celulas adoptivas o activadoras de celulas t biespecificas para tratar el cancer.
CN202280091741.8A CN118695868A (zh) 2021-12-16 2022-12-16 包含il-10的双细胞因子融合蛋白与过继细胞疗法或双特异性t细胞衔接器用于治疗癌症
CA3240977A CA3240977A1 (en) 2021-12-16 2022-12-16 Use of dual cytokine fusion proteins comprising il-10 and adoptive cell therapies or bispecific t-cell engagers to treat cancer
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EP4482512A4 (en) * 2022-02-22 2026-03-11 Deka Biosciences Inc METHOD FOR REDUCING A BISPECIFIC T LYMPHOCYTE RECRUITER OR A CHIMERICAL ANTIGEN RECEPTOR T LYMPHOCYTE CYTOKINE RELEASE SYNDROME USING INTERLEUKIN-4, -10, OR AN ASSOCIATED FUSION PROTEIN

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CA3186753A1 (en) * 2020-07-20 2022-01-27 John Mumm Dual cytokine fusion proteins comprising il-10
CN117327184B (zh) * 2023-12-01 2024-03-05 赛奥斯博生物科技(北京)有限公司 一种靶向msln的嵌合抗原受体及其应用

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CA3186753A1 (en) * 2020-07-20 2022-01-27 John Mumm Dual cytokine fusion proteins comprising il-10

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EP4482512A4 (en) * 2022-02-22 2026-03-11 Deka Biosciences Inc METHOD FOR REDUCING A BISPECIFIC T LYMPHOCYTE RECRUITER OR A CHIMERICAL ANTIGEN RECEPTOR T LYMPHOCYTE CYTOKINE RELEASE SYNDROME USING INTERLEUKIN-4, -10, OR AN ASSOCIATED FUSION PROTEIN

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