Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/208—IL-12
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2839—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
  • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5434—IL-12
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/626—Diabody or triabody
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

• compositions comprising a recombinant protein comprising interleukin- 12 and an antibody binding the extra-domain B of fibronectin
• the present application relates to dosage units and regimen, uses, methods or formulations of compositions comprising a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target-binding fragment or derivative thereof.
• IL-12 interleukin- 12
• ED-B extra-domain B
• IL-12 is a heterodimeric cytokine comprising two disulfide-linked subunits, p35 and p40.
• IL- 12 stimulates the production of IFNy from T-cells and natural killer cells, and also induces i differentiation of Thl helper cells.
• IL-12 is a key mediator of innate and cell-mediated immunity, with the potential for anti-cancer and anti-metastatic activity.
• IL-12 Like many other cytokines, however, the administration of IL-12 is associated with severe toxicity (Car et ah, 1999), even at doses as low as lpg per kg per day, discouraging its development as an anticancer drug.
• cytokines have shown beneficial effects in preclinical animal models of cancer and immune disorders and represent promising agents for therapy.
• drugs e.g., interleukin 2 (IL2, Proleukin®), tumor necrosis factor (TNF, Beromun®), interferon alpha (IFNa, Roferon A® and Intron A®)).
• IL2 interleukin 2
• TNF tumor necrosis factor
• Beromun® tumor necrosis factor
• IFNa interferon alpha
• IFNa Interferon A® and Intron A®
• Current indications in cancer include metastatic renal cell cancer, malignant melanoma, hairy cell leukemia, chronic myeloid lymphoma, sarcoma and multiple myeloma, either as single agents or in combination with chemotherapy.
• certain cytokines are used for the treatment of viral and bacterial infections in the clinic and are administered to patients suffering from chronic inflammatory conditions. Unfortunately, considerable toxicities can be observed at low doses, which prevent escalation
• the formulation of recombinant proteins for therapeutic use is a complex optimization process utilizing unique pharmaceutical additives to address the varying demands of storage and route of administration necessary for the clinical application.
• Immunocytokines are recombinant proteins comprising a protein-based binding molecule, mostly an antibody, and a cytokine.
• immunocytokines vary significantly, in their chemi co-physical properties, from antibodies. This applies to, for example, the domain structure, the molecular weight, or the number of inter- and intrachain disulfide bridges, and so on.
• solubility, the aggregation behavior, and the pharmacodynamics of immunocytokines may vary significantly from those known from antibodies. Lessons learned from antibodies can hence not simply be transferred to immunocytokines.
• IL12-L19L19 designates the recombinant protein comprising (i) interleukin- 12 (IL- 12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof, which is provided in the single chain diabody format (also called “tandem diabody”) as explained herein.
• the recombinant protein comprises the amino acid according to SEQ ID NO: 9.
• IL12-L19L19 Formulation Study SEC analysis of the different formulated samples, before and after the concentration step. Table 4. IL12-L19L19 Formulation Study. Stability study under stress conditions: evaluation of the loss in A280 nm after the 3 rd cycle of freezing/thawing and classification of the samples from the best to the worst, based on this loss.
• FIG. 3 Schematic drawing of IL12-L19L19.
• the molecule consists of a single-chain polypeptide consisting of the two subunits of the immunomodulatory payload IL12 fused to a human vascular targeting antibody in a single-chain diabody.
• the present invention provides, among other things, dosage units, uses, methods or formulations of compositions comprising a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof.
• a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof.
• a dosage unit comprising > 0.1 pg kg 1 , relative to a human patient’s body weight, of a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof.
• a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof.
• AS1409 is disclosed, which is a fusion protein comprising the humanised antibody BC1 in IgG format linked to interleukin- 12 (IL-12). It is designed to deliver IL-12 to tumor-associated vasculature using an antibody targeting the ED-B variant of fibronectin.
• IL-12 interleukin- 12
• a phase 1 trial of weekly infusional AS 1409 was carried out in renal carcinoma and malignant melanoma patients.
• Safety, efficacy, markers of IL-12-mediated immune response, and pharmacokinetics were evaluated. Doses of 15pg/kg and 25pg/kg were studied. The study demonstrated the safety of this approach, and provided pharmacodynamic support for the proposed mechanism of action.
• efficacy against metastatic melanoma it was stipulated that the experiments provided a rationale for progression to a phase II trial. However, no phase II trial was ever launched.
• immunocytokines have a potentiating effect on the immune system - with each cytokine being capable of stimulating two or more immune cells - finding of a safe and efficient dosage is much more difficult than e.g. in antibody therapy, where the abundance of a given target - either a ligand that is to be inactivated, or a receptor that is to be blocked - is known, and a clear stochiometric relationship between antibody and target can be calculated.
• NHS-IL12 Tumor-Targeted Cytokine
• the NHS-IL12 immunocytokine is composed of two IL-12 heterodimers, each fused to one of the H chains of the anti-histone antibody NHS76.
• MTD maximum tolerated dose
• the fusion protein consists of murine interleukin- 12 in single-chain format, sequentially fused to the anti EDB antibody L19 in a single-chain diabody (also defined as “tandem diabody”) format.
• L19-mIL12 was very well tolerated at a dose of 12 pg, which is equivalent to the human dose of 2 mg, under consideration of the body surface scaling factor - a dose which is by far higher that what has been reported by Rudman et al. and Straus et al.
• a dosage unit or regimen comprising ⁇ 100 pg kg 1 , relative to a human patient’s body weight, of a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof.
• the dosage unit or regimen comprises > 0,1 pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises > 0,25 pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises > 0,5 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 1 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 2 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 3 pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises > 4 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 5 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 6 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 7 pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises > 8 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 9 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 10 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 11 pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises > 12 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 13 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 14 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 15 pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises > 16 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 17 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 18 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 19 pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises > 20 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 21 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 22 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 23 pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises > 24 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 25 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 26 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 27 pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises > 28 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 29 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises > 30 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein.
• the dosage unit or regimen comprises ⁇ 95 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises ⁇ 90 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises ⁇ 85 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises ⁇ 80 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein.
• the dosage unit or regimen comprises ⁇ 75 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises ⁇ 70 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises ⁇ 65 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises ⁇ 60 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein.
• the dosage unit or regimen comprises ⁇ 55 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises ⁇ 50 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises ⁇ 45 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises ⁇ 40 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein.
• the dosage unit or regimen comprises ⁇ 35 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises ⁇ 32 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises ⁇ 30 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises ⁇ 25 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein.
• the dosage unit or regimen comprises between > 2 and ⁇ 20pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 3 and ⁇ 25 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 4 and ⁇ 30pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 5 and ⁇ 35pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises between > 6 and ⁇ 40pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 7 and ⁇ 45 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 8 and ⁇ 50pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 9 and ⁇ 55pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises between > 10 and ⁇ 60pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 11 and ⁇ 65 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 12 and ⁇ 70pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 13 and ⁇ 75pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises between > 14 and ⁇ 80pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 15 and ⁇ 85 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 16 and ⁇ 90pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 17 and ⁇ 95 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 18 and ⁇ lOOpg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises between > 2 and ⁇ 30 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 4 and ⁇ 35 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 6 and ⁇ 40 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 8 and ⁇ 45 pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises between > 10 and ⁇ 50 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 12 and ⁇ 55 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 14 and ⁇ 60 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 16 and ⁇ 65 pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises between > 18 and ⁇ 70 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 20 and ⁇ 75 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 22 and ⁇ 80 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 24 and ⁇ 85 pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises between > 26 and ⁇ 90 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 28 and ⁇ 95 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 30 and ⁇ 100 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 32 and ⁇ 105 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 34 and ⁇ 110 pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises between > 2 and ⁇ 30pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 6 and ⁇ 35 pg kg 1 , relative to a human patient’ s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 10 and ⁇ 40pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 14 and ⁇ 45 pg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises between > 18 and ⁇ 50pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 22 and ⁇ 55pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 26 and ⁇ 60pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between
• the dosage unit or regimen comprises between > 34 and ⁇ 70pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 38 and ⁇ 75pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 42 and ⁇ 80pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between
• the dosage unit or regimen comprises between > 50 and ⁇ 90pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 54 and ⁇ 95 pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 58 and ⁇ lOOpg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• the dosage unit or regimen comprises between > 62 and ⁇ 105pg kg 1 , relative to a human patient’s body weight of the recombinant protein. In some embodiments, the dosage unit or regimen comprises between > 66 and ⁇ 1 lOpg kg 1 , relative to a human patient’s body weight of the recombinant protein.
• a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof, for use in the treatment of a patient being diagnosed for, or suffering from, cancer, wherein the recombinant protein is administered in a dose according to the above description.
• IL-12 interleukin- 12
• ED-B extra-domain B
• a method of treating a patient comprising administering to the patient a composition comprising a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof, in a dose according to the dosage unit of according to the above description.
• a composition comprising a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof, in a dose according to the dosage unit of according to the above description.
• IL-12 interleukin- 12
• ED-B extra-domain B
• the dose is administered to the patient daily. According to some embodiments of the recombinant protein for use or the method of treating the dose is administered to the patient halfweekly. According to some embodiments of the recombinant protein for use or the method of treating the dose is administered to the patient weekly. According to some embodiments of the recombinant protein for use or the method of treating the dose is administered to the patient, biweekly. According to some embodiments of the recombinant protein for use or the method of treating the dose is administered to the patient triweekly. According to some embodiments of the recombinant protein for use or the method of treating the dose is administered to the patient monthly. According to some embodiments of the recombinant protein for use or the method of treating, the dose is administered to the patient bimonthly.
• a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof, relative to a human patient’s body weight, provide a good compromise between good efficacy and reduced side effects.
• IL-12 interleukin- 12
• ED-B extra-domain B
• any one of the above dosage regimens can be administered lx, 2x, 3x, 4x, 5x, 6x, 7x or 8x, based on the suggested interval of half weekly to monthly.
• a dosage unit or regimen comprising between > 0,5 pg kg 1 and ⁇ 4 pg kg 1 , relative to a human patient’s body weight, of a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof, is provided.
• a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof.
• the following table shows total dosages for patients with different body weights (kg) per each administration in pg, based on the different dosage units/dosage regimen (pg/kg _1 ).
• a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof, for (use in) the treatment of a patient being diagnosed for, or suffering from, cancer.
• the cancer is advanced/metastatic immunotherapy responsive solid carcinoma or lymphoma.
• a method of treating a patient comprising administering to the patient a composition comprising, in a therapeutically sufficient dose, a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof.
• a composition comprising, in a therapeutically sufficient dose, a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof.
• IL-12 interleukin- 12
• ED-B extra-domain B
• the carcinoma or lymphoma is malignant melanoma.
• the carcinoma or lymphoma is non-small cell lung cancer (NSCLC).
• NSCLC non-small cell lung cancer
• the carcinoma or lymphoma is renal cell carcinoma.
• the carcinoma or lymphoma is urothelial carcinoma.
• the carcinoma or lymphoma is head and neck squamous cell carcinoma (HNSCC).
• HNSCC head and neck squamous cell carcinoma
• the carcinoma or lymphoma is microsatellite instability -high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer.
• MSI-H microsatellite instability -high
• dMMR mismatch repair deficient metastatic colorectal cancer
• the carcinoma or lymphoma is hepatocellular cancer.
• the carcinoma or lymphoma is gastric cancer.
• the carcinoma or lymphoma is squamous cell carcinoma of the skin.
• the carcinoma or lymphoma is cervical cancer.
• the carcinoma or lymphoma is diffuse large B-cell lymphoma (DLBCL).
• the carcinoma or lymphoma has progressed on immune checkpoint-blockade therapy
• the carcinoma or lymphoma has an Eastern cooperative oncology group (ECOG) performance status ⁇ 2
• the carcinoma or lymphoma is characterized by at least one unidimensionally measurable lesion either by computed tomography (CT), MRI or PET/CT as defined by RECIST (v. 1.1) for solid tumors or by LUGANO criteria for malignant lymphoma.
• CT computed tomography
• MRI magnetic resonance imaging
• PET/CT magnetic resonance imaging
• RECIST v. 1.1
• the patient has received an immune checkpoint blockade therapy-based regimen as immediate prior treatment.
• the patient has had clinical benefit (CR/PR/SD) while on immune checkpoint blockade therapy defined as > 3 month free from progression from initial imaging documenting metastatic disease followed by radiographic disease progression after immune checkpoint blockade therapy.
• the patient has received >2 prior systemic therapies, when being diagnosed for, or suffering from, DLCBL.
• the patient has been negatively tested for HIV, HBV and HCV.
• CR/PR/SD relate to responses as defined by the Response Evaluation Criteria In Solid Tumors (RECIST) criteria.
• Complete response (CR) Disappearance of all target lesions
• Partial response (PR) at least a 30% decrease in the sum of the LD of target lesions, taking as reference the baseline sum LD
• Stable disease (SD) Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum LD since the treatment started.
• the term casualECOG performance status is a score which describes a patient’s level of functioning in terms of their ability to care for themselves, daily activity, and physical ability (walking, working, etc.), as developed by the Eastern Cooperative Oncology Group.
• the term broughtimmune checkpoint-blockade therapy“ as used herein, relates to a therapy that uses medications known as immune checkpoint inhibitors to address several types of cancer. Specifically, these medications can help the body's immune system recognize and attack cancerous cells.
• Immune checkpoint inhibitors include inhibitors or antagonists to inter alia CTLA-4, PD-1, PD-L1, LAG 3, TIM3 and 0X40.
• Some well-established immune checkpoint inhibitors are Ipilimumab, Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab and Cemiplimab.
• a pharmaceutical formulation comprising a recombinant protein comprising (i) interleukin- 12 (IL-12) and (ii) an antibody binding the extra-domain B (ED-B) of fibronectin, or a target binding fragment or derivative thereof.
• the formulation can comprise Histidine.
• the formulation can comprise Sucrose.
• the formulation can comprise EDTA.
• the formulation can comprise Histidine and Sucrose and optionally EDTA.
• the formulation can comprise citric acid. In one embodiment, the formulation can comprise sodium citrate. In one embodiment, the formulation can comprise Sucrose. In one embodiment, the formulation can comprise Glycerol. In one embodiment, the formulation can comprise EDTA. In one embodiment, the formulation can comprise citric acid and sodium citrate and optionally at least one of Sucrose, Glycerol and EDTA.
• the formulation can comprise HEPES. In one embodiment, the formulation can comprise NaCl. In one embodiment, the formulation can comprise Mannitol. In one embodiment, the formulation can comprise Glycerol. In one embodiment, the formulation can comprise EDTA. In one embodiment, the formulation can comprise HEPES and NaCl and optionally at least one of Mannitol, Glycerol and, EDTA.
• the formulation can comprise Histidine, Sucrose and EDTA, adjusted to have a pH of 8.0 ⁇ 0.3.
• the formulation can comprise citric acid, sodium citrate, Sucrose, Glycerol, EDTA, adjusted to have a pH of 6.0 ⁇ 0.3.
• the formulation can comprise Hepes, NaCl, Mannitol, Glycerol, EDTA, adjusted to have a pH of 7.0 ⁇ 0.3.
• AS 1409 (Rudman etal ., discussed above), was supplied as a lmg/ml solution in aqueous buffer at pH 6.0. It was administered to patients following a 1 to 1 dilution with 0.9% sodium chloride.
• HS-IL12 Strauss etal ., discussed above
• L19- mIL12 was diluted in phosphate buffer saline.
• Application W02018011404A1 assigned to the applicant of the present invention discloses a formulation for an immunocytokine comprising the antibody L19 and the cytokine TNF.
• example 4 disclosed therein on pages 25 - 27, the following formulations were inter alia investigated:
• Hist-1 comprises 20 mM histidine at pH 6.0, 8.5% Sucrose (w/v), 130 mM EDTA.
• Hist-2 comprises 20 mM histidine at pH 8.0, 8.5% Sucrose (w/v), 130 mM EDTA.
• Hist-3 comprises 20 mM histidine at pH 9.0, 8.5% Sucrose (w/v), 130 mM EDTA.
• Citrate-1 comprises 5.6 g/L sodium Citrate, 0.21 g/L citric acid, 70 g/L trehalose dihydrate, 0.2 g/L polysorbate80, 1% (w/v) glycerol, 5mM EDTA, pH 6.6.
• Hepes buffers prepared at pH 7.5 and 8.0 • Hepes-1 comprises 30 mM Hepes at pH 7.5, 5 mM EDTA, 75 mM mannitol and 1.8% glycerol (w/v)
• Hepes-2 comprises 30 mM Hepes at pH 7.5, 5 mM EDTA, 75 mM mannitol, 1.8% glycerol (w/v) and 0.1% polysorbate20
• Hepes-3 comprises 15 mM Hepes at pH 8.0, 5 mM EDTA, 75 mM mannitol and 1.8% glycerol (w/v)
• Hepes-4 comprises 15 mM Hepes at pH 8.0, 5 mM EDTA, 75 mM mannitol, 1.8% glycerol (w/v) and 0.005% polysorbate20
• Hepes-5 comprises 15 mM Hepes at pH 8.0, 5 mM EDTA, 75 mM mannitol, 1.8% glycerol (w/v) and 0.01% polysorbate20
• Hepes- 6 comprises 15 mM Hepes at pH 8.0, 5 mM EDTA, 75 mM mannitol, 1.8% glycerol (w/v) and 0.05% polysorbate20
• Histidine buffers no acceptance criteria were met.
• citrate buffer both the visual clarity and A280 stability criteria were met but the purity criteria was not met indicating particles in suspension or aggregation of the trimer. Histidine and citrate buffers showed particles in suspension and were not considered for further investigation.
• the recombinant protein for use, the method of treatment or the formulation of the invention comprises at least one single-chain Fv (scFv) antibody fragment, optionally a single chain diabody.
• scFv single-chain Fv
• single chain diabody relates to a construct of two single chain Fv (scFv) antibodies with a short linker, preferably 3 - 10 amino acids long, more preferably 5 amino acid long (also known as “diabodies”), joined to one another by a longer linker, preferably 5 - 20 amino acids long, more preferably 15 amino acid long, according to the following scheme (N->C orientation): L19VH-linker-L19VL-linker-L19VH-linker- L19VL.
• the recombinant protein for use, the method of treatment or the formulation of the invention comprises a p40 subunit and a p35 subunit, linked by a linker.
• the recombinant protein for use, the method of treatment or the formulation of the invention comprise the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3, respectively.
• the recombinant protein for use, the method of treatment or the formulation of the invention is the anti-EDB antibody L19, comprising the amino acid sequence according to SEQ ID NO: 5 as VL domain and SEQ ID NO: 7 as VH domain.
• the recombinant protein for use, the method of treatment or the formulation of the invention at least one of the antibody, the IL-12 and/or the linker connecting the two is one disclosed in WO2019154986, optionally wherein the recombinant protein is one disclosed in WO2019154986.
• WO2019154986 describes technical and physiological properties of the recombinant protein that is subject to the present invention.
• the content of WO2019154986A1 is incorporated by reference herein.
• the recombinant protein for use, the method of treatment or the formulation of the invention comprises
• the recombinant protein for use, the method of treatment or the formulation of the invention comprises, optionally consists of, the amino acid sequence according to SEQ ID NO: 9.
• the recombinant protein for use, the method of treatment or the formulation of the invention administration is done intravenously or subcutaneously.
• the dosage unit or recombinant protein for use is provided in a formulation according to the above description.
• the formulation according to the above description comprises a dosage unit or recombinant protein according to the above description.
• kits of parts comprising a) the dosage unit or recombinant protein for use according to the above description b) an apparatus for administering the dosage unit or recombinant protein, and, optionally c) instructions for use. is provided.
• the extradomain B of fibronectin which is targeted by the L19 antibody in IL12-L19L19, is a very well conserved domain across species and is 100 % identical in man and monkeys.
• the homology between human and monkey IL12 is 98% and 95% for p40 and p35, respectively, and human IL12 is active both in man and monkey.
• the activity of IL12-L19L19 has been tested on human and monkey peripheral blood mononuclear cells (PBMCs).
• PBMCs peripheral blood mononuclear cells
• IL12- L19L19 and recombinant human IL12 have comparable activities with regard to IFNy increase on both human and monkey PBMCs.
• both IL12-L19L19 and recombinant IL12 are lOx less active in monkey than in human PBMCs.
• the concentration of the formulated protein is done by using Amicon device (lOkDa cut-off) and centrifugation.
• the product is formulated at 2mg/ml.
• L19L19 it was expressed by TGE procedure (i.e. Transient Gene expression) and purified on Protein A resin by eluting with TEA lOOmM native pH, then it was desalted in PBS as described in WO2019154896.
• the total yield (i.e., formulation + concentration steps) was between 86.2% and 90.2%, so very comparable data were recorded among the different formulation conditions investigated.
• the single chain diabody purity was between 95.49% and 96.11% after the formulation step. Considering that the purity of the input sample was 96.06%, the recorded data after the formulation attest that no aggregates formation was induced by the buffer exchange. After concentration up to 2 mg/ml, the SEC results show the % of single chain diabody between 93.15% and 94.89% showing a small increase of the high MW form ( ⁇ 3%), due to the concentration procedure itself.
• Figures 1 and 2 show the SEC profiles of the sample after formulation and after concentration.
• Table 3 reports the recorded results during the stability study, for each formulated sample.
• IL12-L19L19 The stability of IL12-L19L19 was investigated in 7 formulation buffers under stress conditions (i.e. 3 cycles of freezing at -80°C and thawing at RT) and after 14 days of storage at 2-8°C. The visual appearance was very good for all samples.
• A280 nm values were used to compare the different formulations and to define the best conditions.
• Table 4 reports the loss in A280 nm value after the 3rd cycles of freezing/thawing, recorded for each sample under study. The samples were classified from the best to the worst based on the loSS in A280nm.
• pH 8.0 (2B) is better than pH 6.0 (2A).
• sucrose 8.5% w/v and 8% w/v, respectively: sucrose seems to assure the stability and the pH is irrelevant.
• IB 15mM Hepes, 30mM NaCl, 50mM Mannitol, 1% w/v Glycerol, 5mM EDTA, pH 7.0
• Table 5 reports the loss in A280 nm value after 14 days of storage at 2-8°C, recorded for each sample under study. The samples were classified from the best to the worst based on the loss in
• Hepes/NaCl buffer (IB) Hepes buffer without NaCl (1 A) showing 2.4-2.5% of protein loss.
• the preferred formulation buffer was the buffer 2B: 20mM Histidine, 8.5% w/v Sucrose, 50mg/L EDTA, pH 8.0.

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