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WO2025172582A1 - Methods for treating melanoma - Google Patents

Methods for treating melanoma

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Publication number
WO2025172582A1
WO2025172582A1 PCT/EP2025/054112 EP2025054112W WO2025172582A1 WO 2025172582 A1 WO2025172582 A1 WO 2025172582A1 EP 2025054112 W EP2025054112 W EP 2025054112W WO 2025172582 A1 WO2025172582 A1 WO 2025172582A1
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WO
WIPO (PCT)
Prior art keywords
immunoconjugate
tnf
melanoma
seq
treatment
Prior art date
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Pending
Application number
PCT/EP2025/054112
Other languages
French (fr)
Inventor
Dario Neri
Giuliano Elia
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Philogen SpA
Original Assignee
Philogen SpA
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Filing date
Publication date
Application filed by Philogen SpA filed Critical Philogen SpA
Publication of WO2025172582A1 publication Critical patent/WO2025172582A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K47/6851—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 determinant of a tumour cell
    • A61K47/6865—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 determinant of a tumour cell the tumour determinant being from skin, nerves or brain cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the antigen-binding site which may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), is the part of the molecule that recognizes and binds to all or part of a target antigen, such as those produced by cancer cells.
  • Monoclonal antibodies are immune system proteins that are created in the lab, for example to target specific cancers or to mark cancer cells so the immune system will recognize and destroy those cells.
  • ipilimumab a monoclonal antibody that binds to cytotoxic T lymphocyte- associated antigen-4 (CTLA-4) and turns off the mechanism that otherwise prevents the immune system from destroying cancer cells, is used as a post-surgical adjuvant for treating melanoma.
  • the intratumoral agents used in methods according to the disclosure are chosen from immunoconjugates, in particular fibronectin-targeted immunoconjugates.
  • the combination of intratumoral agents comprises (i) at least one tumor necrosis factor alpha (TNF-a) immunoconjugate, and (ii) at least one IL2 immunoconjugate.
  • TNF-a immunoconjugate comprises TNF-a linked to an antibody molecule comprising L19 complementarity determining regions (CDRs)
  • the IL2 immunoconjugate comprises IL2 linked to an antibody molecule comprising L19 CDRs.
  • the antibody comprises the L19 VH domain amino acid sequence of SEQ ID NO: 7.
  • the antibody comprises the amino acid sequence of scFv L19 set forth in SEQ ID NO: 9.
  • the methods comprise (a) treating one or more melanoma lesions of said patient by administering (i) at least one TNF-a immunoconjugate, and (ii) at least one IL2 immunoconjugate to one or more melanoma lesions of a patient, and (b) resecting the treated melanoma lesion(s), wherein (i) and (ii) are administered together or separately, and wherein administration of (i) and (ii) may occur simultaneously and/or sequentially in any order.
  • the treatment of step (a) is completed prior to step (b).
  • the methods comprise (a) treating one or more melanoma lesions of said patient by administering to one or more melanoma lesions of a patient: (i) at least one TNF-a immunoconjugate comprising an amino acid sequence with at least about 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with L19-TNF-a (SEQ ID NO: 15), and/or (ii) at least one IL2 immunoconjugate comprising an amino acid sequence with at least about 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with L19-IL2 (SEQ ID NO: 14), and (b) resecting the treated melanoma lesion(s).
  • the methods comprise administering the intratumoral agents where a total amount of TNF-a immunoconjugates administered to the melanoma lesions per treatment session ranges from about 100 pg to about 600 pg, such as from about 200 pg to about 550 pg, from about 300 pg to about 500 pg, from about 350 pg to about 450 pg, or from about 375 pg to about 425 pg, or is about 400 pg, and/or where a total amount of IL2 immunoconjugates administered to the melanoma lesions per treatment session ranges from about 0.75 mg to about 6 mg, such as from about 1 mg to about 4 mg, from about 1.5 mg to about 3 mg, or from about 2 mg to about 2.5 mg, or is about 2.2 mg or 2.17 mg (13 MiolU).
  • the total dose of the IL2 immunoconjugate injected into the melanoma lesion(s) per treatment session is about 2.2 mg or 2.17 mg (13 MiolU), and the total dose of the TNF-a immunoconjugate injected into a melanoma lesion per treatment session is about 100 pg, about 200 pg, about 300 pg, or about 400 pg, and preferably is about 400 pg.
  • a treatment session comprises injecting about 2.2 mg or 2.17 mg (13 MiolU) of L19-IL2 (SEQ ID NO: 14), and about 100 pg, about 200 pg, about 300 pg, or about 400 pg, preferably about 400 pg, of L19-TNF-a (SEQ ID NO: 15) into the melanoma lesion(s).
  • the intratumoral agents may be administered to the melanoma lesion(s) together or separately, and administration may occur simultaneously and/or sequentially in any order.
  • the intratumoral agents may be present in a single composition which can be injected into the lesion(s) via subcutaneous or intratumoral injection.
  • the intratumoral agents may be present in separate compositions, which may be injected at or about the same time into the lesion(s), or may be injected with a rest period between injection of each individual intratumoral agent.
  • the compositions comprising the intratumoral agent(s) can include additional pharmaceutically acceptable components, for example carriers, buffers, etc.
  • the melanoma lesion(s) that may be treated with methods described herein may be Stage III or Stage IV; preferably Stage III, more preferably Stage 11 IB or Stage I IIC.
  • the melanoma lesion(s) may be Stage III or Stage IV; preferably Stage III, more preferably Stage IIIB or Stage IIIC defined according to according to American Joint Committee on Cancer (AJCC) Cancer Staging Manual 7 th Edition.
  • the melanoma lesion(s) may be Stage III or Stage IV; preferably Stage III, more preferably Stage IIIB, Stage IIIC, or Stage HID defined according to according to American Joint Committee on Cancer (AJCC) Cancer Staging Manual 8 th Edition.
  • the lesion(s) may, in various embodiments, be cutaneous, subcutaneous, and/or nodal.
  • the size of the lesion(s) may vary, and may, for example, have at least one diameter ranging from about 5 mm to about 20 cm, such as from about 10 mm to about 15 mm.
  • the lesion(s) may or may not be ulcerated just prior to treatment, and preferably are not ulcerated.
  • the melanoma may have any BRAF mutation status, but is preferably wild type.
  • the melanoma may or may not have metastasized, although the methods may be particularly useful for treating patients with metastasized melanoma.
  • the patient may have an ECOG performance status of 0 or 1 just prior to treatment.
  • a patient treated according to methods described herein may have had previous anti-cancer treatment for melanoma.
  • a treatment regimen may comprise (a) administering (i) at least one TNF-a immunoconjugate, and (ii) at least one IL2 immunoconjugate to one or more melanoma lesions of a patient one or more times per week, for example once per week.
  • administering the intratumoral agents occurs about 5-10 days, preferably about 6-8 days, more preferably about 7 days before and/or after a subsequent and/or previous treatment session.
  • Treatment step (a) may occur over a period of time as needed, for example from about one week to about ten weeks, from about one week to about six weeks, or from about one week to about four weeks.
  • a useful treatment regimen may comprise (a) administering (i) at least one TNF-a immunoconjugate, and (ii) at least one IL2 immunoconjugate to one or more melanoma lesions of a about once per week for a period of time ranging from one week to four weeks.
  • Step (b) preferably occurs within about six weeks, more preferably within about four weeks, of completion of step (a). If possible, it may be preferable to obtain complete tumor excision (RO resection) in step (b).
  • patients treated according to methods described herein may undergo subsequent adjuvant therapy after completion of step (b).
  • the methods may further comprise administering one or more adjuvant therapies such as radiotherapy, chemotherapy, immunotherapy, targeted therapy, cytokine therapy, etc., after step (b).
  • recurrence free survival has been shown to be improved by an increase in median RFS and/or DMFS by a factor greater than 1 , such as at least about 1.25, at least about 1.5, at least about 1.75, at least about 2, at least about 2.25, or at least about 2.5 after step (b), compared to the same treatment without step (a).
  • recurrence free survival has been shown to be improved by about at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 1-year after step (b), at least a 40%, at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 2-years after step (b), and/or at least a 55%, at least a 50%, at least a 45%, at least a 40%, at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 3-years after step (b), compared to the same treatment without step (a), and/or distant metastasis free survival has been shown to be improved by about at least a 15%, at least a 14%, at least a
  • the disclosure also provides a method for improving recurrence free survival and/or distant metastasis free survival in a patient with resectable melanoma, the method comprising (a) administering a means for intratumoral treatment of melanoma to one or more melanoma lesions, and subsequently (b) resecting the treated melanoma lesion(s).
  • the means for intratumoral treatment of melanoma includes the combination of intratumoral agents described herein.
  • FIG. 1A shows clinical trial results of recurrence free survival from randomization for the treatment group (ARM 1) and the control group (ARM 2) (blinded review).
  • FIG. 1 B shows clinical trial results of recurrence free survival from randomization for the treatment group (ARM 1 ) and the control group (ARM 2) (investigator assessment).
  • FIG. 1 H shows clinical trial results of recurrence free survival from randomization for the treatment group (ARM 1 ) and the control group (ARM 2) for patients without prior systemic therapy.
  • Intratumoral agents that can be used according to the disclosure are immunoconjugates, particularly fibronectin-targeted immunoconjugates.
  • the IL2 comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 18, or a variant thereof.
  • the IL2 may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18.
  • the immunoconjugate is an IL2 immunoconjugate comprising an amino acid sequence with at least about 80% identity with L19-IL2 (SEQ ID NO: 14), and/or a TNF-a immunoconjugate comprising an amino acid sequence with at least about 80% identity with L19-TNF-a (SEQ ID NO: 15).
  • the IL2 immunoconjugate comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identity with L19-IL2 (SEQ ID NO: 14), and/or the TNF-a immunoconjugate comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identity with L19-TNF-a (SEQ ID NO: 15).
  • the cytokine and antibody of the immunoconjugate may be conjugated via a linker (linked), e.g. a peptide linker.
  • the peptide linker connecting the antibody and cytokine in an immunoconjugate described herein may, for example, be chosen from flexible peptide linkers. Suitable examples of peptide linker sequences are known.
  • the linker may, in various embodiments, be 10-20 amino acids in length, such as 10-15 amino acids in length or 15-18 amino acids in length. Preferably, the linker is 11-15 amino acids in length or 16-18 amino acids in length, for example 17 amino acids in length.
  • the linker may have the sequence set forth in SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 17, or may be a variant thereof.
  • the peptide linker connecting the antibody and cytokine in an immunoconjugate described herein has the sequence set forth in SEQ ID NO: 17.
  • the immunoconjugates chosen for combinations and methods according to the disclosure comprise, consist essentially of, or consist of IL2 and/or TNF-a immunoconjugates comprising an L19 antibody, for example having the CDRs of antibody L19 as set forth in SEQ ID NOs: 1 to 6.
  • Particular amino acid sequence variants may differ from a reference sequence by insertion, addition, substitution, or deletion of 1 or more amino acids, for example 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 5-10 amino acids, 10-20 amino acids, or 20-30 amino acids.
  • a variant sequence may comprise the reference sequence with 1 or more residues, for example 2 residues, 3 residues, 4 residues, 5 residues, 6 residues, 7 residues, 8 residues, 9 residues, or 10 or more residues inserted, deleted, or substituted.
  • residues for example 2 residues, 3 residues, 4 residues, 5 residues, 6 residues, 7 residues, 8 residues, 9 residues, or 10 or more residues inserted, deleted, or substituted.
  • up to 15 residues, up to 20 residues, up to 30 residues, or up to 40 residues may be inserted, deleted, or substituted.
  • intratumoral agents according to the disclosure comprise IL2 immunoconjugates, in particular one or more IL2 immunoconjugates and one or more TNF-a immunoconjugates.
  • combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates comprising an anti-EDB antibody and one or more TNF-a immunoconjugates comprising an anti-EDB antibody.
  • combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates comprising an L19 antibody and one or more TNF-a immunoconjugates comprising an L19 antibody.
  • combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and one or more TNF-a immunoconjugates where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10.
  • intratumoral agents comprise one or more IL2 immunoconjugates, where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and one or more TNF-a immunoconjugates, where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16.
  • combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates comprising an antifibronectin antibody in scFv form and one or more TNF-a immunoconjugates comprising an anti-fibronectin antibody in scFv form.
  • combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof, and one or more TNF-a immunoconjugates comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof.
  • combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates comprising an anti-fibronectin antibody in scFv form, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof, and one or more TNF-a immunoconjugates comprising an anti-fibronectin antibody in scFv form, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof.
  • TNF-a immunoconjugates where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, and comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof, and one or more IL2 immunoconjugates, where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof.
  • combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates, where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and comprising an anti-fibronectin antibody in scFv form, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof, and one or more TNF-a immunoconjugates, where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and comprising an anti-fibronectin antibody in scFv form, having the amino acid sequence set
  • intratumoral agents comprise one or more IL2 immunoconjugates, where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and comprising an anti-fibronectin antibody in scFvform, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof, and one or more TNF-a immunoconjugates, where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, and comprising an anti-fibronectin antibody in scFvform, having the amino acid sequence set forth in SEQ ID NO: 9, or
  • intratumoral agents comprise one or more IL2 immunoconjugates, where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and comprising an anti-fibronectin antibody in scFv form, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof, and one or more TNF-a immunoconjugates, where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16, and comprising an anti- fibronectin antibody in scFv form, having the amino acid sequence set forth
  • compositions typically administered in the form of pharmaceutical compositions, which may optionally comprise at least one component in addition to the intratumoral agent(s).
  • Pharmaceutical compositions may comprise, for example, at least one pharmaceutically acceptable excipient, carrier, buffer, stabilizer, and/or other material suitable for use in a pharmaceutical composition.
  • the precise nature of the carrier or other material will vary, and may depend on the route of administration, which may, for example, be by subcutaneous or intratumoral injection. It is within the ability of those skilled in the art to choose types and amounts of suitable components for pharmaceutical compositions according to the disclosure.
  • compositions for administering intratumoral agents according to the disclosure comprise therapeutically effective amounts of one or more immunoconjugates chosen from IL2 and/or TNF-a immunoconjugates.
  • the compositions are in a form suitable for administration directly to the area to be treated, e.g. the melanoma lesion.
  • compositions for administering intratumoral agents according to the disclosure comprise an amount of IL2 immunoconjugates ranging from about 0.5 mg to about 8 mg, for example from about 0.75 mg to about 6 mg, from about 0.75 mg to about 3 mg, from about 0.75 mg to about 2 mg, from about 0.75 mg to about 1.25 mg, from about 1 mg to about 5 mg, from about 1 mg to about 4 mg, from about 1 mg to about 3 mg, from about 1 mg to about 2 mg, from about 1 mg to about 1 .5 mg, from about 1.5 mg to about 3 mg, or from about 2 mg to about 2.5 mg.
  • IL2 immunoconjugates ranging from about 0.5 mg to about 8 mg, for example from about 0.75 mg to about 6 mg, from about 0.75 mg to about 3 mg, from about 0.75 mg to about 2 mg, from about 0.75 mg to about 1.25 mg, from about 1 mg to about 5 mg, from about 1 mg to about 4 mg, from about 1 mg to about 3 mg, from about 1 mg to about 2 mg, from about 1 mg to about
  • the compositions comprise an amount of IL2 immunoconjugates ranging from about 0.9 mg to about 2.3 mg, such as from about 1 mg to about 2.2 mg. In more preferred embodiments, the compositions comprise an amount of IL2 immunoconjugates of 1 mg ( ⁇ 10%), such as about 1.08 mg, or an amount of IL2 immunoconjugates of 2 mg ( ⁇ 10%), such as about 2.17 mg.
  • compositions for administering intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof, in an amount ranging from about 0.5 mg to about 8 mg, for example from about 0.75 mg to about 6 mg, from about 0.75 mg to about 3 mg, from about 0.75 mg to about 2 mg, from about 0.75 mg to about 1.25 mg, from about 1 mg to about 5 mg, from about 1 mg to about 4 mg, from about 1 mg to about 3 mg, from about 1 mg to about 2 mg, from about 1 mg to about 1 .5 mg, from about 1 .5 mg to
  • compositions comprise an amount of these IL2 immunoconjugates ranging from about 0.9 mg to about 2.3 mg, such as from about 1 mg to about 2.2 mg. In even more preferred embodiments, the compositions comprise an amount of these IL2 immunoconjugates of about 1.08 mg, or about 2.17 mg.
  • compositions for administering intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and comprising an anti-fibronectin antibody in scFv form, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof, in an amount ranging from about 0.5 mg to about 8 mg, for example from about 0.75 mg to about 6 mg, from about 0.75 mg to about 3 mg, from about 0.75 mg to about 2 mg, from about 0.75 mg to about 1.25 mg, from about 1 mg to about 5 mg, from about 1 mg to about 4 mg, from about 1 mg to about 3 mg, from about 1 mg to about 2 mg, from about 1 mg to about 1 .5 mg, from about 1
  • compositions comprise an amount of these IL2 immunoconjugates ranging from about 0.9 mg to about 2.3 mg, such as from about 1 mg to about 2.2 mg. In even more preferred embodiments, the compositions comprise an amount of these IL2 immunoconjugates of about 1.08 mg, or about 2.17 mg.
  • compositions for administering intratumoral agents according to the disclosure comprise L19-IL2 in an amount ranging from about 0.5 mg to about 8 mg, for example from about 0.75 mg to about 6 mg, from about 0.75 mg to about 3 mg, from about 0.75 mg to about 2 mg, from about 0.75 mg to about 1.25 mg, from about 1 mg to about 5 mg, from about 1 mg to about 4 mg, from about 1 mg to about 3 mg, from about 1 mg to about 2 mg, from about 1 mg to about 1 .5 mg, from about 1 .5 mg to about 3 mg, or from about 2 mg to about 2.5 mg, such as 1 mg ( ⁇ 10%), or 2 mg ( ⁇ 10%).
  • compositions for administering intratumoral agents according to the disclosure comprise an amount of TNF-a immunoconjugates ranging from about 50 pg to about 600 pg, such as from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 150 pg to about 450 pg, from about 150 pg to about 400 pg, from about 200 pg to about 400 pg, from about 150 pg to about 300 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 250 pg to about 450 pg, from about 300 pg to about 450 pg, from about 350 pg to about 500 pg, or from about 350 pg to about 450 pg.
  • the compositions comprise an amount of TNF-a immunoconjugates ranging from about 150 pg to about 450 pg, for example from about 175 pg to about 425 pg, or from about 200 pg to about 400 pg. In other preferred embodiments, the compositions comprise an amount of TNF- a immunoconjugates ranging from about 175 pg to about 225 pg, for example about 200 pg, or from about 375 pg to about 425 pg, for example about 400 pg.
  • compositions for administering intratumoral agents according to the disclosure comprise one or more TNF-a immunoconjugates where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof, in an amount ranging from about 50 pg to about 600 pg, such as from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 150 pg to about 450 pg, from about 150 pg to about 400 pg, from about 200 pg to about 400 pg, from about 150 pg to about 300 pg, from about 150 pg to about 250 pg
  • the compositions comprise an amount of these TNF-a immunoconjugates ranging from about 150 pg to about 450 pg, for example from about 175 pg to about 425 pg, or from about 200 pg to about 400 pg. In even more preferred embodiments, the compositions comprise an amount of these TNF-a immunoconjugates ranging from about 175 pg to about 225 pg, for example about 200 pg, or from about 375 pg to about 425 pg, for example about 400 pg.
  • compositions for administering intratumoral agents according to the disclosure comprise one or more TNF-a immunoconjugates where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, and comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof, in an amount ranging from about 50 pg to about 600 pg, such as from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 150 pg to about 450 pg, from about 150 pg to about 400 pg, from about 200 pg to about 400 pg, from about 150 pg to about 300 pg, from about 150 pg to about 250 pg
  • the compositions comprise an amount of these TNF-a immunoconjugates ranging from about 150 pg to about 450 pg, for example from about 175 pg to about 425 pg, or from about 200 pg to about 400 pg. In even more preferred embodiments, the compositions comprise an amount of these TNF-a immunoconjugates ranging from about 175 pg to about 225 pg, for example about 200 pg, or from about 375 pg to about 425 pg, for example about 400 pg.
  • the compositions comprise an amount of these TNF-a immunoconjugates ranging from about 150 pg to about 450 pg, for example from about 175 pg to about 425 pg, or from about 200 pg to about 400 pg. In even more preferred embodiments, the compositions comprise an amount of these TNF-a immunoconjugates ranging from about 175 pg to about 225 pg, for example about 200 pg, or from about 375 pg to about 425 pg, for example about 400 pg-
  • compositions for administering intratumoral agents according to the disclosure comprise one or more TNF-a immunoconjugates where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, and comprising an antifibronectin antibody in scFv form, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof, in an amount ranging from about 50 pg to about 600 pg, such as from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 150 pg to about 450 pg, from about 150 pg to about 400 pg, from about 200 pg to about 400 pg, from about 150 pg to about 300 pg, from about 150 pg to about
  • compositions for administering intratumoral agents according to the disclosure comprise L19-TNF-a in an amount ranging from about 50 pg to about 600 pg, such as from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 150 pg to about 450 pg, from about 150 pg to about 400 pg, from about 200 pg to about 400 pg, from about 150 pg to about 300 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 250 pg to about 450 pg, from about 300 pg to about 450 pg, from about 350 pg to about 500 pg, or from about 350 pg to about 450 pg, such as about 200 pg, or about 400 pg.
  • the compositions comprise an amount of L19-TNF-a ranging from about 150 pg to about 450 pg, for example from about 175 pg to about 425 pg, or from about 200 pg to about 400 pg. In even more preferred embodiments, the compositions comprise an amount of L19-TNF-a ranging from about 175 pg to about 225 pg, for example about 200 pg, or from about 375 pg to about 425 pg, for example about 400 pg. Preferably, the compositions comprise about 200 pg or about 400 pg of L19- TNF-a.
  • the compositions may comprise from about 1 mg to about 5 mg of IL2 immunoconjugates, and from about 100 g to about 500 pg of TNF-a immunoconjugates.
  • the compositions may comprise from about 1 mg to about 5 mg L19-IL2 and from about 100 pg to about 500 pg L19- TNFa.
  • the composition comprises from about 1 mg to about 2.2 mg L19-I L2, and from about 200 pg to about 400 pg L19-TNFa.
  • the composition comprises about 1.08 mg L19-IL2 and about 200 pg L19-TNFa, in another preferred embodiment, the composition comprises about 2.17 mg L19-IL2 and about 100 pg L19-TNFa, in another preferred embodiment, the composition comprises about 2.17 mg L19-IL2 and about 200 pg L19-TNFa, in another preferred embodiment, the composition comprises about 2.17 mg L19-IL2 and about 300 pg L19-TNFa, and in yet another preferred embodiment, the composition comprises about 2.17 mg L19-IL2 and about 400 pg L19-TNFa.
  • intratumoral agents e.g. combinations of intratumoral agents
  • methods of treatment provided herein is equally applicable to intratumoral agents (e.g. combinations of intratumoral agents) for use in such methods.
  • methods of treatment provided herein is equally applicable to the use of intratumoral agents (e.g. combinations of intratumoral agents) in the manufacture of a medicament, wherein the medicament is for the treatment of melanoma according to such methods.
  • Methods according to the disclosure comprise (a) administering combinations of intratumoral agents as described herein to a patient (also referred to as an “intratumoral treatment” or “preceding intratumoral treatment”), followed by (b) tumor resection and/or excision.
  • the methods can be used to treat cutaneous, subcutaneous, and/or nodal melanoma, for example in Stage III, (e.g. Stage 11 IB, Stage 11 IC, Stage HID) or Stage IV patients.
  • the patient treated using methods according to the disclosure has metastasized melanoma.
  • the treatment methods surprisingly increase the recurrence free survival and/or distant metastasis free survival in treated patients.
  • the methods comprise treatment regimens that include administering the combination of intratumoral agents to the patient subcutaneously or intratumorally.
  • the dosages, routes, and timing of administering the intratumoral agents can vary.
  • dosages administered to a patient may correspond to an amount of the antitumoral agent(s) present in a composition comprising the agent(s), or may correspond to a multiple of an amount of the agent(s) present in the composition.
  • a method of administering an intratumoral agent at a dosage of 400 pg can be accomplished by administering a composition having 400 pg of the agent, or can be accomplished by administering two compositions having 200 pg of the agent.
  • methods according to the disclosure comprise administering intratumoral agents as described herein according to a dosing regimen that permits at least about 24 hours, such as at least about 48 hours, or at least about 72 hours between intratumoral treatment sessions where one or more intratumoral agents are administered to a patient.
  • various methods comprise administering one or a combination of intratumoral agents as described herein approximately or exactly every 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or approximately or exactly every 1 , 2, or 3 weeks, for a specified period of time.
  • administering the intratumoral agents occurs about 5-10 days, preferably about 6-8 days, more preferably about 7 days before and/or after a subsequent and/or previous treatment session.
  • Dosages of IL2 immunoconjugates that can be administered in methods according to the disclosure include those amounts disclosed as useful in compositions according to the disclosure, for example may range from about 0.25 mg to about 8 mg.
  • dosages of IL2 immunoconjugates that can be administered per intratumoral treatment session in various methods according to the disclosure range from about 0.5 mg to about 8 mg, for example from about 0.75 mg to about 6 mg, from about 0.75 mg to about 3 mg, from about 0.75 mg to about 2 mg, from about 0.75 mg to about 1.25 mg, from about 1 mg to about 5 mg, from about 1 mg to about 4 mg, from about 1 mg to about 3 mg, from about 1 mg to about 2 mg, from about 1 mg to about 1 .5 mg, from about 1.5 mg to about 3 mg, or from about 2 mg to about 2.5 mg.
  • the dosage of the IL2 immunoconjugates administered ranges from about 0.9 mg to about 2.3 mg, such as from about 1 mg to about 2.2 mg, at approximately or exactly 1 week intervals. In more preferred embodiments, the dosage of the IL2 immunoconjugates administered is 1 mg ( ⁇ 10%), such as about 1.08 mg, or 2 mg ( ⁇ 10%), such as about 2.17 mg, at approximately or exactly 1 week intervals.
  • the dosage of an IL2 immunoconjugate administered in methods according to the disclosure may alternatively be defined with reference to International Units (IU) of IL2 immunoconjugates administered to the patient.
  • IU International Units
  • the IU of an IL2 immunoconjugate may be determined by any suitable means.
  • the dosage of IL2 immunoconjugates that can be administered per intratumoral treatment session according to the disclosure range from about 1.6 million IU (MiolU) to about 15 MiolU, such as from about 2 MiolU to about 15 MiolU, from about 3 MiolU to about 15 MiolU, from about 4 MiolU to about 15 MiolU, from about 5 MiolU to about 15 MiolU, from about 6 MiolU to about 13 MiolU, or from about 6.5 MiolU to about 13 MiolU.
  • the dosage of IL2 immunoconjugates is about 6.5 MiolU, and in other preferred embodiments the dosage of IL2 immunoconjugates is about 13 MiolU.
  • methods according to the disclosure comprise injecting a composition comprising one or more IL2 immunoconjugates into a melanoma lesion, where the dosage is defined by the IU of the IL2 immunoconjugate injected into the lesion as a function of the longest diameter or the longest distance between two vertices of the lesion (e.g. lU/cm or lU/cm lesion diameter).
  • the lesion diameter refers to the longest diameter of a melanoma lesion.
  • the lesion diameter is the line passing through the center of the circle or spheroid melanoma lesion.
  • the lesion diameter is the major axis of the ellipse (i.e. the longest diameter).
  • the lesion diameter is the longest distance between two vertices of the melanoma lesion.
  • the dosage of IL2 immunoconjugates injected into a melanoma lesion per intratumoral treatment session can range from about 0.25 MiolU/cm lesion diameter to about 100 Mioll/cm lesion diameter, such as from about 0.5 MiolU/cm lesion diameter to about 90 MiolU/cm lesion diameter, about 0.75 MiolU/cm lesion diameter to about 80 MiolU/cm lesion diameter, about 1 MiolU/cm lesion diameter to about 80 MiolU/cm lesion diameter, about 1.25 MiolU/cm lesion diameter to about 70 MiolU/cm lesion diameter, about 1.5 MiolU/cm lesion diameter to about 60 MiolU/cm lesion diameter, about 1.75 MiolU/cm lesion diameter to about 50 MiolU/cm lesion diameter, about 2 MiolU/cm lesion diameter to about 40 MiolU/cm lesion
  • the dosage of IL2 immunoconjugate injected into a melanoma lesion per treatment session ranges from about 3.25 MiolU/cm lesion diameter to about 16.25 MiolU/cm lesion diameter, at approximately or exactly 1 week intervals.
  • dosages of TNF-a immunoconjugates that can be administered per intratumoral treatment session in various methods according to the disclosure range from about 50 pg to about 600 pg, such as from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 150 pg to about 450 pg, from about 150 pg to about 400 pg, from about 200 pg to about 400 pg, from about 150 pg to about 300 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 250 pg to about 450 pg, from about 300 pg to about 450 pg, from about 350 pg to about 500 pg, or from about 350 pg to about 450 pg.
  • the dosage of TNF-a immunoconjugates administered ranges from about 150 g to about 450 pg, for example from about 175 pg to about 425 pg, or from about 200 pg to about 400 pg, at approximately or exactly 1 week intervals. In other preferred embodiments, the dosage of TNF-a immunoconjugates administered ranges from about 175 pg to about 225 pg, for example about 200 pg, or from about 375 pg to about 425 pg, for example about 400 pg, at approximately or exactly 1 week intervals. Preferably, the dosage of TNF-a immunoconjugates administered is about 200 pg or about 400 pg, more preferably about 200 pg or about 400 pg of L19-TNF-a.
  • the dose of the TNF-a immunoconjugate is reduced, e.g. to about 100 pg, about 200 pg, or about 300 pg per treatment session following a grade > 3 adverse events (Aes) recorded after the first dose administration.
  • the dosage adjustment can be determined depending on the patient’s tolerance.
  • methods according to the disclosure comprise injecting a composition comprising one or more TNF-a immunoconjugates into a melanoma lesion, where the dosage is defined as the mass of TNF-a immunoconjugate injected into the lesion as a function of the longest diameter or the longest distance between two vertices of the lesion (e.g. pg/cm or pg/cm lesion diameter).
  • the lesion diameter refers to the longest diameter of a melanoma lesion.
  • the lesion diameter is the line passing through the center of the circle or spheroid melanoma lesion.
  • the lesion diameter is the major axis of the ellipse (i.e. the longest diameter).
  • the lesion diameter is the longest distance between two vertices of the melanoma lesion.
  • the dosage of TNF-a immunoconjugates injected into a melanoma lesion per intratumoral treatment session can range from about 12.5 pg/cm lesion diameter to about 1 mg/cm lesion diameter, such as from about 25 pg/cm lesion diameter to about 900 pg/cm lesion diameter, about 37.5 pg/cm lesion diameter to about 800 pg/cm lesion diameter, about 50 pg/cm lesion diameter to about 750 pg/cm lesion diameter, about 62.5 pg/cm lesion diameter to about 700 pg/cm lesion diameter, about 75 pg/cm lesion diameter to about 600 pg/cm lesion diameter, about 87.5 pg/cm lesion diameter to about 550 pg/cm lesion diameter, or about 100 pg/cm lesion diameter to about 500 pg/cm lesion diameter.
  • the dosage of TNF-a immunoconjugate injected into a melanoma lesion per intratumoral treatment session ranges from about 100 pg/cm lesion diameter to about 500 pg/cm lesion diameter, at approximately or exactly 1 week intervals.
  • the immunoconjugates can be administered together or separately.
  • the immunoconjugates can be formulated into a single combined composition or into separate compositions. If separate administration is contemplated, the immunoconjugates may be administered in a single treatment session or in separate treatment sessions.
  • the two compositions may be combined at or near the time when the intratumoral agent will be administered, e.g. just prior to administration.
  • the compositions may be combined into a single syringe prior to injecting the combination into a melanoma lesion.
  • compositions useful in methods according to the disclosure comprise at least one TNF-a immunoconjugate as described herein, and at least one IL2 immunoconjugate as described herein.
  • the intratumoral compositions can be administered by injection at or near a melanoma lesion.
  • the methods comprise injecting the intratumoral composition into the targeted melanoma lesion.
  • the composition injected into the lesion may comprise from 0.25 mg to 5 mg L19-IL2 and from 50 pg to 500 pg L19-TNFa.
  • the composition injected into the lesion comprises from 1 mg to 2.2 mg L19-IL2 and from 200 pg to 400 pg L19-TNFa.
  • the composition injected into the lesion comprises 0.27 mg L19-IL2 and 50 pg L19-TNFa.
  • the composition injected into the lesion comprises 0.54 mg L19-IL2 and 100 pg L19-TNFa. In other preferred embodiments, the composition injected into the lesion comprises 1.08 mg L19-IL2 and 200 pg L19-TNFa. In still other preferred embodiments, the composition injected into the lesion comprises 1.627 mg L19-IL2 and 300 pg L19-TNFa. In particularly preferred embodiments, the composition injected into the lesion comprises 2.17 mg (13 MiolU) L19-IL2 and 400 pg L19-TNFa.
  • the methods may comprise injecting an intratumoral composition into the melanoma lesion comprising from 0.25 mg to 5 mg L19-IL2 and from 25 pg to 250 pg L19-TNFa, such as from 1 mg to 2.2 mg L19- IL2 and from 100 pg to 200 pg L19-TNFa.
  • the composition injected into the melanoma lesion may comprise 0.27 mg L19-IL2 and 25 pg L19-TNFa.
  • the composition injected into the lesion comprises 0.54 mg L19-IL2 and 50 pg L19-TNFa. In other preferred embodiments, the composition injected into the lesion comprises 1.08 mg L19-IL2 and 100 pg L19-TNFa. In still other preferred embodiments, the composition injected into the lesion comprises 1.627 mg L19-IL2 and 150 pg L19-TNFa. In other preferred embodiments still, the composition injected into the lesion comprises 2.17 mg (13 MiolU) L19-IL2 and 200 pg L19-TNFa.
  • the intratumoral treatment may continue for a period of time up to about 12 weeks, such as up to about 10 weeks, up to about 8 weeks, up to about 6 weeks, up to about 5 weeks, up to about 4 weeks, up to about 3 weeks, or up to about 2 weeks before treatment with the intratumoral agent is discontinued.
  • treatment with the intratumoral agents lasts for a period of time ranging from about 1 week to about 6 weeks, such as about 1 week to about 5 weeks, or about 1 week to about 4 weeks. More preferably, treatment with the intratumoral agents lasts for a period of 4 weeks.
  • An exemplary treatment regimen may comprise administering a combination of intratumoral agents as described herein about or exactly 3 times per week, such as about or exactly 2 times per week, or about or exactly 1 time per week, for a period of time ranging from about 1 week to about 8 weeks, such as from about 1 week to about 6 weeks, from about 1 week to about 4 weeks, from about 1 week to about 3 weeks, or from about 1 week to about 2 weeks, preferably once per week for 1 -4 weeks, more preferably once per week for 4 weeks.
  • an intratumoral treatment regimen may comprise injecting a combination of one or more TNF-a immunoconjugates and one or more IL2 immunoconjugates into one or more melanoma lesions, where the TNF-a immunoconjugates and/or IL2 immunoconjugates may be administered to the patient approximately or exactly once a week for a period of time ranging from one week to four weeks, in a dosage of TNF-a immunoconjugates ranging from about 150 pg to about 450 pg, for example from about 175 pg to about 425 pg, or from about 200 pg to about 400 pg, and/or a dosage of IL2 immunoconjugates ranging from about 0.75 mg to about 3 mg, such as from about 1 mg to about 2.2 mg.
  • a preferred intratumoral treatment regimen may comprise injecting one or more IL2 immunoconjugates and one or more TNF-a immunoconjugates into one or more melanoma lesions approximately or exactly once a week for a period of time ranging from one week to four weeks, where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16.
  • the IL2 immunoconjugates and/or TNF-a immunoconjugates comprise the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8.
  • the dose of L19-TNF-a ranges from about 350 pg to about 450 pg, for example from about 375 pg to about 425 pg, or is about 400 pg
  • the dose of L19-IL2 ranges from about 0.75 mg to about 3 mg, such as from about 1 mg to about 2.2 mg, or is about 2.17 mg (13 MiolU).
  • an intratumoral treatment regimen according to the disclosure may comprise injecting an IL2 immunoconjugate comprising an amino acid sequence with at least about 70% identity with L19-IL2 (SEQ ID NO: 14), and/or a TNF-a immunoconjugate comprising an amino acid sequence with at least about 70% identity with L19-TNF-a (SEQ ID NO: 15) into one or more melanoma lesions approximately or exactly once a week for a period of time ranging from one week to four weeks, where the dose of L19-TNF-a is about 400 pg, and the dose of L19-IL2 is about 2.17 mg (13 MiolU).
  • an L19-TNF-a immunoconjugate as described herein is formulated for injection as a composition comprising 15mM NaH2PO4(2H 2 O), 10mM Na 2 HPO4(2H2O), 1.5 mM KCI, 75 mM mannitol, 30 mM NaCI, 1 % (w/v) glycerol, 5mM EDTA, and 0.01 % (v/v) polysorbate 20 (Tween® 20) at pH of about 8, and is suitable for intratumoral injection
  • an L19-IL2 immunoconjugate as described herein is formulated for injection as a composition comprising 6.7 mM NaH2PO4 (2H2O), 1.8 mM KCI, 133 mM mannitol, 20 mM NaCI, 1 % (w/v) glycerol, and 0.3% (v/v) polysorbate 80 (Tween® 80) at pH of about 6.3, and is suitable
  • an IL2 immunoconjugate for use in a method of improving recurrence free survival and/or distant metastasis free survival in a patient with resectable melanoma, wherein the method comprises: (a) treating at least one melanoma lesion of said patient by administering to said lesion(s) a combination of intratumoral agents, wherein the combination of intratumoral agents comprises: (i) the IL2 immunoconjugate, and (ii) a TNF-a immunoconjugate, and (b) resecting the treated melanoma lesion(s); wherein recurrence free survival and/or distant metastasis free survival is improved compared to the same treatment without step (a).
  • the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18.
  • intratumoral agents are administered to the melanoma lesion(s) together or separately by subcutaneous or intratumoral injection.
  • step (a) comprises administering the combination of intratumoral agents together or separately in one or more treatment sessions, wherein each treatment session occurs from one day to two weeks before and/or after a subsequent and/or previous treatment session.
  • step (a) comprises injecting the combination of intratumoral agents together or separately into the melanoma lesion(s) during one to four treatment sessions, wherein each treatment session occurs about 5-10 days, preferably about 6-8 days, more preferably about 7 days, before and/or after a subsequent and/or previous treatment session.
  • the total dose of the IL2 immunoconjugate(s) injected into a melanoma lesion per treatment session ranges from about 0.5 mg to about 8 mg, preferably from about 0.75 mg to about 6 mg, from about 1 mg to about 5 mg, from about 1 .25 mg to about 4 mg, or from about 1.5 mg to about 3 mg, most preferably ranging from about 2 mg to about 2.5 mg, or is about 2.2 mg or about 2.17 mg.
  • the total dose of the TNF-a immunoconjugate(s) injected per treatment session into a melanoma lesion ranges from about 50 pg to about 600 pg, preferably from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 200 to about 450 pg, or from about 350 pg to about 450 pg, most preferably ranging from about 375 pg to about 425 pg, or is about 100 pg, about 200 pg, about 300 pg, or about 400 pg.
  • the total dose of the IL2 immunoconjugate(s) injected into a melanoma lesion per treatment session is about 2.17 mg
  • the total dose of the TNF-a immunoconjugate(s) injected into a melanoma lesion per treatment session is about 100 pg, about 200 pg, about 300 pg, or about 400 pg, preferably about 400 pg.
  • step (a) comprises administering the combination of intratumoral agents together or separately at least once a week for up to 8 weeks, up to 6 weeks, up to 5 weeks, up to 4 weeks, up to 3 weeks, or up to 2 weeks.
  • step (a) The method according to any one of the preceding embodiments, wherein the patient is assessed as having an ECOG performance status of 0 or 1 at the beginning of the treatment of step (a).
  • step (a) 22.
  • the melanoma has a wild type BRAF mutation status at the beginning of the treatment of step (a).
  • the melanoma lesion(s) treated in step (a) are cutaneous, subcutaneous, and/or nodal.
  • step (b) occurs within 8 weeks, preferably within 6 weeks, more preferably within 4 weeks after completion of the treatment of step (a).
  • step (b) comprises complete tumor excision (RO resection) of one or more melanoma lesion(s) treated in step (a).
  • step (a) wherein the patient received one or more treatments for melanoma chosen from tumor resection, radiotherapy, chemotherapy, immunotherapy, targeted therapy, cytokine therapy, or combinations thereof, prior to step (a).
  • a method of improving distant metastasis free survival in a patient with resectable melanoma comprising:
  • the TNF-a immunoconjugate comprises TNF-a linked to an antibody molecule comprising L19 CDRs
  • the IL2 immunoconjugate comprises IL2 linked to an antibody molecule comprising L19 CDRs
  • the L19 CDRs are:
  • step (b) resecting the treated melanoma lesion(s); wherein distant metastasis free survival is improved compared to the same treatment without step (a).
  • step (a) is completed prior to step (b).
  • the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18.
  • TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16.
  • TNF-a comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 10 or SEQ ID NO: 16; and wherein the IL2 comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 18.
  • IL2 immunoconjugate comprises an amino acid sequence with at least about 70% identity with L19-IL2 (SEQ ID NO: 14), and/or the TNF-a immunoconjugate comprises an amino acid sequence with at least about 70% identity with L19-TNF-0 (SEQ ID NO: 15).
  • IL2 immunoconjugate comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with L19-IL2 (SEQ ID NO: 14).
  • TNF-a immunoconjugate comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with L19-TNF (SEQ ID NO: 15).
  • step (a) comprises injecting the combination of intratumoral agents together or separately into the melanoma lesion(s) during one to four treatment sessions, wherein each treatment session occurs about 5-10 days, preferably about 6-8 days, more preferably about 7 days, before and/or after a subsequent and/or previous treatment session.
  • the total dose of the TNF-a immunoconjugate(s) injected per treatment session into a melanoma lesion ranges from about 50 pg to about 600 pg, preferably from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 200 to about 450 pg, or from about 350 pg to about 450 pg, most preferably ranging from about 375 pg to about 425 pg, or is about 100 pg, about 200 pg, about 300 pg, or about 400 pg.
  • step (a) comprises administering the combination of intratumoral agents together or separately at least once a week for up to 8 weeks, up to 6 weeks, up to 5 weeks, up to 4 weeks, up to 3 weeks, or up to 2 weeks.
  • step (a) The method according to any one of embodiments 35-54, wherein the melanoma lesion(s) are not ulcerated at the beginning of the treatment of step (a).
  • melanoma lesion(s) treated in step (a) are cutaneous, subcutaneous, and/or nodal.
  • step (b) occurs within 8 weeks, preferably within 6 weeks, more preferably within 4 weeks after completion of the treatment of step (a).
  • step (b) comprises complete tumor excision (RO resection) of one or more melanoma lesion(s) treated in step (a).
  • step (a) The method according to any one of embodiments 35-61 , wherein the patient received one or more treatments for melanoma chosen from tumor resection, radiotherapy, chemotherapy, immunotherapy, targeted therapy, cytokine therapy, or combinations thereof, prior to step (a).
  • DMFS distant metastasis free survival
  • a “day” is intended to be a 24-hour period
  • a “week” is intended to be a 7-day period.
  • blindded review is intended to mean the reading of medical images, reviewed by an independent readers blinded to the results of study treatment, the investigator assessment, and some pre-defined clinical information to independently verify endpoints and control bias that might result from errors in response or progression assessments.
  • Prior anti-tumor treatment for the primary melanoma lesion including surgery and approved adjuvant treatments (e.g., radiotherapy, immune checkpoint inhibitors, BRAF/MEK inhibitors, etc.) was allowed.
  • adjuvant treatments e.g., radiotherapy, immune checkpoint inhibitors, BRAF/MEK inhibitors, etc.
  • a total of 256 patients were selected and randomly assigned into two groups.
  • ARM 1 the treatment group, included 127 patients.
  • ARM 2 the control group, included 129 patients.
  • Table 1 below presents the general characteristics of patients in each group.
  • Table 2 below presents information regarding the tumors treated.
  • the disease stage of tumors was classified according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual 7 th Edition (second row), and subsequently according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual 8 th Edition (third row).
  • the 8 th edition of the AJCC introduced Stage HID melanoma for the first time, and several tumors classified as Stage HIX (unspecified stage III patient) according to the AJCC 7 th edition were classified as Stage HID under the AJCC 8 th edition.
  • stage III melanoma Regardless of the specific staging criteria used, these data demonstrate that all substages of stage III melanoma were successfully treated, including the most advanced stage III melanoma substage (the most advanced Stage III substage according to the AJCC 7 th Edition is Stage 11 IC, and the most advanced Stage III substage according to the AJCC 8 th Edition is Stage HID).
  • the intratumoral treatment composition was prepared by mixing a 1 mL composition comprising 2.17 mg L19-IL2, 6.7 mM NaH 2 PO 4 (2H 2 O), 1 .8 mM KCI, 133 mM mannitol, 20 mM NaCI, 1 % (w/v) glycerol, and 0.3% (v/v) polysorbate 80 (Tween® 80) at pH of about 6.3, with a volume ranging from 0.25 mL to 1 mL of a composition comprising 400 pg of L19-TNF-a, 15mM NaH 2 PO4(2H 2 O), 10mM Na 2 HPO 4 (2H 2 O), 1.5 mM KCI, 75 mM mannitol, 30 mM NaCI, 1 % (w/v) glycerol, 5mM EDTA, and 0.01 % (v/v) polysorbate 20 (Tween® 20) at pH of about 8.
  • L19-TNF-a was 400 pg
  • the investigator was permitted to reduce the dosage to amounts ranging from 100 pg to 400 pg (i.e. 100 pg, 200 pg, 300 pg, or 400 pg), as deemed appropriate based on the size and number of lesions and the patient’s tolerability to the treatment.
  • the intratumoral treatment composition was distributed among all injectable lesions.
  • Surgical treatment focused on complete tumor removal (R0 resection) to ensure the patient was free of any detectable tumor lesion by physical examination, imaging diagnostics, and/or histopathological analysis.
  • FIG. 1A shows, the 1-year, 2-year, and 3-year recurrence free survival rates were 57.47%, 41.57%, and 25.47% respectively for ARM 1 , and 39.46%, 23.60%, and 10.33% respectively for ARM 2.
  • FIG. 1 B shows, the 1-year, 2-year, and 3-year recurrence free survival rates were 61.95%, 50.44%, and 38.15% respectively for ARM 1 , and 46.54%, 30.89%, and 21 .38% respectively for ARM 2.
  • Table 4C and FIG. 1 C show the recurrence free survival from surgery for the treatment group (ARM 1) and the control group (ARM 2).
  • the 1 -year and 2-year recurrence free survival rates were 54.75% and 37.85% respectively for ARM 1 , and 36.66% and 23.36% respectively for ARM 2.
  • Tables 4A-4C and FIGS. 1A-1 C show that the median RFS for ARM 1 (treatment) was considerably longer than the median RFS for ARM 2 (control). These data show a statistically significant increase in median RFS with methods of treatment according to the disclosure.
  • Table 4E shows the distribution by ARM of number and type of post-surgical adjuvant therapy.
  • Table 4F and FIG. 1 D show the median RFS from randomization for ARM 1 (treatment) and ARM 2 (control) with and without post-surgical adjuvant therapy.
  • FIG. 1 D shows, the 1 -year and 2-year recurrence free survival rates were 60.07% and 47.20% respectively for ARM 1 (with adjuvant therapy), 51 .34% and 31 .74% respectively for ARM 1 (without adjuvant therapy), 49.88% and 35.21 % respectively for ARM 2 (with adjuvant therapy), and 25.35% and 13.31 % respectively for ARM 2 (without adjuvant therapy).
  • FIG. 1 E shows, the 1-year, 2-year, and 3-year recurrence free survival rates were 58.13%, 43.23%, and 26.49% respectively for ARM 1 , and 39.83%, 23.10%, and 9.60% respectively for ARM 2.
  • Table 4H and FIG. 1 F show the median RFS from randomization for ARM 1 (treatment) and ARM 2 (control) for patients who underwent prior immunotherapy for melanoma before the study.
  • Table 4I and FIGS. 1 G-1 H show the median RFS from randomization for ARM 1 (treatment) and ARM 2 (control) for patients with or without prior systemic therapy for melanoma before the study.
  • Tables 5A-5C present hazard ratio data for recurrence free survival based on patient demographics, tumor, prior anti-cancer treatment, and post-surgical adjuvant therapy.
  • the 1-year, 2-year, and 3-year survival rates were 69.84%, 59.41 %, and 36.4% respectively for ARM 1 , and 60.83%, 38.81 %, and 16.98% respectively for ARM 2.
  • Amino acid sequence of the L19-huTNF conjugate (SEQ ID NO: 15) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSG

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Abstract

The disclosure relates to methods for treating melanoma with combinations of intratumoral agents and surgery, and methods for improving recurrence free survival and distant metastasis free survival in treated patients.

Description

METHODS FOR TREATING MELANOMA
[0001] This application claims priority from US 63/553,777 filed 15 February 2024, the contents and elements of which are herein incorporated by reference for all purposes.
TECHNICAL FIELD
[0002] The disclosure relates to methods for treating melanoma. In particular, the disclosure relates to methods for treating melanoma with combinations of intratumoral agents and surgery, and methods for improving recurrence free survival and distant metastasis free survival in treated patients.
BACKGROUND
[0003] Melanoma is a form of skin cancer that begins in the cells (melanocytes) that control the pigment in skin. Cutaneous malignant melanoma (CMM) is a very aggressive form of skin cancer that kills about 46,000 patients per year worldwide. In advanced stages, CMM has a poor prognosis with a median survival time of about 8 months.
[0004] Typically, treatment of Stage III or Stage IV melanoma is based primarily on surgical removal of the cancerous lesions. However, not all melanoma tumors are resectable. In addition, patients with Stage III or Stage IV melanoma are exposed to a high-risk of recurrency, even with complete tumor removal. Therefore, even when surgery is possible it may need to be followed by adjuvant therapy, for example with monoclonal antibodies. The term “antibody” describes an immunoglobulin which may be natural or may be partly or wholly synthetically produced, as well as polypeptides or proteins comprising an antibody antigen-binding site. The antigen-binding site, which may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), is the part of the molecule that recognizes and binds to all or part of a target antigen, such as those produced by cancer cells. Monoclonal antibodies are immune system proteins that are created in the lab, for example to target specific cancers or to mark cancer cells so the immune system will recognize and destroy those cells. For example, ipilimumab, a monoclonal antibody that binds to cytotoxic T lymphocyte- associated antigen-4 (CTLA-4) and turns off the mechanism that otherwise prevents the immune system from destroying cancer cells, is used as a post-surgical adjuvant for treating melanoma.
[0005] In view of the high risk that melanoma will recur in patients with Stage III or Stage IV melanoma, there is an ongoing need for studies to find effective treatments to reduce the risk of recurrence and death.
SUMMARY
[0006] The disclosure relates to compositions and methods for treating cutaneous, subcutaneous, and/or nodal malignant melanoma, using combinations of intratumoral agents and surgery to improve recurrence free survival and/or distant metastasis free survival in patients with melanoma. The disclosure also relates to intratumoral agents (e.g. combinations of intratumoral agents), for use in such methods, as well as the use of intratumoral agents (e.g. combinations of intratumoral agents) in the manufacture of a medicament, wherein the medicament is for the treatment of melanoma according to such methods.
[0007] All description of methods of treatment provided herein is equally applicable to intratumoral agents (e.g. combinations of intratumoral agents) for use in such methods. Similarly, all description of methods of treatment provided herein is equally applicable to the use of intratumoral agents (e.g. combinations of intratumoral agents) in the manufacture of a medicament, wherein the medicament is for the treatment of melanoma according to such methods.
[0008] The intratumoral agents used in methods according to the disclosure are chosen from immunoconjugates, in particular fibronectin-targeted immunoconjugates. The combination of intratumoral agents comprises (i) at least one tumor necrosis factor alpha (TNF-a) immunoconjugate, and (ii) at least one IL2 immunoconjugate. In particular embodiments, the TNF-a immunoconjugate comprises TNF-a linked to an antibody molecule comprising L19 complementarity determining regions (CDRs), and the IL2 immunoconjugate comprises IL2 linked to an antibody molecule comprising L19 CDRs. In some embodiments, the antibody comprises the L19 VH domain amino acid sequence of SEQ ID NO: 7. In some embodiments, the L19 VL domain amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises the amino acid sequence of scFv L19 set forth in SEQ ID NO: 9.
[0009] The methods comprise (a) treating one or more melanoma lesions of said patient by administering (i) at least one TNF-a immunoconjugate, and (ii) at least one IL2 immunoconjugate to one or more melanoma lesions of a patient, and (b) resecting the treated melanoma lesion(s), wherein (i) and (ii) are administered together or separately, and wherein administration of (i) and (ii) may occur simultaneously and/or sequentially in any order. Preferably, the treatment of step (a) is completed prior to step (b).
[0010] In another aspect, the methods comprise (a) treating one or more melanoma lesions of said patient by administering to one or more melanoma lesions of a patient: (i) at least one TNF-a immunoconjugate wherein the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16, and/or (ii) at least one IL2 immunoconjugate wherein the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and (b) resecting the treated melanoma lesion(s).
[0011] In yet another aspect, the methods comprise (a) treating one or more melanoma lesions of said patient by administering to one or more melanoma lesions of a patient: (i) at least one TNF-a immunoconjugate comprising an amino acid sequence with at least about 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with L19-TNF-a (SEQ ID NO: 15), and/or (ii) at least one IL2 immunoconjugate comprising an amino acid sequence with at least about 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with L19-IL2 (SEQ ID NO: 14), and (b) resecting the treated melanoma lesion(s).
[0012] The methods comprise administering the intratumoral agents where a total amount of TNF-a immunoconjugates administered to the melanoma lesions per treatment session ranges from about 100 pg to about 600 pg, such as from about 200 pg to about 550 pg, from about 300 pg to about 500 pg, from about 350 pg to about 450 pg, or from about 375 pg to about 425 pg, or is about 400 pg, and/or where a total amount of IL2 immunoconjugates administered to the melanoma lesions per treatment session ranges from about 0.75 mg to about 6 mg, such as from about 1 mg to about 4 mg, from about 1.5 mg to about 3 mg, or from about 2 mg to about 2.5 mg, or is about 2.2 mg or 2.17 mg (13 MiolU). For example, in a preferred embodiment, the total dose of the IL2 immunoconjugate injected into the melanoma lesion(s) per treatment session is about 2.2 mg or 2.17 mg (13 MiolU), and the total dose of the TNF-a immunoconjugate injected into a melanoma lesion per treatment session is about 100 pg, about 200 pg, about 300 pg, or about 400 pg, and preferably is about 400 pg. In a more preferred embodiment, a treatment session comprises injecting about 2.2 mg or 2.17 mg (13 MiolU) of L19-IL2 (SEQ ID NO: 14), and about 100 pg, about 200 pg, about 300 pg, or about 400 pg, preferably about 400 pg, of L19-TNF-a (SEQ ID NO: 15) into the melanoma lesion(s).
[0013] The intratumoral agents may be administered to the melanoma lesion(s) together or separately, and administration may occur simultaneously and/or sequentially in any order. For example, the intratumoral agents may be present in a single composition which can be injected into the lesion(s) via subcutaneous or intratumoral injection. As an alternate example, the intratumoral agents may be present in separate compositions, which may be injected at or about the same time into the lesion(s), or may be injected with a rest period between injection of each individual intratumoral agent. The compositions comprising the intratumoral agent(s) can include additional pharmaceutically acceptable components, for example carriers, buffers, etc. [0014] The melanoma lesion(s) that may be treated with methods described herein may be Stage III or Stage IV; preferably Stage III, more preferably Stage 11 IB or Stage I IIC. The melanoma lesion(s) may be Stage III or Stage IV; preferably Stage III, more preferably Stage IIIB or Stage IIIC defined according to according to American Joint Committee on Cancer (AJCC) Cancer Staging Manual 7th Edition. Alternatively, the melanoma lesion(s) may be Stage III or Stage IV; preferably Stage III, more preferably Stage IIIB, Stage IIIC, or Stage HID defined according to according to American Joint Committee on Cancer (AJCC) Cancer Staging Manual 8th Edition. The lesion(s) may, in various embodiments, be cutaneous, subcutaneous, and/or nodal. The size of the lesion(s) may vary, and may, for example, have at least one diameter ranging from about 5 mm to about 20 cm, such as from about 10 mm to about 15 mm. The lesion(s) may or may not be ulcerated just prior to treatment, and preferably are not ulcerated. The melanoma may have any BRAF mutation status, but is preferably wild type. The melanoma may or may not have metastasized, although the methods may be particularly useful for treating patients with metastasized melanoma. Preferably, the patient may have an ECOG performance status of 0 or 1 just prior to treatment. Optionally, a patient treated according to methods described herein may have had previous anti-cancer treatment for melanoma.
[0015]A treatment regimen may comprise (a) administering (i) at least one TNF-a immunoconjugate, and (ii) at least one IL2 immunoconjugate to one or more melanoma lesions of a patient one or more times per week, for example once per week. In some aspects, administering the intratumoral agents occurs about 5-10 days, preferably about 6-8 days, more preferably about 7 days before and/or after a subsequent and/or previous treatment session. Treatment step (a) may occur over a period of time as needed, for example from about one week to about ten weeks, from about one week to about six weeks, or from about one week to about four weeks. As an example, a useful treatment regimen may comprise (a) administering (i) at least one TNF-a immunoconjugate, and (ii) at least one IL2 immunoconjugate to one or more melanoma lesions of a about once per week for a period of time ranging from one week to four weeks. [0016] Step (b) preferably occurs within about six weeks, more preferably within about four weeks, of completion of step (a). If possible, it may be preferable to obtain complete tumor excision (RO resection) in step (b). Optionally, patients treated according to methods described herein may undergo subsequent adjuvant therapy after completion of step (b). For example, the methods may further comprise administering one or more adjuvant therapies such as radiotherapy, chemotherapy, immunotherapy, targeted therapy, cytokine therapy, etc., after step (b).
[0017] Surprisingly, methods according to the disclosure have shown statistically significant improvement in recurrence free survival and distant metastasis free survival rates. For example, recurrence free survival has been shown to be improved by an increase in median RFS and/or DMFS by a factor greater than 1 , such as at least about 1.25, at least about 1.5, at least about 1.75, at least about 2, at least about 2.25, or at least about 2.5 after step (b), compared to the same treatment without step (a). Further, recurrence free survival has been shown to be improved by about at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 1-year after step (b), at least a 40%, at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 2-years after step (b), and/or at least a 55%, at least a 50%, at least a 45%, at least a 40%, at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 3-years after step (b), compared to the same treatment without step (a), and/or distant metastasis free survival has been shown to be improved by about at least a 15%, at least a 14%, at least a 13%, at least a 12%, at least an 11 %, at least a 10%, at least a 9%, at least an 8%, at least a 7%, at least a 6%, or at least a 5% lower risk of recurrence or death 1-year after step (b), at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 2-years after step (b), and/or at least a 50%, at least a 45%, at least a 40%, at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 3-years after step (b), compared to the same treatment without step (a). [0018] The disclosure also provides a method for improving recurrence free survival and/or distant metastasis free survival in a patient with resectable melanoma, the method comprising (a) administering a means for intratumoral treatment of melanoma to one or more melanoma lesions, and subsequently (b) resecting the treated melanoma lesion(s). The means for intratumoral treatment of melanoma includes the combination of intratumoral agents described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1A shows clinical trial results of recurrence free survival from randomization for the treatment group (ARM 1) and the control group (ARM 2) (blinded review).
[0020] FIG. 1 B shows clinical trial results of recurrence free survival from randomization for the treatment group (ARM 1 ) and the control group (ARM 2) (investigator assessment).
[0021] FIG. 1C shows clinical trial results of recurrence free survival from surgery for the treatment group (ARM 1) and the control group (ARM 2).
[0022] FIG. 1 D shows clinical trial results of recurrence free survival from randomization for the treatment group (ARM 1) and the control group (ARM 2) with and without post- surgical adjuvant therapy.
[0023] FIG. 1 E shows clinical trial results of recurrence free survival from randomization for the treatment group (ARM 1 ) and the control group (ARM 2) for patients with prior anticancer treatment.
[0024] FIG. 1F shows clinical trial results of recurrence free survival from randomization for the treatment group (ARM 1) and the control group (ARM 2) for patients with prior immunotherapy.
[0025] FIG. 1G shows clinical trial results of recurrence free survival from randomization for the treatment group (ARM 1) and the control group (ARM 2) for patients with prior systemic therapy.
[0026] FIG. 1 H shows clinical trial results of recurrence free survival from randomization for the treatment group (ARM 1 ) and the control group (ARM 2) for patients without prior systemic therapy.
[0027] FIG. 2 shows clinical trial results of distant metastasis free survival from randomization for the treatment group (ARM 1 ) and the control group (ARM 2) (blinded review).
DETAILED DESCRIPTION
[0028] The disclosure relates to the treatment of cutaneous, subcutaneous, and/or nodal melanoma using combinations of intratumoral agents and subsequent tumor resection and/or excision. The treatment methods surprisingly improve the rate of recurrence free survival and/or distant metastasis free survival in human patients.
I. INTRATUMORAL AGENTS
[0029] Intratumoral agents that can be used according to the disclosure are immunoconjugates, particularly fibronectin-targeted immunoconjugates.
[0030] The immunoconjugates that can be used according to the disclosure are immunocytokines comprising antibody-cytokine fused or linked proteins.
Antibodies
[0031] As is known, antibodies can be modified in a number of ways; thus, the term "antibody” as used herein is intended to include any specific binding member or substance having an antibody antigen-binding site with the required specificity and/or binding. As such, as used herein this term covers antibody fragments, in particular antigen-binding fragments, and derivatives, including any polypeptide comprising an antibody antigen-binding site, whether natural or wholly or partially synthetic. Chimeric molecules comprising an antibody antigen-binding site, or equivalent, fused to another polypeptide (e.g. belonging to another antibody class or subclass) are also included.
[0032] The immunoconjugates according to the disclosure are fibronectin-targeted immunoconjugates. Specifically, the immunoconjugates target fibronectin via an antibody (L19) specific for the extra domain-B of fibronectin (B-FN or ED-B), which is a domain that is rarely found in healthy adults and is almost exclusively expressed by newly formed blood vessels in tumors. Fibronectin is subject to alternative splicing, and a number of alternative isoforms of fibronectin are known. For example, alternatively spliced isoforms A-FN and B-FN, comprising domains ED-A or ED-B respectively, are markers of angiogenesis and are selectively expressed in the neovasculature. Fibronectin-targeted immunoconjugates that can be used are those that selectively bind to fibronectin isoform B-FN, for example extra domain B (e.g. ED-B). The antibodies that can be used in fibronectin-targeted immunoconjugates according to the disclosure are therefore chosen from anti-fibronectin antibodies, preferably anti-EDB antibodies, more preferably L19 antibody.
[0033] As is known, an antibody typically contains 3 heavy chain complementarity determining regions (CDRs) and 3 light chain CDRs. As is also known, CDRs are hypervariable regions of the heavy and light chains. An antigen binding site may be provided by means of arrangement of CDRs. The structure for carrying a CDR or a set of CDRs will generally be an antibody heavy or light chain sequence or substantial portion thereof in which the CDR or set of CDRs is located at a location corresponding to the CDR or set of CDRs of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. Determining the structures and locations of these variable domains can be done by known methods.
[0034] An immunoconjugate according to the disclosure may specifically bind to the ED- B of fibronectin, and thus also B-FN. Anti-ED-B antibodies and conjugates comprising anti-EDB antibodies such as the L19 antibody are known. In preferred embodiments, immunoconjugates chosen for combinations according to the disclosure comprise the CDRs of antibody L19 as set forth in SEQ ID NOs: 1 to 6.
[0035] As mentioned above, fragments of a whole antibody can perform the function of binding an antigen. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL, and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment, which consists of a VH or a VL domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; (viii) bispecific single chain Fv dimers; (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion, and (x) a single chain diabody format wherein each of the VH and VL domains within a set is connected by a short or 'non-flexible' peptide linker. Fv, scFv or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains. A single chain Fv (scFv) may be comprised within a mini-immunoglobulin or small immunoprotein (SIP). A SIP may comprise an scFv molecule fused to the CH4 domain of the human IgE secretory isoform IgE S2 (sS2- CH4) forming a homo-dimeric mini-immunoglobulin antibody molecule. Minibodies comprising a scFv joined to a CH3 domain may also be made. Other examples of binding fragments are Fab’, which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region, and Fab’-SH, which is a Fab’ fragment in which the cysteine residue(s) of the constant domains bear a free thiol group.
[0036] A fibronectin-targeted immunoconjugate as described herein may comprise an antibody that specifically binds to fibronectin (an anti-fibronectin antibody), such as a scFv. scFvs do not comprise an antibody Fc region, thus potentially reducing the effects of anti-idiotypic reaction. Thus, in preferred embodiments, the anti-fibronectin antibody for use in the immunoconjugates described herein is a scFv.
[0037] When the anti-fibronectin antibody is a scFv, the VH and VL domains of the antibody can be linked by a 10 to 20 amino acid linker, preferably by a 14 to 20 amino acid linker, more preferably by a 10 to 14 amino acid linker. Suitable linkers are known in the art. Preferably, the linker has the amino acid sequence set forth in SEQ ID NO: 11 , or is a variant thereof.
[0038] Preferably, an antibody for use as described herein may comprise the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof. Yet more preferably, an antibody for use as described herein comprises the VH and VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof. The L19 antibody is preferably in scFv format. Where the L19 antibody is in scFv format, the antibody molecule for use as described herein preferably has the amino acid sequence set forth in SEQ ID NO: 9, or is a variant thereof.
[0039] Variants of an antibody disclosed herein may be produced and used as an antifibronectin antibody as described herein. The techniques required to make substitutions within amino acid sequences of CDRs, antibody VH or VL domains, in particular the framework regions of the VH and VL domains, and antibody molecules generally, are known. Variant sequences may be made and tested for ability to bind antigen, such as B-FN and/or the ED-B of fibronectin, and/or for any other desired property, by known methods.
[0040] It is contemplated that from 1 to 5 amino acid alterations, for example from 1 to 4 amino acid alterations, from 1 to 3 amino acid alterations, or from 1 to 2 amino acid alterations, such as 1 , 2, 3, 4, or 5 amino acid alterations (addition, deletion, substitution, and/or insertion of an amino acid residue) may be made in one or more of the CDRs and/or the VH domain and/or the VL domain of an antibody molecule as described herein. For example, an anti-fibronectin antibody may comprise the CDRs and/or the VH domain and/or the VL domain of antibody L19 described herein with 5 or fewer amino acid alterations, 4 or fewer amino acid alterations, 3 or fewer amino acid alterations, or 2 or fewer amino acid alterations, for example, 5 amino acid alterations, 4 amino acid alterations, 3 amino acid alterations, 2 amino acid alterations, or 1 amino acid alteration within the CDRs and/or the VH domain and/or the VL domain. As a further example, an antibody which binds the B-FN, may comprise the VH domain and/or the VL domain of antibody L19 described herein with 5 or fewer amino acid alterations, 4 or fewer amino acid alterations, 3 or fewer amino acid alterations, or 2 or fewer amino acid alterations, for example, 5 amino acid alterations, 4 amino acid alterations, 3 amino acid alterations, 2 amino acid alterations, or 1 amino acid alteration within the framework region of the VH and/or VL domain.
[0041] An antibody that binds the B-FN or ED-B of fibronectin, as referred to herein, thus may comprise the VH domain shown in SEQ ID NO: 7 and/or the VL domain shown in SEQ ID NO: 8 with 5 or fewer amino acid alterations, 4 or fewer amino acid alterations, 3 or fewer amino acid alterations, or 2 or fewer amino acid alterations, for example, 5 amino acid alterations, 4 amino acid alterations, 3 amino acid alterations, 2 amino acid alterations, or 1 amino acid alteration within the framework region of the VH and/or VL domain. Such an antibody molecule may bind the ED-B isoform or ED-B of fibronectin with the same or substantially the same affinity as an antibody molecule comprising the VH domain shown in SEQ ID NO: 7 and the VL domain shown in SEQ ID NO: 8, or may bind the ED-B isoform or ED-B of fibronectin with a higher affinity than an antibody molecule comprising the VH domain shown in SEQ ID NO: 7 and the VL domain shown in SEQ ID NO: 8.
[0042] As one example, an anti-fibronectin antibody as described herein may comprise a VH and/or VL domain that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the L19 antibody VH and/or VL domains set forth in SEQ ID NOs: 7 and 8, respectively. As another example, an anti-fibronectin antibody as described herein may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH and VL amino acid sequence of the L19 antibody set forth in SEQ ID NOs: 7 and 8, respectively. In further embodiments, the anti-fibronectin antibody may comprise the VH and VL CDR sequences of the L19 antibody set forth in SEQ ID NOs: 1 to 6, respectively, such that the variation may be in the framework regions.
Cytokines [0043] The cytokines that can be used in immunoconjugates according to the disclosure are chosen from IL2 or TNF-a.
[0044] In some embodiments, the immunoconjugates as described herein comprise TNF- a. In some embodiments, TNF-a is preferably human TNF-a.
[0045] Human TNF-a consists of a 35 amino acid cytoplasmic domain, a 20 amino acid transmembrane domain, and a 177 amino acid extracellular domain. The 177 amino acid extracellular domain is cleaved to produce a 157 amino acid soluble form, which is biologically active, and which forms a non-covalently linked trimer in solution. Human TNF-a may, in various embodiments, be the soluble form of the extracellular domain of human TNF-a, or may be the extracellular domain of human TNF-a.
[0046] The sequence of the soluble form of the extracellular domain of human TNF-a is shown in SEQ ID NO: 10. In various embodiments of the disclosure, the TNF-a comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 10, or a variant thereof. For example, in various embodiments the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10.
[0047] The sequence of the extracellular domain of human TNF-a is shown in SEQ ID NO: 16. Thus, in other embodiments, the TNF-a may comprise, consist essentially of, or consist of the sequence set forth in SEQ ID NO: 16, or a variant thereof. For example, the TNF-a may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16.
[0048] In some embodiments, TNF-a in immunoconjugates according to the disclosure may retain a biological activity of human TNF-a, e.g. the ability to inhibit cell proliferation. In other embodiments, TNF-a may be a wild type human TNF-a or a TNF-a mutant which retains biological function of human TNF-a, e.g. the ability to inhibit cell proliferation but has a reduced activity relative to the wild-type human TNF-a.
[0049] In some embodiments, the immunoconjugates as described herein comprise IL2. The IL2 may in some embodiments be human IL2.
[0050] In various embodiments, the IL2 comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 18, or a variant thereof. For example, the IL2 may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18.
[0051] In some embodiments, IL2 in immunoconjugates according to the disclosure may retain a biological activity of human IL2, e.g. the ability to inhibit cell proliferation.
[0052] In various preferred embodiments, the immunoconjugate is an IL2 immunoconjugate comprising an amino acid sequence with at least about 80% identity with L19-IL2 (SEQ ID NO: 14), and/or a TNF-a immunoconjugate comprising an amino acid sequence with at least about 80% identity with L19-TNF-a (SEQ ID NO: 15). In particularly preferred embodiments, the IL2 immunoconjugate comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identity with L19-IL2 (SEQ ID NO: 14), and/or the TNF-a immunoconjugate comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identity with L19-TNF-a (SEQ ID NO: 15).
Linkers
[0053] In various embodiments, the cytokine and antibody of the immunoconjugate may be conjugated via a linker (linked), e.g. a peptide linker. The peptide linker connecting the antibody and cytokine in an immunoconjugate described herein may, for example, be chosen from flexible peptide linkers. Suitable examples of peptide linker sequences are known. The linker may, in various embodiments, be 10-20 amino acids in length, such as 10-15 amino acids in length or 15-18 amino acids in length. Preferably, the linker is 11-15 amino acids in length or 16-18 amino acids in length, for example 17 amino acids in length. The linker may have the sequence set forth in SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 17, or may be a variant thereof. Preferably, the peptide linker connecting the antibody and cytokine in an immunoconjugate described herein has the sequence set forth in SEQ ID NO: 17.
[0054] In other embodiments, however, the immunoconjugate does not include a linker, but rather the cytokine and antibody are conjugated directly (fused), e.g. via a chemical bond. The chemical bond can be, for example, a covalent or ionic bond. Examples of covalent bonds include peptide bonds (amide bonds) and disulphide bonds. The cytokine and antibody may be covalently linked, for example by peptide bonds (amide bonds). Thus, the antibody molecule, e.g. a scFv portion of an antibody molecule, and cytokine, e.g. IL2 or TNF-a, may be a fusion protein. By “fusion protein” it is meant a polypeptide that is a translation product resulting from the fusion of two or more genes or nucleic acid coding sequences into one open reading frame (ORF).
Fibronectin-Targeted Immunoconjuciates
[0055]As described herein, immunoconjugates that can be used in combinations and methods according to the disclosure comprise a cytokine chosen from IL2 or TNF-a that is connected, e.g. fused or linked, to an anti-fibronectin antibody, for example an anti- EDB antibody. In preferred embodiments, the immunoconjugates comprise a cytokine chosen from IL2 and TNF-a that is connected, e.g. fused or linked, to an antibody molecule comprising L19 CDRs. More preferably, the immunoconjugates comprise IL2 or TNF-a and the CDRs of antibody L19 as set forth in SEQ ID NOs: 1 to 6.
[0056] In preferred embodiments, the immunoconjugates chosen for combinations and methods according to the disclosure comprise, consist essentially of, or consist of IL2 and/or TNF-a immunoconjugates comprising an L19 antibody, for example having the CDRs of antibody L19 as set forth in SEQ ID NOs: 1 to 6.
[0057] In preferred embodiments, the immunoconjugates used in combinations and methods according to the disclosure are chosen from IL2 or TNF-a immunoconjugates comprising the L19 VH domain amino acid sequence of SEQ ID NO: 7 and the L19 VL domain amino acid sequence of SEQ ID NO: 8.
[0058] A preferred immunoconjugate that can be used in combinations and methods according to the disclosure may comprise, consist essentially of, or consist of the sequence shown in SEQ ID NO: 14, or may be a variant thereof. A further preferred immunoconjugate that can be used in combinations and methods according to the disclosure may comprise, consist essentially of, or consist of the sequence shown in SEQ ID NO: 15, or may be a variant thereof. In some particularly preferred embodiments, combinations according to the disclosure comprise a TNF-a immunoconjugate comprising or consisting of the sequence shown in SEQ ID NO: 15 and an IL2 immunoconjugate comprising or consisting of the sequence shown in SEQ ID NO: 14.
[0059]A variant of a reference sequence, such as an antibody, IL2, TNF-a, linker, or immunoconjugate sequence as described herein, may have an amino acid sequence having at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the reference amino acid sequence. Amino acid sequence identity is generally defined with reference to the algorithm GAP, but other known algorithms may be used, e.g. BLAST, psiBLAST, or TBLASTN.
[0060] Particular amino acid sequence variants may differ from a reference sequence by insertion, addition, substitution, or deletion of 1 or more amino acids, for example 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 5-10 amino acids, 10-20 amino acids, or 20-30 amino acids. In some embodiments, a variant sequence may comprise the reference sequence with 1 or more residues, for example 2 residues, 3 residues, 4 residues, 5 residues, 6 residues, 7 residues, 8 residues, 9 residues, or 10 or more residues inserted, deleted, or substituted. For example, up to 15 residues, up to 20 residues, up to 30 residues, or up to 40 residues may be inserted, deleted, or substituted. [0061] In some preferred embodiments, a variant may differ from a reference sequence by 1 or more substitutions, for example 2 substitutions, 3 substitutions, 4 substitutions, 5 substitutions, 6 substitutions, 7 substitutions, 8 substitutions, 9 substitutions, or 10 or more substitutions, which are preferably conservative substitutions. “Conservative substitutions” involve the replacement of an amino acid with a different amino acid having similar properties. For example, an aliphatic residue may be replaced by another aliphatic residue, a non-polar residue may be replaced by another non-polar residue, an acidic residue may be replaced by another acidic residue, a basic residue may be replaced by another basic residue, a polar residue may be replaced by another polar residue, or an aromatic residue may be replaced by another aromatic residue. Conservative substitutions may, for example, be between amino acids within the following groups:
(i) alanine and/or glycine;
(ii) glutamic acid, aspartic acid, glutamine, and/or asparagine
(iii) arginine and/or lysine;
(iv) asparagine, glutamine, glutamic acid, and/or aspartic acid
(v) isoleucine, leucine, and/or valine;
(vi) phenylalanine, tyrosine, and/or tryptophan;
(vii) serine, threonine, and/or cysteine.
[0062] Thus, various combinations of intratumoral agents according to the disclosure comprise IL2 immunoconjugates, in particular one or more IL2 immunoconjugates and one or more TNF-a immunoconjugates. In preferred embodiments, combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates comprising an anti-EDB antibody and one or more TNF-a immunoconjugates comprising an anti-EDB antibody. In more preferred embodiments, combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates comprising an L19 antibody and one or more TNF-a immunoconjugates comprising an L19 antibody.
[0063] In yet further preferred embodiments, combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and one or more TNF-a immunoconjugates where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10. Other combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates, where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and one or more TNF-a immunoconjugates, where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. Still other preferred embodiments of combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates, where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and one or more TNF-a immunoconjugates, where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16.
[0064] In further preferred embodiments, combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates comprising an antifibronectin antibody in scFv form and one or more TNF-a immunoconjugates comprising an anti-fibronectin antibody in scFv form.
[0065] In still further preferred embodiments, combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof, and one or more TNF-a immunoconjugates comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof.
[0066] In yet further preferred embodiments, combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates comprising an anti-fibronectin antibody in scFv form, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof, and one or more TNF-a immunoconjugates comprising an anti-fibronectin antibody in scFv form, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof.
[0067] In particularly preferred embodiments, combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates, where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof, and one or more TNF-a immunoconjugates, where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof. Other combinations of intratumoral agents according to the disclosure comprise one or more TNF-a immunoconjugates, where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, and comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof, and one or more IL2 immunoconjugates, where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof. Other preferred embodiments of combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates, where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof, and, and one or more TNF-a immunoconjugates, where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16, and comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof.
[0068] In further particularly preferred embodiments, combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates, where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and comprising an anti-fibronectin antibody in scFv form, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof, and one or more TNF-a immunoconjugates, where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and comprising an anti-fibronectin antibody in scFv form, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof. Other combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates, where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and comprising an anti-fibronectin antibody in scFvform, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof, and one or more TNF-a immunoconjugates, where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, and comprising an anti-fibronectin antibody in scFvform, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof. Still other combinations of intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates, where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and comprising an anti-fibronectin antibody in scFv form, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof, and one or more TNF-a immunoconjugates, where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16, and comprising an anti- fibronectin antibody in scFv form, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof.
II. COMPOSITIONS
[0069] The intratumoral agents described herein are typically administered in the form of pharmaceutical compositions, which may optionally comprise at least one component in addition to the intratumoral agent(s). Pharmaceutical compositions may comprise, for example, at least one pharmaceutically acceptable excipient, carrier, buffer, stabilizer, and/or other material suitable for use in a pharmaceutical composition. The precise nature of the carrier or other material will vary, and may depend on the route of administration, which may, for example, be by subcutaneous or intratumoral injection. It is within the ability of those skilled in the art to choose types and amounts of suitable components for pharmaceutical compositions according to the disclosure.
[0070] Compositions for administering intratumoral agents according to the disclosure comprise therapeutically effective amounts of one or more immunoconjugates chosen from IL2 and/or TNF-a immunoconjugates. The compositions are in a form suitable for administration directly to the area to be treated, e.g. the melanoma lesion.
[0071] In various embodiments, compositions for administering intratumoral agents according to the disclosure comprise an amount of IL2 immunoconjugates ranging from about 0.5 mg to about 8 mg, for example from about 0.75 mg to about 6 mg, from about 0.75 mg to about 3 mg, from about 0.75 mg to about 2 mg, from about 0.75 mg to about 1.25 mg, from about 1 mg to about 5 mg, from about 1 mg to about 4 mg, from about 1 mg to about 3 mg, from about 1 mg to about 2 mg, from about 1 mg to about 1 .5 mg, from about 1.5 mg to about 3 mg, or from about 2 mg to about 2.5 mg. In preferred embodiments, the compositions comprise an amount of IL2 immunoconjugates ranging from about 0.9 mg to about 2.3 mg, such as from about 1 mg to about 2.2 mg. In more preferred embodiments, the compositions comprise an amount of IL2 immunoconjugates of 1 mg (± 10%), such as about 1.08 mg, or an amount of IL2 immunoconjugates of 2 mg (± 10%), such as about 2.17 mg.
[0072] In some preferred embodiments, compositions for administering intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof, in an amount ranging from about 0.5 mg to about 8 mg, for example from about 0.75 mg to about 6 mg, from about 0.75 mg to about 3 mg, from about 0.75 mg to about 2 mg, from about 0.75 mg to about 1.25 mg, from about 1 mg to about 5 mg, from about 1 mg to about 4 mg, from about 1 mg to about 3 mg, from about 1 mg to about 2 mg, from about 1 mg to about 1 .5 mg, from about 1 .5 mg to about 3 mg, or from about 2 mg to about 2.5 mg, such as 1 mg (± 10%), or 2 mg (± 10%). In more preferred embodiments, the compositions comprise an amount of these IL2 immunoconjugates ranging from about 0.9 mg to about 2.3 mg, such as from about 1 mg to about 2.2 mg. In even more preferred embodiments, the compositions comprise an amount of these IL2 immunoconjugates of about 1.08 mg, or about 2.17 mg.
[0073] In other preferred embodiments, compositions for administering intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and comprising an anti-fibronectin antibody in scFv form, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof, in an amount ranging from about 0.5 mg to about 8 mg, for example from about 0.75 mg to about 6 mg, from about 0.75 mg to about 3 mg, from about 0.75 mg to about 2 mg, from about 0.75 mg to about 1.25 mg, from about 1 mg to about 5 mg, from about 1 mg to about 4 mg, from about 1 mg to about 3 mg, from about 1 mg to about 2 mg, from about 1 mg to about 1 .5 mg, from about 1 .5 mg to about 3 mg, or from about 2 mg to about 2.5 mg, such as 1 mg (± 10%), or 2 mg (± 10%). In more preferred embodiments, the compositions comprise an amount of these IL2 immunoconjugates ranging from about 0.9 mg to about 2.3 mg, such as from about 1 mg to about 2.2 mg. In even more preferred embodiments, the compositions comprise an amount of these IL2 immunoconjugates of about 1.08 mg, or about 2.17 mg.
[0074] In still further preferred embodiments, compositions for administering intratumoral agents according to the disclosure comprise L19-IL2 in an amount ranging from about 0.5 mg to about 8 mg, for example from about 0.75 mg to about 6 mg, from about 0.75 mg to about 3 mg, from about 0.75 mg to about 2 mg, from about 0.75 mg to about 1.25 mg, from about 1 mg to about 5 mg, from about 1 mg to about 4 mg, from about 1 mg to about 3 mg, from about 1 mg to about 2 mg, from about 1 mg to about 1 .5 mg, from about 1 .5 mg to about 3 mg, or from about 2 mg to about 2.5 mg, such as 1 mg (± 10%), or 2 mg (± 10%). In more preferred embodiments, the compositions comprise an amount of L19-IL2 ranging from about 0.9 mg to about 2.3 mg, such as from about 1 mg to about 2.2 mg. In even more preferred embodiments, the compositions comprise an amount of L19-IL2 of about 1.08 mg, or about 2.17 mg.
[0075] In various embodiments, compositions for administering intratumoral agents according to the disclosure comprise an amount of TNF-a immunoconjugates ranging from about 50 pg to about 600 pg, such as from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 150 pg to about 450 pg, from about 150 pg to about 400 pg, from about 200 pg to about 400 pg, from about 150 pg to about 300 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 250 pg to about 450 pg, from about 300 pg to about 450 pg, from about 350 pg to about 500 pg, or from about 350 pg to about 450 pg. In preferred embodiments, the compositions comprise an amount of TNF-a immunoconjugates ranging from about 150 pg to about 450 pg, for example from about 175 pg to about 425 pg, or from about 200 pg to about 400 pg. In other preferred embodiments, the compositions comprise an amount of TNF- a immunoconjugates ranging from about 175 pg to about 225 pg, for example about 200 pg, or from about 375 pg to about 425 pg, for example about 400 pg.
[0076] In some preferred embodiments, compositions for administering intratumoral agents according to the disclosure comprise one or more TNF-a immunoconjugates where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof, in an amount ranging from about 50 pg to about 600 pg, such as from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 150 pg to about 450 pg, from about 150 pg to about 400 pg, from about 200 pg to about 400 pg, from about 150 pg to about 300 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 250 pg to about 450 pg, from about 300 pg to about 450 pg, from about 350 pg to about 500 pg, or from about 350 pg to about 450 pg, such as about 200 pg, or about 400 pg. In more preferred embodiments, the compositions comprise an amount of these TNF-a immunoconjugates ranging from about 150 pg to about 450 pg, for example from about 175 pg to about 425 pg, or from about 200 pg to about 400 pg. In even more preferred embodiments, the compositions comprise an amount of these TNF-a immunoconjugates ranging from about 175 pg to about 225 pg, for example about 200 pg, or from about 375 pg to about 425 pg, for example about 400 pg.
[0077] In other preferred embodiments, compositions for administering intratumoral agents according to the disclosure comprise one or more TNF-a immunoconjugates where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, and comprising the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8, or variants thereof, in an amount ranging from about 50 pg to about 600 pg, such as from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 150 pg to about 450 pg, from about 150 pg to about 400 pg, from about 200 pg to about 400 pg, from about 150 pg to about 300 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 250 pg to about 450 pg, from about 300 pg to about 450 pg, from about 350 pg to about 500 pg, or from about 350 pg to about 450 pg, such as about 200 pg, or about 400 pg. In more preferred embodiments, the compositions comprise an amount of these TNF-a immunoconjugates ranging from about 150 pg to about 450 pg, for example from about 175 pg to about 425 pg, or from about 200 pg to about 400 pg. In even more preferred embodiments, the compositions comprise an amount of these TNF-a immunoconjugates ranging from about 175 pg to about 225 pg, for example about 200 pg, or from about 375 pg to about 425 pg, for example about 400 pg.
[0078] In further preferred embodiments, compositions for administering intratumoral agents according to the disclosure comprise one or more TNF-a immunoconjugates where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and comprising an antifibronectin antibody in scFv form, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof, in an amount ranging from about 50 pg to about 600 pg, such as from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 150 pg to about 450 pg, from about 150 pg to about 400 pg, from about 200 pg to about 400 pg, from about 150 pg to about 300 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 250 pg to about 450 pg, from about 300 pg to about 450 pg, from about 350 pg to about 500 pg, or from about 350 pg to about 450 pg, such as about 200 pg, or about 400 pg. In more preferred embodiments, the compositions comprise an amount of these TNF-a immunoconjugates ranging from about 150 pg to about 450 pg, for example from about 175 pg to about 425 pg, or from about 200 pg to about 400 pg. In even more preferred embodiments, the compositions comprise an amount of these TNF-a immunoconjugates ranging from about 175 pg to about 225 pg, for example about 200 pg, or from about 375 pg to about 425 pg, for example about 400 pg-
[0079] In still further preferred embodiments, compositions for administering intratumoral agents according to the disclosure comprise one or more TNF-a immunoconjugates where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, and comprising an antifibronectin antibody in scFv form, having the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof, in an amount ranging from about 50 pg to about 600 pg, such as from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 150 pg to about 450 pg, from about 150 pg to about 400 pg, from about 200 pg to about 400 pg, from about 150 pg to about 300 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 250 pg to about 450 pg, from about 300 pg to about 450 pg, from about 350 pg to about 500 pg, or from about 350 pg to about 450 pg, such as about 200 pg, or about 400 pg. In more preferred embodiments, the compositions comprise an amount of these TNF-a immunoconjugates ranging from about 150 pg to about 450 pg, for example from about 175 pg to about 425 pg, or from about 200 pg to about 400 pg. In even more preferred embodiments, the compositions comprise an amount of these TNF-a immunoconjugates ranging from about 175 pg to about 225 pg, for example about 200 pg, or from about 375 pg to about 425 pg, for example about 400 pg-
[0080] In even further preferred embodiments, compositions for administering intratumoral agents according to the disclosure comprise L19-TNF-a in an amount ranging from about 50 pg to about 600 pg, such as from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 150 pg to about 450 pg, from about 150 pg to about 400 pg, from about 200 pg to about 400 pg, from about 150 pg to about 300 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 250 pg to about 450 pg, from about 300 pg to about 450 pg, from about 350 pg to about 500 pg, or from about 350 pg to about 450 pg, such as about 200 pg, or about 400 pg. In more preferred embodiments, the compositions comprise an amount of L19-TNF-a ranging from about 150 pg to about 450 pg, for example from about 175 pg to about 425 pg, or from about 200 pg to about 400 pg. In even more preferred embodiments, the compositions comprise an amount of L19-TNF-a ranging from about 175 pg to about 225 pg, for example about 200 pg, or from about 375 pg to about 425 pg, for example about 400 pg. Preferably, the compositions comprise about 200 pg or about 400 pg of L19- TNF-a.
[0081] It should be understood that, in various embodiments, compositions for administering intratumoral agents according to the disclosure comprise one or more IL2 immunoconjugates and one or more TNF-a immunoconjugates. In those embodiments, the compositions can comprise amounts of IL2 immunoconjugates and TNF-a immunoconjugates as described above.
[0082] For example, the compositions may comprise from about 1 mg to about 5 mg of IL2 immunoconjugates, and from about 100 g to about 500 pg of TNF-a immunoconjugates. In some preferred embodiments, the compositions may comprise from about 1 mg to about 5 mg L19-IL2 and from about 100 pg to about 500 pg L19- TNFa. In some preferred embodiments, the composition comprises from about 1 mg to about 2.2 mg L19-I L2, and from about 200 pg to about 400 pg L19-TNFa. For example, in one preferred embodiment, the composition comprises about 1.08 mg L19-IL2 and about 200 pg L19-TNFa, in another preferred embodiment, the composition comprises about 2.17 mg L19-IL2 and about 100 pg L19-TNFa, in another preferred embodiment, the composition comprises about 2.17 mg L19-IL2 and about 200 pg L19-TNFa, in another preferred embodiment, the composition comprises about 2.17 mg L19-IL2 and about 300 pg L19-TNFa, and in yet another preferred embodiment, the composition comprises about 2.17 mg L19-IL2 and about 400 pg L19-TNFa.
III. METHODS
[0083] All description of methods of treatment provided herein is equally applicable to intratumoral agents (e.g. combinations of intratumoral agents) for use in such methods. Similarly, all description of methods of treatment provided herein is equally applicable to the use of intratumoral agents (e.g. combinations of intratumoral agents) in the manufacture of a medicament, wherein the medicament is for the treatment of melanoma according to such methods.
[0084] Methods according to the disclosure comprise (a) administering combinations of intratumoral agents as described herein to a patient (also referred to as an “intratumoral treatment” or “preceding intratumoral treatment”), followed by (b) tumor resection and/or excision. The methods can be used to treat cutaneous, subcutaneous, and/or nodal melanoma, for example in Stage III, (e.g. Stage 11 IB, Stage 11 IC, Stage HID) or Stage IV patients. In preferred embodiments, the patient treated using methods according to the disclosure has metastasized melanoma. The treatment methods surprisingly increase the recurrence free survival and/or distant metastasis free survival in treated patients. [0085] The methods comprise treatment regimens that include administering the combination of intratumoral agents to the patient subcutaneously or intratumorally. The dosages, routes, and timing of administering the intratumoral agents can vary.
[0086] It will be understood that dosages administered to a patient may correspond to an amount of the antitumoral agent(s) present in a composition comprising the agent(s), or may correspond to a multiple of an amount of the agent(s) present in the composition. For example, a method of administering an intratumoral agent at a dosage of 400 pg can be accomplished by administering a composition having 400 pg of the agent, or can be accomplished by administering two compositions having 200 pg of the agent.
[0087] In various embodiments, methods according to the disclosure comprise administering intratumoral agents as described herein according to a dosing regimen that permits at least about 24 hours, such as at least about 48 hours, or at least about 72 hours between intratumoral treatment sessions where one or more intratumoral agents are administered to a patient. For example, various methods comprise administering one or a combination of intratumoral agents as described herein approximately or exactly every 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or approximately or exactly every 1 , 2, or 3 weeks, for a specified period of time. In preferred embodiments, administering the intratumoral agents occurs about 5-10 days, preferably about 6-8 days, more preferably about 7 days before and/or after a subsequent and/or previous treatment session.
[0088] Dosages of IL2 immunoconjugates that can be administered in methods according to the disclosure include those amounts disclosed as useful in compositions according to the disclosure, for example may range from about 0.25 mg to about 8 mg. Thus, in various embodiments, dosages of IL2 immunoconjugates that can be administered per intratumoral treatment session in various methods according to the disclosure range from about 0.5 mg to about 8 mg, for example from about 0.75 mg to about 6 mg, from about 0.75 mg to about 3 mg, from about 0.75 mg to about 2 mg, from about 0.75 mg to about 1.25 mg, from about 1 mg to about 5 mg, from about 1 mg to about 4 mg, from about 1 mg to about 3 mg, from about 1 mg to about 2 mg, from about 1 mg to about 1 .5 mg, from about 1.5 mg to about 3 mg, or from about 2 mg to about 2.5 mg. In preferred embodiments, the dosage of the IL2 immunoconjugates administered ranges from about 0.9 mg to about 2.3 mg, such as from about 1 mg to about 2.2 mg, at approximately or exactly 1 week intervals. In more preferred embodiments, the dosage of the IL2 immunoconjugates administered is 1 mg (± 10%), such as about 1.08 mg, or 2 mg (± 10%), such as about 2.17 mg, at approximately or exactly 1 week intervals.
[0089] The dosage of an IL2 immunoconjugate administered in methods according to the disclosure may alternatively be defined with reference to International Units (IU) of IL2 immunoconjugates administered to the patient. The IU of an IL2 immunoconjugate may be determined by any suitable means.
[0090] Thus, in some embodiments, the dosage of IL2 immunoconjugates that can be administered per intratumoral treatment session according to the disclosure range from about 1.6 million IU (MiolU) to about 15 MiolU, such as from about 2 MiolU to about 15 MiolU, from about 3 MiolU to about 15 MiolU, from about 4 MiolU to about 15 MiolU, from about 5 MiolU to about 15 MiolU, from about 6 MiolU to about 13 MiolU, or from about 6.5 MiolU to about 13 MiolU. In some preferred embodiments, the dosage of IL2 immunoconjugates is about 6.5 MiolU, and in other preferred embodiments the dosage of IL2 immunoconjugates is about 13 MiolU.
[0091] In other embodiments, methods according to the disclosure comprise injecting a composition comprising one or more IL2 immunoconjugates into a melanoma lesion, where the dosage is defined by the IU of the IL2 immunoconjugate injected into the lesion as a function of the longest diameter or the longest distance between two vertices of the lesion (e.g. lU/cm or lU/cm lesion diameter). The lesion diameter refers to the longest diameter of a melanoma lesion. For example, where the melanoma lesion is substantially circular or spheroid, the lesion diameter is the line passing through the center of the circle or spheroid melanoma lesion. Where the melanoma lesion is, for example, elliptical the lesion diameter is the major axis of the ellipse (i.e. the longest diameter). Where the melanoma lesion is non-uniform or polygonal in shape, the lesion diameter is the longest distance between two vertices of the melanoma lesion.
[0092]As non-limiting examples, the dosage of IL2 immunoconjugates injected into a melanoma lesion per intratumoral treatment session can range from about 0.25 MiolU/cm lesion diameter to about 100 Mioll/cm lesion diameter, such as from about 0.5 MiolU/cm lesion diameter to about 90 MiolU/cm lesion diameter, about 0.75 MiolU/cm lesion diameter to about 80 MiolU/cm lesion diameter, about 1 MiolU/cm lesion diameter to about 80 MiolU/cm lesion diameter, about 1.25 MiolU/cm lesion diameter to about 70 MiolU/cm lesion diameter, about 1.5 MiolU/cm lesion diameter to about 60 MiolU/cm lesion diameter, about 1.75 MiolU/cm lesion diameter to about 50 MiolU/cm lesion diameter, about 2 MiolU/cm lesion diameter to about 40 MiolU/cm lesion diameter, about 2.25 MiolU/cm lesion diameter to about 37.5 MiolU/cm lesion diameter, about 2.5 MiolU/cm lesion diameter to about 35 MiolU/cm lesion diameter, about 3 MiolU/cm lesion diameter to about 33 MiolU/cm lesion diameter, or about 3.25 MiolU/cm lesion diameter to about 16.25 MiolU/cm lesion diameter. In preferred embodiments, the dosage of IL2 immunoconjugate injected into a melanoma lesion per treatment session ranges from about 3.25 MiolU/cm lesion diameter to about 16.25 MiolU/cm lesion diameter, at approximately or exactly 1 week intervals.
[0093] Dosages of TNF-a immunoconjugates that can be administered in methods according to the disclosure include those amounts disclosed as useful in compositions according to the disclosure. Thus, in various embodiments, dosages of TNF-a immunoconjugates that can be administered per intratumoral treatment session in various methods according to the disclosure range from about 50 pg to about 600 pg, such as from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 150 pg to about 450 pg, from about 150 pg to about 400 pg, from about 200 pg to about 400 pg, from about 150 pg to about 300 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 250 pg to about 450 pg, from about 300 pg to about 450 pg, from about 350 pg to about 500 pg, or from about 350 pg to about 450 pg. In preferred embodiments, the dosage of TNF-a immunoconjugates administered ranges from about 150 g to about 450 pg, for example from about 175 pg to about 425 pg, or from about 200 pg to about 400 pg, at approximately or exactly 1 week intervals. In other preferred embodiments, the dosage of TNF-a immunoconjugates administered ranges from about 175 pg to about 225 pg, for example about 200 pg, or from about 375 pg to about 425 pg, for example about 400 pg, at approximately or exactly 1 week intervals. Preferably, the dosage of TNF-a immunoconjugates administered is about 200 pg or about 400 pg, more preferably about 200 pg or about 400 pg of L19-TNF-a.
[0094] In some embodiments where the first dose is higher, the dose of the TNF-a immunoconjugate is reduced, e.g. to about 100 pg, about 200 pg, or about 300 pg per treatment session following a grade > 3 adverse events (Aes) recorded after the first dose administration. The dosage adjustment can be determined depending on the patient’s tolerance.
[0095] In other embodiments, methods according to the disclosure comprise injecting a composition comprising one or more TNF-a immunoconjugates into a melanoma lesion, where the dosage is defined as the mass of TNF-a immunoconjugate injected into the lesion as a function of the longest diameter or the longest distance between two vertices of the lesion (e.g. pg/cm or pg/cm lesion diameter). The lesion diameter refers to the longest diameter of a melanoma lesion. For example, where the melanoma lesion is substantially circular or spheroid, the lesion diameter is the line passing through the center of the circle or spheroid melanoma lesion. Where the melanoma lesion is, for example, elliptical the lesion diameter is the major axis of the ellipse (i.e. the longest diameter). Where the melanoma lesion is non-uniform or polygonal in shape, the lesion diameter is the longest distance between two vertices of the melanoma lesion.
[0096] As non-limiting examples, the dosage of TNF-a immunoconjugates injected into a melanoma lesion per intratumoral treatment session can range from about 12.5 pg/cm lesion diameter to about 1 mg/cm lesion diameter, such as from about 25 pg/cm lesion diameter to about 900 pg/cm lesion diameter, about 37.5 pg/cm lesion diameter to about 800 pg/cm lesion diameter, about 50 pg/cm lesion diameter to about 750 pg/cm lesion diameter, about 62.5 pg/cm lesion diameter to about 700 pg/cm lesion diameter, about 75 pg/cm lesion diameter to about 600 pg/cm lesion diameter, about 87.5 pg/cm lesion diameter to about 550 pg/cm lesion diameter, or about 100 pg/cm lesion diameter to about 500 pg/cm lesion diameter. In preferred embodiments, the dosage of TNF-a immunoconjugate injected into a melanoma lesion per intratumoral treatment session ranges from about 100 pg/cm lesion diameter to about 500 pg/cm lesion diameter, at approximately or exactly 1 week intervals.
[0097] In various embodiments where TNF-a immunoconjugates and IL2 immunoconjugates are both administered, the immunoconjugates can be administered together or separately. Thus, the immunoconjugates can be formulated into a single combined composition or into separate compositions. If separate administration is contemplated, the immunoconjugates may be administered in a single treatment session or in separate treatment sessions.
[0098] For example, in some embodiments the TNF-a immunoconjugate and the IL2 immunoconjugate may be formulated as two separate compositions. Separate composition preparations may be useful, for example, to facilitate separate and sequential (in any order) or simultaneous administration, and may also allow administration of the compositions by different routes. Separate compositions may also permit the use of different excipients, stabilizers, buffers, etc. The separate compositions can be administered into the tumor in any order.
[0099] Optionally, when the TNF-a immunoconjugate and the IL2 immunoconjugate are formulated as separate compositions, the two compositions may be combined at or near the time when the intratumoral agent will be administered, e.g. just prior to administration. For example, the compositions may be combined into a single syringe prior to injecting the combination into a melanoma lesion.
[0100] In other embodiments, the TNF-a immunoconjugate and the IL2 immunoconjugate may be formulated together as a single, combined composition. A combined composition may facilitate co-administration of the IL2 and TNF-a immunoconjugates. Thus, in preferred embodiments, compositions useful in methods according to the disclosure comprise at least one TNF-a immunoconjugate as described herein, and at least one IL2 immunoconjugate as described herein.
[0101] In various embodiments, the intratumoral compositions can be administered by injection at or near a melanoma lesion. Preferably, the methods comprise injecting the intratumoral composition into the targeted melanoma lesion. For example, the composition injected into the lesion may comprise from 0.25 mg to 5 mg L19-IL2 and from 50 pg to 500 pg L19-TNFa. Preferably, the composition injected into the lesion comprises from 1 mg to 2.2 mg L19-IL2 and from 200 pg to 400 pg L19-TNFa. In some preferred embodiments, the composition injected into the lesion comprises 0.27 mg L19-IL2 and 50 pg L19-TNFa. In other preferred embodiments, the composition injected into the lesion comprises 0.54 mg L19-IL2 and 100 pg L19-TNFa. In other preferred embodiments, the composition injected into the lesion comprises 1.08 mg L19-IL2 and 200 pg L19-TNFa. In still other preferred embodiments, the composition injected into the lesion comprises 1.627 mg L19-IL2 and 300 pg L19-TNFa. In particularly preferred embodiments, the composition injected into the lesion comprises 2.17 mg (13 MiolU) L19-IL2 and 400 pg L19-TNFa.
[0102] In embodiments where the dose of the TNF-a immunoconjugate per treatment session is reduced following a grade > 3 adverse event, the methods may comprise injecting an intratumoral composition into the melanoma lesion comprising from 0.25 mg to 5 mg L19-IL2 and from 25 pg to 250 pg L19-TNFa, such as from 1 mg to 2.2 mg L19- IL2 and from 100 pg to 200 pg L19-TNFa. For example, in some preferred embodiments the composition injected into the melanoma lesion may comprise 0.27 mg L19-IL2 and 25 pg L19-TNFa. In other preferred embodiments, the composition injected into the lesion comprises 0.54 mg L19-IL2 and 50 pg L19-TNFa. In other preferred embodiments, the composition injected into the lesion comprises 1.08 mg L19-IL2 and 100 pg L19-TNFa. In still other preferred embodiments, the composition injected into the lesion comprises 1.627 mg L19-IL2 and 150 pg L19-TNFa. In other preferred embodiments still, the composition injected into the lesion comprises 2.17 mg (13 MiolU) L19-IL2 and 200 pg L19-TNFa.
[0103] In various embodiments, the intratumoral treatment may continue for a period of time up to about 12 weeks, such as up to about 10 weeks, up to about 8 weeks, up to about 6 weeks, up to about 5 weeks, up to about 4 weeks, up to about 3 weeks, or up to about 2 weeks before treatment with the intratumoral agent is discontinued. Preferably, treatment with the intratumoral agents lasts for a period of time ranging from about 1 week to about 6 weeks, such as about 1 week to about 5 weeks, or about 1 week to about 4 weeks. More preferably, treatment with the intratumoral agents lasts for a period of 4 weeks.
[0104] An exemplary treatment regimen may comprise administering a combination of intratumoral agents as described herein about or exactly 3 times per week, such as about or exactly 2 times per week, or about or exactly 1 time per week, for a period of time ranging from about 1 week to about 8 weeks, such as from about 1 week to about 6 weeks, from about 1 week to about 4 weeks, from about 1 week to about 3 weeks, or from about 1 week to about 2 weeks, preferably once per week for 1 -4 weeks, more preferably once per week for 4 weeks.
[0105] By way of example, an intratumoral treatment regimen according to the disclosure may comprise injecting a combination of one or more TNF-a immunoconjugates and one or more IL2 immunoconjugates into one or more melanoma lesions, where the TNF-a immunoconjugates and/or IL2 immunoconjugates may be administered to the patient approximately or exactly once a week for a period of time ranging from one week to four weeks, in a dosage of TNF-a immunoconjugates ranging from about 150 pg to about 450 pg, for example from about 175 pg to about 425 pg, or from about 200 pg to about 400 pg, and/or a dosage of IL2 immunoconjugates ranging from about 0.75 mg to about 3 mg, such as from about 1 mg to about 2.2 mg.
[0106] A preferred intratumoral treatment regimen may comprise injecting one or more IL2 immunoconjugates and one or more TNF-a immunoconjugates into one or more melanoma lesions approximately or exactly once a week for a period of time ranging from one week to four weeks, where the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, and where the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16. In some cases, the IL2 immunoconjugates and/or TNF-a immunoconjugates comprise the VH and/or VL domains of antibody L19 set forth in SEQ ID NOs: 7 and 8. Preferably, the dose of L19-TNF-a ranges from about 350 pg to about 450 pg, for example from about 375 pg to about 425 pg, or is about 400 pg, and the dose of L19-IL2 ranges from about 0.75 mg to about 3 mg, such as from about 1 mg to about 2.2 mg, or is about 2.17 mg (13 MiolU).
[0107] In a particularly preferred embodiment, an intratumoral treatment regimen according to the disclosure may comprise injecting an IL2 immunoconjugate comprising an amino acid sequence with at least about 70% identity with L19-IL2 (SEQ ID NO: 14), and/or a TNF-a immunoconjugate comprising an amino acid sequence with at least about 70% identity with L19-TNF-a (SEQ ID NO: 15) into one or more melanoma lesions approximately or exactly once a week for a period of time ranging from one week to four weeks, where the dose of L19-TNF-a is about 400 pg, and the dose of L19-IL2 is about 2.17 mg (13 MiolU).
[0108] In one particularly preferred embodiment, an L19-TNF-a immunoconjugate as described herein is formulated for injection as a composition comprising 15mM NaH2PO4(2H2O), 10mM Na2HPO4(2H2O), 1.5 mM KCI, 75 mM mannitol, 30 mM NaCI, 1 % (w/v) glycerol, 5mM EDTA, and 0.01 % (v/v) polysorbate 20 (Tween® 20) at pH of about 8, and is suitable for intratumoral injection, and an L19-IL2 immunoconjugate as described herein is formulated for injection as a composition comprising 6.7 mM NaH2PO4 (2H2O), 1.8 mM KCI, 133 mM mannitol, 20 mM NaCI, 1 % (w/v) glycerol, and 0.3% (v/v) polysorbate 80 (Tween® 80) at pH of about 6.3, and is suitable for intratumoral injection. Optionally, the L19-IL2 immunoconjugate composition and the L19-TNF-a immunoconjugate composition are mixed together before injection into a melanoma lesion.
[0109] It should be understood that methods according to the disclosure contemplate treating any size of melanoma lesion using the intratumoral agents described. However, it may be particularly advantageous to treat lesions that are about 5 mm or larger in diameter. It is contemplated that lesions to be treated can have a diameter of about 7.5 mm or larger, such as about 10 mm or larger. While no upper limit on the size of the lesion to be treated is contemplated, it may be particularly advantageous to treat lesions having a largest diameter of no more than about 20 cm, such as no more than about 15 cm. In order to maximize the benefit of intratumoral treatments in methods according to the disclosure, it may be advantageous to inject the intratumoral agents into multiple locations within larger lesions, e.g. by injecting 1/4 of the composition(s) into each of four quadrants of the lesion. In preferred embodiments, all resectable melanoma lesions having a diameter of about 5 mm or greater, about 7.5 mm or greater, or about 10 mm or greater are treated with intratumoral agents as described herein.
[0110] Methods according to the disclosure further comprise surgical treatment of the treated melanoma lesion(s). Surgical treatment can include tumor excision or resection. Preferably, total excision of a treated lesion is performed (R0 resection); however, it will be understood that in some instances, total excision is not possible for one or more treated lesions. In preferred embodiments, all melanoma lesions treated with intratumoral agents as described herein are partially or totally removed.
[0111] Surgical treatment can be performed once the intratumoral treatment regimen is completed. Typically, surgical treatment will be performed at least 24 hours, such as at least 48 hours, or at least 72 hours after completion of the intratumoral treatment regimen. For example, tumor resection and/or excision can be performed approximately or exactly 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or approximately or exactly 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks, after completion of the intratumoral treatment regimen. Preferably, surgical treatment occurs more than one day and less than 8 weeks, more preferably less than 6 weeks, more preferably still less than 5 weeks, most preferably less than 4 weeks after completion of the intratumoral treatment regimen.
[0112] Surprisingly, methods of treating cutaneous, subcutaneous, and nodal melanoma as described herein have been shown to improve recurrence free survival and/or distant metastasis free survival rates in treated patients compared to patients undergoing surgical treatment without preceding intratumoral treatment. Improvements in recurrence free survival (RFS) and/or distant metastasis free survival (DMFS) have been shown to include increased median RFS and/or DMFS, and significantly fewer patients presenting with recurrence at a particular time point, e.g. 3 months, 6 months, 12 months, 18 months, 24 months, 30 months, 36 months, etc., post-surgical treatment. A significant reduction in the hazard ratio for recurrence has also been shown.
[0113] For example, recurrence free survival has been shown to be improved by at least about a 30% reduction in the risk of recurrence or death 1-year post-surgical treatment compared to patients 1-year post-surgical treatment when no intratumoral treatment was administered. The 1 -year improvement may, for example, be a reduction in risk of at least about 25%, at least about 20%, at least about 15%, or at least about 10%, such as from about: 10-30%, 10-25%, 10-20%, 10-15%, 15-30%, 15-25%, 15-20%, 20-30%, 20-25%, or 25-30%, compared to patients 1-year post-surgical treatment without preceding intratumoral treatment.
[0114] Recurrence free survival has been shown to be improved by at least about a 40% reduction in the risk of recurrence or death 2-years post-surgical treatment compared to patients 2-years post-surgical treatment when no intratumoral treatment was administered. The 2-year improvement may, for example, be a reduction in risk of at least about 35%, at least about 30%, at least about 25%, at least about 20%, at least about 15%, or at least about 10%, such as from about: 10-40%, 10-35%, 10-30%, 10-25%, 10- 20%, 10-15%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-40%, 20-35%, 20-30%, 20-25%, 25-40%, 25-35%, 25-30%, 30-40%, 30-35%, or 35-40%, compared to patients 2-years post-surgical treatment without preceding intratumoral treatment.
[0115] Recurrence free survival has been shown to be improved by at least about a 55% reduction in the risk of recurrence or death 3-years post-surgical treatment compared to patients 3-years post-surgical treatment when no intratumoral treatment was administered. The 3-year improvement may, for example, be a reduction in risk of at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20%, at least about 15%, or at least about 10%, such as from about: 10-55%, 10-50%, 10-45%, 10-40%, 10-35%, 10-30%, 10-25%, 10- 20%, 10-15%, 15-55%, 15-50%, 15-45%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-55%, 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20-25%, 25-55%, 25-50%, 25- 45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55%, compared to patients 3-years post-surgical treatment without preceding intratumoral treatment.
[0116] Further, distant metastasis free survival has been shown to be improved by at least about a 30% reduction in the risk of distant metastasis or death 1-year post-surgical treatment compared to patients 1 -year post-surgical treatment when no intratumoral treatment was administered. The 1-year improvement may, for example, be a reduction in risk of at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11 %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, or at least about 5%, such as from about: 5-15%, 5-14%, 5- 13%, 5-12%, 5-11%, 5-10%, 6-15%, 6-14%, 6-13%, 6-12%, 6-11%, 6-10%, 7-15%, 7- 14%, 7-13%, 7-12%, 7-11 %, 7-10%, 8-15%, 8-14%, 8-13%, 8-12%, 8-11 %, 8-10%, 10- 15%, 10-14%, 10-13%, 10-12%, 12-15%, 12-14%, 12-13%, 13-15%, 13-14%, or 14-15%, compared to patients 1-year post-surgical treatment without preceding intratumoral treatment.
[0117] Distant metastasis free survival has been shown to be improved by at least about a 35% reduction in the risk of distant metastasis or death 2-years post-surgical treatment compared to patients 2-years post-surgical treatment when no intratumoral treatment was administered. The 2-year improvement may, for example, be a reduction in risk of at least about 30%, at least about 25%, at least about 20%, at least about 15%, or at least about 10%, such as from about: 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-35%, 15-30%, 15-25%, 15-20%, 20-35%, 20-30%, 20-25%, 25-35%, 25-30%, or 30-35%, compared to patients 2-years post-surgical treatment without preceding intratumoral treatment.
[0118] Distant metastasis free survival has been shown to be improved by at least about a 50% reduction in the risk of distant metastasis or death 3-years post-surgical treatment compared to patients 3-years post-surgical treatment when no intratumoral treatment was administered. The 3-year improvement may, for example, be a reduction in risk of at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20%, at least about 15%, or at least about 10%, such as from about: 10-50%, 10-45%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-50%, 15- 45%, 15-40%, 15-35%, 15-30%, 15-25%, 15-20%, 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20-25%, 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-50%, 30-45%, 30- 40%, 30-35%, 35-50%, 35-45%, 35-40%, 40-50%, 40-45%, or 45-50%, compared to patients 3-years post-surgical treatment without preceding intratumoral treatment.
[0119] Further, recurrence free survival and distant metastasis free survival have been shown to be improved by an increase in median RFS and/or DMFS by a factor greater than 1 , such as greater than about 1.1. For example, the increased median RFS and/or DMFS may be at least about 1 .2 times longer in patients treated with methods according to the disclosure compared to patients undergoing surgical treatment without preceding intratumoral treatment. For example, the median RFS and/or DMFS time may be at least 1.25 times longer, at least 1.3 times longer, at least 1.35 times longer, at least 1.4 times longer, at least 1.45 times longer, at least 1.5 times longer, at least 1.55 times longer, at least 1.6 times longer, at least 1.65 times longer, at least 1.7 times longer, at least 1.75 times longer, at least 1 .8 times longer, at least 1 .85 times longer, at least 1 .9 times longer, at least 1.95 times longer, at least 2 times longer, at least 2.1 times longer, at least 2.15 times longer, at least 2.2 times longer, at least 2.25 times longer, at least 2.3 times longer, at least 2.35 times longer, at least 2.4 times longer, at least 2.45 times longer, or at least 2.5 times longer post-surgical treatment in treated patients compared to patients not undergoing intratumoral treatment according to the disclosure. Thus, in various embodiments, the median RFS may be from about 1 .2-2.5, from about 1 .2-2.4, from about 1 .2-2.3, from about 1 .2-2.2, from about 1 .2-2.1 , from about 1.2-2, from about 1 .2-1 .9, from about 1 .2-1 .8, from about 1 .2-1 .7, from about 1 .2-1 .6, from about 1.2-1 .5, from about 1 .2-
1.4, from about 1.2-1.3, from about 1.3-2.5, from about 1.3-2.4, from about 1.3-2.3, from about 1.3-2.2, from about 1.3-2.1 , from about 1.3-2, from about 1.3-1.9, from about 1.3- 1.8, from about 1.3-1.7, from about 1.3-1 .6, from about 1.3-1.5, from about 1.3-1 .4, from about 1 .4-2.5, from about 1 .4-2.4, from about 1 .4-2.3, from about 1.4-2.2, from about 1 .4- 2.1 , from about 1.4-2, from about 1.4-1.9, from about 1.4-1.8, from about 1.4-1.7, from about 1 .4-1 .6, from about 1 .4-1 .5, from about 1 .5-2.5, from about 1.5-2.4, from about 1 .5-
2.3, from about 1.5-2.2, from about 1.5-2.1 , from about 1.5-2, from about 1.5-1.9, from about 1 .5-1 .8, from about 1 .5-1 .7, from about 1 .5-1 .6, from about 1.6-2.5, from about 1 .6-
2.4, from about 1.6-2.3, from about 1.6-2.2, from about 1.6-2.1 , from about 1.6-2, from about 1 .6-1 .9, from about 1 .6-1 .8, from about 1 .6-1 .7, from about 1.7-2.5, from about 1 .7-
2.4, from about 1.7-2.3, from about 1.7-2.2, from about 1.7-2.1 , from about 1.7-2, from about 1 .7-1 .9, from about 1 .7-1 .8, from about 1 .9-2.5, from about 1.9-2.4, from about 1 .9-
2.3, from about 1.9-2.2, from about 1.9-2.1 , from about 1 .9-2, from about 2-2.5, from about 2-2.4, from about 2-2.3, from about 2-2.2, from about 2-2.1 , from about 2.1-2.5, from about 2.1 -2.4, from about 2.1-2.3, from about 2.1-2.2, from about 2.2-2.5, from about 2.2-
2.4, from about 2.2-2.3, from about 2.3-2.5, from about 2.3-2.4, or from about 2.4-2.5 times longer in patients treated with methods according to the disclosure compared to patients with the same staging of melanoma but without preceding intratumoral treatment.
[0120] In various embodiments, a patient treated using methods according to the disclosure may have undergone one or more previous anti-cancer treatments for melanoma, and/or may undergo one or more post-surgical adjuvant treatments, such as, for example, cytokine therapy, radiation therapy, immunotherapy, targeted therapy (such as BRAF inhibitors and/or c-Kit inhibitors), and/or chemotherapy. Notably, however, methods of treating melanoma described herein improve recurrence free survival rates and/or distant metastasis free survival in treated patients regardless of any such previous or subsequent therapies.
[0121] The methods of treating melanoma described herein are particularly useful for treating patients having Stage IIIB, Stage IIIC, Stage HID and/or Stage IV malignant melanoma. The methods are preferably used when the lesion(s) are not ulcerated and/or are BRAF wild type, however the methods can also be used when the lesion(s) are ulcerated and/or BRAF mutated, or if the ulceration and/or BRAF mutation status is unknown. The methods are particularly useful for treating patients with clinically low or normal lactate dehydrogenase (LDH) levels, but can also be used for patients with clinically high LDH levels. In addition, the methods are particularly preferrable for treating patients with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.
[0122] In a first aspect there is provided a method of improving recurrence free survival and/or distant metastasis free survival in a patient with resectable melanoma, wherein the method comprises: (a) treating at least one melanoma lesion of said patient by administering to said lesion(s) a combination of intratumoral agents comprising: (i) at least one TNF-a immunoconjugate, and (ii) at least one IL2 immunoconjugate, and (b) resecting the treated melanoma lesion(s); wherein recurrence free survival and/or distant metastasis free survival is improved compared to the same treatment without step (a).
[0123] In a second aspect there is provided a combination of intratumoral agents for use in a method of improving recurrence free survival and/or distant metastasis free survival in a patient with resectable melanoma, wherein the method comprises: (a) treating at least one melanoma lesion of said patient by administering to said lesion(s) the combination of intratumoral agents, wherein the combination of intratumoral agents comprises: (i) at least one TNF-a immunoconjugate, and (ii) at least one IL2 immunoconjugate, and (b) resecting the treated melanoma lesion(s); wherein recurrence free survival and/or distant metastasis free survival is improved compared to the same treatment without step (a).
[0124] In a third aspect there is provided a use of a combination of intratumoral agents in the manufacture of a medicament, wherein the medicament is for use in a method of improving recurrence free survival and/or distant metastasis free survival in a patient with resectable melanoma, wherein the method comprises: (a) treating at least one melanoma lesion of said patient by administering to said lesion(s) the combination of intratumoral agents, wherein the combination of intratumoral agents comprises: (i) at least one TNF- a immunoconjugate, and (ii) at least one IL2 immunoconjugate, and (b) resecting the treated melanoma lesion(s); wherein recurrence free survival and/or distant metastasis free survival is improved compared to the same treatment without step (a).
[0125] In a fourth aspect there is provided a TNF-a immunoconjugate for use in a method of improving recurrence free survival and/or distant metastasis free survival in a patient with resectable melanoma, wherein the method comprises: (a) treating at least one melanoma lesion of said patient by administering to said lesion(s) a combination of intratumoral agents, wherein the combination of intratumoral agents comprises: (i) the TNF-a immunoconjugate, and (ii) an IL2 immunoconjugate, and (b) resecting the treated melanoma lesion(s); wherein recurrence free survival and/or distant metastasis free survival is improved compared to the same treatment without step (a).
[0126] In a fifth aspect there is provided an IL2 immunoconjugate for use in a method of improving recurrence free survival and/or distant metastasis free survival in a patient with resectable melanoma, wherein the method comprises: (a) treating at least one melanoma lesion of said patient by administering to said lesion(s) a combination of intratumoral agents, wherein the combination of intratumoral agents comprises: (i) the IL2 immunoconjugate, and (ii) a TNF-a immunoconjugate, and (b) resecting the treated melanoma lesion(s); wherein recurrence free survival and/or distant metastasis free survival is improved compared to the same treatment without step (a). [0127] In a sixth aspect there is provided a use of a TNF-a immunoconjugate in the manufacture of a medicament, wherein the medicament is for use in a method of improving recurrence free survival and/or distant metastasis free survival in a patient with resectable melanoma, wherein the method comprises: (a) treating at least one melanoma lesion of said patient by administering to said lesion(s) a combination of intratumoral agents, wherein the combination of intratumoral agents comprises: (i) the TNF-a immunoconjugate, and (ii) an IL2 immunoconjugate, and (b) resecting the treated melanoma lesion(s); wherein recurrence free survival and/or distant metastasis free survival is improved compared to the same treatment without step (a).
[0128] In a seventh aspect there is provided a use of an IL2 immunoconjugate in the manufacture of a medicament, wherein the medicament is for use in a method of improving recurrence free survival and/or distant metastasis free survival in a patient with resectable melanoma, wherein the method comprises: (a) treating at least one melanoma lesion of said patient by administering to said lesion(s) a combination of intratumoral agents, wherein the combination of intratumoral agents comprises: (i) the IL2 immunoconjugate, and (ii) a TNF-a immunoconjugate, and (b) resecting the treated melanoma lesion(s); wherein recurrence free survival and/or distant metastasis free survival is improved compared to the same treatment without step (a).
[0129] In an eighth aspect there is provided a TNF-a immunoconjugate and an IL2 immunoconjugate for use in a method of improving recurrence free survival and/or distant metastasis free survival in a patient with resectable melanoma, wherein the method comprises: (a) treating at least one melanoma lesion of said patient by administering to said lesion(s) a combination of intratumoral agents, wherein the combination of intratumoral agents comprises: (i) the TNF-a immunoconjugate and (ii) the IL2 immunoconjugate, and (b) resecting the treated melanoma lesion(s); wherein recurrence free survival and/or distant metastasis free survival is improved compared to the same treatment without step (a). [0130] In a ninth aspect there is provided a use of a TNF-a immunoconjugate and an IL2 immunoconjugate in the manufacture of a medicament, wherein the medicament is for use in a method of improving recurrence free survival and/or distant metastasis free survival in a patient with resectable melanoma, wherein the method comprises: (a) treating at least one melanoma lesion of said patient by administering to said lesion(s) a combination of intratumoral agents, wherein the combination of intratumoral agents comprises: (i) the TNF-a immunoconjugate, and (ii) the IL2 immunoconjugate, and (b) resecting the treated melanoma lesion(s); wherein recurrence free survival and/or distant metastasis free survival is improved compared to the same treatment without step (a).
[0131] In each of aspects 1-9 listed above, the TNF-a immunoconjugate comprises TNF-a linked to an antibody molecule comprising L19 CDRs; the IL2 immunoconjugate comprises IL2 linked to an antibody molecule comprising L19 CDRs; and the L19 CDRs are:
CDR1 VH SFSMS (SEQ ID NO: 1),
CDR2 VH SISGSSGTTYYADSVKG (SEQ ID NO: 2),
CDR3 VH PFPYFDY (SEQ ID NO: 3),
CDR1 VL RASQSVSSSFLA (SEQ ID NO: 4),
CDR2 VL YASSRAT (SEQ ID NO: 5),
CDR3 VL QQTGRIPPT (SEQ ID NO: 6).
[0132] In addition, embodiments of any of aspects 1-9 listed above may have any one or more of the following optional features.
[0133] The treatment of step (a) may be completed prior to step (b). The antibody may comprise the amino acid sequence of scFv L19 set forth in SEQ ID NO: 9. The IL2 may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18. The TNF-a may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16. The TNF-a may comprise an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 10 or SEQ ID NO: 16; and the IL2 may comprise an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 18. The IL2 immunoconjugate may comprise an amino acid sequence with at least about 70% identity with L19-IL2 (SEQ ID NO: 14), and/or the TNF-a immunoconjugate may comprise an amino acid sequence with at least about 70% identity with L19-TNF-a (SEQ ID NO: 15). The IL2 immunoconjugate may comprise an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with L19-IL2 (SEQ ID NO: 14). The TNF-a immunoconjugate may comprise an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with L19-TNF (SEQ ID NO: 15).
[0134] The intratumoral agents may be administered to the melanoma lesion(s) together or separately. For example, the intratumoral agents may be administered to the melanoma lesion(s) together or separately by subcutaneous or intratumoral injection.
[0135] The melanoma may be Stage III or Stage IV melanoma. Preferably, the melanoma is Stage III, more preferably Stage II IB or Stage IIIC. For example, the melanoma may be: Stage I II B or Stage IIIC melanoma defined according to according to American Joint Committee on Cancer (AJCC) Cancer Staging Manual 7th Edition; or Stage 11 IB, Stage IIIC, or Stage HID melanoma defined according to according to American Joint Committee on Cancer (AJCC) Cancer Staging Manual 8th Edition.
[0136] The longest diameter of melanoma lesions treated may range from about 5 mm to about 20 cm, preferably from about 10 mm to about 15 cm.
[0137] The treatment of step (a) may comprise administering the combination of intratumoral agents together or separately in one or more treatment sessions, wherein each treatment session occurs from one day to two weeks before and/or after a subsequent and/or previous treatment session. The treatment of step (a) may comprise injecting the combination of intratumoral agents together or separately into the melanoma lesion(s) during one to four treatment sessions, wherein each treatment session occurs about 5-10 days, preferably about 6-8 days, more preferably about 7 days, before and/or after a subsequent and/or previous treatment session.
[0138] The total dose of the IL2 immunoconjugate(s) injected into a melanoma lesion per treatment session may range from about 0.5 mg to about 8 mg, preferably from about 0.75 mg to about 6 mg, from about 1 mg to about 5 mg, from about 1.25 mg to about 4 mg, or from about 1.5 mg to about 3 mg, most preferably from about 2 mg to about 2.5 mg, or is about 2.2 mg or about 2.17 mg. The total dose of the TNF-a immunoconjugate(s) injected per treatment session into a melanoma lesion may range from about 50 pg to about 600 pg, preferably from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 200 to about 450 pg, or from about 350 pg to about 450 pg, most preferably from about 375 pg to about 425 pg, or is about 100 pg, about 200 pg, about 300 pg, or about 400 pg. The total dose of the IL2 immunoconjugate(s) injected into a melanoma lesion per treatment session may be about 2.17 mg, and the total dose of the TNF-a immunoconjugate(s) injected into a melanoma lesion per treatment session may be about 100 pg, about 200 pg, about 300 pg, or about 400 pg, preferably about 400 pg.
[0139] The treatment of step (a) may comprise administering the combination of intratumoral agents together or separately at least once a week for up to 8 weeks, up to 6 weeks, up to 5 weeks, up to 4 weeks, up to 3 weeks, or up to 2 weeks.
[0140] The patient may be assessed as having an ECOG performance status of 0 or 1 at the beginning of the treatment of step (a). The melanoma lesion(s) may not be ulcerated at the beginning of the treatment of step (a). The melanoma may have a wild type BRAF mutation status at the beginning of the treatment of step (a). The melanoma lesion(s) treated in step (a) may be cutaneous, subcutaneous, and/or nodal. The melanoma may be metastasized.
[0141] Step (b) may occur within 8 weeks, preferably within 6 weeks, more preferably within 4 weeks after completion of the treatment of step (a). [0142] Step (b) may comprise complete tumor excision (RO resection) of one or more melanoma lesion(s) treated in step (a).
[0143] The patient may have received one or more treatments for melanoma prior to step (a). For example, the patient may have received one or more treatments for melanoma chosen from tumor resection, radiotherapy, chemotherapy, immunotherapy, targeted therapy, cytokine therapy, or combinations thereof, prior to step (a).
[0144] In embodiments, the method further comprises administering one or more adjuvant therapies to the patient after step (b). For example, the method may further comprise administering one or more adjuvant therapies chosen from radiotherapy, chemotherapy, immunotherapy, targeted therapy, cytokine therapy, or combinations thereof, to the patient after step (b).
[0145] In embodiments, recurrence free survival may be improved by an increase in median RFS by a factor greater than 1 , preferably at least about 1 .2, at least about 1 .25, at least about 1.3, at least about 1.35, at least about 1.4, at least about 1.45, at least about 1.5, at least about 1.55, at least about 1.6, at least about 1.65, at least about 1.7, at least about 1.75, at least about 1.8, at least about 1.85, at least about 1.9, at least about 1.95, at least about 2, at least about 2.1 , at least about 2.15, at least about 2.2, at least about 2.25, at least about 2.3, at least about 2.35, at least about 2.4, at least about 2.45, or at least about 2.5 after step (b), compared to the same treatment without step (a).
[0146] In embodiments, recurrence free survival may be improved by an increase in median RFS by a factor greater than 1 , preferably at least 1.2, at least 1 .25, at least 1 .3, at least 1.35, at least 1.4, at least 1.45, at least 1.5, at least 1.55, at least 1.6, at least 1.65, at least 1.7, at least 1.75, at least 1.8, at least 1.85, at least 1.9, at least 1.95, at least 2, at least 2.1 , at least 2.15, at least 2.2, at least 2.25, at least 2.3, at least 2.35, at least 2.4, at least 2.45, or at least 2.5 after step (b), compared to the same treatment without step (a). For example, recurrence free survival may be improved by an increase in median RFS by a factor of at least 1.9, at least 1.95, at least 2, at least 2.1 , at least 2.15, at least 2.2, at least 2.25, at least 2.3, at least 2.35, at least 2.4, at least 2.45, or at least 2.5 after step (b), compared to the same treatment without step (a).
[0147] In embodiments, the patient has at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 1-year after step (b), compared to the same treatment without step (a).
[0148] In embodiments, the patient has at least a 40%, at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 2-years after step (b), compared to the same treatment without step (a).
[0149] In embodiments, the patient has at least a 55%, at least a 50%, at least a 45%, at least a 40%, at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 3-years after step (b), compared to the same treatment without step (a).
[0150] In embodiments, distant metastasis free survival is improved by an increase in median DMFS by a factor greater than 1 , preferably at least about 1 .2, at least about 1 .25, at least about 1.3, at least about 1.35, at least about 1.4, at least about 1.45, at least about 1 .5, at least about 1 .55, at least about 1 .6, at least about 1 .65, at least about 1 .7, at least about 1.75, at least about 1.8, at least about 1.85, at least about 1.9, at least about 1.95, at least about 2, at least about 2.1 , at least about 2.15, at least about 2.2, at least about 2.25, at least about 2.3, at least about 2.35, at least about 2.4, at least about 2.45, or at least about 2.5 after step (b), compared to the same treatment without step
(a).
[0151] In embodiments, the patient has at least a 15%, at least a 14%, at least a 13%, at least a 12%, at least an 11 %, at least a 10%, at least a 9%, at least an 8%, at least a 7%, at least a 6%, or at least a 5% lower risk of distant metastasis or death 1-year after step
(b), compared to the same treatment without step (a). [0152] In embodiments, the patient has at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of distant metastasis or death 2-years after step (b), compared to the same treatment without step (a).
[0153] In embodiments, the patient has at least a 50%, at least a 45%, at least a 40%, at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of distant metastasis or death 3-years after step (b), compared to the same treatment without step (a).
[0154] The present disclosure provides the following numbered embodiments:
1. A method of improving recurrence free survival in a patient with resectable melanoma, the method comprising:
(a) treating at least one melanoma lesion of said patient by administering to said lesion(s) a combination of intratumoral agents comprising:
(i) at least one TNF-a immunoconjugate, and
(ii) at least one IL2 immunoconjugate, wherein: the TNF-a immunoconjugate comprises TNF-a linked to an antibody molecule comprising L19 CDRs; the IL2 immunoconjugate comprises IL2 linked to an antibody molecule comprising L19 CDRs; and the L19 CDRs are:
CDR1 VH SFSMS (SEQ ID NO: 1),
CDR2 VH SISGSSGTTYYADSVKG (SEQ ID NO: 2),
CDR3 VH PFPYFDY (SEQ ID NO: 3),
CDR1 VL RASQSVSSSFLA (SEQ ID NO: 4),
CDR2 VL YASSRAT (SEQ ID NO: 5),
CDR3 VL QQTGRIPPT (SEQ ID NO: 6), and (b) resecting the treated melanoma lesion(s); wherein recurrence free survival is improved compared to the same treatment without step (a).
2. The method according to embodiment 1 , wherein the treatment of step (a) is completed prior to step (b).
3. The method according to embodiment 1 or embodiment 2, wherein the antibody comprises the amino acid sequence of scFv L19 set forth in SEQ ID NO: 9.
4. The method according to any one of the preceding embodiments, wherein the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18.
5. The method according to any one of the preceding embodiments, wherein the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16.
6. The method according to any one of the preceding embodiments, wherein the TNF-a comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 10 or SEQ ID NO: 16; and wherein the IL2 comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 18.
7. The method according to any one of the preceding embodiments, wherein the IL2 immunoconjugate comprises an amino acid sequence with at least about 70% identity with L19-IL2 (SEQ ID NO: 14), and/or the TNF-a immunoconjugate comprises an amino acid sequence with at least about 70% identity with L19-TNF-a (SEQ ID NO: 15).
8. The method according to any one of the preceding embodiments, wherein the IL2 immunoconjugate comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with L19-IL2 (SEQ ID NO: 14). 9. The method according to any one of the preceding embodiments, wherein the TNF-a immunoconjugate comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with L19- TNF (SEQ ID NO: 15).
10. The method according to any one of the preceding embodiments, wherein the intratumoral agents are administered to the melanoma lesion(s) together or separately.
11. The method according to any one of the preceding embodiments, wherein the intratumoral agents are administered to the melanoma lesion(s) together or separately by subcutaneous or intratumoral injection.
12. The method according to any one of the preceding embodiments, wherein the melanoma is Stage 11 IB, Stage 11 IC, or Stage IV melanoma.
13. The method according to any one of the preceding embodiments, wherein the longest diameter of melanoma lesions treated ranges from about 5 mm to about 20 cm, preferably from about 10 mm to about 15 cm.
14. The method according to any one of the preceding embodiments, wherein the treatment of step (a) comprises administering the combination of intratumoral agents together or separately in one or more treatment sessions, wherein each treatment session occurs from one day to two weeks before and/or after a subsequent and/or previous treatment session.
15. The method according to any one of the preceding embodiments, wherein the treatment of step (a) comprises injecting the combination of intratumoral agents together or separately into the melanoma lesion(s) during one to four treatment sessions, wherein each treatment session occurs about 5-10 days, preferably about 6-8 days, more preferably about 7 days, before and/or after a subsequent and/or previous treatment session.
16. The method according to any one of the preceding embodiments, wherein the total dose of the IL2 immunoconjugate(s) injected into a melanoma lesion per treatment session ranges from about 0.5 mg to about 8 mg, preferably from about 0.75 mg to about 6 mg, from about 1 mg to about 5 mg, from about 1 .25 mg to about 4 mg, or from about 1.5 mg to about 3 mg, most preferably ranging from about 2 mg to about 2.5 mg, or is about 2.2 mg or about 2.17 mg.
17. The method according to any one of the preceding embodiments, wherein the total dose of the TNF-a immunoconjugate(s) injected per treatment session into a melanoma lesion ranges from about 50 pg to about 600 pg, preferably from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 200 to about 450 pg, or from about 350 pg to about 450 pg, most preferably ranging from about 375 pg to about 425 pg, or is about 100 pg, about 200 pg, about 300 pg, or about 400 pg.
18. The method according to any one of the preceding embodiments, wherein the total dose of the IL2 immunoconjugate(s) injected into a melanoma lesion per treatment session is about 2.17 mg, and the total dose of the TNF-a immunoconjugate(s) injected into a melanoma lesion per treatment session is about 100 pg, about 200 pg, about 300 pg, or about 400 pg, preferably about 400 pg.
19. The method according to any one of the preceding embodiments, wherein the treatment of step (a) comprises administering the combination of intratumoral agents together or separately at least once a week for up to 8 weeks, up to 6 weeks, up to 5 weeks, up to 4 weeks, up to 3 weeks, or up to 2 weeks.
20. The method according to any one of the preceding embodiments, wherein the patient is assessed as having an ECOG performance status of 0 or 1 at the beginning of the treatment of step (a).
21. The method according to any one of the preceding embodiments, wherein the melanoma lesion(s) are not ulcerated at the beginning of the treatment of step (a).
22. The method according to any one of the preceding embodiments, wherein the melanoma has a wild type BRAF mutation status at the beginning of the treatment of step (a). 23. The method according to any one of the preceding embodiments, wherein the melanoma lesion(s) treated in step (a) are cutaneous, subcutaneous, and/or nodal.
24. The method according to any one of the preceding embodiments, wherein the melanoma is metastasized.
25. The method according to any one of the preceding embodiments, wherein step (b) occurs within 8 weeks, preferably within 6 weeks, more preferably within 4 weeks after completion of the treatment of step (a).
26. The method according to any one of the preceding embodiments, wherein step (b) comprises complete tumor excision (RO resection) of one or more melanoma lesion(s) treated in step (a).
27. The method according to any one of the preceding embodiments, wherein the patient received one or more treatments for melanoma prior to step (a).
28. The method according to any one of the preceding embodiments, wherein the patient received one or more treatments for melanoma chosen from tumor resection, radiotherapy, chemotherapy, immunotherapy, targeted therapy, cytokine therapy, or combinations thereof, prior to step (a).
29. The method according to any one of the preceding embodiments, further comprising administering one or more adjuvant therapies to the patient after step (b).
30. The method according to any one of the preceding embodiments, further comprising administering one or more adjuvant therapies chosen from radiotherapy, chemotherapy, immunotherapy, targeted therapy, cytokine therapy, or combinations thereof, to the patient after step (b).
31. The method according to any one of the preceding embodiments, wherein recurrence free survival is improved by an increase in median RFS by a factor greater than 1 , preferably at least about 1 .2, at least about 1 .25, at least about 1 .3, at least about 1 .35, at least about 1 .4, at least about 1 .45, at least about 1 .5, at least about 1 .55, at least about 1 .6, at least about 1 .65, at least about 1 .7, at least about 1 .75, at least about 1 .8, at least about 1 .85, at least about 1 .9, at least about 1 .95, at least about 2, at least about 2.1 , at least about 2.15, at least about 2.2, at least about 2.25, at least about 2.3, at least about 2.35, at least about 2.4, at least about 2.45, or at least about 2.5 after step (b), compared to the same treatment without step (a).
32. The method according to any one of the preceding embodiments, wherein the patient has at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 1 -year after step (b), compared to the same treatment without step (a).
33. The method according to any one of the preceding embodiments, wherein the patient has at least a 40%, at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 2-years after step (b), compared to the same treatment without step (a).
34. The method according to any one of the preceding embodiments, wherein the patient has at least a 55%, at least a 50%, at least a 45%, at least a 40%, at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 3-years after step (b), compared to the same treatment without step (a).
35. A method of improving distant metastasis free survival in a patient with resectable melanoma, the method comprising:
(a) treating at least one melanoma lesion of said patient by administering to said lesion(s) a combination of intratumoral agents comprising:
(i) at least one TNF-a immunoconjugate, and
(ii) at least one IL2 immunoconjugate, wherein: the TNF-a immunoconjugate comprises TNF-a linked to an antibody molecule comprising L19 CDRs; the IL2 immunoconjugate comprises IL2 linked to an antibody molecule comprising L19 CDRs; and the L19 CDRs are:
CDR1 VH SFSMS (SEQ ID NO: 1),
CDR2 VH SISGSSGTTYYADSVKG (SEQ ID NO: 2),
CDR3 VH PFPYFDY (SEQ ID NO: 3),
CDR1 VL RASQSVSSSFLA (SEQ ID NO: 4),
CDR2 VL YASSRAT (SEQ ID NO: 5),
CDR3 VL QQTGRIPPT (SEQ ID NO: 6), and
(b) resecting the treated melanoma lesion(s); wherein distant metastasis free survival is improved compared to the same treatment without step (a).
36. The method according to embodiment 35, wherein the treatment of step (a) is completed prior to step (b).
37. The method according to embodiment 35 or embodiment 36, wherein the antibody comprises the amino acid sequence of scFv L19 set forth in SEQ ID NO: 9.
38. The method according to any one of embodiments 35-37, wherein the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18.
39. The method according to any one of embodiments 35-38, wherein the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16.
40. The method according to any one of embodiments 35-39, wherein the TNF-a comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 10 or SEQ ID NO: 16; and wherein the IL2 comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 18.
41. The method according to any one of embodiments 35-40, wherein the IL2 immunoconjugate comprises an amino acid sequence with at least about 70% identity with L19-IL2 (SEQ ID NO: 14), and/or the TNF-a immunoconjugate comprises an amino acid sequence with at least about 70% identity with L19-TNF-0 (SEQ ID NO: 15).
42. The method according to any one of embodiments 35-41 , wherein the IL2 immunoconjugate comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with L19-IL2 (SEQ ID NO: 14).
43. The method according to any one of embodiments 35-42, wherein the TNF-a immunoconjugate comprises an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with L19-TNF (SEQ ID NO: 15).
44. The method according to any one of embodiments 35-43, wherein the intratumoral agents are administered to the melanoma lesion(s) together or separately.
45. The method according to any one of embodiments 35-44, wherein the intratumoral agents are administered to the melanoma lesion(s) together or separately by subcutaneous or intratumoral injection.
46. The method according to any one of embodiments 35-45, wherein the melanoma is Stage I II B, Stage 11 IC, or Stage IV melanoma.
47. The method according to any one of embodiments 35-46, wherein the longest diameter of melanoma lesions treated ranges from about 5 mm to about 20 cm, preferably from about 10 mm to about 15 cm.
48. The method according to any one of embodiments 35-47, wherein the treatment of step (a) comprises administering the combination of intratumoral agents together or separately in one or more treatment sessions, wherein each treatment session occurs from one day to two weeks before and/or after a subsequent and/or previous treatment session.
49. The method according to any one of embodiments 35-48, wherein the treatment of step (a) comprises injecting the combination of intratumoral agents together or separately into the melanoma lesion(s) during one to four treatment sessions, wherein each treatment session occurs about 5-10 days, preferably about 6-8 days, more preferably about 7 days, before and/or after a subsequent and/or previous treatment session.
50. The method according to any one of embodiments 35-49, wherein the total dose of the IL2 immunoconjugate(s) injected into a melanoma lesion per treatment session ranges from about 0.5 mg to about 8 mg, preferably from about 0.75 mg to about 6 mg, from about 1 mg to about 5 mg, from about 1.25 mg to about 4 mg, or from about 1 .5 mg to about 3 mg, most preferably ranging from about 2 mg to about 2.5 mg, or is about 2.2 mg or about 2.17 mg.
51. The method according to any one of embodiments 35-50, wherein the total dose of the TNF-a immunoconjugate(s) injected per treatment session into a melanoma lesion ranges from about 50 pg to about 600 pg, preferably from about 75 pg to about 550 pg, from about 100 pg to about 500 pg, from about 200 to about 450 pg, or from about 350 pg to about 450 pg, most preferably ranging from about 375 pg to about 425 pg, or is about 100 pg, about 200 pg, about 300 pg, or about 400 pg.
52. The method according to any one of embodiments 35-51 , wherein the total dose of the IL2 immunoconjugate(s) injected into a melanoma lesion per treatment session is about 2.17 mg, and the total dose of the TNF-a immunoconjugate(s) injected into a melanoma lesion per treatment session is about 100 pg, about 200 pg, about 300 pg, or about 400 pg, preferably about 400 pg.
53. The method according to any one of embodiments 35-52, wherein the treatment of step (a) comprises administering the combination of intratumoral agents together or separately at least once a week for up to 8 weeks, up to 6 weeks, up to 5 weeks, up to 4 weeks, up to 3 weeks, or up to 2 weeks.
54. The method according to any one of embodiments 35-53, wherein the patient is assessed as having an ECOG performance status of 0 or 1 at the beginning of the treatment of step (a).
55. The method according to any one of embodiments 35-54, wherein the melanoma lesion(s) are not ulcerated at the beginning of the treatment of step (a).
56. The method according to any one of embodiments 35-55, wherein the melanoma has a wild type BRAF mutation status at the beginning of the treatment of step (a).
57. The method according to any one of embodiments 35-56, wherein the melanoma lesion(s) treated in step (a) are cutaneous, subcutaneous, and/or nodal.
58. The method according to any one of embodiments 35-57, wherein the melanoma is metastasized.
59. The method according to any one of embodiments 35-58, wherein step (b) occurs within 8 weeks, preferably within 6 weeks, more preferably within 4 weeks after completion of the treatment of step (a).
60. The method according to any one of embodiments 35-59, wherein step (b) comprises complete tumor excision (RO resection) of one or more melanoma lesion(s) treated in step (a).
61. The method according to any one of embodiments 35-60, wherein the patient received one or more treatments for melanoma prior to step (a).
62. The method according to any one of embodiments 35-61 , wherein the patient received one or more treatments for melanoma chosen from tumor resection, radiotherapy, chemotherapy, immunotherapy, targeted therapy, cytokine therapy, or combinations thereof, prior to step (a).
63. The method according to any one of embodiments 35-62, further comprising administering one or more adjuvant therapies to the patient after step (b). 64. The method according to any one of embodiments 35-63, further comprising administering one or more adjuvant therapies chosen from radiotherapy, chemotherapy, immunotherapy, targeted therapy, cytokine therapy, or combinations thereof, to the patient after step (b).
65. The method according to any one of embodiments 35-64, wherein distant metastasis free survival (DMFS) is improved by an increase in median DMFS by a factor greater than 1 , preferably at least about 1.2, at least about 1.25, at least about 1.3, at least about 1.35, at least about 1.4, at least about 1.45, at least about 1.5, at least about 1 .55, at least about 1 .6, at least about 1 .65, at least about 1 .7, at least about 1 .75, at least about 1.8, at least about 1.85, at least about 1.9, at least about 1.95, at least about 2, at least about 2.1 , at least about 2.15, at least about 2.2, at least about 2.25, at least about 2.3, at least about 2.35, at least about 2.4, at least about 2.45, or at least about 2.5 after step (b), compared to the same treatment without step (a).
66. The method according to any one of embodiments 35-65, wherein the patient has at least a 15%, at least a 14%, at least a 13%, at least a 12%, at least an 11 %, at least a 10%, at least a 9%, at least an 8%, at least a 7%, at least a 6%, or at least a 5% lower risk of distant metastasis or death 1-year after step (b), compared to the same treatment without step (a).
67. The method according to any one of embodiments 35-66, wherein the patient has at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of distant metastasis or death 2-years after step (b), compared to the same treatment without step (a).
68. The method according to any one of embodiments 35-67, wherein the patient has at least a 50%, at least a 45%, at least a 40%, at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of distant metastasis or death 3-years after step (b), compared to the same treatment without step (a).
[0155] The features disclosed in the foregoing description, or in the following claims, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.
[0156] Having described various embodiments of the disclosure in detail, it will be apparent that modifications and variations are possible without departing from the scope of the disclosure defined in the appended claims. Furthermore, it should be appreciated that the examples are provided as non-limiting examples and are, therefore, not to be taken as limiting the various aspects so illustrated. It is to be understood that all definitions herein are provided for the present disclosure only.
[0157] In this application, the use of the singular includes the plural unless specifically stated otherwise. The singular forms “a,” “an,” “the,” and “at least one” are understood to encompass the plural as well as the singular unless the context clearly dictates otherwise. The expression “one or more” means “at least one” and thus includes individual components as well as mixtures/combinations. Likewise, the term “a derivative thereof’ also relates to “derivatives thereof,” and so on.
[0158] For purposes of the present disclosure, it should be noted that to provide a more concise description, some of the quantitative expressions given herein are not qualified with the terms “about” or “approximately,” which are used interchangeably. It is understood that whether the term “about” is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including approximations due to the experimental and/or measurement conditions for such given value, unless otherwise stated. The term “about” in relation to a numerical value is optional and means, for example, +/- 10%, +/- 9%, +/- 8%, +/- 7%, +/- 6%, +/- 5%, +/- 4%, +/- 3%, +/- 2%, +/- 1 %, +/- 0.5%, +/- 0.1 %, or +/- 0.01 %. Unless otherwise specified, “about” means +/- 5%.
[0159] Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particularly recited value forms another embodiment; as such, “about 2” also expressly encompasses “exactly 2.”
[0160] Unless otherwise expressly stated, it is in no way intended that any method be construed as requiring that its steps be performed in a specific order. Accordingly, where a method does not expressly recite an order to be followed by its steps or it is not specifically stated that the steps are to be limited to a specific order, it not intended that any particular order be inferred.
[0161] The term “and/or” should be understood to include both the conjunctive and the disjunctive.
[0162] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
[0163] For purposes of this disclosure, the term “recurrence” includes the appearance of local, regional, distant, and/or primary melanoma lesions post-treatment, confirmed by histopathological analysis.
[0164] Herein, “recurrence free survival” and “distant metastasis free survival” are measured from a specified event to the earlier of the appearance of local, regional, distant, or primary melanoma lesions or death. Recurrence free survival (RFS) and distant metastasis free survival (DMFS), for example median RFS and median DMFS, are preferably measured in months. Thus, an increase in median RFS (mRFS) preferably refers to an increase in the medium number of months of RFS.
[0165]As used herein, the terms “improving” or “increasing” the rate of recurrence free survival, distant metastasis free survival, and variations thereof, include preventing or delaying recurrence of melanoma in a patient and/or death, or increasing the likelihood that a patient will be recurrence free at a specified point in time, post-treatment.
[0166]As used herein, a “treatment session” refers to one clinical session, e.g. one visit of a patient to a clinical center, doctor office, hospital, etc., where the agent is administered.
[0167] As used herein, a “day” is intended to be a 24-hour period, and a “week” is intended to be a 7-day period.
[0168] Herein, the terms “tumor resection,” “tumor excision,” and the like are intended to mean surgical tumor removal. Tumor resection is graded depending on the absence or presence of residual tumor after surgery (RO = complete removal; R1 = microscopic residual tumor; R2 = macroscopic residual tumor). Tumor excision is considered to be complete tumor removal (RO).
[0169] As used herein, the term “blinded review” is intended to mean the reading of medical images, reviewed by an independent readers blinded to the results of study treatment, the investigator assessment, and some pre-defined clinical information to independently verify endpoints and control bias that might result from errors in response or progression assessments.
EXAMPLES
[0170] The following examples detail a clinical trial to evaluate benefits of treating melanoma according to the disclosed methods.
Patient Selection and Grouping
[0171] Patients aged over 18 diagnosed with malignant melanoma of the skin with locally advanced disease as defined by clinical Stage 11 IB and Stage 11 IC according to AJCC 7th Ed., having at least one injectable cutaneous, subcutaneous, or nodal melanoma lesion (> 10 mm in longest diameter, or with multiple injectable lesions that in aggregate have a longest diameter of > 10 mm) eligible for complete surgical resection, ECOG PS < 1 , and a life expectance of at least 24 months, were selected.
[0172] Prior anti-tumor treatment for the primary melanoma lesion, including surgery and approved adjuvant treatments (e.g., radiotherapy, immune checkpoint inhibitors, BRAF/MEK inhibitors, etc.) was allowed.
[0173] Patients with uveal melanoma, mucosal melanoma, or melanoma with unknown primary lesions were excluded, as were patients with evidence of distant metastases. Also excluded were patients with previous or concurrent cancer that is distinct in primary site or histology from the cancer being evaluated in this study except cervical carcinoma in situ, treated basal cell carcinoma, superficial bladder tumors, second primary melanoma in situ, or any cancer curatively treated > 5 years prior to study entry.
[0174] A total of 256 patients were selected and randomly assigned into two groups. ARM 1 , the treatment group, included 127 patients. ARM 2, the control group, included 129 patients.
[0175]Table 1 below presents the general characteristics of patients in each group.
TABLE 1 - Patients
[0176] Table 2 below presents information regarding the tumors treated.
TABLE 2A - Tumors of randomized patients
[0177] Of the 256 patients randomized, the study proceeded with 122 patients in ARM 1 and 124 patients in ARM 2. Five patients in each arm were enrolled after the data cut-off date and, therefore, removed from further analysis as they were too early to be analysed. Consequently, a total of 246 patients were subject to evaluation, as summarised in Tables 2B and 2C.
TABLE 2B - Summary of tumors of evaluated patients classified according to AJCC (7th edition)
TABLE 2C - Summary of tumors of evaluated patients classified according to AJCC (8th edition)
[0178] As shown in Table 2B and Table 2C, the disease stage of tumors was classified according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual 7th Edition (second row), and subsequently according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual 8th Edition (third row). The 8th edition of the AJCC introduced Stage HID melanoma for the first time, and several tumors classified as Stage HIX (unspecified stage III patient) according to the AJCC 7th edition were classified as Stage HID under the AJCC 8th edition. Regardless of the specific staging criteria used, these data demonstrate that all substages of stage III melanoma were successfully treated, including the most advanced stage III melanoma substage (the most advanced Stage III substage according to the AJCC 7th Edition is Stage 11 IC, and the most advanced Stage III substage according to the AJCC 8th Edition is Stage HID).
[0179]Table 3 below presents information regarding prior anti-cancer treatments.
TABLE 3 - Prior Treatment
Administration of Intratumoral Agents
[0180] Patients in ARM 1 had a composition comprising 13 MiolU of L19-IL2 (antibody
L19 conjugated to human IL2; INN = bifikafusp alfa) and 400 pg of L19-TNF-Q (antibody L19 conjugated to human TNF in homotrimer format; INN = onfekafusp alfa) administered by intratumoral injection into the melanoma lesion(s) once weekly. The intratumoral treatment composition was prepared by mixing a 1 mL composition comprising 2.17 mg L19-IL2, 6.7 mM NaH2PO4 (2H2O), 1 .8 mM KCI, 133 mM mannitol, 20 mM NaCI, 1 % (w/v) glycerol, and 0.3% (v/v) polysorbate 80 (Tween® 80) at pH of about 6.3, with a volume ranging from 0.25 mL to 1 mL of a composition comprising 400 pg of L19-TNF-a, 15mM NaH2PO4(2H2O), 10mM Na2HPO4(2H2O), 1.5 mM KCI, 75 mM mannitol, 30 mM NaCI, 1 % (w/v) glycerol, 5mM EDTA, and 0.01 % (v/v) polysorbate 20 (Tween® 20) at pH of about 8. Although the recommended dose of L19-TNF-a was 400 pg, the investigator was permitted to reduce the dosage to amounts ranging from 100 pg to 400 pg (i.e. 100 pg, 200 pg, 300 pg, or 400 pg), as deemed appropriate based on the size and number of lesions and the patient’s tolerability to the treatment. The intratumoral treatment composition was distributed among all injectable lesions.
[0181]The once-weekly intratumoral treatment regimen lasted for four weeks or until all injectable tumors disappeared, intolerance to study treatment was observed, or in the opinion of the investigator immediate surgical resection or other treatment for melanoma was warranted, whichever occurred first. Newly-occurring injectable lesions observed during the four-week period were also treated, but the treatment regimen was not extended beyond the four-week, pre-defined period.
Surgical Treatment
[0182] Of the 246 patients evaluated in the study, 104 patients in ARM 1 and 116 patients in ARM 2 underwent surgical treatment.
[0183] Patients in ARM 1 underwent surgery within four weeks after the intratumoral treatment regimen was completed. The median time to surgery for ARM 1 was 6.57 weeks after randomization. Patients in ARM 2 did not receive intratumoral treatment prior to surgery. The median time to surgery for ARM 2 was 2.29 weeks after randomization.
[0184] Surgical treatment focused on complete tumor removal (R0 resection) to ensure the patient was free of any detectable tumor lesion by physical examination, imaging diagnostics, and/or histopathological analysis.
[0185] Follow-up visits for each patient occurred every three months.
Example 1 - Recurrence Free Survival
[0186] This Example details results of the study demonstrating improvement in recurrence free survival (RFS) with methods of treating melanoma according to the disclosure.
[0187] The recurrence free survival data was calculated for both arms and the Kaplan Meier estimator was applied. A two-sided log rank test with alpha equal to 5% was used to reject the hypothesis of equality between recurrence free survival in the treatment group (ARM 1 ) versus the control group (ARM 2).
[0188]Table 4A and FIG. 1A show the recurrence free survival from randomization for the treatment group (ARM 1 ) and the control group (ARM 2) (blinded review). Table 4B and FIG. 1 B show the recurrence free survival from randomization for the treatment group (ARM 1 ) and the control group (ARM 2) (investigator assessment).
TABLE 4A - Median RFS From Randomization (Blinded Review)1
1 Log-Rank p-value: 0.005
[0189] As FIG. 1A shows, the 1-year, 2-year, and 3-year recurrence free survival rates were 57.47%, 41.57%, and 25.47% respectively for ARM 1 , and 39.46%, 23.60%, and 10.33% respectively for ARM 2.
TABLE 4B - Median RFS From Randomization (Investigator Assessment)2
2Log-Rank p-value: 0.018
[0190] As FIG. 1 B shows, the 1-year, 2-year, and 3-year recurrence free survival rates were 61.95%, 50.44%, and 38.15% respectively for ARM 1 , and 46.54%, 30.89%, and 21 .38% respectively for ARM 2.
[0191]Table 4C and FIG. 1 C show the recurrence free survival from surgery for the treatment group (ARM 1) and the control group (ARM 2).
TABLE 4C - Median RFS From Surgery3
3Log-Rank p-value: 0.023
[0192] As FIG. 1 C shows, the 1 -year and 2-year recurrence free survival rates were 54.75% and 37.85% respectively for ARM 1 , and 36.66% and 23.36% respectively for ARM 2.
[0193] Tables 4A-4C and FIGS. 1A-1 C show that the median RFS for ARM 1 (treatment) was considerably longer than the median RFS for ARM 2 (control). These data show a statistically significant increase in median RFS with methods of treatment according to the disclosure.
[0194] Of the 220 patients who underwent surgical treatment (ARM 1 : N=104; ARM 2: N=116), 78 received post-surgical adjuvant therapy, while 142 received no post-surgical adjuvant treatment (45 experienced recurrence during the study period, and 33 experienced no recurrence during the study period) (p-value: 0.2929). Table 4D shows the distribution by ARM of adjuvant and recurrence match. TABLE 4D - Distribution by ARM of Adjuvant Therapy and Recurrence
[0195]Table 4E shows the distribution by ARM of number and type of post-surgical adjuvant therapy.
TABLE 4E - Distribution by ARM of Number and Type of Adjuvant Therapy
[0196]Table 4F and FIG. 1 D show the median RFS from randomization for ARM 1 (treatment) and ARM 2 (control) with and without post-surgical adjuvant therapy. TABLE 4F - Median RFS From Surgery With and Without Adjuvant Therapy4
4Log-Rank p-value: 0.003
[0197] As FIG. 1 D shows, the 1 -year and 2-year recurrence free survival rates were 60.07% and 47.20% respectively for ARM 1 (with adjuvant therapy), 51 .34% and 31 .74% respectively for ARM 1 (without adjuvant therapy), 49.88% and 35.21 % respectively for ARM 2 (with adjuvant therapy), and 25.35% and 13.31 % respectively for ARM 2 (without adjuvant therapy).
[0198] As Table 4F and FIG. 1 D show, the median RFS for ARM 1 (treatment) was considerably longer than the median RFS for ARM 2 (control), regardless of whether post- surgical adjuvant therapy was used. These data show a statistically significant increase in median RFS with methods of treating melanoma according to the disclosure even when post-surgical adjuvant therapy was not used, compared to surgical resection of the melanoma tumor without prior intratumoral treatment, either with or without post-surgical adjuvant therapy.
[0199]Table 4G and FIG. 1 E show the median RFS from randomization for ARM 1 (treatment) and ARM 2 (control) for patients who underwent any prior anti-cancer treatment for melanoma before the study.
TABLE 4G - Median RFS With Prior Anti-Cancer Treatment5
5Log-Rank p-value: 0.004
[0200] As FIG. 1 E shows, the 1-year, 2-year, and 3-year recurrence free survival rates were 58.13%, 43.23%, and 26.49% respectively for ARM 1 , and 39.83%, 23.10%, and 9.60% respectively for ARM 2.
[0201] Table 4H and FIG. 1 F show the median RFS from randomization for ARM 1 (treatment) and ARM 2 (control) for patients who underwent prior immunotherapy for melanoma before the study.
TABLE 4H - Median RFS With Prior Immunotherapy6
6Log-Rank p-value: 0.047
[0202] As FIG. 1 F shows, the 1 -year, 2-year, and 3-year recurrence free survival rates for ARM 1 were 53.81 %, 33.54%, and 26.84% respectively, and the 1-year and 2-year recurrence free survival rates for ARM 2 were 29.63% and 14.81 % respectively.
[0203] Table 4I and FIGS. 1 G-1 H show the median RFS from randomization for ARM 1 (treatment) and ARM 2 (control) for patients with or without prior systemic therapy for melanoma before the study.
TABLE 4I - Median RFS With and Without Prior Systemic Therapy7
[0204]The data shown in Tables 4F-4I and FIGS. 1 D-1 H show that the median RFS for ARM 1 (treatment) was considerably longer than the median RFS for ARM 2 (control). These data show a statistically significant increase in median RFS with methods of treatment according to the disclosure whether or not other treatments such as prior and/or adjuvant therapies are considered.
[0205] Tables 5A-5C present hazard ratio data for recurrence free survival based on patient demographics, tumor, prior anti-cancer treatment, and post-surgical adjuvant therapy.
TABLE 5A - RFS Hazard Ratio Based on Patient Demographics
TABLE 5B - RFS Hazard Ratio Based on Tumor
TABLE 5C - RFS Hazard Ratio Based on Prior Therapy / Adjuvant Therapy [0206] The data in Tables 5A-5C demonstrate that methods of treatment according to the disclosure provide a significant reduction in the hazard ratio for recurrence free survival, compared to surgical resection of the melanoma tumor without preceding intratumoral treatment, either with or without prior therapy and/or post-surgical adjuvant therapy.
[0207] The results shown in Example 1 , which are statistically significant, are surprising. Cancer treatment is highly unpredictable, and to date there has been no evidence to suggest that using a combination of TNF and IL2 immunoconjugates, and L19-IL2 and L19-TNF-a in particular, would provide such significantly improved recurrence free survival when used with surgical tumor removal to treat resectable melanoma in human patients.
Example 2 - Distant Metastasis Free Survival
[0208] This Example details results of the study demonstrating improvement in distant metastasis free survival (DMFS) with methods of treating melanoma according to the disclosure.
[0209] The distant metastasis free survival data was calculated for both arms and the Kaplan Meier estimator was applied. A two-sided log rank test with alpha equal to 5% was used to reject the hypothesis of equality between distant metastasis free survival in the treatment group (ARM 1) versus the control group (ARM 2).
[0210] Table 6 and FIG. 2 show the distant metastasis free survival for the treatment group (ARM 1) and the control group (ARM 2) (blinded review). The hazard ratio was 0.60, with a 95% Cl of 0.37-0.95. In accordance with the protocol, data were obtained only for the first disease recurrence, whether local or distant.
TABLE 6 - Median DMFS From Randomization (Blinded Review)8
8Log-Rank p-value: 0.029 HR = 0.60 [0.37-0.95]
[0211] As FIG. 2 shows, the 1-year, 2-year, and 3-year survival rates were 69.84%, 59.41 %, and 36.4% respectively for ARM 1 , and 60.83%, 38.81 %, and 16.98% respectively for ARM 2.
[0212] The results shown in Example 2, which are statistically significant, are also surprising. There has been no evidence to suggest that using a combination of TNF and IL2 immunoconjugates, and L19-IL2 and L19-TNF-a in particular, would provide such significantly improved distant metastasis free survival when used with surgical tumor removal to treat resectable melanoma in human patients.
Example 3 - Overall Survival
[0213] Additional studies are continuing to assess the overall survival (OS) rate when treating melanoma according to the methods described herein.
[0214] Although results are preliminary, based on the data obtained to date and discussed in Examples 1-2, it is contemplated that the OS rate may be similarly surprisingly improved compared to treatment with surgery in the absence of preceding intratumoral treatment as described herein.
SEQUENCE LISTING
Amino acid sequences of L19 CDRs
L19 CDR1 VH SFSMS (SEQ ID NO: 1)
L19 CDR2 VH SISGSSGTTYYADSVKG (SEQ ID NO: 2)
L19 CDR3 VH PFPYFDY (SEQ ID NO: 3)
L19 CDR1 VL RASQSVSSSFLA (SEQ ID NO: 4)
L19 CDR2 VL YASSRAT (SEQ ID NO: 5)
L19 CDR3 VL QQTGRIPPT (SEQ ID NO: 6)
Amino acid sequence of the L19 VH domain (SEQ ID NO: 7)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSG
TTYYADSVKGRFTISRDNSKN
TLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSS
Amino acid sequence of the L19 VL domain (SEQ ID NO: 8)
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGI
PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK
Amino acid sequence of L19 scFV (SEQ ID NO: 9)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSG
TTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTV
SSGDGSSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQ APRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQG
TKVEIK
Amino acid sequence of the soluble form of the extracellular domain of human TNF
(huTNF) (SEQ ID NO: 10)
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYL
IYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYE
PIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL
Amino acid sequence of linker (SEQ ID NO: 11)
GDGSSGGSGGAS
Amino acid sequence of linker (SEQ ID NO: 12)
SSSSGSSSSGSSSSG
Amino acid sequence of linker (SEQ ID NO: 13)
GGGGSGGGGSGGGG
Amino acid sequence of the L19-hulL2 conjugate (SEQ ID NO: 14)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSG
TTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTV
SSGDGSSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQ
APRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQG
TKVEIKEFSSSSGSSSSGSSSSGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT
RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK
GSETTFMCEYADETATIVEFLNRWITFCQSIISTLT
Amino acid sequence of the L19-huTNF conjugate (SEQ ID NO: 15) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSG
TTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTV
SSGDGSSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQ
APRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQG
TKVEIKEFSSSSGSSSSGSSSSGVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRAN
ALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLL
SAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYF
GIIAL
Amino acid sequence of the extracellular domain of human TNF (huTNF) [extracellular domain] (SEQ ID NO: 16)
GPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALL
ANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAI
KSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIA L
Amino acid sequence of linker (SEQ ID NO: 17)
EFSSSSGSSSSGSSSSG
Amino acid sequence of human interleukin 2 (hu IL2) (SEQ ID NO: 18)
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL
EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR
WITFCQSIISTLT

Claims

1. A TNF-a immunoconjugate and an IL2 immunoconjugate for use in a method of improving recurrence free survival in a patient with resectable melanoma, wherein the melanoma is Stage III melanoma, and the method comprises:
(a) treating at least one melanoma lesion of said patient by administering to said lesion(s) a combination of intratumoral agents, wherein the combination of intratumoral agents comprises:
(i) the TNF-a immunoconjugate, and
(ii) the IL2 immunoconjugate, wherein: the TNF-a immunoconjugate comprises TNF-a linked to an antibody molecule comprising L19 CDRs; the IL2 immunoconjugate comprises IL2 linked to an antibody molecule comprising L19 CDRs; and the L19 CDRs are:
CDR1 VH SFSMS (SEQ ID NO: 1),
CDR2 VH SISGSSGTTYYADSVKG (SEQ ID NO: 2),
CDR3 VH PFPYFDY (SEQ ID NO: 3),
CDR1 VL RASQSVSSSFLA (SEQ ID NO: 4),
CDR2 VL YASSRAT (SEQ ID NO: 5),
CDR3 VL QQTGRIPPT (SEQ ID NO: 6), and
(b) resecting the treated melanoma lesion(s); wherein recurrence free survival (RFS) is improved compared to the same treatment without step (a).
2. A TNF-a immunoconjugate for use in a method of improving recurrence free survival in a patient with resectable melanoma, wherein the melanoma is Stage III melanoma, and the method comprises:
(a) treating at least one melanoma lesion of said patient by administering to said lesion(s) a combination of intratumoral agents, wherein the combination of intratumoral agents comprises:
(i) the TNF-a immunoconjugate, and
(ii) an IL2 immunoconjugate, wherein: the TNF-a immunoconjugate comprises TNF-a linked to an antibody molecule comprising L19 CDRs; the IL2 immunoconjugate comprises IL2 linked to an antibody molecule comprising L19 CDRs; and the L19 CDRs are:
CDR1 VH SFSMS (SEQ ID NO: 1),
CDR2 VH SISGSSGTTYYADSVKG (SEQ ID NO: 2),
CDR3 VH PFPYFDY (SEQ ID NO: 3),
CDR1 VL RASQSVSSSFLA (SEQ ID NO: 4),
CDR2 VL YASSRAT (SEQ ID NO: 5),
CDR3 VL QQTGRIPPT (SEQ ID NO: 6), and
(b) resecting the treated melanoma lesion(s); wherein recurrence free survival (RFS) is improved compared to the same treatment without step (a).
3. An IL2 immunoconjugate for use in a method of improving recurrence free survival in a patient with resectable melanoma, wherein the melanoma is Stage III melanoma, and the method comprises:
(a) treating at least one melanoma lesion of said patient by administering to said lesion(s) a combination of intratumoral agents, wherein the combination of intratumoral agents comprises:
(i) a TNF-a immunoconjugate, and
(ii) the IL2 immunoconjugate, wherein: the TNF-a immunoconjugate comprises TNF-a linked to an antibody molecule comprising L19 CDRs; the IL2 immunoconjugate comprises IL2 linked to an antibody molecule comprising L19 CDRs; and the L19 CDRs are:
CDR1 VH SFSMS (SEQ ID NO: 1),
CDR2 VH SISGSSGTTYYADSVKG (SEQ ID NO: 2),
CDR3 VH PFPYFDY (SEQ ID NO: 3),
CDR1 VL RASQSVSSSFLA (SEQ ID NO: 4),
CDR2 VL YASSRAT (SEQ ID NO: 5),
CDR3 VL QQTGRIPPT (SEQ ID NO: 6), and
(b) resecting the treated melanoma lesion(s); wherein recurrence free survival (RFS) is improved compared to the same treatment without step (a).
4. The TNF-a immunoconjugate and/or IL2 immunoconjugate for use according to any one of claims 1 to 3, wherein RFS is improved by an increase in median RFS by a factor of at least about 1 .9 after step (b), compared to the same treatment without step (a).
5. The TNF-a immunoconjugate and/or IL2 immunoconjugate for use according to any one of the preceding claims, wherein:
(i) the antibody comprises the amino acid sequence of scFv L19 set forth in SEQ ID NO: 9;
(ii) the IL2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18;
(iii) the TNF-a has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 16;
(iv) the IL2 immunoconjugate comprises an amino acid sequence with at least about 70% identity to the amino acid sequence of L19-IL2 set forth in SEQ ID NO: 14;
(v) the TNF-a immunoconjugate comprises an amino acid sequence with at least about 70% identity to the amino acid sequence of L19-TNF-a set forth in SEQ ID NO: 15; and/or
(vi) the treatment of step (a) is completed prior to step (b).
6. The TNF-a immunoconjugate and/or IL2 immunoconjugate for use according to any one of the preceding claims, wherein the intratumoral agents are administered by injection at or near the melanoma lesion(s), optionally wherein the intratumoral agents are administered by injection into the melanoma lesion(s).
7. The TNF-a immunoconjugate and/or IL2 immunoconjugate for use according to any one of the preceding claims, wherein the intratumoral agents are administered to the melanoma lesion(s) together or separately, optionally by subcutaneous or intratumoral injection.
8. The TNF-a immunoconjugate and/or IL2 immunoconjugate for use according to claim 7, wherein the intratumoral agents are administered to the melanoma lesion(s) together, and wherein the TNF-a immunoconjugate and the IL2 immunoconjugate are combined into a single syringe prior to injecting the combination of intratumoral agents into the melanoma lesion(s).
9. The TNF-a immunoconjugate and/or IL2 immunoconjugate for use according to any one of the preceding claims, wherein the melanoma is Stage IIIB or Stage IIIC melanoma.
10. The TNF-a immunoconjugate and/or IL2 immunoconjugate for use according to any one of the preceding claims, wherein the treatment of step (a) comprises administering the combination of intratumoral agents together or separately in one or more treatment sessions, wherein each treatment session occurs from one day to two weeks before and/or after a subsequent and/or previous treatment session, optionally wherein the treatment of step (a) comprises injecting the combination of intratumoral agents together or separately into the melanoma lesion(s) during one to four treatment sessions, wherein each treatment session occurs 5-10 days, preferably 6-8 days, more preferably 7 days, before and/or after a subsequent and/or previous treatment session.
11. The TNF-a immunoconjugate and/or IL2 immunoconjugate for use according to claim 10, wherein:
(i) the total dose of the IL2 immunoconjugate(s) injected into the melanoma lesion(s) per treatment session ranges from 0.5 mg to 8 mg, preferably from 0.75 mg to 6 mg, from 1 mg to 5 mg, from 1.25 mg to 4 mg, or from 1.5 mg to about 3 mg, most preferably ranging from 2 mg to 2.5 mg, optionally wherein the total dose of the IL2 immunoconjugate(s) injected into the melanoma lesion(s) per treatment session is 2.17 mg; and/or
(ii) the total dose of the TNF-a immunoconjugate(s) injected per treatment session into the melanoma lesion(s) ranges from 50 pg to 600 pg, preferably from 75 pg to 550 pg, from 100 pg to 500 pg, from 200 to 450 pg, or from 350 pg to 450 pg, most preferably ranging from 375 pg to 425 pg, or is 100 pg, 200 pg, 300 pg, or 400 pg.
12. The TNF-a immunoconjugate and/or IL2 immunoconjugate for use according to claim 11 , wherein the total dose of the IL2 immunoconjugate(s) injected into the melanoma lesion(s) per treatment session is 2.17 mg, and the total dose of the TNF-a immunoconjugate(s) injected into the melanoma lesion(s) per treatment session is 100 pg, 200 pg, 300 pg, or 400 pg, preferably 400 pg.
13. The TNF-a immunoconjugate and/or IL2 immunoconjugate for use according to any one of the preceding claims, wherein the treatment of step (a) comprises administering the combination of intratumoral agents together or separately at least once a week for up to 8 weeks, up to 6 weeks, up to 5 weeks, up to 4 weeks, up to 3 weeks, or up to 2 weeks.
14. The TNF-a immunoconjugate and/or IL2 immunoconjugate for use according to any one of the preceding claims, wherein:
(i) the patient is assessed as having an ECOG performance status of 0 or 1 at the beginning of the treatment of step (a);
(ii) the melanoma lesion(s) are not ulcerated at the beginning of the treatment of step (a);
(iii) the melanoma has a wild type BRAF mutation status at the beginning of the treatment of step (a);
(iv) the melanoma lesion(s) treated in step (a) are cutaneous, subcutaneous, and/or nodal;
(v) the melanoma is metastasized; and/or
(vi) the patient received one or more treatments for melanoma prior to step (a), optionally wherein the one or more treatments for melanoma are chosen from, radiotherapy, chemotherapy, immunotherapy, targeted therapy, cytokine therapy, or combinations thereof.
15. The TNF-a immunoconjugate and/or IL2 immunoconjugate for use according to any one of the preceding claims, wherein:
(i) step (b) occurs within 8 weeks, preferably within 6 weeks, more preferably within 4 weeks after completion of the treatment of step (a); and/or
(ii) step (b) comprises complete tumor excision (RO resection) of one or more melanoma lesion(s) treated in step (a).
16. The TNF-a immunoconjugate and/or IL2 immunoconjugate for use according to any one of the preceding claims, further comprising administering one or more adjuvant therapies to the patient after step (b), optionally wherein the one or more adjuvant therapies are chosen from radiotherapy, chemotherapy, immunotherapy, targeted therapy, cytokine therapy, or combinations thereof.
17. The TNF-a immunoconjugate and/or IL2 immunoconjugate for use according to any one of the preceding claims, wherein:
(i) the patient has at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 1 -year after step (b), compared to the same treatment without step (a);
(ii) the patient has at least a 40%, at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 2-years after step (b), compared to the same treatment without step (a); and/or
(iii) the patient has at least a 55%, at least a 50%, at least a 45%, at least a 40%, at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of recurrence or death 3-years after step (b), compared to the same treatment without step (a).
18. The TNF-a immunoconjugate and/or IL2 immunoconjugate for use according to any one of the preceding claims, wherein:
(i) distant metastasis free survival (DMFS) is improved by an increase in median DMFS by a factor greater than 1 , preferably at least about 1 .2, at least about 1 .25, at least about 1 .3, at least about 1 .35, at least about 1 .4, at least about 1 .45, at least about 1 .5, at least about 1.55, at least about 1.6, at least about 1.65, at least about 1.7, at least about 1 .75, at least about 1 .8, at least about 1 .85, at least about 1 .9, at least about 1 .95, at least about 2, at least about 2.1 , at least about 2.15, at least about 2.2, at least about 2.25, at least about 2.3, at least about 2.35, at least about 2.4, at least about 2.45, or at least about 2.5 after step (b), compared to the same treatment without step (a);
(ii) the patient has at least a 15%, at least a 14%, at least a 13%, at least a 12%, at least an 11%, at least a 10%, at least a 9%, at least an 8%, at least a 7%, at least a 6%, or at least a 5% lower risk of distant metastasis or death 1-year after step (b), compared to the same treatment without step (a);
(iii) the patient has at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of distant metastasis or death 2-years after step (b), compared to the same treatment without step (a); and/or
(iv) the patient has at least a 50%, at least a 45%, at least a 40%, at least a 35%, at least a 30%, at least a 25%, at least a 20%, at least a 15%, or at least a 10% lower risk of distant metastasis or death 3-years after step (b), compared to the same treatment without step (a).
PCT/EP2025/054112 2024-02-15 2025-02-14 Methods for treating melanoma Pending WO2025172582A1 (en)

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