TNF alpha and interleukin-2 combination therapy for non-melanoma skin cancer

Abstract

The present application relates to the treatment of cancers, including non-melanoma skin cancer. The present invention relates to the use of a tumor necrosis factor alpha (TNF alpha) immunoconjugate and an interleukin 2 (IL2) immunoconjugate in combination therapy for the treatment of non-melanoma skin cancer.

Description

[0001] This application claims priority to EP22193432.6 filed on September 1, 2022, EP23163294.4 filed on March 21, 2023, and EP23168600.7 filed on April 18, 2023, the contents and elements of which are incorporated herein by reference for all purposes. Field of the Invention

[0002] The present invention relates to a combination therapy for the treatment of non-melanoma skin cancer, wherein a tumor necrosis factor alpha (TNFα) immunoconjugate and an interleukin 2 (IL2) immunoconjugate are administered intratumorally. Background Art

[0003] Many cytokines have demonstrated potent antitumor activity in preclinical experiments and represent potential candidates for cancer therapy. Tumor necrosis factor alpha (TNFα) is a cytokine produced by many cell types, primarily activated monocytes and macrophages. It is expressed as a 26 kDa intact transmembrane precursor protein, which is proteolytically cleaved to release a mature protein of approximately 17 kDa. Soluble, bioactive TNFα is a homotrimer that binds to cell surface receptors. TNFα has been shown to induce necrosis of solid tumors. It acts primarily on endothelial cells of tumor-associated blood vessels, producing effects that include increased vascular permeability, upregulation of tissue factor, fibrin deposition and thrombosis, and extensive destruction of endothelial cells.

[0004] Interleukin-2 (IL2), a four-alpha-helical bundle cytokine produced by T helper cells, plays a key role in the activation phase of both adaptive and innate immune responses. Although this cytokine is not thought to have direct cytotoxic effects on cancer cells, it has been reported to induce tumor regression by stimulating cell-mediated immune responses. In order to increase the therapeutic index of certain cytokines, antibody-cytokine fusion proteins (also known as "immunocytokines" or "immunoconjugates") have been developed. In these immunoconjugates, the antibody acts as a "vehicle" to promote the selective accumulation of the cytokine at the disease site, while the cytokine payload is responsible for exerting therapeutic activity (Pasche & Neri, 2012).

[0005] The applicant of the present application described in WO01 / 062298 the immunoconjugates L19-huIL2 and L19-huTNFα, which respectively comprise human interleukin 2 (huIL2) and human tumor necrosis factor α (huTNFα), each fused to the L19 antibody (disclosed in WO99 / 058570) in the form of a single chain variable fragment (scFv). L19 specifically binds to the ED-B domain of fibronectin, one of the best-known markers of angiogenesis (US 10 / 382,107; WO01 / 62298). ED-B is an additional domain of 91 amino acids that accumulates around new blood vessel structures in invasive tumors and other tissues where angiogenesis occurs (such as the proliferative endometrium and some ocular structures in pathological conditions), but cannot be detected in healthy adult tissues.

[0006] The applicant of the present application describes various uses of L19-huIL2 and L19-huTNFα for treating cancer in combination with a variety of other cancer therapies. For example, WO2007 / 115837, WO2009 / 089858, WO2013 / 010749, WO2017 / 178562, WO2018 / 154517, WO2019 / 185792 and WO2021 / 234178 respectively report L19-huIL2 combined with gemcitabine, antibodies against B cell progenitor cells, CTLA-4 blockers, CAR-T cells, certain small molecule drug conjugates, certain checkpoint inhibitors and chemotherapy to treat cancer.

[0007] In addition, L19-huIL2 and L19-huTNFα have been used in combination in cancer therapy. For example, L19-huIL2 and L19-huTNFα have been shown to cure neuroblastoma in a fully syngeneic mouse model of the disease, whereas the individual immunoconjugates used as single agents did not result in eradication of the disease (Balza et al., 2010). The combination of IL2 and TNFα payloads has also shown promising results in clinical trials. L19-huIL2 and L19-huTNFα have been shown to have a strong synergistic effect in intralesional treatment of certain solid tumors in mice (Schwager et al., 2013). The corresponding fully human fusion protein has been administered intratumorally to patients with stage IIIC melanoma (Danielli et al., 2015), showing better results than intratumoral administration of interleukin-2 (Weide et al., 2011) or L19-IL2 (Weide et al., 2014). In WO 2013 / 045125, the applicants of the present application describe the administration of a single dose of L19-huIL2 and L19-huTNFα for the treatment of skin tumors.

[0008] Non-melanoma skin cancer (NMSC) refers to a group of cancers that develop slowly in the upper layers of the skin. The term "non-melanoma" distinguishes these common types of skin cancer from the less common but potentially more serious skin cancer called melanoma. Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC) account for 99% of NMSC. Other NMSC include Merkel cell carcinoma (MCC), Kaposi sarcoma (KS), cutaneous T-cell lymphoma (CTCL), keratoacanthoma (KA), and malignant adnexal tumors of the skin (MATS). Specifically, other NMSC include Merkel cell carcinoma (MCC), Kaposi sarcoma (KS), cutaneous T-cell lymphoma (CTCL), and malignant adnexal tumors of the skin (MATS).

[0009] Complete surgical resection with histologically controlled margins remains the treatment option of choice for BCC and cSCC due to their high five-year recurrence-free survival rates. However, surgery is not always feasible depending on the anatomical location, and patients often do not undergo surgery because it may cause disfigurement, physical deformation, and adversely affect quality of life. Alternative treatment options for BCC are topical therapies (such as imiquimod or 5-fluorouracil), photodynamic therapy, or radiotherapy, but these therapies do not provide a significant benefit in terms of recurrence-free survival. For cSCC, topical treatment with imiquimod or 5-fluorouracil or photodynamic therapy is not recommended, further limiting nonsurgical treatment options.

[0010] Although WO2013 / 045125 describes the intratumoral administration of a single dose of L19-huIL2 and L19-huTNFα for the treatment of skin tumors, in particular malignant skin tumors, melanomas and carcinomas, it does not mention the treatment of NMSCs, especially for patients who are at risk of postoperative disfigurement, i.e., patients in whom surgery would produce unacceptable cosmetic results.

[0011] Thus, there remains a need in the art for effective treatments for NMSC, particularly treatments that do not result in the disfigurement typically associated with surgical treatment.

[0012] The present invention is designed in view of the above considerations. Summary of the invention

[0013] Here we report the unexpected therapeutic effect of intratumoral administration of a combination of L19-huTNFα and L19-huIL2 in a patient with disfiguring BCC tumors on the nose. Surprisingly, the tumors showed a complete clinical and pathological response to the treatment (Example 1).

[0014] Patients received four intratumoral injections of a mixture of 1.08 mg L19-huIL2 and 200 μg L19-huTNFα, each in a volume of 1 mL. In the second injection, the dose of L19-huIL2 was unexpectedly doubled to 2.17 mg. This is not expected to have a substantial impact on the treatment outcome, and patients treated with the 1.08 mg L19-huIL2 dose throughout the course of treatment are expected to have the same results. In response to treatment, tumors decreased in size, gradually transformed into necrotic crusts, then formed crusts, and eventually sloughed off. Two months after the first injection, the tumors showed a complete clinical and pathological response to treatment, with the tumors replaced by fully epithelialized scars.

[0015] The complete clinical and pathological response of the tumor to treatment was completely unexpected and was observed in the first human trial of this combination therapy for the treatment of non-melanoma skin cancer. Due to the complete response of the tumor to treatment, the patient did not need to undergo nasal amputation. At follow-up 6 months after the first injection, the patient showed no recurrence of cancer.

[0016] The complete response of BCC as a result of intratumoral injection of the combination of L19-huIL2 and L19-huTNFα demonstrates the surprising efficacy of this combination for the treatment of non-melanoma skin cancer.

[0017] In a first aspect, the present invention therefore relates to a composition comprising a combination of L19-TNFα and L19-IL2 for use in a method of treating non-melanoma skin cancer in a patient by therapy.

[0018] The present invention also relates to a method of treating non-melanoma skin cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of a combination of L19-TNFα and L19-IL2.

[0019] The present invention also relates to L19-TNFα for use in a method of treating non-melanoma skin cancer in a patient by therapy, wherein the method comprises administering a combination of L19-TNFα and L19-IL2.

[0020] The present invention further relates to L19-IL2 for use in a method of treating non-melanoma skin cancer in a patient by therapy, wherein the method comprises administering L19-IL2 in combination with L19-TNFα.

[0021] Preferably, the method of treating non-melanoma skin cancer in a patient comprises intratumorally injecting the composition.

[0022] Also disclosed is the use of a combination of L19-TNFα and L19-IL2 for preparing a medicament for treating non-melanoma skin cancer.

[0023] Another aspect of the present invention provides a therapeutic kit comprising the composition as described herein.

[0024] Preferably, the method comprises injecting a dose of a composition. For example, the dose of the composition may include 1 to 5 mg L19-IL2 and 100 to 500 μg L19-TNFα. Preferably, the dose of the composition includes 1 to 2 mg L19-IL2 and 200 to 400 μg L19-TNFα. More preferably, the dose of the composition includes 1 to 2.2 mg L19-IL2 and 200 to 400 μg L19-TNFα. In some preferred embodiments, the dose of the composition includes 1.08 mg L19-IL2 and 200 μg L19-TNFα. In other preferred embodiments, the dose of the composition includes 2.17 mg L19-IL2 and 400 μg L19-TNFα.

[0025] Alternatively, the dosage of L19-IL2 can also be expressed in international units (IU). For example, the dosage of the composition may include 5 million to 15 million IU L19-IL2 and 100 to 500 μg L19-TNFα. Preferably, the dosage of the composition includes 6 million to 13 million IU L19-IL2 and 200 to 400 μg L19-TNFα. More preferably, the dosage of the composition may include 6.5 million to 13 million IU L19-IL2 and 200 to 400 μg L19-TNFα. 1.08 mg L19-IL2 is equivalent to 6.5 million IU of L19-IL2. 2.17 mg L19-IL2 is equivalent to 13 million IU L19-IL2. Thus, in some preferred embodiments, the dosage of the composition comprises 6.5 million IU L19-IL2 and 200 μg L19-TNFα. In some alternative preferred embodiments, the dosage of the composition comprises 13 million IU L19-IL2 and 400 μg L19-TNFα.

[0026] Preferably, L19-TNFα is L19-huTNFα. More preferably, L19-huTNFα comprises or consists of the amino acid sequence shown in SEQ ID NO:16.

[0027] Preferably, L19-IL2 is L19-huIL2. More preferably, L19-huIL2 comprises or consists of the amino acid sequence shown in SEQ ID NO:13.

[0028] Preferably, the method of treating a patient's non-melanoma skin cancer comprises intratumoral injection of the composition. In some preferred embodiments, a single dose of the composition is divided into multiple intratumoral injections, which are administered to the same tumor. This method of administration delivers doses to different locations of the tumor.

[0029] Therefore, the composition of a single dose can be administered by one or more intratumoral injections. Preferably, the composition of a single dose is administered by multiple intratumoral injections, and it is applied to the same tumor, wherein each intratumoral injection is applied to different parts of the tumor so that the dosage is distributed on the tumor surface. The purpose is to evenly distribute the dosage on the tumor surface. Therefore, the number of injections can be selected according to the surface area of ​​the tumor. For example, the composition of a single dose can be administered by 1 to 30 intratumoral injections, for example, by 1 to 20 times, 1 to 15 times, 1 to 10 times, 1 to 9 times, 1 to 8 times, 1 to 7 times, 1 to 6 times, 1 to 5 times, 1 to 4 times, 1 to 3 times or 1 to 2 intratumoral injections. In an example, a single dose can be administered by 1 to 6 intratumoral injections. When the composition of a single dose is divided into two intratumoral injections, for example, each intratumoral injection can be applied to different parts (preferably different halves) of the tumor surface.

[0030] The non-melanoma skin cancer is preferably selected from the group consisting of basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), Merkel cell carcinoma (MCC), Kaposi's sarcoma (KS), cutaneous T-cell lymphoma (CTCL), malignant adnexal tumors of the skin (MATS) and keratoacanthoma (KA). More preferably, the non-melanoma skin cancer is BCC or cSCC. Most preferably, the non-melanoma skin cancer is BCC. In a preferred embodiment, BCC is stage IIA, IIB, IIIA or IIIB BCC as defined by the EADO classification system (Grob et al., 2021). Most preferably, BCC is stage IIIB BCC as defined by the EADO classification system (Grob et al., 2021).

[0031] Preferably, the non-melanoma skin cancer is non-metastatic. In a preferred embodiment, the non-melanoma skin cancer is located on the patient's head. In a more preferred embodiment, the non-melanoma skin cancer is located on the patient's scalp, face or ears, particularly on the patient's face or ears, most preferably on the patient's face.

[0032] Thus, in a preferred embodiment, the non-melanoma skin cancer is a non-metastatic BCC. In a preferred embodiment, the BCC is located on the patient's head. In a more preferred embodiment, the BCC is located on the patient's scalp, face or ear, particularly on the patient's face or ear, and most preferably on the patient's face.

[0033] Similarly, in alternative preferred embodiments, the non-melanoma skin cancer is cSCC, wherein the cSCC is non-metastatic. In preferred embodiments, the cSCC is located on the patient's head. In more preferred embodiments, the cSCC is located on the patient's scalp, face or ear, particularly on the patient's face or ear, and most preferably on the patient's face.

[0034] Preferably, the methods described herein for treating non-melanoma skin cancer in a patient reduce tumor size.

[0035] Preferably, tumor size is measured using tumor volume or tumor area on the patient's skin surface. Therefore, the method may optionally include a step of observing the patient's tumor size at a certain time point after administering the first dose of L19-TNFα and L19-IL2 to the patient, for example, at least 10 days, at least two weeks, at least one month, at least two months and / or at least 3 months after administering the first dose of L19-TNFα and L19-IL2 to the patient, for example, between 1 and 3 months, such as two months, after administering the first dose of L19-TNFα and L19-IL2 to the patient. In one example, the method may optionally include a step of observing the patient's tumor size at least 6 weeks, at least 9 weeks, at least 12 weeks, at least 20 weeks, at least 28 weeks, at least 36 weeks, at least 44 weeks and / or at least 52 weeks after administering the first dose of L19-TNFα and L19-IL2 to the patient. Preferably, a reduction in tumor size is observed at one or more of these time points. In a preferred embodiment, the method includes a step of observing the patient's tumor size at least 6 weeks after administering the first dose of L19-TNFα and L19-IL2, for example, between 6 and 9 weeks, such as 9 weeks. For example, the method may include a step of observing the patient's tumor size at least 36 days after administering the first dose of L19-TNFα and L19-IL2 to the patient, for example, between 36 and 63 days, such as 63 days.

[0036] Preferably, the tumor exhibits a complete clinical response to treatment. Most preferably, the tumor exhibits a complete pathological response to treatment. A complete clinical and / or pathological response is advantageous because it eliminates the need for surgical removal of the tumor, which may result in disfigurement due to the size and / or location of the tumor, and / or adversely affect the patient's quality of life. Surgery is particularly problematic in the case of treating tumors located on the patient's head, particularly on the patient's face, because surgical removal of tumors on the head and / or face may be particularly disfiguring. Thus, in one embodiment, the method comprises injecting the composition at the site of a non-melanoma skin cancer tumor, wherein the tumor is located on the patient's head, optionally wherein the tumor is located on the patient's face.

[0037] In some embodiments, the non-melanoma skin cancer is inoperable.

[0038] The method for treating non-melanoma skin cancer may also include making a decision on further therapy after viewing the results of the above-mentioned tumor size observations, and optionally performing subsequent steps of the further therapy. Those skilled in the art can easily use techniques such as caliper measurements to make this observation. Further therapy may include further administration of the composition of the present invention. Additionally or alternatively, further therapy may include chemotherapy, radiotherapy and / or surgery. Preferably, further therapy includes surgery. Therefore, in some embodiments, the treatment may be neoadjuvant therapy. In some embodiments, the method includes performing surgery on the patient or performing a step of surgery at least 6 weeks, for example, between 6 and 12 weeks after administering a first dose of L19-TNFα and L19-IL2 to the patient.

[0039] In some embodiments, the patient is one who has not received prior IL-2 or TNFα treatment.

[0040] Preferably, the method comprises intratumorally injecting more than one dose of the composition. For example, the method may comprise intratumorally injecting an initial dose of the composition and one or more additional doses of the composition. Preferably, the method comprises an initial dose of the composition and three additional doses of the composition. Preferably, the dose is administered once a week.

[0041] In some embodiments, one or more of the additional doses are the same as the initial dose (ie, contain the same amount of L19-IL2 and / or L19-TNFα).

[0042] In some optional embodiments, one or more other doses include more or less L19-IL2 and / or L19-TNFα than the initial dose. If the patient experiences side effects such as inflammation after applying the initial dose, it may be desirable to reduce the dose. Alternatively, the initial dose can be reduced to observe whether the patient tolerates the treatment, and if the patient shows good tolerance, the subsequent dose is increased. In an example, one or more other doses may include twice or half the amount of L19-IL2 and L19-TNFα relative to the amount of L19-IL2 and L19-TNFα in the initial dose.

[0043] In some embodiments, one or more further doses contain more L19-IL2 and / or L19-TNFα than the initial dose. Preferably, one or more further doses contain twice the amount of L19-IL2 and / or L19-TNFα relative to the initial dose. For example, all further doses may contain twice the amount of L19-IL2 and / or L19-TNFα relative to the initial dose.

[0044] In some embodiments, one or more further doses contain less L19-IL2 and / or L19-TNFα than the initial dose. Preferably, one or more further doses contain half the amount of L19-IL2 and / or L19-TNFα relative to the initial dose. For example, all further doses may contain half the amount of L19-IL2 and / or L19-TNFα relative to the initial dose.

[0045] If the patient has multiple non-melanoma skin cancer tumors, such as multiple BCC tumors, the total dose may be distributed between two or more of the tumors by intratumoral injection.

[0046] A patient as referred to herein is preferably a human patient.

[0047] The present invention includes any combination of the above-described aspects and preferred features, unless such a combination is obviously infeasible or clearly avoided. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Embodiments and experiments illustrating the principles of the present invention will now be discussed in conjunction with the accompanying drawings, in which:

[0049] Figure 1 Shown is the response of a patient with stage IIIB basal cell carcinoma (BCC) located in the nose, at baseline and after 4 intratumoral injections of L19-huTNFα and L19-huIL2 administered once weekly, as described in Example 1.

[0050] Figure 2 A fully epithelialized scar on the nose of the patient described in Example 1 is shown, indicating no recurrence of cancer at follow-up four months after the first injection.

[0051] Figure 3 Shown are histochemical stains of needle biopsies from the primary tumor site of the patient described in Example 1. The needle biopsies were taken before treatment (left) and on day 63 after the first injection (right). The absence of any residual tumor cells in the biopsy taken on day 63, as determined by histological analysis, indicates that the patient achieved a complete pathological response. DETAILED DESCRIPTION OF THE INVENTION

[0053] Various aspects and embodiments of the present invention will now be discussed in conjunction with the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

[0054] Immunoconjugates

[0055] The composition of the present invention comprises L19-IL2 and L19-TNFα. L19-IL2 is an immunoconjugate comprising IL2, IL2 is connected to the antibody molecule L19, and L19 binds to the ED-B domain of fibronectin. L19-TNFα is an immunoconjugate comprising TNFα, and TNFα is connected to the antibody molecule L19.

[0056] Preferably, L19 comprises the complementarity determining regions (CDRs) of the L19 antibody as shown below.

[0057] VH CDR 1 SFSMS SEQ ID NO: 1

[0058] VH CDR 2 SISGSSGTTYYADSVKG SEQ ID NO:2

[0059] VH CDR 3 PFPYFDY SEQ ID NO: 3

[0060] VL CDR 1 RASQSVSSSFLA SEQ ID NO:4

[0061] VL CDR 2 YASSRAT SEQ ID NO:5

[0062] VL CDR 3 QQTGRIPPT SEQ ID NO:6

[0063] Preferably, L19 comprises a L19 VH domain and / or a L19 VL domain. The amino acid sequences of the L19 VH and L19 VL domains are listed in SEQ ID NO: 7 and SEQ ID NO: 9, respectively.

[0064] Preferably, the antibody molecule is a single-chain Fv (scFv), a diabody or other antibody fragments of low molecular weight and / or lacking an Fc region. These properties contribute to the targeting and tissue penetration of the immunoconjugate at the tumor site. Therefore, L19-IL2 and L19-TNFα preferably comprise scFv-L19, which is a scFv comprising an L19VH domain and an L19VL domain, wherein VH and VL are connected in a single polypeptide chain by a peptide linker sequence (such as a 12 amino acid residue linker listed in SEQ ID NO: 8). Preferably, scFv-L19 comprises or consists of the amino acid sequence listed in SEQ ID NO: 10.

[0065] The antibody molecule may comprise a VH domain having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence of the L19 VH domain shown in SEQ ID NO: 7, and / or a VL domain having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence of the L19 VL domain shown in SEQ ID NO: 9. Preferably, the antibody molecule is L19 in scFv form, comprising the L19 VH domain listed in SEQ ID NO: 7 and the L19 VL domain shown in SEQ ID NO: 9. In a preferred embodiment, the antibody molecule is a L19 scFv having the amino acid sequence shown in SEQ ID NO: 10. The antibody molecule may comprise or consist of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence of L19scFv having the amino acid sequence shown in SEQ ID NO:10.

[0066] The composition of the present invention may comprise a modified form of the L19 VH and / or VL domain, for example, the antibody molecule may comprise the L19 VH or L19 VL domain, wherein 1, 2, 3, 4 or 5 amino acid substitutions are made in the CDR and / or framework regions, while retaining specific binding to fibronectin ED-B. In addition, the antibody molecule may comprise a linker, which has undergone 1, 2, 3, 4 or 5 amino acid substitutions relative to the amino acid sequence shown in SEQ ID NO: 8. Such amino acid substitutions are preferably conservative, for example, one hydrophobic residue is replaced by another hydrophobic residue, one polar residue is replaced by another polar residue, arginine is replaced by lysine, glutamic acid is replaced by aspartic acid, or glutamine is replaced by asparagine.

[0067] Molecular linkers such as peptides can be used to connect cytokines to antibody molecules, which helps to express all or part of the immunoconjugate as a fusion protein. Examples of suitable linkers are shown in SEQ ID NO: 12 and SEQ ID NO: 15. The immunoconjugate may include such a linker, which has undergone 1, 2, 3, 4 or 5 amino acid substitutions relative to the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 15. When the antibody molecule is a single-chain molecule such as scFv, the entire immunoconjugate polypeptide chain can be conveniently produced as a fusion protein. Without wishing to be bound by theory, for TNFα immunoconjugates, the fusion protein is considered to be assembled into a trimer, allowing TNFα to adopt its normal trimeric form.

[0068] L19-IL2 preferably comprises human IL2. Human IL2 preferably comprises or consists of the amino acid sequence shown in SEQ ID NO: 11. The antibody molecule is preferably a human or humanized antibody molecule. The L19-huIL2 conjugate may comprise or consist of the amino acid sequence shown in SEQ ID NO: 13. The immunoconjugate molecule may comprise or consist of a polypeptide having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence of L19-huIL2 shown in SEQ ID NO: 13. The immunoconjugate may comprise or consist of a polypeptide having 1, 2, 3, 4 or 5 amino acid substitutions relative to the amino acid sequence shown in SEQ ID NO: 13.

[0069] L19-TNFα preferably comprises human TNFα. More preferably, L19-TNFα comprises a soluble form of the extracellular domain of human TNFα. Human TNFα preferably comprises or consists of the amino acid sequence shown in SEQ ID NO:14. The antibody molecule is preferably a human or humanized antibody molecule. The L19-huTNFα conjugate may comprise or consist of the amino acid sequence shown in SEQ ID NO:16. The immunoconjugate molecule may comprise or consist of a polypeptide having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence of L19-huTNFα shown in SEQ ID NO:16. The immunoconjugate may comprise or consist of a polypeptide having 1, 2, 3, 4 or 5 amino acid substitutions relative to the amino acid sequence shown in SEQ ID NO:16.

[0070] Indications

[0071] The cancer to be treated according to the present invention is non-melanoma skin cancer (NMSC). NMSC includes basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), Merkel cell carcinoma (MCC), Kaposi's sarcoma (KS), cutaneous T-cell lymphoma (CTCL), keratoacanthoma (KA) and malignant adnexal tumor of the skin (MATS). In one example, NMSC can be basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), Merkel cell carcinoma (MCC), Kaposi's sarcoma (KS), cutaneous T-cell lymphoma (CTCL) and malignant adnexal tumor of the skin (MATS).

[0072] Preferably, the NMSC is BCC or cSCC. More preferably, the NMSC is BCC.

[0073] The BCC or cSCC may be a localized (non-metastatic, node-negative, unifocal or multifocal) BCC or cSCC that is amenable to intratumoral injection. The BCC or cSCC may be a high-risk BCC or cSCC.

[0074] BCC can be classified according to the disease stage in the EADO classification system version 1 described in Grob et al., 2021. In a preferred embodiment, the BCC is stage IIA, IIB, IIIA or IIIB BCC, most preferably stage IIIB BCC. Stage IIA, IIB, IIIA or IIIB of BCC is preferably defined according to the EADO classification system version 1 (Grob et al., 2021).

[0075] Preferably, the non-melanoma skin cancer is non-metastatic. In some embodiments, the non-melanoma skin cancer is located on the patient's head. In some embodiments, the non-melanoma skin cancer is located on the patient's face or ears, preferably the face.

[0076] Thus, in some preferred embodiments, the non-melanoma skin cancer is a BCC, wherein the BCC is non-metastatic. In some preferred embodiments, the non-melanoma skin cancer is a BCC, wherein the BCC is located on the head of the patient. In some preferred embodiments, the non-melanoma skin cancer is a BCC, wherein the BCC is located on the face or ear of the patient, preferably on the face of the patient. In some preferred embodiments, the non-melanoma skin cancer is a non-metastatic BCC located on the head of the patient. In some preferred embodiments, the non-melanoma skin cancer is a non-metastatic BCC located on the face, scalp or ear of the patient, preferably on the face or ear, more preferably on the face.

[0077] In some preferred embodiments, the non-melanoma skin cancer is cSCC, wherein the cSCC is non-metastatic. In some preferred embodiments, the non-melanoma skin cancer is cSCC, wherein the cSCC is located on the head of the patient. In some preferred embodiments, the non-melanoma skin cancer is cSCC, wherein the cSCC is located on the face or ear of the patient, preferably on the face of the patient. In some preferred embodiments, the non-melanoma skin cancer is a non-metastatic cSCC located on the head of the patient. In some preferred embodiments, the non-melanoma skin cancer is a non-metastatic cSCC located on the face, scalp, or ear of the patient, preferably on the face or ear of the patient, more preferably on the face of the patient.

[0078] In some preferred embodiments, the treatment reduces the size of a non-melanoma skin cancer tumor. For example, the method can reduce the size of a non-melanoma skin cancer tumor by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Preferably, the treatment reduces the size of a non-melanoma skin cancer tumor by at least 50%. More preferably, the method reduces the size of a non-melanoma skin cancer tumor by at least 90%. In this context, tumor size can refer to tumor volume. Alternatively, tumor size can refer to the area of ​​the tumor visible on the patient's skin surface.

[0079] More preferably, the non-melanoma skin cancer tumor shows a clinical complete response to treatment. Most preferably, the non-melanoma skin cancer tumor shows a pathological complete response to treatment. The determination of a clinical or pathological complete response is within the capabilities of those skilled in the art. A clinical complete response (cCR) may refer to the absence of detectable tumor as determined by imaging such as computed tomography (CT). A pathological complete response (pCR) may refer to the absence of detectable cancer cells in a tissue sample obtained from the primary tumor site. The presence of cancer cells in a tissue sample may be determined by histological analysis.

[0080] Application

[0081] The dosage is the amount of TNFα and IL2 cytokines in the form of an immunoconjugate as described above, which is effective for treating a tumor in a combination therapy according to the invention.

[0082] In some embodiments, the dosage of the composition comprises 0.5 to 5 mg L19-IL2 and 100 to 500 μg L19-TNFα. In some embodiments, the dosage of the composition comprises 0.5 to 3 mg L19-IL2 and 150 to 450 μg L19-TNFα. In some embodiments, the dosage of the composition comprises 1 to 2.2 mg L19-IL2 and 200 to 400 μg L19-TNFα. For example, the dosage of the composition may comprise 1, 1.08, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.17 or 2.2 mg L19-IL2. The dosage of the composition may further comprise 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg, 300 μg, 310 μg, 320 μg, 330 μg, 340 μg, 350 μg, 360 μg, 370 μg, 380 μg, 390 μg or 400 μg L19-TNFα. Preferably, the dosage of the composition comprises 1.08 mg L19-IL2 and 200 μg L19-TNFα. In a preferred embodiment for selection, the dosage of the composition comprises 2.17 mg L19-IL2 and 400 μg L19-TNFα.

[0083] Alternatively, the dosage of L19-IL2 can also be expressed in international units (IU). For example, the dosage of the composition can include 5 million to 15 million IU L19-IL2 and 100 to 500 μg L19-TNFα. For example, the dosage of the composition can include 5 million, 5.5 million, 6 million, 6.5 million, 7 million, 7.5 million, 8 million, 8.5 million, 9 million, 9.5 million, 10 million, 10.5 million, 11 million, 11.5 million, 12 million, 12.5 million, 13 million, 13.5 million, 14 million, 14.5 million or 15 million IU L19-IL2. Preferably, the dosage of the composition includes 6 million to 13 million IU L19-IL2 and 200 to 400 μg L19-TNFα. More preferably, the dosage of the composition comprises 6.5 million to 13 million IU L19-IL2 and 200 to 400 μg L19-TNFα. 1.08 mg L19-IL2 is equivalent to 6.5 million IU L19-IL2. 2.17 mg L19-IL2 is equivalent to 13 million IU L19-IL2. Therefore, in some preferred embodiments, the dosage of the composition comprises 6.5 million IU L19-IL2 and 200 μg L19-TNFα. In some alternative preferred embodiments, the dosage of the composition comprises 13 million IU L19-IL2 and 400 μg L19-TNFα.

[0084] In some embodiments, the non-melanoma skin cancer is selected from the list consisting of Merkel cell carcinoma (MCC), Kaposi's sarcoma (KS), cutaneous T-cell lymphoma (CTCL), malignant adnexal tumor of the skin (MATS), and keratoacanthoma (KA), and the dose of the composition comprises 2.17 mg L19-IL2 and 400 μg L19-TNFα.

[0085] L19-IL2 can optionally be formulated as a total volume of 1.0 mL at a dose of 2.17 mg, while L19-TNFα can optionally be formulated as a total volume of 1.0 mL at a dose of 400 μg. To prepare a solution for intratumoral injection, the entire volume of L19-IL2 can be transferred to the vial containing L19-TNFα (or vice versa), resulting in a solution of 2.17 mg L19-IL2 + 400 μg L19-TNFα in a total volume of 2.0 mL.

[0086] After gently mixing the solution, a single syringe with a 30-gauge needle can be used to draw 1.0 mL of a solution containing 1.08 mg L19-IL2 + 200 μg L19-TNFα for intratumoral injection, and the remaining solution can be discarded. Alternatively, a single syringe with a 30-gauge needle can be used to draw 2.0 mL of a solution containing 2.17 mg L19-IL2 + 400 μg L19-TNFα for intratumoral injection, and the remaining solution can be discarded.

[0087] Preferably, the method of treating a non-melanoma skin cancer in a patient comprises injecting the composition at the site of a non-melanoma skin cancer tumor, preferably by intratumoral injection. Intratumoral injection is injection into the tumor. Peritumoral injection, such as local intradermal injection, is another suitable method of locally administering the composition to the tumor site.

[0088] In some preferred embodiments, a single dose of the composition is divided and injected intratumorally at multiple sites into a single non-melanoma skin cancer tumor. In this case, the total volume of the dose is distributed over the surface area of ​​the tumor or the tumor mass (e.g., tumor mass).

[0089] In other preferred embodiments, the method comprises injecting a dose of the composition into more than one non-melanoma skin cancer tumor in the patient, wherein the dose is distributed among the tumors. The total volume of the dose can be distributed among the tumors according to the following scheme:

[0090] Tumor number Volume injected per tumor 1 100% L19-IL2+L19-TNFα volume 2 50% of L19-IL2+L19-TNFα volume 3 33% of L19-IL2+L19-TNFα volume

[0091] Thus, in some embodiments, the method comprises injecting the dose into two non-melanoma skin cancer tumors, wherein 50% of the dose is injected into each tumor. In some embodiments, the method comprises injecting the dose into three non-melanoma skin cancer tumors, wherein 33% of the dose is injected into each tumor. In this case, the total volume of the dose can be evenly distributed among all tumors, regardless of tumor size.

[0092] In patients with four or more injectable non-melanoma skin cancer tumors, tumor priority can be determined based on tumor size. Thus, in some embodiments, where the patient has more than three non-melanoma skin cancer tumors, the method comprises injecting a dose into the three largest tumors, wherein 33% of the dose is injected into each of the three largest tumors. In this case, other smaller tumors may remain uninjected. In some embodiments, the three largest tumors, regardless of their size, may receive the same dose in each tumor, so that the dose for each tumor remains constant. In other embodiments, the largest tumor may be injected first, and then all other tumors may be injected sequentially in order of decreasing tumor size until the total volume is injected. The size of a non-melanoma skin cancer tumor preferably refers to the longest (i.e., largest) diameter of the tumor on the surface of the patient's skin (referred to herein as the "longest diameter").

[0093] New non-melanoma skin cancer tumors that appear during the 4-week treatment period may also be treated as described above. For new tumors, the treatment period may not extend beyond the 4-week treatment period starting from the first intratumoral injection of L19-IL2 and L19-TNFα.

[0094] In some embodiments, the method comprises injecting a dose of a composition comprising a maximum of 2.2 mg of L19-huIL2 and a maximum of 400 μg of L19-TNFα. In some preferred embodiments, the method comprises injecting a dose of a composition comprising a maximum of 1.08 mg of L19-IL2 and a maximum of 200 μg of L19-TNFα. In some other preferred embodiments, the method comprises injecting a dose of a composition comprising a maximum of 2.17 mg of L19-IL2 and a maximum of 400 μg of L19-TNFα. The dose of the composition injected into the patient's non-melanoma skin cancer tumor can be adjusted according to the tumor size (such as the longest diameter) and / or the location of the tumor on the patient's body. For example, the dose of the composition injected into the patient's non-melanoma skin cancer tumor can be reduced relative to one of the maximum amounts of L19-IL2 and L19-TNFα described above.

[0095] In some patients, inflammation was observed around the injection site after administration of the composition. Surprisingly, the inventors observed that adjusting (lowering) the dose of the composition injected into the tumor, based on the tumor size (e.g., longest diameter) and / or the location of the tumor on the patient's body, can reduce inflammation around the injection site while still achieving a therapeutic effect of the tumor, such as a complete clinical and / or pathological response, but preferably a pathological complete response.

[0096] For example, 50% of the maximum amount of L19-IL2 and L19-TNFα (in this case 1.08 mg L19-IL2 and 200 μg L19-TNFα) was injected intratumorally into a cSCC tumor located on the patient's eyelid. Surprisingly, despite receiving half the maximum amount of the combination (i.e., 0.54 mg L19-IL2 and 100 μg L19-TNFα), the tumor still showed a complete pathological response to the treatment.

[0097] The inventors have also demonstrated that the dose of the composition injected intratumorally can be reduced based on the size (eg, longest diameter) of the patient's BCC tumor while still achieving a pathological complete response.

[0098] In cases where tumor treatment is prioritized based on tumor size, the volume of the dose may be administered according to the following regimen:

[0099] Tumor-longest diameter (cm) Volume to be injected Diameter>3.0 100% L19-IL2+L19-TNFα volume 2.0 < diameter ≤ 3.0 75% of L19-IL2+L19-TNFα volume 1.0 < diameter ≤ 2.0 50% of L19-IL2+L19-TNFα volume 0 < diameter ≤ 1.0 25% L19-IL2+L19-TNFα volume

[0100] Thus, in some embodiments, the method comprises injecting a dose into a non-melanoma skin cancer tumor with a longest diameter greater than 3 cm, wherein 100% of the dose is injected into the tumor. In some embodiments, the method comprises injecting a dose into a non-melanoma skin cancer tumor with a longest diameter greater than 2 cm and less than or equal to 3 cm, wherein 75% of the dose is injected into the tumor. In some embodiments, the method comprises injecting a dose into a non-melanoma skin cancer tumor with a longest diameter greater than 1 cm and less than or equal to 2 cm, wherein 50% of the dose is injected into the tumor. In some embodiments, the method comprises injecting a dose into a non-melanoma skin cancer tumor with a longest diameter greater than 0 cm (e.g., at least 0.1 cm, such as between 0.1 cm and 1 cm), wherein 25% of the dose is injected into the tumor.

[0101] In some embodiments, the method comprises injecting a dose into a non-melanoma skin cancer tumor located on the patient's nose or in the area surrounding the orifice of the head. The area surrounding the orifice of the patient's head can be determined by a clinician, for example, according to Pons et al., 2022. In patients with non-melanoma skin cancer tumors located on the patient's nose or in the area surrounding the orifice of the head, the volume of the dose of the composition injected into the tumor can be adjusted according to the size of the tumor (such as the longest diameter) according to the following scheme:

[0102]

[0103] Thus, in some embodiments, the method comprises injecting into a non-melanoma skin cancer tumor located on the patient's nose or in the area surrounding the orifice of the head, the method comprising injecting a dose of a composition comprising:

[0104] (a) 25% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the tumor surface area is >1 cm and ≤2 cm;

[0105] (b) 37.5% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the tumor surface area is >2 cm and ≤3 cm;

[0106] (c) 50% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the tumor surface area is >3 cm and ≤4 cm;

[0107] (d) 62.5% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the surface area of ​​the tumor is >4 cm and ≤5 cm; or

[0108] (e) 75% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the tumor surface area is >5 cm.

[0109] Alternatively, in patients with non-melanoma skin cancer tumors located on the nose or in the area surrounding the orifice of the head, the volume of the dose of the composition injected into the tumor may be adjusted according to the size of the tumor (e.g., longest diameter) according to the following alternative schemes:

[0110]

[0111] Thus, in some alternative embodiments, the method comprises injecting into a non-melanoma skin cancer tumor located on the patient's nose or in the area surrounding the orifice of the head, the method comprises injecting a dose of a composition comprising:

[0112] (a) 25% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the tumor surface area is >1 cm and ≤2 cm;

[0113] (b) 37.5% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the tumor surface area is >2 cm and ≤3 cm;

[0114] (c) 50% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the surface area of ​​the tumor is >3 cm and ≤5 cm; or

[0115] (d) 62.5% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the tumor surface area is >5 cm.

[0116] In some embodiments, the method comprises injecting a dose into a non-melanoma skin cancer tumor in a location other than on the nose or in the area around the orifice of the head of the patient. For example, the method may comprise injecting a dose into a non-melanoma skin cancer tumor in an area other than on the nose or in the area around the orifice of the patient's head (e.g., the forehead, cheek, chin, or scalp of the patient). In patients with non-melanoma skin cancer tumors located in locations other than on the nose or in the area around the orifice of the head of the body, the volume of the dose of the composition injected into the tumor may be adjusted according to the size of the tumor (e.g., the longest diameter) according to the following scheme:

[0117]

[0118]

[0119] Thus, in some embodiments, the method comprises injecting into a non-melanoma skin cancer tumor on a patient's body (e.g., head) other than on the nose or in the area around the orifice of the head, the method comprising injecting a dose of a composition comprising:

[0120] (a) 37.5% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the tumor surface area is >1 cm and ≤2 cm;

[0121] (b) 50% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the tumor surface area is >2 cm and ≤3 cm;

[0122] (c) 62.5% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the tumor surface area is >3 cm and ≤4 cm;

[0123] (d) 75% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the surface area of ​​the tumor is >4 cm and ≤5 cm; or

[0124] (e) 100% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the tumor surface area is >5 cm.

[0125] Alternatively, in patients with non-melanoma skin cancer tumors located on a site on the body (e.g., head) other than on the nose or in the area around the orifice of the head, the volume of the dose of the composition injected into the tumor may be adjusted according to the size of the tumor (e.g., longest diameter) according to the following alternative schemes:

[0126]

[0127] Thus, in some alternative embodiments, the method comprises injecting into a non-melanoma skin cancer tumor located on a patient's body (e.g., head) other than on the nose or in the area around the orifice of the head, the method comprises injecting a dose of a composition comprising:

[0128] (a) 37.5% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the tumor surface area is >1 cm and ≤2 cm;

[0129] (b) 50% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the tumor surface area is >2 cm and ≤3 cm;

[0130] (c) 75% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the tumor surface area is >3 cm and ≤5 cm; or

[0131] (d) 100% of the maximum amount of L19-IL2 and L19-TNFα as described above, when the longest diameter of the tumor surface area is >5 cm.

[0132] In patients with multiple non-melanoma skin cancer tumors, tumor priority can be determined based on tumor size. Therefore, in some embodiments, where the patient has multiple non-melanoma skin cancer tumors, the method includes injecting a dose of the composition into each tumor according to one of the above-mentioned dosage regimens in descending order of tumor size as measured by the longest diameter of the tumor surface area until: (i) the maximum amount of L19-IL2 and L19-TNFα as described above is injected; or (ii) all non-melanoma skin cancer tumors of the patient have been injected. In case (i), other smaller non-melanoma skin cancer tumors of the patient may not have been injected yet. In case (ii), the remaining dose volume can be discarded.

[0133] In patients with a single non-melanoma skin cancer tumor where the volume to be injected into the patient's tumor according to one of the above regimens is <100% of the total dose volume, the remaining dose volume may be discarded.

[0134] Thus, in patients with single or multiple non-melanoma skin cancer tumors, where the total volume injected intratumorally according to one of the above regimens (i.e., the sum of the volumes injected into each tumor of the patient) is < 100% of the total volume of the dose, the remaining dose is discarded. Thus, in some embodiments, the method comprises intratumorally injecting 25%, 37.5%, 50%, 62.5%, 75%, 87.5% or 100% of the maximum amount of L19-IL2 and L19-TNFα as described above.

[0135] Thus, in some embodiments, the method comprises injecting a dose of a composition comprising 0.25 mg to 2.2 mg L19-IL2 and 50 μg to 400 μg L19-TNFα.

[0136] In some preferred embodiments, the method comprises injecting a dose of a composition comprising 0.27 mg to 1.08 mg L19-IL2 and 50 μg to 200 μg L19-TNFα. For example, the dose may comprise: 0.27 mg L19-IL2 and 50 μg L19-TNFα; 0.41 mg L19-IL2 and 75 μg L19-TNFα; 0.54 mg L19-IL2 and 100 μg L19-TNFα; 0.68 mg L19-IL2 and 125 μg L19-TNFα; 0.81 mg L19-IL2 and 150 μg L19-TNFα; 0.95 mg L19-IL2 and 175 μg L19-TNFα; or 1.08 mg L19-IL2 and 200 μg L19-TNFα. In other words, the dose may contain 25%, 37.5%, 50%, 62.5%, 75%, 87.5% or 100% of the maximum amount of L19-IL2 and L19-TNFα, 1.08 mg L19-IL2 and 200 μg L19-TNFα.

[0137] In other preferred embodiments, the method comprises injecting a dose of a composition comprising 0.54 mg to 2.17 mg L19-IL2 and 100 μg to 400 μg L19-TNFα. For example, the dose may comprise: 0.54 mg L19-IL2 and 100 μg L19-TNFα; 0.81 mg L19-IL2 and 150 μg L19-TNFα; 1.08 mg L19-IL2 and 200 μg L19-TNFα; 1.36 mg L19-IL2 and 250 μg L19-TNFα; 1.63 mg L19-IL2 and 300 μg L19-TNFα; 1.90 mg L19-IL2 and 350 μg L19-TNFα; or 2.17 mg L19-IL2 and 400 μg L19-TNFα. In other words, the dose may contain 25%, 37.5%, 50%, 62.5%, 75%, 87.5% or 100% of the maximum amount of L19-IL2 and L19-TNFα, 2.17 mg L19-IL2 and 400 μg L19-TNFα.

[0138] In some embodiments, the method comprises intratumoral injection of an initial dose of the composition and one or more additional doses of the composition. Preferably, these doses are administered once a week. For example, the dose may be administered once a week for one to four weeks, such as one, two, three or four consecutive weeks. In a preferred embodiment, the dose is injected once a week for four weeks. In one embodiment, the dose is administered for no more than four weeks. The dose administered at each treatment point can be determined according to one of the above schemes.

[0139] In some embodiments, the method comprises intratumorally injecting an initial dose of a composition and one or more additional doses of a composition, and wherein the one or more additional doses comprise the same amount of L19-IL2 and / or L19-TNFα as the initial dose. For example, in the context of the above regimen, if the initial dose is determined based on the location and size (such as the longest diameter) of the tumor, one or more additional doses may comprise the same amount of L19-IL2 and / or L19-TNFα as the initial dose.

[0140] Alternatively, in some embodiments, the method comprises intratumorally injecting an initial dose of a composition and one or more additional doses of a composition, and wherein the one or more additional doses comprise more or less L19-IL2 and / or L19-TNFα than the initial dose. For example, in the context of the above regimen, if the initial dose is determined based on the location and size (such as the longest diameter) of the tumor, one or more additional doses may comprise more or less L19-IL2 and / or L19-TNFα than the initial dose, wherein the one or more additional doses administered do not exceed the maximum amount of L19-IL2 and L19-TNFα described above. For example, one or more additional doses may comprise twice or half the amount of L19-IL2 and / or L19-TNFα relative to the initial dose, wherein the one or more additional doses administered do not exceed the maximum amount of L19-IL2 and L19-TNFα described above.

[0141] In the case where the initial dose is well tolerated, a dose increase in subsequent administrations may be desirable. In some embodiments, the method includes intratumoral injection of an initial dose of the composition and one or more other doses, and wherein the one or more other doses include more L19-IL2 and / or L19-TNFα than the initial dose, wherein the one or more other doses administered do not exceed the maximum amount of L19-IL2 and L19-TNFα described above. Preferably, one or more other doses include twice the amount of L19-IL2 and / or L19-TNFα relative to the initial dose, wherein the one or more other doses administered do not exceed the maximum amount of L19-IL2 and L19-TNFα described above.

[0142] In the case of adverse reactions (such as inflammation) at the injection site, a reduction in dose may be desirable in subsequent administrations. In some embodiments, the method includes intratumoral injection of an initial dose of a composition and one or more other doses, and wherein the one or more other doses include less L19-IL2 and / or L19-TNFα than the initial dose. Preferably, the method includes intratumoral injection of an initial dose of a composition and one or more other doses, and wherein the one or more other doses include L19-IL2 and / or L19-TNFα relative to half the amount of the initial dose.

[0143] Some embodiments of the present invention relate to the use of a combination of IL2 and TNFα immunoconjugates formulated into a pharmaceutical composition. The pharmaceutical composition may include a pharmaceutically acceptable "excipient" composed of a material that is considered safe and effective. "Pharmaceutically acceptable" refers to those molecular entities and compositions that are "generally regarded as safe", for example, when administered to humans, they are physiologically tolerable and generally do not produce allergic or similar adverse reactions, such as stomach discomfort, etc. Excipients may include solvents, solubility enhancers, suspending agents, buffers, isotonic agents, antioxidants or antimicrobial preservatives. Therefore, the present invention also provides a pharmaceutical composition comprising a combination of L19-huTNFα and L19-huIL2 for use in a method of treating non-melanoma skin cancer in a patient, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0144] Reagent test kit

[0145] Another aspect of the present invention provides a therapeutic kit for use in treating a disease or condition as described herein, the therapeutic kit comprising a composition as described herein. The composition of the kit is preferably sterile and placed in a sealed vial or other container. In some embodiments, the therapeutic kit comprises a composition as described herein, wherein L19-TNFα and L19-IL2 are contained separately, for example, in separate sealed vials. In some alternative embodiments, the therapeutic kit comprises a composition as described herein, wherein L19-TNFα and L19-IL2 are contained together, for example, in the same sealed vial.

[0146] The kit may also include instructions for using the components in the methods described herein.The components of the kit may be contained or packaged in a container, such as a bag, box, can, tin or blister pack.

[0147] The features disclosed in the preceding description, or in the following claims or in the accompanying drawings, expressed in their specific form or as a means for performing the disclosed function, or as a method or process for obtaining the disclosed result, may be used alone or in any combination of these features, as the case may be, to realize the invention in multiple forms.

[0148] Although the present invention is described in conjunction with the above exemplary embodiments, after obtaining the present disclosure, many equivalent modifications and changes will be apparent to those skilled in the art. Therefore, the exemplary embodiments of the present invention described above are considered to be illustrative rather than restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the present invention.

[0149] To avoid any doubt, any theoretical explanations provided herein are intended to help readers better understand. The inventors do not wish to be bound by any of these theoretical explanations.

[0150] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0151] Throughout the specification (including the appended claims), unless the context requires otherwise, the words “comprise”, “include” and variations such as “comprises”, “comprising”, “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.

[0152] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. 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 use of the antecedent "about," it is understood that the particular value constitutes another embodiment. The word "about" with respect to a value is optional, for example meaning ±10%. Example

[0153] Example 1 - Intratumoral combination of L19-huIL2 and L19-huTNFα in BCC patients

[0154] 1.1 Experimental process

[0155] A 55-year-old man presented with an ulcerated, crusted, nonpigmented tumor measuring 27 × 25 mm on the left side of his nose, causing disfigurement similar to a saddle nose ( Figure 1 Histopathological examination of the needle biopsy specimens revealed solid and partially (approximately 30%) morphea-like basal cell carcinomas (BCCs) with basosquamous cell components ( Figure 3, left). Surgical resection of the tumor would result in further disfigurement of the patient's face. This BCC tumor was classified as stage IIIB as defined by the EADO classification system (Grobet al., 2021).

[0156] Patients received four weekly intratumoral injections of a mixture of 1.08 mg L19-huIL2 and 200 μg L19-huTNFα, each in a volume of 1 mL. In the second injection, the dose of L19-huIL2 was unexpectedly increased to 2.17 mg. This is not expected to have a substantial impact on the treatment outcome, and patients treated with the 1.08 mg L19-huIL2 dose throughout treatment are expected to have the same results.

[0157] For each injection, the total injection volume was spread throughout the tumor mass by performing approximately five to six injections at different sites in the tumor.

[0158] 1.2 Results

[0159] In response to treatment, the tumors decreased in size, gradually transformed into necrotic crusts, and then formed a crust that eventually fell off. In addition, a transient increase in edema of the lesions at the tumor site and the facial skin surrounding the lesions was observed. Two months after the first injection (day 63), the tumors showed a clinical complete response and a pathological complete response to treatment and were replaced by a fully epithelialized scar. At a follow-up visit four months after the first injection, the fully epithelialized scar was still present, indicating that the cancer had not recurred ( Figure 2 ). The pathological complete response was confirmed by histological analysis of tissue samples obtained from the primary tumor site. Specifically, histochemical staining of a needle biopsy sample obtained 63 days after the first injection showed the presence of residual inflammatory lymphohistiocytic infiltrates, some of which were granulomatous, but without basaloid or morphea-like epithelial tumor cells ( Figure 3 , right). At follow-up 6 months after the first injection, the patient showed no recurrence of the cancer.

[0160] Treatment was well tolerated. Patients experienced mild to moderate chills and elevated body temperatures up to 38.5°C, which lasted up to 36 hours after injection. To alleviate these cytokine-related symptoms, patients were premedicated with 1 g of midazolam (metamizole) one hour before injection, starting with the second treatment cycle. Locally, patients experienced transient facial edema that lasted three to four days after injection.

[0161] The results of intratumoral injection of the combination of L19-huIL2 and L19-huTNFα resulted in clinical and pathological complete responses in basal cell carcinoma (BCC) tumors, which was unexpected and demonstrated the surprising efficacy of this combination in the treatment of non-melanoma skin cancers.

[0162] Sequence Listing

[0163] SEQ ID NO: 1 - Amino acid sequence of L19 CDR1 VH

[0164] SFSMS

[0165] SEQ ID NO:2-L19 CDR2 VH amino acid sequence

[0166] SISGSSGTTYYADSVKG

[0167] SEQ ID NO:3-L19 CDR3 Amino acid sequence of VH

[0168] PFPYFDY

[0169] SEQ ID NO:4-L19 Amino acid sequence of CDR1 VL

[0170] RASQSVSSSFLA

[0171] SEQ ID NO:5-L19 CDR2 Amino acid sequence of VL

[0172] YASSRAT

[0173] SEQ ID NO:6-L19 Amino acid sequence of CDR3 VL

[0174] QQTGRIPPT

[0175] SEQ ID NO: 7-L19 Amino acid sequence of VH domain

[0176] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSS

[0177] SEQ ID NO:8—Amino acid sequence of the linker between VH and VL

[0178] GDGSSGGSGGAS

[0179] SEQ ID NO:9-L19 Amino acid sequence of the VL domain

[0180] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK

[0181] SEQ ID NO:10-L19 Amino acid sequence of scFv

[0182] The linker sequence is underlined.

[0183] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSS GDGSSGGSGGAS EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK

[0184] SEQ ID NO:11-Amino acid sequence of human interleukin 2 (huIL2)

[0185] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0186] SEQ ID NO:12—Amino acid sequence of the linker between L19 scFv and IL2

[0187] EFSSSSGSSSSGSSSSG

[0188] SEQ ID NO:13 - Amino acid sequence of L19-huIL2 conjugate

[0189] The linker sequence is underlined.

[0190] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSS GDGSSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK EFSSSSGSSSSGSSSSG APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0191] SEQ ID NO:14 - Amino acid sequence of the soluble form of the extracellular domain of human TNFα (huTNFα)

[0192] VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0193] SEQ ID NO:15-L19 Amino acid sequence of the linker between scFv and TNFα

[0194] EFSSSSGSSSSGSSSSG

[0195] SEQ ID NO: Amino acid sequence of 16-L19-huTNFα conjugate

[0196] The linker sequence is underlined.

[0197] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSS GDGSSGGSGGAS EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK EFSSSSGSSSSGSSSSGVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0198] References

[0199] In order to more fully describe and disclose the present invention and the prior art in the field to which the present invention belongs, some publications are cited above. The complete citation information of these publications is as follows.

[0200] Balza et al.,2010,Int.J.Cancer,127,101

[0201] Danielli et al.,2015,Cancer Immunol.Immunother.64,113-121

[0202] Grob et al., "Position Statement on classification of Basal cellcarcinomas.Part2: EADO proposal for new operational Staging System adapted to Basal cell carcinomas", J Eur Acad Dermatol Venereol, 2021.

[0203] Pasche&Neri,2012,Drug Discov.Today,17,583

[0204] Pons et al., 2022, "Observational Study of a Series of Basal cellcarcinomas: Evaluation of Location as a risk factor for recurrence", J StomatolOral Maxillofac Surg 123, 655-659

[0205] Schwager et al.,2013,J.Invest.Dermatol.133,751-758

[0206] Stratigos et al., Eur J Cancer, 2020.

[0207] Weide et al.,2011,Cancer-116,4139-4146

[0208] Weide et al.,2014,Cancer Immunol.Immunother.2,668-678

[0209] For standard molecular biology techniques, see Sambrook, J., Russel, DW Molecular Cloning, A Laboratory Manual. 3ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

Claims

1. A composition comprising a combination of L19-huTNFα and L19-huIL2 for use in a method of treating non-melanoma skin cancer in a human patient, wherein the L19-huTNFα comprises or consists of the sequence shown in SEQ ID NO: 16, and the L19-huIL2 comprises or consists of the sequence shown in SEQ ID NO: 13, and The method comprises intratumoral injection of the composition.

2. The composition for use according to claim 1, wherein the non-melanoma skin cancer is basal cell carcinoma (BCC).

3. The composition for use of claim 2, wherein the BCC is stage IIA, IIB, IIIA or IIIB BCC as defined by the EADO classification system.

4. The composition for use of claim 3, wherein the BCC is stage IIIB BCC as defined according to the EADO classification system.

5. The composition for use of claim 1, wherein the non-melanoma skin cancer is cutaneous squamous cell carcinoma (cSCC).

6. The composition for use of claim 1, wherein the non-melanoma skin cancer is selected from the group consisting of Merkel cell carcinoma (MCC), malignant adnexal tumor of the skin (MATS), tumors from cutaneous T-cell lymphoma (CTCL), Kaposi's sarcoma (KS), and keratoacanthoma (KA).

7. A composition for use according to any one of the preceding claims, wherein the method comprises injecting a dose of the composition comprising: (i) a maximum amount of 1.08 mg of L19-huIL2 and a maximum amount of 200 μg of L19-huTNFα; or (ii) A maximum amount of 2.17 mg of L19-huIL2 and a maximum amount of 400 μg of L19-huTNFα.

8. The composition for use according to claim 7, wherein the dosage comprises: (i) L19-huIL2 in an amount of 0.27 mg to 1.08 mg and L19-huTNFα in an amount of 50 μg to 200 μg; or (ii) L19-huIL2 in an amount ranging from 0.54 mg to 2.17 mg and L19-huTNFα in an amount ranging from 100 μg to 400 μg.

9. The composition for use according to any one of claims 1 to 8, wherein the dose comprises 1.08 mg L19-huIL2 and 200 μg L19-huTNFα.

10. The composition for use according to any one of claims 1 to 8, wherein the dose comprises 2.17 mg L19-huIL2 and 400 μg L19-huTNFα.

11. The composition for use of claim 7, wherein the method comprises injecting into a non-melanoma skin cancer tumor located on the nose or in the peri-orificial area of ​​the head of the patient, the method comprising injecting a dose of the composition comprising: (a) 25% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii), when the longest diameter of the surface area of ​​the tumor is >1 cm and ≤2 cm; (b) 37.5% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii), when the longest diameter of the surface area of ​​the tumor is >2 cm and ≤3 cm; (c) 50% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii), when the longest diameter of the surface area of ​​the tumor is >3 cm and ≤4 cm; (d) 62.5% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii), when the longest diameter of the surface area of ​​the tumor is >4 cm and ≤5 cm; or (e) 75% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii) when the longest diameter of the surface area of ​​the tumor is >5 cm.

12. The composition for use of claim 7, wherein the method comprises injecting into a non-melanoma skin cancer tumor located in the peri-orificial area of ​​the nose or head of the patient, the method comprising injecting a dose of the composition comprising: (a) 25% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii), when the longest diameter of the surface area of ​​the tumor is >1 cm and ≤2 cm; (b) 37.5% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii), when the longest diameter of the surface area of ​​the tumor is >2 cm and ≤3 cm; (c) 50% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii), when the longest diameter of the surface area of ​​the tumor is >3 cm and ≤5 cm; or (d) 62.5% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii) when the longest diameter of the surface area of ​​the tumor is >5 cm.

13. The composition for use of claim 7, wherein the method comprises injecting into a non-melanoma skin cancer tumor in a patient's body other than on the nose or in the area around the orifice of the head, the method comprising injecting a dose of the composition comprising: (a) 37.5% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii), when the longest diameter of the surface area of ​​the tumor is >1 cm and ≤2 cm; (b) 50% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii), when the longest diameter of the surface area of ​​the tumor is >2 cm and ≤3 cm; (c) 62.5% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii), when the longest diameter of the surface area of ​​the tumor is >3 cm and ≤4 cm; (d) 75% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii), when the longest diameter of the surface area of ​​the tumor is >4 cm and ≤5 cm; or (e) 100% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii), when the longest diameter of the surface area of ​​the tumor is >5 cm; Optionally, wherein the non-melanoma skin cancer tumor is located on an area of ​​the patient's head other than on the nose or in the area around the orifice, optionally wherein the non-melanoma skin cancer tumor is located on the forehead, cheek, chin or scalp of the patient.

14. The composition for use of claim 7, wherein the method comprises injecting into a non-melanoma skin cancer tumor in a patient's body other than on the nose or in the area around the orifice of the head, the method comprising injecting a dose of the composition comprising: (a) 37.5% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii), when the longest diameter of the surface area of ​​the tumor is >1 cm and ≤2 cm; (b) 50% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii), when the longest diameter of the surface area of ​​the tumor is >2 cm and ≤3 cm; (c) 75% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii) when the longest diameter of the tumor surface area is >3 cm and ≤5 cm; or (d) 100% of the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii), when the longest diameter of the tumor surface area is >5 cm; Optionally, wherein the non-melanoma skin cancer tumor is located in an area of ​​the patient's head other than on the nose or in the area around the orifice, optionally wherein the non-melanoma skin cancer tumor is located on the forehead, cheek, chin or scalp of the patient.

15. A composition for use according to any one of claims 11 to 14, wherein the patient has a plurality of non-melanoma skin cancer tumors, wherein the method comprises injecting a dose of a composition as described in parts (a) to (e) of claims 11 or 13, or parts (a) to (d) of claims 12 or 14, into each tumor in descending order of tumor size as measured by the longest diameter of the tumor surface area, until: (i) the maximum amount of L19-huIL2 and L19-huTNFα as described in claim 7(i) or claim 7(ii) has been injected; or (ii) All of the patient's non-melanoma skin cancer tumors have been injected.

16. The composition for use of any one of the preceding claims, wherein the method comprises injecting more than one dose of the composition into a non-melanoma skin cancer tumor, and wherein the doses are administered to the patient once a week.

17. The composition for use of claim 16, wherein the method comprises intratumorally injecting an initial dose of the composition and one or more further doses of the composition, and wherein the one or more further doses comprise more or less L19-huIL2 and / or L19-huTNFα than the initial dose.

18. A composition for use according to claim 17, wherein one or more further doses comprises twice or half the amount of L19-huIL2 and / or L19-huTNFα relative to the initial dose, wherein the one or more further doses administered do not exceed the maximum amount of L19-IL2 and L19-TNFα described in claim 7(i) or claim 7(ii).

19. A composition for use according to any one of the preceding claims, wherein the method is neoadjuvant therapy.

20. The composition for use according to any one of the preceding claims, wherein the method reduces the size of a non-melanoma skin cancer tumor, such as tumor volume and / or visible tumor area.

21. The composition for use of any one of the preceding claims, wherein the non-melanoma skin cancer tumor exhibits a clinical complete response to treatment.

22. The composition for use of any one of the preceding claims, wherein the non-melanoma skin cancer tumor exhibits a pathological complete response to treatment.

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