Effect of CTX-CNF1 on the switch of macrophages towards a pro- inflammatory phenotype

Abstract

The invention concerns a chimeric protein CTX-CNF1, or a pharmaceutical composition comprising it, for use in stimulating the immune system in immunogenic and non- immunogenic tumors. A pharmaceutical kit comprising the chimeric protein CTX-CNF1 and uses thereof are also described.

Description

[0001] EFFECT OF CTX-CNF1 ON THE SWITCH OF MACROPHAGES TOWARDS A PRO- INFLAMMATORY PHENOTYPE

[0002] FIELD OF THE INVENTION

[0003] The invention concerns a chimeric protein CTX-CNF1 , or a pharmaceutical composition comprising it, for use in stimulating the immune system in immunogenic and non-immunogenic tumors. A pharmaceutical kit comprising the chimeric protein CTX-CNF1 and uses thereof are also described.

[0004] STATE OF THE ART

[0005] In recent years, cancer research has made great strides, particularly in understanding the differences between immunogenic and non-immunogenic tumors. These two groups of tumors have distinctive characteristics that influence treatment strategies and response to therapies.

[0006] Immunogenic tumors, often referred to as “hot tumors,” are characterized by a strong activation of the immune system.

[0007] These tumors, such as melanoma or some types of lung cancer, tend to have a high mutational burden. This means they produce many neoantigens, i.e. altered proteins that the immune system recognizes as foreign and potentially dangerous. The presence of these neoantigens increases the likelihood that the body will respond positively to treatments such as immune checkpoint inhibitors. Drugs such as pembrolizumab and nivolumab have been shown to be effective in patients with these types of tumors, leading to significant clinical responses.

[0008] However, some tumors can develop mechanisms to evade recognition by the immune system. This phenomenon can reduce the effectiveness of immunological therapies. On the other hand, non-immunogenic tumors, or “cold tumors,” present a completely different picture. These tumors, such as glioblastoma multiforme (GBM), glioma, pancreatic cancer, or some types of bladder cancer, have a very low mutational burden and exhibit a limited immune response. This makes it difficult for the immune system to recognize and attack tumor cells.

[0009] As a result, traditional immunological therapies tend to be less effective. However, their genetic stability can make the response to traditional treatments, such as chemotherapy, more predictable. However, the main issue remains resistance to immunological treatments, which can lead to higher mortality rates.

[0010] Currently, there are several therapeutic approaches to treat immunogenic and non-immunogenic tumors. The main ones are:

[0011] 1. Immunological therapies: development of new immune checkpoint inhibitors and cell therapies such as CAR-T cells;

[0012] 2. Combinatorial therapies: combinations of treatments such as immunotherapy along with chemotherapy or radiotherapy;

[0013] 3. Targeting tumor microenvironments: modifying the tumor microenvironment to promote an immune response also to cold tumors.

[0014] A promising direction is represented by integration of targeted and immunological therapies, tailoring approaches based on the tumor immunogenic profile.

[0015] Recent studies have shown that the bacterial protein CNF1 (cytotoxic necrotizing factor 1 ) can reduce the tumor mass and protect the peritumoral tissue from tissue dysfunction; however, for example, it is unable to cross the blood-brain barrier (BBB), which could hinder its translationality to all tumor types.

[0016] The aim of the present invention is therefore to provide a chimeric protein capable of treating both immunogenic and non-immunogenic tumors and possibly crossing the BBB to “awaken” the immune system, which is generally inactive in cold tumors and poorly effective in warm tumors.

[0017] SUMMARY OF THE INVENTION

[0018] The lack of effective therapies, together with a growing number of cases, confirms that cancer treatment is a rapidly expanding field of research.

[0019] Of all cancers, Glioblastoma Multiforme (GBM) represents a huge risk to public health. Its development is rapid and destructive, resulting in extremely severe and disabling symptoms, making GBM a significant social and economic burden on national healthcare systems. The current standard of care is ineffective, and the survival rate for GBM patients is of approximately 15 months after diagnosis.

[0020] Therefore, there is an urgent need to find effective therapeutic approaches aimed at counteracting the progression of GBM and, more generally, of immunogenic and non-immunogenic tumors, to improve patients’ quality of life and increase their life expectancy.

[0021] In a first aspect, the invention therefore concerns a pharmaceutical kit comprising: - a chimeric protein CTX-CNF1 ; and

[0022] - a checkpoint inhibitor, selected from the group consisting of anti-PD-1 , anti-PDL-1 , anti-CTLA-4, anti-TIGET, anti-LAG 3, and anti-TIM-3,

[0023] for simultaneous, separate, or sequential use, wherein the chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

[0024] In a second aspect, the invention describes a pharmaceutical kit for use as a medicament comprising:

[0025] - a chimeric protein CTX-CNF1 ; and

[0026] - a checkpoint inhibitor, selected from the group consisting of anti-PD-1 , anti-PDL-1 , anti-CTLA-4, anti-TIGET, anti-LAG 3, and anti-TIM-3,

[0027] for simultaneous, separate, or sequential use, wherein the chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

[0028] In a third aspect, the present invention relates to a pharmaceutical kit for use in the treatment of cancer comprising:

[0029] - a chimeric protein CTX-CNF1 ; and

[0030] - a checkpoint inhibitor, selected from the group consisting of anti-PD-1 , anti-PDL-1 , anti-CTLA-4, anti-TIGET, anti-LAG 3, and anti-TIM-3,

[0031] for simultaneous, separate, or sequential use, wherein the chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

[0032] In a fourth aspect, the invention concerns the use of a chimeric protein CTX-CNF1 in the treatment of cancer by stimulating immune system cells, wherein the chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

[0033] In a fifth aspect, a pharmaceutical composition comprising a chimeric protein CTX-CNF1 and a pharmaceutically acceptable excipient for use in the treatment of cancer by stimulating immune system cells, wherein the chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1 , is described herein.

[0034] In the present invention, when the term “chimeric protein CTX-CNF1” or “CTX-CNF1” is used, it is intended to comprise a peptide of 36 amino acids (CTX - chlorotoxin) fused with the bacterial protein CNF1 (cytotoxic necrotizing factor 1, of about 1,014 amino acids) to form a chimeric protein of about 1,050 amino acids (SEQ ID NO: 1).

[0035] In a sixth aspect, the invention relates to a pharmaceutical kit for use in the treatment of recurrent cancer, comprising:

[0036] - a chimeric protein CTX-CNF1 ; and

[0037] - a checkpoint inhibitor, selected from the group consisting of anti-PD-1 , anti-PDL-1 , anti-CTLA-4, anti-TIGET, anti-LAG 3, and anti-TIM-3,

[0038] for simultaneous, separate, or sequential use, wherein the chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

[0039] The dependent claims describe particular embodiments of the invention.

[0040] DESCRIPTION OF THE FIGURES

[0041] The invention will now be described in detail, and with reference to the attached Figures.

[0042] Figure 1: In vivo experiments in mice with GL261 glioma. A) Experimental protocol used. B) GL261 mice treated with CTX-CNF1 showed restoration of motor function compared to untreated mice. C) MRI images of untreated (above) and CTX-CNF1 (below) GL261 mice. It is noted that the tumor mass reduces in size with treatment. D) MRI quantification and survival analysis of untreated (light gray) and CTX-CNF1 -treated (dark gray) GL261 mice. Treated mice show a significant reduction in tumor mass and increased survival. E) MRI image of a mouse surviving 180 days after tumor induction. Note that in this case the tumor mass is absent. F) CTX-CNF1 -treated mice that fully recovered from GBM were less susceptible to developing another GBM.

[0043] Figure 2: In vivo experiments in CT-2A mice. A) Experimental protocol used. MRI quantification and survival analysis of untreated (light gray) and CTX-CNF1 -treated (dark gray) CT-2A mice. Treated mice showed a significant reduction in tumor mass and increased survival.

[0044] B) CT-2A mice treated with CTX-CNF1 showed improved motor function compared to untreated mice.

[0045] Figure 3: Effect of the chimeric protein CTX-CNF1 in GBM mice. A) Number of GFAP and F4 / 80 cells in untreated (left column) and CTX-CNF1 -treated (right column) GL261 mice. Treated mice show a reduction in F4 / 80+ cells in the peritumoral tissue. B) Number of CD4+ and CD8+ cells in untreated (left column) and CTX-CNF1 -treated (right column) GL261 mice. Treated mice show an increase in CD8+ cells. C) Number of granzyme B- and perforin-positive CD8+ cells in untreated (left column) and CTX-CNF1-treated (right column) GL261 mice. Treated mice show an increase in these markers. D, E) Example images of granzyme B (D) and perforin (E) in CTX-CNF1 -untreated GL261 mice (left) and in untreated GL261 mice (right). F) CD8 depletion blocks the efficacy of CTX-CNF1 in GL261 mice.

[0046] Figure 4: Safety of the chimeric protein CTX-CNF1 in GBM mice. Mice weight and internal organ weights (kidneys, spleen, thymus, and liver) in untreated (left column) and CTX-CNF1 -treated GBM GL261 mice (middle column: acutely, i.e., 48 hours after administration of the recombinant protein; right column: chronically, i.e., 48 hours after three treatments of the recombinant protein administration). There were no significant changes either in the weight of the mice or their internal organs.

[0047] Figure 5: Effect of CTX-CNF1 on macrophages in vivo. A, B, C) Immunofluorescence analyses show a significant decrease in arginase-1 (A) and a significant increase in Cd11 c (B, C) in glioma-bearing animals treated with CTX-CNF1. This indicates that the macrophages are in the M1 state(proinflammatory phenotype).

[0048] Figure 6: Effect of CTX-CNF1 on murine macrophages. A) CTX-CNF1 increases pro-inflammatory cytokines in MO BMDMs (bone marrow-derived macrophages). (B, C) CTX-CNF1 influences cytokines in M1 (B) and M2 (C) BMDMs. (D, E) CTX-CNF1 shifts BMDMs towards a pro-inflammatory phenotype (D: ELISA analysis; E: mRNA analysis, confirming the cytokine results in A).

[0049] Figure 7: Effect of CTX-CNF1 on human macrophages. The shift towards pro-inflammatory macrophages is also confirmed in humans. A) MO macrophages; B) M1 macrophages; C) M2 macrophages.

[0050] Figure 8: Combined administration of CTX-CNF1 and anti-PD-1. Above, experimental protocol. Below, survival analysis. It is noted that the combined administration of intravenous CTX-CNF1 and intraperitoneal anti-PD-1 results in a significant increase in survival (vs. vehicle / untreated mice and vs. CTX-CNF1 alone).

[0051] Figure 9: Effect of CTX-CNF1 on different tumoral cell lines. Clonogenic assay on (A) B16-F10 murine melanoma cells, (B) MIA PaCa-2 human pancreatic cells, (C, D) GL261 and CT-2A murine glioma cells, and (E) SF8628 human pediatric glioma cells. (F) Trypan blue assay on SF8628 human pediatric glioma cells.

[0052] Figure 10: Survival (%) of mice subjected to tumor re-challenge with GL261 cells after initial treatment. The curve shows the cumulative survival percentage starting from the day of tumor reinoculation (day 0). The dotted-dashed line corresponds to animals previously treated with anti PD-1 + CTX-CNF1, the dashed line identifies animals previously treated with CTX-CNF1 and the solid line indicates age-matched naive controls (without previous tumor, then treated with vehicle solution).

[0053] Art. 170-bis, paragraphs 2, 3 and 4 of the Italian Industrial Property Code (IPC) With regard to the provisions of Art. 170-bis, paragraphs 2, 3 and 4 IPC, please note the following: the origin of the biological material used in the present patent application is specified in the application itself, and the handling of said biological material has been carried out in compliance with the obligations arising from national and Ell regulations.

[0054] DETAILED DESCRIPTION OF THE INVENTION

[0055] For the purpose of the present invention, a chimeric protein CTX-CNF1 was developed, conjugating the protein CNF1 with antitumor capacity, and the ability to stimulate the immune system towards a pro-inflammatory phenotype, with chlorotoxin (CTX), a protein capable of crossing the BBB when necessary and selectively binding to tumor cells. This chimeric protein has proven to be a surprising activator of the pro-inflammatory response.

[0056] The chimeric CTX-CNF1 protein has the following structure: chlorotoxin is localized at the N-terminal end of the recombinant molecule and is followed by CNF1 , consisting of its three domains (binding domain, translocation domain, and catalytic domain) (Vannini E. et al. “CTX-CNF1 Recombinant Protein Selectively Targets Glioma Cells In vivo." Toxins 2021 Mar 8; 13(3): 194.).

[0057] Amino acid sequence of the chimeric protein CTX-CNF1 (SEQ ID NO:1):

[0058] MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCRWDPMGNQWQQKYLLEYN ELVSNFPSPERVVSDYIKNCFKTDLPWFSRIDPDNAYFICFSQNRSNSRSYTGWDHL GKYKTEVLTLTQAALINIGYRFDVFDDANSSTGIYKTKSADVFNEENEEKMLPSEYLHF LQKCDFAGVYGKTLSDYWSKYYDKFKLLLKNYYISSALYLYKNGELDEREYNFSMNA LNRSDNISLLFFDIYGYYASDIFVAKNNDKVMLFIPGAKKPFLFKKNIADLRLTLKELIKD SDKQQLLSQHFSLYSRQDGVSYAGVNSVLHAIENDGNFNESYFLYSNKTLSNKDVFD AIAISVKKRSFSDGDIVIKSNSEAQRDYALTILQTILSMTPIFDIWPEVSVPLGLGIITSS MGISFDQLINGDTYEERRSAIPGLATNAVLLGLSFAIPLLISKAGINQEVLSSVINNEGR TLNETNIDIFLKEYGIAEDSISSTNLLDVKLKSSGQHVNIVKLSDEDNQIVAVKGSSLSGI YYEVDIETGYEILSRRIYRTEYNNEILWTRGGGLKGGQPFDFESLNIPVFFKDEPYSAV TGSPLSFINDDSSLLYPDTNPKLPQPTSEMDIVNYVKGSGSFGDRFVTLMRGATEEE AWNIASYHTAGGSTEELHEILLGQGPQSSLGFTEYTSNVNSADAASRRHFLWIKVHV KYITNNNVSYVNHWAIPDEAPVEVLAVVDRRFNFPEPSTPPDISTIRKLLSLRYFKESIE STSKSNFQKLSRGNIDVLKGRGSISSTRQRAIYPYFEAANADEQQPLFFYIKKDRFDN HGYDQYFYDNTVGLNGIPTLNTYTGEIPSDSSSLGSTYWKKYNLTNETSIIRVSNSAR GANGIKIALEEVQEGKPVIITSGNLSGCTTIVARKEGYIYKVHTGTTKSLAGFTSTTGVK KAVEVLELLTKEPIPRVEGIMSNDFLVDYLSENFEDSLITYSSSEKKPDSQITIIRDNVSV FPYFLDNIPEHGFGTSATVLVRVDGNWVRSLSESYSLNADASEISVLKVFSKKF

[0059] The protein CNF1 triggers long-lasting activation of intracellular Rho GTPases, leading to multinucleation, senescence, and ultimately death of both murine and human glioma cells. When administered in vivo, CNF1 increases the survival of glioma-bearing mice and improves neuronal function and plasticity, sparing neuronal responses in the peritumoral areas. Despite promising results obtained in preclinical models, the potential use of CNF1 in the clinic is limited by its inability to cross the blood-brain barrier (BBB). To overcome this limitation, a novel chimeric protein was developed by fusing chlorotoxin (CTX) with CNF1. CTX, a 36-amino acid peptide derived from the venom of the scorpion Leiurus quinquestriatus, is able to cross the BBB and recognize and target glioma cells.

[0060] Surprisingly, the combined administration of CTX-CNF1 and anti-PD-1 (an immune checkpoint inhibitor) was observed to have a synergistic effect on the survival of tumor-affected animals.

[0061] In a first aspect, the invention relates to a pharmaceutical kit comprising:

[0062] - a chimeric protein CTX-CNF1 ; and - a checkpoint inhibitor, selected from the group consisting of anti-PD-1 , anti-PDL-1 , anti-CTLA-4, anti-TIGET, anti-LAG-3, and anti-TIM-3,

[0063] for simultaneous, separate, or sequential use, wherein said chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

[0064] In a second aspect, the invention describes a pharmaceutical kit for use as a medicament comprising:

[0065] - a chimeric protein CTX-CNF1 ; and

[0066] - a checkpoint inhibitor, selected from the group consisting of anti-PD-1 , anti-PDL-1 , anti-CTLA-4, anti-TIGET, anti-LAG-3, and anti-TIM-3,

[0067] for simultaneous, separate, or sequential use, wherein said chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

[0068] In a third aspect, the present invention relates to a pharmaceutical kit for use in the treatment of cancer comprising:

[0069] - a chimeric protein CTX-CNF1 ; and

[0070] - a checkpoint inhibitor, selected from the group consisting of anti-PD-1 , anti-PDL-1 , anti-CTLA-4, anti-TIGET, anti-LAG-3, and anti-TIM-3,

[0071] for simultaneous, separate, or sequential use, wherein said chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

[0072] Overall, the results described herein highlight that CTX-CNF1 could revolutionize the treatment of immunogenic and non-immunogenic tumors, as this protein is able to ‘awaken’ the immune system, which is generally inactive in this type of tumor.

[0073] In one embodiment, in the use of the pharmaceutical kit described herein, the cancer is selected from the group consisting of glioblastoma multiforme, melanoma, glioma, pancreatic cancer, lung cancer, non-small cell lung cancer (NSCLC), kidney cancer, Hodgkin’s and non-Hodgkin’s lymphomas, squamous cell carcinoma of the head and neck (SCCHN), urothelial cancer, bladder and urinary tract cancer, pleural mesothelioma, colon cancer, rectal cancer, esophageal cancer and esophageal squamous cell carcinoma, gastric adenocarcinoma, gastroesophageal adenocarcinoma, and esophageal adenocarcinoma.

[0074] Preferably, the cancer is selected from the group consisting of glioblastoma multiforme, melanoma, glioma, and pancreatic cancer. CTX-CNF1 could be used in various tumor types and could be administered in combination with dietary supplements.

[0075] It could also be administered in conjunction with radiotherapy or immune checkpoint inhibitors, given the synergistic action of the chimeric protein of the present invention with the anti-PDL-1 antibody, which results in increased survival in GBM mice.

[0076] Testing of the chimeric protein CTX-CNF1 as a potential therapeutic agent, alone or in combination with the anti-PDL-1 antibody, on other tumoral cell lines (melanoma, pancreatic cancer, two glioma lines, and one pediatric glioma line) has surprisingly shown that it is capable of blocking the proliferation and survival of all these tumoral cell lines. After treatment with CTX-CNF1, as shown in the examples, these tumoral cell lines appear multinucleated - this is a sign that CNF1 has entered their cytoplasm, blocking their cytokinesis.

[0077] The combined treatment of the chimeric protein CTX-CNF1 with the anti-PDL-1 antibody showed an enhanced effect, improving the result obtained with treatment with anti-PDL-1 alone.

[0078] In general, CTX-based bioconjugates are currently used clinically as tumor dyes and drug carriers.

[0079] In the present invention, the chimera CTX-CNF1 was developed to cross the BBB and selectively target glioma cells.

[0080] The significant increase in survival and progressive reduction in tumor volume in GBM-affected mice following systemic administration of the chimeric protein CTX-CNF1 in a clinically relevant therapeutic window ( / .e., after GBM diagnosis by MRI) demonstrates the high translational value of the chimera of the present invention.

[0081] In a fourth aspect, the invention concerns the use of a chimeric protein CTX-CNF1 in the treatment of cancer by stimulating the immune system cells, wherein the chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

[0082] In one embodiment, the cancer is selected from the group consisting of glioblastoma multiforme, melanoma, glioma, pancreatic cancer, lung cancer, non-small cell lung cancer (NSCLC), kidney cancer, Hodgkin’s and non-Hodgkin’s lymphomas, squamous cell carcinoma of the head and neck (SCCHN), urothelial cancer, bladder and urinary tract cancer, pleural mesothelioma, colon cancer, rectal cancer, esophageal cancer and esophageal squamous cell carcinoma, gastric adenocarcinoma, gastroesophageal adenocarcinoma, and esophageal adenocarcinoma.

[0083] In a preferred embodiment, the cancer is selected from the group consisting of melanoma, pancreatic cancer, lung cancer, non-small cell lung cancer (NSCLC), kidney cancer, Hodgkin’s and non-Hodgkin’s lymphomas, squamous cell carcinoma of the head and neck (SCCHN), urothelial cancer, bladder and urinary tract cancer, pleural mesothelioma, colon cancer, rectal cancer, esophageal cancer and esophageal squamous cell carcinoma, gastric adenocarcinoma, gastroesophageal adenocarcinoma, and esophageal adenocarcinoma.

[0084] In another preferred embodiment, the cancer to be treated is selected from the group consisting of glioblastoma multiforme, melanoma, glioma, and pancreatic cancer.

[0085] Remarkably, 50% of CTX-CNF1 -treated mice are still alive 180 days after tumor induction (as compared to untreated mice, which have a median survival of 26 days after induction) and are less susceptible to developing another GBM than animals of similar age, suggesting the possibility that some sort of immune memory has been established in them.

[0086] In a preferred embodiment, the protein for use according to the invention acts on immune system cells selected from the group consisting of macrophages and T lymphocytes.

[0087] Specifically, in a preferred embodiment, the stimulation increases the number and functionality of T lymphocytes and is responsible for the switch of macrophages towards a proinflammatory phenotype.

[0088] Preferably, the immune system cells are peritumoral tissue cells.

[0089] In fact, although GBM is typically poorly immunogenic (and for this reason is also referred to as “cold”), significantly increased number and functionality of killer T lymphocyte cells were found in the peritumoral tissue of GBM animals treated with CTX-CNF1.

[0090] Furthermore, without being tied to any theory, the protective effect observed after CTX-CNF1 administration is caused not only by tumoral cell death, but also by an increased antitumoral inflammatory response in the brain. Specifically, CTX-CNF1 is capable of shifting the macrophage phenotype toward a proinflammatory state. The macrophage population represents a major component of the microenvironment and plays a key role in tumor resistance and progression mechanisms.

[0091] Specifically, tumoral cells have the ability to recruit circulating monocytes and macrophages from the microenvironment which result to be polarized towards a M2 phenotype (the so-called Tumor Associated Macrophages - TAMs), which are involved in cancer progression, immunosuppression, and metastasis.

[0092] The present invention fits into this context and is fully aligned with pharmacological strategies aimed at modulating the microenvironment through a process of macrophage polarization, from M2 (anti-inflammatory and pro-tumor phenotype) towards M1 state (pro-inflammatory and anti-tumor phenotype).

[0093] The experimental treatment surprisingly showed the potential to act by increasing the anti-tumor immune response through a significant modulation of the function of the macrophage component.

[0094] This aspect is of significant importance for developing the treatment in line with international research trends aimed at identifying and developing products capable of reducing anti-cancer drug resistance to immunotherapy drugs, enhancing the immune response, and acting synergistically with immunotherapy drugs.

[0095] In a fifth aspect, a pharmaceutical composition comprising a chimeric protein CTX-CNF1 and a pharmaceutically acceptable excipient is described herein for use in the treatment of cancer by stimulating the immune system cells, wherein the chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

[0096] In one embodiment, said cancer is selected from the group consisting of glioblastoma multiforme, melanoma, glioma, pancreatic cancer, lung cancer, non-small cell lung cancer (NSCLC), kidney cancer, Hodgkin’s and non-Hodgkin’s lymphomas, squamous cell carcinoma of the head and neck (SCCHN), urothelial cancer, bladder and urinary tract cancer, pleural mesothelioma, colon cancer, rectal cancer, esophageal cancer and esophageal squamous cell carcinoma, gastric adenocarcinoma, gastroesophageal adenocarcinoma, and esophageal adenocarcinoma, more preferably the cancer to be treated is selected from the group consisting of glioblastoma multiforme, melanoma, glioma, and pancreatic cancer.

[0097] In one embodiment, the pharmaceutical composition for use according to the invention has an effect on immune system cells selected from the group consisting of macrophages and T lymphocytes.

[0098] Specifically, in a preferred form, the stimulation of the immune system increases the number and functionality of killer T lymphocytes and is responsible for the switch of macrophages towards a pro-inflammatory phenotype.

[0099] Preferably, the immune system cells are peritumoral tissue cells.

[0100] The chimeric protein CTX-CNF1 , in fact, activates the immune system, which is usually inactive in cold tumors, particularly in GBM.

[0101] It is believed that a non-active immune system is, in fact, pro-tumor and one of the causes of the lack of efficacy of GBM therapies.

[0102] The fact that CTX-CNF1 activates T cells and causes macrophages to switch phenotype towards a pro-inflammatory (and therefore anti-tumor) phenotype makes the protein described herein fundamental in therapeutic strategies for treating tumors wherein the immune system is silent.

[0103] Specifically, in GBM peritumoral tissue, the treatment with the chimeric protein CTX-CNF1 resulted in killer T lymphocytes’ numerical and functional increase and the stimulation towards a macrophages’ pro-inflammatory phenotype.

[0104] In a sixth aspect, the invention relates to a pharmaceutical kit for use in the treatment of recurrent cancer comprising:

[0105] - a chimeric protein CTX-CNF1 ; and

[0106] - a checkpoint inhibitor, selected from the group consisting of anti-PD-1 , anti-PDL-1 , anti-CTLA-4, anti-TIGET, anti-LAG-3, and anti-TIM-3,

[0107] for simultaneous, separate, or sequential use, wherein said chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

[0108] In the present invention, when the definition “recurrent cancer” is used, it is intended to comprise forms of neoplasm that recur after a period of remission following an initial treatment deemed effective (surgery, chemotherapy, radiotherapy, or targeted therapies). Recurrence may occur at the same site as the original tumor (local recurrence), in surrounding tissues (regional recurrence), or in distant organs (metastatic recurrence).

[0109] In a preferred embodiment, in the use of the pharmaceutical kit described herein, the recurrent cancer is selected from the group consisting of glioblastoma multiforme, melanoma, glioma, pancreatic cancer, lung cancer, non-small cell lung cancer (NSCLC), kidney cancer, Hodgkin and non-Hodgkin lymphomas, squamous cell carcinoma of the head and neck (SCCHN), urothelial cancer, bladder and urinary tract cancer, pleural mesothelioma, colon cancer, rectal cancer, esophageal cancer and esophageal squamous cell carcinoma, gastric adenocarcinoma, gastroesophageal adenocarcinoma, and esophageal adenocarcinoma.

[0110] Surprisingly, animals re-inoculated with GL261 cells (re-challenge) following treatment with anti-PD-1 in combination with the chimeric protein CTX-CNF1 did not show any lethal events until >140 days after re-challenge, indicating the development of protective immune memory. Furthermore, animals treated with the chimeric protein CTX-CNF1 alone showed prolonged survival, with a progressive reduction in the number of surviving animals until day ~110. In the experimental part, it will be even more apparent how surprisingly the protein CTX-CNF1 :

[0111] i) Has a synergistic effect when administered with anti-PD-1 ;

[0112] ii) Allows to increase the number and function of killer T cells;

[0113] iii) Is responsible for the switch of macrophages towards a pro-inflammatory phenotype;

[0114] iv) Allows for a reduction in tumor mass and maintenance of motor function, a reduced susceptibility to cancer recurrence, and a significant increase in survival in cancer-affected animals; and

[0115] v) Promotes the development of protective immune memory.

[0116] These findings are of fundamental importance because they are highly relevant for managing the shaping of the immune system towards inflammation in immunogenic and non-immunogenic tumors.

[0117] EXAMPLES

[0118] Example 1 : the chimeric protein CTX-CNF1 increases survival and reduces tumor mass in GL261 mice.

[0119] A therapeutic protocol was developed in which treatment with CTX-CNF1 or inactivated CTX-CNF1 (vehicle) was initiated after the diagnosis of glioblastoma multiforme (GBM), which was performed 12 days after tumor induction by MRI. After GBM diagnosis by MRI, the mice were randomly divided into two groups: one was treated with CTX-CNF1 , while the other received the vehicle solution. CTX-CNF1 (n = 11) or inactivated CTX-CNF1 (vehicle, n = 12) were administered intravenously via the tail vein (20 pL, 80 nM). The treatment / vehicle administration was performed once a week for three weeks. It is worth noting that this administration method resembles traditional chemotherapy. Animal survival was assessed along with motor function (monitored longitudinally by specific motor tests) and GBM growth (monitored longitudinally by MRI scans).

[0120] CTX-CNF1 was shown to be effective in prolonging the lifespan of mice bearing GL261 glioma; surprisingly, 50% of GL261 animals treated with CTX-CNF1 survived for as long as 180 days after tumor induction (vs. the mean survival of GL261 vehicle mice, which is 26 days after tumor induction). Consistently, longitudinal MRI acquisitions showed that CTX-CNF1 treatment progressively reduced tumor mass in mice, while vehicle mice showed a progressive increase in glioma mass over time.

[0121] Example 2: CTX-CNF1 increases survival and reduces tumor mass also in CT-2A mice.

[0122] Since more than one type of glioma model is typically needed to validate therapies and scientific studies, it was assessed whether CTX-CNF1 was also effective in animals bearing CT-2A glioma. In this case, GBM diagnosis by MRI was possible as early as 7 days after tumor induction, as this is a more invasive model. After GBM diagnosis, the mice were randomly divided into two groups: one was treated with CTX-CNF1 (n=8), while the other received the vehicle solution (n=7). Also in this case, the treatment with CTX-CNF1 was shown to be effective in prolonging the lifespan of mice bearing CT-2A glioma; consistently, animals treated with CTX-CNF1 also showed a reduction in GBM mass compared to vehicle mice.

[0123] Example 3: Safety of the chimeric protein CTX-CNF1 in GBM mice.

[0124] Because tumor induction was performed in the primary motor cortex, the motor performance of the mice could also be tested through specific motor tasks (Grip Strength and Grid Walk). For both tests, each animal performance was normalized to their baseline performance, before tumor induction. In the Grip Strength test, the two experimental groups differed significantly only 28 days after tumor induction (two-way ANOVA, p < 0.05). In the Grid Walk test, starting 14 days after tumor induction (and 48 hours after the first treatment with CTX-CNF1), GL261 glioma-bearing vehicle animals showed significantly worse performance than mice treated with CTX-CNF1. Indeed, while motor performance was significantly impaired in animals treated with the vehicle solution, mice treated with GL261 maintained their baseline motor performance even in the later stages of the disease. The fact that CTX-CNF1 preserved the motor function of GL261 -bearing mice is the first sign that the treatment was safe for the mice. Furthermore, no significant differences in body weight or changes in the weight of internal organs were observed (neither after a single acute treatment with CTX-CNF1 nor at the end of three chronic treatments), demonstrating the safety of the treatment.

[0125] Example 4: GL261 glioma-bearing mice treated with CTX-CNF1 that are completely cured of their glioma are less susceptible to GBM relapse.

[0126] To assess immune system involvement after CTX-CNF1 treatment, it was decided to attempt to induce a new tumor in GL261 mice that were completely healed ( / .e., in the 50% of mice that were still alive 180 days after tumor implantation). Therefore, GL261 cells were reimplanted into the contralateral motor cortex of surviving mice. As a control, mice of the same age ( / .e. age-matched) were injected with GL261 cells. Importantly, mice previously treated with CTX-CNF1 and cured appear to be less susceptible to developing another GBM, thus suggesting the possibility of immune memory existence in CTX-CNF1 -treated animals.

[0127] Example 5: CTX-CNF1 increases the number and function of CD8+ cells in GBM mice.

[0128] To verify possible T cell recruitment following CTX-CNF1 administration, immunohistochemical analysis was performed 48 hours after the first administration of CTX-CNF1 / vehicle. Animals treated with CTX-CNF1 showed a significantly increased number of CD8 cells in the peritumoral tissue, while the number of CD4 cells was the same across groups. Importantly, the number of CD8 cells was not only upregulated in GBM animals treated with CTX-CNF1, but these cells were also more functional, as demonstrated by the functional markers Granzyme B and Perforin.

[0129] Example 6: Effect of CTX-CNF1 on murine and human macrophages.

[0130] To better clarify what the mechanism underlying this immune response could be, ex vivo experiments were performed to assess the direct interaction between CTX-CNF1 and immune cells.

[0131] Bone marrow-derived macrophages (BMDMs) were generated from the bone marrow and then polarized towards the different macrophage phenotypes present in GBM: i) MO, undifferentiated; ii) M1, anti-tumor; or iii) M2, pro-tumor phenotypes. After 24 hours of polarization, the cells were treated for an additional 24 hours with CTX-CNF1 or the mutated / inactive control CTX-CNF1-C866S. CTX-CNF1 was used at 25 nM, since the same concentration in vitro is sufficient to reduce glioma cell viability by almost 50%. Interestingly, CTX-CNF1, but not CTX-CNF1-C866S, significantly increased the expression of pro-inflammatory genes (e.g., II1[3, II6, and 1112), while significantly decreasing the expression of anti-inflammatory genes (e.g., 1110) in MO BMDMs. Furthermore, CTX-CNF1 treatment was able to shift the already polarized M2 BMDMs toward an M1 gene expression profile. Specifically, CTX-CNF1 significantly increased the expression of the pro-inflammatory genes 111(3 and II6 in M2 BMDMs. Consistent with qPCR data, a significant increase in TNF-alpha and IL-12 proteins was found in MO BMDMs supernatants by ELISA. Finally, a similar effect was observed in human macrophages, highlighting the translational potential of our recombinant protein.

[0132] Example 7: Effect of CTX-CNF1 on macrophages in vivo.

[0133] To verify that macrophage shift also occurs in vivo in mice treated with CTX-CNF1, an immunohistochemical analysis was performed 48 hours after the first administration of CTX-CNF1 / vehicle. CTX-CNF1 -treated animals were seen to show a significantly increased number of M1 macrophages (F4 / 80+ / cd11c+ cells) in tumor tissue and a significantly decreased number of M2 macrophages (F4 / 80+ / Arginase1 cells) in peritumoral tissue. This finding is crucial, as GBM is defined as a cold tumor, with inactive T cells and anti-inflammatory macrophages that allow tumor cells to grow unchecked. Therefore, the results further support the potential of CTX-CNF1 as an innovative therapeutic approach to contrast GBM.

[0134] Example 8: Combined systemic administration of CTX-CNF1 and anti-PD-1.

[0135] To understand whether a combined treatment with CTX-CNF1 and immune checkpoint inhibitors (e.g., anti-PD-1, anti-PD-L1) could be even more effective in combating GBM, the survival of mice bearing GL261 glioma after administration of these treatments was evaluated. Therefore, after GBM diagnosis by MRI, the mice were randomly divided into four groups: one was treated with CTX-CNF1 and anti-PD-1 , one was treated with CTX-CNF1 and anti-PD-L1, while the others received only anti-PD-1 or anti-PD-L1, respectively. As can be seen from the survival curve, animals treated with anti-PD-1 and CTX-CNF1 survived longer than all other groups of mice. This confirms that the combination of the immune checkpoint inhibitor anti-PD-1 with the recombinant protein described herein leads to increased survival in glioma mice.

[0136] Example 9: Effect of CTX-CNF1 on different tumoral cell lines.

[0137] To evaluate the ability of CTX-CNF1 to influence cell replication and the resulting ability of cells to form clones, the clonogenic assay was used on various cell lines derived from tumors of different anatomical origin.

[0138] Specifically, increasing concentrations of CTX-CNF1 (1, 5, 12.5, 25, and 50 nM) were tested, quantifying the different sensitivity of the cell lines examined to form colonies after the treatment. This assay allowed to demonstrate that CTX-CNF1 is capable of inhibiting the formation of cell clones in different tumor types, originating from different anatomical regions.

[0139] Example 10: Survival (%) over time of mice subjected to tumor re-challenge with GL261 cells after initial treatment.

[0140] To assess the presence of an anti-tumor memory immune response, animals previously treated with CTX-CNF1, with or without anti-PD-1 antibody, and surviving the first tumor implantation, were re-inoculated with GL261 cells (re-challenge).

[0141] GL261 cells were stereotaxically inoculated into the contralateral cortex. The number of cells injected was the same as the primary inoculation. The animals received no further post-re-challenge treatments. As a control, a group of age-matched naive mice was simultaneously inoculated with the same number of tumor cells. Death or achievement of ethical endpoints was considered an event for the calculation of Kaplan-Meier curves. As shown in Figure 10, animals previously treated with anti-PD-1 + CTX-CNF1 (dashed-dotted line) show no lethal events observed up to >140 days after rechallenge, indicative of protective immune memory. Animals treated with CTX-CNF1 alone (dashed line) show prolonged survival, with a progressive reduction in the number of surviving animals up to day ~110, while animals previously administered with the control (age-matched naive controls) (solid line) show rapid cancer progression, with all animals dying within ~35 days of rechallenge.

[0142] MATERIALS AND METHODS

[0143] Cloning and preparation of CTX-CNF1

[0144] The sequence encoding chlorotoxin (36 amino acids, 108 nucleotides) was cloned as a G block (with BamH1-Bglll restriction sides) in frame into the pGEX2TGL+2-CNF1 vector opened with BamH1 and dephosphorylated (corresponding to an N -terminal extension of CNF1). Correct cloning was verified by sequencing (Vannini E. et al. “CTX-CNF1 Recombinant Protein Selectively Targets Glioma Cells In vivo." Toxins 2021 Mar 8; 13(3): 194.).

[0145] GST-CTX-CNF1 or mutated GST-CTX-CNF1 ( / .e., vehicle) proteins were expressed in Escherichia coli BL21 cells transformed with pGEX plasmids carrying the respective genes. Bacteria were grown in Luria-Bertani (LB) medium at 37°C and induced with 0.2 mM isopropyl-[3-D-thiogalactopyranoside (IPTG) to an optical density of 0.6. Cells were harvested after 6 h, and proteins were purified by affinity chromatography with glutathione Sepharose beads (Amersham Pharmacia Biotech). Loaded beads were washed twice in wash buffer A (20 mM Tris / HCI pH 7.4, 10 mM NaCI, 5 mM MgCh) and wash buffer B (150 mM NaCI, 50 mM Tris / HCI, pH 7.5) at 4°C. Proteins were eluted with 10 mM glutathione in 50 mM Tris / HCI pH 8.0. GST fusion proteins were lyophilized and stored frozen. Proteins were used as GST fusion proteins due to their greater stability. Inactive CTX-CNF1, administered to glioma-bearing mice (vehicle), has a mutation (C866S) in the catalytic domain that impairs its proper functioning.

[0146] Cell Culture

[0147] Murine Glioma Cells

[0148] GL261 and CT-2A syngeneic murine glioma cells were grown as monolayers in Dulbecco’s Modified Eagle’s Medium (DMEM / F12) containing 10% Fetal Calf Serum (FCS), 4.5 g / L glucose, 2 mM L-glutamine, 100 ILI / mL penicillin, and 100 mg / mL streptomycin at 37°C in 5% CO2. The medium was changed three times weekly once 80-90% confluence was reached.

[0149] Murine Bone Marrow-Derived Macrophages (BMDMs)

[0150] Twelve-week-old mice were euthanized in a CO2 chamber, and death was confirmed by cervical dislocation. Bone marrow was harvested by flushing DMEM through the femur, tibia, and hip bones of mice with a 25-gauge needle. Bone marrow was suspended in red blood cell lysis buffer (Sigma) for 3 min before being centrifuged, suspended, and passed through a 70 pm filter. Cells were plated in 10-cm Petri dishes in DMEM containing 2 mM L-glutamine, FCS (10%), penicillin-streptomycin (1%), and 10% L929 supernatant, and cultured at 37°C in a 5% CO2 incubator for 6 days. On day 6, cells were scraped, suspended in complete DMEM, and seeded with polarizing factors in 24-well plates at 5x105cells / mL for 24 h. BMDMs were polarized towards M0 (without added polarizing factors), M1 (+LPS 100 ng / mL, Merck, and + INF-y 20 ng / mL, BioLegend), or M2 (+IL-4 20 ng / mL, BioLegend). After 24 hours of polarization, cells were treated for an additional 24 hours with CTX-CNF1 or the mutated control (both 25 nM), after which the BMDM supernatant and RNA were collected.

[0151] Isolation and culture of human macrophages

[0152] Buffy coats were obtained from the Department of Transfusion Medicine at the Medical University of Graz, where blood donors signed informed consent prior to blood collection. These buffy coats were purchased from the Division of Immunology and processed anonymously. Peripheral blood mononuclear cells (PBNCs) were isolated, and monocyte isolation was performed using human CD14 microbeads (Miltenyi). For macrophage generation, purified cells were seeded in 24-well plates at 1 x6cells / mL for 6 days in RPMI 1640 supplemented with 10% FBS, 2.5 mmol / L GlutaMAX (Invitrogen, Grand Island, NY), 125 U / mL penicillin / streptomycin (PAA, Pasching, Austria), 100 ng / mL M-CSF (Peprotech), and incubated at 37°C and 5% CO2. Cells were cultured for 6 days, with removal and replacement of the medium and M-CSF on day 3 of culture. On day 6, macrophages were polarized towards MO, M1, or M2 by simply changing the medium and adding the same polarizing factors used for BMDMs. Twenty-four hours after polarization, cells were treated with CTX-CNF1 or the mutated control (both at 25 nM) for an additional 24 hours. Subsequently, the supernatant and RNA from human macrophages were collected.

[0153] B16F10 murine melanoma cells

[0154] B16F10 murine melanoma cells were grown as monolayers in Dulbecco’s modified Eagle’s Complete Medium (DMEM / F12) containing 10% FCS, 4.5 g / L glucose, 2 mM L-glutamine, 100 lll / ml penicillin, and 100 mg / ml streptomycin at 37°C in 5% CO2. The medium was changed three times weekly once 80-90% confluence was reached, as recommended by ATCC guidelines.

[0155] MIA PaCa-2 human pancreatic cancer cells

[0156] MIA PaCa-2 human pancreatic cancer cells were grown as monolayers in Dulbecco’s Modified Eagle’s Complete Medium (DMEM / F12) containing 10% fetal bovine serum (FBS), 2.5% horse serum (HS), 1 mM sodium pyruvate, 1 ,500 mg / L sodium bicarbonate, 4.5 g / L glucose, 4 mM L-glutamine, 100 lll / ml penicillin, and 100 mg / ml streptomycin at 37°C in 5% CO2. The medium was changed three times weekly once 80-90% confluence was reached, as recommended by ATCC guidelines.

[0157] SF8628 Human pediatric glioma cells

[0158] SF8628 Human pediatric glioma cells were grown as monolayers in Dulbecco’s Modified Eagle’s Complete Medium (DMEM / F12) containing 10% FCS, 4.5 g / L glucose, 2 mM L-glutamine, 100 lU / mL penicillin, and 100 mg / mL streptomycin at 37°C in 5% CO2. The medium was changed three times weekly once 80-90% confluence was reached, as recommended by Sigma-Aldrich guidelines.

[0159] Clonogenic and Trypan Blue Assays

[0160] For clonogenic assays carried out on different cell types (B16-F10, MIA PaCa-2, GL261, CT-2A, and SF8628), on day 0, 300 cells per well were seeded in 12-well plates using the respective culture media. The following day, the medium was replaced and increasing concentrations of CTX-CNF1 (1, 5, 12.5, 25, and 50 nM) were added to the culture medium in specific wells (the experiment was carried out in duplicate). Colony formation was monitored daily, and when the colonies in the control wells (not treated with CTX-CNF1) reached approximately 50 cells each, the clonogenic assay was completed according to the protocol described in Franken, N., Rodermond, H., Stap, J. et al. Clonogenic assay of cells in vitro. Nat Protoc 1 , 2315-2319 (2006).

[0161] For the trypan blue assay, SF8628 cells were treated with CTX-CNF1 (1 and 5 nM) for 24 hours, after which the trypan blue assay was performed following BPS Bioscience protocol.

[0162] RNA extraction and qPCR

[0163] The medium supernatant was removed, cells were washed with PBS and lysed in 350 pL of RNA lysis buffer (Invitrogen). A volume of 70% EtOH was added before transferring to Invitrogen RNA spin columns. Purification was performed according to the manufacturer’s instructions (Invitrogen PureLink RNA kit, ThermoFisher). Eluted RNA was quantified using a Nanodrop 2000 spectrophotometer, and each RNA sample was diluted to the lowest yield before reverse transcriptase PCR. cDNA was prepared using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s instructions. qRT-PCR was then performed using the Powerllp SYBR Green Master on a 7500 Fast thermal cycler (Applied Biosystems), and Ct values were converted to 2(-AACt)using the Ct of the housekeeping gene 18s (BMDM) or hprt (human macrophages).

[0164] ELISA

[0165] DuoSet ELISA kits for IL-12, TNFc, and IL-6 were purchased from R&D Systems and performed according to the manufacturer’s instructions with appropriately diluted cell supernatants added to each plate in duplicate or triplicate. Absorbance at 450 nm was then quantified using a FLUOstar Optima plate reader. Corrected absorbance values were calculated by subtracting background absorbance, and cytokine concentrations were subsequently obtained by extrapolation from a standard curve plotted on GraphPad Prism 8.0.

[0166] Animals

[0167] This study used adult wild-type C57BL / 6J mice (age > 60 postnatal days, both sexes) bred in our animal facility. Mice were housed on a 12-hour light / dark cycle, with food and water available ad libitum. All experimental procedures compliant with European Council Directive 86 / 609 / EEC were approved by the Italian Ministry of Health (1204 / 2020-PR, prot. B4BB8.30, 02 / 10 / 2020).

[0168] Tumor induction

[0169] To induce glioma formation, C57BL / 6 mice subjected to a ketamine / xylazine cocktail (100 / 10 mg / kg i.p.) received a stereotaxically guided injection of 50,000 CT-2A cells (25,000 cells / pL phosphate-buffered saline; Riva et al. 2019) or 40,000 GL261 cells (20,000 cells / pL phosphate-buffered saline) into the primary motor cortex (1750 pm lateral to midline and 500 pm anterior to bregma) or primary visual cortex (2.5 mm lateral to midline and corresponding to lambda). Injections were performed in the left hemisphere of the brain. The cell solution was slowly administered to a depth of 0.9-1 mm from the pial surface using a Hamilton syringe driven by an automated pump (KdScientific, USA). Postoperatively, animals were assisted in recovery with a thermostated electric blanket (37.0°C). To prevent dehydration, a subcutaneous injection of saline (0.9% NaCI, 1 mL) was administered at the end of the procedure. Seven or 12 days later (for animals injected with CT-2A or GL261 , respectively), gliomabearing mice were divided into two groups: one group received CTX-CNF1 (80 nM, 20 pL) intravenously via the tail vein, while the other group (vehicle) received inactivated and mutated CTX-CNF1 (80 nM, 20 pL) via the same route. The administration protocol was repeated once a week for a total of three weeks. Animals’ weight was monitored daily, and when weight loss reached 30% of baseline weight, they were sacrificed. Animals whose treatment eradicated the tumor were subject to rechallenge. Then, 6 months after tumor induction, the surviving animals underwent a new injection of GL261 cells into the contralateral primary motor cortex ( / .e., 1750 pm lateral to the midline and 500 pm anterior to the bregma hemisphere; 40,000 GL261 cells (20,000 cells / pL of phosphate-buffered saline, in the motor cortex)). Animals’ weight was monitored daily, and when weight loss reached 30% of baseline weight, they were sacrificed.

[0170] Organ harvesting in mice

[0171] Glioma-bearing mice were euthanized in a CO2 chamber, and death was confirmed by cervical dislocation. Kidneys, spleen, thymus, and liver were harvested and weighed in vehicle and in CTX-CNF1 mice acutely, i.e., 48 hours after one CTX-CNF1 administration, and chronically, i.e., 48 hours after three CTX-CNF1 administrations.

[0172] Magnetic Resonance Imaging (MRI)

[0173] Tumor growth in mice was monitored weekly by MRI using a Bruker III Avance HD 300 Hz Ultra Shield, a 7T vertical bore preclinical scanner equipped for microimaging. Glioma-bearing mice were scanned in vivo with optimized parameters and without contrast medium administration. During imaging, mice were anesthetized with ketamine / xylazine (100 / 10 mg / kg i.p.). MRI sessions were performed every 7 days starting from day 7 and day 12 after tumor induction in CT-2A and GL261 animals, respectively. For each animal, the adopted MRI sequence protocol was as follows: a) rapid acquisition with low-resolution FLASH sequences, to anatomically localize the tumor mass in the sagittal, coronal, and axial planes. Field of view (FOV) = 20 x 20 mm; matrix size = 128 x 128; i.e. image resolution= 20 / 128 = 156 ==> 156 x 156 urn; slice thickness = 1 mm; flip angle = 30°; b) TURBORARE T2-weighted sequences (T2-w) were implemented with axial slices corresponding to the tumor region; FOV = 20x20 mm; Matrix = 256 x 256; i.e. image resolution = 78 x 78 pm; section thickness = 500 pm; scan time = 8 min. Acquisitions were performed using a 1 H quadrature volume coil with a 30 mm internal diameter, capable of accommodating the mouse bed. Animals were positioned prone on the mouse bed with their brains located at the isocenter of the magnet (the region with the greatest magnetic field homogeneity). Body temperature was maintained close to 37°C by a flow of warm air directed at the animals, and respiration was monitored using a respiration sensor immobilized under the belly (Respiration Pillow Sensor, SA Instruments, Inc). Each MRI session lasted 30 min. For each coronal section, the tumor area was measured with Mango software (version 4.1). The area was then reprocessed using the software’s Analysis function to obtain the volume, expressed in voxels.

[0174] Motor Tests Mice were tested in two different motor tests: Grip Strength and Grid Walk. Each animal performed all tests twice before glioma cell injection (baseline measurement) and twice weekly starting on day 7 or day 10 after tumor injection (for animals injected with CT-2A or GL261, respectively). To rule out any influence of circadian rhythms on physical performance, all motor tests were performed during the same time window each day (10:00-12:00). All tests and analyses were performed blindly to the experimental conditions, and the results of each test obtained across the different days were normalized to baseline performance.

[0175] - Grip Strength

[0176] The animal was placed on a base plate facing a (trapeze-shaped) grip bar, the height of which was adjustable. The bar was mounted on a force transducer connected to a Peak Amplifier (Ugo Basile S.R.L, Varese, Italy). When pulled by the tail, the animal grasped the bar (rodents instinctively grasp anything they can reach to try and stop this involuntary backward movement) until the pulling force exceeded their grip strength. After the animal lost its grip on the grip bar, the Peak Amplifier automatically stored the maximum pulling force achieved by the forelimbs and displayed it on a liquid crystal display. Three trials per day were performed for each animal and averaged. The values obtained were normalized to the baseline.

[0177] - Grid Walk

[0178] The animal was placed on a grid (32 cm x 20 cm with an 11 mm x 11 mm mesh). To walk correctly, the mouse had to place its paws on the wires of the grid; these were considered as correct steps. However, if one of the paws fell into a hole in the grid, it was considered as a “foot fault”. The test lasted 5 minutes for each animal and was filmed using a camera positioned in front of the grid. A mirror placed under the grid helped determine which limb performed the foot fault. Video analysis was performed using a frame-by-frame playback. Scoring was carried out separately for the affected (e.g., contralateral to the glioma cell injection) and unaffected (ipsilateral) limbs. Performance with the affected limbs (contralateral to tumor induction) was calculated as follows: % of foot faults of the affected limb = (foot faults of the affected I im b / total steps of the affected limb) x 100.

[0179] CD8+ cell depletion and immune checkpoint inhibitor (ICI) administration Mice affected by glioma were treated intraperitoneally with anti-PD-1 (200 pg / mouse, BioXcell, BE0146) and anti-PD-L1 (200 pg / mouse, BioXcell, BE0101) twice weekly starting on day 12 through day 26 after tumor induction. Anti-CD8 for CD8+ cell depletion (250 pg / mouse, BioXcell, BE0117) was administered intraperitoneally twice weekly starting on day 11 and continuing until day 25 after tumor induction.

[0180] Immunofluorescence

[0181] 18 days after tumor induction, animals were deeply anesthetized with chloral hydrate (10 mg / kg) and transcardially perfused with phosphate-buffered saline (PBS; Sigma Aldrich, USA), followed by fixation solution (4% paraformaldehyde, 0.1 M sodium phosphate, pH 7.4). Brains were gently removed, post-fixed for 4 hours in the same fixation solution at 4°C, then cryoprotected by immersion in 30% sucrose, and sliced using a sliding microtome (Leica, Germany) to obtain 50-pm-thick coronal sections. Only brains with visible tumor mass were considered. Sections were stained for CD4 (1 :500; Abeam), CD8 (1:500; Abeam), CD8 and Perforin (1:500 and 1:100; Abeam, respectively), CD8 and Granzyme B (1:500; Abeam and 1:100; Bio-Techne, respectively), F4 / 80 and GFAP (1:500; Abeam and 1:800; Agilent, respectively), F4 / 80 and cd11 c (1 :500; Abeam and 1 : 100; Synaptic Systems GmbH, respectively), F4 / 80 and Arginasel (1:500; Abeam and 1:100; Thermofisher, respectively). Subsequently, they were incubated with fluorophore-conjugated secondary antibodies (Jackson Immunoresearch, USA) and Hoechst stain (1:500; Bisbenzemide, Sigma Aldrich, USA) for nuclear visualization. For CD4, CD8 and Perforin, CD8 and Granzyme B, F4 / 80 and Arginasel and F4 / 80 and cd11c, the sections were incubated with 1X antigen retrieval solution (Antigen Retrieval Buffer 100X Tris-EDTA Buffer, pH 9.0; Abeam) for 5 min at 95°C before the blocking phase. 2.8 Image Acquisition

[0182] Fluorescent images were acquired using a Zeiss Axio Oberver microscope equipped with a Zeiss AxioCam MRm camera (Carl Zeiss MicroImaging GmbH, Jena, Germany) and using a Zeiss LSM 900 with Airyscan2.

[0183] Statistical Analysis

[0184] GraphPad 8 software was used for statistical analyses. Differences between two groups were assessed using the t-test, and among three or more groups using one-way ANOVA. Longitudinal motor tests were analyzed using a two-way ANOVA for repeated measures. Survival analyses were performed using Kaplan-Meier statistics (LogRank). From the detailed description and the Examples reported above, the advantages achieved by the chimeric protein of the present invention and its combination with the antibody anti-PD-1 are apparent.

Claims

CLAIMS1. A pharmaceutical kit comprising:- a chimeric protein CTX-CNF1 ; and- a checkpoint inhibitor, selected from the group consisting of anti-PD-1 , anti-PDL-1 , anti-CTLA-4, anti-TIGET, anti-LAG-3, and anti-TIM-3,for simultaneous, separate, or sequential use, wherein said chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

2. A pharmaceutical kit for use as a medicament comprising:- a chimeric protein CTX-CNF1 ; and- a checkpoint inhibitor, selected from the group consisting of anti-PD-1 , anti-PDL-1 , anti-CTLA-4, anti-TIGET, anti-LAG-3, and anti-TIM-3,for simultaneous, separate, or sequential use, wherein said chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

3. A pharmaceutical kit for use in the treatment of cancer comprising:- a chimeric protein CTX-CNF1 ; and- a checkpoint inhibitor, selected from the group consisting of anti-PD-1 , anti-PDL-1 , anti-CTLA-4, anti-TIGET, anti-LAG-3, and anti-TIM-3,for simultaneous, separate, or sequential use, wherein said chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

4. The pharmaceutical kit for use according to claim 3 comprising:- a chimeric protein CTX-CNF1 ; and- a checkpoint inhibitor, selected from the group consisting of anti-PD-1 , anti-PDL-1 , anti-CTLA-4, anti-TIGET, anti-LAG-3, and anti-TIM-3,for simultaneous, separate, or sequential use,wherein said cancer is selected from the group consisting of glioblastoma multiforme, melanoma, glioma, and pancreatic cancer.

5. A chimeric protein CTX-CNF1 for use in the treatment of cancer by stimulatingimmune system cells, wherein said chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

6. The protein for use according to claim 5, wherein said cancer is selected from the group consisting of glioblastoma multiforme, melanoma, glioma, pancreatic cancer, lung cancer, non-small cell lung cancer (NSCLC), kidney cancer, Hodgkin and non-Hodgkin lymphomas, squamous cell carcinoma of the head and neck (SCCHN), urothelial cancer, bladder and urinary tract cancer, pleural mesothelioma, colon cancer, rectal cancer, esophageal cancer and esophageal squamous cell carcinoma, gastric adenocarcinoma, gastroesophageal adenocarcinoma, and esophageal adenocarcinoma.

7. The protein for use according to claim 5 or 6, wherein said cancer is selected from the group consisting of melanoma, pancreatic cancer, lung cancer, non-small cell lung cancer (NSCLC), kidney cancer, Hodgkin and non-Hodgkin lymphomas, squamous cell carcinoma of the head and neck (SCCHN), urothelial cancer, bladder and urinary tract cancer, pleural mesothelioma, colon cancer, rectal cancer, esophageal cancer and esophageal squamous cell carcinoma, gastric adenocarcinoma, gastroesophageal adenocarcinoma, and esophageal adenocarcinoma.

8. The protein for use according to any one of claims 5 or 6, wherein said cancer is selected from the group consisting of glioblastoma multiforme, melanoma, glioma, and pancreatic cancer.

9. The protein for use according to any one of claims 5 to 8, wherein said immune system cells are selected from the group consisting of macrophages and T lymphocytes.

10. The protein for use according to any one of claims 5 to 9, wherein the stimulation increases the number and functionality of said T lymphocytes.

11. The protein for use according to claim 10, wherein the stimulation is responsible forthe switch of said macrophages towards a proinflammatory phenotype.

12. The protein for use according to any one of claims 5 to 11, wherein said immune system cells are peritumoral tissue cells.

13. A pharmaceutical composition comprising a chimeric protein CTX-CNF1 and a pharmaceutically acceptable excipient, for use in the treatment of cancer by stimulating immune system cells, wherein said chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

14. The pharmaceutical composition for use according to claim 13, wherein said cancer is selected from the group consisting of glioblastoma multiforme, melanoma, glioma, and pancreatic cancer.

15. The pharmaceutical composition for use according to any one of claims 13 or 14, wherein said immune system cells are selected from the group consisting of macrophages and T lymphocytes.

16. The pharmaceutical composition for use according to claim 15, wherein the stimulation increases the number and functionality of said T lymphocytes.

17. The pharmaceutical composition for use according to claim 15, wherein the stimulation is responsible for the switch of said macrophages towards a proinflammatory phenotype.

18. The pharmaceutical composition for use according to any one of claims 13 to 17, wherein said immune system cells are peritumoral tissue cells.

19. A pharmaceutical kit for use in the treatment of recurrent cancer comprising:- a chimeric protein CTX-CNF1 ; and- a checkpoint inhibitor, selected from the group consisting of anti-PD-1 , anti-PDL-1 ,anti-CTLA-4, anti-TIGET, anti-LAG-3, and anti-TIM-3,for simultaneous, separate, or sequential use, wherein said chimeric protein CTX-CNF1 has an amino acid sequence of SEQ ID NO: 1.

20. The pharmaceutical kit for use according to claim 19 comprising:- a chimeric protein CTX-CNF1 ; and- a checkpoint inhibitor, selected from the group consisting of anti-PD-1 , anti-PDL-1 , anti-CTLA-4, anti-TIGET, anti-LAG-3, and anti-TIM-3,for simultaneous, separate, or sequential use,wherein said recurrent cancer is selected from the group consisting of glioblastoma multiforme, melanoma, glioma, pancreatic cancer, lung cancer, non-small cell lung cancer (NSCLC), kidney cancer, Hodgkin and non-Hodgkin lymphomas, squamous cell carcinoma of the head and neck (SCCHN), urothelial cancer, bladder and urinary tract cancer, pleural mesothelioma, colon cancer, rectal cancer, esophageal cancer and esophageal squamous cell carcinoma, gastric adenocarcinoma, gastroesophageal adenocarcinoma, and esophageal adenocarcinoma.

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