WO2025224605A1 - Sequential administration of anti-melanoma vaccine and anti-pd-1 monoclonal antibodies - Google Patents

Abstract

Methods for enhancing an anti-tumor immune response to treatment in a subject who has cutaneous melanoma (CM). In particular, the subject is treated with anti-PD-1 monoclonal antibodies (MAbs) following treatment with a vaccine generating an immune response against antigens of CM cells.

Description

SEQUENTIAL ADMINISTRATION OF ANTI-MELANOMA VACCINE AND ANTI-PD-1

MONOCLONAL ANTIBODIES

FIELD OF THE INVENTION

This disclosure relates generally to methods of enhancing an immune response in subjects treated for cutaneous melanoma (CM).

BACKGROUND OF THE INVENTION

Cutaneous melanoma (CM) is still a serious disease, due to its increasing incidence and its generally rapid growth and dissemination. Fortunately, during the last two decades, great advances have been made in its treatment. Such advances stem from the exploitation of two CM Achilles' heels: its elevated tumor mutational burden (TMB), which gives rise to a large number of neoantigens (neoAgs), and the presence of mutated driver oncogenes, such as BRAF^V600, in 50% of the patients. As a consequence, new treatments have become available, such as inhibitors of the MAPK pathway and of monoclonal antibodies (MAbs) inhibitors of the CTLA4/CD80 and PD- Ll/PD-1 immune checkpoints inhibitors (ICI). With respect to immunotherapy treatments, the mechanisms involved in their effectiveness and resistance are still incompletely understood. Concerning effectiveness, in a study of 655 metastatic CM patients treated in monotherapy with the anti-PD-1 MAb pembrolizumab the overall survival (OS) after 5 years of follow-up was 34% for the total population. Besides, the complete response (CR) rate was only 16%, whereas the partial response (PR) rate was 25%, and stable disease (SD) accounted for 24% of the patients. Thus, 75% of the responding patients still had residual disease and were more prone to recurrence than those who attained CR. Thus, there is an unsatisfied medical need to ameliorate this situation. Due to its high TMB, it is probable that during CM growth different cell clones are generated, which in turn express a great variety of neoAgs, only some of which are capable of triggering effective immune responses. Anti-PD-1 MAbs are usually administered without any previous treatment, and it is accepted that they act on already existing anti-tumor reactive T cell clones by reversing their exhaustion and increasing their cytotoxicity. Consequently, there remains a need to widen the immune system reactivity against a greater number of Tumor-Associated Antigens (TAA) and neoAgs for the treatment of CM. SUMMARY OF THE INVENTION

This disclosure relates to a method for enhancing an immune response, more specifically, an anti -tumor immune response in a subject carrying a cutaneous melanoma (CM) who was treated with a vaccine generating an immune response against antigens of CM cells, the method comprising administering a therapeutically effective dose of an anti-PD-1 monoclonal antibody (MAb) following treatment with the vaccine generating an immune response against antigens of CM cells.

In an embodiment, the subject is carrying a cutaneous melanoma (CM) and was treated with a vaccine generating an immune response against a repertoire of antigens of CM cells.

In an embodiment, the anti-PD-1 MAb administered following treatment with the vaccine generating an immune response against antigens of CM cells is selected from the group consisting of nivolumab, pembrolizumab, MEDI0680, spartalizumab, dostarlimab and tislelizumab. Preferably, the anti-PD-1 Mab is administered at a dose of 3 mg/kg or about 240 mg every two weeks or about 480 mg every four weeks in the case of nivolumab, and at a dose of about 2 mg/kg or about 200 mg every three weeks in the case of pembrolizumab.

In an embodiment, the subject is administered the anti-PD-1 MAb about 10 to about 50 months following treatment with the vaccine generating an immune response against antigens of CM cells.

In an embodiment, the vaccine generating an immune response against antigens of CM cells comprises at least one CM cell line which is incapable of proliferating. Preferably, the vaccine comprises at least one CM cell line which is incapable of proliferating due to have been irradiated. More preferably, the vaccine comprises at least one CM cell line selected from the group consisting of (a) Mel-XYI (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2830), (b) Mel-XY2 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2831), (c) Mel-XY3 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2832), and (d) Mel-XX4 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2829), wherein said cell lines have been irradiated, and are incapable of proliferating. In a particularly preferred embodiment of the invention, the subject carrying a cutaneous melanoma (CM) was treated with a vaccine generating an immune response against antigens of CM cells, wherein the vaccine comprises: the CM cell lines of the group consisting of (a) Mel-XYI (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2830), (b) Mel-XY2 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2831), (c) Mel-XY3 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2832), and (d) Mel-XX4 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2829), wherein said cell lines have been irradiated, and are incapable of proliferating; and BCG (Bacillus Calmette-Guerin) and GM-CSF (granulocyte-macrophage colony- stimulating factor) as adjuvants.

In an embodiment, the subject is a human.

In another aspect of the invention, provided is a method for treating cutaneous melanoma (CM) in a subject, comprising administering to the subject 3 mg/kg or 240 mg every two weeks or about 480 mg every four weeks in the case of anti-PD-1 monoclonal antibody nivolumab and about 2 mg/kg or about 200 mg every three weeks in the case of anti-PD-1 monoclonal antibody pembrolizumab following treatment with a vaccine generating an immune response against antigens of CM cells.

In another aspect of the invention, provided is an anti-PD-1 Mab for use in enhancing an anti -tumor immune response in a subject carrying a CM, wherein the subject has been previously treated with a vaccine generating an immune response against antigens of CM cells.

In an embodiment of this aspect of the invention, the anti-PD-1 MAb for use in enhancing an anti -tumor immune response in a subject carrying a CM is selected from the group consisting of nivolumab, pembrolizumab, MEDI0680, spartalizumab, dostarlimab and tislelizumab. In a preferred embodiment, the anti-PD-1 MAb for use in enhancing an anti-tumor immune response in a subject carrying a CM is nivolumab and it is administered at a dose of about 3 mg/kg or 240 mg every two weeks or 480 mg every four weeks. In another preferred embodiment, the anti-PD- 1 MAb for use in enhancing an anti -tumor immune response in a subject carrying a CM is pembrolizumab and it is administered at a dose of about 2 mg/kg or about 200 mg every three weeks.

In an embodiment of this aspect of the invention, the subject is administered the anti-PD- 1 MAb about 10 to about 50 months following treatment with the vaccine generating an immune response against antigens of CM cells.

In an embodiment of this aspect of the invention, the vaccine generating an immune response against antigens of CM cells comprises at least one CM cell line which is incapable of proliferating. Preferably, the vaccine comprises at least one CM cell line which is incapable of proliferating due to have been irradiated. More preferably, the vaccine comprises at least one CM cell line selected from the group consisting of (a) Mel-XYI (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2830), (b) Mel-XY2 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2831), (c) Mel-XY3 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2832), and (d) Mel-XX4 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2829), wherein said cell lines have been irradiated, and are incapable of proliferating.

In a particularly preferred embodiment of the invention, the vaccine generating an immune response against antigens of CM cells comprises: the CM cell lines of the group consisting of (a) Mel-XYI (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2830), (b) Mel-XY2 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2831), (c) Mel-XY3 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2832), and (d) Mel-XX4 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2829), wherein said cell lines have been irradiated, and are incapable of proliferating; and BCG (Bacill us Calmette-Guerin) and GM-CSF (granulocyte-macrophage colonystimulating factor) as adjuvants.

It is yet another aspect of the invention to provide an anti-PD-1 Mab for use in treating CM in a subject in need thereof, wherein the subject has been previously treated with a vaccine generating an immune response against antigens of CM cells, wherein said use comprises: administering to the subject about 3 mg/kg or about 240 mg every two weeks or about 480 mg every four weeks in the case of anti-PD-1 monoclonal antibody nivolumab, and about 2 mg/kg or about 200 mg every three weeks in the case of anti-PD-1 monoclonal antibody pembrolizumab, following treatment with the vaccine generating an immune response against antigens of CM cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 shows a timeline from all case reports. Course of the disease, expressed in months, and treatments received by each patient are illustrated with a swimmer plot. Relevant dates are indicated within the timeline. Number references for each kind of event are indicated in the legend. Case#l: after pembrolizumab treatment, this patient attained complete remission in 04/2018 (POST) and remained NED until present. Case#2: After pembrolizumab treatment, this patient attained complete remission (09/2021) and remained NED until present. Case#3: Although this patient achieved a rapid CR to nivolumab (05/2018), had to shift to dabrafenib/trametinib, because of a supraventricular arrhythmia (12/2018), with further progression. Case#4: After pembrolizumab treatment, this patient attained complete remission (07/2020) and remained NED until present (09/2023). Case#5: this patient remained NED 46 months after CR to nivolumab (10/2020), further described (Mordoh A et al. 2022).

FIGS. 2A-2C are immunohistochemistry (IHC) and CT images from patient #1 and patient #4. FIG. 2A shows an IHC of an inguinal LN metastasis excised from Case#l intra-VACCIMEL treatment; CD8+ and CDl lc+ populations are shown. IHC was performed as previously described (Aris M, et al., 2019). Original magnification 400X; scale bars 50pm. FIG. 2B is a PET/CAT scan from case#l (PRE, 01/2015) that shows hypercaptating right lung nodules and a parasplenic nodule. After pembrolizumab treatment, this patient attained complete remission in 04/2018 (POST) and remained NED until present (09/2023). FIG. 2C is a CT from Case #4, dated/

FIGS. 3A-3C depict data pertaining to Patient #2 case. FIG. 3A shows histopathological analysis of a tumor-draining LN and cutaneous metastases excised post-VACCIMEL immunization and 4 years before anti-PD-1 treatment; CD8⁺ populations as determined by IHC are shown. Original magnification 400X; scale bars 50pm. FIG. 3B is an IFN-y ELISPOT to analyze T-cell response induced by VACCIMEL (Podaza E et al., 2020). PRE: blood extracted at the selection process; POST-3: blood extracted 24 months after protocol start; unspecific: corresponds to PBMC only stimulated with culture medium. Bars indicate quantification of the spots normalized to 10⁵ stimulated PBMC (effector cells). Peptides tested for this case are shown in the table. FIG. 3C depicts a PRE: CAT scan showing a lung metastasis (red arrow), non- detectable after ICI treatment with pembrolizumab (POST).

FIGS. 4A-4C depict data pertaining to Patient #3 case. FIG. 4A shows histopathological analysis of a LN metastasis excised prior to VACCIMEL immunization (03/2010). HLA-I⁺ and CDl lc⁺ populations determined by IHC are shown. Original magnification 200X; scale bars 100pm. FIG.4B shows T-cell response induced by VACCIMEL protocol to HLA-restricted peptides from shared melanoma associated antigens detected by IFN-y ELISPOT. This was performed as described (Podaza E et al., 2020 ). Pre: blood extracted at the selection process; Post 1, 2 and 3 (Pl, P2, P3): blood extracted 6, 12 and 24 months after protocol start; unspecific: corresponds to PBMC only stimulated with culture medium. Bars indicate quantification of the spots normalized to 10⁵ stimulated PBMC (effector cells). Peptides tested for this case are shown in the table. FIG. 4C depicts a PET/CAT scan performed in 03/2018 that showed multiple metastatic lesions (PRE), non-detectable after ICI treatment with nivolumab (09/2018) (POST).

FIG. 5 shows the result of an ELISPOT assay for Patient #2. Bars represent IFN-gamma spots normalized to 10⁵ PBMC plus standard deviation; each point was performed in triplicate. **= p<0.005; ****=p<0.0001 (2-way Anova, Tukey's multiple comparison test).

FIG. 6 shows the PMEL/gplOO expression present in patient # 2 's tumor as detected by immunohistochemical staining with HMB45 monoclonal antibody. Original magnification 400 X.

FIG. 7 shows the results of an ELISPOT assay for Patient #2. Bars represent IFN-gamma spots normalized to 10⁵ PBMC plus standard deviation; each point was performed in triplicate. Ns=non-significant **= p<0.005; ****=p<0.0001 (2-way Anova, Tukey's multiple comparison test).

FIGS 8A-8B. show the results of an ELISPOT assay. Bars represent IFN-gamma spots normalized to 10⁵ PBMC plus standard deviation; each point was performed in triplicate. FIG 8A. shows the results of PRE, POST1 and POST2-PBMC were tested. FIG 8B. shows the results of P0STt3-PBMC were tested. ns=non-significant; **=p<0.005; ****=p<0.0001 (2-way Anova, Tukey's multiple comparison test). In the case of MAGE-B2 peptide was only tested for POST1 and POST2-PBMC, due to insufficient PRE-PBMC sample.

FIGS. 9A-9B shows the results of Cytotoxic response triggered by VACCIMEL in patient #005 PBMC samples potentiated by the addition of anti-PD-1. MEL-XY3 melanoma cells (target cells, T) lysis was evaluated by the in vitro calcein release assay as described. As effector cells (E), patient #5 PBMC were stimulated during 12 days with antigenic peptides (with or without nivolumab addition) as described and different effector: target ratios were tested in quadruplicate. FIG. 9A shows the results for PRE, POST1 and POST3-PBMC samples; FIG. 9B shows the results for POST3 -PBMC samples. *p<0.05; ** p<0.005; *** p<0.0005. (2-way Anova, Tukey's multiple comparison test).

DETAILED DESCRIPTION OF THE INVENTION

This disclosure relates to methods for enhancing an anti-tumor immune response to treatment in subjects with CM who have been treated with a vaccine generating an immune response against antigens of CM cells. Also provided are methods for treating CM in a subject previously treated with a vaccine generating an immune response against antigens of CM cells. In one aspect, the subject is treated with an anti-PD-1 MAb following treatment with the vaccine generating an immune response against antigens of CM cells.

Anti-PD-1 MAb

Any anti-PD-1 MAb can be used following treatment with a cancer vaccine in a subject in need thereof. Examples of anti-PD-1 MAbs include, but are not limited to, nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), MEDI0680 (Astrazeneca), SPARTALIZUMAB (Novartis), DOSTARLIMAB (JEMPERLI, (GSK)), and tislelizumab (BEIGENE).

Vaccine generating an immune response against antigens of CM cells

The anti-PD-1 MAb can be administered to a subject which has been treated with any vaccine which is effective in generating an immune response in a subject against antigens of CM. For instance, such a vaccine may comprise inactivated CM cells which provide the antigens necessary for triggering the subject’s immune response. By “inactivated”, it should be understood that the CM cells are incapable of proliferating. Such inactivation may be achieved for instance, by irradiating the cells.

U.S. Patent 8,501,168 describes a series of human melanoma cell lines which are effective for generating an effective immune response against CM: (a) Mel-XYI (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2830), (b) Mel-XY2 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2831), (c) Mel-XY3 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2832), and (d) Mel- XX4 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2829). Correspondingly, in an embodiment of the invention, prior to the administration of the anti-PD-1 Mab, the subject has been treated with a vaccine comprising at least one of the aforementioned cell lines, wherein said cell lines have been irradiated, and are incapable of proliferating. The contents of the disclosure of U.S. Patent 8,501,168 are hereby incorporated by reference.

A vaccine particularly suitable to be followed up with an anti-PD-1 MAb according to the present invention is the vaccine known as VACCIMEL, which comprises all four irradiated Mel- XYI, Mel-XY2, Mel-XY3 and Mel-XY4 cell lines, as well as BCG (Bacillus Calmette-Guerin) and GM-CSF (granulocyte-macrophage colony-stimulating factor) as adjuvants.

Method of Treatment

In one aspect, the subject has been treated with a vaccine which is effective in generating an immune response in a subject against antigens of CM for at least two years such as 2 years, 3 years, 4 years, 5 years, or 6 years prior to the administration of the anti-PD-1 MAb. The subject is treated with the vaccine as specified for the corresponding treatment regime.

In one aspect, the subject is treated with an anti-PD-1 MAb about 10 to about 60 months following treatment with a vaccine which is effective in generating an immune response in a subject against antigens of CM (such as VACCIMEL). For example, the subject is treated with anti-PD-1 MAbs following 10 months, 15 months, 20 months, 25 months, 30 months, 40 months, 45 months, 50 months or 60 months after treatment with the vaccine. Preferably, the subject is treated with the anti-PD-1 MAb about 21 to about 57 months following treatment with the vaccine. In one aspect, the subject is treated with an anti-PD-1 MAb 21 months, 29 months, 40 months, or 57 months after treatment with the vaccine. In one aspect, the subject is intravenously administered anti-PD-1 MAbs.

Treatment with anti-PD-1 MAbs acting on pre-existing tumor-reactive lymphocytes, induces clinical responses in CM patients, albeit in a fraction of treated patients.

In one aspect, the subject is treated with about 2 mg/kg to about 3 mg/kg of anti-PD-1 MAb such as 2 mg/kg, 2.5 mg/kg, 3 mg/kg of anti-PD-1 MAbs or any value in-between. In one aspect, the subject is administered the anti-PD-1 MAb every three weeks. In one aspect, the subject is administered the anti-PD-1 MAb every three weeks for at least six months. In one aspect, the subject is administered the anti-PD-1 MAb until detection of cancer recurrence.

In one aspect, the sequential treatment of the cancer vaccine and anti-PD-1 MAbs results in a complete response (CR) ranging from about 3 months to about 80 months such as 3 months, 5 months, 10 months, 15 months, 20 months, 25 months, 30 months, 35 months, 40 months, 45 months, 50 months, 55 months, 60 months, 65 months, 70 months, 75 months, 80 months, or any value in-between. In one aspect the Anti-PD-1 MAbs are usually administered without any previous treatment.

The current proposed mechanism accepts that the existing anti-tumor-reactive T cell clones act by reversing their exhaustion and increasing their cytotoxicity. Consequently, there remains a need to widen the immune system's reactivity against a greater number of Tumor- Associated Antigens (TAA) and neoAgs for the treatment of CM.

A method for enhancing the anti-tumor immune response in a subject carrying a cutaneous melanoma (CM) who was treated with a vaccine generating an immune response against a repertoire of antigens of CM cells, the method comprising administering a therapeutically effective dose of an anti-PD-1 monoclonal antibody (MAb) following treatment with the vaccine generating an immune response against antigens of CM cells.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entireties.

As used herein, the term “subject” refers to an animal, preferably a mammal such as a human. Individuals and patients are also subjects herein.

As used herein and in the appended claims, the singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise.

The term “about” refers to a range of values which would not be considered by a person of ordinary skill in the art as substantially different from the baseline values. For example, the term “about” may refer to a value that is within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value, as well as values intervening such stated - values.

The following example serves to further illustrate the methods of the present disclosure. EXAMPLES

Example 1. Cases descriptions

To widen the immune system reactivity against a greater number of Tumor- Associated Antigens (TAA) and neoAgs, the inventors developed the CM allogeneic vaccine VACCIMEL, with BCG and GM-CSF as adjuvants. Two Phase I trials were performed on stages IIB-IV CM patients, which demonstrated that the combination did not induce any high-grade toxicity (Barrio MM et al. 2006). Also, a randomized phase II study (CASVAC- 0401) comparing VACCIMEL versus IFN-alpha2b in adjuvancy was performed in stages IIB, IIC and III CM patients (Mordoh J et al., 2017}. After 48 months of follow-up, the vaccinated patients had a significantly higher median distant-metastases free survival (DMFS) than those treated with IFN-alpha2b (96 months and 13 months, respectively). Also, VACCIMEL-treated stage III CM patients attained, at 48 months follow up, similar DMFS than patients treated with anti-PD-1 MAbs (Mordoh A etal., 2022).

A case study was conducted with patients #1 to #5 who were previously treated with VACCIMEL in different Phase I and Phase II clinical studies, approved by the Institutional Review Board of the Alexander Fleming Institute. The goal of this study was to determine if the combination of VACCIMEL with anti-PD-1 MAbs increased the clinical responses or provoked special toxi cities. All patients provided written informed consent for publication of his/her results. With the exception of patient #1, who had participated in a previous Phase I study, the rest of the patients were selected from a 30 patients cohort treated with VACCIMEL (Mordoh A et al., 2022}. From those patients, five progressor patients to metastatic disease were selected to obtain detailed clinical stories and the pertinent imaging studies. Those patients were treated at some point with anti-PD-1 MAbs, some of them among other treatments. The details of the disease course and treatments received are summarized in a swimmer plot (FIG. 1). The following examples detail four patients. A fifth patient (case #5) has been previously described (Mordoh A et al., 2022}. A summary of the detailed clinical data of the patient's tumors is described in Table 1. The sequence of patients’ evolution is described in Table 2. Table 1. Characteristics of the primary tumors from patients. NA: Not Available.

Table 2. Main events of the disease and treatments received by all patients. ICI: Immune Checkpoint Inhibitors; PR: Partial Response: CR: Complete Response: PD: Progressive Disease.

*Time of diagnosis of the primary tumor, except for Case#l, who received VACCIMEL treatment following resection of an intestinal metastasis. **Duration of CR to anti-PD-1 treatment.

Example 2. Case #1-Case#4

2A) Case #1

Case #1 is a 65-y ear-old white Caucasian female to whom in 1993 an inferior left dorsal CM was excised. In 04/2009 (FIG.l) she started vomiting, and in 05/2009 a segmentary resection of the small intestine was performed. An intramural CM metastasis was found and 3/9 analyzed lymph nodes (LNs) were also metastatic. Histological analysis of the small intestine metastasis revealed an amelanotic CM without lymphoid infiltration. Therefore, the patient was at stage IV of her disease. A PET scan performed in 06/2009 revealed residual disease at cervical LNs, mesenteric root LNs, and subcutaneous metastasis in the left gluteus. In 07/2009 the patient entered a phase I clinical study of autologous dendritic cells (DC) loaded with VACCIMEL, but she could only receive 3 doses of 10 x 10⁶ DC loaded with apoptotic-necrotic tumor cells, 2 weeks apart, since the yield of PBMC after leukapheresis was low. The patient was therefore shifted to receive VACCIMEL plus BCG plus GM-CSF every two months. In 02/2010 an intra-treatment inguinal adenopathy appeared that was resected. Histologic analysis revealed a metastatic lymph node with brisk tumor infiltration with CD8⁺ lymphocytes and a high number of CD1 lc⁺ DC (FIG. 2A). In 12/2010 and 03/2012 a para-psoas LN and a gluteus dermic metastases were excised. Between 07/2009 and 09/2014 the patient received 23 vaccinations. In 01/2015 a PET/CT scan revealed several hypercaptating nodules at both lungs and two hypermetabolic para-splenic and para- pancreatic nodules (FIG. 2B, PRE). Since the patient's tumor had the BRAF^V600E mutation, she was treated with vemurafenib between 01/2015 and 05/2015, at which time it was permanently suspended because of grade III dermic toxicity and tumor progression. Starting in 09/2015 she received five courses of Ipilimumab (3 mg/kg every three weeks), and in 06/2016 she started treatment with pembrolizumab (2 mg/kg every three weeks), which she continued until 09/2019, without significant toxicity (see timeline in FIG. 1). The patient attained CR in 04/2018 and remains disease-free until 09/2023 (65 months+) (FIG. 2B, POST). The period between the end of vaccination and the start of pembrolizumab was 21 months.

2B) Case #2

Case #2 is a 59-year-old, white Caucasian male. Since 2006, he has been allergic to undefined allergens, with sneezing and dyspnea. In 01/2016 an epithelioid CM was excised from his back. The lesion was in a vertical growth phase, had a Breslow index of 2.3 mm, it was not ulcerated and had an intermediate mitotic index. Lymphoid infiltration was not detected. A sentinel lymph node biopsy (SLNB) in the right axilla was performed and a micro-metastatic LN was found, which contained gpl00⁺ and Melan-A⁺ cells. The patient was at stage IIIA and radical LN dissection was not performed at that time. In 04/2016 he signed the informed consent to enter the CASVAC-0401 study (FIG. 1). In 08/2016 two cutaneous metastatic lesions close to the primary tumor scar appeared and were excised. The patient continued in the study as allowed per protocol. From 11/2016 onward the BCG doses in VACCIMEL were reduced to 2x10⁵ CFU per vaccination due to intense local reaction. In 03/2017, a dermic nodule in the upper right scapular area and an enlarged right axillary LN were detected and biopsied (FIG. 3A). In both cases, metastases were found and excised in 05/2017, after a PET/CT scan revealed o distant metastases.

Histopathological analysis of LN revealed brisk CD8⁺ infiltration and the excised metastasis revealed a portion heavily infiltrated by CD8⁺PD1⁺ cells and another part of the same nodule with almost no lymphoid infiltration (Aris Met al., 2019). The patient continued in the study and ended his participation in 07/2018 without further events. Noteworthy, peripheral blood mononuclear cells (PBMC) increased IFN-y release to TAA following immunization (FIG. 3B). In 1/2021, a CAT-Scan revealed a right paracardiac nodule (17 mm diameter) and a micronodule (3 mm diameter) in the upper left lung lobule. The patient, whose tumor was WT for BRAF oncogene, started treatment in 01/2021 with pembrolizumab (200 mg every 3 weeks). Therefore, 29 months elapsed since the end of VACCIMEL treatment and start of pembrolizumab treatment. In 05/2021 the right paracardiac nodule reached 2.4 cm diameter and the pulmonary nodule increased to 1.2 cm, as detected by PET-CAT scan (FIG. 3C PRE). After 12 infusions, a new CAT-Scan was performed in 09/2021 and a CR of the lung (FIG. 3C POST) and paracardiac nodules was observed. The patient has remained disease-free until 09/2023, when data were locked for this study. Thus, CR has so far lasted 24 months.

2C) Case #3

Case #3 was a 49-year-old white Caucasian male, to whom in 01/2010 a dorsal melanoma was excised. The tumor was an ulcerated nodular polypoid melanoma, in vertical growth phase; Breslow index 4.1 mm; 2 mitosis/sq mm. Lymphoid infiltration was non-brisk, and satellitosis was present. In 03/2010, SLNB was performed, and 1/3 LN was found to be metastatic. This was followed by radical lymphadenectomy, which was negative for metastases (0/11 LN). Therefore, the patient was at stage IIIC of the disease. Histological analysis of the LN metastasis revealed scarce infiltration of tumor nests by CD8⁺ cells; HLA-I negative tumor cells were abundant (FIG. 4A left) and CD11C⁺ cells were scarce (FIG. 4A right). This patient had therefore bad prognostic features (Bravo Al et al., 2023) On 26/04/2010 the patient entered the CASVAC 0401 study (Mordoh J et al, 2017), and he was randomized to the vaccine arm (FIG. 1). After receiving 13 vaccinations with VACCIMEL plus BCG plus GM-CSF, the patient ended NED (no-evidence of disease) the 2-year protocol in 05/2012 and received two additional vaccinations in 11/2012 and 06/2013. Reactivity to TAA increased throughout immunization, as determined by IFN-y release from PBMC response in ELISPOT assays, thus revealing immune capabilities (FIG. 4B). He remained NED until 03/2018 (52 months later), when clinical progression with left supraclavicular adenopathies, muscular and subcutaneous metastases, a nodule in the basal right lung and several small brain metastases were detected (FIG. 4C, PRE). Three small brain lesions were treated in 03/2018 with gamma-knife surgery, and he started nivolumab treatment at the dose of 3 mg/kg every three weeks, which he continued until 12/2018. A new PET/CAT Scan performed in 09/2018 revealed that the patient achieved a CR of the previous lesions (FIG. 4C POST); the etiology of an increased isotope uptake observed at the root of the ascending aorta and the right atrium was not ascertained. In 12/2018 the patient's brain metastases recurred and since the patient's tumor had the BRAF^V600E mutation, the treatment was shifted to dabrafenib/trametinib in 01/2019. The duration of the CR was therefore 3 months and the patient passed away in April 2020. The period between the end of vaccination and start of nivolumab treatment was 57 months.

2D) Case #4

Case #4 is a 56 years-old white Caucasian male to whom in 2006 a melanoma in the retro- auricular right pavilion was excised (FIG. 1). The lesion had a 6mm Breslow index and it was ulcerated. SLNB was performed, and a micrometastasis was found. Radical cervical LN resection was performed and no further metastatic LN were found (0/15 LN). A CAT-Scan revealed no metastatic disease. Therefore, this patient was at stage IIIC of his disease. In 07/2006 VACCIMEL treatment was started with the addition of GM-CSF 120 mg id for four days and BCG (0.5 x 10⁶ CFU per vaccination). From 08/2007 BCG doses were reduced and then suspended due to ulceration at the vaccination site. Between 08/2007 and 07/2008, he continued vaccination every three months without BCG, and then once a year until 05/2018, having no evidence of disease, twelve years after SLNB. However, a control CAT scan performed in 04/2019 revealed a 41 x 22 mm right lung hilar adenopathy determining an interval of 11 months between the end of vaccination and the appearance of metastasis. A PET scan confirmed the lesion but no other metastases were found. In 07/2019 a VATS surgery was performed, a biopsy was obtained and revealed melanoma metastasis, which had the BRAF^V600E mutation. Due to the COVID-19 pandemic, this patient could only start on 02/2020 treatment with pembrolizumab (200 mg every 3 weeks), of which he could only receive three infusions because of travel restrictions (FIG. 2C, PRE) Therefore, the interval between the last vaccination and the start of pembrolizumab was 21 months. In 07/2020 a new CAT-Scan was performed and revealed CR of the hilar adenopathy (FIG. 2C, POST) He received no further treatment and remains disease- free for 38 months until the time this report is written. E) Case #5

Case #5 is a 47-year-old, white Caucasian female, to whom in 02/2008 a CM was excised from her right leg. Two years after surgery, satellite lesions appeared and were excised; inguinal LN were spared. On 07/10 the patient entered the CASVAC 0401 study and she was randomized to the VACCIMEL arm (FIG. 1). This patient’s case was described in detail in a previous paper from the inventors (Mordoh A et al.; 2022). After responding to VACCIMEL for 30 months, the patient progressed locally and was treated with vemurafenib, and ipilimumab, which response was an increase in the number of reddish lesions in her right leg and the treatment was interrupted. The patient also received hyperthermic perfusion with a CR, followed one year later by reappearance of the lesions. After relapse, she received nivolumab, administered at 300 mg every three weeks, which induced a CR that lasted 48 months before recurrence, which incidentally took place in the posterior part of the leg whereas previous lesions were mostly located in the inner part. Thirteen years after entering the CASVAC 0401 study, the patient is alive and well, with small lesions still confined to her right leg.

Conclusions

Five out of five vaccinated patients who progressed even years after ending vaccination, responded with CR to anti-PD-1 treatment. Patients #1 and #5 received ipilimumab prior to anti- PD-1; in the case of patient #5 this was accompanied by local disease progression. Bulk tumors were present in patients #1 and #3; the rest of the patients had small lesions. The case of patient #2 is of special interest since it was shown that different metastases simultaneously excised had diverging lymphoid infiltration (FIGS. 3A-3C). This patient may have a few dominant tumor clones with the ability of metastasize: some clones would express the adequate Ags repertoire that makes them sensitive to cytotoxic lymphocytes; other clones would be “silent” with respect to Ags expression and therefore resistant to CD8⁺ cells.

With respect to patient #3, who has the shortest CR, it should be mentioned that his tumor cells had a quite low HLA-I expression; therefore, it is not surprising that even when the patient was able to build immunity, the HLA-I^neg target cells would be resistant (FIGS. 4A-4C).

It is generally accepted that anti-PD-1 MAbs act by relieving immune suppression exerted by tumor cells or by the immune microenvironment through the PD-l/PDL-1 axis (Lee J and Kim EH, 2023). As it refers to the intratumoral mechanism of action of anti-PD-1 MAbs in vivo, the evidence is contradictory. Ahmazadeh el al. (Ahmadzadeh M et al., 2009) analyzed in 28 pretreated metastatic CM patients, lymphocytes purified from dissociated tumors (tumor infiltrating lymphocytes, TIL) and from normal adjacent tissues. They found that the majority of TIL, opposite to lymphocytes from normal tissues, expressed PD-l⁺CTLA-4⁺HLA- DR⁺Ki67⁺CD127‘, a phenotype that the authors assume characterizes exhausted lymphocytes. Divergently, Ribas et al. (Ribas A et al., 2016) studied 102 biopsies from 53 metastatic CM patients, obtained before treatment (basal) and during treatment with pembrolizumab. They found that PD-1 blockade increased the frequency of T cells, B cells, and myeloid-derived suppressor cells in

They found that the presence of WT PD-1 in CD8⁺ cells led to a restriction of cell proliferation in the early effector phase, this restriction leading to a high amount of memory cells. Instead, KO PD-1 CD8⁺ lymphocytes originated a stronger proliferation in the effector phase but a diminution of memory cells, and therefore a lesser recall activity. These findings suggest that a complicated interrelationship between the length of anti-PD-1 treatment and clinical results in patients may exist.

A puzzling feature of anti-PD-1 treatment is why, in cases in which SD or PR are the clinical outcomes, the tumor control is maintained even when the antibody has disappeared from the blood (Bonilla FA. 2008)). This outcome may have a reasonable explanation if a pathological CR is obtained, since the disappearance of every tumor cell could explain the lack of recurrences. However, as previously mentioned (Hamid O et al., 2019) in the pembrolizumab KeyNote 001 study, the ORR (overall response rate) was composed of 16% of patients attaining CR; 24% attaining PR and 25% attaining SD. Response was ongoing in 89% of patients who achieved CR and in 63% of patients who achieved PR. This suggests that even in patients who attain PR or SD, in whom tumor cells are presumably still present, immune activity is present long after pembrolizumab was cleared from blood, since the plasmatic concentration of pembrolizumab has a 11/2 of about three weeks. These results also highlight the importance of achieving CR in treated patients. As to the mechanism by which prior vaccination can enhance the effect of anti-PD-1 MAbs, the inventors previously demonstrated that VACCIMEL treatment induces T cell clones directed against TAA, such as MD- Ags and neoAgs, and which were usually non-detected before vaccination (Podaza E et al. 2020; Aris M et al. 2019, Aris M et al., 2018).

Every 3 days, fresh CTL medium with IL-2 was added. At day 10, additional Patient #2 PBMC samples were thawed, the percentage of CD20⁺ and CD14⁺ cells (Ag presenting cells, APC) were recorded by flow cytometry, and cells were pulsed with peptides during 48 h. At day 12, APC were treated with mitomycin C (50pg/ml) during 2hr, washed twice with PBS and resuspended in RPMI 1640 supplemented with 10% FBS. In addition, effector cells were collected, washed with PBS and resuspended in RPMI 1640 supplemented with 10% FBS. Effector cells (6 x 10⁴) were seeded in 96- well plates (previously coated with 5 pg/ml mouse anti-human IFN-y) and APC were added in a 10: 1 ratio, APC/well (0.6 x 10⁴ CD20⁺ plus CD14⁺ cells) and cultured O.N. As a positive control PBMC (6 xlO⁴) were seeded and stimulated with 30 ng/ml OKT3 plus 1/1,000 PHA (M form, Gibco Life Technologies). As a negative control, non-stimulated cells were co-cultured with non-pulsed APC. Each experimental condition was performed in triplicate. ELISPOT plates were developed as previously described by Mordoh J et al., 2017. Plates were scanned using an AIDzSPOT ELR088IFL analyzer to quantify the number of spots per well; 350 spots/well were set as the maximum quantification limit. As observed in Figure 5, no T cells recognizing TAAs were detected in Pt #2 PBMC before VACCIMEL (PRE-PBMC). After 2 years of VACCIMEL treatment (POST 3-PBMC) T cells producing IFN-gamma were detected in response to TYR2, PMEL87 and PMEL95 peptides from PMEL/gplOO and Tyrosinase TAAs, demonstrating immunization against melanoma- associated antigens. Thus, vaccination induced an antitumor immune response, not spontaneously present. PMEL/gplOO antigen was expressed in the tumor of patient #2, as shown in Figure 6.

In vitro incubation with anti-PD-1 antibody increased PBMC response to some TAA. In PRE-PBMC a low frequency of T cells reacting to TYR3 peptide was detected. In the case of POST3-PBMC, addition of anti-PDl to the culture increased the T cell responses to TYR1, TYR3 and PMEL95 peptides, but decreased the T cell responses to TYR2 and PMEL87 peptides. However, the responses found in post-vaccination PBMC samples (either with or without Nivolumab) were always higher than those detected in the PRE-PBMC sample.

In a different assay, three peripheral blood mononuclear cells (PBMC) samples from patient #2 were studied: PRE-PBMC, obtained before VACCIMEL treatment, POST 3-PBMC, obtained after 2 years of VACCIMEL treatment (13 vaccinations) and a PBMC sample obtained after receiving 10 months of Pembrolizumab treatment (1-11-2021 (from now on named anti-PDl treatment-PBMC). The ELISPOT assay was performed as described before, but no Nivolumab was added to the PBMC cultures.

As observed in Figure 7, after 10 months of pembrolizumab treatment (anti-PDl treatment-PBMC), T cells reactive to TYR2 and PMEL87 were still detected in peripheral blood. These T cells were induced by VACCIMEL treatment since they were not detected in PRE sample but were present in POST-3-PBMC. Of note, the frequency of T cells recognizing TAA in the ELISPOT assay present in the Anti-PDl treatment and POST3-PBMC samples, were of the same order. POST-3 and Anti-PDl treatment PBMC responses were all significantly higher than PRE-PBMC response (p<0.0001).

Supporting our disclosure, the immune response to TAA expressed in the patient's tumor was not detected previously to vaccination, it was induced by VACCIMEL immunization and was detected in PBMC during anti-PDl treatment. This fact is relevant since, despite the long time from the last VACCIMEL dose and the Anti-PDl treatment sample (40 months), T cells recognizing TAA were still detectable in PBMC. Our results suggest that the expansion of these T cells could have been involved in the anti-tumor clinical response observed in Patient#2.

Example 4 - T cell phenotype after in vitro stimulation with TAA peptides of PRE, POST 3 and Anti-PDl Treatmen t-PBMC samples.

In another experiment, PBMC samples were incubated with a TAA peptide pool (TYR2, TYR3 PMEL95, PMEL87) for 12 days, as described before, starting from 0.5xl0⁶ cells. After culture, the T cell phenotype (memory populations, proliferation, activation and exhaustion markers) was analyzed by flow cytometry. Results are shown in Table 4.

Results at baseline:

Anti-PDl treatmen t-PBMC were the more proliferating as assed by Ki67 staining (both CD4+ and CD8+ T cells).

Anti-PDl in vivo PBMC, contained less exhausted T cells (both CD4+ and CD8+) than PRE and POST 3-PBMC.

Regarding activation markers, Anti-PDl treatmen t-PBMC showed a higher proportion of CD137+ and CD69+ T cells (both CD4+ and CD8+) than PRE and POST3-PBMC, Anti-PDl treatment-PBMC showed a higher proportion of HLA-DR+ CD4+ T cells, than PRE and POST 3-PBMC, indicating that anti-PD-1 treatment rendered T cells in a more activated state.

Results after 12 days of culture:

After in vitro stimulation with the TAA peptide pool, POST3-PBMC and Anti-PDl treatment- PBMC showed a higher activation as compared to PRE-PBMC, as evidenced by the increment in the proportion and expression of CD137+, HLA-DR+ and CD69+T cells.

In the case of exhaustion markers, TAA peptide stimulation of anti-PDl treatment-PBMC achieved the lowest proportion of exhausted PD1+ T cells (both CD4+ and CD8+). Table 4: Patient #2 PBMC phenotype

Example 5 - T Cell Memory Phenotype

Also, PBMC memory populations were assessed by CCR7 and CD45RO staining, which allows to discriminate them as follows (Table 5):

• CCR7+CD45RO- Naive T cells

• CCR7+CD45RO+ Central memory T cells

• CCR7-CD45RO+ Effector Memory T cells

• CCR7-CD45RO- Temra (terminally differentiated effector memory) T cells

Table 5: Patient#! PBMC samples. T cells memory populations

At Baseline Anti-PDl treatment-PBMC had the highest proportion of effector memory T cells (CD45RO+CCR7-), and the lowest proportion of TEMRA (terminally differentiated effector memory T cells, (CD45RO-CCR7-), as compared to PRE and POST3 T cells, suggesting that anti-PDl treatment allowed a higher expansion of proliferative effector memory cells.

POST-3 and Anti-PDl treatment-PBMC expanded in response to the TAA peptide pool in vitro displayed a similar pattern, with an increase in the proportion of effector memory T cells (especially CD8+) and a reduction of naive and central memory T cells, different from PRE- PBMC

After 12 days of stimulation with the TAA peptide pool, PRE -PBMC showed the highest proportion of naive T cells (CD4+ and CD8+) as compared to POST3-PBMC and Anti- PDl treatment-PBMC.

The TEMRA population present at baseline did not expand and disappeared from cultures in all the PBMC samples, due to their lack of proliferating capacity. Example 6 - VACCIMEL T cell response developed in Patient #2

In a previously published work, the inventors analyzed the T cell receptor dynamics studied by TCR0 sequencing. (Aris M et al., 2019).

The CSF-470/VACCIMEL cellular vaccine plus BCG and rhGM-CSF increased distant metastases-free survival in cutaneous melanoma patients stages IIB-IIC-III relative to medium dose IFN-a2b (CASVAC-0401 study). Patient #2 developed a mature vaccination site (VAC- SITE) and a regional cutaneous metastasis (C-MTS), which were excised during the protocol, remaining disease-free 36 months from vaccination start.

CDR3-TCRb repertoire sequencing in PBMC and tissue samples, along with skin-DTH score and IFN-g ELISPOT assay, were performed to analyze the T-cell immune response dynamics throughout the immunization protocol. Histopathological analysis of the VAC-SITE revealed a highly-inflamed granulomatous structure encircled by CDl lc+ nested-clusters, brisk CD8+ and scarce FOXP3+, lymphocytes with numerous Langhans multinucleated-giant-cells and macrophages.

A large tumor-regression area fulfilled the C-MTS with brisk lymphocyte infiltration, mainly composed of CD8+PD1+ T-cells, CD20+ B-cells, and scarce FOXP3+ cells. Increasing DTH score and IFN-g ELISPOT assay signal against the CSF-470/VACCIMEL vaccine- lysate was evidenced throughout immunization. TCRb repertoire analysis revealed for the first time the presence of common clonotypes between a VAC-SITE and a C-MTS; most of them persisted in blood by the end of the immunization protocol. In vitro boost with vaccine-lysate revealed the expansion of persistent clones that infiltrated the VAC-SITE and/or the C-MTS; other persistent clones expanded in the patient’s blood as well.

We propose that expansion of such persistent clonotypes might derive from two different although complementary mechanisms: the proliferation of specific clones as well as the expansion of redundant clones, which increased the number of nucleotide rearrangements per clonotype, suggesting a functional antigenic selection. In this patient, immunization with the CSF- 470/VACCIMEL vaccine plus BCG and rhGM-CSF induced a T-cell repertoire at the VAC-SITE that was able to infiltrate an emerging C-MTS, which resulted in the expansion of a T-cell repertoire that persisted in blood by the end of the 2-year treatment.

Example 7 - Results from patient #5

Immune response to tumor-associated antigens (TAA) and Neoantigens (NeoAgs) assessed by IFN-gamma ELISPOT after in vitro treatment with anti-PD-1 antibody

In a first assay, peripheral blood mononuclear cells (PBMC) samples from patient #5 were studied: PRE-PBMC, POST1-PBMC (obtained 6 months after VACCIMEL treatment, 5 vaccinations) and POST2-PBMC (obtained after 12 months of VACCIMEL treatment /10 doses). In a second assay, POST 3-PBMC, (obtained after 2 years of VACCIMEL treatment, 13 vaccinations) were tested. PBMC were thawed and seeded (0.35-0.4 xlO⁶) in 1ml of CTL medium consisting of RPMI 1640 supplemented with 10% heat-inactivated human AB sera, 2 mM glutamine, 100 U/mL penicillin, 100 pg/ml streptomycin, 2.5 mM HEPES, and 50 U/mL IL-2, in 24-well plates. PBMC were stimulated with peptides (10 pg/ml) derived from TAA and NeoAgs (Table 4), and cultured at 37°C, in 5% CO2 for 12 days (effector cells). Effector cells were incubated either with anti-PD-1 antibody (Nivolumab, 10 pg/ml) or without anti-PD-1.

Table 6: Neo Ag peptides were predicted as described in Carri I et al., 2023. TAA HLA-class I restricted peptides were selected from the TANTIGEN DataBase (http://projects.met- hilab. org/tadb/). Selected peptides were either T cell epitopes previously identified in functional assays (in vitro and/or in vivo) orHLA ligands as determined by physical detection

Every 3 days, fresh CTL medium with IL-2 was added. At day 10, additional Patient #5 PBMC samples were thawed, the percentage of CD20⁺ and CD14⁺ cells (Ag presenting cells, APC) were recorded by flow cytometry and cells were pulsed with peptides during 48 h. At day 12, APC were treated with mitomycin C (50pg/ml) during 2hr, washed twice with PBS and resuspended in RPMI 1640 supplemented with 10% FBS. In addition, effector cells were collected, washed and resuspended in RPMI 1640 10% FBS. Effector cells (4 x 10⁴) were seeded in 96- well plates (previously coated with 5 pg/ml mouse anti-human IFN-y) and APC were added in a 10: 1 ratio, APC/well (0.4 x 10⁴ CD20⁺ plus CD14⁺ cells) and cultured O.N. As a positive control PBMC (4 xlO⁴) were seeded and stimulated with 30 ng/ml OKT3 plus 1/1,000 PHA (M form, Gibco Life Technologies). As a negative control, non-stimulated cells were co-cultured with non-pulsed APC. Each experimental condition was performed in triplicate. ELISPOT plates were developed as previously described (Podaza E et al., 2020). Plates were scanned using an AIDzSPOT ELR088IFL analyzer to quantify the number of spots per well; 350 spots/well were set as the maximum quantification limit. As observed in Figure 8A, treatment with VACCIMEL induced T cells that were able to produce IFN-gamma upon recognition of neoantigens and TAA peptides, mostly in POST2-PBMC, but these T cells were not detected in PRE- vaccination PBMC. In vitro addition of nivolumab during the 12-day culture increased the number of T cells recognizing PMEL2, TYR1 and MAGEB2 TAA peptides, although not reaching statistical significance.

In vitro incubation of POST 3-PBMC (Figure 8B) with anti-PD-1 antibody (nivolumab) during the 12-day culture, significantly increased the number of T cells producing IFN-gamma upon peptide stimulation in case of neoantigen MGAT4A (p=0.0016) and PMEL2 (pO.OOOl) peptides.

Example 8 - Tumor Cell Lysis Assay

One relevant point to assess is if T cells induced by VACCIMEL treatment were able to kill melanoma cells, and if anti-PDl treatment increases the ability of these T cells to destroy tumor cells.

In the case of patient #5, the inventors performed an in vitro assay to assess melanoma cell lysis by PBMC (obtained before and after VACCIMEL treatment) on the MEL-XY3 melanoma cell line (HLA-A*02:01+), which expresses the three TAA, used as target tumor cells. This cell line was used since autologous tumor cells were not available. In a first assay PRE-PBMC, POST1- PBMC and POST2-PBMC were stimulated in vitro with TAA peptides (SOX2, PMEL 2, TYR 5, all HLA-A*02:01 restricted) for 12 days with or without nivolumab (lOpg/ml) as described before, and cultured for additional 24 hs in RPMI 1640 supplemented with 10% FBS (effector cells). In a second assay, POST3-PBMC were used.

Tumor cell lysis assay was performed as previously described (Podaza E et al., 2020 ). Briefly, target cells were labeled with 16pM calcein-acetoxymethyl for 30min at 37°C, washed twice and resuspended in serum-free assay medium for T cells (AIMV, Life Technologies). Target cells were centrifuged, resuspended in assay medium and seeded in 96- well plates (5 x 10³ cells/well). Effector cells from each PBMC sample (PRE-PBMC, POST1-PBMC, POST2-PBMC or POST3-PBMC) were obtained as a pool of cell cultures stimulated with each TAA peptide. Different effector: target ratios were tested in quadruplicate. For spontaneous and maximum release, targets were incubated without effector cells in assay medium alone or assay medium plus 2% Triton X-100, respectively. Plates were incubated for 4 h at 37°C in 5% CO2, centrifuged, and calcein release was quantified in supernatants in a fluorimeter (485/520 nm OPTIMA, BMG Labtech). The specific lysis (%) was calculated as: (experimental release — spontaneous release)/(maximum release — spontaneous release) x 100.

As observed in Fig. 9A, PRE-VACCIMEL PBMC had a very low capacity to lyse melanoma cells as compared to POST-1 and POST2-PBMC. The addition of nivolumab during culture increased POST-l-PBMC lytic capacity. E: T 30:1, p=0.0245). POST3-PBMC stimulated with TAA peptides (Fig. 9B) could kill MEL-XY3 melanoma cells and in vitro incubation with nivolumab, increased their lytic capacity. E:T 20: 1 p= 0.0032; E:T 30: 1 p= 0.0006.

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Claims

1. A method for enhancing an anti -tumor immune response in a subject carrying a cutaneous melanoma (CM), the method comprising administering a therapeutically effective dose of an anti-PD-1 monoclonal antibody (MAb), wherein the subject was previously treated with a vaccine generating an immune response against antigens of CM cells.

2. The method of claim 1, wherein the anti-PD-1 MAb is selected from nivolumab, pembrolizumab, MEDI0680, spartalizumab, dostarlimab and tislelizumab.

3. The method of claim 2, wherein the anti-PD-1 Mab is nivolumab or pembrolizumab.

4. The method of any one of claims 1 to 3, wherein the anti-PD-1 Mab is intravenously administered.

5. The method of any one of claims 1 to 4, wherein the anti-PD-1 MAb is nivolumab and it is administered at a dose of about 3 mg/kg or about 240 mg every two weeks or about 480 mg every four weeks.

6. The method of any one of claims 1 to 4, wherein the anti-PD-1 MAb is pembrolizumab and it is administered at a dose of about 2 mg/kg or about 200 mg every three weeks.

7. The method of any one of claims 1 to 6, wherein the subject is administered the anti-PD-1 MAb about 10 to about 50 months after being treated with the vaccine generating an immune response against antigens of CM cells.

8. The method of any one of claims 1 to 7, wherein the vaccine generating an immune response against antigens of CM cells comprises at least one CM cell line which is incapable of proliferating.

9. The method of claim 8, wherein the at least one CM cell line which is incapable of proliferating has been irradiated.

10. The method of any one of claims 8 or 9, wherein the vaccine comprises at least one CM cell line selected from the group consisting of (a) Mel-XYI (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2830), (b) Mel-XY2 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2831), (c) Mel-XY3 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2832), and (d) Mel-XX4 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2829), wherein said cell lines have been irradiated, and are incapable of proliferating.

11. The method of any one of claims 8 to 10, wherein the vaccine generating an immune response against antigens of CM cells comprises: the CM cell lines of the group consisting of (a) Mel-XYI (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2830), (b) Mel-XY2 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2831), (c) Mel-XY3 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2832), and (d) Mel-XX4 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2829), wherein said cell lines have been irradiated, and are incapable of proliferating; and BCG (Bacillus Calmette-Guerin) and GM-CSF (granulocyte- macrophage colony-stimulating factor) as adjuvants.

12. The method of any one of claims 1 to 12, wherein the subject is human.

13. A method for treating cutaneous melanoma (CM) in a subject, comprising administering to the subject about 3 mg/kg or about 240 mg every two weeks or about 480 mg every four weeks of nivolumab, or about 2 mg/kg or about 200 mg every three weeks of pembrolizumab, wherein the subject was previously treated with a vaccine generating an immune response against antigens of CM cells.

14. An anti-PD-1 Mab for use in enhancing an anti -tumor immune response in a subject carrying a CM, wherein the subject was previously treated with a vaccine generating an immune response against antigens of CM cells.

15. The anti-PD-1 Mab for use of claim 14, wherein the anti-PD-1 MAb is selected from the group consisting of nivolumab, pembrolizumab, MEDI0680, spartalizumab, dostarlimab and tislelizumab.

16. The anti-PD-1 Mab for use of claim 15, wherein the anti-PD-1 MAb is nivolumab or pembrolizumab.

17. The anti-PD-1 Mab for use of any one of claims 14 to 16, wherein the anti-PD-1 Mab is intravenously administered.

18. The anti-PD-1 Mab for use of any one of claims 14 to 17, wherein the anti-PD-1 MAb is nivolumab and it is administered at a dose of about 3 mg/kg or about 240 mg every two weeks or about 480 mg every four weeks.

19. The anti-PD-1 Mab for use of any one of claims 14 to 17, wherein the anti-PD-1 MAb is pembrolizumab and it is administered at a dose of about 2 mg/kg or about 200 mg every three weeks.

20. The anti-PD-1 Mab for use of any one of claims 14 to 19, wherein the subject is administered the anti-PD-1 MAb about 10 to about 50 months following treatment with the vaccine generating an immune response against antigens of CM cells.

21. The anti-PD-1 Mab for use of any one of claims 14 to 20, wherein the vaccine generating an immune response against antigens of CM cells comprises at least one CM cell line which is incapable of proliferating.

22. The anti-PD-1 Mab for use of claim 21, wherein the at least one CM cell line which is incapable of proliferating has been irradiated.

23. The anti-PD-1 Mab for use of any one of claims 21 or 22, wherein the vaccine comprises at least one CM cell line selected from the group consisting of (a) Mel- XYI (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2830), (b) Mel-XY2 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2831), (c) Mel-XY3 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2832), and (d) Mel-XX4 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2829), wherein said cell lines have been irradiated, and are incapable of proliferating.

24. The anti-PD-1 Mab for use of any one of claims 21 to 23, wherein the vaccine generating an immune response against antigens of CM cells comprises: the CM cell lines of the group consisting of (a) Mel-XYI (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2830), (b) Mel-XY2 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2831), (c) Mel-XY3 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2832), and (d) Mel-XX4 (deposited at German Collection of Microorganisms and Cell Cultures DSMZ under access number DSMACC2829), wherein said cell lines have been irradiated, and are incapable of proliferating; and BCG (Bacillus Calmette-Guerin) and GM-CSF (granulocyte- macrophage colony-stimulating factor) as adjuvants.