EP4701650A2

EP4701650A2 - Methods of treating cancer by administering immunogenic compositions and a pd-1 inhibitor

Info

Publication number: EP4701650A2
Application number: EP24720029.8A
Authority: EP
Inventors: Cornelis Johannes Maria Melief; Leonard Willem Frederik Hooftman; Israel Lowy; Frank SEEBACH
Original assignee: Isabella Pharma BV; Regeneron Pharmaceuticals Inc
Current assignee: Isabella Pharma BV; Regeneron Pharmaceuticals Inc
Priority date: 2023-04-26
Filing date: 2024-04-10
Publication date: 2026-03-04
Also published as: WO2024223299A3; WO2024223299A2

Abstract

The present disclosure relates to methods of treating or inhibiting head and neck cancer comprising administering a combination of a PD-1 inhibitor and one or more immunogenic compositions capable of inducing an immune response against cells expressing HPV16 antigens. Furthermore, the disclosure relates to treatment of a subject afflicted with a solid tumor, comprising administering one or more immunogenic compositions comprising one or more tumor antigens to said subject, wherein a tumor sample of said subject exhibits a high level of PD-1 expression.

Description

METHODS OF TREATING CANCER BY ADMINISTERING IMMUNOGENIC COMPOSITIONS AND A PD-1 INHIBITOR

FIELD OF THE INVENTION

The present disclosure relates to novel methods of treating or inhibiting head and neck cancer, said methods comprising administering a combination of a PD-1 inhibitor and one or more immunogenic compositions capable of inducing an immune response against cells expressing HPV16 antigens. In a further aspect, the disclosure relates to treatment of a subject afflicted with a solid tumor, comprising administering one or more immunogenic compositions comprising one or more tumor antigens to said subject, wherein a tumor sample of said subject exhibits a high level of PD-L1 expression.

BACKGROUND OF THE INVENTION

Infection with oncogenic HPV types, such as HPV type 16, is quite common in both men and women (Koutsky et al. 1997 Am J Med 102:3-8). Only a minor fraction of infected individuals will develop persistent virus infections and eventually virus-induced disease. The majority of infections spontaneously resolve, most likely through an HPV-specific adaptive immune response. A previous study has demonstrated that HPV16 E6 specific T cell immunity is frequently detected in healthy individuals, suggesting that the immune system plays a role in the protection against persistent HPV infections (Welters et al. 2003 Cancer Res 63:636-641). However, in some cases, the initiation of an HPV16 specific T cell immune response fails or is ineffective, which may result in a persistent HPV16 infection and consequently HPV-induced malignancies, such as cervical cancer and head and neck cancer, for example oropharyngeal cancer. It has been shown that the majority of cervical cancer patients have absent or impaired HPV16 specific T cell immune responses (de Jong et al. 2004 Cancer Res 64:5449-5455).

Massarelli et al. (2019 JAMA Oncol 5(l):67-73) have described a clinical trial for patients with incurable HPV-16-related cancers, combining immune checkpoint blockade (nivolumab, an anti-PD-1 antibody) and a tumor-specific vaccine (ISA 101, a synthetic long-peptide based vaccine) (ClinicalTrials.gov identifier: NCT02426892). The clinical trial excluded patients that had prior therapy with an anti-PD-1, anti-PD-Ll, anti-PD-L2, anti- CD137 or anti-CTLA-4 antibody, or any other antibody or drug specifically targeting T-cell co-stimulation or checkpoint pathways.

Head and neck cancer develops from tissues in the lip and oral cavity, larynx, salivary glands, nose, sinuses or the skin of the face. Oropharyngeal cancer, also termed oropharyngeal squamous cell carcinoma, is amongst the most common forms of head and neck cancer and comprises cancers of the tonsils, base of tongue, soft palate and uvula. Current treatment options include primary surgery, radiotherapy, primary chemoradiotherapy, including platinum-based compounds such as cisplatin, other types of chemotherapy such as taxanes or a combination of a taxane and a platin compound like cis- or carboplatin and the anti-EGFR monoclonal antibody cetuximab and variants or equivalents thereof.

While significant progress has been made in the treatment of head and neck cancer, such as HPV16-positive oropharyngeal cancer, there is still a need for improved treatment regimens that can be used in broader patient populations and induces more rapid, stronger and/or prolonged responses and treatment outcomes while minimizing adverse effects.

Furthermore, in the field of solid tumor cancers generally, there is a need for improved methods of differentiating between patients and for improved methods for identification of subgroups of patients that benefit or benefit more from certain treatment regimens.

SUMMARY OF THE INVENTION

In a first main aspect, the invention relates to a method of treating or inhibiting the growth of a tumor or improving overall survival of a head and neck cancer patient, comprising: a) selecting a patient with HPV16-positive head and neck cancer who progressed on prior immune checkpoint modulatory therapy; b) administering to the patient an effective amount of one or more immunogenic compositions capable of inducing a T cell immune response against cells expressing HPV16 antigens, preferably E6, E7 and/ or E2 antigen; and c) administering to the patient a therapeutically effective amount of a PD-1 inhibitor.

In a second main aspect, the invention relates to a method of treating or inhibiting the growth of a tumor or improving overall survival of a head and neck cancer patient, comprising: a) selecting a patient with HPV16-positive head and neck cancer; b) administering to the patient an effective amount of one or more immunogenic compositions capable of inducing a T cell immune response against cells expressing HPV16 antigens, preferably E6, E7 and/ or E2 antigen; and c) administering to the patient a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof that comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) and a light chain variable region comprising three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein : HCDR1 has an amino acid sequence of SEQ ID NO: 13; HCDR2 has an amino acid sequence of SEQ ID NO: 14; HCDR3 has an amino acid sequence of SEQ ID NO: 15; LCDR1 has an amino acid sequence of SEQ ID NO: 16; LCDR2 has an amino acid sequence of SEQ ID NO: 17; and LCDR3 has an amino acid sequence of SEQ ID NO: 18.

In a third main aspect, the invention relates to a method for the treatment of a solid tumor in a subject, such as a human subject, comprising identifying a subject afflicted with a solid tumor having a PD-L1 expression level above a predefined cut-off value and administering one or more immunogenic compositions comprising one or more tumor antigens to said subject. Accordingly, the invention also relates to a method for the treatment of a solid tumor in a subject, comprising i) identifying a subject afflicted with a solid tumor, wherein, in a tumor sample of said subject, PD-L1 is expressed in at least 5% of the viable tumor cells and/or the CPS is at least 5 and/or the TPS is at least 5 and/or the TAP score is at least 5%, and ii) administering one or more immunogenic compositions comprising one or more tumor antigens to said subject.

DESCRIPTION OF THE FIGURES

Figure 1. Timing of ISAlOlb and cemiplimab doses

Figure 2. Waterfall plot showing Best Change from Baseline in the Target Lesions (%). Data of patients with at least one on-study tumor assessment are shown.

Figure 3. Spiderplot showing Change in sum of Target Lesions. The target lesion of patient B increased initially. The absolute increase was <5 mm, meaning that this increase did not meet the criteria of PD per RECIST 1.1.

Figure 4. Study design. Abbreviations: DOR = duration of response; ECOG = Eastern Cooperative Oncology Group; OPC = oropharyngeal cancer; ORR = overall response rate; OS = overall survival; PD = progressive disease; PFS = progression-free survival; PS = performance status; R = randomization; TTR = time to response.

Figure 5. Timing of ISAlOlb or placebo and cemiplimab doses

Figure 6. Kaplan Meier OS curves in the per protocol subgroup (PPS) of all patients

Figure 7. Kaplan Meier OS curves of those patients with a pre-treatment CPS score of > 20 in the per protocol subgroup (PPS) of all patients.

Figure 8. Timing of ISAlOlb and cemiplimab doses DETAILED DESCRIPTION OF THE INVENTION

As described above, in a first aspect, the invention relates to a method of treating or inhibiting the growth of a tumor or improving overall survival of a head and neck cancer patient, comprising: a) selecting a patient with HPV16-positive head and neck cancer who progressed on prior immune checkpoint modulatory therapy; b) administering to the patient an effective amount of one or more immunogenic compositions capable of inducing a T cell immune response against cells expressing HPV16 antigens, preferably E6, E7 and/ or E2 antigen; and c) administering to the patient a therapeutically effective amount of a PD-1 inhibitor.

Similarly, in a further aspect, the invention relates to a PD-1 inhibitor for use in a method of treating or inhibiting the growth of a tumor or improving overall survival of a head and neck cancer patient, the method comprising: a) selecting a patient with HPV16-positive head and neck cancer who progressed on prior immune checkpoint modulatory therapy; b) administering to the patient an effective amount of one or more immunogenic compositions capable of inducing a T cell immune response against cells expressing HPV16 antigens, preferably E6, E7 and/ or E2 antigen; and c) administering to the patient a therapeutically effective amount of a PD-1 inhibitor.

Similarly, in a further aspect, the invention relates to one or more immunogenic compositions capable of inducing an immune response against HPV16 for use in a method of treating or inhibiting the growth of a tumor or improving overall survival of a head and neck cancer patient, the method comprising: a) selecting a patient with HPV16-positive head and neck cancer who progressed on prior immune checkpoint modulatory therapy; b) administering to the patient an effective amount of one or more immunogenic compositions capable of inducing a T cell immune response against cells expressing HPV16 antigens, preferably E6, E7 and/ or E2 antigen; and c) administering to the patient a therapeutically effective amount of a PD-1 inhibitor.

As used herein, the terms "treating", "treat", or the like, mean to alleviate or reduce the severity of at least one symptom or indication, to eliminate the causation of symptoms either on a temporary or permanent basis, to delay or inhibit tumor growth, to reduce tumor cell load or tumor burden, to promote tumor regression, to cause tumor shrinkage, necrosis and/or disappearance, to prevent tumor recurrence, to prevent or inhibit metastasis, to inhibit metastatic tumor growth, to eliminate the need for radiation or surgery, and/or to increase duration of survival of the subject. In many embodiments, the terms "tumor", "lesion," "tumor lesion," "cancer," and "malignancy" are used interchangeably and refer to one or more cancerous growths.

The term "advanced" when used herein in the context of the cancer or disease does not refer to cancers that are classified as locally advanced.

In one embodiment, the patient progressed on anti-PD-1, anti-PD-Ll, anti-PD-L2, anti-CD137, or anti-CTLA-4 therapy. Such therapy may for example be antibody-based, peptide-based or small molecule based. In one embodiment, the patient progressed on anti-PD-1 antibody, anti-PD-Ll antibody, anti-PD-L2 antibody, anti-CD137 antibody, anti- CTLA-4 or anti-LAG 3 antibody therapy.

In one embodiment, the head and neck cancer is advanced, recurrent, persistent, primary refractory/ refractory, incurable, unresectable and/or metastatic.

The head and neck cancer may still be responsive to prior treatments or not, or no longer, be responsive to prior treatments. The patient may have had one, two, three or more prior treatments for the same indication. The patient may have never achieved a complete response or remission from any prior therapy or even never have achieved partial response or remission from any prior therapy.

In one embodiment, the head and neck cancer is oropharyngeal cancer and the oropharyngeal cancer is advanced, recurrent, persistent, primary refractory/refractory, incurable, unresectable and/or metastatic.

In one embodiment, the patient progressed, for example was refractory or resistant to prior PD-1 therapy. In one embodiment, the patient progressed on prior platinum- containing therapy. In one embodiment, the patient progressed, i.e. was refractory or resistant to prior PD-1 therapy and progressed on prior platinum-containing therapy.

In one embodiment, the patient has:

(1) recurrent or metastatic HPV16-positive OPC and relapsed following primary surgical or chemoradiation treatment, wherein the patient preferably did not receive prior treatment with anti-PDl/anti-PDLl antibody or therapeutic HPV16 comprising vaccines, or

(2) recurrent or metastatic HPV16-positive OPC and has progressed on prior platinumbased therapy, wherein the patient preferably did not receive prior treatment with anti-PDl/anti-PDLl antibody or therapeutic HPV16 comprising vaccines, or (3) previously untreated metastatic HPV-16 OPC, wherein the patient preferably did not receive prior treatment with anti-PDl/anti-PDLl antibody or therapeutic HPV16 antigen(s) comprising vaccines.

In one embodiment, the cancer is PD-L1 positive. In one embodiment, the cancer exhibits elevated expression of PD-L1 protein. In one embodiment, the cancer or cancer- associated leukocytes exhibit elevated expression of PD-L1 mRNA. In one embodiment, the cancer-associated or tumor-draining lymph node cells T cells exhibit elevated expression of PD-1 protein. In one embodiment, the cancer or cancer-associated leukocytes exhibit elevated expression of PD-1 mRNA. In one embodiment, the cancer is PD-L1 positive and the Combined Positivity Score is > 1.

In one embodiment, the patient is not a candidate for curative surgery or curative radiation.

In one embodiment, step a) comprises selecting a patient with HPV16-positive head and neck cancer: i) who progressed on prior immune checkpoint modulatory therapy, such as anti-PD-1, anti-PD-Ll, anti-PD-L2, anti-CD137, or anti-CTLA-4 therapy, for example anti-PD-1, anti- PD-L1, anti-PD-L2, anti-CD137, anti-CTLA-4 or anti-LAG-3 antibody therapy, and ii) wherein, in a tumor sample of said subject, PD-L1 is expressed in at least 5% of the viable tumor cells and/or the CPS is at least 5 and/or the TPS is at least 5 and/or the TAP score is at least 5%.

The Combined Positive Score (CPS) is the number of viable PD-L1 expressing cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100 (wherein values above 100 are adjusted to 100).

The Tumor Proportion Score (TPS) is the number of viable PD-L1 expressing tumor cells divided by the total number of viable tumor cells, multiplied by 100.

The Tumor Area Positivity (TAP) score is the area covered by PD-L1 positive tumor cells and tumor-associated immune cells relative to the total tumor area.

In one embodiment hereof, the tumor sample is a tumor biopsy sample. In another further embodiment, in a tumor sample of said subject,

• PD-L1 is expressed in at least 6% of the viable tumor cells and/or the CPS is at least 6 and/or the TPS is at least 6 and/or the TAP score is at least 6%,

• PD-L1 is expressed in at least 7% of the viable tumor cells and/or the CPS is at least 7 and/or the TPS is at least 7 and/or the TAP score is at least 7%,

• PD-L1 is expressed in at least 8% of the viable tumor cells and/or the CPS is at least 8 and/or the TPS is at least 8 and/or the TAP score is at least 8%, • PD-L1 is expressed in at least 9% of the viable tumor cells and/or the CPS is at least 9 and/or the TPS is at least 9 and/or the TAP score is at least 9%,

• PD-L1 is expressed in at least 10% of the viable tumor cells and/or the CPS is at least 10 and/or the TPS is at least 10 and/or the TAP score is at least 10%,

• PD-L1 is expressed in at least 5% of the viable tumor cells and/or the CPS is at least 10 and/or the TPS is at least 5 and/or the TAP score is at least 10%,

• PD-L1 is expressed in at least 8% of the viable tumor cells and/or the CPS is at least 10 and/or the TPS is at least 8 and/or the TAP score is at least 10%,

• PD-L1 is expressed in at least 11% of the viable tumor cells and/or the CPS is at least 11 and/or the TPS is at least 11 and/or the TAP score is at least 11%,

• PD-L1 is expressed in at least 12% of the viable tumor cells and/or the CPS is at least 12 and/or the TPS is at least 12 and/or the TAP score is at least 12%,

• PD-L1 is expressed in at least 13% of the viable tumor cells and/or the CPS is at least 13 and/or the TPS is at least 13 and/or the TAP score is at least 13%,

• PD-L1 is expressed in at least 14% of the viable tumor cells and/or the CPS is at least 14 and/or the TPS is at least 14 and/or the TAP score is at least 14%,

• PD-L1 is expressed in at least 15% of the viable tumor cells and/or the CPS is at least 15 and/or the TPS is at least 15 and/or the TAP score is at least 15%,

• PD-L1 is expressed in at least 16% of the viable tumor cells and/or the CPS is at least 16 and/or the TPS is at least 16 and/or the TAP score is at least 16%,

• PD-L1 is expressed in at least 17% of the viable tumor cells and/or the CPS is at least 17 and/or the TPS is at least 17 and/or the TAP score is at least 17%,

• PD-L1 is expressed in at least 18% of the viable tumor cells and/or the CPS is at least 18 and/or the TPS is at least 18 and/or the TAP score is at least 18%,

• PD-L1 is expressed in at least 19% of the viable tumor cells and/or the CPS is at least 19 and/or the TPS is at least 19 and/or the TAP score is at least 19%,

• PD-L1 is expressed in at least 20% of the viable tumor cells and/or the CPS is at least 20 and/or the TPS is at least 20 and/or the TAP score is at least 20%, or

• PD-L1 is expressed in at least 10% of the viable tumor cells and/or the CPS is at least 20 and/or the TPS is at least 10 and/or the TAP score is at least 20%.

• PD-L1 is expressed in at least 15% of the viable tumor cells and/or the CPS is at least 20 and/or the TPS is at least 15 and/or the TAP score is at least 20%.

In one embodiment, the patient that is selected is a patient with advanced, recurrent and/or metastatic HPV16-positive oropharyngeal cancer who has experienced disease progression with prior anti-PD-1 therapy. In a further embodiment, the recurrent or metastatic HPV16 positive oropharyngeal cancer is histologically confirmed.

In a further embodiment, HPV16 positivity is confirmed, for example using the method described in Einstein et al 2010 Gynecol Oncol. 2010; 118(2): 116-122.

In a further embodiment, the patient has received a minimum total dose of 600 mg of pembrolizumab or 960 mg of nivolumab or equivalent anti-PD-1 antibody with or without chemotherapy for only 1st or 2nd line recurrent/metastatic HPV16 positive oropharyngeal cancer. In a further embodiment, the last dose of anti-PD-1 has not been more than 6 months prior to the first dose of the treatment of the invention.

In one embodiment, progressive disease has been diagnosed in the patient during or after anti-PD-1 therapy (but not longer than 6 months after the last dose), and anti PD- 1 therapy (as 1st or 2nd line for recurrent/metastatic HPV16 positive OPC) should have been the last treatment regimen that the patient received before entry into the treatment of the invention.

In one embodiment, the patient did not undergo invasive surgery (defined as surgical intervention requiring general or spinal anaesthesia and hospital admission) within 4 weeks prior to start of the treatment of the invention. In one embodiment, the patient did not, after progressing on anti-PD-1 therapy, receive additional anti-cancer therapy (chemotherapy, radiotherapy, experimental TKI's, immunotherapy, anti-EGFR antibodies, surgery).

In a further embodiment, the one or more immunogenic compositions are ISAlOlb (peltopepimut-S) and the PD-1 inhibitor is cemiplimab (see below).

As described above, in a further main second aspect, the invention relates to a method of treating or inhibiting the growth of a tumor or improving overall survival of a head and neck cancer patient, comprising: a) selecting a patient with HPV16-positive head and neck cancer; b) administering to the patient an effective amount of one or more immunogenic compositions capable of inducing a T cell immune response against cells expressing HPV16 antigens, preferably E6, E7 and/ or E2 antigen; and c) administering to the patient a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof that comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) and a light chain variable region comprising three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein : HCDR1 has an amino acid sequence of SEQ ID NO: 13; HCDR2 has an amino acid sequence of SEQ ID NO: 14; HCDR3 has an amino acid sequence of SEQ ID NO: 15; LCDR1 has an amino acid sequence of SEQ ID NO: 16; LCDR2 has an amino acid sequence of SEQ ID NO: 17; and LCDR3 has an amino acid sequence of SEQ ID NO: 18.

Similarly, in a further aspect, the invention relates to a PD-1 inhibitor for use in a method of treating or inhibiting the growth of a tumor or improving overall survival of a head and neck cancer patient, the method comprising: a) selecting a patient with HPV16-positive head and neck cancer; b) administering to the patient an effective amount of one or more immunogenic compositions capable of inducing a T cell immune response against cells expressing HPV16 antigens, preferably E6, E7 and/ or E2 antigen; and c) administering to the patient a therapeutically effective amount of a PD-1 inhibitor, wherein the PD-1 inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof that comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) and a light chain variable region comprising three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein : HCDR1 has an amino acid sequence of SEQ ID NO: 13; HCDR2 has an amino acid sequence of SEQ ID NO: 14; HCDR3 has an amino acid sequence of SEQ ID NO: 15; LCDR1 has an amino acid sequence of SEQ ID NO: 16; LCDR2 has an amino acid sequence of SEQ ID NO: 17; and LCDR3 has an amino acid sequence of SEQ ID NO: 18.

Similarly, in a further aspect, the invention relates to one or more immunogenic compositions capable of inducing an immune response against HPV16 for use in a method of treating or inhibiting the growth of a tumor or improving overall survival of a head and neck cancer patient, the method comprising: a) selecting a patient with HPV16-positive head and neck cancer; b) administering to the patient an effective amount of one or more immunogenic compositions capable of inducing a T cell immune response against cells expressing HPV16 antigens, preferably E6, E7 and/ or E2 antigen; and c) administering to the patient a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof that comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) and a light chain variable region comprising three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein : HCDR1 has an amino acid sequence of SEQ ID NO: 13; HCDR2 has an amino acid sequence of SEQ ID NO: 14; HCDR3 has an amino acid sequence of SEQ ID NO: 15; LCDR1 has an amino acid sequence of SEQ ID NO: 16; LCDR.2 has an amino acid sequence of SEQ ID NO: 17; and LCDR.3 has an amino acid sequence of SEQ ID NO: 18.

In one embodiment, the patient that is selected is a patient diagnosed with histologically confirmed recurrent or metastatic HPV16 positive oropharyngeal cancer, whose tumors express PD-L1 (Combined Positive Score [CPS] > 1). The CPS for PD-L1 expression may, for example, be determined using the 22C3 pharmDx or SP263 assay, described in Pagni et al. Int J Mol Sci. 2019 20(21) : 5452 (incorporated by reference).

In one embodiment, the patient that is selected is a patient with recurrent or metastatic HPV16 positive oropharyngeal cancer with disease progression on or after platinum containing chemotherapy.

In one embodiment of the second main aspect, the patient that is selected is a patient with recurrent or metastatic HPV16 positive oropharyngeal cancer with disease progression on or after on prior platinum-containing therapy, but who did not receive prior immune checkpoint modulatory therapy, in particular did not receive prior treatment with an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, cemiplimab), anti-PL-Ll, anti- PD-L2, anti-CTLA-4 or anti-LAG 3 antibody, or any other antibody or drug specifically targeting T-cell co stimulation or immune checkpoint pathways.

In a further embodiment, prior curative radiation therapy, if any, was completed at least 8 weeks prior to initiation of the treatment of the invention. In a further embodiment, prior focal palliative radiotherapy, if any, was completed at least 2 weeks before initiation of the treatment of the invention. In a further embodiment, the patient did not receive prior treatment with more than one chemotherapy regimen for the management of metastatic oropharyngeal cancer.

In one embodiment, step a) comprises selecting a patient with HPV16-positive head and neck cancer, wherein, in a tumor sample of said subject, PD-L1 is expressed in at least 5% of the viable tumor cells and/or the CPS is at least 5 and/or the TPS is at least 5 and/or the TAP score is at least 5%.

• PD-L1 is expressed in at least 19% of the viable tumor cells and/or the CPS is at least 19 and/or the TPS is at least 19 and/or the TAP score is at least 19%, or

• PD-L1 is expressed in at least 20% of the viable tumor cells and/or the CPS is at least 20 and/or the TPS is at least 20 and/or the TAP score is at least 20%.

In a further main aspect, the invention relates to a method of treating or inhibiting the growth of a tumor or improving overall survival of a head and neck cancer patient, comprising: a) selecting a patient with HPV16-positive head and neck cancer; b) administering to the patient an effective amount of one or more immunogenic compositions capable of inducing a T cell immune response against cells expressing HPV16 antigens, wherein said one or more immunogenic compositions comprise the 12 different immunogenic peptides set forth in SEQ ID NO: 1-12 and no further immunogenic peptides; and c) administering to the patient a therapeutically effective amount of a PD-1 inhibitor. In one embodiment, the treatment of the present disclosure promotes tumor regression, reduces tumor cell load, reduces tumor burden, and/or prevents tumor recurrence in the patient.

In one embodiment, the treatment of the present disclosure leads to at least one improvement selected from increase in overall survival, progression free survival, overall response rate, complete response, partial response, and stable disease, as compared to patients treated with chemotherapy and/or compared to patients treated with a PD-1 inhibitor, such as cemiplimab, alone.

As described above, in a third main aspect, the invention relates to a method for the treatment of a solid tumor in a subject, such as a human subject, comprising identifying a subject afflicted with a solid tumor having a PD-L1 expression level above a predefined cut-off value and administering one or more immunogenic compositions comprising one or more tumor antigens to said subject. Said one or more tumor antigens are typically ones that may be present or have been demonstrated to be present in said solid tumor.

As shown in the Examples herein, it has surprisingly been observed that better responses to immunisation with tumor antigens were observed in a patient subpopulation that had a high expression of PD-L1 in the tumor. Thus, without being bound by any specific theory, the data suggest that tumors with high PD-L1 expression may be more sensitive and/or reactive to immunisation with tumor antigens. Thus, patients may be selected for therapeutic immunisation based on tumor antigens (vaccination) on the basis of PD-L1 expression levels in a tumor sample.

For measurement of PD-L1 expression in tumor tissues several immunohistochemistry assays/scoring algorithms have been developed as companion diagnostic assays (CDx). The assays are commonly used to assess, by immunohistology staining, the proportion of viable tumor cells (TC) and/or the proportion of immune cells (IC) that express PD-L1 in tumor tissue specimens. Either by cell-counting methods or tumor area percentage scoring methods.

TC-only algorithms assess the proportion of viable tumor cells showing expression of PD-L1 relative to all viable tumor cells present in a specimen (depicted as %TC or tumor proportion score, TPS) or relative to the tumor area (depicted as tumor area percentage (%TC)).

IC-only algorithms assess the proportion of viable immune cells showing expression of PD-L1 in a tumor tissue sample as area percentage (%IC).

TC-only and IC-only algorithms may be combined and depicted as a double percentage score. Alternatively, the proportion of PD-L1 positive TC an IC in a tumor tissue specimen may be assessed by a combination of TC and IC area percentages (Tumor Associated Positivity) or as combination of the number of viable PD-L1 expressing tumor cells plus viable PD-L1 expressing (mononuclear) immune cells relative to all viable TCs present in the tumor tissue specimen times 100 (Combined proportion score, CPS). Although the result of the CPS calculation can exceed 100, the maximum score is defined as CPS 100.

Well-known assays that serve as CDx's are the 28-8 pharmDx assay (Dako-Agilent), the 22C3 pharmDx assay (Dako-Agilent) or the SP263 assay (Ventana-Roche). The following table provides a summary of on-market FDA-approved PD-L1 IHC scoring algorithms and associated testing assays (adapted from Liu et al 2023 Diagnostic Pathology 18:48) :

In general, TC-only scoring methods have been favorably adopted by the pathology community, whereas IC scoring alone or sequential TC/IC scoring have been perceived as not sufficiently predicting. CPS is the only FDA-approved method that combines TC and IC (e.g. the pharmDx 22C3 scoring method). It is a cell counting-based approach where the number of PD-L1- stained cells (TC, mononuclear IC (i.e. lymphocytes and macrophages)) is divided by the total number of viable TC, multiplied by 100.

In practice, pathologist may apply preferred scoring algorithms with non-associated PD-L1 expression testing assays. E.g. apply the CPS scoring algorithm with the SP263 IHC assay.

Recent studies have shown that for the higher scores the CPS and TPS assessments yield similar clinical results and that for lower PD-L1 scores the CPS score may be more sensitive (Emancipator et al 2021 Modern Pathology 34; De Marchi et al 2021 J Clin Pathol 74). This means that e.g. CPS >20 is similar to TPS > 20.

In a comparison of the different assays to measure PD-L1 expression in cancer tissues, it was noted that the analytical concordance between 28-8 (%TC), 22C3 (TPS), and SP263 (%TC)-based assays was high when used to assess the % of PD-L1 expressing tumor cells in several cancer tissues, including SCCHN. This means that these assays are interchangeable. Analytical concordance for assessment of PD-L1 expression on ICs was variable and generally lower than for PD-L1 expression on TCs (Prince et al 2021 ICO Precis Oncol 5).

The recently developed TAP scoring algorithm, based on the proportion of PD-L1 positive TC and IC (all types) of a defined tumor area, was shown to be highly reproducible with a high concordance rate between TAP score and CPS using the same SP263 IHC assay for both methods on a same sample set (Liu et al 2023 Diagnostic Pathology 18). This study indicated that the TAP scoring method is as effective as the CPS method to assess the proportion of PD-L1 positive TC and IC in a tumor tissue specimen.

In one embodiment, the present invention relates to a method for the treatment of a solid tumor in a subject, comprising selecting a subject afflicted with a solid tumor having a PD- L1 expression level above a predefined cut-off value and administering one or more immunogenic compositions comprising one or more tumor antigens to said subject. In one embodiment, the immunogenic compositions are only administered to the subject if the PD-L1 expression is above the predefined cut-off value.

In another embodiment, the invention relates to a method for the selection of a patient afflicted with a solid tumor for therapeutic immunisation, comprising determining PD-L1 expression in a sample of said tumor and selecting the patient for therapeutic immunisation if the number of PD-L1 expressing cells in said tumor is above a predetermined value.

In another embodiment, the invention relates to a method for predicting the response to therapeutic immunisation of a patient afflicted with a solid tumor, comprising determining PD-L1 expression in a sample of said tumor, wherein the patients is predicted to respond to therapeutic immunisation if the number of PD-L1 expressing cells in said tumor is above a predetermined value.

Similarly, in one embodiment, the invention relates to a method for determining the eligibility for therapeutic immunisation of a patient afflicted with a solid tumor, comprising determining PD-L1 expression in a sample of said tumor, wherein the patient is eligible for therapeutic immunisation if the number of PD-L1 expressing cells in said tumor is above a predetermined value.

In one embodiment, the predetermined cut-off value in each of the above- mentioned methods is a value reflecting the proportion of PD-L1 expressing cells in a tumor sample relative to the total number of cells in the sample. In a further embodiment, the predetermined cut-off value in each of the above- mentioned methods is a value reflecting the proportion of viable PD-L1 expressing cells in a tumor sample relative to the total number of viable cells in the sample.

In a further embodiment, the predetermined cut-off value in each of the above- mentioned methods is a value reflecting the proportion of viable PD-L1 expressing tumor cells in a tumor sample relative to the total number of viable tumor cells in the sample.

In a further embodiment, the predetermined cut-off value in each of the above- mentioned methods is a value reflecting the proportion of the total of: viable PD-L1 expressing tumor cells + viable PD-L1 expressing tumor infiltrating lymphocytes + PD-L1 expressing tumor infiltrating macrophages cells in a tumor sample relative to the total number of viable tumor cells in the sample.

In some embodiments, the value is determined directly by cell counting. In other embodiments, the value for PD-L1 expressing cells is determined indirectly by measuring PD-L1 positive staining of a selected area of the tumor sample. In one embodiment, the value is a CPS, a TPS or a TAP score.

Accordingly, the invention also relates to a method for the treatment of a solid tumor in a subject, comprising i) identifying a subject afflicted with a solid tumor, wherein, in a tumor sample of said subject, PD-L1 is expressed in at least 5% of the viable tumor cells and/or the CPS is at least 5 and/or the TPS is at least 5 and/or the TAP score is at least 5%, and ii) administering one or more immunogenic compositions comprising one or more tumor antigens to said subject.

In one embodiment hereof, the tumor sample is a tumor biopsy sample. Thus, the method typically involves a step of determining the CPS, the TPS, the TAP score and/or the percentage of PD-L1 expression tumor cells in a tumor biopsy sample of the subject.

In another further embodiment, in a tumor sample of said subject,

• PD-L1 is expressed in at least 8% of the viable tumor cells and/or the CPS is at least 8 and/or the TPS is at least 8 and/or the TAP score is at least 8%,

• PD-L1 is expressed in at least 9% of the viable tumor cells and/or the CPS is at least 9 and/or the TPS is at least 9 and/or the TAP score is at least 9%,

• PD-L1 is expressed in at least 10% of the viable tumor cells and/or the CPS is at least 10 and/or the TPS is at least 10 and/or the TAP score is at least 10%, • PD-L1 is expressed in at least 11% of the viable tumor cells and/or the CPS is at least 11 and/or the TPS is at least 11 and/or the TAP score is at least 11%,

In one embodiment of this third aspect of the invention, said one or more tumor antigens are capable of inducing a CD8+ and/or CD4+ T cell response.

In one embodiment of this aspect of the invention, said one or more tumor antigens are protein-based, such as peptide-based, or polynucleotide-based, for example based on DNA or RNA.

In one embodiment of this aspect of the invention, said one or more tumor antigens are peptides, preferably having a length of 20-40 amino acids.

In one embodiment of this third aspect of the invention, said solid tumor is a tumor of cancer of the cervix, vulva, vagina, anus, penis, aerodigestive tract, and head and neck (including cancer of the tongue, nasal cavity, oral cavity, salivary gland, paranasal sinuses, larynx, pharynx (nasopharynx, oropharynx, hypopharynx).

In one embodiment of this third aspect of the invention, said solid tumor is a squamous cell carcinoma head and neck cancer tumor (such as an oropharyngeal tumor) or a cervical cancer tumor of the squamous cell type.

In one embodiment of this third aspect of the invention, said solid tumor is a cervical intraepithelial neoplasia (CIN), Vulvar intraepithelial neoplasia (VIN), vaginal intraepithelial neoplasia (ValN), anal intraepithelial neoplasia (AIN), penal intraepithelial neoplasia (PIN) or adenocarcinoma in situ (AIS).

In one embodiment of this third aspect of the invention, said solid tumor is a squamous cell carcinoma head and neck cancer tumor (such as an oropharyngeal tumor) and the subject has not received prior treatment.

In one embodiment of this third aspect of the invention, said solid tumor is a primary tumor.

In one embodiment of this third aspect of the intention, said solid tumor is a secondary tumor or metastasis of a primary tumor.

In one embodiment of this third aspect of the invention, said solid tumor is an HPV positive tumor.

In one embodiment of this third aspect of the invention, said solid tumor is surgically removed and the subject is treated according to the method of the invention prior or after surgery.

In one embodiment of this third aspect of the invention, said cancer is locally advanced, advanced, recurrent, persistent, primary refractory or refractory, incurable, unresectable and/or metastatic.

In one embodiment of this third aspect of the invention, said solid tumor is a squamous cell carcinoma head and neck cancer tumor (such as an oropharyngeal tumor) and the cancer is advanced, recurrent, persistent, primary refractory or refractory, incurable, unresectable and/or metastatic.

In one embodiment of this third aspect of the invention, said solid tumor is an HPV positive squamous cell carcinoma head and neck cancer tumor or an HPV positive cervical cancer tumor and wherein said one or more tumor antigens are HPV antigens, such as E2, E6 or E7 antigens of HPV16, HPV18, HPV45, HPV33, HPV31, HPV52, HPV58, HPV35, HPV39, HPV59, HPV73, HPV51, HPV56 or HPV68. Suitable tumor antigens have e.g. been described in WO2008/147187, WO2017/220463 and WO2021/156404 (ISA Pharmaceuticals) (all incorporated by reference).

In one embodiment of this third aspect of the invention, said solid tumor is an HPV16 positive squamous cell carcinoma head and neck cancer tumor or an HPV16 positive cervical cancer tumor and wherein said one or more tumor antigens are HPV16 antigens.

In one embodiment of this third aspect of the invention, said solid tumor is a squamous cell carcinoma head and neck cancer tumor or a cervical cancer tumor and wherein said one or more tumor antigens are peptides based on the E6 and E7 proteins of HPV16, comprising 12 synthetic long peptides of 25 to 35 amino acid in length, most preferably the peptides set forth in SEQ ID NO: 1 to SEQ ID NO: 12. In one embodiment of this third aspect of the invention, said solid tumor is a cervical cancer tumor, such as a cervical cancer tumor of the squamous cell type or of the adenocarcinoma or adenosquamous cell type. Preferably, the solid tumor is of the squamous cell type.

In one embodiment of this third aspect of the invention, the tumor disease is recurrent or metastatic.

In one embodiment of this third aspect of the invention, the subject has received prior therapy for the tumor disease, wherein optionally the tumor disease has progressed after said prior therapy. In a further embodiment hereof, the prior therapy is chemotherapy, such as platinum-containing therapy, optionally in combination with taxane and/or bevacizumab therapy.

In one embodiment of this third aspect of the invention, said solid tumor is a cervical cancer tumor, preferably of the squamous cell type, the tumor disease is recurrent or metastatic and the subject has received prior therapy for the tumor disease, wherein the prior therapy is chemotherapy, such as platinum-containing therapy, optionally in combination with taxane and/or bevacizumab therapy. In a further embodiment hereof, the subject did not receive prior treatment with an agent that blocks the PD-1/PD-L1 pathway. In another further embodiment hereof, the subject did not receive prior treatment with other systemic immune-modulating agents, such as therapeutic vaccines, cytokine treatments (other than granulocyte colony stimulating factor, thrombopoietin analogues, or erythropoietin), or agents that target cytotoxic T lymphocyte antigen 4 (CTLA-4), 4-1BB (CD137), PI 3-K-delta, LAG3, or OX-40.

In one embodiment of this third aspect of the invention, said immunogenic composition(s) is/are administered two or more times. In such embodiments, when two or more administrations are performed the timing of the second administration is such that the second dose induces a booster effect. In one embodiment, the immunogenic composition(s) are administered two or three times with intervals of between 2 and 5 weeks, such as 3 or 4 weeks. In one embodiment, a further (e.g. third or fourth) administration of the immunogenic composition(s) is performed between 4 and 8 months after the second or third initial dose.

In one embodiment of this third aspect of the invention, said solid tumor is a cervical cancer tumor, preferably of the squamous cell type, the tumor disease is recurrent or metastatic, the subject has received prior therapy for the tumor disease, wherein the prior therapy is chemotherapy, such as platinum-containing therapy, optionally in combination with taxane and/or bevacizumab therapy and the immunogenic composition(s) are administered two or three times with intervals of between 2 and 5 weeks, such as 3 or 4 weeks. In one embodiment of this third aspect of the invention, said immunogenic composition(s) is/are administered intravenously or subcutaneously.

In one embodiment of this third aspect of the invention, the treatment further comprises administration of an adjuvant.

In one embodiment of this third aspect of the invention, the method does not comprise administration of a PD-1 inhibitor or PD-L1 inhibitor.

In one embodiment of this third aspect of the invention, the method does not comprise administration of an immune checkpoint inhibitor.

In one embodiment of this third aspect of the invention, the method further comprises administration of an immune checkpoint inhibitor, wherein preferably, the immune checkpoint inhibitor is a PD-1 or a PD-L1 inhibitor, such as an antibody that binds PD-1 or PD-L1, for example selected from the group consisting of: cemiplimab, nivolumab and pembrolizumab.

In one embodiment of this third aspect of the invention, the method further comprises administration of one or more chemotherapeutic agents and/or radiation therapy.

Immunogenic compositions and adjuvants

The methods disclosed herein include administering an effective amount of one or more immunogenic compositions capable of inducing an immune response, for example against cells expressing HPV16 antigens.

When used herein, the term "inducing" in the context of an immune response includes inducing or triggering an immune response but also activating, i.e. boosting a previous or existing immune response. The immune response may include a CD8+ response and/or a CD4+ response and/or an antibody response (such as an IgG response).

Immunogenic compositions used in the methods of the present disclosure may be protein-based, such as peptide-based, or polynucleotide-based, for example based on DNA, RIMA or viral vectors.

Suitable methods for polynucleotide-based vaccination have e.g. been described in Trimble et al. 2015 Lancet 386:2078; Kranz et al. 2016 Nature 534:396; WO2011015656A2; Kratzer, et al. 2018 AASLD, The Liver Meeting 2018, abstract #426; W02017080920; and Boni et al. 2019 Int J Mol Sci 20(ll):2754.

In certain embodiments, the induction of immune response against cells expressing HPV16 antigens is based on the use of immunogenic peptides. Thus, the one or more immunogenic compositions are peptide-based composition(s).

When used herein, the term "immunogenic peptide" refers to a peptide capable of inducing (including triggering or boosting) an immune response (such as a local and/or systemic CD4+ and/or CD8+ T cell response and/or an antibody response) in a host organism, typically a mammalian host organism, such as a human host. Likewise, the term "immunogenic composition" means a composition capable of inducing an immune response. An immunogenic peptide may for example have a length of from 8 to 100 amino acids, e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 55, 60, 70, 80, 90 amino acids or more. Preferably, a peptide is a synthetic long peptide (SLP), i.e. prepared synthetically (e.g. using solid phase synthesis) having a length of 18 to 100 amino acids, more preferably a peptide having a length of 20-45, e.g. 20-35, amino acid residues. In one embodiment, an immunogenic peptide used in the invention is from 20 to 45 amino acid residues in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acids in length.

In the context of describing peptides, the term "in length", for example a peptide of 20 to 45 amino acids or amino acid residues "in length" refers to the number of amino acid residues in the linear peptide chain.

An immunogenic peptide may comprise or consist of a fragment of a protein, typically a protein which is a suitable target for prophylactic or therapeutic immunisation. For example, proteins that are specifically expressed by infected, pre-cancerous and/or cancerous cells are suitable targets for therapeutic immunization. Suitable target protein antigens from HPV16 include L2 protein (e.g. UniProtKB P03107; P06793), E2 protein (e.g. UniProtKB P03120, P06790), E6 oncoprotein (e.g. UniProtKB P03126; P06463) and E7 oncoprotein (e.g. UniProtKB P03129; P06788).

"Fragment" refers to a sequence of consecutive amino acids that corresponds to, i.e. is identical to, a part of a protein sequence. This does not exclude, however, that the immunogenic peptide or the fragment, while preserving immunogenic properties, may be further modified. For example, the immunogenic peptide of the invention may or may not comprise modified amino acids and/or non-naturally occurring amino acids and/or covalently linked groups.

In one embodiment, the immunogenic peptide used in the invention comprises at least one MHC class I ligand and/or least one MHC class II ligand.

The term "MHC class I ligand" refers to a peptide sequence that can bind to and be presented by an MHC class I molecule. MHC (major histocompatibility complex) class I molecules (in humans including HLA-A, HLA-B and HLA-C) are one of two classes of (MHC) molecules found on the cell surface of nucleated cells. Their function is to present peptide fragments of proteins to cytotoxic T cells, thus trigger an immune response. MHC class I ligands typically have a length of 8-11 amino acids. Proteins or long peptides, such as long peptides of the invention, that comprise MHC class I ligands typically require intracellular processing for the MHC class I ligand to be generated and made available to bind to the MHC Class I molecule and be presented on the cell surface. The intracellular processing typically occurs via proteasomal cleavage in the cytosol.

The term "MHC class II ligand" refers to a peptide sequence that can bind to and be presented by an MHC class II molecule. MHC (major histocompatibility complex) class II molecules (in humans including HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA- DR) are one of two classes of (MHC) molecules found on the cell surface of nucleated cells. Their function is to present peptide fragments of proteins to T helper cells, thus trigger an immune response. MHC class II ligands typically have a length of 11-16 amino acids. Proteins or long peptides, such as long peptides of the invention, that comprise MHC class II ligands typically require intracellular processing for the MHC class II ligand to be generated and made available to bind to the MHC Class II molecule and be presented on the cell surface. The intracellular processing of MHC class II ligands typically occurs via endocytosis and endosomal digestion.

As mentioned above, in certain embodiments, the target protein is HPV16 E6 or E7 and thus, in certain embodiments, one or more immunogenic compositions are peptide- based compositions based on the E6 and E7 proteins of HPV16. Suitable HPV16-derived immunogenic peptides have been described in WO2017220463 (ISA Pharmaceuticals) and WOOO/75336 (INSERM).

In some embodiments, peptides are peptides comprising or consisting of a contiguous sequence within an immunogenic region represented by any one of SEQ ID NO:24-30. In certain embodiments, one or more peptides to be used in the method of the invention comprises a CTL epitope selected from the group represented by SEQ ID NO: 31- 71.

Other suitable peptides and peptide mixes derived from HPV E2, E6 and E7 proteins are as defined in W02002/070006 and W02002/090382, which are incorporated herein by reference. For example, suitable peptides are peptides comprising or consisting of a contiguous sequence within the following HPV immunogenic regions E2 (31-120); E2 (151- 195); E2 (271-365); E6 (81-158); E7 (31-77) and defined herein by SEQ ID NO:72-76, respectively.

In some embodiments, one or more of the peptides comprise a Th epitope that is selected from DR1/E2 351-365, DR2/E2 316-330, DR2/E2 346-355, DR4/E2 51-70, E2 61- 76, DQ6/E2 311-325, DR15/E7 50-62, DR3/E7 43-77, DQ2/E7 35-50 and DR1/E6 127-142 (represented herein by SEQ ID NO: 77-86, respectively).

In some embodiments, the peptides comprise at least one T cell epitope that is recognized by a T cell that infiltrates a cervical neoplastic lesion or by a T cell that is present in or isolated from a lymph node from the pelvic region, that is draining from the cervical neoplastic lesion. Preferably, the T cell epitope is present in or isolated from a draining lymph node comprising metastatic tumor cells. Such epitopes are disclosed in e.g., WO2008/147187, US20060182762, W02006013336, W02009148230, WO2009148229 and W02002044384 which are incorporated herein by reference.

In some embodiments, the peptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NO:87-108, which have been proven to be T-cell epitopes that are recognized by a T cells that infiltrates a cervical neoplastic lesion or by a T cell from a draining lymph node.

A suitable class II CD4⁺ Th cell epitope comprised in a peptide to be used in the method of the invention is selected from the group consisting of the amino acid sequences represented by SEQ ID NO:87-103.

A suitable class I CD8⁺ CTL cell epitope comprised in a peptide to be used in the method of the invention is selected from the group consisting of the amino acid sequences represented by SEQ ID NO:89, 86, 104-108.

In some embodiments, the one or more immunogenic compositions in total comprise 12 synthetic long peptides, each of 25 to 35 amino acids in length.

Preferred peptides to be used in the method of the present disclosure comprise or consist of a contiguous amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NO: 1-12. Preferably, the method of the invention comprises administration of one, two, three, four, five, six, seven, eight, nine, ten, eleven or all twelve of the peptides set forth in SEQ ID NO: 1-12.

In a preferred embodiment, the one or more immunogenic compositions (together) comprise all of the peptides set forth in SEQ ID NO: 1 to SEQ ID NO: 12. In a further embodiment, the one or more immunogenic compositions do not comprise other peptides than SEQ ID NO: 1 to SEQ ID NO: 12. In another embodiment, the one or more immunogenic compositions further comprise the peptide set forth in SEQ ID NO:23.

Preferably, the method of the present disclosure further comprises administration of an adjuvant. The term "adjuvant" is used herein to refer to substances that have immune-potentiating effects and are added to or co-formulated with an antigenic agent in order to enhance, induce, elicit, and/or modulate the immunological response against the antigenic agent when administered to a subject.

In certain embodiments, the adjuvant is an oil-based adjuvant. Oil-based adjuvants can be used to form emulsions (e.g. water-in-oil or oil-in-water emulsions) and are appreciated in the art to enhance and direct the immune response. The oil-based adjuvant may be any mineral or non-mineral oil-based adjuvant known in the art. In one embodiment, the oil-based adjuvant is a mineral oil-based adjuvant. Non-limiting examples of oil-based adjuvants are bio-based oil adjuvants (based on vegetable oil / fish oil, etc.), squalene-based adjuvant (e.g. MF59), Syntex Adjuvant Formulation (SAF; Lidgate, Deborah M, Preparation of the Syntex Adjuvant Formulation (SAF, SAF-m, and SAF-1), In: Vaccine Adjuvants, Volume 42 of the series Methods in Molecular Medicine™ p229-237, ISSN1543-1894), Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvant (FIA), adjuvants based on peanut oil e.g. Adjuvant 65) , Lipovant Byars, N.E., Allison, A.C., 1990. Immunologic adjuvants: general properties, advantages, and limitations. In: Zola, H. (Ed.), Laboratory Methods in Immunology. p39-51), ASO4 A. Tagliabue, R. Rappuoli Vaccine adjuvants: the dream becomes real Hum. Vaccine, 4 (5), 2008, p347-349'), Montanide adjuvants, which are based on purified squalene and squalene emulsified with highly purified mannide mono-oleate (e.g. Montanide ISA 25 VG, 28 VG, 35 VG, 50 V, 50 V2, 51 VG, 61 VG, 70 VG, 70 M VG, 71 VG, 720 VG, 760 VG, 763 A VG, 775 VG, 780 VG, 201 VG, 206 VG, 207 VG). Preferably the adjuvant is Montanide ISA 51VG (Seppic), which is a mixture of Drakeol VR and mannide monooleate.

As described above, the method of the present disclosure comprises administration of one or more immunogenic compositions. Immunogenic peptides to be used in the method of the present disclosure may be combined in a single composition. However, in particular when a larger number of immunogenic peptides is to be used, they may advantageously be distributed over two or more immunogenic compositions. Distribution over more than one immunogenic composition may for example improve product stability and/or give more flexibility with respect to dosing or site of administration.

Accordingly, in some embodiments, the method of the disclosure comprises the use of a kit or vaccine product, comprising two or more parts, e.g. two or more vials, wherein a plurality of immunogenic peptides of the invention are distributed over said two or more parts, e.g. distributed over two or more vials. In such embodiments, the compositions may be mixed before administration of the vaccine to the patient or the compositions may be administered separately. In one embodiment, the kit or vaccine product comprises two or more compositions comprising dried or lyophilized peptides and the vaccine further comprises a reconstitution solution and optionally an adjuvant, wherein the adjuvant may be comprised within the reconstitution solution or be provided in a further separate vial.

In one embodiment, the method of the invention comprises administration of two immunogenic compositions, a first composition comprising the five peptides set forth in SEQ ID NO: 1-5 and a second composition comprising the seven peptides set forth in SEQ ID NO:6-12.

The combination of two immunogenic compositions, a first composition comprising the five peptides set forth in SEQ ID NO: 1-5 and a second composition comprising the seven peptides set forth in SEQ ID NO:6-12 is also termed ISAlOlb or peltopepimut-S herein. ISAlOlb (peltopepimut-S) consists of nine overlapping long E6 peptides (five 32- mer and four 25-mer E6 peptides) and three 35-mer E7 peptides. These peptides overlap by 10 to 18 residues and cover the complete sequence of HPV16 E6 oncoprotein. The E7 oncoprotein sequence is almost completely represented by the peptide sequences, only amino acids 57-63 are not covered.

Preferably, the different peptides in the compositions are present in the pharmaceutical composition in substantially equal ratios. In further embodiments, said first and second compositions are each mixed to make an emulsion in a 1: 1 (v/v) ratio with Montanide ISA 51 VG before administration.

Formulation of immunogenic compositions

Immunogenic compositions used in the invention, such as ISAlOlb, may be prepared (formulated) by any suitable method. In some embodiments, the immunogenic composition(s) are prepared from dried, preferably lyophilized, immunogenic peptides.

For example, the composition may be prepared by a method comprising the following steps: a) providing a vial comprising dried, preferably lyophilized, peptides; b) thawing the peptides, preferably for about 5-30 min; c) adding a reconstitution composition to the vial comprising the peptides, preferably without swirling the vial; d) allowing to admix, preferably for about 0.5-5 minutes; and e) swirling until a clear solution is obtained, preferably for about 1-3 minutes. Steps b) to e) may be performed at room temperature.

In one embodiment, said vial comprises peptides in an amount for injection as a single volume in a method of treatment as defined herein, i.e. a single pharmaceutical dosage unit, or part thereof in case of multiple injections at difference locations of the subject's body at substantially the same time point.

In one embodiment, the reconstitution composition of step c) comprises or consists of DMSO and/or water-for-injection. In another embodiment, the reconstitution composition of step c) of the method for reconstituting peptides comprises or consists of about 60-80% v/v aqueous solution comprising an organic acid, about 5-10% v/v propylene glycol (CAS no. 57-55-6), about 10-20% v/v lower alcohol and about 5-10% v/v non-ionic hydrophilic surfactant. In one embodiment, the organic acid is citric acid and the citric acid is present in the aqueous solution in a concentration of about 0.05 - 0.1M. In one embodiment, the lower alcohol is ethanol. In one embodiment, the non-ionic hydrophilic surfactant: a. is a mono-, di or triglyceride, preferably an ethoxylated triglyceride, and/or b. has a hydrophilic-lipophilic balance (HLB) value between 9 and 14.

In a further embodiment, the non-ionic hydrophilic surfactant is ethoxylated castor oil, preferably polyoxyethyleneglyceroltriricinoleate 35 (CAS no. 61791-12-6).

In one embodiment, the composition comprises or consists of about 75% v/v aqueous solution comprising about 0.1M citric acid, about 6.25% v/v propylene glycol (CAS no. 57-55-6), about 12.5% v/v ethanol and about 6.25% v/v polyoxyethyleneglyceroltriricinoleate 35 (CAS no. 61791-12-6).

In certain embodiments, the amount of reconstitution composition in step c) is in a range of from about 0.5 and 2 mL, such as 1 mL.

In one embodiment, the reconstituted composition comprises or consists of about 1-2 mg/mL peptides, 0.038M citric acid, about 3.13% v/v propylene glycol (CAS no. 57- 55-6), about 6.25% v/v ethanol, about 3.13% v/v polyoxyethyleneglyceroltriricinoleate 35 (CAS no. 61791-12-6) and about 50% of an oil-based adjuvant, preferably Montanide ISA 51 VG (Seppic), in water.

Dried peptides may be peptides free of further constituents but may also comprise buffer components such as trifluoroacetic acid (TFA), salts such as sodium, potassium or phosphate salts (e.g. NaCI, KCI and NaPCk). The amount of further constituents is preferably less than 30%, more preferably less than 25%, of the total weight of the dry peptides to be reconstituted. Dried peptides to be reconstituted may be in a physical dried state as can be obtained by processes such as, but not limited to, rotor evaporation, lyophilization (freeze drying) and spray drying.

Administration and dosing of immunogenic compositions

Various delivery systems are known and can be used to administer the immunogenic compositions of the disclosure. Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous and subcutaneous routes. The composition may be administered by any convenient route, for example by bolus injection. Injectable preparations may include dosage forms for intravenous, subcutaneous, intracranial, intraperitoneal and intramuscular injections, etc. These injectable preparations may be prepared by methods publicly known. An immunogenic composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe.

In some embodiments, the method comprises administration or two or more immunogenic compositions that are administered by injection, for example subcutaneous injection, in two or more different limbs. If two or more immunogenic compositions are administered, the administrations may be performed essentially simultaneously or sequentially, for example within a time interval of 24 hrs or less, such as 2 hrs or less, e.g. 1 hr or less, such as 30 min or less.

In some embodiments, an immunogenic composition applied to a subject at a given time point, comprises an amount of each peptide in the range from 0.1 pg to 20 mg. Thus, the dose of each peptide may be 0.1 pg, 0.5 pg, 1 pg, 5 pg, 10 pg, 15 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 150 pg, 200 pg, 500 pg, 1 mg, 20 mg or any value in between. In some embodiment, an immunogenic composition applied to a subject at a given time point, comprises an amount of each peptide in the range from 1 pg to 500 pg, for example from 10 pg to 200 pg, such as from 50 u pg to 200 pg.

A single injection volume (i.e. volume applied on one location at a certain time point) may for example be between 50 pL and 1 ml_. The single injection volume may be 50 pL, 75 pL, 100 pL, 200 pL, 300 pL, 400 pL, 500 pL, 600 pL, 700 pL, 800 pL, or any value in between. In one embodiment, the volume is between 75 pL and 250 pL or between 50 pL and 75 pL.

In a preferred embodiment, the method of the invention comprises administration of two compositions ("HPV-DP-5P" and "HPV-DP-7P") having the following compositions:

Table 1: Quantitative composition of HPV-DP-5P 2.00 mg/vial

SEQ ID Peptide sequence Amount net Amount net

NO: peptide per peptide per vial dose mg pg

1 DKCLKFYSKISEYRHYCYSLYGTTL 0.40 100

2 LYCYEQLNDSSEEEDEIDGPAGQAEP 0.40 100

DRAHYNIVT

3 MHGDTPTLHEYMLDLQPETTDLYCYE 0.40 100

QLNDSSEEE

4 RCINCQKPLCPEEKQRHLDKKQRFHN 0.40 100

IRGRWT

5 LRLCVQSTHVDIRTLEDLLMGTLGIVC 0.40 100

PICSQKP

Total 2.00 500 weight Table 2: Quantitative composition of HPV-DP-7P 2.80 mg/vial

SEQ ID Peptide sequence Amount net Amount net

NO: peptide per peptide per vial dose mg pg

6 DKKQRFHNIRGRWTGRCMSCCRSSRT 0.40 100

RRETQL

7 HYCYSLYGTTLEQQYNKPLCDLLIR 0.40 100

8 KQQLLRREVYDFAFRDLCIVYRDGN 0.40 100

9 LPQLCTELQTTIHDIILECVYCKQQLLRR 0.40 100

EVY

10 MHQKRTAMFQ 0.40 100

QERPRKLPQ LCTE LQTTI H D

11 RDLCIVYRDGNPYAVCDKCLKFYSKI 0.40 100

12 YGTTLEQQYNKPLCDLLIRCINCQKPLC 0.40 100

PEEK

Total 2.80 700 weight

In certain embodiments, the methods disclosed herein include administering the immunogenic composition(s) (e.g. HPV-DP-5P and HPV-DP-7P) in multiple doses, e.g., as part of a specific dosing regimen. For example, the dosing regimen may comprise administering each of the immunogenic composition(s) twice, three times, four times or five or more times.

The immunogenic composition(s), such as ISAlOlb, may be administered with intervals of between 1 week and 6 months, such as intervals of 1 week and 2 months, for example intervals between 2 and 5 weeks, such as 3 or 4 weeks.

In one embodiment, the immunogenic composition(s), such as ISAlOlb, are administered three times with intervals of between 2 and 5 weeks, such as 3 or 4 weeks.

In one embodiment, a further (booster) dose is administered between 2 and 18 months, such as between 4 and 8 months, e.g. 6 months, after the initial administration(s).

In one embodiment, a first administration of the immunogenic composition(s) is after 24-32 days followed by a second administration of the immunogenic composition(s), which after 17-25 days is followed by a third administration of the immunogenic composition(s).

In one embodiment, a first administration of HPV-DP-5P and HPV-DP-7P on day 1 is after 17-32 days, such as 24-32 days, e.g. 28 days followed by a second administration of HPV-DP-5P and HPV-DP-7P.

In a further embodiment, a first administration of HPV-DP-5P and HPV-DP-7P on day 1 is after 17-32 days, such as 24-32 days, e.g. 28 days followed by a second administration of HPV-DP-5P and HPV-DP-7P, which after 17-32 days, 17-25 days, e.g. 21 days is followed by a third administration of the HPV-DP-5P and HPV-DP-7P.

In a further embodiment, a first administration of HPV-DP-5P and HPV-DP-7P on day 1 is after 28 days followed by a second administration of HPV-DP-5P and HPV-DP-7P, which after 21 days is followed by a third administration of the HPV-DP-5P and HPV-DP- 7P.

PD-1 inhibitors

The methods disclosed herein in some embodiments include administering a therapeutically effective amount of a PD-1 inhibitor.

As used herein, a "PD-1 inhibitor" refers to any molecule capable of inhibiting, blocking, abrogating or interfering with the activity or expression of PD-1. In some embodiments, the PD-1 inhibitor can be an antibody, a small molecule compound, a nucleic acid, a polypeptide, or a functional fragment or variant thereof. Non-limiting examples of suitable PD-1 inhibitor antibodies include anti-PD-1 antibodies and antigen-binding fragments thereof, anti-PD-Ll antibodies and antigen-binding fragments thereof, and anti- PD-L2 antibodies and antigen-binding fragments thereof. Other non-limiting examples of suitable PD-1 inhibitors include RNAi molecules such as anti-PD-1 RNAi molecules, anti- PD-Ll RNAi, and an anti-PD-L2 RNAi, antisense molecules such as anti-PD-1 antisense RNA, anti-PD-Ll antisense RNA, and anti-PD-L2 antisense RNA, and dominant negative proteins such as a dominant negative PD-1 protein, a dominant negative PD-L1 protein, and a dominant negative PD-L2 protein. Some examples of the foregoing PD-1 inhibitors are described in e.g., US 9308236, US 10011656, and US 20170290808, the portions of which that identify PD-1 inhibitors are hereby incorporated by reference.

The term "antibody," as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds (i.e., "full antibody molecules"), as well as multimers thereof (e.g. IgM) or antigen-binding fragments thereof. Each heavy chain is comprised of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (comprised of domains CHI, CH2 and CH3). Each light chain is comprised of a light chain variable region ("LCVR or "VL") and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FRs of the antibody (or antigen binding fragment thereof) may be identical to the human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. The term "antibody," as used herein, also includes antigen-binding fragments of full antibody molecules.

As used herein, the terms "antigen-binding fragment" of an antibody, "antigenbinding portion" of an antibody, and the like, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigenbinding fragment," as used herein.

An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non- limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) VH- CH I; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH 1-CH2; (V) VH-CH 1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH 1; (ix) VL-CH2; (X) VL-CH3; (xi) VL-CH 1-CH2; (xii) VL-CH 1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigenbinding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).

The antibodies used in the methods disclosed herein may be human antibodies. As used herein, the term "human antibody" refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or sitespecific mutagenesis in vitro or by somatic mutation in vivo , for example in the CDRs and in particular CDR3. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The antibodies used in the methods disclosed herein may be recombinant human antibodies. As used herein, the term "recombinant human antibody" includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

Anti-PD-1 Antibodies and Antigen-Binding Fragments Thereof

In some embodiments, PD-1 inhibitors used in the methods disclosed herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1. The term "specifically binds," or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that "specifically binds" PD-1, as used in the context of the present disclosure, includes antibodies that bind PD-1 or a portion thereof with a KD of less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM or less than about 0.5 nM, as measured in a surface plasmon resonance assay. An isolated antibody that specifically binds human PD-1 may, however, have cross-reactivity to other antigens, such as PD-1 molecules from other (non-human) species.

According to certain exemplary embodiments, the anti-PD-1 antibody, or antigenbinding fragment thereof comprises a heavy chain variable region (HCVR), light chain variable region (LCVR), and/or complementarity determining regions (CDRs) comprising the amino acid sequences of any of the anti-PD-1 antibodies set forth in US 9987500, which is hereby incorporated by reference in its entirety. In certain exemplary embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof that can be used in the context of the present disclosure comprises the heavy chain complementarity determining regions (HCDRs) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 19 and the light chain complementarity determining regions (LCDRs) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:20. According to certain embodiments, the anti-PD-1 antibody or antigenbinding fragment thereof comprises three HCDRs (HCDR1, HCDR2 and HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 13; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 14; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 15; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 16; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 17; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 18. In yet other embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises an HCVR comprising SEQ ID NO: 19 and an LCVR comprising SEQ ID NO:20. In certain embodiments, the methods of the present disclosure comprise the use of an anti-PD-1 antibody, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:21. In some embodiments, the anti-PD-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 22. An exemplary antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:20 is the fully human anti-PD-1 antibody known as cemiplimab (also known as REGN2810; LIBTAYO®).

According to certain exemplary embodiments, the methods of the present disclosure comprise the use of cemiplimab or a bioequivalent thereof. As used herein, the term "bioequivalent" refers to anti-PD-1 antibodies or PD-l-binding proteins or fragments thereof that are pharmaceutical equivalents or pharmaceutical alternatives whose rate and/or extent of absorption do not show a significant difference with that of a reference antibody (e.g., cemiplimab) when administered at the same molar dose under similar experimental conditions, either single dose or multiple doses. In the context of the present disclosure, the term "bioequivalent" includes antigen-binding proteins that bind to PD-1 and do not have clinically meaningful differences with cemiplimab with respect to safety, purity and/or potency.

According to certain embodiments of the present disclosure, the anti-human PD-1, or antigen-binding fragment thereof, comprises a HCVR having 90%, 95%, 97% or 98% sequence identity to SEQ ID NO: 19.

According to certain embodiments of the present disclosure, the anti-human PD-1, or antigen-binding fragment thereof, comprises a LCVR having 90%, 95%, 97% or 98% sequence identity to SEQ ID NO:20.

Sequence identity may be measured by methods known in the art (e.g., GAP, BESTFIT, and BLAST).

According to certain embodiments of the present disclosure, the anti-human PD-1, or antigen-binding fragment thereof, comprises a HCVR comprising an amino acid sequence of SEQ ID NO: 19 having no more than 5 amino acid substitutions. According to certain embodiments of the present disclosure, the anti-human PD-1, or antigen-binding fragment thereof, comprises a LCVR comprising an amino acid sequence of SEQ ID NO:20 having no more than 2 amino acid substitutions.

The present disclosure also includes use of anti-PD-1 antibodies or antigen-binding fragments thereof in methods to treat head and neck cancer, wherein the anti-PD-1 antibodies or antigen-binding fragments thereof comprise variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein having one or more conservative amino acid substitutions. For example, the present disclosure includes use of anti-PD-1 antibodies or antigen-binding fragments thereof having HCVR, LCVR and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

Other anti-PD-1 antibodies or antigen-binding fragments thereof that can be used in the context of the methods of the present disclosure include, e.g., the antibodies referred to and known in the art as nivolumab, pembrolizumab, MEDI0608, pidilizumab, BI 754091, spartalizumab (also known as PDR001), camrelizumab (also known as SHR-1210), JNJ- 63723283, MCLA-134, or any of the anti-PD-1 antibodies set forth in US Patent Nos. 6808710, 7488802, 8008449, 8168757, 8354509, 8609089, 8686119, 8779105, 8900587, and 9987500, and in patent publications WO 2006/121168, WO 2009/114335. The portions of all of the aforementioned publications that identify anti-PD-1 antibodies are hereby incorporated by reference.

Anti-PD-Ll Antibodies and Antigen-Binding Fragments Thereof

In some embodiments, PD-1 inhibitors used in the methods disclosed herein are antibodies or antigen-binding fragments thereof that specifically bind PD-L1. For example, an antibody that "specifically binds" PD-L1, as used in the context of the present disclosure, includes antibodies that bind PD-L1 or a portion thereof with a KD of about lxlO^-8 M or less (e.g., a smaller KD denotes a tighter binding). A "high affinity" anti-PD-Ll antibody refers to those mAbs having a binding affinity to PD-L1, expressed as KD of at least IO^-8 M, such as IO^-9 M, 10 ¹⁰ M, 10 ¹¹ M, or 10 ¹² M, as measured by surface plasmon resonance, e.g., BIACORE™ or solution-affinity ELISA. An isolated antibody that specifically binds human PD-L1 may, however, have cross-reactivity to other antigens, such as PD-L1 molecules from other (non-human) species.

According to certain exemplary embodiments, the anti-PD-Ll antibody or antigenbinding fragment thereof comprises a heavy chain variable region (HCVR), light chain variable region (LCVR), and/or complementarity determining regions (CDRs) comprising the amino acid sequences of any of the anti-PD-Ll antibodies set forth in US 9938345, which is hereby incorporated by reference in its entirety. An exemplary anti-PD-Ll antibody in the context of the present methods is REGN3504.

Other anti-PD-Ll antibodies that can be used in the context of the methods of the present disclosure include, e.g., the antibodies referred to and known in the art as MDX- 1105, atezolizumab (TECENTRIQ™), durvalumab (IMFINZI™), avelumab (BAVENCIO™), LY3300054, FAZ053, STI-1014, CX-072, KN035 (Zhang et al., Cell Discovery, 3, 170004 (March 2017)), CK-301 (Gorelik et al., American Association for Cancer Research Annual Meeting (AACR), 2016-04-04 Abstract 4606), or any of the other anti-PD-Ll antibodies set forth in patent publications US 7943743, US 8217149, US 9402899, US 9624298, US 9938345, WO 2007/005874, WO 2010/077634, WO 2013/181452, WO 2013/181634, WO 2016/149201, WO 2017/034916, or EP3177649. The portions of all of the aforementioned publications that identify anti-PD-Ll antibodies are hereby incorporated by reference.

Pharmaceutical Compositions and Administration of PD-1 inhibitors

The present disclosure provides therapeutic pharmaceutical compositions comprising a PD-1 inhibitor, such as a PD-1 antibody, such as cemiplimab. Such pharmaceutical compositions may be formulated with suitable pharmaceutically acceptable carriers, excipients, buffers, and other agents that provide suitable transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations" PDA, J Pharm Sci Techno! 52:238-311 (1998).

In one embodiment, the PD-1 inhibitor is cemiplimab and is formulated in an aqueous buffered solution at pH 6.0 containing 10 mM histidine, 5% (w/v) sucrose, 1.5% (w/v) L proline, and 0.2% (w/v) polysorbate 80. In one embodiment, the cemiplimab concentration is 50 mg/mL.

The dose of PD-1 inhibitor (e.g., anti-PD-1 antibody, such as cemiplimab, or antigen-binding fragment thereof) may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. When a PD-1 inhibitor, e.g., anti-PD-1 antibody, such as cemiplimab, of the present disclosure is used for treating or inhibiting the growth of head and neck cancer or improving overall survival of a head and neck cancer patient, it may be advantageous to administer the PD-1 inhibitor, e.g., anti-PD-1 antibody, such as cemiplimab, at a single dose of about 0.1 to about 100 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. In certain embodiments, the PD-1 inhibitor, e.g., anti-PD-1 antibody, such as cemiplimab, can be administered as an initial dose of at least about 0.1 mg to about 1500 mg, about 1 to about 1000 mg, about 3 to about 800 mg, about 5 to about 500 mg, or about 10 to about 400 mg. In certain embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of the PD-1 inhibitor, e.g., anti-PD-1 antibody, such as cemiplimab, in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks. 20. In one embodiment, the PD-1 inhibitor, e.g., anti-PD-1 antibody, such as cemiplimab, or antigen-binding fragment thereof, is administered with intervals of between 2 and 5 weeks, such as 3 weeks.

Various delivery systems are known and can be used to administer the pharmaceutical composition of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al. (1987) 7. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see, e.g., Langer (1990) Science 249: 1527-1533).

The use of nanoparticles to deliver the PD-1 inhibitor, e.g., anti-PD-1 antibody, such as cemiplimab, is also contemplated herein. Antibody-conjugated nanoparticles may be used both for therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods of preparation and use are described in detail by Arruebo et al., 2009, "Antibody-conjugated nanoparticles for biomedical applications," J. Nanomat., Vol. 2009, Article ID 439389, 24 pages. Nanoparticles may be developed and conjugated to antibodies contained in pharmaceutical compositions to target cells. Nanoparticles for drug delivery have also been described in, for example, US 8257740, or US 8246995.

In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose.

The injectable preparations may include dosage forms for intravenous, subcutaneous, intracranial, intraperitoneal and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. A pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 1500 mg per dosage form in a unit dose.

In certain embodiments, the present disclosure provides a pharmaceutical composition or formulation comprising a therapeutic amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody, such as cemiplimab, or antigen-binding fragment thereof) and a pharmaceutically acceptable carrier. Non-limiting examples of pharmaceutical compositions comprising an anti-PD-1 antibody that can be used in the context of the present disclosure are disclosed in US 2019/0040137.

The present disclosure also provides kits comprising a PD-1 inhibitor (e.g., an anti- PD-1 antibody, such as cemiplimab, or antigen-binding fragment thereof) for therapeutic uses as described herein. Kits typically include a label indicating the intended use of the contents of the kit and instructions for use. As used herein, the term "label" includes any writing, or recorded material supplied on, in or with the kit, or which otherwise accompanies the kit. Accordingly, this disclosure provides a kit for treating a patient afflicted with head and neck cancer, the kit comprising: (a) a therapeutically effective dosage of a PD-1 inhibitor (e.g., an anti-PD-1 antibody, such as cemiplimab, or antigenbinding fragment thereof); and (b) instructions for using the PD-1 inhibitor, e.g., an anti- PD-1 antibody, such as cemiplimab, in any of the methods disclosed herein.

PD-1 inhibitor administration Regimens In certain embodiments, the methods disclosed herein include administering to the tumor of a subject in need thereof a therapeutically effective amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody, such as cemiplimab, or antigen-binding fragment thereof) in multiple doses, e.g., as part of a specific therapeutic dosing regimen. For example, the therapeutic dosing regimen may comprise administering one or more doses of a PD-1 inhibitor to the subject at a frequency of about once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, once a month, once every two months, once every three months, once every four months, twice a day, twice every two days, twice every three days, twice every four days, twice every five days, twice every six days, twice a week, twice every two weeks, twice every three weeks, twice every four weeks, twice every five weeks, twice every six weeks, twice every eight weeks, twice every twelve weeks, twice a month, twice every two months, twice every three months, twice every four months, three times a day, three times every two days, three times every three days, three times every four days, three times every five days, three times every six days, three times a week, three times every two weeks, three times every three weeks, three times every four weeks, three times every five weeks, three times every six weeks, three times every eight weeks, three times every twelve weeks, three times a month, three times every two months, three times every three months, three times every four months or less frequently or as needed so long as a therapeutic response is achieved. In one embodiment, one or more doses of a PD-1 inhibitor as set forth herein are administered once every three weeks. In one embodiment, one or more doses of a PD-1 inhibitor as set forth herein are administered once every six weeks.

In certain embodiments, the one or more doses are administered in at least one treatment cycle. The methods, according to this aspect, comprise administering to a subject in need thereof at least one treatment cycle comprising administration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of a PD-1 inhibitor (e.g., an anti-PD-1 antibody, such as cemiplimab, or antigen-binding fragment thereof). In one embodiment, a treatment cycle comprises 12 doses of a PD-1 inhibitor. In one embodiment, a treatment cycle comprises 24 doses of a PD-1 inhibitor.

PD-1 inhibitor Dosage

The amount of PD-1 inhibitor (e.g., an anti-PD-1 antibody, such as cemiplimab, or antigenbinding fragment thereof) administered to a subject according to the methods disclosed herein is, generally, a therapeutically effective amount. As used herein, the term "therapeutically effective amount" means an amount of a PD-1 inhibitor that results in one or more of: (a) a reduction in the severity or duration of a symptom or an indication of head and neck cancer - e.g., a tumor lesion; (b) inhibition of tumor growth, or an increase in tumor necrosis, tumor shrinkage and/or tumor disappearance; (c) delay in tumor growth and development; (d) inhibition of tumor metastasis; (e) prevention of recurrence of tumor growth; (f) increase in survival of a subject with a cancer; and/or (g) a reduction in the use or need for conventional anti-cancer therapy (e.g., elimination of need for surgery or reduced or eliminated use of chemotherapeutic or cytotoxic agents) as compared to an untreated subject or a subject treated with platinum based chemotherapy or other SOC therapy such as those disclosed herein.

In certain embodiments, a therapeutically effective amount of the PD-1 inhibitor (e.g., an anti-PD-1 antibody or antigen-binding fragment thereof, such as cemiplimab or a bioequivalent thereof) can be from about 0.05 mg to about 1500 mg, from about 1 mg to about 800 mg, from about 5 mg to about 600 mg, from about 10 mg to about 550 mg, from about 50 mg to about 400 mg, from about 75 mg to about 350 mg, or from about 100 mg to about 300 mg of the antibody. For example, in various embodiments, the amount of the PD-1 inhibitor is about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about

160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about

220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about

280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about

340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about

400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about

460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about

520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about

580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about

640 mg, about 650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg, about

700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about 750 mg, about

760 mg, about 770 mg, about 780 mg, about 790 mg, about 800 mg, about 810 mg, about

820 mg, about 830 mg, about 840 mg, about 850 mg, about 860 mg, about 870 mg, about

880 mg, about 890 mg, about 900 mg, about 910 mg, about 920 mg, about 930 mg, about

940 mg, about 950 mg, about 960 mg, about 970 mg, about 980 mg, about 990 mg, about

1000 mg, about 1010 mg, about 1020 mg, about 1030 mg, about 1040 mg, about 1050 mg, about 1060 mg, about 1070 mg, about 1080 mg, about 1090 mg, about 1200 mg, about 1210 mg, about 1220 mg, about 1230 mg, about 1240 mg, about 1250 mg, about 1260 mg, about 1270 mg, about 1280 mg, about 1290 mg, about 1300 mg, about 1310 mg, about 1320 mg, about 1330 mg, about 1340 mg, about 1350 mg, about 1360 mg, about 1370 mg, about 1380 mg, about 1390 mg, about 1400 mg, about 1410 mg, about 1420 mg, about 1430 mg, about 1440 mg, about 1450 mg, about 1460 mg, about 1470 mg, about 1480 mg, about 1490 mg, or about 1500 mg.

The amount of a PD-1 antibody, such as cemiplimab, contained within an individual dose may be expressed in terms of milligrams of antibody per kilogram of subject body weight (/.e., mg/kg). In certain embodiments, the PD-1 antibody used in the methods disclosed herein may be administered to a subject at a dose of about 0.0001 to about 100 mg/kg of subject body weight. In certain embodiments, an anti-PD-1 antibody may be administered at dose of about 0.1 mg/kg to about 20 mg/kg of a patient's body weight. In certain embodiments, the methods of the present disclosure comprise administration of a PD-1 inhibitor (e.g., an anti-PD-1 antibody, such as cemiplimab, or antigen-binding fragment thereof) at a dose of about 1 mg/kg to 3 mg/kg, 1 mg/kg to 5 mg/kg, 1 mg/kg to 10 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg, or 10 mg/kg of a patient's body weight.

In certain embodiments, an individual dose amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody, such as cemiplimab, or antigen-binding fragment thereof) administered to a patient may be less than a therapeutically effective amount, /.e., a subtherapeutic dose. For example, if the therapeutically effective amount of a PD-1 inhibitor, e.g., an anti-PD-1 antibody, such as cemiplimab, or antigen-binding fragment thereof, comprises 3 mg/kg, a subtherapeutic dose comprises an amount less than 3 mg/kg, e.g., 2 mg/kg, 1.5 mg/kg, 1 mg/kg, 0.5 mg/kg or 0.3 mg/kg. As defined herein, a "subtherapeutic dose" refers to an amount of the PD-1 inhibitor that does not lead to a therapeutic effect by itself. However, in certain embodiments, multiple subtherapeutic doses of a PD-1 inhibitor are administered to collectively achieve a therapeutic effect in the subject.

In certain embodiments, each dose comprises 0.1 - 10 mg/kg (e.g., 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 10 mg/kg) of the subject's body weight. In certain other embodiments, each dose comprises 5 - 1500 mg of the PD-1 inhibitor (such as an anti-PD-1 antibody, for example cemiplimab, or antigen-binding fragment thereof), e.g., 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 45 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1100 mg, 1150 mg, 1200 mg, 1550 mg, 1300 mg, 1350 mg, 1400 mg, 1450 mg, or 1500 mg of the PD-1 inhibitor.

In certain embodiments, the PD-1 inhibitor (e.g., an anti-PD-1 antibody, such as cemiplimab, or antigen-binding fragment thereof) is administered in combination with radiation therapy provided in one or more doses of 2 - 100 Gray (Gy). In certain embodiments, the radiation therapy comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 23, 25, 27, 30, 35, 40, or 45 Gy. In certain other embodiments, the radiation therapy comprises 50 - 100, 60 - 90, or 70-80 Gy. In certain embodiments, the radiation therapy is administered in fractions (hypofractionated radiation therapy). Hypofractionated radiation therapy (hfRT) refers to radiation therapy in which a radiation dose is comprised in 2 or more fractions. In various embodiments, each fraction comprises 2 - 20 Gy. For example, a radiation dose of 50 Gy may be split up into 10 fractions, each comprising 5 Gy. In certain embodiments, the 2 or more fractions are administered on consecutive or sequential days. In certain other embodiments, the 2 or more fractions are administered over a period of time comprising once in 2 days, once in 3 days, once in 4 days, once in 5 days, once in 6 days, once in 7 days, twice in 3 days, twice in one week, three times in one week, or a combination thereof.

Combination regimens

In certain embodiments of the method of the invention, treatment with the one or more immunogenic compositions capable of inducing an immune response against cells expressing HPV16 antigens and treatment with the PD-1 inhibitor is performed simultaneously or in overlapping time intervals. In one embodiment, the treatment with the one or more immunogenic composition is initiated before the treatment with the PD-1 inhibitor. In another embodiment, the treatment with the PD-1 inhibitor is initiated before the treatment with the one or more immunogenic compositions.

In one embodiment, the PD-1 inhibitor, such as the PD-1 antibody, for example cemiplimab, is administered with intervals of 2 to 4 weeks, such as 3 weeks and the one or more immunogenic compositions, such as ISAlOlb, are also administered with intervals of 2 to 4 weeks, such as 3 weeks.

In one embodiment, the PD-1 inhibitor, such as the PD-1 antibody, for example cemiplimab, is administered with intervals of 2 to 4 weeks, such as 3 weeks and the one or more immunogenic compositions, such as ISAlOlb, are also administered with intervals of 2 to 4 weeks, such as 3 weeks and the one or more immunogenic compositions are in total administered twice or three times within a 6-12 weeks period.

In one embodiment, the PD-1 inhibitor, such as the PD-1 antibody, for example cemiplimab, is administered with intervals of 2 to 4 weeks, such as 3 weeks and the one or more immunogenic compositions, such as ISAlOlb, are also administered with intervals of 2 to 4 weeks, such as 3 weeks, wherein the first administration of the one or more immunogenic compositions, such as ISAlOlb, is initiated between 1 day and 2 months, for example between 2 days and 1 months, before the initiation of the PD-1 inhibitor therapy. In one embodiment, the combination therapy follows the regimen set forth in Figure 1 or Figure 3, wherein it is understood that the exact day of administration may deviate somewhat, for example 1,2 or 3 days. For example, the administration at Day 29 could alternatively be performed on day 26, 27, 28, 30, 31 or 32.

In some embodiments, the method of the present disclosure comprises further forms of therapy, such as chemotherapy, further antibody therapy, radiotherapy and/or surgery.

In some embodiments, the method of the present disclosure comprises further forms of therapy, such as chemotherapy, further antibody therapy, radiotherapy and/or surgery for patients having a lower expression of PD-L1 (Combined Positive Score [CPS] < 1).

In some embodiments, the method of the present disclosure comprises further forms of antibody therapy wherein the antibody is a target that is different from PD-1, PD- L1 or PD-L2.

In one embodiment, the PD-1 inhibitor, such as cemiplimab, and the immunogenic composition(s) (e.g. ISAlOlb) are administered on the same day (on some days of the regimen). In one such embodiment, cemiplimab is infused first and ISASlOlb afterwards, for example at least 30 min later, e.g. at least 1 hour later.

Further aspects

In a further aspect, the disclosure relates to a kit comprising a PD-1 inhibitor, such as an anti-PD-1 antibody, such as cemiplimab, in combination with written instructions for use of a therapeutically effective amount of the PD-1 inhibitor for treating or inhibiting the growth of a tumor or improving overall survival of a patient with head and neck cancer.

In a further aspect, the disclosure relates to a kit comprising one or more immunogenic compositions, such as ISAlOlb, capable of inducing an immune response against HPV16 in combination with written instructions for use of a therapeutically effective amount of the one or more immunogenic compositions for treating or inhibiting the growth of a tumor or improving overall survival of a patient with head and neck cancer.

Table 3: Sequence listing

All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present disclosure and are not intended to limit the scope of what the inventors regard as their invention. Likewise, the disclosure is not limited to any particular preferred embodiments described herein. Indeed, modifications and variations of the embodiments may be apparent to those skilled in the art upon reading this specification and can be made without departing from its spirit and scope. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, room temperature is about 25°C, and pressure is at or near atmospheric.

EXAMPLE 1:

Clinical trial of cemiplimab and ISAlOlb vaccine (peltopepimut-S) in patients with recurrent/metastatic HPV16-positive Oropharyngeal Cancer who have experienced disease progression with prior anti-PD-1 therapy

This study is an open-label, single arm, phase 2 study of ISAlOlb plus cemiplimab in HPV16-positive OPC patients with disease progression on prior anti-PD-1 therapy

Study Objectives

A primary objective of the study is to estimate the clinical benefit of ISAlOlb plus cemiplimab after progression on prior anti-PD-1 therapy, as assessed by objective response rate (ORR) based on radiographic response, according to RECIST 1.1 for HPV16- positive Oropharyngeal Cancer (OPC) patients who have experienced disease progression with prior anti-PD-1 therapy. Secondary objectives of the study performed among HPV16- positive Oropharyngeal Cancer (OPC) patients who have experienced disease progression with prior anti-PD-1 therapy include: (1) to characterize the safety profile of ISAlOlb plus cemiplimab, as measured by incidence and severity of TEAEs/AESIs/SAEs and >3 grade laboratory abnormalities during the treatment period and up to 105 days after the last dose of study treatment; (2) to assess preliminary efficacy of ISAlOlb plus cemiplimab as measured by multiple criteria, such as (a) Duration of Response (DOR); (b) Progression- free Survival (PFS); (c) Overall Survival (OS) until death, loss to follow-up or termination of the study by the Sponsor. Exploratory objectives of the study performed among HPV16- positive Oropharyngeal Cancer (OPC) patients who have experienced disease progression with prior anti-PD-1 therapy include: (1) to assess the association of clinical efficacy with baseline tumor tissue immune biomarkers including PD-L1, immune cell subsets, MHC class 1 1 II, gene expression profile and tumor mutational burden; (2) to assess the association of clinical efficacy with the changes in frequency and clonal repertoire of HPV antigen specific T cells in peripheral blood and with ISAlOlb-specific IgG antibodies in serum/ plasma; (3) to assess the association of clinical efficacy with the changes in pro- and antiinflammatory serum cytokine biomarkers and immune cell subsets; (4) to assess the association of clinical efficacy with changes in tumor infiltrating T cells, T cell receptor clonal repertoire, and other tumor biomarkers (PD-L1, tumor gene expression and mutational profile); (5) to assess the association of clinical efficacy endpoints with posttreatment changes in tumor tissue and peripheral blood immune cell subsets (Teff, Treg, myeloid, dendritic and NK cells) and serum cytokine levels. (6) to explore novel candidate baseline predictive and pharmacodynamic biomarkers associated with clinical responses, including but not limited to cellular, gene expression and genomic parameters in tumor tissue and peripheral blood, including circulating tumor-derived HPV DNA; (7) to assess the immunogenicity of cemiplimab when combined with ISAlOlb; (8) to assess the presence of absence of anti-drug antibodies (ADA) against cemiplimab.

Study Design:

This is an open-label, single arm, phase 2 study of ISAlOlb plus cemiplimab in HPV16- positive OPC patients with disease progression on prior anti-PD-1 therapy.

Number of Patients:

At least 60 patients with recurrent or metastatic HPV16-positive OPC who have progressed on prior anti-PD-1 therapy.

Treatment Regimen:

All patients receive the following treatment regimen:

• ISAlOlb 100 pg/peptide by subcutaneous (SC) injection on Day 1, Day 29, and Day 50 and booster injection with ISAlOlb (100 pg/peptide, SC) 6 months after the first dose of ISAlOlb (a total of 4 doses). Cemiplimab 350 mg given by intravenous (IV) infusion over 30 minutes on Day 8, and then Q3W (Day 29, Day 50, etc) for up to 24 months, unless disease progression occurs, or treatment is withdrawn for toxicity or any other reason.

The timing of doses is summarized in Figure 1.

Study Duration: Study duration (after optional pre-screening*) includes up to 3 weeks of screening, 3 doses of ISAlOlb over 7 weeks, a booster ISAlOlb injection at 6 months, up to a total of 24 months of cemiplimab treatment, a follow-up visit 30 days after treatment discontinuation, and follow-up for long-term survival until death or end of study.

Patients are assessed for tumor response according to RECIST 1.1, at week 7, then every 9 weeks thereafter for up to 12 months, and then every 12 weeks for up to 24 months, until progressive disease (PD), unacceptable toxicity, or treatment withdrawal for any other reason, Those who achieve a complete response (CR), partial response (PR) or stable disease (SD) according to modified response evaluation criteria in solid tumors (RECIST) 1.1 will continue on study. Cemiplimab treatment continues for up to a total of 24 months.

*A pre-screening consent allows potential patients with recurrent/ metastatic OPC who meet inclusion criteria 1, 2, 4 and 6, to submit archival tumor tissue to be pre-screened for HPV16.

End of Study Definition : The end of study is defined as the date of the last contact of the last patient in the study.

Inclusion Criteria:

1. Men and women > 18 years of age.

2. Histologically confirmed recurrent or metastatic HPV16-positive Oropharyngeal Cancer (OPC)*. Patients with squamous cell carcinoma (SCC) of occult primary site, presenting with lymph node(s) limited only to the neck, are also eligible. Patients should have HPV16 positivity confirmed before being considered a candidate for this study, see inclusion criteria 3.

* Recurrent or metastatic disease in the context of this study is defined as recurrent, metastatic or advanced disease.

3. HPV16 genotyping as determined by a specified central reference laboratory with an established polymerase chain reaction (PCR)-based assay. If local specific HPV16 genotype assessment (PCR or in situ hybridization (ISH) has been performed, the patient can be enrolled if the result shows HPV16 positivity. Confirmation of HPV16 positive status then subsequently has to be performed by the central laboratory.

4. Patients who have received a minimum total dose of 600 mg of pembrolizumab or 960 mg of nivolumab or equivalent anti-PD-1 antibody with or without chemotherapy for only 1st or 2nd line recurrent/ metastatic HPV16 positive OPC. The last dose of anti-PD-1 must have been no more than 6 months prior to the first dose of study drug. Progressive disease (PD) must have been diagnosed during or after anti-PD-1 therapy (but not longer than 6 months after the last dose), and anti PD-1 therapy (as 1st or 2nd line for recurrent/metastatic HPV16 positive OPC) should have been the last treatment regimen that the patient received before entry into the current trial.

5. Ability to provide archived tumor sample (preferably tumor block or otherwise at least 10 unstained slides) to allow determination of HPV16- status by the central laboratory -or a local laboratory if applicable- using an investigational use only assay.

6. At least one measurable lesion by computed tomography (CT) or magnetic resonance imaging (MRI) per RECIST version 1.1 criteria. Target lesions may be located in a previously irradiated field if there is documented (radiographic) disease progression in that site.

7. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

8. Adequate organ and bone marrow function documented by: a. Hemoglobin > 8 g/dL b. Absolute neutrophil count > 1.5 x 109/L c. Platelet count > 75 x 109/L d. Serum creatinine < 1.5 x upper limit of normal (ULN) or creatinine clearance (CrCI) > 30 mL/min (using the Cockcroft-Gault formula):

Female CrCI = (140 - age in years) x weight in kg x 0.85

72 x serum creatinine in mg/dL

Male CrCI = (140 - age in years) x weight in kg x 1.00

72 x serum creatinine in mg/dL e. Adequate hepatic function: i. Total bilirubin < 1.5 x upper limit of normal (ULN) (<3 x ULN if tumor liver involvement) ii. Aspartate Aminotransferase (AST) < 2.5 x ULN (<3 x ULN if tumor liver involvement) iii. Alanine Aminotransferase (ALT) < 2.5 x ULN (<3 x ULN if tumor liver involvement) iv. Alkaline Phosphatase (ALP) < 2.5 x ULN (<3 x ULN if tumor liver or bone involvement)

NOTES: (i) In patients with tumor liver involvement if levels of AST >3 x ULN or ALT >3 x ULN, and bilirubin levels >2 x ULN, the patient is excluded regardless of the above criteria (ii) Patients with Gilbert's syndrome and total bilirubin up to 3 x ULN may be eligible if total bilirubin is < 3.0 mg/dL. Gilbert's syndrome must be documented appropriately as past medical history.

Exclusion criteria:

1. Invasive surgery (defined as surgical intervention requiring general or spinal anaesthesia and hospital admission) within 4 weeks prior to start of study treatment.

2. Patients who, after progressing on anti-PD-1, received additional anti-cancer therapy (chemotherapy, radiotherapy, experimental TKI's, immunotherapy, anti-EGFR antibodies, surgery). The following palliative treatments are allowed: palliative radiotherapy (but NOT for target lesions) palliative surgery bone resorptive therapy such as bisphosphonates and denosumab but only if patients have been on stable doses for > 4 weeks prior to first dose of test treatment

3. Patients who have permanently discontinued anti-cancer immune modulating therapies due to drug-related toxicity.

4. Another malignancy that is progressing or requires active treatment and/or history of malignancy other than oropharyngeal cancer within 3 years of date of first planned dose of study drug, except: a. Non-melanoma skin cancer that has undergone potentially curative therapy b. In situ cervical carcinoma c. Ductal carcinoma in situ of the breast d. In-situ prostate cancer with non-detectable prostate specific antigen e. Any tumor that has been deemed to be effectively treated with definitive local control (with or without continued adjuvant hormonal therapy), and the patient is deemed to be in complete remission for at least 2 years prior to randomization, and no additional therapy is required during the study period. 5. Any condition that requires ongoing/continuous corticosteroid therapy (> 10 mg prednisone/day or anti-inflammatory equivalent) within 1 week prior to the first dose of study therapy. Patients who require a brief course of steroids (up to 2 days in the week before enrolment) or physiologic replacement are not excluded.

6. Ongoing or recent (within 5 years) evidence of significant autoimmune disease that required treatment with systemic immunosuppressive treatments. The following are not exclusionary: vitiligo, childhood asthma that has resolved, endocrinopathies (such as hypothyroidism or type 1 diabetes) that require only hormone replacement, or psoriasis that does not require systemic treatment.

7. Subjects with known brain metastases or leptomeningeal metastases.

Subjects with brain metastases are eligible if i) these have been treated, ii) there is no MRI (or CT scan where MRI is contraindicated) evidence of progression for at least 4 weeks after completion of the last treatment, iii) absence of new neurological signs/symptoms, and iv) there is no need for corticosteroid use for management of these lesions. Base of skull involvement without definitive evidence of dural or parenchymal (brain) involvement is acceptable.

8. Encephalitis, meningitis, organic brain disease (e.g. Parkinson's disease) or uncontrolled seizures in the year prior to first dose of study therapy

9. Known history of, or any evidence of interstitial lung disease, or active, non- infectious pneumonitis (in the past 5 years). A history of radiation pneumonitis in the radiation field is permitted.

10. Has participated in a study of an investigational agent or an investigational device, unless approved by the Sponsor

11. Prior treatment with therapeutic anti-HPV vaccines including ISA101 or ISAlOlb. Patients who have received a preventive HPV vaccine are allowed

12. Grade 1 or greater toxicities attributed to systemic prior anti-cancer therapy other than alopecia, fatigue (NCI CTCAE), radiation dermatitis, laboratory abnormalities that are not considered clinically significant by the treating physician, before administration of study drug. Patients with residual grade 1 toxicities or toxicities attributed to systemic prior anticancer therapy that have become chronic and are not expected to resolve, such as neuropathy after platinum-based therapy or chronic skin eruptions/dermatitis due to anti- EGFR therapy, can be included in this study. 13. Uncontrolled infection with human immunodeficiency virus, hepatitis B or hepatitis C infection; or diagnosis of immunodeficiency.

NOTES:

Patients are tested for hepatitis C virus (HCV) and hepatitis B virus (HBV) at screening.

Patients with known HIV infection who have controlled infection (undetectable viral load (HIV RNA PCR) and CD4 count above 350 either spontaneously or on a stable antiviral regimen) are permitted. For patients with controlled HIV infection, monitoring is performed per local standards.

Patients with hepatitis B (HepBsAg + ) who have controlled infection (serum hepatitis B virus DNA PCR that is below the limit of detection AND receiving anti-viral therapy for hepatitis B) are permitted. Patients with controlled infections must undergo periodic monitoring of HBV DNA.

Patients must remain on anti-viral therapy for at least 6 months beyond the last dose of investigational study drug.

Patients who are hepatitis C antibody positive (HCV Ab+) who have controlled infection (undetectable HCV RNA by PCR either spontaneously or in response to a successful prior course of anti-HCV therapy) are permitted.

14. Any infection requiring hospitalization or treatment with IV anti-infectives within 2 weeks of first dose of study therapy.

15. Receipt of a live vaccine within 4 weeks of planned start of study drug.

16. Allogeneic stem cell transplantation, or autologous stem cell transplantation.

17. Recipients of organ transplants at any time unless discussed with and approved by the medical monitor.

18. Known allergy or hypersensitivity to components of study drug or excipients.

19. Known psychiatric or substance abuse disorders that would interfere with participation with the requirements of the study.

20. Any medical condition, co-morbidity, physical examination finding, or metabolic dysfunction, or clinical laboratory abnormality that, in the opinion of the investigator, renders the patient unsuitable for participation in a clinical trial due to high safety risks and/or potential to affect interpretation of results of the study. 21. Clinically significant cardiac disease, including left ventricular ejection fraction < 40%, unstable angina, acute myocardial infarction within 6 months before Cycle 1 Day 1, New York Heart Association Class III or IV congestive heart failure, and uncontrolled arrhythmia.

22. Member of the clinical site study team or his/her immediate family, unless prior approval granted by the Sponsor.

23. Women with a positive serum hCG pregnancy test at the screening/baseline visit. If positive, pregnancy must be ruled out by a second sensitive hCG pregnancy test for patient to be eligible. Breastfeeding women are also excluded.

24. Women of childbearing potential (WOCBP)*, or sexually active men, who are unwilling to practice highly effective contraception prior to the initial dose/start of the first treatment, during the study, and for at least 6 months after the last dose.

Main study parameters/endpoints and statistical considerations:

Analysis of primary endpoint

ORR per RECIST version 1.1 is summarized for each stage using descriptive statistics, along with 2-sided 95% confidence interval. The statistical inference is based on number of responders.

Analysis of secondary endpoints

Treatment-emergent AEs/AESIs/SAEs and grade 3 or greater laboratory abnormalities are summarized using descriptive statistics.

OS, PFS, and DOR are summarized using Kaplan-Meier method.

Exploratory Biomarker Assessments

Tumor biopsies (optional) and blood samples are requested for the 26 study patients participating in the substudy. If tumor biopsies are obtained, they are used to assess increased entry of T cells into tumor tissue and determination of their phenotype compared to a pre-treatment tumor tissue sample. Peripheral blood immune monitoring on cryopreserved peripheral blood mononuclear cells (PBMC) and serum cytokine analysis are performed to assess systemic immune activation, as exploratory correlative parameters of immune safety and pharmacodynamic activity.

Blood samples for PBMC biomarkers and detection of circulating tumor-derived HPV DNA are collected for all patients before and after ISAlOlb booster injection to assess the (re)activation of the systemic immune system after the booster injection of ISAlOlb. PD- L1 expression is evaluated in archival tissue samples for all patients in the study.

Analysis of all exploratory tissue or PBMC/ serum biomarker assessments is descriptive.

Primary Efficacy Analysis

The primary efficacy analyses use ORR based on radiographic response, measured by RECIST version 1.1. The tumor response is assessed by the investigator/treating physician, with the option for central review for all patients in the study at the discretion of the Sponsor. As per RECIST criteria a confirmation tumor assessment of the CR or PR is performed 4 weeks after the CR or PR has been demonstrated.

The primary analysis of efficacy is based on the binomial exact confidence interval approach, i.e., whether the lower limit of 95% confidence interval will exclude a historical control ORR. The 95% binomial exact confidence interval is computed using the Clopper- Pearson method for observed ORR.

The study is designed as a two-stage single-arm historically-controlled response evaluation assessing for deciding between the null hypothesis of a < 0.10 response proportion against the specific alternative hypothesis of a > 0.25 response proportion. The design is based on a target of 86 patients with the first 26 evaluable patients analyzed for stage 1 evaluation. Evidence of either futility or early evidence of efficacy will be assessed using stage 1 data with no pause in accrual. Finding evidence of efficacy from the stage 1 analysis will not necessarily induce a termination of accrual because the response proportion estimate will be strong and additional safety data will then become available. For the designed trial the false positive probability is 0.0214 and the power probability is 0.9446.

Investigational Products

Preparation and labelling of the investigational products is done in accordance with Good Manufacturing Practice guidelines, Good Clinical Practice (GCP) guidelines and applicable local regulations.

Identity of Investigational Products

ISAlOlb

The DP ISAlOlb is provided in 2 vials, HPV-DP-5P containing 5 peptides and HPV-DP-7P containing 7 peptides. Bulk preparation, sterile filtration, filling, and lyophilization have been developed for each DP. Before SC injection, the content of the 2 DP vials are dissolved separately in reconstitution solution. Subsequently, the two solutions, each containing a dissolved DP are mixed to make an emulsion (white appearance) in a 1 : 1 (v/v) ratio Montanide ISA 51 VG. The final vaccine composition has a pH of approximately 3.

The vaccine is administered in two different limbs. It is recommended to rotate between limbs e.g. first vaccination in the upper-leg, second vaccination in the upper-arm and third vaccination in the upper-leg.

Cemiplimab

Cemiplimab (REGN2810) is supplied as a liquid in sterile, single use vials. Each vial contains cemiplimab at a concentration of 50 mg/mL. Cemiplimab is administered IV at a dose of 350 mg over 30 minutes Q3W.

Investigational Product Administration

All patients receive the following treatment regimen:

• ISAlOlb 100 pg/peptide by SC injection on Day 1, Day 29, and Day 50 and a booster injection with ISAlOlb (100 pg/peptide, SC) 6 months after the first dose of ISAlOlb (a total of 4 doses). The term 'booster injection' refers to an additional dose of ISAlOlb given post-primary vaccination. Tumors that have escaped the control of anti-PD- 1 therapy once before may undermine this immunity again. An extra dose of ISAlOlb at 6 months is given to explore if the booster injection will improve immune memory and protection against HPV16 induced cancer in those patients who have not developed medically important breakthrough disease at that time.

• Cemiplimab 350 mg given by IV infusion over 30 minutes on Day 8, and then Q3W (Day 29, Day 50, etc) for up to 24 months, unless disease progression occurs, or treatment is withdrawn for any other reason.

Procedures and assessments

Screening

The screening visit takes place from Day -21 to Day 1 before the first administration of ISAlOlb. During the screening visit several assessments are performed.

Duration of Treatment

Patients are considered to be on treatment from administration of the first dose of study drug until the day they are withdrawn from study treatment (EOT). Treatment duration includes 3 doses of ISAlOlb over 7 weeks, with a booster injection with ISAlOlb at 6 months from the first dose of ISAlOlb and up to 24 months of treatment with cemiplimab, every 3 weeks. Patients return 30 days after the last dose of study drug for a follow-up visit. Patients are then followed for PFS and OS for up to 12 months following EOT, see below.

Patients are considered to be on study from the signing of the ICF until death or the completion of the follow-up period.

Efficacy Evaluation

Tumor Assessments for Response

Efficacy assessments include tumor response assessed by RECIST 1.1 using CT scan or MRI imaging studies and survival data.

At screening tumor assessments for response include a CT or MRI of the head and neck, chest abdomen, and all other known sites of disease. If radiology assessment has been performed as a standard of care procedure within 28 days prior to enrolment, this assessment may be used as screening assessment if fulfils the required study criteria. An MRI of the brain (or CT scan with contrast if MRI is contraindicated) is performed for patients with known or suspected brain metastases (base of skull covered on CT scan of head/neck or MRI of head/neck).

Tumor assessments are performed in the same manner at baseline, at week 7 (before the fourth cemiplimab dose) and then every 9 weeks (± 7 days) or as clinically indicated for up to 12 months, and then every 12 weeks up to 24 months, until disease progression or treatment is discontinued (whichever occurs later), performing a CT scan or MRI of the head and neck, chest, and abdomen, and all know sites of disease by the same imaging method used at screening/baseline. As per RECIST criteria a confirmation tumor assessment of the CR or PR is to be performed 4 weeks after the CR or PR has been demonstrated. Patients with a history of brain metastasis should also have a brain MRI (or CT scan with contrast if MRI is contraindicated) approximately every 9 weeks, or sooner if clinically indicated.

Patients who finish treatment without radiological PD continue with the tumor assessment every 12 weeks (± 7 days) from enrolment until PD, start of new antitumor therapy, death, or date of end of study. After radiological PD is documented or new antitumor therapy is started, patients are followed for survival every 12 weeks (± 2 weeks) from the EOT visit until death or date of end of study, whichever occurs first. Follow-up for survival, after radiological PD is documented or new therapy is started, may be made by telephone calls. Pharmacokinetic Variables

Cemiplimab

Blood samples for the assessment of cemiplimab concentrations in serum are collected from all patients as follows:

• Pre-dose and within 10 minutes after end-of-infusion at Day 8 (after the first dose of cemiplimab)

• Pre-dose at Day 50 (Ctrough).

• Pre-dose (Ctrough) at Day 176 (before 9th dose of cemiplimab).

• Pre-dose (Ctrough) every 6 months (24 weeks) thereafter during therapy; and predose at EOT, the follow-up visit.

Immunogenicity variables for cemiplimab:

The immunogenicity variables are ADA status, titer, and time-point/visit.

Pharmacodynamic Variables

Pharmacodynamic assessments include analysis of blood and tissue samples.

Biomarkers

Tumor and blood samples are collected for the 26 patients enrolled in the substudy at baseline and following the initiation of study treatment

• Assessment of tumor biopsies at baseline to explore PD-L1 expression.

• Assessment of on treatment biopsy for exploratory endpoints.

• Assessment of peripheral blood biomarkers (plasma, serum and PBMC samples)

Blood samples for PBMC biomarkers and detection of circulating tumor-derived HPV DNA are collected for all patients before and after ISAlOlb booster injection (at Day 176 and Day 197) to assess the (re)activation of the systemic immune system by the booster ISAlOlb injection. PD-L1 expression are evaluated in archival tissue samples for all patients in the study.

Results

ISAlOlb plus cemiplimab treatment response in patients with OPC who have progressed on prior anti-PDl monotherapy. An interim analysis was performed at the end of Stage 1, when 26 patients with a centrally confirmed HPV16 positive tumor, who had received at least one dose of ISAlOlb had been followed for 6 months.

As shown in Table 4, 26 patients (mean age 60.7 ±12.9 years; 22 (84.6%) male and 4 (15.4%) female) enrolled in Stage 1. Median follow-up was 16.1 weeks (range 1.3-84.7). In all patients, the anti-PD-1 used before entry into the current trial was either pembrolizumab or nivolumab. Four patients had a confirmed Partial Response (PR) (15.4%) (Table 5 and Figure 2). In 12 patients (46.2%) Best Overall Response (BOR) was Stable Disease (SD); in 8 patients (30.8%) BOR was PR. Two (7.7%) patients did not have a tumor assessment after baseline: one patient died on day 8 due to OPC and one patient withdrew consent shortly after enrolment. The Clinical Benefit Ratio was 61.5%. Nine (34.6%) patients received a booster injection. At the time of the booster, 2 patients had PR and 5 patients had meaningful (> 6 months) SD. Time to response ranged between 127 and 525 days, with a duration of response ranging from 25 to 250 days. The spider plot (Figure 3) depicts the change in sum of Target Lesions during the study.

Table 6 shows time to response (TTR) and duration of response (DOR) for four responding patients. Patient A (Figure 2, Figure 3, Table 6) was a 73-year old male, initially diagnosed with stage IV OPC. He was treated with surgery, followed by first line carboplatin, 5FU and cetuximab. His BOR on this chemo regimen was PR. His second line treatment consisted of nivolumab, of which he received 7 doses in total (1680mg). The BOR of the nivolumab treatment was Progressive Disease (PD). Subsequently, the patient started with study treatment. He reached PR after 167 days in the trial and has received a booster injection after 6 months. The response is still ongoing with currently a Duration of Response (DOR) of 439 days. The sum of the target lesions decreased with 85% compared to baseline.

Patient B (Figure 2, Figure 3, Table 6) was a 64-year old male who presented with a T3N3Nx tumor. He was initially treated with surgery and radiotherapy, followed by cisplatin (BOR not evaluable). About 18 months after the end of the cisplatin treatment, he developed metastasized disease and was treated with pembrolizumab in combination with carboplatin and paclitaxel (the latter two stopped for toxicity). In total, he received 1600mg pembrolizumab in 8 doses with a PR as BOR. Eventually, this patient progressed and started with study treatment. As a target lesion, a metastasis in the liver was chosen with a diameter of 12 mm. Additionally, this patient had 2 non-target lesions in the lungs and head and neck region, respectively. As shown in Figure 3, the target lesion initially increased. The absolute increase was smaller than 5 mm, meaning that this increase did not meet the criteria of PD according to RECIST 1.1. After the increase, the lesion started decreasing. The patient received a booster injection, and after 525 days in the trial, this patient had a PR.

The median duration of study treatment was 13.1 weeks (range 1.1-81.9), with 3 patients still on treatment at the data cut-off. Median progressive-free survival and overall survival were 3.9 months (95% CI 1.6 to 5.9) and 11.6 months (95% CI 6.5 to x), respectively.

There were two grade 3 adverse events (AEs) related to ISAlOlb: erythema at the injection site and diarrhea. Two patients (7.7%) had grade 3 immune checkpoint inhibitor-related auto-immune events. Grade 4-5 AEs related to study treatment did not occur.

Table 4. Baseline characteristics

Characteristic Number of subjects (total n = 26)

Mean (±SD) 60.7 (12.9)

Male 22 (84.6)

Time from diagnosis to study entry, months

Treatment line in current study

Third line 10 (38.5)

Median (range) 16.1 (1.3 - 84.7)

Table 5. Efficacy

Characteristic Number of subjects (total n = 26) [24]

Complete response 0 Stable disease 12 (46.2) [50]

Objective response rate 4 (15.4) [16.7]

Disease control rate at 6 months, n (%)** 7 (26.9) [29.2]

*Two (7.7%) patients did not have a tumor assessment after baseline: one patient died on day 8 due to OPC and one patient withdrew consent shortly after enrolment. **Disease control rate = BOR CR + PR + SD

Table 6. Time to response (TTR)* and duration of response (DOR)**

Patient Baseline sum target lesions (mm) TTR (days) DOR (days)

B 12 525 25

D 66 307 135

* Calculated until first tumor assessment showing response. ** DOR calculated from first tumor assessment showing response until last tumor assessment showing response, progression or date of last contact if no progression and no death

EXAMPLE 2

Clinical trial of cemiplimab versus the combination of cemiplimab with ISAlOlb in the treatment of subjects with HPV16-positive Oropharyngeal Cancer (OPC).

This study is a randomized, double-blind, placebo-controlled, Phase 2 Study of cemiplimab versus the combination of cemiplimab with ISAlOlb (peltopepimut-S) in the treatment of subjects with HPV16-positive Oropharyngeal Cancer (OPC) who relapsed following primary surgical or chemoradiation treatment or who have progressed on prior platinum-based therapy and did not receive prior treatment with anti-PDl/anti-PDLl antibody or therapeutic HPV16 comprising vaccines.

Study Objectives

A primary efficacy objective is to evaluate if the addition of ISAlOlb to cemiplimab results in improved overall response rate (ORR) compared to cemiplimab alone, according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 by independent review for subject with HPV16-positive OPC. A primary safety objective is to assess the safety and tolerability of the combination of cemiplimab plus ISAlOlb compared to placebo plus cemiplimab in subject with HPV16-positive OPC.

Secondary objectives of the study performed in subject with HPV16-positive OPC include: (1) Duration of response (DOR) by independent review in subjects randomized to receive ISAlOlb plus cemiplimab compared to placebo plus cemiplimab; (2) Time to response (TTR) by independent review in subjects randomized to receive ISAlOlb plus cemiplimab compared to placebo plus cemiplimab; (3) Prog cession -free survival (PFS) by independent review in subjects randomized to receive ISAlOlb plus cemiplimab compared to placebo plus cemiplimab. The PFS rate at 6 months will also be compared; (4) Median overall survival (OS) in subjects randomized to receive ISAlOlb plus cemiplimab compared to placebo plus cemiplimab. The OS rate will also be compared.

Exploratory objectives of the study performed in subject with HPV16-positive OPC include: (1) to compare the HPV16 specific immune responses and clinical responses in the 2 treatment arms, and determine if enhanced HPV16 immune responses are associated with improved clinical outcomes, e.g., ORR, PFS, and OS; (2) to assess tumor biopsies at baseline and during treatment to explore PD-L1 expression, and lymphocyte and myeloid cell distribution, and specific gene and protein expression; (3) to assess correlative biomarkers to treatment response or adverse events (AEs)

Blood samples at baseline and during treatment for serum cytokines, whole blood for control of tumor mutation analysis, cell-free nucleic acids in plasma, peripheral blood mononuclear cell (PBMC) characterization with profiles by multichannel fluorescence activated cell sorting (FACS), RNA sequence expression analysis (RNAseq), and HPV and control recall antigen-specific immune responses.

Study Design:

This is a double-blind, placebo-controlled, randomized, Phase 2 study in which subjects are randomly assigned in a 1 : 1 fashion to cemiplimab plus ISAlOlb placebo or cemiplimab plus ISAlOlb. The randomization is stratified by smoking status (non-smoker vs. smoker and the number of prior treatment lines (1 vs. 2). The study design is summarized in the Figure 4.

Number of Patients:

199 patients with (1) recurrent or metastatic HPV16-positive OPC and relapsed following primary surgical or chemoradiation treatment, wherein the patient did not receive prior treatment with anti-PDl/anti-PDLl antibody or therapeutic HPV16 comprising vaccines, or

(2) recurrent or metastatic HPV16-positive OPC and has progressed on prior platinumbased therapy, wherein the patient did not receive prior treatment with anti- PDl/anti-PDLl antibody or therapeutic HPV16 comprising vaccines, or

(3) previously untreated metastatic HPV-16 OPC, wherein the patient did not receive prior treatment with anti-PDl/anti-PDLl antibody or therapeutic HPV16 comprising vaccines.

Treatment regimen:

Arm 1 (Control :

• Matching placebo for ISAlOlb by subcutaneous (SC) injection on Day 1, Day 29, and Day 50 (total of 3 doses).

• Cemiplimab 350 mg given by intravenous (IV) infusion on Day 8 and then every 3 weeks (Q3W) (Day 29, Day 50, etc), for up to 24 months unless disease progression occurs or treatment is withdrawn for any other reason.

Arm 2 (Experimental Arm):

• ISAlOlb 100 pg/peptide by SC injection on Day 1, Day 29, and Day 50 (total of 3 doses).

• Cemiplimab 350 mg given by IV infusion on Day 8, and then Q3W (Day 29, Day 50, etc) for up to 24 months unless disease progression occurs or treatment is withdrawn for any other reason.

The timing of doses is summarized in Figure 5.

Study Duration:

Study duration (after optional pre-screening) includes up to 3 weeks of screening, 3 doses of ISAlOlb over 7 weeks, up to a total of 24 months of cemiplimab treatment, a follow-up visit 30 days after treatment discontinuation, and follow-up for long-term survival until death or end of study. Subjects are assessed for tumor response at Week 7, then every 9 weeks for up to 12 months, and then every 12 weeks for up to 24 months, until progressive disease (PD), unacceptable toxicity, or treatment withdrawal for any other reason. As per RECIST criteria a confirmation tumor assessment of the CR or PR is performed 4 weeks after the CR or PR has been demonstrated. It is anticipated that ORR for the study are evaluable at approximately 41 months after the first subject is enrolled, and the OS analysis is performed at approximately 3.5 years after the first subject is enrolled.

Study Population:

Inclusion criteria:

1. Males and females, > 18 years of age.

2. Subjects with histologically confirmed recurrent or metastatic* HPV16 positive OPC not amenable to any therapy with curative intent, whose tumors express PD-L1 (Combined Positive Score [CPS] > 1) and who are candidates for first line therapy with an PD-1 blocking antibody, AND subjects with recurrent or metastatic HPV16 positive OPC with disease progression on or after platinum containing chemotherapy not amenable to any therapy with curative intent.

* Recurrent or metastatic disease in the context of this study if defined as recurrent, metastatic, or advanced disease.

3. HPV16 genotyping determined by a specified central reference laboratory with an established polymerase chain reaction (PCR)-based assay. If local HPV16 specific genotype assessment or in situ hybridization (ISH) assessment has been performed, the subject can be enrolled if the result shows HPV16 positivity. Confirmation of HPV16 positive status is performed retrospectively by the central laboratory.

4. Eastern Cooperative Oncology Group (ECOG) performance status of 0-1.

5. Measurable disease, defined as at least 1 lesion that can be accurately measured in at least 1 dimension with a minimum size of 10 mm by computed tomography (CT) scan or magnetic resonance imaging (MRI) per RECIST 1.1 criteria. Indicator lesions must not have been previously treated with surgery, radiation therapy, or radiofrequency ablation, unless there is documented progression after therapy.

6. Prior curative radiation therapy must have been completed at least 8 weeks prior to study drug administration. Prior focal palliative radiotherapy must have been completed at least 2 weeks before study drug administration.

7. Screening laboratory values must meet the following criteria and should be obtained within 14 days prior to randomization: i) White blood cell count (WBC) > 2 x 109/L ii) Absolute neutrophil count (ANC) > 1.5 x 109/L iii) Platelets > 100 x 109/L iv) Hemoglobin > 8.0 g/dL v) Serum creatinine < 1.5 x upper limit of normal (ULN) or creatinine clearance (CrCI) > 40 mL/min (using the Cockcroft-Gault formula):

Female CrCI = (140 - age in years) x weight in kg x 0.85

72 x serum creatinine in mg/dL

Male CrCI = (140 - age in years) x weight in kg x 1.00

72 x serum creatinine in mg/dL vi) Hepatic function: a. Total bilirubin < 1.5 x ULN (if liver metastases < 3 x ULN). Subjects with Gilbert's Disease and total bilirubin up to 3 x ULN may be eligible if total bilirubin < 3.0 mg/dL. b. Transaminases (ALT and AST) < 3 x ULN (or < 5.0 x ULN, if liver metastases) c. Alkaline phosphatase < 2.5 x ULN (or < 5.0 x ULN, if liver or bone metastases)

Note for subjects with hepatic metastases: If transaminase levels (aspartate aminotransferase [AST] and/or alanine aminotransferase [ALT]) > 3 x but < 5 x ULN, total bilirubin must be < 1.5 x ULN. If total bilirubin > 1.5 x but < 3 x ULN, both transaminases (AST and ALT) must be < 3 x ULN.

8. Subjects must have baseline oxygen saturation by pulse oximetry of > 92% at rest on room air.

9. Women of childbearing potential (WOCBP) must have a negative serum or urine pregnancy test (minimum sensitivity 25 IU/L or equivalent units of human chorionic gonadotropin [HCG]) within 24 hours prior to the start of study drug.

10. Women must not be breastfeeding.

11. WOCBP must agree to follow instructions for method(s) of contraception from the time of enrolment for the duration of treatment with study drug(s), and then for 6 months post treatment completion. 12. Males who are sexually active with WOCBP must agree to follow instructions for methods of contraception for the duration of treatment with study drugs, and then for a total of 6 months post treatment completion.

13. Azoospermic males and WOCBP who are continuously not heterosexually active are exempt from contraceptive requirements. However, WOCBP still must undergo pregnancy testing.

Investigators shall counsel WOCBP and male subjects who are sexually active with WOCBP on the importance of pregnancy prevention and the implications of an unexpected pregnancy. Investigators shall advise WOCBP and male subjects who are sexually active with WOCBP on the use of highly effective methods of contraception. Highly effective methods of contraception have a failure rate of < 1% when used consistently and correctly.

Exclusion criteria:

1. Subjects with previously untreated metastatic or unresectable, recurrent HPV16 positive OPC whose tumors do not express PD-L1 (CPS < 1) and who are therefore not candidates for monotherapy with an anti-PD-1 antibody.

2. Subjects with known brain metastases or leptomeningeal metastases.

3. Any serious or uncontrolled medical disorder that, in the opinion of the investigator, may increase the risk associated with study participation or study drug administration, impair the ability of the subject to receive protocol therapy, or interfere with the interpretation of study results.

4. History of other malignancy < 3 years prior to entry into this trial with the exception of basal cell or squamous cell skin carcinoma(s) which were treated with local resection only, OR carcinoma in situ of the cervix, prostate or breast, OR low grade non-muscle invasive superficial bladder cancer (TaLG)/carcinoma in situ of the bladder.

5. Subjects with active, known, diagnosed or suspected auto-immune disease. Subjects suffering from vitiligo, type I diabetes mellitus, residual hypothyroidism due to auto-immune thyroiditis only requiring thyroid hormone replacement therapy, or psoriasis not requiring systemic treatment can be enrolled.

6. Patients diagnosed with active interstitial lung disease (ILD)/pneumonitis or a history of ILD/pneumonitis within the last 5 years, or another condition requiring immunosuppressive doses of medication such as systemic corticosteroids or absorbed topical corticosteroids (doses > 10 mg/day prednisone or equivalent) or other immunosuppressive medications within 14 days of study drug administration. Inhaled or topical corticosteroids and adrenal replacement doses < 10 mg daily prednisone or equivalent are permitted.

7. Subjects requiring maintenance treatment with immunosuppressive doses of systemic corticosteroids. Subjects being treated with a short course of corticosteroids (> 10 mg/day prednisone equivalents) should discontinue this therapy at least 2 weeks prior to start of study treatment.

8. Prior treatment with an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, cemiplimab), as well as anti-PD-Ll, anti-PD-L2, anti-CTLA-4 antibody, or any other antibody or drug specifically targeting T-cell co stimulation or immune checkpoint pathways.

9. Prior treatment with more than one chemotherapy regimen for the management of metastatic OPC.

10. Prior treatment with therapeutic anti-HPV vaccines including ISA101 or ISAlOlb. Subjects who have received a preventive HPV vaccine are allowed.

11. Grade 1 or greater toxicities attributed to systemic prior anti-cancer therapy other than alopecia, fatigue (NCI CTCAE), radiation dermatitis, laboratory abnormalities that are not considered clinically significant by the treating physician, before administration of study drug. Subjects with residual grade 1 toxicities or toxicities attributed to systemic prior anticancer therapy that have become chronic and are not expected to resolve, such as neuropathy after platinum based therapy, can be included in this trial.

12. Prior treatment with other immune-modulating agents that was

(a) administered within 4 weeks (28 days) prior to the first dose of cemiplimab, OR

(b) associated with immune-mediated adverse events that were > Grade 1 within 90 days prior to the first dose of cemiplimab, OR (c) associated with toxicity that resulted in discontinuation of the immune-modulating agent.

13. Invasive surgery (defined as surgical intervention requiring general or spinal anaesthesia, and hospital admission) within 28 days prior to start of study treatment.

14. Subjects being treated or requiring treatment with: chemotherapy, for cancer, biological therapy for cancer, the PI3-kinase inhibitor idelalisib (Zydelig ®); any other investigational therapy; unless five half-lives have elapsed after the last dose of the above listed medication.

15. Administration of any live vaccine within 30 days before first dose of study drug.

16. Uncontrolled infection with human immunodeficiency virus (HIV), hepatitis B virus or hepatitis C virus, except for:

• Subjects with HIV whose infection is controlled (undetectable viral load and CD4 count above 350 cells/mm3, either spontaneously or on a stable antiviral regimen) are permitted.

• Subjects with hepatitis B (HepBsAg + ) virus whose infection is controlled (serum hepatitis B virus DNA PCR that is below the limit of detection AND receiving anti-viral therapy for hepatitis B) are permitted.

• Subjects who are hepatitis C virus antibody positive (HCV Ab +) whose infection is controlled (undetectable HCV RNA by PCR either spontaneously or in response to a successful prior course of anti-HCV therapy) are permitted.

17. Known history of allergy to any of the drug components of ISAlOl/lOlb: e.g. ISAlOl/lOlb, Montanide, or Macrogolglycerol Ricinoleate, also known as cremophore.

18. Known history of allergy to any of the drug components of cemiplimab and its excipients.

19. Known history of allergy to tetracycline or doxycycline.

20. History of severe hypersensitivity reaction to any human monoclonal antibody. 21. Severe concurrent illness(es) including psychiatric conditions that would limit compliance with study requirements.

22. Serious systemic infection requiring hospital treatment with intravenous antibiotics within 14 days prior to start of study treatment (last dose of antibiotic given at least 14 days prior to start of study treatment).

23. Subjects with an uncontrolled or significant heart disease such as long QT syndrome with QTcF > 480 ms on baseline ECG, NYHA III/IV or uncontrolled arrhythmia.

24. History of organ transplantation, including allogenic peripheral stem cell or bone marrow transplantation.

Efficacy Assessments:

Efficacy assessments will include tumor response rates as assessed by RECIST 1.1 using CT or MRI imaging studies and survival data. Irrespective of treatment arm, radiological and clinical tumor assessment will be performed symmetrically at baseline, at week 7 and every 9 weeks (± 7 days) thereafter for up to 12 months, and then every 12 weeks (± 7 days) for up to 24 months, or until evidence of PD.

Subjects who finish treatment without radiological PD will continue with tumor assessments every 12 weeks (± 7 days) until PD, start of new antitumor therapy, death, or end of study. After radiological PD is documented or new antitumor therapy is started, subjects will be followed for survival every 12 weeks (± 2 weeks) from the end-of-treatment (EOT) visit until death or end of study, whichever occurs first.

Pharmacokinetic Assessments:

Pharmacokinetic assessments will include analysis of serum samples for determination of cemiplimab concentrations.

Safety Assessments:

Safety assessments include incidence, type, and severity of AEs, the relationship of AEs to study drug, vital signs, physical examination findings, laboratory parameters, and antidrug antibody (ADA) to cemiplimab.

Identity of Investigational Products

ISAlOlb

The drug product for ISAlOlb is provided in 2 vials, HPV-DP-5P containing 5 peptides and HPV-DP-7P containing 7 peptides. Each of the 2 vials of drug product (HPV-DP-5P and HPV-DP-7P) is individually reconstituted with reconstitution solution and then mixed with the adjuvant Montanide, leading to the preparation of the ISAlOlb vaccine. Each individual vaccine (i.e. HPV-DP-5P and HPV-DP- 7P vaccine) is separately injected subcutaneously into a separate (upper) arm or (upper) leg of the patient. The final vaccine composition has a pH of approximately 3.

For ISAlOlb placebo the same preparation procedure is followed, with the exception that the actual drug product is not added: reconstitution solution is mixed in a 1: 1 (v/v) ratio with Montanide adjuvant prior to administration.

Cemiplimab

Cemiplimab (also known as REGN2810) is manufactured at 50 mg/mL and formulated in an aqueous buffered solution at pH 6.0 containing 10 mM histidine, 5% (w/v) sucrose, 1.5% (w/v) L proline, and 0.2% (w/v) polysorbate 80. Cemiplimab drug product is supplied as a sterile liquid solution of 5.0 mL or 7.0 mL in a 10 mL glass vial for IV administration.

Investigational Product Administration

Arm 1 (Control Arm):

• Matching placebo for ISAlOlb by SC injection on Day 1, Day 29 and Day 50 (total of 3 doses).

Arm 2 (Experimental Arm):

• Cemiplimab 350 mg given by IV infusion on Day 8, and then Q3W (Day 29, Day 50 etc) for up to 24 months unless disease progression occurs or treatment is withdrawn for any other reason.

Timing of doses is summarized in Figure 3.

Following ISAlOlb or ISAlOlb placebo administration, subjects must be monitored in the outpatient clinic for 4 hours as follows:

• Vital signs are measured and the injection site is checked at least every hour after vaccination and before discharge. • Subjects should be evaluated by qualified medical staff before discharge to verify whether there are any signs or symptoms of a systemic allergic reaction (e.g., urticaria, rash, flushing, respiratory symptoms, or hypotension).

On the first day (D8) where cemiplimab is administered alone, vital signs should be assessed and documented before the infusion and then 2 hours after the end of infusion.

On days where cemiplimab and ISAlOlb or ISAlOlb placebo are administered on the same day (namely at dose 2 and dose 3), cemiplimab should be infused first and the subject observed for 1 hour after completion of infusion. Then ISAlOlb or ISAlOlb placebo should be injected followed by an observation period of 4 hours.

On the days after ISAlOlb or ISAlOlb placebo completion (D71 and onwards) where cemiplimab is administered alone, vital signs should be assessed and documented before the infusion and then approximately 30 minutes after the infusion. If the vital signs are stable, the subject can be discharged.

PD-L1 Diagnostic Test

Subjects who are candidates for first line therapy with a PD-l-blocking antibody should be positive for PD-L1 expression (CPS > 1) and this should have been/be confirmed with a validated assay. PD-L1 expression in OPC tumor biopsies can be assessed using immunohistochemical (IHC) testing with antibodies that bind specifically to the PD-L1 protein. Testing will have been/will be performed on a biopsy sample that was collected between the last dose of the previous systemic anti-cancer therapy and the first dose of study medication/vaccine (archival tissue) or on a contemporaneous biopsy sample (within 60 days of first dose of study medication).

All subjects who have failed platin based chemotherapy and who are candidates to be treated in this trial with cemiplimab (+ ISAlOlb) as 2nd line therapy can be enrolled into this trial regardless their CPS score, and a PD-L1 expression analysis is therefore optional.

Timing of study procedures

Subjects provide written informed consent before any study-related procedures are performed. Subjects are evaluated at scheduled visits during 3 study periods: Screening, Treatment, and Follow-up.

Subjects who finish treatment without radiological PD continue with tumor assessments every 12 weeks (± 7 days) from randomization until PD, start of new antitumor therapy, death, or end of study. After radiological PD is documented or new antitumor therapy is started, subjects are followed for survival every 12 weeks (± 2 weeks) from the EOT visit until death or end of study, whichever occurs first. Death dates may be found by checking death registries.

Duration of Treatment

Subjects are considered to be on treatment from administration of the first dose of study drug until the day they are withdrawn from study treatment (EOT). Treatment duration includes 3 doses of ISAlOlb/ISAlOlb placebo over 7 weeks and up to 24 months of treatment with cemiplimab. Subjects return 30 days after the last dose of study drug for a follow-up visit. Subjects are then followed for PFS and OS following EOT until death or end of study.

Subjects are considered to be on study from the signing of the ICF until death or the completion of the follow-up period.

Treatment Beyond Progression

For a small percentage of cancer subjects treated with immunotherapy such as ISAlOlb or anti-PD-1 therapy, unconventional responses may occur in which radiologic evidence of progression on therapy occurs before subsequent radiologic response. To account for this possibility, treatment with either study drug beyond progression (determined by local assessment) is allowed if the following conditions are met:

• The subject has stable performance status

• The subject does not have rapid progression of disease

• The subject has not experienced AEs that would require permanent discontinuation of cemiplimab or ISAlOlb/ISAlOlb placebo

Efficacy, Pharmacokinetics, Pharmacodynamics, and Safety Measurements Assessed

Efficacy Variables

Tumor Assessments for Response

Tumor assessments for response include a CT or MRI of the head and neck, chest abdomen, and all other known sites of disease within 28 days before randomization. An MRI of the brain (or CT scan with contrast if MRI is contraindicated) is performed for subjects with known or suspected brain metastases (base of skull covered on CT scan of head/neck or MRI of head/neck).

Tumor assessments are performed at Week 7 and then every 9 weeks (± 7 days) or as clinically indicated for up to 12 months, and then every 12 weeks up to 24 months, until disease progression or treatment is discontinued (whichever occurs later), performing a CT scan or MRI of the head and neck, chest, and abdomen, and all knows sites of disease by the same imaging method used at screening/baseline. As per RECIST criteria a confirmation tumor assessment of the CR or PR is performed 4 weeks after the CR or PR has been demonstrated. Subjects with a history of brain metastasis should also have a brain MRI (or CT scan with contrast if MRI is contraindicated) approximately every 9 weeks, or sooner if clinically indicated.

Irrespective of treatment arm, radiological and clinical tumor assessment are performed symmetrically at baseline, at 7 weeks, and every 9 weeks (± 7 days) thereafter from randomization for up to 12 months, then every 12 weeks (± 7 days) for up to 24 months, or until evidence of PD (criteria for continuing treatment beyond progression are provided in this Example) Subjects who finish treatment without radiological PD continue with the tumor assessment every 12 weeks (± 7 days) from randomization until PD, start of new antitumor therapy, death, or end of study. After radiological PD is documented or new antitumor therapy is started, subjects are followed for survival every 12 weeks (± 2 weeks) from the EOT visit until death or end of study, whichever occurs first. Follow-up for survival, after radiological PD is documented or new therapy is started, may be made by telephone calls.

Definition of Primary Efficacy Endpoint

The ORR is defined as the percentage of subjects who are observed to have a best overall response of confirmed CR or PR using RECIST vl.l, as determined by independent review. Subjects for which response cannot be ascertained are assumed to have no response in the primary analysis.

Definition of Secondary and Exploratory Endpoints

Duration of response is defined as the time between the date of first response of CR or PR to the date of first documented tumor progression per RECIST vl.l or death due to any cause. This is defined only for subjects demonstrating a response, with response and progression status both determined by independent review. If a subject has not had a progression event at the time of the analysis cut-off, or at the time of study withdrawal, or at the start of any new antineoplastic therapy, DOR is censored at the date of last adequate tumor assessment.

Time to response is defined as the time between the start of treatment to the time of overall response of CR or PR using RECIST vl.l, as determine by independent review. If a subject has not had a response at the time of the analysis cut-off, or at the time of study withdrawal, or at the start of any new antineoplastic therapy, TTR is censored at the date of last adequate tumor assessment.

PFS is defined as the time from randomisation to objective tumor progression or death from any cause, whichever occurs first. If a subject has not had a PFS event at the time of the analysis cut-off, or at the time of study withdrawal, or at the start of any new antineoplastic therapy, PFS is censored at the date of last adequate tumor assessment.

OS in is defined as the time from randomisation to death from any cause. If a death has not been observed by the date of the analysis cut-off, OS is censored at the date of last contact.

Molecular profiling of tumor tissue at baseline and following the initiation of study treatment is performed, including but not limited to whole exome DNA sequencing, genome wide RNA sequencing, histological analysis of tumor infiltrating immune cell subsets (effector/regulatory T-cells, dendritic cells, myeloid cell subpopulations, NK cells), immune- regulatory molecule (i.e., PD-L1, MHC-I, MHC-II, PD-1, Lag3, CTLA-4, GITR, 0X40, and 4- 1-BB) and HPV oncoprotein expression, by IHC and/or RNA Scope assays.

Pharmacokinetic Variables

Blood samples are collected at various time points during the study for the assessment of PK of cemiplimab. Samples for immunogenicity testing (serum and PBMC) are collected at various time points during the study for assessment of pharmacodynamic parameters. Tumor tissue and blood samples are collected in a subset of subjects for biomarker assessment including soluble biomarkers, immunophenotyping, ex vivo functional assay, gene expression, and single nucleotide polymorphisms.

Cemiplimab

Serum samples for cemiplimab are collected from all subjects as follows:

• Pre-dose and within 10 minutes after end-of-infusion at Day 8 (after the first dose of cemiplimab) and pre-dose at Day 29 (Ctrough after first dose). • Pre-dose (Ctrough) every 6 months (24 weeks) thereafter during therapy; and predose at EOT and the follow-up visit.

Samples are packaged and shipped as described in the separate Laboratory Manual.

Pharmacodynamic Variables

Pharmacodynamic assessments include analysis of blood and tissue samples.

Biomarkers

• Assessment of tumor biopsies at baseline and during treatment to explore PD-L1 expression, and lymphocyte and myeloid cell distribution and specific gene and protein expression.

• Assessment of correlative biomarkers as described in this example

Safety Assessments

This study assesses the safety and tolerability of the combination of cemiplimab plus ISAlOlb compared to cemiplimab alone using the following safety variables: AEs, physical examination findings, ECOG performance status, vital signs measurements, oxygen saturation, laboratory test results, and ADA.

Safety assessments include monitoring and recording AEs, including SAEs; measurement of protocol-specified safety laboratory assessments; measurement of protocol-specified vital signs; anti-drug antibodies (ADA) to cemiplimab; and other protocol-specified tests that are deemed critical to the safety evaluation of the study.

Results

An analysis was performed when 198 patients had received at least one dose of ISAlOlb had been followed for at least 6 months.

As shown in Table 7, 198 patients (mean age 62.8 ± 9.6 years; 173 (87%) male and 25 (13%) female; 104 (52.5%) first, 57 (28.8%) second line and 37 (18.7%) not stratified were randomized (Full Analysis Set (FAS)). In total, 140 patients received 3 vaccinations (with cemiplimab) or placebo (cemiplimab only), were confirmed HPV16 positive by a central lab and underwent at least one post baseline scan/target lesion assessment (the per protocol set (PPS)) of which 61 patients received the combination and 79 the placebo.

Grade >3 SAEs occurred in 30% of patients in the combination arm vs 34% in the control arm, with injection site reactions observed most frequently. Based on all SAEs there are no apparent differences in frequency, severity and type of events between the two arms.

Table 8 depicts the ORR in the FAS set. I.e. all patients that received at least one dose of ISAlOlb/placebo, including patients that did not receive 3 vaccinations and/or were not confirmed to be HPV16 positive, excluding the patients for which the response could not be evaluated by independent review. When analysing the ORR in this full analysis set group (n = 187), no difference was observed between the two arms. In analysing the effect of CPS in this set, a significant improvement of ORR was observed for the combination-arm in the group of patients for which a CPS >20 was recorded before the start of the treatment (P=0.078).

When focusing on the patients that received the full vaccination treatment (PPS) a clear difference is observed. The ORR in the PPS-combination arm was 38.9% compared to 27.3% in the PPS-control arm at time of analysis (Table 9). This difference is statistically significant, according to the pre-defined value of p< 0.1. The highest ORR and an even more significant increase in ORR of the combination with ISAlOlb was observed in the subgroup of the PPS with a CPS >20 before study entry, wherein the ORR was 61.9% for the combination versus 28.0% in the control arm (Table 8). The Overall Survival (OS) rate between the combination and control arm in the per protocol set did not demonstrate a significant difference with an HR(CI 95%) of 1.21 (0.74, 1.99) (Figure 6).

Surprisingly the OS rate in the subgroup of the PPS with a CPS >20 at baseline demonstrated a substantial increase in OS of the combination with ISAlOlb versus the control with an HR (CI 95%) of 0.23 (0.07, 0.72) (Figure 7). In the combination arm, median OS had not yet been reached.

Table 7. Baseline characteristics

Characteristic Number of subjects (total n = 198)

Mean (±SD) 62.8 (9.6)

Gender,

Male 173 (87.4)

Treatment line in current study

Second line 57 (28.8)

Not stratified 37 (18.7)

Retrospectively assigned 22 (11.1) Not collected 15 (7.6)

Table 8. Primary endpoint - ORR (FAS)

Cemiplimab + placebo Cemiplimab +ISA101b

Independent review, n 22 (22.9) 23 (25.3)

*P-value = 0.078*

*Statistically significant, according to predefined value of P< 0.1

Table 9. Primary endpoint - ORR (PPS)

Cemiplimab + placebo Cemiplimab +ISA101b (n = 79) (n = 61)

Independent review, n (%) 21 (27.3) 21 (38.9)*

*P-value = 0.064* n (%) 7 (28.0) 13 (61.9)*

*P-value = 0.026*

*Statistically significant, according to predefined value of P< 0.1

EXAMPLE 3

A Phase 2 Study of Cemiplimab, an Anti-PD-1 Monoclonal Antibody, and ISAlOlb Vaccine in Patients with Recurrent/ Metastatic HPV16 Cervical Cancer Who Have Experienced Disease Progression after First Line Chemotherapy

This study is a single-arm, phase 2, global study of treatment with cemiplimab + ISAlOlb (peltopepimut-S) in HPV16-positive cervical cancer patients with disease progression on first line chemotherapy in the recurrent or metastatic setting. Study treatment and duration include cemiplimab every 3 weeks (with 3 doses of ISAlOlb on days 1, 29, and 50) until progression or any reason for early discontinuation. The study is hereinafter referred to as the 1981 study.

Study Objectives and endpoints

Overview of Study Design:

This was a single-arm, phase 2, global study to evaluate the treatment effect of cemiplimab + ISAlOlb in patients with HPV16 positive cervical cancer with SCC or adenocarcinoma/adenosquamous (AC) histology, who had progressed on first-line chemotherapy in the recurrent or metastatic setting. Approximately 105 patients were planned to be enrolled in the study worldwide.

After a screening period of 28 days, all eligible patients (who met all the inclusion and none of the exclusion criteria) received the following treatment regimen:

• ISAlOlb 100 pg/peptide by subcutaneous (SC) injection on days 1, 29, and 50 (total of 3 doses)

• Cemiplimab 350 mg given by intravenous (IV) infusion over 30 minutes every 3 weeks (Q3W) on days 8 and 29 in cycle 1, on days 1 and 22 in cycles 2 through 4, and on days 1, 22, and 43 in all subsequent cycles until disease progression or discontinuation of study drug for any other reason.

(Note: On days 29 and 50, ISAlOlb administration followed approximately 1 hour after the end of the cemiplimab infusion. Patients were observed for 4 hours after each ISAlOlb administration). The study flow including the timing of doses is shown in Figure 8.

Cycle 1 was 7 weeks duration; cycles 2 through 4 were 6 weeks duration; and all the subsequent cycles were 9 weeks duration.

Patients were assessed for response at week 7, then every 6 weeks for 3 cycles, then every 9 weeks in all subsequent cycles or until disease progression or discontinuation of study drug for any other reason. Patients with confirmed complete response (CR) after a minimum of 48 weeks of treatment could decide to discontinue treatment and continue with all relevant study assessments (e.g., efficacy assessments). An end-of-treatment (EOT) visit was conducted 30 days after the last dose of study drug.

A 90-day safety follow-up followed the last dose of cemiplimab. Patients who discontinued the study drug for reasons other than progression were followed approximately every 4 months by scans until disease progression or until the patient received another anticancer systemic therapy, whichever occurred first. After progression, survival follow-up occurred approximately every 4 months.

The total study duration from start of screening to final analysis of OS was expected to be about 36 months (about 12 months of accrual, about 12 months of treatment, and about 12 months of follow-up).

Study Population:

Inclusion criteria:

A patient had to meet the following criteria to be eligible for inclusion in the study:

1. Adult patients > 18 years of age (or the legal age of adults to consent to participate in a clinical study per country specific regulations).

2. Has histologically confirmed recurrent or metastatic HPV16 positive cervical cancer as determined by an investigational HPV16 PCR assay by Qiagen, who have experienced disease progression after treatment with platinum containing therapy (must have been used to treat metastatic, persistent, or recurrent cervical cancer). NOTE: Platinum-therapy given in other settings (e.g., concurrent with radiation therapy as part of curative-intent therapy, after radiation [or chemoradiation] as adjuvant treatment in a patient with no evidence of disease) does not satisfy the eligibility requirement regarding prior platinum therapy.

Acceptable histology is squamous carcinoma and adenocarcinoma/adenosquamous carcinomas. Sarcomas and neuro-endocrine carcinomas are not eligible histologies.

3. Patient must be determined to be positive for HPV16 genotype, as determined by a specified central reference laboratory.

4. Patient must have measurable disease as defined by RECIST 1.1. Measurable disease is defined as at least 1 lesion that can be accurately measured in at least 1 dimension (longest dimension to be recorded). Each lesion must be >10 mm when measured by computed tomography (CT), magnetic resonance imaging (MRI), or caliper measurement by clinical exam or must be >20 mm when measured by chest x-ray. Lymph nodes must be > 15 mm in short axis when measured by CT or MRI. Tumors within a previously irradiated field will be designated as non-measurable lesions unless progression is documented or a biopsy is obtained to confirm persistence at least 90 days following completion of radiation therapy. 5. Patients must meet at least 1 of the following criteria regarding prior bevacizumab therapy: a. Received prior bevacizumab-containing therapy and experienced subsequent progression of disease b. Received prior bevacizumab-containing therapy which was discontinued due to toxicity c. Was unsuitable for bevacizumab due to i) unacceptable risk of fistula formation, ii) poorly controlled hypertension, iii) low risk disease according to Moore criteria d. Refused prior bevacizumab e. Did not have access to bevacizumab

6. Patients must meet at least 1 of the following criteria regarding prior taxol therapy a. Received prior taxol-containing therapy and experienced subsequent progression of disease b. Received prior taxol-containing therapy which was discontinued due to toxicity c. Was unsuitable for taxol therapy due to i) neuropathy, ii) allergy or intolerance to taxol or one of its components d. Refused prior taxol

7. ECOG performance status of 0 or 1.

8. Has adequate organ and bone marrow function documented by: a. Hemoglobin >8 g/dL b. Absolute neutrophil count >1.0 x 109/L c. Platelet count >75 x 109/L d. Serum creatinine <1.5 x upper limit of normal (ULN) or estimated glomerular filtration rate >30 mL/min/1.73m2. A 24-hour urine creatinine collection may substitute for the calculated creatinine clearance to meet eligibility criteria. e. Adequate hepatic function :

(i) Total bilirubin <1.5 x ULN (<3 x ULN if tumor liver involvement)

(ii) Aspartate Aminotransferase (AST) <2.5 x ULN (<3 x ULN if tumor liver involvement)

(iii) Alanine Aminotransferase (ALT) <2.5 x ULN (<3 x ULN if tumor liver involvement)

(iv) Alkaline Phosphatase (ALP) <2.5 x ULN (<3 x ULN if tumor liver or bone involvement)

NOTES: • Patients with tumor liver involvement if levels of AST >3 x ULN or ALT >3 x ULN, and bilirubin levels >2 x ULN are excluded regardless of the above criteria

• Patients with Gilbert's syndrome do not need to meet total bilirubin requirements provided their total bilirubin is not greater from their historical level. Gilbert's syndrome must be documented appropriately as past medical history

Exclusion criteria:

A patient who meets any of the following criteria will be excluded from the study:

1. Prior treatment with an agent that blocks the PD-1/PD-L1 pathway.

2. Prior treatment with other systemic immune-modulating agents that was a. less than 4 weeks (28 days) of the enrolment date, or b. associated with imAEs of any grade within 90 days prior to enrolment, or c. associated with toxicity that resulted in discontinuation of the immune- modulating agent.

Examples of immune-modulating include therapeutic vaccines, cytokine treatments (other than granulocyte colony stimulating factor, thrombopoietin analogues, or erythropoietin), or agents that target cytotoxic T lymphocyte antigen 4 (CTLA-4), 4-1BB (CD137), PI 3-K- delta, LAG3, or OX-40.

3. Major surgery or radiation therapy within 14 days of first administration of study drug

4. Has received treatment with an approved systemic therapy within 4 weeks of first dose of study drug, or has not yet recovered (i.e., grade < 1 or baseline) from any acute toxicities except for laboratory changes as described in inclusion criteria and as below: a. Neuropathy of grade <2 b. Alopecia of any grade

5. Has another malignancy that is progressing or requires active treatment and/or history of malignancy other than cervical cancer within 3 years of date of first planned dose of study drug, except: a. Non-melanoma skin cancer that has undergone potentially curative therapy b. Ductal carcinoma in situ of the breast c. Any tumor that has been deemed to be effectively treated with definitive local control (with or without continued adjuvant hormonal therapy), and the patient is deemed to be in complete remission for at least 2 years prior to first dose of study drug, and no additional therapy is required during the study period. Note: Patients with hematologic malignancies (e.g., chronic lymphocytic leukemia) are excluded.

Study Procedures:

HPV16 Diagnostic Test

For HPV16 determination, a formalin-fixed, paraffin-embedded tissue block or unstained slides of tumor samples must be provided after the ICF is signed. Tumor biopsies should be of sufficient size or quantity to ensure an adequate amount of tissue for analysis. A validated assay at a central laboratory will be used for determination of HPV16 genotyping in patient samples and to determine eligibility based on HPV16 positivity.

Efficacy Assessments:

Computed Tomography and/or Magnetic Resonance Imaging

Diagnostic quality contrast-enhanced CT and contrast-enhanced MRI are the preferred imaging modalities for assessing radiographic tumor response. In patients whom contrast is strictly contraindicated, non-contrast MRI scans and CT chest scans are acceptable. The chest, abdomen, and pelvis must be imaged along with any other known or suspected sites of disease. If more than one imaging modality is used at screening for the same anatomies, the most accurate imaging modality according to RECIST 1.1 should be used when recording data. The same imaging modality and techniques used at screening should be used for all subsequent assessments. At screening, MRI of the brain with contrast or without contrast if contrast is contraindicated, or CT with contrast when MRI is contraindicated, should be performed in patients with a known history of treated brain metastasis. Additional sites of known disease (including CNS) should be imaged at screening. Refer to the Site Imaging Manual for details of imaging requirements.

After the baseline tumor assessment, radiographic tumor assessments will be obtained in all patients at the time points indicated in Table 2 (with a window of ±7 days). During safety follow-up, radiographic evaluation will be performed every 4 months until RECIST 1.1-defined progressive disease, withdrawal of consent, death, or initiation of another anticancer treatment.

Tumor assessments should be performed even if dosing is interrupted. Weeks and months are in reference to the calendar week and month and should not be adjusted due to dosing delays/interruptions. Cycle 1 day 1 date is the reference for all visits including tumor assessments visits.

Tumor Burden Assessments Tumor measurements will be performed in accordance with RECIST 1.1. Criteria (Eisenhauer, 2009) (Appendix 3) and should be done by the same investigator or radiologist for each assessment of a patient, to the extent feasible.

Investigators will assess response to therapy using RECIST 1.1 criteria. RECIST 1.1-defined progressive disease determined by the investigator will be used for clinical management of the patient. RECIST 1.1-based tumor burden assessments will be used for evaluation of efficacy endpoints.

Efficacy assessments will include tumor response rates as assessed by RECIST 1.1 using CT or MRI imaging studies and survival data. Radiological and clinical tumor assessment will be performed symmetrically at baseline, at week 7 and every 9 weeks (± 7 days) thereafter for up to 12 months, and then every 12 weeks (± 7 days) for up to 24 months, or until evidence of PD.

Drug Concentration, Pharmacokinetic and Immunogenicity Measurements:

Pharmacokinetic assessments will include analysis of serum samples for determination of cemiplimab concentrations and ADA assessment.

Pharmacodynamic and Exploratory Biomarker Procedures

In this study, research assessments will be performed to better understand the baseline predictive biomarkers of cemiplimab + ISAlOlb response that may include but are not limited to tumor tissue analysis of PD-L1 expression and immune cell subsets by IHC or RNAScope, tumor DNA and RNA sequencing. Pharmacodynamic and tumor biomarker samples will be collected and measurements will be performed to determine treatment effect on the disease and relevant physiological processes. The biomarkers studied are believed to be relevant to the pathophysiology of indication target engagement, mechanism of action of cemiplimab and possible toxicities.

Biomarker assays may include, but will not be limited to, the following:

• Tumor specific and HPV gene panel (circulating tumor DNA sequence profile)

• T cell effector and innate immune cytokine panel (serum cytokines) • T cell, myeloid cell subset analysis, functional HPV E6/E7 specific T cell assays

Peripheral Blood Biomarkers

Samples for peripheral blood (plasma, serum, and PBMC samples) biomarker assessments will be collected.

PBMCs from patients receiving cemiplimab may be used for characterization of immune cell subsets including T cells, B cells, natural killer cells, monocytes, dendritic cells, and subsets of these cell types. Peripheral blood mononuclear cell samples may also be used to assess immune cell function, including T cell activation and proliferation.

Safety Assessments:

Identity of Investigational Products

ISAlOlb

The drug product for ISAlOlb is provided in 2 vials, HPV-DP-5P containing 5 peptides and HPV-DP-7P containing 7 peptides.

Each of the 2 vials of drug product (HPV-DP-5P and HPV-DP-7P) is individually reconstituted with reconstitution solution and then mixed with the adjuvant Montanide, leading to the preparation of the ISAlOlb vaccine. Each individual vaccine (i.e. HPV-DP-5P and HPV-DP- 7P vaccine) is separately injected subcutaneously into a separate (upper) arm or (upper) leg of the patient. The final vaccine composition has a pH of approximately 3.

Cemiplimab

Investigational Product Administration • ISAlOlb 100 pg/peptide by SC injection on Day 1, Day 29, and Day 50 (total of 3 doses).

Timing of doses is summarized in Figure 8.

Results

A total of 113 patients were enrolled in the study to receive ISAlOlb + Cemiplimab at 19 sites in 6 countries.

Below the clinical efficacy and safety findings of the above described phase 2 study of cemiplimab + ISAlOlb vaccine in patients with recurrent/metastatic HPV16 cervical cancer who had disease progression after first-line chemotherapy.

This single arm study cohort consisted of 113 patients (Table 10). Median duration of follow-up was 4.9 months and median duration of treatment exposure was 10.0 weeks for ISAlOlb and 18.2 weeks for cemiplimab. Ninety patients discontinued treatment for reasons including disease progression (66.4%), death (7.1%), or an adverse event (AE; 3.5%). At data cut-off, 23 (20.4%) patients were still on treatment and 24 patients were ongoing in the study, of which 7 patients longer than 365 days.

Table 10 Demographic and baseline characteristics

Demographics and baseline characteristics, n (%) unless otherwise All patients (N = 113) specified

Age, years, mean (SD) 49.4 (12.0)

ECOG PS 0 66 (58.4)

ECOG PS 1 47 (41.6)

Adenocarcinoma 17 (15.0)

Squamous cell carcinoma 96 (85.0)

FIGO stage at initial diagnosis

IIB 21 (18.6)

IVA 11 (9.7)

IVB 30 (26.5) Prior and concomitant therapy

Prior systemic therapies

All 113 patients (100%) received at least one prior anti-cancer related systemic therapy including 97 patients (85.8%) who had received prior therapy for metastatic disease. The median number of prior lines of anticancer systemic therapy in any setting was 2 (range: 1 to 7). The frequency of receiving prior lines of anticancer systemic therapy (median value) for recurrent or metastatic disease was 1.0 (range: 1 to 4).

The most frequently received prior antineoplastic therapies included the standard first-line therapy of platinum compounds in 113 patients (100.0%), taxanes in 105 patients (92.9%), and VEGF/VEGFR inhibitors in 68 patients (60.2%). All patients were required to have progressed on firstline chemotherapy in the recurrent or metastatic setting.

Prior surgery

All 113 patients (100%) underwent at least one prior cancer-related surgery including 66 patients (58.4%) patients and 48 patients (42.5%) of cervix and uterus, respectively. The frequency of undergoing prior cancer related surgery (median value) was 2 (range: 1 to 14).

Prior radiotherapy

A total of 87 patients (77.0%) received prior cancer related radiotherapy including 56 patients (49.6%) who received radiotherapy alone and 71 patients (62.8%) who received radiotherapy with chemotherapy. The frequency of receiving prior cancer-related radiotherapy (median value) was 2.0 (range: 1 to 5).

Concomitant medications and procedures

All 113 (100.0%) patients received at least one concomitant medication during the study. The most used (>20% patients) medications were anilides (55 patients, 48.7%), protonpump inhibitors (39 patients, 34.5%), natural opium alkaloids (31 patients, 27.4%), gabapentinoids (29 patients, 25.7%), glucocorticoids (28 patients, 24.8%), benzodiazepine derivatives (25 patients, 22.1%), and osmotically acting laxatives (24 patients, 21.2%).

A total of 55 (48.7%) patients underwent at least one concomitant procedure/surgery during the study. The most commonly reported (>5% patients) concomitant procedures/surgery included nephrostomy (9 patients, 8.0%), CT thorax (7 patients, 6.2%), chest X-ray (6 patients, 5.3%), CT abdomen (6 patients, 5.3%), ultrasound doppler (6 patients, 5.3%), and ureteral stent insertion (6 patients, 5.3%).

Post-treatment anti-cancer systemic therapies and procedures

A total of 48 (42.5%) patients received at least one post-treatment antineoplastic systemic therapies. The most commonly (>5% patients) received medications included pyrimidine analogues (19 patients, 16.8%), platinum compounds (12 patients, 10.6%), taxanes (10 patients, 8.8%), and vinca alkaloids and analogues (9 patients, 8.0%).

Twelve (10.6%) patients underwent post-treatment anti-cancer procedures including radiotherapy received by 8 (7.1%) patients.

Efficacy

The primary efficacy endpoint was ORR based on the radiographic response measured by RECIST version 1.1. All 113 patients in FAS were analyzed for ORR (Table 11). ORR (95% CI) was 16.8% (9.9-23.7). Of the 19 responders, 3 patients (2.7%) experienced complete response (CR) and 16 patients (14.2%) experienced partial response to study intervention. Stable disease was reported in 43 patients (38.1%). All responders had SCC histology with an ORR of 19.8% (95% CI: 11.8%, 27.8%). There were no responders in the AC histology subgroup.

ORR by PD-L1 expression in tumor cells was 12.5% for patients with PD-L1 < 1% (TPS) and 22.4% for patients with PD-L1 > 1% (TPS), per investigator assessment (Table 11). Overall, the DCR was 54.9% (95% CI: 45.7% to 64.0%). The proportion of patients with CR, PR or stable disease for at least 12 weeks without PD (durable DCR) was 37.2% (95% CI: 28.3% to 46.1%). Median (95% CI) DOR was 5.6 (3.5-not estimable) months.

Median (95% CI) OS and PFS were 13.3 (10.8-16.3) months and 3.0 (1.7-4.0) months, respectively. At 6 and 12 months, the estimated survival probabilities (95% CI) were 80.5% (71.7-86.9) and 54.5% (42.1-65.3), respectively, for OS, and 20.5% (13.4-28.6) and 7.8% (3.3-15.0), respectively, for PFS. Treatment-emergent AEs occurred in 92.9% of patients, with the most common being injection-site reaction (38.9%), anemia (25.7%), and nausea (22.1%). Six (5.3%) patients died due to a treatment-emergent AE. Table 11: Best Overall Tumor Response Rate per RECIST 1.1 by Investigator Assessment (Full Analysis Set)

Data cut-off as of May 22, 2023.

[a] CR/PR must be confirmed by repeated Assessments no less than 4 weeks apart.

[b] SD criteria must be met at least once after a minimum duration of 42 days after first dose date.

[c] Not evaluable response includes the missing and unknown tumor response.

[d] Wald (normal approximation to binomial distribution) confidence interval. Conclusions: Results from this phase 2 study evaluating the clinical efficacy and safety of cemiplimab + ISAlOlb vaccine in patients with recurrent/metastatic HPV16 cervical cancer, show clinical benefit especially in PDLl-expressing patients and no unexpected safety signals.

Comparison 1981 with 1676 results

The 1981 study has been conducted as a follow-up to the, so called, 1676 study which compared cemiplimab alone versus chemotherapy (Tewari et al 2022 NEJM 386: 544-55, NCT03257267) with the same investigators and at the same hospitals. The patient populations treated with the combination of Cemiplimab plus ISAlOlb (1981) and Cemiplimab only (1676) were selected applying the same eligibility criteria and assessments.

Besides the fact that the 1981 study was conducted immediately after closure of the 1676 study with full overlap of study parameters, the similarity of the demographics, baseline characteristics and baseline tumor characteristics between both trials justify a direct comparison between the two groups (i.e. cemiplimab + ISAlOlb (1981) versus cemiplimab (1676 study arm)). Table 12 and table 13 demonstrate comparable baseline characteristics of patients and consistency of the study population with secondline cervical cancer.

Table 12: Demographics and Baseline Characteristics (Full Analysis Set) comparison

Tablel3: Demographics and Baseline Characteristics (Full Analysis Set) comparison

Comparison of efficacy Overall, the ORR between the two groups (cemiplimab + ISAlOlb vs cemiplimab) were comparable, i.e. 16.8% (19/113) vs 16.4% (50/304), respectively. All responders in 1981 had SCO histology and most, although not all, responders in 1676 had SCC histology.

In both studies, PD-L1 tumor expression status at baseline was detected with the use of the SP263 monoclonal antibody (Ventana) or a functional equivalent thereof. PD-L1 expression status (measured as the tumor proportion score (TPS (%) or %TC) [the percentage of tumor cells expressing PD-L1]) was categorized as a) either less than 1%, b) greater than or equal to 1% or c) greater than or equal to 20%] .

Similar tumor response was observed across the PD-L1 subsets (TC), with greater activity in PD-L1 > 1% group compared to ORR in PD-L1 < 1% group for both interventions (Table 14).

When dividing the patient population into three subgroups according to PD-L1 expression levels (< 1%, > 1% and >20%), it was observed that in the PD-L1 >20% subgroup, the increase in ORR was larger in the combination arm (1981, from 22.4% to 33.3%) versus cemiplimab (1676, from 18.3% to 22.2%) (Table 14). While the numbers are small, this confirms the trend observed in the Opcemisa study outcome (Example 2

Table 14: Best Overall Tumor Response Rate by Investigator Assessment by PD- L1 (Tumor Cells) (Full Analysis Set)

Claims

1. A method for the treatment of a solid tumor in a subject, comprising i) identifying a subject afflicted with a solid tumor, wherein, in a tumor sample, such as a tumor biopsy sample, of said subject, PD-L1 is expressed in at least 5% of the viable tumor cells and/or the CPS is at least 5 and/or the TPS is at least 5 and/or the TAP score is at least 5%, and ii) administering one or more immunogenic compositions comprising one or more tumor antigens to said subject.

2. The method according to claim 1, wherein the solid tumor is classified as a squamous cell carcinoma, non- squamous cell carcinoma or adenocarcinoma.

3. The method according to claim 1 or 2, wherein, in a tumor sample of said subject, PD- L1 is expressed in at least 6% of the viable tumor cells and/or the CPS is at least 6 and/or the TPS is at least 6 and/or the TAP score is at least 6%,

PD-L1 is expressed in at least 7% of the viable tumor cells and/or the CPS is at least 7 and/or the TPS is at least 7 and/or the TAP score is at least 7%,

PD-L1 is expressed in at least 8% of the viable tumor cells and/or the CPS is at least 8 and/or the TPS is at least 8 and/or the TAP score is at least 8%,

PD-L1 is expressed in at least 9% of the viable tumor cells and/or the CPS is at least 9 and/or the TPS is at least 9 and/or the TAP score is at least 9%,

PD-L1 is expressed in at least 10% of the viable tumor cells and/or the CPS is at least 10 and/or the TPS is at least 10 and/or the TAP score is at least 10%,

PD-L1 is expressed in at least 11% of the viable tumor cells and/or the CPS is at least 11 and/or the TPS is at least 11 and/or the TAP score is at least 11%,

PD-L1 is expressed in at least 12% of the viable tumor cells and/or the CPS is at least 12 and/or the TPS is at least 12 and/or the TAP score is at least 12%,

PD-L1 is expressed in at least 13% of the viable tumor cells and/or the CPS is at least 13 and/or the TPS is at least 13 and/or the TAP score is at least 13%,

PD-L1 is expressed in at least 14% of the viable tumor cells and/or the CPS is at least 14 and/or the TPS is at least 14 and/or the TAP score is at least 14%,

PD-L1 is expressed in at least 15% of the viable tumor cells and/or the CPS is at least 15 and/or the TPS is at least 15 and/or the TAP score is at least 15%,

PD-L1 is expressed in at least 16% of the viable tumor cells and/or the CPS is at least 16 and/or the TPS is at least 16 and/or the TAP score is at least 16%, PD-L1 is expressed in at least 17% of the viable tumor cells and/or the CPS is at least 17 and/or the TPS is at least 17 and/or the TAP score is at least 17%,

PD-L1 is expressed in at least 18% of the viable tumor cells and/or the CPS is at least 18 and/or the TPS is at least 18 and/or the TAP score is at least 18%,

PD-L1 is expressed in at least 19% of the viable tumor cells and/or the CPS is at least 19 and/or the TPS is at least 19 and/or the TAP score is at least 19%, or

PD-L1 is expressed in at least 20% of the viable tumor cells and/or the CPS is at least 20 and/or the TPS is at least 20 and/or the TAP score is at least 20%.

4. The method according to any one of claims 1 to 3, wherein said one or more tumor antigens are capable of inducing a CD8+ and/or CD4+ T cell response.

5. The method according to any one of claims 1 to 4, wherein said one or more tumor antigens are peptides, preferably having a length of 20-40 amino acids.

6. The method according to any one of claims 1 to 5, wherein said solid tumor is a squamous cell carcinoma head and neck cancer tumor, a non-squamous cell carcinoma head and neck cancer tumor, an adenocarcinoma head and neck cancer tumor or a cervical cancer tumor, such as a cervical cancer tumor of the squamous cell type.

7. The method according to any one of claims 1 to 6, wherein said solid tumor is an HPV positive tumor, such as an HPV positive squamous cell carcinoma head and neck cancer tumor, a non-squamous cell carcinoma head and neck cancer tumor, an adenocarcinoma head and neck cancer tumor or an HPV positive cervical cancer tumor and wherein said one or more tumor antigens are HPV antigens.

8. The method according to any one of claims 1 to 7, wherein said solid tumor is an HPV16 positive tumor, such as HPV16 positive squamous cell carcinoma head and neck cancer tumor, a non-squamous cell carcinoma head and neck cancer tumor, an adenocarcinoma head and neck cancer tumor or an HPV16 positive cervical cancer tumor and wherein said one or more tumor antigens are HPV16 antigens.

9. The method according to any one of claims 1 to 8, wherein said solid tumor is a squamous cell carcinoma head and neck cancer tumor, a non-squamous cell carcinoma head and neck cancer tumor, an adenocarcinoma head and neck cancer tumor or a cervical cancer tumor and wherein said one or more tumor antigens are peptides based on the E6 and E7 proteins of HPV16, preferably comprising 12 synthetic long peptides of 25 to 35 amino acid in length, most preferably the peptides set forth in SEQ ID NO: 1 to SEQ ID NO: 12.

10. The method according to any one of claims 1 to 8, wherein said solid tumor is a cervical cancer tumor, such as a cervical cancer tumor of the squamous cell type or of the adenocarcinoma or adenosquamous cell type.

11. The method according to claim 10, wherein the tumor disease is recurrent or metastatic.

12. The method according to claim 10 or 11, wherein the subject has received prior therapy for the tumor disease, wherein optionally the tumor disease has progressed after said prior therapy.

13. The method according to claim 12, wherein the prior therapy is chemotherapy, such as platinum-containing therapy, optionally in combination with taxane and/or bevacizumab therapy.

14. The method according to any one of claims 1 to 13, wherein said subject is human.

15. The method according to any one of claims 1 to 14, wherein said immunogenic composition(s) is/are administered two, three or more times.

16. The method according to any one of claims 1 to 15, further comprising administration of an adjuvant.

17. The method according to any one of claims 1 to 16, wherein the method does not comprise administration of a PD-1 inhibitor or PD-L1 inhibitor.

18. The method according to any one of claims 1 to 17, wherein the method does not comprise administration of an immune checkpoint inhibitor.

19. The method according to any one of claims 1 to 16, wherein the method further comprises administration of an immune checkpoint inhibitor.

20. The method according to claim 19, wherein the immune checkpoint inhibitor is a PD-1 or a PD-L1 inhibitor.

21. The method according to claim 20, wherein the immune checkpoint inhibitor is an antibody that binds PD-1 or PD-L1.

22. The method according to claim 21, wherein the immune checkpoint inhibitor is selected from the group consisting of: cemiplimab, nivolumab and pembrolizumab.

23. The method according to any one of claims 1 to 22, wherein the method further comprises administration of one or more chemotherapeutic agents and/or radiation therapy.

24. A method for the selection of a patient afflicted with a solid tumor for therapeutic immunisation, comprising determining PD-L1 expression in a sample of said tumor and selecting the patient for therapeutic immunisation if the number of PD-L1 expressing cells in said tumor is above a predetermined value preferably one of the values described in claim 1 or claim 3.

25. A method of treating or inhibiting the growth of a tumor or improving overall survival of a head and neck cancer patient, comprising: a) selecting a patient with HPV16-positive head and neck cancer who progressed on prior immune checkpoint modulatory therapy, such as anti-PD-1, anti-PD-Ll, anti-PD- L2, anti-CD137, or anti-CTLA-4 therapy, for example anti-PD-1, anti-PD-Ll, anti-PD- L2, anti-CD137 or anti-CTLA-4 antibody therapy; b) administering to the patient an effective amount of one or more immunogenic compositions capable of inducing a T cell immune response against cells expressing HPV16 antigens, preferably E6, E7 and/ or E2 antigens; and c) administering to the patient a therapeutically effective amount of a PD-1 inhibitor.

26. The method according to claim 25, wherein the head and neck cancer is advanced, recurrent, persistent, primary refractory or refractory, incurable, unresectable and/or metastatic.

27. The method according to claim 25 or 26, wherein the head and neck cancer is an oropharyngeal cancer.

28. The method according to claim 25, 26 or 27, wherein the tumor is a squamous cell carcinoma head and neck cancer tumor, a non-squamous cell carcinoma head and neck cancer tumor or an adenocarcinoma head and neck cancer tumor

29. The method according to any one of claims 25 to 28, wherein the patient also progressed i.e. refractory or resistant, on prior PD-1 therapy.

30. The method according to any one of claims 25 to 29, wherein the patient progressed on prior platinum-containing therapy.

31. The method according to any one of claims 25 to 30, wherein the cancer is PD-L1 positive, preferably wherein the Combined Positivity Score is > 1.

32. The method according to any one of claims 25 to 31, wherein step a) comprises selecting a patient with HPV16-positive head and neck cancer i) who progressed on prior immune checkpoint modulatory therapy, such as anti-PD-1, anti-PD-Ll, anti-PD-L2, anti-CD137, anti-CTLA-4 or anti-LAG 3 therapy, for example anti- PD-1, anti-PD-Ll, anti-PD-L2, anti-CD137 or anti-CTLA-4 antibody therapy, and ii) wherein, in a tumor sample of said subject, PD-L1 is expressed in at least 5% of the viable tumor cells and/or the CPS is at least 5 and/or the TPS is at least 5 and/or the TAP score is at least 5%.

33. The method according to claim 32, wherein the tumor sample is a tumor biopsy sample.

34. The method according to claim 32 or 33, wherein, in a tumor sample of said subject,

PD-L1 is expressed in at least 6% of the viable tumor cells and/or the CPS is at least 6 and/or the TPS is at least 6 and/or the TAP score is at least 6%,

PD-L1 is expressed in at least 11% of the viable tumor cells and/or the CPS is at least 11 and/or the TPS is at least 11 and/or the TAP score is at least 11%, PD-L1 is expressed in at least 12% of the viable tumor cells and/or the CPS is at least 12 and/or the TPS is at least 12 and/or the TAP score is at least 12%,

PD-L1 is expressed in at least 16% of the viable tumor cells and/or the CPS is at least 16 and/or the TPS is at least 16 and/or the TAP score is at least 16%,

PD-L1 is expressed in at least 17% of the viable tumor cells and/or the CPS is at least 17 and/or the TPS is at least 17 and/or the TAP score is at least 17%,

35. The method according to any one claims 25 to 34, wherein the one or more immunogenic compositions are peptide-based composition(s).

36. The method according to any one of claims 25 to 35, wherein the one or more immunogenic compositions are peptide-based compositions based on the E6 and E7 proteins of HPV16, wherein preferably the one or more immunogenic compositions in total comprise 12 synthetic long peptides of 25 to 35 amino acid in length.

37. The method according to claim 36, wherein the one or more immunogenic compositions (together) comprise one or more of all of the peptides set forth in SEQ ID NO: 1 to SEQ ID NO: 12.

38. The method according to claim 37, further comprising administration of an adjuvant.

39. The method according to any one of claims 25 to 38, wherein the PD-1 inhibitor is selected from an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-Ll antibody or antigen-binding fragment thereof, and an anti-PD-L2 antibody or antigenbinding fragment thereof.

40. The method according to any one of claims 25 to 39, wherein the PD-1 inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof that comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) and a light chain variable region comprising three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein: HCDR1 has an amino acid sequence of SEQ ID NO: 13; HCDR2 has an amino acid sequence of SEQ ID NO: 14; HCDR3 has an amino acid sequence of SEQ ID NO: 15; LCDR1 has an amino acid sequence of SEQ ID NO: 16; LCDR2 has an amino acid sequence of SEQ ID NO: 17; and LCDR3 has an amino acid sequence of SEQ ID NO: 18.

41. The method according to any one of claims 25 to 40, wherein the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein

• the heavy chain variable region comprising or consists of an amino acid sequence of SEQ ID NO: 19 and/or the light chain variable region comprises or consists of an amino acid sequence of SEQ ID NO:20, or

• the heavy chain variable region comprising or consists of an amino acid sequence with 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 19 and/ or the light chain variable region comprises or consists of an amino acid sequence with 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO:20.

42. The method according to any one of claims 25 to 41, wherein the anti-PD-1 antibody comprises a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence of SEQ ID NO:21 and the light chain has an amino acid sequence of SEQ ID NO:22.

43. The method according to any one of claims 25 to 42, wherein the one or more immunogenic compositions are peptide-based compositions and wherein the composition(s) are administered at a dose of between 10 and 200 micrograms per peptide.

44. The method according to claim 43, wherein the immunogenic composition(s) are administered three times with intervals of between 2 and 5 weeks, such as 3 or 4 weeks.

45. The method according to claim 44, wherein a further administration of the immunogenic composition(s) is performed between 4 and 8 months after the third initial dose.

46. The method according to any one of claims 25 to 45, wherein the immunogenic composition(s) are administered intravenously or subcutaneously.

47. The method according to any one of claims 25 to 46, wherein the PD-1 inhibitor is administered at a dose of 5 mg to 1500 mg, such as at a dose of 200 mg, 250 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, 750 mg, 800 mg, 1000 mg, 1050 mg, or 1200 mg.

48. The method according to any one of claims 25 to 47, wherein the PD-1 inhibitor is administered with intervals of between 2 and 5 weeks, such as 3 weeks.

49. The method according to any one of claims 25 to 48, wherein the PD-1 inhibitor is administered intravenously or subcutaneously.

50. A method of treating or inhibiting the growth of a tumor or improving overall survival of a head and neck cancer patient, comprising: a) selecting a patient with HPV16-positive head and neck cancer; b) administering to the patient an effective amount of one or more immunogenic compositions capable of inducing a T cell immune response against cells expressing HPV16 antigens, preferably E6, E7 and/ or E2 antigen; and c) administering to the patient a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof that comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) and a light chain variable region comprising three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein : HCDR1 has an amino acid sequence of SEQ ID NO: 13; HCDR2 has an amino acid sequence of SEQ ID NO: 14; HCDR3 has an amino acid sequence of SEQ ID NO: 15; LCDR1 has an amino acid sequence of SEQ ID NO: 16; LCDR2 has an amino acid sequence of SEQ ID NO: 17; and LCDR3 has an amino acid sequence of SEQ ID NO: 18.

51. The method according to claim 50, wherein step a) comprises selecting a patient with HPV16-positive head and neck cancer, wherein, in a tumor sample of said subject, PD-L1 is expressed in at least 5% of the viable tumor cells and/or the CPS is at least 5 and/or the TPS is at least 5 and/or the TAP score is at least 5%.

52. The method according to claim 51, wherein the tumor sample is a tumor biopsy sample.

53. The method according to claim 51 or 52, wherein, in a tumor sample of said subject, PD-L1 is expressed in at least 6% of the viable tumor cells and/or the CPS is at least 6 and/or the TPS is at least 6 and/or the TAP score is at least 6%,

54. The method according to any one of claims 50 to 53, wherein the patient has

(1) recurrent or metastatic HPV16-positive OPC and relapsed following primary surgical or chemoradiation treatment and wherein the patient preferably did not receive prior treatment with anti-PDl/anti-PDLl antibody or therapeutic HPV16 comprising vaccines, or (2) recurrent or metastatic HPV16-positive OPC and has progressed on prior platinumbased therapy, and wherein the patient preferably did not receive prior treatment with anti-PDl/anti-PDLl antibody or therapeutic HPV16 comprising vaccines, or

(3) previously untreated metastatic HPV-16 OPC and wherein the patient preferably did not receive prior treatment with anti-PDl/anti-PDLl antibody or therapeutic HPV16 comprising vaccines.

55. The method according to any one of claims 50 to 54, comprising one or more of the further features of any one of claims 26-49.