Combination therapy using LAG-3 protein and Anti-PD-1 inhibitor with radiotherapy for treating cancer

Abstract

Treatment of Cancer Methods for treatment of cancer, in particular soft tissue sarcomas (STS), are described, as well as compounds, compositions, and combined preparations for use in such methods. An effective amount of: a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules; a programmed cell death protein-1 (PD-1) pathway inhibitor; and radiotherapy, is administered to a subject. Optionally the subject has undergone, is undergoing, or is to undergo, surgery for treatment of the cancer. A clinical trial study is described in which a triple combination of LAG-3 derivative eftilagimod alpha, a PD-1 pathway inhibitor, and radiotherapy was administered. The combination was surprisingly effective in the treatment of STS in patients who have completed 10 weeks of treatment, followed by surgery 2-3 weeks later. A substantial number of near complete pathological responses were observed, which are rarely seen in STS patients with standard therapeutic approaches including radiotherapy.

Description

[0001] COMBINATION THERAPY USING LAG-3 PROTEIN AND ANTI-PD-1 INHIBITOR WITH RADIOTHERAPY FOR TREATING CANCER

[0002] This invention relates to methods for the prevention, treatment, and amelioration of cancer, in particular soft tissue sarcomas (STS), and to compounds, compositions, and combined preparations for use in such methods.

[0003] Over the past decade, PD-1 and CTLA-4 immune checkpoint inhibitors such as OPDIVO (nivolumab), KEYTRUDA (pembrolizumab) and YERVOY (ipilimumab) have become the standard of care therapies for many forms of cancer, however unfortunately, many patients still fail to respond to these modern medicines. In some cases, these new medicines are combined with chemotherapy (chemo-IO) to improve response rates, although this can lead to undesirable toxic effects. In other cases, combinations of immune checkpoint inhibitors (IO-IO) are used, but this can also lead to undesirable toxic effects.

[0004] To improve patient outcomes, significant work has been undertaken to investigate other immune checkpoints, such as LAG-3, TIM-3, VISTA, CD47, IDO, and TIGIT. LAG-3 in particular has emerged as a promising checkpoint and several companies are developing new inhibitors that target this checkpoint. The aim of a LAG-3 inhibitor, as with the currently approved PD-1 and CTLA-4 inhibitors, is to block the down-regulation of the immune system i.e. take the “brakes off the body’s immune processes. Significant work has also been undertaken to explore combinations of PD-1 and CTLA-4 immune checkpoint inhibitors with other approved or experimental therapies.

[0005] Another type of active immunotherapy being investigated are the antigen presenting cell (APC) activators. APC activators bind to antigen presenting cells such as dendritic cells, monocytes and macrophages via MHC II molecules. This activates the APCs causing them to become professional antigen presenting cells, thereby presenting antigen to the adaptive immune system. This leads to activation and proliferation of CD4+ (helper) and CD8+ (cytotoxic) T cells. Thus, the aim of APC activators is to “push the gas” on the body’s immune system.

[0006] Eftilagimod alpha (IMP321 , INN: eftilagimod alfa, or efti), a soluble dimeric recombinant form of LAG-3, is a first-in-class APC activator under clinical development. By stimulating dendritic cells and other APCs through MHC class II molecules, IMP321 induces a powerful anticancer T cell response. IMP321 is described in WO 2009 / 044273, which also describes the use of IMP321 alone and in combination with a chemotherapy agent for the treatment of cancer. In addition, WO 2016 / 110593 describes the use of IMP321 in combination with a PD- 1 pathway inhibitor for the treatment of cancer and infectious disease.

[0007] There remains a need in the art for improved cancer therapies and treatment regimens leading to better outcomes for patients. This is especially so for cancers where the prognosis for patients undertaking treatment with current approved medicines is poor and / or where current medicines are poorly tolerated. One such example are soft tissue sarcomas (STS).

[0008] STS are a diverse group of rare tumors. The incidence of STS varies in different regions, with approximately 23,400 cases annually and a crude incidence of 4.7 per 100,000 in Europe, according to the RARECARE project. In the United States, the number of new cases is estimated to be 13,400 annually with 5,140 deaths, according to the American Cancer Society. Currently, over 100 STS subtypes have been characterized, and this number is continuously growing. Liposarcoma and leiomyosarcoma are the most common STS subtypes in adults, and are followed by undifferentiated pleomorphic sarcoma and synovial sarcoma. Surgery is the mainstay of treatment of primary localized soft tissue sarcoma (STS), independently of histological subtype. However, despite optimal surgical resection, disease recurrence is common. Patients with extremity and trunk wall STS with high malignancy grade and size > 5 cm have a high propensity for distant spread, which leads to death from metastatic disease in up to 50% of patients.

[0009] To improve treatment outcomes, preoperative treatment such as radiotherapy (RT), chemotherapy (ChT), or chemoradiotherapy (CRT) is used, especially in high-risk patients. For example, in patients with high-grade localised STS of the extremity / trunk, and tumor size > 5 cm, radiation therapy (RT) (also referred to as radiotherapy herein) is added to reduce local recurrence. According to the international guidelines, perioperative RT is the standard treatment of high-grade (G2-3) lesions that are over 5 cm in size. The timing of radiotherapy (pre- or post-operatively) varies between institutions, but there is a general shift towards the use of perioperative RT, especially when it is crucial to preserve critical structures. Neoadjuvant RT in STS may be prescribed in conventional and altered fractions. The conventional fractionation is 50-50.4 Gy in 25-28 fractions for 5-6 weeks. However, the quality of evidence is low and is not confirmed in randomized trials.

[0010] Adjuvant / neoadjuvant anthracycline plus ifosfamide (Al) ChT may be used to improve survival, but its efficacy is limited to patients with poor prognosis. Recently, immunotherapy (ITH) has been widely studied in patients with metastatic STS, however response rates are modest - its efficacy and impact on tumor microenvironment remain unclear. There is no uniform use of adjuvant and neoadjuvant ChT in resectable, localized STSs of extremities and the trunk wall. Formally, adjuvant and neoadjuvant Al ChT is not a standard treatment. The results of published clinical controlled trials are conflicting. Some controlled trials and subgroups analyses of larger trials suggest that neoadjuvant or adjuvant Al ChT may improve relapse free-survival and overall survival in high-risk patients. International guidelines recommend discussing the option of chemotherapy with patients affected by a primary high- risk STS of extremity and trunk wall within a shared decision. While radiotherapy is widely accepted as standard of care, chemotherapy can be discussed, especially in patients with a 10-year predicted overall survival (OS) < 60%.

[0011] Thus, STS is a hard-to-treat orphan disease with poor prognosis and high unmet medical need. There is an urgent need for effective therapies and treatment regimens leading to better outcomes for patients with STS.

[0012] We have found that a novel triple combination of eftilagimod alpha (also known as “efti” or “IMP321"), a PD-1 pathway inhibitor, and radiotherapy is well-tolerated, and surprisingly effective in the treatment of STS. A clinical trial study, described in Examples 1 to 4 below (EFTISARC-NEO study), is the first to evaluate efti in a neoadjuvant setting, which takes place before intended surgery, and is the first to combine efti with radiotherapy. Importantly, the neoadjuvant setting allows for the impact of this novel combination to be assessed in the tumor microenvironment (TME). The chemotherapy-free combination has revealed no new safety findings and has been well tolerated in the first 21 patients who have completed the ten weeks of treatment, followed by surgery two to three weeks later. A substantial number of near complete pathological responses (according to EORTC-STBSDG) have been observed, which are rarely seen in STS patients with standard therapeutic approaches including radiotherapy. We have appreciated that such triple combination therapy may also be effective in treating other cancers, especially where there is a requirement for radiotherapy and / or surgery in conventional treatments.

[0013] According to the invention there is provided a triple combination therapy, comprising a LAG- 3 protein or derivative thereof, a PD-1 pathway inhibitor, and radiotherapy, for use in the prevention, treatment, or amelioration of a cancer in a subject.

[0014] According to the invention there is provided a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, and a programmed cell death protein-1 (PD-1 ) pathway inhibitor, for use in the prevention, treatment, or amelioration of a cancer in a subject who has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

[0015] There is also provided according to the invention use of a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, and a programmed cell death protein- 1 (PD-1 ) pathway inhibitor, in the manufacture of a medicament for the prevention, treatment, or amelioration of a cancer in a subject who has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

[0016] There is further provided according to the invention a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, for use in the prevention, treatment, or amelioration of a cancer in a subject, wherein the LAG-3 protein, or derivative, is to be administered simultaneously or sequentially with a programmed cell death protein-1 (PD-1 ) pathway inhibitor to the subject, and wherein the subject has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

[0017] There is also provided according to the invention use of a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, in the manufacture of a medicament for the prevention, treatment, or amelioration of a cancer in a subject, wherein the LAG-3 protein, or derivative, is to be administered simultaneously or sequentially with a programmed cell death protein-1 (PD-1 ) pathway inhibitor to the subject, and wherein the subject has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

[0018] There is further provided according to the invention a programmed cell death protein-1 (PD- 1 ) pathway inhibitor for use in the manufacture of a medicament for the prevention, treatment, or amelioration of a cancer in a subject, wherein the PD-1 pathway inhibitor is to be administered simultaneously or sequentially with a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, to the subject, and wherein the subject has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

[0019] There is further provided according to the invention a programmed cell death protein-1 (PD- 1 ) pathway inhibitor for use in the prevention, treatment, or amelioration of a cancer in a subject, wherein the PD-1 pathway inhibitor is to be administered simultaneously or sequentially with a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, to the subject, and wherein the subject has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

[0020] There is also provided according to the invention use of a programmed cell death protein-1 (PD-1 ) pathway inhibitor in the prevention, treatment, or amelioration of a cancer in a subject, wherein the PD-1 pathway inhibitor is to be administered simultaneously or sequentially with a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, to the subject, and wherein the subject has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

[0021] There is also provided according to the invention use of a programmed cell death protein-1 (PD-1 ) pathway inhibitor in the manufacture of a medicament for the prevention, treatment, or amelioration of a cancer in a subject, wherein the PD-1 pathway inhibitor is to be administered simultaneously or sequentially with a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, to the subject, and wherein the subject has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

[0022] The LAG-3 protein, or derivative thereof, and the PD-1 pathway inhibitor for use according to the invention may be provided as a combined preparation, or as a mixture in a pharmaceutical composition.

[0023] Accordingly, there is further provided according to the invention a combined preparation comprising:

[0024] (a) a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules; and

[0025] (b) a programmed cell death protein-1 (PD-1) pathway inhibitor; for use in the prevention, treatment, or amelioration of a cancer in a subject who has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

[0026] There is also provided according to the invention use of a combined preparation in the manufacture of a medicament for the prevention, treatment, or amelioration of a cancer in a subject who has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer, wherein the combined preparation comprises: (a) a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules; and

[0027] (b) a programmed cell death protein-1 (PD-1) pathway inhibitor.

[0028] There is further provided according to the invention a pharmaceutical composition comprising:

[0029] (a) a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules;

[0030] (b) a programmed cell death protein-1 (PD-1) pathway inhibitor; and

[0031] (c) a pharmaceutically acceptable carrier, excipient, or diluent; for use in the prevention, treatment, or amelioration of a cancer in a subject who has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

[0032] There is also provided according to the invention use of a pharmaceutical composition in the manufacture of a medicament for the prevention, treatment, or amelioration of a cancer in a subject who has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer, wherein the pharmaceutical composition comprises:

[0033] (a) a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules;

[0034] (b) a programmed cell death protein-1 (PD-1) pathway inhibitor; and

[0035] (c) a pharmaceutically acceptable carrier, excipient, or diluent.

[0036] There is further provided according to the invention a method for preventing, treating, or ameliorating a cancer in a subject in need thereof, which comprises administering to the subject an effective amount of:

[0037] (a) a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules;

[0038] (b) a programmed cell death protein-1 (PD-1) pathway inhibitor; and (c) radiotherapy.

[0039] There is further provided according to the invention a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, for use in the prevention, treatment, or amelioration of a cancer in a subject who has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

[0040] There is also provided according to the invention use of a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, in the manufacture of a medicament for the prevention, treatment, or amelioration of a cancer in a subject who has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

[0041] There is also provided according to the invention a method for preventing, treating, or ameliorating a cancer in a subject in need thereof, which comprises administering to the subject an effective amount of a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, and radiotherapy.

[0042] Exemplary cancers that may be treated according to the invention include, but are not limited to, breast cancer, skin cancer, lung cancer (for example NSCLC or SCLC), ovarian cancer, renal cancer (for example renal cell carcinoma), colon cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, bladder cancer, urothelial cancer, liver cancer, melanoma (for example, metastatic malignant melanoma), prostate cancer (for example hormone refractory prostate adenocarcinoma), head and neck cancer (for example, head and neck squamous cell carcinoma), cervical cancer, endometrial cancer, uterine cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma (for example, a B cell lymphoma or Hodgkin lymphoma), adrenal gland cancer, AIDS-associated cancer, alveolar soft part sarcoma, astrocytic tumor, bone cancer, brain and spinal cord cancer, metastatic brain tumor, carotid body tumor, chondrosarcoma, chordoma, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasia of the bone, gallbladder or bile duct cancer, gestational trophoblastic disease, germ cell tumor, haematological malignancy, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, lipoma / benign lipomatous tumor, liposarcoma / malignant lipomatous tumor, medulloblastoma, meningioma, Merkel cell carcinoma, multiple endocrine neoplasia, multiple myeloma, myelodysplasia syndrome, neuroblastoma, neuroendocrine tumor, papillary thyroid carcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, phaeochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematologic disorder, rhabdoid tumor, rhabdomysarcoma, sarcoma, soft-tissue sarcoma, squamous cell cancer, synovial sarcoma, mesothelioma, cutaneous squamous cell carcinoma, testicular cancer, thymic carcinoma, thymoma, and thyroid metastatic cancer.

[0043] Optionally the cancer is a sarcoma.

[0044] Preferably the cancer is a soft tissue sarcoma (STS).

[0045] Optionally the STS is undifferentiated pleomorphic sarcoma (UPS).

[0046] Optionally the STS is fibrosarcoma.

[0047] Optionally the STS is myxofibrosarcoma (MF).

[0048] Optionally the STS is liposarcoma.

[0049] Optionally the STS is pleomorphic liposarcoma (PLS).

[0050] Optionally the STS is dedifferentiated liposarcoma (DDLPS).

[0051] Optionally the STS is myxoid and round cell liposarcoma (MRCLPS).

[0052] Optionally the STS is myxoid liposarcoma.

[0053] Optionally the STS is epithelioid sarcoma (ES).

[0054] Optionally the STS is angiosarcoma (AS).

[0055] Optionally the STS is soft tissue sarcoma NOS.

[0056] Optionally the STS is leiomyosarcoma (LMS).

[0057] Optionally the STS is malignant peripheral nerve sheath tumor (MPNST).

[0058] Optionally the STS is undifferentiated pleomorphic sarcoma (UPS).

[0059] Optionally the STS is gastrointestinal stromal tumor (GIST).

[0060] Optionally the STS is synovial sarcoma

[0061] Optionally the STS is rhabdomyosarcoma (RMS). Optionally the STS is dermatofibrosarcoma protuberans (DFSP).

[0062] Optionally the STS is Kaposi sarcoma.

[0063] Optionally the STS is Ewing sarcoma.

[0064] Optionally the STS is uterine sarcoma.

[0065] Optionally the STS is solitary fibrous tumor (SFT).

[0066] Optionally the STS is epithelioid sarcoma.

[0067] Optionally the STS is alveolar soft part sarcoma (ASPS).

[0068] Optionally the STS is myxoid chondrosarcoma.

[0069] Optionally the STS is clear cell sarcoma (CCS).

[0070] Optionally the STS is extrarenal rhabdoid tumor (ERRT).

[0071] Optionally the STS is extraskeletal osteosarcoma (ESOS).

[0072] Optionally the STS is mesenchymal chondrosarcoma (MCS).

[0073] Optionally the STS is paraganglioma.

[0074] Optionally the STS is atypical fibroxanthoma (AFX).

[0075] Optionally the cancer is a PD-L1 -positive or a PD-L2-positive cancer. Methods for identifying PD-L1 -positive and PD-L2-positive cancers are known to the skilled person. An example of a suitable method involves looking at tumor tissue under a microscope using immunohistochemical (IHC) staining.

[0076] PD-L1 and / or PD-L2 may be found on some normal cells and in higher-than-normal amounts on some types of cancer cells. When PD-L1 or PD-L2 binds to PD-1 (a protein found on T cells), it keeps T cells from killing the PD-L1- or PD-L2-containing cells, including the cancer cells. PD-1 pathway inhibitors bind to PD-1 , PD-L1 or PD-L2 and block binding of PD-1 to PD-L1 and / or PD-L2. This releases the “brakes” on the immune system and leaves T cells free to kill cancer cells.

[0077] Examples of PD-L1 -positive cancers include non-small cell lung cancer (NSCLC), melanoma, Hodgkin lymphoma, bladder cancer, kidney cancer, breast cancer, head and neck squamous cell carcinoma (HNSCC), esophageal squamous cell carcinoma (ESCC), stomach adenocarcinoma, and cervical cancer.

[0078] Examples of PD-L2-positive cancers include head and neck squamous cell carcinomas (HNSCC), lung squamous cell cancer (LUSC), renal cell carcinoma (RCC), pancreatic ductal adenocarcinoma (PDAC), and cervical cancer.

[0079] Optionally the cancer is a PD-L1 -negative or a PD-L2-negative cancer.

[0080] Optionally the cancer is a PD-L1 -negative cancer.

[0081] Optionally the cancer is a cancer that is treatable by radiotherapy and / or surgery.

[0082] Radiotherapy is a treatment used for many types of cancers, including anal cancer, bladder cancer, breast cancer, central nervous system tumors, gastro-oesophageal cancer, gynaecological cancers, head and neck cancer, hepato-pancreato-biliary cancer, lung cancer, lymphoma, paediatric cancer, penile cancer, prostate cancer, rectal cancer, renal cancer, sarcoma, seminoma and skin cancer (The Royal College of Radiologists, “Clinical Oncology Radiotherapy dose fractionation”, Fourth edition, January 2024).

[0083] Central nervous system tumors treatable with radiotherapy include glioma, meningioma and pituitary adenoma. Gynaecological cancers treatable with radiotherapy include cervical cancer, endometrial cancer, vulval cancer and vaginal carcinoma. Head and neck cancers treatable with radiotherapy include head and neck squamous cell carcinomas. Hepato- pancreato-biliary cancers treatable with radiotherapy include pancreatic cancer and liver cancers such as hepatocellular carcinoma and biliary tract cancer (including intrahepatic cholangiocarcinoma). Lung cancers treatable with radiotherapy include non-small cell lung cancer, small cell lung cancer and mesothelioma. Lymphomas treatable with radiotherapy include Hodgkin lymphoma and non-Hodgkin lymphoma. Paediatric cancers treatable with radiotherapy include leukaemia (such as acute lymphoblastic leukaemia, acute myeloid leukaemia and chronic myeloid leukaemia), Hodgkin lymphoma, neuroblastoma, nephroblastoma, rhabdomyosarcoma, Ewing sarcoma, central nervous system tumors (such as glioma, ependymoma and medulloblastoma) and intracranial germ cell tumors. Sarcomas treatable with radiotherapy include soft tissue sarcomas, such as extremity soft tissue sarcomas and retroperitoneal soft tissue sarcomas. Other sarcomas treatable with radiotherapy include bone sarcomas, such as osteosarcoma, chondrosarcoma and chordoma. Skin cancers treatable with radiotherapy include squamous cell carcinoma, basal cell carcinoma, melanoma and Merkel cell carcinoma.

[0084] Radiotherapy can also be used in the treatment of metastatic cancer, including bone metastases, brain metastases, extracranial oligometastases and metastatic spinal cord compression.

[0085] Whether a cancer, such as a sarcoma, is resectable is dependent on the stage of the cancer, its location and any patient comorbidities. The aim of any resection is to completely remove the tumor with clear margins. Radical margins, when the tumor is removed en bloc with the affected compartment, or wide margins, where the entire tumor is removed but not the entire compartment are desired, but may not be possible, for example due to anatomical constraints. In cases of positive margins, re-excisions should be undertaken.

[0086] Currently, surgery is the primary treatment in the majority of soft tissue sarcoma (STS). Surgery is often used in cases of localised STS, and is considered to be the standard treatment (Dangoor, Adam, et al. "UK guidelines for the management of soft tissue sarcomas." Clinical sarcoma research 6 (2016): 1-26.). Surgery is used more rarely in the case of metastatic disease, and may be a palliative approach.

[0087] Optionally the subject has undergone, is undergoing, or is to undergo, surgery for treatment of the cancer, for example STS. Optionally the subject is to undergo surgery for treatment of the cancer, for example STS after administration of the LAG-3 protein, or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy. Optionally the subject may undergo surgery within six months, within four months, within two months, or within a month of administration of the LAG-3 protein, or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy. Optionally the subject may undergo surgery within a month of administration of the LAG-3 protein, or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy. A method of the invention may further comprise performing surgery on the subject to treat the cancer, for example STS. Optionally the surgery is performed after administration of the LAG-3 protein, or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy. Optionally the surgery is performed within six months, within four months, within two months, or within a month of administration of the LAG-3 protein, or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy. Optionally the surgery is performed within six months, within four months, within two months, or within a month of administration of a final dose of a course comprising a plurality of doses of the LAG-3 protein, or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy. Optionally the surgery is performed within a month of administration of a final dose of a course comprising a plurality of doses of the LAG-3 protein, or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy. Optionally the surgery is performed within 1 week, within 2 weeks, within 3 weeks, or within 4 weeks, of administration of a final dose of a course comprising a plurality of doses of the LAG-3 protein, or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy. Optionally the surgery is performed about 2 to about 3 weeks after administration of a final dose of a course comprising a plurality of doses of the LAG-3 protein, or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy.

[0088] LAG-3 Protein and Derivatives

[0089] The LAG-3 protein may be an isolated natural or recombinant LAG-3 protein. The LAG-3 protein may comprise an amino acid sequence of LAG-3 protein from any suitable species, such as a primate or murine LAG-3 protein, but preferably a human LAG-3 protein. The amino acid sequence of human and murine LAG-3 protein is provided in Figure 1 of Huard et al (Proc. Natl. Acad. Sci. USA, 11 : 5744-5749, 1997). The sequence of human LAG-3 protein is repeated in Figure 7 herein (SEQ ID NO: 1 ). The amino acid sequences of the four extracellular Ig superfamily domains (D1 , D2, D3, and D4) of human LAG-3 are also identified in Figure 1 of Huard et al., at amino acid residues: 1-149 (D1 ); 150-239 (D2); 240-330 (D3); and 331-412 (D4).

[0090] Derivatives of LAG-3 protein include soluble fragments, variants, or mutants of LAG-3 protein that are able to bind to MHC class II molecules. Several derivatives of LAG-3 protein are known that are able to bind to MHC class II molecules. Many examples of such derivatives are described in Huard et al (Proc. Natl. Acad. Sci. USA, 11 : 5744-5749, 1997). This document describes characterization of the MHC class II binding site on LAG-3 protein. Methods for making mutants of LAG-3 are described, as well as a quantitative cellular adhesion assay for determining the ability of LAG-3 mutants to bind to class Il-positive Daudi cells. Binding of several different mutants of LAG-3 to MHC class II molecules was determined. Some mutations were able to reduce class II binding, while other mutations increased the affinity of LAG-3 for class II molecules. Many of the residues essential for binding of LAG-3 to MHC class II proteins are clustered at the base of a large 30 amino acid extra-loop structure in the LAG-3 D1 domain. The amino acid sequence of the extra-loop structure of the D1 domain of human LAG-3 protein is GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO:2). The amino acid sequence of the extra-loop structure of the D1 domain of human LAG-3 protein is shown underlined in bold in Figure 7.

[0091] Optionally the derivative of LAG-3 protein comprises the 30 amino acid extra-loop sequence of the human LAG-3 D1 domain, or a variant of such sequence with one or more amino acid substitutions (e.g. a conservative amino acid substitution). The variant may comprise an amino acid sequence that has at least 70%, 80%, 90%, or 95% amino acid identity with the 30 amino acid extra-loop sequence of the human LAG-3 D1 domain.

[0092] The derivative of LAG-3 protein may comprise an amino acid sequence of domain D1 , domain D1 and optionally D2, or domains D1 and D2, of LAG-3 protein, preferably human LAG-3 protein.

[0093] The derivative of LAG-3 protein may comprise an amino acid sequence that has at least 70%, 80%, 90%, or 95% amino acid identity with domain D1 , domain D1 and optionally D2, or domains D1 and D2, of LAG-3 protein, preferably human LAG-3 protein.

[0094] Optionally the derivative of LAG-3 protein comprises an amino acid sequence that has at least 70% amino acid identity with domain D1 , and optionally domain D2, of LAG-3 protein, preferably human LAG-3 protein.

[0095] The derivative of LAG-3 protein may comprise an amino acid sequence of domains D1 , D2, and D3, domains D1 , D2, D3 and optionally D4, or domains D1 , D2, D3 and D4, of LAG-3 protein, preferably human LAG-3 protein.

[0096] The derivative of LAG-3 protein may comprise an amino acid sequence that has at least 70%, 80%, 90%, or 95% amino acid identity with domains D1 , D2 and D3, domains D1 , D2, D3 and optionally D4, or with domains D1 , D2, D3 and D4, of LAG-3 protein, preferably human LAG-3. Optionally the derivative of LAG-3 protein comprises an amino acid sequence that has at least 70% amino acid identity with domains D1 , D2 and D3, and optionally domain D4, of LAG-3 protein, preferably human LAG-3 protein.

[0097] Sequence identity between amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties.

[0098] Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector. Examples include MatGat (Campanella et al., 2003, BMC Bioinformatics 4: 29; program available from http: / / bitincka.com / ledion / matgat), Gap (Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453), FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410; program available from http: / / www.ebi.ac.uk / fasta), Clustal W 2.0 and X 2.0 (Larkin et al., 2007, Bioinformatics 23: 2947-2948; program available from http: / / www.ebi.ac.uk / tools / clustalw2) and EMBOSS Pairwise Alignment Algorithms (Needleman & Wunsch, 1970, supra; Kruskal, 1983, In: Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Sankoff & Kruskal (eds), pp 1-44, Addison Wesley; programs available from http: / / www.ebi.ac.uk / tools / emboss / align). All programs may be run using default parameters.

[0099] For example, sequence comparisons may be undertaken using the “needle” method of the EMBOSS Pairwise Alignment Algorithms, which determines an optimum alignment (including gaps) of two sequences when considered over their entire length and provides a percentage identity score. Default parameters for amino acid sequence comparisons (“Protein Molecule” option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: Blosum 62.

[0100] The sequence comparison may be performed over the full length of the reference sequence. The derivative of LAG-3 protein may be fused to Immunoglobulin Fc amino acid sequence, preferably human IgG 1 Fc amino acid sequence, optionally by a linker amino acid sequence.

[0101] The ability of a derivative of LAG-3 protein to bind to MHC class II molecules may be determined using a quantitative cellular adhesion assay as described in Huard et al (Proc. Natl. Acad. Sci. USA, 11 : 5744-5749, 1997). The affinity of a derivative of LAG-3 protein for MHC class II molecules may be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the affinity of human LAG-3 protein for MHC class II molecules.

[0102] Preferably, the affinity of a derivative of LAG-3 protein for MHC class II molecules is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the affinity of human LAG-3 protein for MHC class II molecules.

[0103] Examples of suitable derivatives of LAG-3 protein that are able to bind to MHC class II molecules include derivatives comprising: amino acid residues 23 to 448 of the human LAG-3 sequence; amino acid sequence of domains D1 and D2 of LAG-3; amino acid sequence of domains D1 and D2 of LAG-3 with an amino acid substitution at one or more of the following positions: position 30 where ASP is substituted with ALA; position 56 where HIS is substituted with ALA; position 73 where ARG is substituted with GLU; position 75 where ARG is substituted with ALA or GLU; position 76 where ARG is substituted with GLU; or position 103 where ARG is substituted with ALA; and a recombinant soluble human LAG-3lg fusion protein (“IMP321”, or “eftilagimod alpha”) - a 160-kDa dimer produced in Chinese hamster ovary cells transfected with a plasmid encoding for the extracellular domain of hLAG-3 fused to the human IgG 1 Fc. The sequence of IMP321 is given in SEQ ID NO: 17 of US 2011 / 0008331 .

[0104] Optionally the derivative of LAG-3 protein is eftilagimod alpha (efti, or IMP321 ).

[0105] Eftilagimod alpha (efti) is a dimeric soluble recombinant LAG-3 protein obtained by replacing the Fab immunoglobulin domains of an lgG1 molecule with the four immunoglobulin-like domains of the extracellular region of LAG-3. The LAG-3 - MHC II interaction controls the signaling between T cells and antigen presenting cells (APCs), which are responsible for the adaptive immune response. In contrast to antagonist anti-LAG-3 antibodies, efti is an agonist stimulating APCs via MHC II.

[0106] Efti mimics the structure of endogenous LAG-3. Through its 2 binding sites, efti has a high avidity (KD of 60 nM at 37°C) to MHC class II receptors expressed on APCs. Efti directly activates its primary target cells (i.e. dendritic cells (DCs) and monocyte subsets) via MHC class II binding. T cells and NK cells (secondary target cells) are subsequently induced, resulting in a Type 1 cytotoxic T cell response, required for efficient anti-tumor immunity. As such, efti is a first-in-class MHC II agonist acting as an APC activator.

[0107] In clinical studies, efti has been shown to induce a significant, sustained and durable increase in primary (monocytes and DCs) and secondary target cells (CD4 and CD8 T cells) together with Th1 biomarkers (interferon-gamma (IFN-y)). The observed effects were linked to improved overall survival OS in MBC patients receiving paclitaxel (Marme F, et al. 171 P Biomarker and multivariate analyses results from AIPAC: A phase lib study comparing eftilagimod alpha (a soluble LAG-3 protein) vs placebo in combination with weekly paclitaxel in HR+ HER2- metastatic breast cancer. Annals of Oncology. 2022;33:S203-S4).

[0108] Without being bound by theory, it is believed that combining efti with radiotherapy (and not metronomic low dose paclitaxel) could have the same effect as radiotherapy also induces the slow release of tumor antigens and therefore facilitates the reactivation of antigenspecific memory T cells and the priming of naive T cells to these tumor antigens by efti- activated APCs. Efti’s targeting and unique activation of dendritic cells, the most potent professional antigen-presenting cells, via MHC Class II agonism leads to broad adaptive and innate immunity to fight cancer, including proliferation of CD8+ cytotoxic T cells that can be armed with radiotherapy-induced tumor antigens. The combination of efti with radiotherapy and anti-PD-1 therapy has the potential to generate a robust anti-tumor immune response in an immunosuppressed tumor microenvironment, such as that of soft tissue sarcoma.

[0109] The LAG-3 protein or derivative thereof is administered in a therapeutically effective amount. A “therapeutically effective amount” refers to an amount of the active ingredient sufficient to have a therapeutic effect upon administration. Effective amounts of the active ingredient may vary, for example, with the particular disease or diseases being treated, the severity of the disease, the duration of the treatment, and characteristics of the patient (e.g. sex, age, height and weight).

[0110] Optionally the LAG-3 protein or derivative thereof is administered at a dose which is a molar equivalent of about 0.1 mg to about 200 mg, about 0.1 mg to about 180 mg, about 0.1 mg to about 150 mg, about 0.1 mg to about 120mg, about 0.1 mg to about 90 mg, about 0.1 mg to about 60 mg, about 6 mg to about 200 mg, about 6 mg to about 180 mg, about 6 mg to about 150 mg, about 6 mg to about 120mg, about 6 mg to about 90 mg, about 6 mg to about 60 mg, about 10 mg to about 50 mg, about 20 mg to about 40 mg, about 25 mg to about 35 mg, or about 30 mg of the LAG-3 derivative LAG-3lg fusion protein IMP321.

[0111] Optionally the LAG-3 protein or derivative thereof is administered at a dose which is a molar equivalent of about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, or about 35 mg of the LAG-3 derivative LAG-3lg fusion protein IMP321.

[0112] Optionally the LAG-3 protein or derivative thereof is administered at a dose which is a molar equivalent of about 30 mg of the LAG-3 derivative LAG-3lg fusion protein IMP321.

[0113] Optionally the LAG-3 protein or derivative thereof is administered at a dose which is a molar equivalent of about 90 mg of the LAG-3 derivative LAG-3lg fusion protein IMP321.

[0114] Optionally the LAG-3 protein or derivative thereof is administered at a dose which is a molar equivalent from about 25 mg to about 60 mg, such as about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, or about 60 mg, of the LAG-3 derivative LAG-3lg fusion protein IMP321.

[0115] Optionally the LAG-3 protein or derivative thereof is IMP321 and is administered at a dose of about 0.1 mg to about 60 mg, about 6 mg to about 60 mg, about 10 mg to about 50 mg, about 20 mg to about 40 mg, about 25 mg to about 35 mg, or about 30 mg.

[0116] Optionally the IMP321 is administered at a dose of about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, or about 35 mg.

[0117] Optionally IMP321 is administered at a dose of about 30 mg.

[0118] Optionally IMP321 is administered at a dose of about 90 mg.

[0119] Optionally IMP321 is administered at a dose from about 25 mg to about 60 mg, such as about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, or about 60 mg. Doses of 6-30 mg per subcutaneous (s.c.) injection of IMP321 have been shown to be safe, and provide an effective systemic exposure based on the results of pharmacokinetics data obtained in cancer patients. A blood concentration of IMP321 superior to 1 ng / ml for at least 24 hours after s.c. injection is obtained in patients injected with IMP321 doses of more than 6 mg.

[0120] Optionally the LAG-3 protein or derivative thereof is administered about once every week to the subject. Optionally the LAG-3 protein or derivative thereof is administered about once every two weeks to the subject. Optionally the LAG-3 protein or derivative thereof is administered about once every three weeks to the subject. Optionally the LAG-3 protein or derivative thereof is administered about once every four weeks to the subject. Optionally the LAG-3 protein or derivative thereof is administered about once every month to the subject. As will be appreciated by those of skill in the art, the precise treatment regimen may vary and be adapted according to the particular cancer being treated and characteristics of the patient.

[0121] Optionally the LAG-3 protein or derivative thereof is administered at least two times to the subject. Optionally the LAG-3 protein or derivative thereof is administered at least three times to the subject. Optionally the LAG-3 protein or derivative thereof is administered at least four times to the subject. Optionally the LAG-3 protein or derivative thereof is administered at least five times to the subject. Optionally the LAG-3 protein or derivative thereof is administered at least six times to the subject. Optionally the LAG-3 protein or derivative thereof is administered at least seven times to the subject. Optionally the LAG-3 protein or derivative thereof is administered at least eight times to the subject.

[0122] Optionally a plurality of doses of the LAG-3 protein, or derivative thereof, is administered to the subject.

[0123] Optionally a plurality of doses of the IMP321 is administered to the subject.

[0124] Optionally a dose of the LAG-3 protein, or derivative thereof, is administered to the subject once every two weeks, preferably up to five times.

[0125] Optionally the LAG-3 protein or derivative thereof is administered once every two weeks for a total of five times to the subject.

[0126] Optionally the IMP321 is administered once every two weeks for a total of five times to the subject. Optionally the LAG-3 protein or derivative thereof is present in the absence of any additional antigen added to the pharmaceutical composition, combined preparation, or medicament.

[0127] Optionally the IMP321 is present in the absence of any additional antigen added to the pharmaceutical composition, combined preparation, or medicament.

[0128] Optionally the subject is a mammal, preferably a human.

[0129] PD-1 Pathway Inhibitor

[0130] The PD-1 pathway inhibitor is an agent that inhibits binding of PD-1 to PD-L1 and / or PD-L2. In particular, the agent may inhibit binding of human PD-1 to human PD-L1 and / or human PD-L2. The agent may inhibit binding of PD-1 to PD-L1 and / or PD-L2 by at least 50%, 60%, 70%, 80%, or 90%. Suitable assays for determining binding of PD-1 to PD-L1 or PD-L2, by Surface Plasmon Resonance (SPR) analysis, or flow cytometry analysis, are described in Ghiotto et al (Int. Immunol. Aug 2010; 22(8): 651-660). The agent may inhibit binding of PD- 1 to PD-L1 and / or PD-L2, for example, by binding to PD-1 , to PD-L1 , or to PD-L2.

[0131] The agent may be an antibody, suitably a monoclonal antibody, such as a human or humanized monoclonal antibody. The agent may be a fragment or derivative of an antibody that retains ability to inhibit binding of PD-1 to PD-L1 and / or PD-L2.

[0132] Exemplary PD-1 pathway inhibitors include, but are not limited to, pembrolizumab, nivolumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, retifanlimab, atezolizumab, avelumab, and durvalumab, or a fragment or derivative thereof that retains ability to inhibit binding of PD-1 to PD-L1 and / or PD-L2.

[0133] Optionally the PD-1 pathway inhibitor is an anti-PD-1 antibody selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, and retifanlimab, or a fragment or derivative thereof that retains ability to inhibit binding of PD-1 to PD-L1 and / or PD-L2.

[0134] Optionally the PD-1 pathway inhibitor is an anti-PD-L1 antibody selected from the group consisting of atezolizumab, avelumab, and durvalumab, or a fragment or derivative thereof that retains ability to inhibit binding of PD-L1 to PD-1 .

[0135] Optionally the PD-1 pathway inhibitor is pembrolizumab, or a fragment or derivative thereof that retains ability to inhibit binding of PD-1 to PD-L1 and / or PD-L2. Optionally the PD-1 pathway inhibitor is pembrolizumab. Optionally the PD-1 pathway inhibitor is nivolumab. Optionally the PD-1 pathway inhibitor is cemiplimab. Optionally the PD-1 pathway inhibitor is spartalizumab. Optionally the PD-1 pathway inhibitor is camrelizumab. Optionally the PD-1 pathway inhibitor is sintilimab. Optionally the PD-1 pathway inhibitor is tislelizumab. Optionally the PD-1 pathway inhibitor is toripalimab. Optionally the PD-1 pathway inhibitor is dostarlimab. Optionally the PD-1 pathway inhibitor is retifanlimab.

[0136] Optionally the PD-1 pathway inhibitor is atezolizumab. Optionally the PD-1 pathway inhibitor is avelumab. Optionally the PD-1 pathway inhibitor is durvalumab.

[0137] Other exemplary PD-1 pathway inhibitors include vopratelimab (JTX-4014), acrixolimab (YBL-006), INCMGA00012, AMP-224, AMP-514, KN035, cosibelimab (CK-301 ), AUNP12, CA-170 and BMS-986189.

[0138] Optionally the LAG-3 derivative is eftilagimod alpha (efti, or IMP321 ), and the PD-1 pathway inhibitor is pembrolizumab, or a fragment or derivative thereof that retains ability to inhibit binding of PD-1 to PD-L1 and / or PD-L2.

[0139] Optionally the LAG-3 derivative is eftilagimod alpha (efti, or IMP321 ), and the PD-1 pathway inhibitor is pembrolizumab.

[0140] The dose of the PD-1 pathway inhibitor will depend on the particular PD-1 pathway inhibitor being used. In general, a typically prescribed dose of a PD-1 pathway inhibitor for a human subject may be 0.1 to 10 mg / kg, for example 0.1 to 1 mg / kg, or 1 to 10 mg / kg. The term "typically prescribed dose" is used herein to include a dose which is the same as the dose, or within the dosage range, that is safe and therapeutically effective for administration to a subject (suitably a human subject).

[0141] Optionally the PD-1 pathway inhibitor is administered to the subject at a dose of about 0.1 to about 10 mg / kg of the PD-1 pathway inhibitor.

[0142] Optionally the PD-1 pathway inhibitor is pembrolizumab, and the pembrolizumab is administered to the subject at a dose of 100-600 mg of the pembrolizumab.

[0143] Optionally a plurality of doses of the PD-1 pathway inhibitor is administered to the subject.

[0144] Optionally the PD-1 pathway inhibitor is administered about once every week to the subject.

[0145] Optionally the PD-1 pathway inhibitor is administered about once every two weeks to the subject. Optionally the PD-1 pathway inhibitor is administered about once every three weeks to the subject. Optionally the PD-1 pathway inhibitor is administered about once every four weeks to the subject. Optionally the PD-1 pathway inhibitor is administered about once every month to the subject. Optionally the PD-1 pathway inhibitor is administered about once every five weeks to the subject. Optionally the PD-1 pathway inhibitor is administered about once every six weeks to the subject. Optionally the PD-1 pathway inhibitor is administered about once every seven weeks to the subject. Optionally the PD-1 pathway inhibitor is administered about once every eight weeks to the subject. Optionally the PD-1 pathway inhibitor is administered about once every two months to the subject.

[0146] Optionally a dose of the PD-1 pathway inhibitor is administered to the subject once every three weeks, preferably up to three times.

[0147] Optionally a dose of the PD-1 pathway inhibitor is administered to the subject once every three weeks for a total of three times.

[0148] As will be appreciated by those of skill in the art, the precise treatment regimen may vary and be adapted according to the particular type of STS being treated and characteristics of the patient.

[0149] Examples of typically prescribed human doses of known PD-1 pathway inhibitors include:

[0150] Pembrolizumab: 200 mg every three weeks or 400 mg every six weeks.

[0151] Nivolumab: 240 mg every two weeks, 360 mg every 3 weeks, or 480 mg every 4 weeks Avelumab: 800 mg every two weeks (or maximum dose 10 mg / kg if weight < 80kg).

[0152] In some embodiments, the PD-1 pathway inhibitor is administered parenterally (including by subcutaneous, intravenous, or intramuscular injection) or orally. Optionally the PD-1 pathway inhibitor is administered intravenously.

[0153] Optionally the LAG-3 derivative is IMP321 , and the PD-1 pathway inhibitor is pembrolizumab. Optionally the IMP321 is administered subcutaneously at a dose of 30 mg every two weeks for a total of five administrations, and the pembrolizumab is administered intravenously at a dose of 200 mg every three weeks for a total of three administrations.

[0154] Optionally the subject is a mammal, preferably a human.

[0155] Radiation therapy (radiotherapy) Radiation therapy or radiotherapy involves administering ionising radiation to a tumor. It is based on the principle that ionising radiation induces DNA damage which leads to cell death. The most common type of radiation therapy is external beam radiation therapy which uses a high-energy beam of radiation, from a source external to the body, to target the tumor. Typically high energy photons, such as X-rays or gamma rays are used.

[0156] Radiation is directed to the area to be treated (target volume) with the aim being to reduce the radiation dosage delivered to healthy tissues. The target volume may be determined by methods including computed tomography (CT) and magnetic resonance imaging (MRI). To ensure accurate administration of the radiotherapy, the patient is carefully positioned and immobilised. In addition, types of external beam radiation therapy including 3-dimensional conformal radiation therapy (3DCRT) and intensity-modulated radiation therapy (IMRT) allow for better tumor targeting.

[0157] The dose of ionising radiation administered is measured in grays (Gy). The total dose will depend on the type and the stage of the cancer, patient comorbidities and whether the radiation is being used in conjunction with other therapies. The total dose of ionising radiation is fractionated, or split, over a period of time. This reduces toxic effects on non-cancerous cells. Fractionation schedules can be individualised.

[0158] Radiation therapy is commonly used in the treatment of soft tissue sarcoma (STS) as an adjunct to surgery. The timing of radiotherapy (preoperatively or postoperatively) varies. A conventional regimen of preoperative external beam radiation in STS is 50-50.4 Gy in 25- 28 fractions for 5-6 weeks (Haas, Rick LM, et al. "Preoperative radiotherapy for extremity soft tissue sarcoma; past, present and future perspectives on dose fractionation regimens and combined modality strategies." Radiotherapy and Oncology 119.1 (2016): 14-21 ).

[0159] Optionally a plurality of doses of radiotherapy are administered to the subject.

[0160] Optionally radiation therapy is administered to the subject about five times a week for at least one to eight weeks. Optionally radiation therapy is administered to the subject about five times a week for at least a week. Optionally radiation therapy is administered to the subject about five times a week for at least two weeks. Optionally radiation therapy is administered to the subject about five times a week for at least three weeks. Optionally radiation therapy is administered to the subject about five times a week for at least four weeks. Optionally radiation therapy is administered to the subject about five times a week for at least five weeks. Optionally radiation therapy is administered to the subject about five times a week for at least six weeks. Optionally radiation therapy is administered to the subject about five times a week for at least seven weeks. Optionally radiation therapy is administered to the subject about five times a week for at least eight weeks.

[0161] Optionally radiation therapy is administered to the subject about five times a week for up to eight weeks. Optionally radiation therapy is administered to the subject about five times a week for up to seven weeks. Optionally radiation therapy is administered to the subject about five times a week for up to six weeks. Optionally radiation therapy is administered to the subject about five times a week for up to five weeks.

[0162] Optionally the radiotherapy is administered to the subject five times a week for five or six weeks.

[0163] Optionally the total radiation dose administered to the subject is about 10-80 Gy, for example 40-60 Gy. Optionally the total radiation dose is about 10 Gy. Optionally the total radiation dose is about 20 Gy. Optionally the total radiation dose is about 30 Gy. Optionally the total radiation dose is about 40 Gy. Optionally the total radiation dose is about 50 Gy. Optionally the total radiation dose is about 60 Gy. Optionally the total radiation dose is about 70 Gy. Optionally the total radiation dose is about 80 Gy.

[0164] Optionally the radiotherapy is administered to the subject in conventional fractionation of 2 Gy / day five times a week. Optionally radiation therapy is administered to the subject in conventional fractionation of 2 Gy / day five times a week for at least a week. Optionally radiation therapy is administered to the subject in conventional fractionation of 2 Gy / day five times a week for at least two weeks. Optionally radiation therapy is administered to the subject in conventional fractionation of 2 Gy / day five times a week for at least three weeks. Optionally radiation therapy is administered to the subject in conventional fractionation of 2 Gy / day five times a week for at least four weeks. Optionally radiation therapy is administered to the subject in conventional fractionation of 2 Gy / day five times a week for at least five weeks. Optionally radiation therapy is administered to the subject in conventional fractionation of 2 Gy / day five times a week for at least six weeks. Optionally radiation therapy is administered to the subject in conventional fractionation of 2 Gy / day five times a week for at least seven weeks. Optionally radiation therapy is administered to the subject in conventional fractionation of 2 Gy / day five times a week for at least eight weeks.

[0165] For example, the total planned dose may be 50 Gy administered in conventional fractionation of 2 Gy / day for 5 days a week for five weeks, with high-energy photons of 6-15 MV.

[0166] Optionally the subject is a mammal, preferably a human. Timing of administration of the components of the triple combination therapy

[0167] The components of a triple combination therapy of the invention (comprising a LAG-3 protein or derivative thereof, a PD-1 pathway inhibitor, and radiotherapy) may be administered to the subject simultaneously, or sequentially in any order.

[0168] Administration of a plurality of doses of the LAG-3 protein or derivative thereof, a plurality of doses of the PD-1 pathway inhibitor, and a plurality of doses of radiotherapy to the subject may overlap with each other.

[0169] The LAG-3 protein or derivative thereof, and the PD-1 pathway inhibitor may be administered to the subject simultaneously, or sequentially in any order.

[0170] The LAG-3 protein or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy may be administered to the subject simultaneously, or sequentially in any order.

[0171] Optionally the LAG-3 protein or derivative thereof and the PD-1 pathway inhibitor are administered to the subject during a course of radiotherapy.

[0172] Optionally a course of administration of the LAG-3 protein or derivative thereof to the subject, comprising administration of a plurality of doses of the LAG-3 protein or derivative thereof to the subject, overlaps with administration of a course of radiotherapy to the subject, comprising administration of a plurality of doses of radiotherapy to the subject.

[0173] Optionally a course of administration of the PD-1 pathway inhibitor to the subject, comprising administration of a plurality of doses of the PD-1 pathway inhibitor to the subject, overlaps with administration of a course of radiotherapy to the subject, comprising administration of a plurality of doses of radiotherapy to the subject.

[0174] Optionally a course of administration of the LAG-3 protein or derivative thereof to the subject, comprising administration of a plurality of doses of the LAG-3 protein or derivative thereof to the subject, overlaps with a course of administration of the PD-1 pathway inhibitor to the subject, comprising administration of a plurality of doses of the PD-1 pathway inhibitor to the subject, and with a course of administration of radiotherapy to the subject, comprising administration of a plurality of doses of radiotherapy to the subject.

[0175] Optionally a course of administration of the LAG-3 protein or derivative thereof to the subject, comprising administration of a plurality of doses of the LAG-3 protein or derivative thereof to the subject over several days or weeks, overlaps with a course of administration of the PD- 1 pathway inhibitor to the subject, comprising administration of a plurality of doses of the PD- 1 pathway inhibitor to the subject over several days or weeks, and with a course of administration of radiotherapy to the subject, comprising administration of a plurality of doses of radiotherapy to the subject over several days or weeks.

[0176] Optionally the LAG-3 protein or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy are administered to the subject prior to surgical treatment for the cancer, for example the STS.

[0177] Optionally the LAG-3 protein or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy are administered to the subject at least one week prior to surgical treatment for the cancer, for example the STS.

[0178] Optionally the LAG-3 protein or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy are administered to the subject at least two weeks prior to surgical treatment for the cancer, for example the STS.

[0179] Optionally the LAG-3 protein or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy are administered to the subject up to six months prior to surgical treatment for the cancer, for example the STS.

[0180] Optionally the LAG-3 protein or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy are administered to the subject up to four months prior to surgical treatment for the cancer, for example the STS.

[0181] Optionally the LAG-3 protein or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy are administered to the subject up to two months prior to surgical treatment for the cancer, for example the STS.

[0182] Optionally the LAG-3 protein or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy are administered to the subject up to one month prior to surgical treatment for the cancer, for example the STS.

[0183] Optionally surgical treatment for the cancer is carried out after administration to the subject of a final dose of a course of administration comprising a plurality of doses of LAG-3 protein, a final dose of a course of administration comprising a plurality of doses of a PD-1 pathway inhibitor, and a final dose of a course of administration comprising a plurality of doses of radiotherapy. Optionally the LAG-3 protein, or derivative thereof, is administered to the subject once every two weeks for a total of five administrations (weeks 1 , 3, 5, 7, 9), and the PD-1 pathway inhibitor is administered to the subject once every three weeks for a total of three administrations (weeks 1 , 4, 7). Optionally the radiation therapy is administered to the subject for 5 days a week from week 2 to week 6. Optionally the radiation therapy is administered to the subject in conventional fractionation of 2 Gy / day. Optionally surgery is carried out about two to about three weeks after the final dose of the LAG-3 protein, or derivative thereof (in week 9), i.e. weeks 11-12.

[0184] Optionally the LAG-3 protein, or derivative thereof, is administered to the subject once every two weeks for a total of five administrations (weeks 1 , 3, 5, 7, 9), the PD-1 pathway inhibitor is administered to the subject once every three weeks for a total of three administrations (weeks 1 , 4, 7), and the radiation therapy is administered to the subject for 5 days a week from week 2 to week 6. Optionally the radiation therapy is administered to the subject in conventional fractionation of 2 Gy / day. Optionally surgery is carried out about two to about three weeks after the final dose of the LAG-3 protein, or derivative thereof (in week 9), i.e. weeks 11-12.

[0185] Optionally the LAG-3 derivative is eftilagimod alpha (IMP321 ), and the PD-1 pathway inhibitor is pembrolizumab. Optionally the eftilagimod alpha (IMP321 ) is administered to the subject once every two weeks for a total of five administrations (weeks 1 , 3, 5, 7, 9), and the pembrolizumab is administered to the subject once every three weeks for a total of three administrations (weeks 1 , 4, 7). Optionally the radiation therapy is administered to the subject for 5 days a week from week 2 to week 6. Optionally the radiation therapy is administered to the subject in conventional fractionation of 2 Gy / day. Optionally surgery is carried out about two to about three weeks after the final dose of eftilagimod alpha (IMP321 ) (in week 9), i.e. weeks 11-12.

[0186] Optionally the LAG-3 derivative is eftilagimod alpha (IMP321 ), and the PD-1 pathway inhibitor is pembrolizumab. Optionally the eftilagimod alpha (IMP321 ) is administered to the subject once every two weeks for a total of five administrations (weeks 1 , 3, 5, 7, 9), the pembrolizumab is administered to the subject once every three weeks for a total of three administrations (weeks 1 , 4, 7), and the radiation therapy is administered to the subject for 5 days a week from week 2 to week 6. Optionally the radiation therapy is administered to the subject in conventional fractionation of 2 Gy / day. Optionally surgery is carried out about two to about three weeks after the final dose of eftilagimod alpha (IMP321 ) (in week 9), i.e. weeks 11-12. Optionally the LAG-3 derivative is eftilagimod alpha (IMP321 ), and the PD-1 pathway inhibitor is pembrolizumab. Optionally the eftilagimod alpha (IMP321 ) is administered to the subject subcutaneously at a dose of 30 mg every two weeks for a total of five administrations (weeks 1 , 3, 5, 7, 9), and the pembrolizumab is administered to the subject intravenously at a dose of 200 mg every three weeks for a total of three administrations (weeks 1 , 4, 7), and the radiation therapy is administered to the subject in conventional fractionation of 2 Gy / day for 5 days a week from week 2 to week 6. Optionally surgery is carried out two to three weeks after the final dose of eftilagimod alpha (IMP321 ) (in week 9).

[0187] In a preferred embodiment, eftilagimod alpha is administered as subcutaneous injection (single anatomical site) in the anterior face of the thigh (it is recommended to rotate the injection site every injection). Alternating injections in deltoid regions or lower abdominal quadrants can be performed in situations when injections in the thigh are not feasible (e.g. due to tumor or in patients after amputation). The injection should be performed slowly to avoid discomfort at the site of injection. Eftilagimod alpha should be given > 30 minutes after pembrolizumab infusion is finished if both drugs are administered the same day.

[0188] Doses of the components of a triple combination therapy

[0189] The doses of the components of a triple combination therapy of the invention (comprising a LAG-3 protein or derivative thereof, a PD-1 pathway inhibitor, and radiotherapy) should be chosen to provide a therapeutically effective amount of the components in combination. An "effective amount" of the triple combination therapy may be an amount that results in a reduction of at least one pathological parameter associated with cancer. For example, in some embodiments, an effective amount of the triple combination therapy is an amount that is effective to achieve a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, in the pathological parameter, compared to the expected reduction in the parameter associated with the cancer without the triple combination therapy. For example, the pathological parameter may be tumor growth, or tumor growth rate.

[0190] For example, for soft tissue sarcoma (STS), the pathological parameter may be percentage of hyalinization and / or fibrosis in the surgical specimen. Historical data from retrospective analyses indicate that the median fibrosis and hyalinization after preoperative radiotherapy for STS is approximately 15% (HO). Following triple combination therapy according to the invention (i.e. therapy with a LAG-3 protein or derivative thereof, a PD-1 pathway inhibitor, and radiotherapy), this is expected to increase to 35% (H1 ). Thus, a reduction in this parameter may be determined as an increase in the percentage of hyalinization and / or fibrosis in the surgical specimen, where 15% fibrosis and hyalinization following treatment represents 0%, and 35% fibrosis and hyalinization following treatment represents 100%.

[0191] Alternatively, an "effective amount" of the triple combination therapy may be an amount that results in an increase in a clinical benefit associated with cancer treatment. For example, in some embodiments, an "effective amount" of the combination therapy is an amount that is effective to achieve an increase of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175% or 200%, in the clinical benefit, compared to the expected clinical benefit without the triple combination therapy. For example, the clinical benefit may be response rate, progression-free survival, overall survival, disease control rate, depth of response, duration of response, quality of life, or increased sensitization to subsequent treatments.

[0192] Alternatively, an "effective amount" of the triple combination therapy may be an amount that results in a change of at least one beneficial parameter relating to cancer treatment. For example, in some embodiments, an "effective amount" of the triple combination therapy is an amount that is effective to achieve a change of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, in the parameter, compared to the expected change in the parameter relating to cancer treatment without the combination therapy. For example, the parameter may be an increase in the number of circulating tumor antigen-specific CD8+T cells, or a reduction in the number of tumor antigen-specific regulatory T cells, or an increase in the number of activated T cells, in particular activated CD8+T cells, a reduction in the number of exhausted antigen-specific CD8+T cells, or an increase in the number of circulating functional (i.e. non-exhausted) antigen-specific CD8+T cells.

[0193] According to the invention, triple combination therapy may be employed to increase the therapeutic effect of the PD-1 pathway inhibitor and / or the radiotherapy, compared with (a) the effect of the PD-1 pathway inhibitor and the radiotherapy as monotherapies or (b) a combination therapy consisting of the PD-1 pathway inhibitor and the radiotherapy.

[0194] Triple combination therapy may also be employed to decrease the doses of the individual components in the combination while preventing or further reducing the risk of unwanted or harmful side effects of the individual components.

[0195] In an embodiment, the dosage of the PD-1 pathway inhibitor and / or radiotherapy is less than a typically prescribed dose for monotherapy with the PD-1 pathway inhibitor or radiotherapy, or below a typically prescribed dose for a combination therapy consisting of the PD-1 pathway inhibitor and radiotherapy, for example, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%, of the typically prescribed dose of the PD-1 pathway inhibitor and / or radiotherapy.

[0196] In another embodiment, the dosage of the PD-1 pathway inhibitor and / or radiotherapy is less than a typically prescribed dose for monotherapy with the PD-1 pathway inhibitor or radiotherapy, or below a typically prescribed dose for a combination therapy consisting of the PD-1 pathway inhibitor and radiotherapy, for example, from about 25% to about 75%, or from about 1 % to about 50%, or from about 0.5% to about 25%, of the typically prescribed dose of the PD-1 pathway inhibitor and / or radiotherapy.

[0197] In yet another embodiment, the dosage of the PD-1 pathway inhibitor and the radiotherapy is in accordance with a prescribed standard of care.

[0198] Suitably, a course of triple combination therapy in accordance with the invention takes place over, for example, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 12 months.

[0199] Similarly, a course of triple combination therapy in accordance with the invention takes place over, for example, about 8 weeks, about 12 weeks, about 16 weeks, about 20 weeks, about 24 weeks, about 28 weeks, about 32 weeks, about 36 weeks, about 40 weeks, about 44 weeks, about 48 weeks or about 52 weeks.

[0200] In one embodiment, a course of triple combination therapy in accordance with the invention takes place over about 8-12 weeks. In one embodiment, a course of triple combination therapy in accordance with the invention takes place over about 16 weeks. In another embodiment, the course of triple combination therapy in accordance with the invention takes place over about 24 weeks.

[0201] In one embodiment, a course of triple combination therapy in accordance with the invention takes place over about 9 weeks.

[0202] In an embodiment, after the subject is treated with a triple combination therapy of the invention, the subject moves to a radiotherapy-free maintenance phase comprising the LAG- 3 protein or derivative thereof and / or the PD-1 pathway inhibitor. The radiotherapy-free maintenance phase may be, for example, for about 3 months, about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, or about 24 months.

[0203] Preferably, there is at least one beneficial effect from a triple combination therapy of the invention, for example, advantageous therapeutic effects (e.g. overall response rate, progression-free survival, overall survival, disease control rate, depth of response or duration of response), fewer side effects, less toxicity, or improved quality of life - compared with an effective dosage of one or two of components (a), (b) and (c) (i.e. the LAG-3 protein or derivative thereof, PD-1 pathway inhibitor, and radiotherapy).

[0204] Combined Preparations

[0205] Optionally, the LAG-3 protein or derivative thereof, and the PD-1 pathway inhibitor are packaged separately. That is, in such embodiments, the LAG-3 protein or derivative thereof, and the PD-1 pathway inhibitor are separate unit dosage forms, which would typically (but not necessarily) be sourced from different suppliers, and then used in accordance with methods of the invention.

[0206] In other embodiments, the LAG-3 protein or derivative thereof, and the PD-1 pathway inhibitor are in the form of a combined preparation.

[0207] The two components of the “combined preparation” may be present: (i) in one combined unit dosage form known as a fixed dose combination (FDC), or (ii) as a first unit dosage form of component (a); and a separate, second unit dosage form of component (b); where the two separate dosage forms are packaged together (known as a kit-of-parts).

[0208] The ratio of the total amounts of the combination components (a) and (b) to be administered in the combined preparation can be varied, for example, in order to cope with the needs of a patient sub-population to be treated, or the needs of the patient, which can be due, for example, to the particular disease, age, sex, or body weight of the patient.

[0209] That is, a combined preparation according to the invention may take the form of a pharmaceutical composition comprising the LAG-3 protein or derivative thereof, and the PD- 1 pathway inhibitor or, alternatively, as a kit-of-parts comprising the LAG-3 protein or derivative thereof, and the PD-1 pathway inhibitor, as separate components, but packaged together.

[0210] Thus, in an embodiment, the invention provides a combined preparation, comprising: (a) a LAG-3 protein, or derivative thereof that is able to bind to MHC class II molecules, and

[0211] (b) a PD-1 pathway inhibitor.

[0212] The combined preparation may comprise a plurality of doses of the LAG-3 protein or derivative thereof, and / or a plurality of doses of the PD-1 pathway inhibitor.

[0213] Pharmaceutical Compositions

[0214] Optionally the LAG-3 protein or derivative thereof, and the PD-1 pathway inhibitor are formulated with a pharmaceutically acceptable carrier, excipient, or diluent to provide a pharmaceutical composition. Typically these will be formulated as separate pharmaceutical compositions, although in the case of a fixed dose combination, the LAG-3 protein or derivative thereof, and the PD-1 pathway inhibitor will be formulated together, along with a pharmaceutically acceptable carrier, excipient, or diluent.

[0215] The separate pharmaceutical compositions may be packaged together in the form of a kit- of-parts or sourced separately for use in the methods of the invention.

[0216] In general, the LAG-3 protein or derivative thereof, and the PD-1 pathway inhibitor may be administered by known means, in any suitable pharmaceutical composition, by any suitable route.

[0217] Suitable pharmaceutical compositions may be prepared using conventional methods known to those in the field of pharmaceutical formulation and described in the relevant texts and literature, for example, in Remington: The Science and Practice of Pharmacy (Easton, Pa.: Mack Publishing Co., 1995).

[0218] It is especially advantageous to formulate compositions of the invention in a unit dosage form for ease of administration and uniformity of dosage. The term “unit dosage form” as used herein refers to physically discrete units suited as unitary dosages for the individuals to be treated. That is, the compositions are formulated into discrete dosage units each containing a predetermined “unit dosage” quantity of an active agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier, excipient or diluent. The specifications of unit dosage forms of the invention are dependent on the unique characteristics of the active agent to be delivered. Dosages can further be determined by reference to the usual dose and manner of administration of the ingredients. It should be noted that, in some cases, two or more individual dosage units in combination provide a therapeutically effective amount of the active agent. Preparations according to the invention for parenteral administration include sterile aqueous and non-aqueous solutions, suspensions, and emulsions. Injectable aqueous solutions contain the active agent in water-soluble form. Examples of non-aqueous solvents or vehicles include fatty oils, such as olive oil and corn oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, low molecular weight alcohols such as propylene glycol, synthetic hydrophilic polymers such as polyethylene glycol, liposomes, and the like. Parenteral formulations may also contain adjuvants such as solubilizers, preservatives, wetting agents, emulsifiers, dispersants, and stabilizers, and aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, and dextran. Injectable formulations may be rendered sterile by incorporation of a sterilizing agent, filtration through a bacteria-retaining filter, irradiation, or heat. They can also be manufactured using a sterile injectable medium. The active agent may also be in dried, e.g., lyophilized, form that may be rehydrated with a suitable vehicle immediately prior to administration via injection.

[0219] Embodiments of the invention are described below, by way of example only, with reference to the accompanying drawings in which:

[0220] Figure 1 shows the role of LAG-3 in physiological situation (A), mechanism of action of soluble LAG-3 protein eftilagimod alpha (efti) (B), mechanism of action of an anti-LAG-3 monoclonal antibody (C);

[0221] Figure 2 shows the mechanism of action of soluble LAG-3 protein - eftilagimod alpha;

[0222] Figure 3 shows the rationale for combining eftilagimod alpha, pembrolizumab and radiotherapy based on cancer-immune cycle;

[0223] Figure 4 shows EFTISARC-NEO trial design;

[0224] Figure 5 shows the design of translation studies within EFTISARC-NEO trial;

[0225] Figure 6 shows the trial schema described in Example 2;

[0226] Figure 7 shows the amino acid sequence of mature human LAG-3 protein. The four extracellular Ig superfamily domains are at amino acid residues: 1-149 (D1 ); 150-239 (D2); 240-330 (D3); and 331-412 (D4). The amino acid sequence of the extra-loop structure of the D1 domain of human LAG-3 protein is shown underlined in bold;

[0227] Figure 8 shows the response to neoadjuvant radio-immunotherapy:

[0228] A - hyalinization and fibrosis in surgical specimens after treatment;

[0229] B - viable tumor cells in surgical specimens after treatments;

[0230] C - the proportion of tumor components after treatment in evaluable patients (N=21 ); D - response grade according to EORTC STBSG criteria; E - pathologic response, defined as >35% hyalinization and fibrosis;

[0231] F - radiologic response according to RECIST 1.1 criteria; and

[0232] Figure 9 shows an example of partial radiologic response per RECIST 1.1 (A) and complete pathologic response (B-E) in a patient with myxofibrosarcoma. B - MF before treatment; C - fibrosis; D - hyalinization; E - cell-free mucous.

[0233] Example 1

[0234] Pembrolizumab in combination with eftilagimod alpha and radiotherapy in neoadjuvant treatment of patients with soft tissue sarcomas - EFTISARC-NEO trial

[0235] Background

[0236] Surgery is the mainstay of treatment of primary localized soft tissue sarcoma (STS). However, despite optimal surgical resection, disease recurrence is common. In patients (pts) with high-grade localised STS of the extremity / trunk, and tumor size > 5 cm, radiation therapy (RT) is added to reduce local recurrence. Adjuvant / neoadjuvant Al ChT may be used to improve survival, but its efficacy is limited to pts with poor prognosis. Recently, immunotherapy (ITH) has been widely studied in pts with metastatic STS, however response rates are modest - its efficacy and impact on the tumor microenvironment remain unclear. Combining ITH with RTH may be a promising strategy for synergistic enhancement of treatment efficacy and the use of such a combination in the preoperative setting provides a unique opportunity to derive biological information related to tumor response.

[0237] As anti-PD-1 therapy is not sufficiently effective in many cancers, novel compounds such as eftilagimod alpha (efti) are being tested in combination with ITH to stimulate antigen- presenting cells and boost the immune response. Efti is a dimeric soluble recombinant LAG- 3 protein. In contrast to antagonist anti-LAG-3 antibodies, efti is an agonist stimulating antigen-presenting cells (APCs) via MHC II. The LAG-3 - MHC II interaction controls the signalling between T cells and APCs, which are responsible for the adaptive immune response (Figure 1 , 2). Anti-PD-1 , such as pembrolizumab, addresses part of the immune escape mechanism i.e., the tumor-induced T cell downregulation. On the other hand, the capacity of immune cells to recognize tumor cells and prime an effector response can be increased by APC activators providing support for the combination of these two drug classes (Figure 3).

[0238] We hypothesize that adding combined ITH to RT prior to surgical resection would be safe and improve pathologic response compared to historical cohorts of patients with localized STS treated with RT alone. The percentage of hyalinisation and fibrosis, as a surrogate of pathological response, appears to be most closely correlated with treatment outcome.

[0239] Study Endpoints

[0240] Primary:

[0241] The primary efficacy endpoint is a percent tumor hyalinization as a marker of response to treatment assessed at the time of surgical resection: HO - 15% (based on Schaefer M. et al.), H1 - 35%.

[0242] Secondary:

[0243] - Incidence of adverse events graded according to Common Terminology Criteria for Adverse Events (CTCAE) version 5.0

[0244] - Number of patients completing neoadjuvant treatment and having a curative surgery according to the protocol

[0245] - Disease-free survival time (DFS)

[0246] - Locoregional disease-free survival (LRFS)

[0247] - Distant metastasis-free survival (DMFS)

[0248] - Overall survival time (OS)

[0249] - Radiologic Response To Neoadjuvant Treatment using RECIST 1.1

[0250] Exploratory:

[0251] - To evaluate changes in the composition of tumor microenvironment, including immune infiltrates, before and after neoadjuvant treatment

[0252] - To evaluate correlations between changes of immune-related biomarkers in the tumor or blood with response to therapy and patients' survival

[0253] - To compare changes in tumor microenvironment caused by neoadjuvant radiotherapy and immunotherapy with pembrolizumab and eftilagimod alpha with changes related to other modalities (radiotherapy alone, radiotherapy with chemotherapy in neoadjuvant settings, or immunotherapy + / - other agents in advanced settings)

[0254] Trial Design

[0255] Key inclusion criteria:

[0256] - > 18 years of age;

[0257] - ECOG 0 or 1 ; - Primary or locally recurrent deep-seated extremities, girdles and / or superficial trunk (thoracic or abdominal wall) tumor;

[0258] - One of the following histologies

[0259] • undifferentiated pleomorphic sarcoma (UPS),

[0260] • myxofibrosarcoma,

[0261] • dedifferentiated liposarcoma (DDLPS),

[0262] • myxoid and round cell liposarcoma (MRCLPS),

[0263] • epithelioid sarcoma (ES),

[0264] • angiosarcoma (AS),

[0265] • soft tissue sarcoma NOS.

[0266] - Grade 2 or 3 tumors according FNCLCC;

[0267] - Size of the primary tumor >5 cm or locally recurrent of any size;

[0268] - Measurable disease based on RECIST 1.1 ;

[0269] - No previous systemic treatment for sarcoma.

[0270] Key Exclusion Criteria:

[0271] - Distant metastases;

[0272] - Previous treatment with eftilagimod alpha, anti-PD-1 or anti-PD-L1 ;

[0273] - Prior radiotherapy to tumor-involved sites;

[0274] - Subjects with active, known or suspected autoimmune disease or inflammatory bowel disease, which might impair the subject’s tolerance of trial treatment.

[0275] References

[0276] • Schaefer l-M et al. Histologic appearance after preoperative radiation therapy for soft tissue sarcoma: assessment of the European Organization for Research and Treatment of Cancer-soft tissue and bone sarcoma group response score. Int J Radiat Oncol Biol Phys. 2017;98(2):375-83.

[0277] • Pasquali S et al. Histopathological response (HR) after neoadjuvant chemotherapy (ChT) for high-risk soft tissue sarcomas (STS): A planned analysis of the ISG-STS-1001 trial. J Clin Oncol 41 , 2023 (suppl 16; abstr 11511).

[0278] • Lai, J.Z. et al. Local Irradiation Sensitized Tumors to Adoptive T Cell Therapy via Enhancing the Cross-Priming, Homing, and Cytotoxicity of Antigen-Specific CD8 T Cells. Front Immunol, 2019. 10: p. 2857. • Atkinson, V., et aL, Eftilagimod alpha, a soluble lymphocyte activation gene-3 (LAG-3) protein plus pembrolizumab in patients with metastatic melanoma. J Immunother Cancer, 2020. 8(2).

[0279] Example 2

[0280] A Phase II, single-arm clinical trial evaluating efficacy and safety of pembrolizumab in combination with a soluble LAG-3 protein, eftilagimod alpha, and radiotherapy in neoadjuvant treatment of patients with soft tissue sarcomas (EFTISARC-NEO)

[0281] 1. Protocol synopsis

[0282] Investigational Product(s), Dose and Mode of Administration, Duration of Treatment with Investigational Product(s):

[0283] Eftilagimod alpha 30 mg sc every two weeks for 5 doses combined with pembrolizumab 200 mg iv every 3 weeks for 3 doses.

[0284] Overall Study Duration:

[0285] 60 months.

[0286] Objectives:

[0287] Primary Objective

[0288] To assess anti-tumor efficacy of pembrolizumab in combination with eftilagimod alpha and radiotherapy in neoadjuvant treatment of patients with resectable soft tissue sarcomas by evaluation of pathologic response at time of definitive surgical treatment.

[0289] Secondary Objectives

[0290] • To evaluate pathologic response to neoadjuvant therapy according to the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group (EORTC-STBSG) criteria.

[0291] • To evaluate the best response according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1 ).

[0292] • To evaluate disease-free survival (DFS), locoregional disease-free survival (LRFS), distant metastasis-free survival (DMFS) and overall survival (OS).

[0293] • To evaluate safety and tolerability of pembrolizumab + eftilagimod alpha. • To assess Health Related Quality of Life (HRQoL) using the European Organisation for Research and Treatment of Cancer Core Quality of Life Questionnaire (EORTC QLQ-C30) and The Functional Assessment of Cancer Therapy - General (FACT-G) questionnaire.

[0294] Exploratory objectives

[0295] To explore potential biomarkers associated with clinical efficacy (pathological response, ORR, DFS and OS) by analyzing biomarker measures within the tumor and in the blood.

[0296] Study design

[0297] This is a phase II single-arm single-stage study evaluating efficacy and safety of pembrolizumab in combination with a soluble LAG-3 protein, eftilagimod alpha (efti) and radiotherapy in neoadjuvant treatment of patients with soft tissue sarcomas. This study will determine the pathologic response rate (defined as percentage of tumor hyalinization / fibrosis) to the combination treatment.

[0298] Systemic therapy with pembrolizumab and eftilagimod alpha and radiotherapy will be administered concurrently. Eftilagimod alpha will be administered subcutaneously at a dose of 30 mg every 2 weeks (14 days) beginning in week 1 of the study (weeks 1 , 3, 5, 7, and 9) for a total of 5 doses. Pembrolizumab will be administered at a dose of 200 mg by intravenous infusion over 30 minutes every 3 weeks (21 days) starting in Week 1 of the study (Weeks 1 , 4, and 7) - 3 administrations in total. Radiation therapy will last for 5 weeks. The total planned dose is 50 Gy administered in conventional fractionation of 2 Gy / day for 5 days a week from week 2 to week 6. Surgery will take place 5-6 weeks after completion of radiation therapy. Patients will be then followed up regularly for a period of 24 months

[0299] Study Population:

[0300] Key Inclusion Criteria

[0301] Willing and able to provide written informed consent for the trial;

[0302] Be > 18 years of age on day of signing informed consent;

[0303] Performance status of 0 to 1 on the ECOG Performance Scale;

[0304] Primary or locally recurrent deep-seated extremities, girdles and / or superficial trunk (thoracic or abdominal wall) soft tissue sarcoma;

[0305] Grade 2 or 3 tumors according FNCLCC; Size of the primary tumor >5 cm at instrumental staging (CT, MRI), or locally recurrent of any size;

[0306] Measurable disease based on RECIST 1.1 ;

[0307] Non-metastatic disease;

[0308] No previous systemic treatment for sarcoma. t Exclusion Criteria

[0309] The subject has any condition that, in the opinion of the Investigator, places the subject at unacceptable risk if they were to participate in the study;

[0310] Ewing sarcoma, Alveolar and embryonal rhabdomyosarcoma;

[0311] Previous treatment with eftilagimod alpha, anti-PD-1 or anti-PD-L1 ;

[0312] Prior radiotherapy to tumor-involved sites;

[0313] Subjects with active, known or suspected autoimmune disease. Subjects with vitiligo, type I diabetes mellitus, residual hypothyroidism due to autoimmune condition only requiring replacement therapy, psoriasis not requiring systemic treatment, or conditions not expected to recur in the absence of an external trigger are permitted to enroll.

[0314] Study Assessments:

[0315] Physical examination, ECOG performance status, weight, height, vital signs Laboratory tests (hematology, serum chemistry, evaluation of thyroid function, coagulation, pregnancy test in women of childbearing potential, urinalysis, HBV,

[0316] HCV, HIV)

[0317] - ECG

[0318] MRI scan of the anatomical region involving the tumor

[0319] CT scan of the chest, abdomen and pelvis

[0320] Core-needle biopsy of the tumor and blood sampling for translational research

[0321] Evaluation during neoadjuvant treatment:

[0322] Physical examination

[0323] Laboratory tests (hematology and serum chemistry)

[0324] Evaluation of adverse events and concomitant medications ECG will be performed before each administration of pembrolizumab and before surgery

[0325] MRI scan of the anatomical region involving the tumor and CT scans of the chest, abdomen and pelvis will be performed between 7 and 14 days before the scheduled surgery

[0326] Response to treatment according to RECIST v1.1 criteria will be assessed by the Investigator in collaboration with the radiologist

[0327] Blood sampling for translational research

[0328] Tumor excision surgery will be performed 35-42 days after completion of radiation therapy

[0329] Follow-up:

[0330] At each follow-up visit (at 2, 4 and 12 weeks after surgery, then every 12 weeks for 2 years), a physical examination will be performed, evaluation of side effects, as well as concomitant medications

[0331] CT scans of the chest, abdomen and pelvis will be performed every 12 weeks from the date of surgery for a period of 2 years

[0332] MRI of the anatomical region including the location of the primary tumor will be performed every 3 months in the 1st year of follow-up and every 6 months in the 2nd year of follow-up and in case of clinical suspicion for local recurrence

[0333] Endpoints

[0334] The primary efficacy endpoint is a percent tumor hyalinization as a marker of response to treatment assessed at the time of surgical resection.

[0335] Incidence of adverse events graded according to Common Terminology Criteria for Adverse Events (CTCAE) version 5.0

[0336] Number of patients completing neoadjuvant treatment and having a curative surgery according to the protocol

[0337] Disease-free survival time (DFS)

[0338] Locoregional disease-free survival (LRFS)

[0339] Distant metastasis-free survival (DMFS) Overall survival time (OS)

[0340] Radiologic Response To Neoadjuvant Treatment using RECIST 1.1 Relationship between biomarkers in blood / tissue and efficacy, including ORR, DFS, and OS

[0341] The primary endpoint of the study is the percentage of fibrosis and hyalinization found in the surgical specimen after preoperative treatment.

[0342] Historical data from retrospective analyses indicate that the median fibrosis and hyalinization after preoperative radiotherapy is approximately 15% (HO). With the use of pembrolizumab and eftilagimod alpha, we expect to increase the rate to 35% (H1 ).

[0343] 2. Background and introduction

[0344] 2.1 Soft Tissue Sarcoma

[0345] Soft tissue sarcomas (STS) are a diverse group of rare tumors with an estimated incidence of 4-6 cases per 100,000 a year. Currently, over 100 STS subtypes have been characterized, and this number is continuously growing (1 , 2). Liposarcoma and leiomyosarcoma are the most common STS subtypes in adults, and are followed by undifferentiated pleomorphic sarcoma and synovial sarcoma.

[0346] Surgery is the mainstay of treatment of primary localized disease, independently of histological subtype. However, despite optimal surgical resection, disease recurrence is common. Patients with extremity and trunk wall STS with high malignancy grade and size > 5 cm have a high propensity for distant spread, which leads to death from metastatic disease in up to 50% of patients.

[0347] To improve treatment outcomes, preoperative treatment such as radiotherapy (RT), chemotherapy (ChT), or chemoradiotherapy (CRT) are used, especially in high-risk patients, and should be discussed upfront in multidisciplinary tumor board (MTB). According to the international guidelines, perioperative RT is the standard treatment of high-grade (G2-3) lesions that are over 5 cm in size (3). The timing of radiotherapy (pre- or postoperatively) varies between institutions, but there is a general shift towards the use of perioperative RT, especially when it’s crucial to preserve critical structures. Neoadjuvant RT in STS may be prescribed in conventional and altered fractions (4). The conventional fractionation is SOSO.4 Gy in 25-28 fractions for 5-6 weeks; however, quality of evidence is low and is not confirmed in randomized trials (3, 5). There is no uniform use of adjuvant and neoadjuvant ChT in respectable, localized STSs of extremities and the trunk wall. Formally, adjuvant and neoadjuvant anthracycline plus ifosfamide (Al) ChT is not a standard treatment (3). The results of published clinical controlled trials are conflicting. Some controlled trials and subgroups analyses of larger trials suggest that neoadjuvant or adjuvant Al ChT may improve relapse free-survival and overall survival in high-risk patients (6, 7). International guidelines recommend discussing the option of chemotherapy with patients affected by a primary high-risk STS of extremity and trunk wall within a shared decision (3). While radiotherapy is widely accepted as standard of care, chemotherapy can be discussed, especially in patients with a 10-year predicted OS < 60% (3).

[0348] 2.2 Background information on pembrolizumab

[0349] Pembrolizumab is a potent and highly selective humanized monoclonal antibody (mAb) of the lgG4 / kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. Pembrolizumab has been approved in European Union for the treatment of patients with melanoma, non-small cell lung cancer (NSCLC), classical Hodgkin lymphoma, urothelial cancer, head and neck squamous cell carcinoma, renal cell carcinoma, oesophageal cancer, triple-negative breast cancer, cervical cancer and endometrial carcinoma. Importantly, in triple-negative breast cancer pembrolizumab is approved in combination with chemotherapy as neoadjuvant treatment. Please refer to approved labeling for detailed background information on pembrolizumab.

[0350] 2.3 Background information on eftilagimod alpha

[0351] Eftilagimod alpha (efti) is a dimeric soluble recombinant LAG-3 protein obtained by replacing the Fab immunoglobulin domains of an lgG1 molecule with the four immunoglobulin-like domains of the extracellular region of LAG-3. The LAG-3 - MHC II interaction controls the signaling between T cells and antigen presenting cells (APCs), which are responsible for the adaptive immune response. In contrast to antagonist anti-LAG-3 antibodies, efti is an agonist stimulating APCs via MHC II. This is a completely new concept, which is currently not covered by any other approved medicine.

[0352] Efti mimics the structure of endogenous LAG-3. Through its 2 binding sites, efti has a high avidity (KD of 60 nM at 37°C) to MHC class II receptors expressed on APCs. Efti directly activates its primary target cells (i.e. dendritic cells (DCs) and monocyte subsets) via MHC class II binding. T cells and NK cells (secondary target cells) are subsequently induced, resulting in a Type 1 cytotoxic T cell response, required for efficient anti-tumor immunity. As such, efti is a first-in-class MHC II agonist acting as an APC activator.

[0353] In clinical studies, efti induced a significant, sustained and durable increase in primary (monocytes and DCs) and secondary target cells (CD4 and CD8 T cells) together with Th1 biomarkers (interferon-gamma (IFN-y)). The observed effects were linked to improved overall survival OS in MBC patients receiving paclitaxel (8). Here, combining efti with radiotherapy (and not metronomic low dose paclitaxel) could have the same effect as radiotherapy induces the slow release of tumor antigens and therefore facilitate the reactivation of antigen-specific memory T cells and the priming of naive T cells to these tumor antigens by efti-activated APCs.

[0354] 2.3.1 Nonclinical studies with efti

[0355] Antigen presenting cells are the primary target cells for efti. In vitro data supports the mode of action of efti as direct APC activation through MHC class II engagement. Efti effects were observed on monocytes or dendritic cells isolated from peripheral blood mononuclear cells (PBMCs). The phenotypic and functional changes induced in APCs (increased expression of co-stimulatory molecules CD80, CD83 or CD86, production of inflammatory cytokines and various chemokines) by efti resulted in secondary activation of cytotoxic T cells and NK cells as evidenced by production of cytokines as IFN-y. These ex-vivo pharmacodynamic data support the direct effect of efti on the activation / maturation of APC toward a more professional APC phenotype. The increase in expression of co-stimulatory molecules and an absence of IL-10 induction by efti favors a more efficient T cell response (e.g., production of IFN-y by activated CD8+ T cells). An immune response involving cytotoxic cells promotes the development of an effective anti-tumor response.

[0356] Administration of a murine LAG-3lg fusion protein (mLAG-3lg) to mice in combination with topotecan reduced the growth of colon cancer lesions when compared to no treatment and topotecan monotherapy. In the same model, mLAG-3lg administration in combination with anti-PD-1 antibody also reduced tumor growth and increased survival, with primary tumor tissue showing increased T cell infiltration after administration of mLAG-3lg.

[0357] Administration of efti as a single ~5 mg / kg dose (100 pg) to mice showed that the maximal plasma concentration was achieved by 2 hours after dosing and circulating levels decreased rapidly after this time. Repeat dosing (at 14-day intervals for three administrations) with 5 mg / kg or 15 mg / kg efti in monkeys showed maximal plasma concentrations within the 4 hours after administration of the first dose, with detectable levels seen up to 24 hours. Systemic exposure was low after subcutaneous dosing, being <1 pg / ml in monkeys dosed at 15 mg / kg. After the second dose there was a notable decrease in circulating efti levels, indicative of the development of anti-drug antibodies (ADAs) to the human protein; efti was immunogenic in both animal test species, with the development of neutralising ADAs limiting exposure.

[0358] No efti safety concerns were noted in in vivo nonclinical studies. Local reactions at the injection site were noted but were expected given the mode of action of efti and the presence of high numbers of Langerhans cells in the dermis. Accordingly, the no observed adverse effect level (NOAEL) in the Cynomolgus monkey was the highest dose administered in the repeat dose study (15 mg / kg); this dose corresponds to 30 times the dose used in clinical trials (30 mg / dose, corresponding to 0.5 mg / kg based on 60 kg body weight).

[0359] 2.3.2 Clinical trials with efti

[0360] Efti is being investigated as a subcutaneous injection administered at doses of up to 30 mg every second week, given as monotherapy or in combination with standard chemotherapy or immune checkpoint inhibitors in patients with advanced metastatic cancer.

[0361] The first phase I trial evaluated subjects with metastatic renal cell cancer who were treated with efti monotherapy to a maximum dose of 30 mg (9). Efti dosed at 30 mg achieved a maximum plasma concentration (Cmax) of 8.0 ng / ml and the area under the plasma concentration-time curve (AUC) was 118±41 ng-hr / ml, highlighting good systemic exposure of efti. Subjects treated with efti 6.25 mg and 30 mg showed a significant increase in the frequency of circulating activated CD8+ T cells. Efti monotherapy administered subcutaneously at up to 30 mg every second week was well-tolerated. Another phase I trial evaluated subjects with metastatic breast cancer with efti in combination with paclitaxel (10). Efti treatment increased the numbers of APCs, NK and activated CD8+ T cells in blood and the proportion of CD8+ T cells with a terminally-differentiated effector memory phenotype. The objective response rate (ORR) with paclitaxel plus efti (at different dose levels) was 47% and the disease control rate (DCR) was 90%.

[0362] Based on the promising phase I data, efti was then evaluated in multiple phase I or II trials in patients with metastatic breast cancer (in combination with paclitaxel); and metastatic melanoma, non-small cell lung cancer (NSCLC), and head and neck squamous cell carcinoma (HNSCC) (all in combination with pembrolizumab).

[0363] When efti was administered with paclitaxel to subjects with metastatic breast cancer it did not significantly prolong median progression-free survival in the overall population compared to paclitaxel plus placebo. The final analysis (based on 73% of events) showed an improved hazard ratio (0.88) and improved median overall survival (20.4 to 17.5 months) for subjects receiving efti. The objective response rate by blinded independent central read was higher for the efti treatment arm (51.4% vs. 40.6% in the placebo arm). Effects in important subgroups (<65 years, low monocytes, no prior taxanes, luminal B subtype, <5 years since diagnosis and high neutrophil-lymphocyte count) were more pronounced.

[0364] Preliminary analyses of the combination efti plus pembrolizumab in subjects with first-line NSCLC unselected for PD-L1 expression estimated the overall response rate with 40.4% as per iRECIST based on blinded independent central review of data from evaluable subjects (11 ). T umor responses were observed in all PD-L1 subgroups, including patients with no / very low expression of PD-L1 (TPS <1 %) where response rate was 31.3%. Such high rates of responses with pembrolizumab or other anti-PD1 antibodies have not been observed before. The median progression duration of response was estimated to be 13+ months.

[0365] Preliminary analysis of combination of efti and pembrolizumab in subjects with second-line HNSCC treated identified the overall response rate for efti with pembrolizumab at 35.5% (95% Cl 19.2%, 54.6%) per iRECIST based on data from evaluable subjects (12). The median duration of response had not been reached. In another trial subjects with metastatic melanoma not benefitting from pembrolizumab monotherapy, addition of efti to the pembrolizumab regimen was associated with a 33% overall response rate; the disease control rate was 66% at 3 months after initiation of combination therapy (13).

[0366] Safety data have shown that efti is generally safe and well tolerated with injection-site reactions (Grade 1 and 2) being the most common side effect. Severe (Grade 3 and 4) immediate systemic hypersensitivity (i.e. , allergic / anaphylactic reaction; 3 reactions reported in 377 subjects [0.8%] exposed to efti) has been identified as an important risk. Until this stage of efti clinical development there has been no indication of a severe cytokine release syndrome; the existing risk appears to represent mild to moderate systemic inflammation (e.g. fever, myalgia).

[0367] 2.4 Rationale for combination of efti with an anti-PD-1

[0368] Immune Checkpoint Inhibitors (ICIs) are generally projected to be particularly successful in tumors with high levels of endogenous PD-L1 expression. This was also shown for pembrolizumab in first-line NSCLC (14). The reasons why patients are not reacting to anti- PD-1 therapy are not fully understood, but contributing factors are thought to include e.g. properties of the tumor microenvironment, such as the lack of tumor-infiltrating effector T cells. For example, in the KEYNOTE-042 trial, Objective Response Rate (ORR) was only -17% in patients receiving pembrolizumab in the PD-L1 tumor proportion score (TPS) 1-49 % population (15). Nonresponding patients have been shown to have little or no tumorinfiltrating lymphocytes (TILs) in the tumor bed or a non-functional immune response (i.e. no intratumoral IFN-y) with PD-1 / PD-L1 negative TILs. Immunogenic signals that induce tumor antigen-loaded DCs to escape peripheral tolerance and better prime and activate effector T cells are expected to reactivate the deficient early steps of the cancer-immunity cycle. Anti- PD-1 , such as pembrolizumab, address part of the immune escape mechanism i.e., the tumor-induced T cell downregulation. On the other hand, the capacity of immune cells to recognize tumor cells and prime an effector response can be increased by APC activators, providing support for the combination of these two drug classes. The addition of an APC activator like efti is thought to help activate the cellular immune response mechanisms to mediate tumor recognition and killing, thus leading to a higher frequency of durable responses in patients, compared to pembrolizumab monotherapy, without adding substantial toxicity.

[0369] First clinical data indicated that combination of efti plus anti-PD-1 is safe, leads to increased ORR with long duration of responses (DoR) and desired pharmacodynamics (PD) effects. As encouraging antitumor activity has been shown in combination with PD-1 / PD-L1 antagonists (e.g. pembrolizumab), the potential benefits of efti administration in combination with PD-1 / PD-L1 antagonists outweigh the risks.

[0370] 2.5 Rationale for the current study

[0371] As in other solid tumors, immunotherapy with immune checkpoint inhibitors (ICI) is extensively studied in sarcoma. This treatment modality has mainly been investigated in metastatic settings after progression on doxorubicin-based chemotherapy. In the SARC028, a phase II trial of advanced soft-tissue and bone sarcoma (NCT02301039), pembrolizumab showed a 17.5% objective response rate (ORR) (16, 17). Going beyond ICI monotherapy, the phase II trial Alliance A091401 trial (NCT02500797) demonstrated that combination therapy using nivolumab and ipilimumab could be effective in metastatic sarcoma, increasing ORR from 5% for nivolumab monotherapy to 16% for combination. The median overall survival on the monotherapy arm was 10.7 months, compared to 14.3 months in the combination arm. The median PFS was 1.7 and 4.1 months, respectively. Results were also confirmed in the expansion cohorts in patients with undifferentiated pleomorphic sarcoma (UPS) and dedifferentiated liposarcoma (DDLPS) (18, 19). The activity of PD-1 checkpoint inhibitors as monotherapies and in combination with anti- CTLA-4 in the neoadjuvant setting is currently under investigation in several trials (NCT03474094, NCT03463408, NCT03092323, NCT03116529). Preliminary results of a phase II study (NCT03307616) of neoadjuvant checkpoint blockade for surgically UPS and DDLPS were presented at the ASCO 2020 meeting (20). Patients were randomized between ipilimumab+nivolumab or nivolumab monotherapy, and the UPS cohort also received concomitant RTH. The primary end-point was pathologic response, defined as the percentage of hyalinization. Pathologic response to nivolumab+ipilimumab was 95% in the UPS cohort, compared with 22.5% in the DDLPS cohort. 33% of patients had > 85% of hyalinization in the specimen. Toxicity was significant and complicated the perioperative course, with 6 of 23 patients (24%) developing grade III colitis (20).

[0372] The low efficacy of immunotherapy in advanced sarcomas can be associated, among others, with immunosuppressive microenvironment and low expression of PD-L1 . It is estimated that only 10-15% of sarcomas have detectable PD-L1 expression (> 1 %) (21 , 22). Moreover, most sarcomas are considered as “cold tumors” with low infiltration of immune cells. Petitprez et al., based on the transcriptome profile, has distinguished five sarcoma immune classes (23). Classes A and B, characterized by the lowest expression of gene signatures related to immune cells (“cold-tumors”) accounted for over 50% of all sarcomas. Classes D and E, described as immune-high (“hot tumors”), accounted for approximately 30% of cases. Sarcoma immune classes correlated also with patients’ survival and response to pembrolizumab, with best outcomes observed in class E. Different strategies could be utilized to overcome this immunosuppressive microenvironment and turn “cold tumors" into “hot tumors”.

[0373] In the cross-talk between immune system and cancer cells, PD-1 antagonists aim to overcome T cell downregulation while the capacity of immune cells to recognize tumor cells and the priming of an efficient effector response can be increased by an APC activator. In patients eligible for anti-PD-1 therapy, the addition of an APC activator such as efti may help to activate cellular immune response mechanisms to optimise tumor cell recognition and killing, and thus may lead to synergistic effects and a higher frequency of durable disease responses. Moreover, preclinical studies proved that radiotherapy increased the release of tumor-associated antigens, which facilitated cross-presentation of tumor-associated antigens by dendritic cells and the priming of antigen-specific T lymphocytes. Additionally, irradiation enhanced the homing of the antigen-specific T cells to tumor tissues via the increased release of various cytokines. Moreover, local radiotherapy combined with anti-PD- 1 antibody or adoptive T-cell therapy showed a synergistic effect on tumor growth inhibition in vivo (24).

[0374] Based on this data, we hypothesize that combined treatment with radiotherapy, an anti-PD- 1 antibody and an APC activator can have a synergistic effect in patients with localized soft tissue sarcoma. Considering the high heterogeneity of STS, studying subtypes with the highest sensitivity to immunotherapy seems to be the most reasonable approach. The neoadjuvant approach provides a unique opportunity to derive biological information related to tumor response. It enables identifying patients with the pathological response, which can serve as a surrogate marker for improved clinical outcomes. Moreover, longitudinally collected biospecimens can help to understand mechanisms of treatment response and resistance.

[0375] 2.6 Risk-benefit assessment

[0376] Patients included in the study have relatively high risk (30-50% depending on the subtype) of disease recurrence or development of distant metastases. In case of recurrence, prognosis is very unfavourable with less than 50% of patients surviving over 2 years, while all patients ultimately die of the disease. New therapy options are awaited eagerly, both in localized and advanced settings. Improvement in treatment of localized disease can significantly reduce risk of recurrence and result in more patients who are cured from the disease. If neoadjuvant treatment is effective patients gain not only prolonged survival but also high level of quality of life. The main risk of neoadjuvant trials is risk of delay of curative surgery. In neoadjuvant treatment of sarcomas a perioperative radiotherapy lasting 5 weeks followed by 4-6 weeks rest period before surgery is a standard of care. A treatment schedule proposed in the current study will be administered concurrently with radiotherapy and during the break before the surgery, thus will not lead to delay in surgery. Pembrolizumab and eftilagimod alpha have been previously evaluated in patients with other tumor types in multiple clinical trials that proved that this combination is safe, even during longer treatment than proposed in the current study. The toxicity profile for both agents is known and appropriate mitigation strategies has been implemented in the study. Additionally, other studies with immunotherapy in the neoadjuvant setting in other cancers, proved that treatment is feasible and responses are observed, leading to improved long-term outcomes. Overall, the potential benefits of administering pembrolizumab and eftilagimod alpha along with radiotherapy in neoadjuvant treatment outweigh the risks given the poor prognosis of this subject population. 3. and outcome measures of the

[0377] 3.1 Study objectives

[0378] To assess anti-tumor efficacy of pembrolizumab in combination with eftilagimod alpha and radiotherapy in neoadjuvant treatment of patients with resectable soft tissue sarcomas by evaluation of pathologic response at time of definitive surgical treatment.

[0379] • To evaluate pathologic response to neoadjuvant therapy according to the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group (EORTC-STBSG) criteria.

[0380] • To evaluate the best response according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1 ).

[0381] • To evaluate disease-free survival (DFS), local recurrence-free survival (LRFS), distant metastasis-free survival (DMFS) and overall survival (OS).

[0382] • To evaluate safety and tolerability of pembrolizumab + eftilagimod alpha.

[0383] • To assess Health Related Quality of Life (HRQoL) using the European Organisation for Research and Treatment of Cancer Core Quality of Life Questionnaire (EORTC QLQ-C30) and The Functional Assessment of Cancer Therapy - General (FACT-G) questionnaire.

[0384] To explore potential biomarkers associated with clinical efficacy (pathological response, ORR, DFS and OS) by analyzing biomarker measures within the tumor and in the blood.

[0385] 3.2 Study endpoints

[0386] The primary endpoint of the study is the percentage of fibrosis and hyalinization found in the surgical specimen after preoperative treatment • Incidence of adverse events (graded according to the Common Toxicity Criteria for the Adverse Events, CTCAE, version 5.0)

[0387] • Number of patients completing neoadjuvant treatment and having a curative surgery according to the protocol

[0388] • Disease-free survival time (DFS)

[0389] • Local recurrence- free survival (LRFS)

[0390] • Distant metastasis-free survival (DMFS)

[0391] • Overall survival (OS)

[0392] • 1-year and 2-year overall survival (OS) rate

[0393] • 1-year and 2-year disease-free survival (DFS) rate

[0394] • Overall response rate (ORR) by RECIST 1.1 criteria

[0395] • Pathologic response according EORTC-STBSG criteria

[0396] • Quality of life

[0397] 3.2.3 Explorative endpoints

[0398] • Relationship between PDL-1 expression and measures of efficacy, including pathological response, DFS, ORR, and OS

[0399] • Relationship between biomarkers in blood / tissue and efficacy, including pathological response, DFS, ORR, and OS

[0400] 3.3 Outcomes measures

[0401] 3.3.1 Pathologic response

[0402] Pathologic response is defined as percentage of hyalinization or fibrosis in the surgical specimen.

[0403] 3.3.2 Disease-free survival

[0404] Disease-free survival time (DFS) will be computed from the date of curative surgery to the date of disease recurrence defined as clinically diagnosed or biopsy-confirmed recurrent sarcoma at a site of the primary tumor, development of nodal metastasis, locoregional recurrence or distant metastases confirmed by imaging or clinical examination, or death without documented recurrence. Patients still alive and without signs of disease recurrence at the analysis cut-off date are censored at the last date known to be alive. 1-year and 2- year DFS rates will be calculated as a proportion of patients that survive more than 1 and 2 years from the curative surgery without signs of disease recurrence. 3.3.3 Overall survival

[0405] Overall survival (OS) will be computed from the date of curative surgery to the date of death from any cause. Patients still alive at the analysis cut-off date are censored at the last date known to be alive. 1-year and 2-year OS rates will be calculated as a proportion of patients that survive more than 1 and 2 years from the curative surgery.

[0406] 3.3.4 Local recurrence-free survival

[0407] Local recurrence-free survival (LRFS) will be computed from the date of curative surgery to the date of local recurrence confirmed by imaging or clinical examination. Patients still alive and without signs of disease recurrence at the analysis cut-off date are censored at the last date known to be alive.

[0408] 3.3.5 Distant metastasis-free survival

[0409] Distant metastasis-free survival (DMFS) will be computed from the date of curative surgery to the date of diagnosis of distant metastases confirmed by imaging or clinical examination. Patients still alive and without signs of distant metastases at the analysis cut-off date are censored at the last date known to be alive.

[0410] 3.3.6 Best Response according to RECIST 1.1

[0411] All patients included in the study must be assessed for their best response to treatment according to RECIST 1.1 , from the start of study treatment until the end of neoadjuvant treatment. Each patient will be assigned one of the following categories: complete response (CR), partial response (PR), stable disease (SD), progressive disease (PD), early death (ED) or not evaluable (NE).

[0412] Response assessments performed after the surgery will not be taken into consideration for the best response designation. In this study, confirmation for response is NOT reguired.

[0413] All patients who have received at least one dose of therapy and have their disease reevaluated will be considered evaluable for response. Patients’ response will be classified as "not evaluable" if insufficient data were collected to allow evaluation per these criteria.

[0414] 3.3.7 Pathologic response to neoadjuvant therapy according to the EORTC-STBSG criteria Pathologic response to neoadjuvant therapy will be assessed according to the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group (EORTC-STBSG) criteria.

[0415] 3.3.8 Completion of curative treatment

[0416] Completion of curative treatment will be assessed as percentage of patients who completed neoadjuvant treatment and underwent a curative surgery according to the protocol.

[0417] 4. Trial design

[0418] 4.1 Trial design

[0419] This is a phase II single-arm single-stage study evaluating efficacy and safety of pembrolizumab in combination with an APC activator eftilagimod alpha (efti) and radiotherapy in neoadjuvant treatment of patients with soft tissue sarcomas. This study will determine the pathologic response rate (defined as percentage of tumor hyalinization / fibrosis) to the combination treatment. The study will also provide opportunity to investigate blood and based factors that may predict response to anti-PD1 antibody in combination with a soluble LAG-3 protein and radiotherapy.

[0420] 4.2 Trial Schema

[0421] The trial schema is shown in Figure 6.

[0422] 4.3 End-of-study definition

[0423] The end of the study is defined as the date of the last visit of the last participant in the study.

[0424] A participant is considered to have completed the study if the participant has completed all periods of the study including the last visit.

[0425] 5. Patient selection criteria

[0426] 5.1 Inclusion criteria

[0427] All of the following criteria have to be met for including the patient in the study:

[0428] 1. Willing and able to provide written informed consent for the trial;

[0429] 2. Be > 18 years of age on day of signing informed consent; 3. Primary or locally recurrent deep-seated extremities, girdles and / or superficial trunk (thoracic or abdominal wall) soft tissue sarcoma;

[0430] 4. Grade 2 or 3 tumors according FNCLCC;

[0431] 5. Size of the primary tumor >5 cm at instrumental staging (CT, MRI), or locally recurrent of any size;

[0432] 6. Measurable disease based on RECIST 1.1 ;

[0433] 7. Non-metastatic disease;

[0434] 8. Disease determined to be surgically resectable and candidates for upfront surgery as agreed by a multidisciplinary consensus (Surgical Oncology, Medical Oncology, Radiation Oncology) after presentation at sarcoma multidisciplinary tumor board. Resectable tumors are defined as having no significant vascular, neural, or bone involvement. Only cases where a complete surgical resection can be safely achieved are defined as resectable;

[0435] 9. Performance status of 0 to 1 on the ECOG Performance Scale;

[0436] 10. Expected life expectancy > 6 months;

[0437] 11. At least one tumor amenable to serial biopsy in a clinic during the neoadjuvant phase of the protocol. Patients must be willing to provide tumor samples at the specified time points;

[0438] 12. No other malignancies, except adequately treated and with a cancer-related lifeexpectancy of more than 5 years;

[0439] 13. No previous systemic treatment for sarcoma;

[0440] 14. No immunosuppressive medications within 1 month prior to study inclusion (steroids equivalent to prednisolone < 10 mg are allowed);

[0441] 15. Adequate organ function defined by the following laboratory tests results, obtained within 14 days prior to initiation of study treatment:

[0442] - white blood cell (WBC) count > 3 x 109 / L with absolute neutrophil count (ANC) > 1.5 * 109 / L;

[0443] - lymphocyte count > 0.5 * 109 / L;

[0444] - platelet count > 100 * 109 / L;

[0445] - hemoglobin > 9 g / dL (patients may be transfused);

[0446] - total bilirubin level < 1.5 * the upper limit of normal range (ULN);

[0447] - an aspartate aminotransferase (AST) level < 2.5 * ULN, and an alanine aminotransferase (ALT) level < 2.5 * ULN;

[0448] - serum creatinine < 1.5 x ULN or creatinine clearance > 50 ml / min on the basis of the Cockroft-Gault glomerular filtration rate estimation: (140-age) x (weight in kg) x (0.85 if female) / 72x(serum creatinine in mg / dl);

[0449] 16. No clinically significant cardiovascular disease: cerebral vascular accident / stroke < 6 months prior to enrollment, myocardial infarction < 6 months prior to enrollment, uncontrolled hypertension (systolic blood pressure >150 mmHg and / or diastolic blood pressure > 100 mmHg), unstable angina, congestive heart failure (> 2 New York Heart Association Classification), ongoing cardiac dysrhythmias of NCI CTCAE version 5.0 Grade > 2, atrial fibrillation > grade 2 not controlled by a pacemaker. All other significant diseases (e.g., inflammatory bowel disease), which, in the opinion of the investigator, might impair the subject’s tolerance of trial treatment;

[0450] 17. Absence of any psychological, familial, sociological or geographical condition potentially hampering compliance with the study protocol and follow-up schedule - those conditions should be discussed with the patient before registration in the trial;

[0451] 18. Effective contraception for both male and female subjects if the risk of conception exists. (Note: The effects of the study drugs on the developing human fetus are unknown. Thus, women of childbearing potential and men must agree to use highly effective contraception from the day of trial medication initiation (or 14 days prior to the initiation of trial medication for oral contraception) throughout the trial period up to 6 months after the last dose of the last trial treatment.

[0452] 5.2 Exclusion criteria

[0453] Patients will be excluded from the study if they meet any of the following criteria:

[0454] 1 . Ewing sarcoma, Alveolar and embryonal rhabdomyosarcoma;

[0455] 2. Distant metastases;

[0456] 3. Previous treatment with eftilagimod alpha, anti-PD-1 or anti-PD-L1 ;

[0457] 4. Prior radiotherapy to tumor-involved sites;

[0458] 5. Concurrent anticancer treatment within 14 days before the start of trial treatment

[0459] (e.g., cytoreductive therapy, radiotherapy, immune therapy, or cytokine therapy except for erythropoietin); use of hormonal agents within 7 days before the start of trial treatment; or use of any investigational drug within 28 days before the start of trial treatment;

[0460] 6. Prior high-dose chemotherapy requiring haematopoietic stem cell rescue;

[0461] 7. Major surgical procedure within 28 days prior to the first study treatment (excluding prior diagnostic biopsy), or anticipation of the need for major surgery during the course of the study treatment (excluding surgery as a part of the trial), or minor surgical procedures, within 24 hours prior to the first study treatment;

[0462] 8. Subjects with active, known or suspected autoimmune disease or inflammatory bowel disease, which, in the opinion of the investigator, might impair the subject’s tolerance of trial treatment. Subjects with vitiligo, type I diabetes mellitus, residual hypothyroidism due to autoimmune condition only requiring replacement therapy, psoriasis not requiring systemic treatment, or conditions not expected to recur in the absence of an external trigger are permitted to enroll; Subjects with a condition requiring systemic treatment with either corticosteroids (> 10 mg daily prednisone equivalents) or other immunosuppressive medications within 14 days of study drug administration except for adrenal replacement steroid. The use of inhaled corticosteroids for chronic respiratory diseases is allowed; Subject who have had an allogenic tissue / solid organ transplant; Subject who has a hypersensitivity to efti and / or any of its excipients; History of idiopathic pulmonary fibrosis (including pneumonitis), drug-induced pneumonitis, organizing pneumonia (i.e., bronchiolitis obliterans, cryptogenic organizing pneumonia), or evidence of active pneumonitis on screening chest CT scan; Patients not receiving anticoagulant medication who have an International Normalized Ratio (INR) > 1.5 or an activated partial thromboplastin time (aPTT) > 1.5 x ULN within 7 days prior to first study treatment. Note: Patients receiving full dose oral or parenteral anticoagulants may be included in the study as long as anticoagulant dosing has been stable for at least 2 weeks prior to study entry and the appropriate coagulation monitoring tests are within local therapeutic limits; Significant acute or chronic infections including, among others: positive testing for human immunodeficiency virus (HIV) or known acquired immunodeficiency syndrome (AIDS); Active hepatitis B virus (HBV) infection (chronic or acute), defined as having a positive hepatitis B surface antigen (HBsAg) test at screening. Patients with past or resolved HBV infection, defined as having a negative HBsAg test and a positive total hepatitis B core antibody (HBcAb) test at screening, are eligible for the study; Active hepatitis C virus (HCV) infection, defined as having a positive HCV antibody test and positive HCV RNA test at screening; Active tuberculosis; Severe infection within 2 weeks prior initiation of study treatment, including, but not limited to, hospitalization for complications of infection, bacteriemia or severe pneumonia; T reatment with a live, attenuated vaccine within 4 weeks prior to initiation of study treatment, or anticipation of need for such a vaccine during the course of the study; 20. Known alcohol or drug abuse;

[0463] 21. Pregnant, breastfeeding or expecting to conceive or father children within the projected duration of the trial starting with screening visit. Woman of childbearing potential must have a negative serum pregnancy test result within 14 days prior to initiation of study treatment.

[0464] 6. Study interventions and concomitant therapy

[0465] Systemic therapy with pembrolizumab and eftilagimod alpha and radiotherapy are administered concurrently. Systemic treatment lasts for 9 weeks (study week 1-9). If treatment is delayed / postponed, the last dose cannot be given later than week 9. Radiation therapy lasts for 5 weeks (5 days per week) in weeks 2-6. Surgery takes place 5-6 weeks after completion of radiation therapy (week 11-12). The surgery is considered to be performed on time if is performed by the end of week 12 counting from first dose of study treatment. The period of planned hospital stay after the surgery is about 3-5 days, depending on the extent of surgery and the course of the recovery period. Any adjuvant treatment (chemotherapy, immunotherapy, radiation therapy) after surgical treatment is not allowed. Patients will be then followed up regularly for a period of 24 months.

[0466] 6.1 Systemic therapy

[0467] Eftilagimod alpha (efti) will be administered subcutaneously at a dose of 30 mg every 2 weeks (14 days) beginning in week 1 of the study (weeks 1 , 3, 5, 7, and 9) for a total of 5 administrations. If treatment is delayed / postponed, the last dose cannot be given later than week 9.

[0468] Efti will be administered as subcutaneous injection (single anatomical site) in the anterior face of the thigh (it is recommended to rotate the injection site every injection). Alternating injections in deltoid regions or lower abdominal quadrants can be performed in situations when injections in the thigh are not feasible (e.g. due to tumor or in patients after amputation). The injection should be performed slowly to avoid discomfort at the site of injection. Eftilagimod alpha is to be given always > 30 minutes after pembrolizumab infusion is finished if both drugs are administered the same day.

[0469] In case a systemic reaction occurs after efti administration that raises the possibility of an allergic reaction, further blood samples are to be collected: serum tryptase, total IgE, C3 and C4 levels are to be assessed in the local laboratory and a serum sample is to be stored for ADA analysis from blood samples collected within 3 hours from the event onset; further to that another sample is to be collected at least 24 hours after resolution of the symptoms and to be sent to local laboratory for serum tryptase assessment.

[0470] Pembrolizumab will be administered at a dose of 200 mg by intravenous infusion over 30 minutes every 3 weeks (21 days) starting in Week 1 of the study (Weeks 1 , 4, and 7) - 3 administrations in total. If treatment is delayed / postponed, the last dose cannot be given later than week 9.

[0471] Trial treatment may be administered up to 3 days before or after the scheduled day due to administrative reasons. Subsequent dosing should be based on the actual date of administration of the previous dose of drug. Every effort should be made to adhere to the protocol treatment schedule of administration. In extenuating circumstances in which the patient cannot make the dosing schedule within the 3-day window, the decision regarding the continuation of the treatment should be made at the discretion of the Principal Investigator. All trial treatments will be administered on an outpatient basis. All doses of trial treatment must be administered by the Investigator or designated trial personnel. The exact times of dosing must be recorded in the source and entered into the electronic CRF.

[0472] Subjects must stay for at least 30 minutes on site under close supervision by staff after end of each efti or pembrolizumab administration, whatever was last, to ensure that symptoms of a potential systemic reaction are promptly recognized and managed. Subjects must be made aware that they are required to report any symptom they may perceive during the post-dosing observation period.

[0473] The trial treatment must be administered in a clinical setting where emergency resuscitative equipment and personnel trained in the management of anaphylaxis are immediately available to treat systemic reactions under the direct supervision of a physician.

[0474] 6.2 Radiation therapy

[0475] Radiation therapy will be given in weeks 2-6.

[0476] Radiation therapy technique:

[0477] • Dose and fractionation: The total planned dose is 50 Gy administered in conventional fractionation of 2 Gy / day for 5 days a week from week 2 to week 6.

[0478] • Energy: High-energy photons of 6-15 MV

[0479] • Target Volume: The target volume will be determined by computed tomography (CT) with eventual fusion with magnetic resonance (MR) images. o GTV (Gross Tumor Volume) includes the primary tumor contoured based on TK and eventually T1 MR. o CTV (Clinical Target Volume) will be created by expanding GTV with adequate margins with additional coverage of tumor-related edema visible on T2 MR images. CTV constitutes a longitudinally margins of 4 cm and radial margins of 1.5-2 cm. In superficial lesions it seems reasonable to add 4 cm margin in each direction. Natural anatomic barriers that limit potential spread of tumor cells (fascia, bone, skin, etc.), should be considered. Bone should be included in the volume if it is infiltrated. PTV (Planned Target Volume) includes the margin automatically added to the CTV volume, depending on the location it is 0.3-1 cm.

[0480] • Isodoses distribution: The isodoses distribution within the target volume should be assessed with V95 PTV (volume covered by an isodose of 95% of the prescribed dose) > 95% or V90 PTV > 95%. Patient positioning is personalized and requires the use of customized masks, mattresses, or other immobilization devices to optimize and repeat treatment and will allow for the most appropriate irradiation techniques. Radiotherapy technique would be 3D conformal radiotherapy or IMRT.

[0481] 6.3 Research biopsy and surgery

[0482] Research biopsy must be large core needle biopsy (Tru-cut). The biopsy track must go directly to the tumor, through the muscle fibers with minimum use of retractors. The biopsy track must contaminate only the anatomical compartment in which the tumor is situated.

[0483] Surgical resection must be performed in week 11 or 12 of the study. Each patient must be evaluated by multidisciplinary tumor board after completion the neoadjuvant therapy and before surgery to confirm tumor resectability.

[0484] Surgery has to be performed according to general rules for sarcoma resection. The incision must always be made along the major axis of the tumor-bearing anatomical compartment and must include the biopsy track en bloc. Resection of adjusted / infiltrated tissues such as muscles or nerves is allowed to achieve adequate resection margins.

[0485] The quality of the surgery will be defined by its worst margin and will be classically classified as follows:

[0486] • Intralesional: when macroscopic tumor residue is left in situ.

[0487] Marginal: when the tumor surface emerges macroscopically at the resection surface, or when microscopic tumor extension is present at the margin of resection, but without evidence of macroscopic residual disease. It will be important for the pathologist to examine the specimen with the surgeon so that correct orientation is ensured for accurate evaluation of the margins. The surgeon must help the pathologist to identify the most critical resection margin and likewise must ensure that points where the tumor emerges only due to muscle retraction following surgical removal are not identified as critical margins.

[0488] • Wide: when the tumor is covered at every point by healthy tissue (the width of the margin varies according to the type of tissue: 1 cm if muscle or fatty tissue, 1 mm or less, if anatomical barrier (such as muscle fascia, adventitia, epineurium, periostium).

[0489] • Radical: when the tumor is removed en bloc with the entire muscular compartment and covered by intact deep fascia. If infiltration of the deep fascia is detected, surgery must be extended to include demolition of the structures involved (adjacent muscle compartment, bone, blood vessel or nerve).

[0490] • Contaminated: when accidental rupture of the tumor pseudocapsule with spillage of material into the operating field occurs, and also when the pseudocapsule has simply emerged at the margin of resection. In these cases spillage of material must be controlled by all means, and then the operating field must be rapidly washing and the resection margins, if possible, widened.

[0491] Only wide or radical resections are defined as adequate. Marginal resections in sites where wider resection is not possible may also be acceptable, provided they are always preceded by radiotherapy. In particular, irrespective of the site: surgery will be largely planned on the basis of imaging findings (CT, MRI) and the least favorable intraoperative situations will be hypothesized; a wide cutaneous incision will be made along traditional lines, including en bloc the scar and the holes of previous biopsies or surgery. Once the skin-fat flaps have been prepared the tumor will be isolated within the tumor-bearing structure, with prompt recognition and careful dissection of the main vascular structures and motor nerves (femoral, sciatic, sciatic-popliteal, external / internal, median, ulnar and radial). These structures must not show tumor infiltration. Care must be taken to avoid contamination of the surgical field, which can also occur if the tumor is allowed to emerge on the surface of resection. In any case surgery in which minimal contamination has occurred will be considered acceptable, but complementary radiation therapy will have to be planned in any case. Once the malignancy has been isolated, it must be removed en bloc with the surrounding soft tissue, covered at every point by at least one centimeter of healthy tissue or less if constituted by an anatomical barrier (see above). Compartmental operations will be performed only if made necessary by the site and dimensions of the tumor. If the lesion is near structures such as the vascular-nervous fascia or bone, it must be cautiously prepared by also removing the fascia covering said structures (muscle fascia, vascular adventitia, epineurium or periostium). If these barriers are also found to be infiltrated, the underlying structures should be resected en bloc with the tumor, assessing the possibility of performing vascular, neurological or bone reconstruction as an alternative to demolitive procedures. Drainage can be introduced if necessary.

[0492] Pathology specimen should be placed in individual container and fixed with 10% buffered formalin and transported to local pathology department within 6 hours. Processing should be carried out according to local pathology guidelines. Additional tumor samples can be obtained during the surgery and immediately snap frozen in liquid nitrogen.

[0493] Postoperative care will be conducted according to general surgical standard.

[0494] 7. Clinical evaluation, laboratory tests, follow-up

[0495] 7.1 Summary of trial procedures

[0496] The screening period will be up to 28 days prior to first study drug administration.

[0497] Blood and urine analyses will be performed at screening to assess eligibility for the trial, during the treatment (before drug administration and before surgery) and during follow-up period. Subjects will be assessed with computed tomography (CT) and magnetic resonance imaging (MRI) at screening, after completion of neoadjuvant therapy and during the posttreatment follow-up period.

[0498] Baseline biopsies and blood sample for genomic and immunologic analyses will be obtained from each patient. Second obligatory biopsy will be obtained after completion of neoadjuvant therapy, before the surgery. Patients may voluntarily agree for additional biopsy at the time of disease recurrence, if applicable.

[0499] Quality-of-life data will be collected during the study with appropriate questionnaires.

[0500] All trials procedures are summarized in Table 1. Table 1. Summary table

[0501] a) Pembrolizumab 200 mg iv Q3W b) Eftilagimod alpha 30 mg sc Q2W c) Radiation therapy 50 Gy in 25 daily fractions of 2 Gy d) Surgery 5-6 weeks after completion of radiation therapy e) Vital signs include measurement of heart rate, respiratory rate, and systolic and diastolic blood pressure while the patient is in a seated position, as well as body temperature. Blood pressure and heart rate measurements will be recorded after a 5-minute rest while the patient is in a seated position. Vital signs should be measured within 60 minutes prior to each pembrolizumab and eftilagimod alpha administration and 30 minutes (+ / - 10 min) after each pembrolizumab and eftilagimod alpha administration f) Hematology: WBC count, RBC count, haemoglobin, haematocrit, platelet count, differential count (neutrophils, eosinophils, basophils, monocytes, lymphocytes) g) Chemistry panel (serum or plasma): sodium, potassium, magnesium, urea, creatinine, albumin, total protein, phosphorus, calcium, total bilirubin, uric acid, creatine kinase, ALP, ALT, AST, LDH, glucose (non-fasting). Fasting blood glucose (minimum of 8-hour fast) at screening only h) TSH, FT3, FT4

[0502] i) Urinalysis: Macroscopic appearance; Laboratory analysis: Protein, creatinine, erythrocytes or haemoglobin, leukocytes, nitrite; Microscopic analysis should be performed if the appearance is turbid, or if protein, leukocytes, erythrocytes, or nitrite are out of normal range j) Women of childbearing potential k) 12 lead ECG must be performed at screening, prior to each pembrolizumab infusion and before surgery l) Scans must be performed within 21 days prior to initiation of study treatment, after completion of neoadjuvant treatment and every 3 months (+ / -2 weeks) during follow-up. CT Chest, abdomen and pelvis with IV contrast is the preferred imaging modality. In case of any contraindications (medical or regulatory), it is allowed to perform a non-contrast CT thorax m) MRI Scans must be performed within 21 days prior to initiation of study treatment, before surgery after completion of neoadjuvant treatment and every 3 months (+ / -2 weeks) in the first 12 months of follow-up, then every 6 months (+ / -2 weeks) (F-up visits Y1-4, Y6, Y8) n) Each assessment should be completed prior to the first dose of study therapy on screening visit or Week 1 , at Week 7, before surgery and at 3-, 6-, and 12-month follow-up o) Research biopsy is mandatory p) Mandatory: FFPE tumor tissue from the surgical specimen: 1 slide from each block for histology review and 2 blocks for translational research and snap-frozen in liquid nitrogen tumor sample (at least 3 cores or incisional sample with minimal size of 5x5x5 mm) q) Blood sample for correlative studies must be obtained at screening, before start of radiotherapy (week 2), after completion of radiation therapy (week 7), 24-48h before surgery and 24h after surgery and at 6 months after surgery r) Optional biopsy at the time of disease recurrence s) X1 and X2 visits are visits for postoperative evaluation. X visits occur as follows: X1 = 14 days (+1-7 days) from the date of surgery, X2 = 28 days (+ / - 7 days) from the date of surgery. Y (Y1-Y8) visits should be performed every 3 months from the date of surgery (+ / - 7 days)

[0503] 7.2 Staging

[0504] 7.2.1 Staging procedures before study entry

[0505] 1 . Physical examination with measurement in cm of at least the largest tumor diameter and definition of the site, anatomical compartment, margins, mobility, consistency, relationship with vessel, nerve and bone structures, presence of satellite lymph node involvement.

[0506] 2. Conventional and dynamic contrast enhanced MRI (contrast enhanced CT only if MRI is contraindicated) of local lesion with evaluation of tumor extension and necrosis.

[0507] 3. Chest, abdomen and pelvis CT scan with contrast.

[0508] 4. Total body bone scintigraphy in case of clinical suspicion.

[0509] 7.2.2 Staging procedures before surgery

[0510] 1 . Physical examination with measurement in cm of at least the largest tumor diameter and definition of the site, anatomical compartment, margins, mobility, consistency, relationship with vessel, nerve and bone structures, presence of satellite lymph node involvement.

[0511] 2. Conventional and dynamic contrast enhanced MRI (contrast enhanced CT only if MRI is contraindicated) of local lesion with evaluation of tumor extension and necrosis.

[0512] 3. Chest, abdomen and pelvis CT scan with contrast.

[0513] 4. Total body bone scintigraphy in case of clinical suspicion.

[0514] 7.2.3 Staging procedures for disease recurrence during follow-up period

[0515] 1 . Physical examination (every 3 months).

[0516] 2. Conventional and dynamic contrast enhanced MRI (contrast enhanced CT only if MRI is contraindicated) for early detection of local relapse should be performed every 3 months for the first 12 months, then every 6 months.

[0517] 3. Chest, abdomen and pelvis CT scan with contrast for early detection of distant relapse should be performed every 3 months.

[0518] 4. Total body bone scintigraphy in case of clinical suspicion.

[0519] Subjects will be followed for drug-related toxicities until these toxicities resolve, return to baseline or are deemed irreversible. All adverse events will be documented for a minimum of 100 days after surgical treatment. After completion of the first two follow-up visits, subjects will be followed every 3 months for survival. 7.3 Laboratory Procedures / Assessments

[0520] Laboratory tests for screening should be performed within 14 days prior to the first dose of treatment. After Week 1 treatment, pre-dose laboratory procedures can be conducted up to 48 hours prior to dosing. Results must be reviewed by the investigator or qualified designee and found to be acceptable prior to each dose of trial treatment. Laboratory tests for hematology, chemistry, urinalysis, and others are specified in Table 2.

[0521] Table 2. Laboratory tests

[0522] 7.4 Tumor Tissue Collection and Correlative Studies Blood Sampling

[0523] Changes in the immune microenvironment of a tumor will be assessed by analysis of immune infiltrates and expression of markers such as LAG-3, PD-L1 , TIM-3, and comparison of pretreatment and surgical specimens. Multiomic profiling, including whole-exome sequencing and transcriptome sequencing, will be conducted in pre-treatment and surgical samples. Peripheral blood samples will be obtained for T-cells profiling and analysis of cytokine profile in different time points.

[0524] Biopsies for research purposes will be performed at baseline (before first dose of study treatment) and immediately before surgery in all patients enrolled in this study. Up to 6 cores from each tumor biopsy site should be taken where feasible and safe to do so. An optional biopsy will be offered to all patients at the time of progression. Biopsy procedures will otherwise be performed as per institution guidelines. Archival tissue sample will be collected if available.

[0525] Cores will be obtained with 18-gauge needles where appropriate and be of at least 1 cm in length. Cores will be representative of tumor, and targeted to the de-differentiated component in the case of de-differentiated liposarcoma (DD-LPS). The goal will be to extract 6 cores, with a minimum of one formalin-fixed, for later paraffin embedding (FFPE) and two flash frozen in liquid nitrogen and transported in dry ice, with the priority for FFPE sample. If any extra cores are obtained they will be flash frozen with liquid nitrogen. Biopsy procedures will otherwise be performed as per institutional guidelines.

[0526] Samples will be labeled using an adherent, liquid nitrogen proof label with the following information:

[0527] 1 . Procurement date

[0528] 2. Study IRB number

[0529] 3. Study patient number

[0530] 4. Time point (baseline or on-treatment)

[0531] 5. Biopsy site Each FFPE sample is to be placed in a pathology sample container pre-filled with formalin and delivered to the central pathology lab. Each flash frozen sample is to be placed within an individual cryovial, labeled, and the vial immediately placed in liquid nitrogen for 2 minutes or longer to snap-freeze the tissue. These vials can be transported in either liquid nitrogen or in dry ice.

[0532] At each routine research blood collection time point the additional samples for correlative studies will be collected a maximum of 40 ml of peripheral venous blood tuned to with heparin’s citrate, sodium citrate or RNA stabilizer.

[0533] Samples will be labeled with the following information:

[0534] 1 . Procurement date

[0535] 2. Study IRB number

[0536] 3. Study patient number

[0537] 4. Time point (baseline or week- X)

[0538] Additionally, 8-10 ml of whole blood for germline DNA testing will be collected within 21 days prior initiation of treatment.

[0539] Patients may voluntary agree to have their blood sample biobanked for future research outside this clinical trial. Additional consent needs to be signed for this purpose.

[0540] 7.5 Quality of life evaluation

[0541] Quality of Life will be assessed using European Organization for Research and Treatment of Cancer Quality Of Life Questionnaire-core 30 (EORTC QLQ-C30) and the Functional Assessment of Cancer Therapy - General (FACT-G) questionnaires, to be completed prior to the first dose of study therapy (on screening visit or Week 1 ), at Week 7, before surgery and at 3-, 6-, and 12-month follow-up post-surgery.

[0542] The EORTC QLQ-C30 is a 30-item instrument that comprises 6 functional scales (physical functioning, cognitive functioning, emotional functioning, social functioning and global quality of life) as well as nine symptom scales (fatigue, pain, nausea / vomiting, dyspnea, insomnia, appetite loss, constipation, diarrhea, and financial difficulties). Except for the overall health status and global quality of life items, responses for all items are 4-point categorical scales ranging from 0 (Not at all) to 4 (Very much). The overall health status / quality of life responses are 7-point Likert scales. The FACT-G questionnaire will be used to assess the effects of disease symptoms on functioning and well-being. The FACT-G includes the 27-item FACT General (FACT-G) to assess physical well-being (PWB; seven items), social / family well-being (SWB; seven items), emotional well-being (EWB; six items), and functional well-being (FWB; seven items). Each FACT-G item is rated on a 5-point scale ranging from 0 (not at all) to 4 (very much).

[0543] 8. Assessment of efficacy

[0544] 8.1 Radiological response evaluation

[0545] Tumor assessments and determination of extent of disease will be performed at screening, just before surgery and in follow-up period, with both conventional and dynamic MRI. CT will be used for tumor imaging only if MRI is contraindicated. Chest, abdomen and pelvis CT with IV contrast will be performed at screening, before surgery and in follow-up period. In case of any contraindications (medical or regulatory), it is allowed to perform a non-contrast CT. All exams will be performed according to standard of care and local protocols.

[0546] Tumor response will be evaluated at each radiological assessment according to Response Evaluation Criteria in Solid Tumors (revised RECIST version 1.1 ) as outlined below. RECIST criteria are summarized in Table 3.

[0547] Complete Response (CR): disappearance of all target and non-target lesions and normalization of tumor markers. Pathological lymph nodes must have short axis measures < 10 mm (Note: continue to record the measurement even if < 10 mm and considered CR). Tumor markers must have normalized. Residual lesions (other than nodes < 10 mm) thought to be non-malignant should be further investigated (by cytology or PET scans) before CR can be accepted.

[0548] Partial Response (PR): at least a 30% decrease in the sum of measures (longest diameter for tumor lesions and short axis measure for nodes) of target lesions, taking as reference the baseline sum of diameters.

[0549] Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD taking as reference the smallest sum of diameters on study.

[0550] Progressive Disease (PD): at least a 20% increase in the sum of diameters of measured lesions taking as references the smallest sum of diameters recorded on study (including baseline) and an absolute increase of > 5 mm. Appearance of new lesions will also constitute PD (including lesions in previously unassessed areas). In exceptional circumstances, unequivocal progression of non-target disease may be accepted as evidence of disease progression, where the overall tumor burden has increased sufficiently to merit discontinuation of treatment, for example where the tumor burden appears to have increased by at least 73% in volume (which is the increase in volume when all dimensions of a single lesion increase by 20%).

[0551] Table 3: Integration of target, non-target and new lesions into response assessment:

[0552] 8.2 Pathological response evaluation

[0553] All the surgical specimens should be sampled according to a standardized protocol. The intact surgical specimen should be oriented, in order to assess margins, ideally in the presence of the surgeon. Size of the specimen and tumor mass should be assessed before cutting in 3 dimensions. The neoplasm should be mapped on its largest section taking about a sample per 1 cm. The identification of the section should be performed in collaboration with the radiologist. In addition, all the macroscopically distinct areas (i.e., solid, cystic, hemorrhagic, necrotic) should be described and separately sampled. The result should reflect the average mapping of the whole tumor mass. Margins should be inked and separately sampled. Distance between tumor and free margins should be measured. The number of blocks of neoplasia taken and evaluated must be specified. Unfixed snap frozen samples should be taken and stored. On histology the following characteristics of the post-treated tumor mass should be assessed:

[0554] Percentage of residual viable tumor (RVT)

[0555] Percentage of hyalinization / fibrosis

[0556] Percentages of the other components of the mass: i.e. necrosis, hemorrhage, fibrohistiocytic reaction with haemosiderin, myxoid component, or any other observed micro / macroscopic changes. The presence of a cystic component needs to be addressed separately as it may affect the estimation of tissue response to the treatment.

[0557] Mitotic index

[0558] In the case of round cell I myxoid liposarcoma the following features should be also assessed:

[0559] The percentages of round cell I classic component

[0560] The amount of lipogenic differentiation as expressed by presence of lipoblasts and / or mature adipocytes

[0561] The presence of damage of blood vessels

[0562] The pathologic response will be assessed according to the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group (EORTC- STBSG) recommendations for pathologic examination and reporting and expressed as a percentage of the total tumor area stained with hematoxylin in representative tissue slabs. Response scores are classified into 5 different categories: (A) no stained tumor cells; (B) single stained tumor cells or small clusters (< 1 % of the whole specimen); (C) > 1 % to < 10% stained tumor cells; (D) > 10% to < 50% stained tumor cells; and (E) > 50% stained tumor cells. Pathologic complete response (pCR) and near pCR are defined as no and < 5% stained tumor cells in the postoperative specimen, respectively.

[0563] 9. List of abbreviations

[0564] ADAs Anti-Drug Antibody

[0565] ADR Adverse Drug Reaction

[0566] AE Adverse Event

[0567] Al Doxorubicin and Ifosfamide

[0568] AIDS Acquired Immunodeficiency Syndrome

[0569] ALP Alkaline phosphatase

[0570] ALT Alanine aminotransferase ANC Absolute Neutrophil Count

[0571] APC Antigen-Presenting Cells aPTT Activated Partial Thromboplastin Time

[0572] ASCO American Society of Clinical Oncology

[0573] AST Aspartate aminotransferase

[0574] AUG Area Under the Curve

[0575] Ca Calcium

[0576] CBC Complete Blood Count

[0577] ChT Chemotherapy

[0578] Cl Confidence Interval

[0579] COVID-19 Coronavirus Disease 2019

[0580] CRT Chemoradiotherapy

[0581] CR Complete Response

[0582] CRF Case Report Form

[0583] Cr Creatinine

[0584] CT Computed Tomography

[0585] CTV Clinical Target Volume

[0586] CTCAE Common Terminology Criteria for Adverse Events

[0587] DCR Disease Control Rate

[0588] DDLPS Dedifferentiated Liposarcoma

[0589] DFS Disease-Free Survival

[0590] DKA Diabetic Ketoacidosis

[0591] DMFS Distant Metastasis-Free Survival

[0592] DRESS Drug Reaction with Eosinophilia and Systemic Symptoms

[0593] ECOG Eastern Cooperative Oncology Group eCRF electronic Case Report Form

[0594] EDCs Electronic Data Capture systems efti eftilagimod alpha

[0595] EMA European Medicine Agency

[0596] EORTC European Organization for Research and Treatment of Cancer

[0597] ES Epithelioid Sarcoma

[0598] ESMO European Society for Medical Oncology

[0599] EU European Union

[0600] FACT-Q Functional Assessment of Cancer Therapy - General

[0601] FISH Fluorescence in situ hybridization

[0602] GBS Guillain-Barre Syndrome

[0603] GCP Good Clinical Practice

[0604] GDPR General Data Protection Regulation

[0605] GTV Gross Tumor Volume HBcAb Hepatitis B core antibody

[0606] HBsAg Hepatitis B surface antigen

[0607] HBV Hepatitis B Virus

[0608] HCV Hepatitis C Virus

[0609] HIV Human Immunodeficiency Virus

[0610] Hgb Hemoglobin

[0611] HNSCC Head and Neck Squamous Cell Carcinoma

[0612] ICI Immune checkpoint inhibitors

[0613] ICH International Conference on Harmonization

[0614] IF Immunofluorescence

[0615] IHC Immunohistochemistry irAE Immune Related Adverse Events

[0616] LAG-3 Lymphocyte-Activation Gene 3 iRECIST Immune Response Evaluation Criteria in Solid Tumors

[0617] K Potassium

[0618] HRQoL Health-Related Quality of Life

[0619] IEC Independent Ethic Committee

[0620] IFN-y Interferon-y

[0621] INR International Normalized Ratio

[0622] IRB Institutional Review Board

[0623] LDH Lactate Dehydrogenase

[0624] LLN Lower Limit of Normal

[0625] LRFS Local Recurrence-Free Survival

[0626] LVEF Left Ventricular Ejection Fraction mAb monoclonal antibody

[0627] MBC Metastatic Breast Cancer

[0628] MDSCs Myeloid-Derived Suppressor Cells

[0629] MG Myasthenia Gravis

[0630] MHC Major Histocompatibility Complex

[0631] MMR Measles, Mumps, Rubella

[0632] MRI Magnetic Resonance Imaging

[0633] MRCLPS Myxoid and Round Cell Liposarcoma

[0634] NCI National Cancer Institute

[0635] NCI-CTCAE National Cancer Institute-Common Terminology Criteria for Adverse Events

[0636] NE Not Evaluable

[0637] NIO-PIB Maria Sklodowska-Curie National Research Institute of Oncology in Warsaw

[0638] NK Natural Killer Cells

[0639] NOAEL No Observed Adverse Effect Level

[0640] NSAiDS Non-Steroidal Anti-Inflammatory Drugs NSCLC Non-Small Cell Lung Cancer

[0641] ORR Objective Response Rate

[0642] OS Overall Survival

[0643] PBMCs Peripheral Blood Mononuclear Cells pCR Pathologic Complete Response

[0644] PD Progressive disease

[0645] PD-1 Programmed Cell Death Protein 1

[0646] PD-L1 Programmed Cell Death-Ligand 1

[0647] PD-L2 Programmed Cell Death-Ligand 2

[0648] PFS Progression Free Survival

[0649] PLT Platelets

[0650] PR Partial Response

[0651] PS Performance Status

[0652] PT Prothrombin Time

[0653] Pts Patients

[0654] PTT Partial Thromboplastin Time

[0655] PTV Planned Target Volume

[0656] QLQ-C30 Cancer Core Quality of Life Questionnaire qRT-PCR quantitative Real-Time Polymerase Chain Reaction

[0657] RECIST Response Evaluation Criteria in Solid Tumors

[0658] RT Radiation Therapy

[0659] SADR Serious Adverse Drug Reaction

[0660] SAE Serious Adverse Event

[0661] SD Stable Disease

[0662] SJS Steven-Johnson Syndrome

[0663] SOC Standard of Care

[0664] STBSG Soft Tissue and Bone Sarcoma Group

[0665] STS Soft Tissue Sarcoma

[0666] STS NOS Soft Tissue Sarcoma Not Otherwise Specified

[0667] SUSAR Suspected, Unexpected Serious Adverse Event

[0668] T3 Tri-iodothyronine

[0669] T4 Thyroxine

[0670] TCR T-cell Receptor

[0671] TEN Toxic Epidermal Necrolysis

[0672] TILs Tumor-Infiltrating Lymphocytes

[0673] TNM Classification of Malignant Tumors (tumor / nodes / metastasis)

[0674] TPS Tumor Proportion Score

[0675] TSH Thyroid-Stimulating Hormone

[0676] TTP Time to T umor Progression Tregs Regulatory T cells

[0677] UPS Undifferentiated Pleomorphic Sarcoma

[0678] ULN Upper Limit of Normal

[0679] VEGF Vascular Endothelial Growth Factor

[0680] WBC White Blood Cells

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[0698] 18. Chen JL, Mahoney MR, George S, Antonescu CR, Liebner DA, Tine BAV, et al. A multicenter phase II study of nivolumab + / - ipilimumab for patients with metastatic sarcoma (Alliance A091401 ): Results of expansion cohorts. Journal of Clinical Oncology. 2020;38(15_suppl):11511-.

[0699] 19. D'Angelo SP, Mahoney MR, Van Tine BA, Atkins J, Milhem MM, Jahagirdar BN, et al. Nivolumab with or without ipilimumab treatment for metastatic sarcoma (Alliance A091401 ): two openlabel, non-comparative, randomised, phase 2 trials. The Lancet Oncology. 2018;19(3):416-26.

[0700] 20. Roland CL, Keung EZ-Y, Lazar AJ, Torres KE, Wang W-L, Guadagnolo A, et al. Preliminary results of a phase II study of neoadjuvant checkpoint blockade for surgically resectable undifferentiated pleomorphic sarcoma (UPS) and dedifferentiated liposarcoma (DDLPS). Journal of Clinical Oncology. 2020;38(15_suppl):1 1505-.

[0701] 21. Italiano A, Bellera C, D'Angelo S. PD1 / PD-L1 targeting in advanced soft-tissue sarcomas: a pooled analysis of phase II trials. J Hematol Oncol. 2020;13(1 ):55.

[0702] 22. Dancsok AR, Gao D, Lee AF, Steigen SE, Blay JY, Thomas DM, et al. Tumor-associated macrophages and macrophage-related immune checkpoint expression in sarcomas. Oncoimmunology. 2020;9(1 ):1747340.

[0703] 23. Petitprez F, de Reynies A, Keung EZ, Chen TW, Sun CM, Calderaro J, et al. B cells are associated with survival and immunotherapy response in sarcoma. Nature. 2020;577(7791 ):556-60.

[0704] 24. Lai JZ, Zhu YY, Ruan M, Chen L, Zhang QY. Local Irradiation Sensitized Tumors to Adoptive T Cell Therapy via Enhancing the Cross-Priming, Homing, and Cytotoxicity of Antigen-Specific CD8 T Cells. Front Immunol. 2019;10:2857. 11. Results

[0705] The chemotherapy-free combination has revealed no new safety findings and has been well tolerated in the first six patients who have completed the 10 weeks of treatment followed by surgery 2-3 weeks later. A substantial number of near complete pathological responses according to EORTC-STBSDG have been observed, which are rarely seen in STS patients with standard therapeutic approaches including radiotherapy. The results are shown in Table 4 below.

[0706] Table 4

[0707] Example 3

[0708] Preliminary results from a phase II EFTISARC-NEO trial of neoadjuvant soluble LAG-3 protein eftilaqimod alpha, pembrolizumab, and concurrent radiotherapy in patients with resectable soft tissue sarcoma

[0709] This example provides an update to the results for the phase II study described in Example 2. The update is based on data available as of 10 June 2024 for the first 13 patients who have completed the 10 weeks of treatment followed by surgery 2-3 weeks later (compared with 6 patients in Example 2).

[0710] Objective:

[0711] Perioperative radiotherapy has led to better local control in localized soft tissue sarcoma (STS), but long-term outcomes remain unsatisfactory. Recent studies have shown that the addition of anti-PD-1 antibodies can improve patient outcomes, but the benefit is limited to a small subset of patients as the majority of STS is considered to be immuno-cold. Eftilagimod alpha (efti) is a dimeric soluble LAG-3 molecule that binds to MHC class II on APCs, thereby inducing T cell proliferation, T helper 1 (Th1 ) response, and NK cell activation. We hypothesize that the addition of efti and anti-PD-1 antibody pembrolizumab (pembro) to neoadjuvant radiotherapy will increase pathological response and thereby improve long-term disease control. EFTISARC-NEO (NCT NCT06128863) is an open-label phase II study evaluating the efficacy and safety of neoadjuvant efti and pembro in combination with RT in patients with STS.

[0712] Methods:

[0713] Key eligibility criteria include primary or locally recurrent extremity, girdles, and / or trunk grade 2 / 3 STS (undifferentiated pleomorphic sarcoma [UPS], myxofibrosarcoma [MF], dedifferentiated liposarcoma [DDLPS], myxoid and round cell liposarcoma [MRCLPS], epithelioid sarcoma, angiosarcoma, soft tissue sarcoma NOS) of > 5 cm. Patients receive efti 30 mg sc Q2W (5 cycles) and pembrolizumab 200 mg iv Q3W (3 cycles) with concurrent RT (25x2Gy), followed by surgery 4-6 weeks after radiotherapy. The primary endpoint is a percentage of tumor hyalinization at the time of surgical resection. The study is powered to detect an increase of hyalinization from 15% (HO) with radiotherapy to 35% (H1 ) with the study protocol. Secondary endpoints include disease-free survival, overall survival, response rate, and incidence of adverse events. Here, we report pathological (percent of hyalinization and per EORTC-STBSG response criteria) and radiological responses (per RECIST 1.1 ) of the first 13 evaluable patients. Results:

[0714] As of June 10, 2024, 24 patients had been screened, and 18 were enrolled in the study. One patient discontinued study treatment due to an SAE (intracranial hematoma) not related to study treatment, 4 patients remained on treatment, and 13 patients completed neoadjuvant therapy, underwent surgery, and were available for efficacy assessment.

[0715] Among 13 evaluable patients, the majority were males (8; 61 .5%), and the median age was 54 years. The most common subtype was MF (6; 54.5%), followed by MRCLPS (3; 27.3%) and DDLPS (2; 18.2%), UPS, and MPNST (1 patient each). The median tumor size was 8.8 cm, and 84.6% (11 ) were in the lower extremity.

[0716] In the surgical specimens, the median percent of hyalinization / fibrosis was 49% (IQR 36-65), and 76.9% (10) of patients had > 35% of hyalinization. Median residual viable tumor was 7% (IQR 4-22). 15.4% (2) of patients achieved response grade B, 46.1 % (6) grade C, 30.8% (4) grade D, and 7.7% (1 ) grade E according to EORTC-STBSG criteria.

[0717] In the pre-surgical assessment by MRI, as per RECIST 1.1 criteria, 11 patients achieved stable disease (84.6%), and 2 patients achieved partial response, leading to an ORR of 15.4%. The median tumor size change was -7.4%. All patients except 2 had a decrease of target lesion size. No grade > 3 TRAEs were observed.

[0718] The chemotherapy-free combination has been well tolerated in the first 13 patients who have completed the 10 weeks of treatment followed by surgery 2-3 weeks later.

[0719] The preliminary data shown from the EFTISARC-NEO study combining efti with pembrolizumab and RT shows a median / mean fibrosis / hyalinization rate of 49 / 51 %. About 77% (10 / 13) patients had a % hyalinization / fibrosis > 35%. This compares to the 10-15% fibrosis achieved in patients in historical studies with radiotherapy alone (i.e. the study result is 3-4 times better than in the historical studies with radiotherapy alone). Interestingly good results were observed in patients with different subtypes of STS like malignant peripheral nerve sheath tumor, dedifferentiated liposarcoma or myxofibrosarcoma. Similarly, according to the EORTC-STBSG grading, about 61.5% (8 / 13) had a very favourable outcome with a category B or C grading. As % of hyalinization / fibrosis is a good early surrogate marker for later DFS and OS, these results are very encouraging.

[0720] In addition, about 38% (5 / 13) patients were considered to have a complete / almost complete pathological response as they had < 5% viable tumor cells in the specimen at surgery. This is rarely seen in STS patients with standard therapeutic approaches including radiotherapy. The results are shown in Tables 5 and 6 below (which represent an update to Table 4 of Example 2).

[0721] Table 5 Results in 13 patients, study EFTISARC-NEO Table 6 Summary of the results of fibrosis and tumor cells, study EFTISARC-NEO (N=13)

[0722] Conclusion:

[0723] Based on the preliminary analysis, combining efti and pembrolizumab with radiotherapy demonstrates significant efficacy in the neoadjuvant settings in patients with resectable STS.

[0724] The combination is safe and leads to higher tumor hyalinization than radiotherapy alone compared to historical data. Example 4

[0725] Further preliminary results from a phase II EFTISARC-NEO trial of neoadjuvant soluble LAG- 3 protein eftilaqimod alpha, pembrolizumab, and concurrent radiotherapy in patients with resectable soft tissue sarcoma

[0726] This example provides a further update to the results for the phase II study described in Example 2 and 3. The update is based on data available as of 20 October 2024 after enrolment of 29 out of 40 patients, with 21 patients available for primary endpoint assessment. This is compared to the data in Example 3, available as of 10 June 2024, for the first 13 patients who have completed the 10 weeks of treatment followed by surgery 2-3 weeks later, and the data in Example 2 with 6 patients.

[0727] Background

[0728] • Surgery is the mainstay of treatment of primary localized soft tissue sarcoma (STS), while in patients (pts) with high-grade localized STS of the extremity / trunk, radiation therapy (RTH) is added to reduce local recurrence.

[0729] • Combining immunotherapy (ITH) with RTH may be a promising strategy for synergistic enhancement of treatment efficacy.

[0730] • Eftilagimod alpha (efti) is a dimeric soluble recombinant LAG-3 protein and MHC Class II agonist stimulating antigen-presenting cells (APCs). The LAG-3 - MHC II interaction controls the signalling between T cells and APCs, which are responsible for the adaptive immune response. Combining efti with anti-PD-1 antibody pembrolizumab can enhance its antitumor activity.

[0731] • We hypothesize that adding combined ITH to RT prior to surgical resection would be safe and improve pathologic response compared to historical cohorts of pts with localized STS treated with RT alone.

[0732] • The percentage of hyalinization and fibrosis, as a surrogate of pathological response, appears to be most closely correlated with treatment outcome.

[0733] Figure 3 shows the rationale for combining eftilagimod, pembrolizumab and radiotherapy based on cancer-immune cycle.

[0734] Materials and methods

[0735] - Single-arm single-center phase II study

[0736] - NCT06128863

[0737] - Planned enrollment - 40 patients - Recruitment period: June 2023 - ongoing (planned completion 12 / 2024)

[0738] Key Inclusion Criteria:

[0739] - > 18 years of age

[0740] - ECOG 0 or 1

[0741] - Primary or locally recurrent deep-seated extremities, girdles and / or superficial trunk (thoracic or abdominal wall) tumor

[0742] Histologic diagnosis of undifferentiated pleomorphic sarcoma (UPS), myxofibrosarcoma, dedifferentiated liposarcoma (DDLPS), myxoid and round cell liposarcoma (MRCLPS), epithelioid sarcoma (ES), angiosarcoma (AS), soft tissue sarcoma NOS

[0743] - Amended on 28 Mar 2024 to allow of STS except for Ewing Sarcoma, alveolar and embryonal rhabdomyosarcoma

[0744] - Grade 2 or 3 tumors according FNCLCC

[0745] - Size of the primary tumor >5 cm or locally recurrent of any size;

[0746] - No distant metastases

[0747] - No Previous treatment with eftilagimod alpha, anti-PD-1 or anti-PD-L1

[0748] - No Prior radiotherapy to tumor-involved sites

[0749] Primary Endpoint:

[0750] - The primary efficacy endpoint is a percent tumor hyalinization as a marker of response to treatment assessed at the time of surgical resection.

[0751] HO - 75% (based on historical data for radiotherapy alone from Schaefer M. et al.), H1 - 35%

[0752] Key Secondary Endpoints:

[0753] - Incidence of adverse events graded according to CTCAE version 5.0

[0754] - Disease-free survival time (DFS), Locoregional disease-free survival (LRFS), Distant metastasis-free survival (DMFS), Overall survival time (OS)

[0755] - Radiologic Response To Neoadjuvant Treatment using RECIST 1.1

[0756] Data cut-off for preliminary results: 20 October 2024, after enrollment of 29 / 40 patients and with 21 patients available for primary endpoint assessment.

[0757] Figure 4 shows the EFTISARC-NEO trial procedures. Results

[0758] Patients disposition flow diagram (as of 20 October 2024):

[0759] Total enrolment planned for 40 patients

[0760] *due to SAE not related to therapy

[0761] Table 7 Patients characteristics.

[0762] Figure 8 shows the response to neoadjuvant radio-immunotherapy: A - Hyalinization and fibrosis in surgical specimens after treatment;

[0763] B - viable tumor cells in surgical specimens after treatments;

[0764] C - the proportion of tumor components after treatment in all evaluable patients;

[0765] D - response grade according to EORTC STBSG criteria;

[0766] E - pathologic response, defined as >35% hyalinization and fibrosis;

[0767] F - radiologic response according to RECIST 1.1 criteria.

[0768] Figure 9 shows an example of partial radiologic response per RECIST 1.1 (A) and complete pathologic response (B-E) in a patient with myxofibrosarcoma. B - MF before treatment; C - fibrosis; D - hyalinization; E - cell-free mucous.

[0769] Conclusions

[0770] Based on the preliminary analysis, combining eftilagimod alpha and pembrolizumab with radiotherapy demonstrates significant efficacy in the neoadjuvant setting in patients with resectable soft tissue sarcoma (STS).

[0771] Median hyalinization / fibrosis was 50% (compared to historical 15% for radiotherapy alone) and the median viable tumor cells was 8%.

[0772] 71 .4% achieved pathologic response defined as > 35% of hyalinization / fibrosis and ORR was 19%.

[0773] 9.5% achieved complete pathologic response.

[0774] In the pre-surgical assessment by MRI, as per RECIST 1.1 criteria, four patients achieved a partial response leading to an ORR of 19%.

[0775] The combination is safe (no grade > 3 toxicities related to eftilagimod alpha and pembrolizumab) and leads to higher tumor hyalinization than radiotherapy alone compared to historical data.

[0776] In the neoadjuvant setting for patients with resectable STS, the combination achieved a greater than three-fold increase in tumor hyalinization / fibrosis (median 50%) at the time of surgical resection as compared to a historical median 15% from standard radiotherapy alone. In addition to being the primary endpoint of the EFTISARC-NEO study, the tumuor hyalinization / fibrosis rate has also been identified as an important predictor of recurrence- free survival (RFS) and overall survival (OS) for STS patients: • Schaefer IM, et al., Histologic Appearance After Preoperative Radiation Therapy for Soft Tissue Sarcoma: Assessment of the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group Response Score. Int J Radiat Oncol Biol Phys. 2017 Jun 1;98(2):375-383. doi: 10.1016 / j.ijrobp.2017.02.087. Epub 2017 Feb 24. PMID: 28463157',

[0777] • Rao SR, et al., Extent of tumor fibrosis / hyalinization and infarction following neoadjuvant radiation therapy is associated with improved survival in patients with soft-tissue sarcoma. Cancer Med. 2022 Jan;11 (1):194-206. doi: 10.1002 / cam4.4428. Epub 2021 Nov 27. PMID: 34837341; PMCID: PMC8704179). Thus, efti in combination with pembrolizumab and radiotherapy demonstrates significant efficacy in the neoadjuvant setting in patients with soft tissue sarcoma. The combination achieved over a three-fold increase in tumor hyalinization, the primary endpoint of the study and an important predictor of overall survival, as compared to historical results from radiotherapy alone.

Claims

Claims1 . A LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, and a programmed cell death protein-1 (PD-1 ) pathway inhibitor, for use in the prevention, treatment, or amelioration of a cancer in a subject who has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

2. Use of a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, and a programmed cell death protein-1 (PD-1 ) pathway inhibitor, in the manufacture of a medicament for the prevention, treatment, or amelioration of a cancer in a subject who has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

3. A LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, for use in the prevention, treatment, or amelioration of a cancer in a subject, wherein the LAG-3 protein, or derivative, is to be administered simultaneously or sequentially with a programmed cell death protein-1 (PD-1 ) pathway inhibitor to the subject, and wherein the subject has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

4. Use of a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, in the manufacture of a medicament for the prevention, treatment, or amelioration of a cancer in a subject, wherein the LAG-3 protein, or derivative, is to be administered simultaneously or sequentially with a programmed cell death protein-1 (PD-1 ) pathway inhibitor to the subject, and wherein the subject has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

5. A programmed cell death protein-1 (PD-1 ) pathway inhibitor for use in the prevention, treatment, or amelioration of a cancer in a subject, wherein the PD-1 pathway inhibitor is to be administered simultaneously or sequentially with a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, to the subject, and wherein the subject has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

6. Use of a programmed cell death protein-1 (PD-1 ) pathway inhibitor in the manufacture of a medicament for the prevention, treatment, or amelioration of a cancer in a subject, wherein the PD-1 pathway inhibitor is to be administered simultaneously or sequentially witha LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules, to the subject, and wherein the subject has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

7. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to claim 1 , a LAG-3 protein, or a derivative thereof, for use according to claim 3, a PD-1 pathway inhibitor for use according to claim 5, or use according to any of claims 2, 4, or 6, wherein the subject has undergone, is undergoing, or is to undergo, surgery for treatment of the cancer.

8. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to claim 1 , a LAG-3 protein, or a derivative thereof, for use according to claim 3, a PD-1 pathway inhibitor for use according to claim 5, or use according to any of claims 2, 4, or 6, wherein the subject is to undergo surgery for treatment of the cancer after administration of the LAG-3 protein, or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy.

9. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , 7, or 8, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, 7, or 8, a PD-1 pathway inhibitor for use according to any of claims 5, 7, or 8, or use according to any of claims 2, 4, or 6 to 8, wherein the derivative of LAG-3 protein comprises an amino acid sequence that has at least 70% amino acid identity with domain D1 , and optionally domain D2, of LAG-3 protein, preferably human LAG-3 protein.

10. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , 7, or 8, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, 7, or 8, a PD-1 pathway inhibitor for use according to any of claims 5, 7, or 8, or use according to any of claims 2, 4, or 6 to 8, wherein the derivative of LAG-3 protein comprises an amino acid sequence that has at least 70% amino acid identity with domains D1 , D2 and D3, and optionally domain D4, of LAG-3 protein, preferably human LAG-3 protein.

11. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , 7, or 8, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, 7, or 8, a PD-1 pathway inhibitor for use according to any of claims 5, 7, or 8, or use according to any of claims 2, 4, or 6 to 8, wherein the derivative of LAG-3 protein is eftilagimod alpha (efti, or IMP321 ).

12. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , or 7 to 11 , a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, or 7 to 11 , a PD-1 pathway inhibitor for use according to any of claims 5, or 7 to 11 , or use according to any of claims 2, 4, or 6 to 11 , wherein the PD-1 pathway inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, retifanlimab, atezolizumab, avelumab, and durvalumab.

13. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , or 7 to 11 , a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, or 7 to 11 , a PD-1 pathway inhibitor for use according to any of claims 5, or 7 to 11 , or use according to any of claims 2, 4, or 6 to 11 , wherein the PD-1 pathway inhibitor is pembrolizumab.

14. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , 7 or 8, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, 7 or 8, a PD-1 pathway inhibitor for use according to any of claims 5, 7, or 8, or use according to any of claims 2, 4, or 6 to 8, wherein the LAG-3 derivative is eftilagimod alpha (efti, or IMP321 ), and the PD-1 pathway inhibitor is pembrolizumab.

15. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , or 7 to 14, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, or 7 to 14, a PD-1 pathway inhibitor for use according to any of claims 5, or 7 to 14, or use according to any of claims 2, 4, or 6 to 14, wherein the LAG-3 protein, or derivative thereof, is to be administered to the subject at a dose which is a molar equivalent of about 0.1 mg to about 200 mg, of the LAG-3 derivative LAG-3lg fusion protein IMP321 (eftilagimod alpha).

16. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , or 7 to 15, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, or 7 to 15, a PD-1 pathway inhibitor for use according to any of claims 5, or 7 to 15, or use according to any of claims 2, 4, or 6 to 15, wherein a plurality of doses of the LAG-3 protein, or derivative thereof, is to be administered to the subject.

17. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to claim 16, a LAG-3 protein, or a derivative thereof, for use according to claim 16, a PD-1 pathway inhibitor for use according to claim 16, or use according to claim 16, whereina dose of the LAG-3 protein, or derivative thereof, is to be administered to the subject once every two weeks, preferably up to five times.

18. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , or 7 to 17, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, or 7 to 17, a PD-1 pathway inhibitor for use according to any of claims 5, or 7 to 17, or use according to any of claims 2, 4, or 6 to 17, wherein the PD-1 pathway inhibitor is to be administered to the subject at a dose of about 0.1 to about 10 mg / kg of the PD-1 pathway inhibitor.

19. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , or 7 to 17, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, or 7 to 17, a PD-1 pathway inhibitor for use according to any of claims 5, or 7 to 17, or use according to any of claims 2, 4, or 6 to 17, wherein the PD-1 pathway inhibitor is pembrolizumab, and the pembrolizumab is to be administered to the subject at a dose of 100-600 mg of the pembrolizumab.

20. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , or 7 to 19, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, or 7 to 19, a PD-1 pathway inhibitor for use according to any of claims 5, or 7 to 19, or use according to any of claims 2, 4, or 6 to 19, wherein a plurality of doses of the PD-1 pathway inhibitor is to be administered to the subject.

21. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to claim 20, a LAG-3 protein, or a derivative thereof, for use according to claim 20, a PD-1 pathway inhibitor for use according to claim 20, or use according to claim 20, wherein a dose of the PD-1 pathway inhibitor is to be administered to the subject once every three weeks, preferably up to three times.

22. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , or 7 to 21 , a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, or 7 to 21 , a PD-1 pathway inhibitor for use according to any of claims 5, or 7 to 21 , or use according to any of claims 2, 4, or 6 to 21 , wherein a plurality of doses of radiotherapy are to be administered to the subject.

23. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to claim 22, a LAG-3 protein, or a derivative thereof, for use according to claim 22,a PD-1 pathway inhibitor for use according to claim 22, or use according to claim 22, wherein the radiotherapy is to be administered to the subject five times a week for five or six weeks.

24. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to claim 22 or 23, a LAG-3 protein, or a derivative thereof, for use according to claim 22 or 23, a PD-1 pathway inhibitor for use according to claim 22 or 23, or use according to claim 22 or 23, wherein the radiotherapy is to be administered to the subject in conventional fractionation of 2 Gy / day five times a week.

25. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , or 7 to 24, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, or 7 to 24, a PD-1 pathway inhibitor for use according to any of claims 5, or 7 to 24, or use according to any of claims 2, 4, or 6 to 24, wherein the total radiation dose to be administered to the subject is 40-60 Gy.

26. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , or 7 to 25, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, or 7 to 25, a PD-1 pathway inhibitor for use according to any of claims 5, or 7 to 25, or use according to any of claims 2, 4, or 6 to 25, wherein the subject is a human subject.

27. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , or 7 to 26, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, or 7 to 26, a PD-1 pathway inhibitor for use according to any of claims 5, or 7 to 26, or use according to any of claims 2, 4, or 6 to 26, wherein the cancer is a cancer that is treatable by radiotherapy and / or surgery.

28. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , or 7 to 27, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, or 7 to 27, a PD-1 pathway inhibitor for use according to any of claims 5, or 7 to 27, or use according to any of claims 2, 4, or 6 to 27, wherein the cancer is a PD-L1 -positive, or a PD-L2-positive cancer.

29. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , or 7 to 28, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, or 7 to 28, a PD-1 pathway inhibitor for use according to any of claims 5, or 7 to 28, or use according to any of claims 2, 4, or 6 to 28, wherein the cancer is a breast cancer, skin cancer, lung cancer (for example NSCLC or SCLC), ovarian cancer, renal cancer (for example renal cell carcinoma), colon cancer, colorectal cancer, gastriccancer, esophageal cancer, pancreatic cancer, bladder cancer, urothelial cancer, liver cancer, melanoma (for example, metastatic malignant melanoma), prostate cancer (for example hormone refractory prostate adenocarcinoma), head and neck cancer (for example, head and neck squamous cell carcinoma), cervical cancer, endometrial cancer, uterine cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma (for example, a B cell lymphoma or Hodgkin lymphoma), adrenal gland cancer, AIDS-associated cancer, alveolar soft part sarcoma, astrocytic tumor, bone cancer, brain and spinal cord cancer, metastatic brain tumor, carotid body tumor, chondrosarcoma, chordoma, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasia of the bone, gallbladder or bile duct cancer, gestational trophoblastic disease, germ cell tumor, haematological malignancy, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, lipoma / benign lipomatous tumor, liposarcoma / malignant lipomatous tumor, medulloblastoma, meningioma, Merkel cell carcinoma, multiple endocrine neoplasia, multiple myeloma, myelodysplasia syndrome, neuroblastoma, neuroendocrine tumor, papillary thyroid carcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, phaeochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematologic disorder, rhabdoid tumor, rhabdomysarcoma, sarcoma, soft-tissue sarcoma, squamous cell cancer, synovial sarcoma, mesothelioma, cutaneous squamous cell carcinoma, testicular cancer, thymic carcinoma, thymoma, and thyroid metastatic cancer.

30. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , or 7 to 29, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, or 7 to 29, a PD-1 pathway inhibitor for use according to any of claims 5, or 7 to 29, or use according to any of claims 2, 4, or 6 to 29, wherein the cancer is a sarcoma.

31. A LAG-3 protein, or a derivative thereof, and a PD-1 pathway inhibitor, for use according to any of claims 1 , or 7 to 30, a LAG-3 protein, or a derivative thereof, for use according to any of claims 3, or 7 to 30, a PD-1 pathway inhibitor for use according to any of claims 5, or 7 to 30, or use according to any of claims 2, 4, or 6 to 30, wherein the cancer is a soft tissue sarcoma (STS).

32. A combined preparation comprising:(a) a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules; and(b) a programmed cell death protein-1 (PD-1) pathway inhibitor; for use in the prevention, treatment, or amelioration of a cancer in a subject who has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

33. Use of a combined preparation in the manufacture of a medicament for the prevention, treatment, or amelioration of a cancer in a subject who has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer, wherein the combined preparation comprises:(a) a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules; and(b) a programmed cell death protein-1 (PD-1) pathway inhibitor.

34. A pharmaceutical composition comprising:(a) a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules;(b) a programmed cell death protein-1 (PD-1) pathway inhibitor; and(c) a pharmaceutically acceptable carrier, excipient, or diluent; for use in the prevention, treatment, or amelioration of a cancer in a subject who has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer.

35. Use of a pharmaceutical composition in the manufacture of a medicament for the prevention, treatment, or amelioration of a cancer in a subject who has been, is being, or is to be, administered radiotherapy for prevention, treatment, or amelioration of the cancer, wherein the pharmaceutical composition comprises:(a) a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules;(b) a programmed cell death protein-1 (PD-1) pathway inhibitor; and(c) a pharmaceutically acceptable carrier, excipient, or diluent.

36. A combined preparation for use according to claim 32, a pharmaceutical composition for use according to claim 34, or use according to claim 33 or 35, wherein the subject has undergone, is undergoing, or is to undergo, surgery for treatment of the cancer.

37. A combined preparation for use according to claim 32, a pharmaceutical composition for use according to claim 34, or use according to claim 33 or 35, wherein the subject is to undergo surgery for treatment of the cancer after administration of the LAG-3 protein, or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy.

38. A combined preparation for use according to any of claims 32, 36, or 37, a pharmaceutical composition for use according to any of claims 34, 36, or 37, or use according to any of claims 33, or 35 to 37, wherein the derivative of LAG-3 protein comprises an amino acid sequence that has at least 70% amino acid identity with domain D1 , and optionally domain D2, of LAG-3 protein, preferably human LAG-3 protein.

39. A combined preparation for use according to any of claims 32, 36, or 37, a pharmaceutical composition for use according to any of claims 34, 36, or 37, or use according to any of claims 33, or 35 to 37, wherein the derivative of LAG-3 protein comprises an amino acid sequence that has at least 70% amino acid identity with domains D1 , D2 and D3, and optionally domain D4, of LAG-3 protein, preferably human LAG-3 protein.

40. A combined preparation for use according to any of claims 32, 36, or 37, a pharmaceutical composition for use according to any of claims 34, 36, or 37, or use according to any of claims 33, or 35 to 37, wherein the derivative of LAG-3 protein is eftilagimod alpha (efti, or lMP321 ).

41. A combined preparation for use according to any of claims 32, or 36 to 40, a pharmaceutical composition for use according to any of claims 34, or 36 to 40, or use according to any of claims 33, or 35 to 40, wherein the PD-1 pathway inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, retifanlimab, atezolizumab, avelumab, and durvalumab.

42. A combined preparation for use according to any of claims 32, or 36 to 40, a pharmaceutical composition for use according to any of claims 34, or 36 to 40, or useaccording to any of claims 33, or 35 to 40, wherein the PD-1 pathway inhibitor is pembrolizumab.

43. A combined preparation for use according to any of claims 32, 36, or 37, a pharmaceutical composition for use according to any of claims 34, 36, or 37, or use according to any of claims 33, or 35 to 37, wherein the LAG-3 derivative is eftilagimod alpha (efti, or IMP321 ), and the PD-1 pathway inhibitor is pembrolizumab.

44. A combined preparation for use according to any of claims 32, or 36 to 43, a pharmaceutical composition for use according to any of claims 34, or 36 to 43, or use according to any of claims 33, or 35 to 43, wherein the LAG-3 protein, or derivative thereof, is to be administered to the subject at a dose which is a molar equivalent of about 0.1 mg to about 200 mg, of the LAG-3 derivative LAG-3lg fusion protein IMP321 (eftilagimod alpha).

45. A combined preparation for use according to any of claims 32, or 36 to 44, a pharmaceutical composition for use according to any of claims 34, or 36 to 44, or use according to any of claims 33, or 35 to 44, wherein a plurality of doses of the LAG-3 protein, or derivative thereof, is to be administered to the subject.

46. A combined preparation for use according to claim 45, a pharmaceutical composition for use according to claim 45, or use according to claim 45, wherein a dose of the LAG-3 protein, or derivative thereof, is to be administered to the subject once every two weeks, preferably up to five times.

47. A combined preparation for use according to any of claims 32, or 36 to 46, a pharmaceutical composition for use according to any of claims 34, or 36 to 46, or use according to any of claims 33, or 35 to 46, wherein the PD-1 pathway inhibitor is to be administered to the subject at a dose of about 0.1 to about 10 mg / kg of the PD-1 pathway inhibitor.

48. A combined preparation for use according to any of claims 32, or 36 to 46, a pharmaceutical composition for use according to any of claims 34, or 36 to 46, or use according to any of claims 33, or 35 to 46, wherein the PD-1 pathway inhibitor is pembrolizumab, and the pembrolizumab is to be administered to the subject at a dose of 100-600 mg of the pembrolizumab.

49. A combined preparation for use according to any of claims 32, or 36 to 48, a pharmaceutical composition for use according to any of claims 34, or 36 to 48, or useaccording to any of claims 33, or 35 to 48, wherein a plurality of doses of the PD-1 pathway inhibitor is to be administered to the subject.

50. A combined preparation for use according to claim 49, a pharmaceutical composition for use according to claim 49, or use according to claim 49, wherein a dose of the PD-1 pathway inhibitor is to be administered to the subject once every three weeks, preferably up to three times.

51. A combined preparation for use according to any of claims 32, or 36 to 50, a pharmaceutical composition for use according to any of claims 34, or 36 to 50, or use according to any of claims 33, or 35 to 50, wherein a plurality of doses of radiotherapy are to be administered to the subject.

52. A combined preparation for use according to claim 51 , a pharmaceutical composition for use according to claim 51 , or use according to claim 51 , wherein the radiotherapy is to be administered to the subject five times a week for five or six weeks.

53. A combined preparation for use according to claim 51 or 52, a pharmaceutical composition for use according to claim 51 or 52, or use according to claim 51 or 52, wherein the radiotherapy is to be administered to the subject in conventional fractionation of 2 Gy / day five times a week.

54. A combined preparation for use according to any of claims 32, or 36 to 53, a pharmaceutical composition for use according to any of claims 34, or 36 to 53, or use according to any of claims 33, or 35 to 53, wherein the total radiation dose to be administered to the subject is 40-60 Gy.

55. A combined preparation for use according to any of claims 32, or 36 to 54, a pharmaceutical composition for use according to any of claims 34, or 36 to 54, or use according to any of claims 33, or 35 to 54, wherein the subject is a human subject.

56. A combined preparation for use according to any of claims 32, or 36 to 55, a pharmaceutical composition for use according to any of claims 34, or 36 to 55, or use according to any of claims 33, or 35 to 55, wherein the cancer is a cancer that is treatable by radiotherapy and / or surgery.

57. A combined preparation for use according to any of claims 32, or 36 to 56, a pharmaceutical composition for use according to any of claims 34, or 36 to 56, or useaccording to any of claims 33, or 35 to 56, wherein the cancer is a PD-L1 -positive, or a PD- L2-positive cancer.

58. A combined preparation for use according to any of claims 32, or 36 to 57, a pharmaceutical composition for use according to any of claims 34, or 36 to 57, or use according to any of claims 33, or 35 to 57, wherein the cancer is a breast cancer, skin cancer, lung cancer (for example NSCLC or SCLC), ovarian cancer, renal cancer (for example renal cell carcinoma), colon cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, bladder cancer, urothelial cancer, liver cancer, melanoma (for example, metastatic malignant melanoma), prostate cancer (for example hormone refractory prostate adenocarcinoma), head and neck cancer (for example, head and neck squamous cell carcinoma), cervical cancer, endometrial cancer, uterine cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma (for example, a B cell lymphoma or Hodgkin lymphoma), adrenal gland cancer, AIDS-associated cancer, alveolar soft part sarcoma, astrocytic tumor, bone cancer, brain and spinal cord cancer, metastatic brain tumor, carotid body tumor, chondrosarcoma, chordoma, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasia of the bone, gallbladder or bile duct cancer, gestational trophoblastic disease, germ cell tumor, haematological malignancy, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, lipoma / benign lipomatous tumor, liposarcoma / malignant lipomatous tumor, medulloblastoma, meningioma, Merkel cell carcinoma, multiple endocrine neoplasia, multiple myeloma, myelodysplasia syndrome, neuroblastoma, neuroendocrine tumor, papillary thyroid carcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, phaeochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematologic disorder, rhabdoid tumor, rhabdomysarcoma, sarcoma, soft-tissue sarcoma, squamous cell cancer, synovial sarcoma, mesothelioma, cutaneous squamous cell carcinoma, testicular cancer, thymic carcinoma, thymoma, and thyroid metastatic cancer.

59. A combined preparation for use according to any of claims 32, or 36 to 58, a pharmaceutical composition for use according to any of claims 34, or 36 to 58, or use according to any of claims 33, or 35 to 58, wherein the cancer is a sarcoma.

60. A combined preparation for use according to any of claims 32, or 36 to 59, a pharmaceutical composition for use according to any of claims 34, or 36 to 59, or use according to any of claims 33, or 35 to 59, wherein the cancer is a soft tissue sarcoma (STS).

61. A method for preventing, treating, or ameliorating a cancer in a subject in need thereof, which comprises administering to the subject an effective amount of:(a) a LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules;(b) a programmed cell death protein-1 (PD-1) pathway inhibitor; and(c) radiotherapy.

62. A method according to claim 61 , wherein the subject has undergone, is undergoing, or is to undergo, surgery for treatment of the cancer.

63. A method according to claim 61 , wherein the subject is to undergo surgery for treatment of the cancer after administration of the LAG-3 protein, or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy.

64. A method according to claim 61 , which further comprises performing surgery on the subject to treat the cancer.

65. A method according to claim 64, wherein the surgery is performed after administration of the LAG-3 protein, or derivative thereof, the PD-1 pathway inhibitor, and the radiotherapy.

66. A method according to any of claims 61 to 65, wherein the derivative of LAG-3 protein comprises an amino acid sequence that has at least 70% amino acid identity with domain D1 , and optionally domain D2, of LAG-3 protein, preferably human LAG-3 protein.

67. A method according to any of claims 61 to 65, wherein the derivative of LAG-3 protein comprises an amino acid sequence that has at least 70% amino acid identity with domains D1 , D2 and D3, and optionally domain D4, of LAG-3 protein, preferably human LAG-3 protein.

68. A method according to any of claims 61 to 65, wherein the derivative of LAG-3 protein is eftilagimod alpha (efti, or IMP321 ).

69. A method according to any of claims 61 to 68, wherein the PD-1 pathway inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, retifanlimab, atezolizumab, avelumab, and durvalumab.

70. A method according to any of claims 61 to 68, wherein the PD-1 pathway inhibitor is pembrolizumab.

71. A method according to any of claims 61 to 65, wherein the LAG-3 derivative is eftilagimod alpha (efti, or IMP321), and the PD-1 pathway inhibitor is pembrolizumab.

72. A method according to any of claims 61 to 71 , wherein the LAG-3 protein, or derivative thereof, is administered to the subject at a dose which is a molar equivalent of about 0.1 mg to about 200 mg, of the LAG-3 derivative LAG-3lg fusion protein IMP321 (eftilagimod alpha).

73. A method according to any of claims 61 to 72, wherein a plurality of doses of the LAG- 3 protein, or derivative thereof, is administered to the subject.

74. A method according to claim 73, wherein a dose of the LAG-3 protein, or derivative thereof, is administered to the subject once every two weeks, preferably up to five times.

75. A method according to any of claims 61 to 74, wherein the PD-1 pathway inhibitor is administered to the subject at a dose of about 0.1 to about 10 mg / kg of the PD-1 pathway inhibitor.

76. A method according to any of claims 61 to 75, wherein the PD-1 pathway inhibitor is pembrolizumab, and the pembrolizumab is administered to the subject at a dose of 100-600 mg of the pembrolizumab.

77. A method according to any of claims 61 to 76, wherein a plurality of doses of the PD- 1 pathway inhibitor is administered to the subject.

78. A method according to claim 77, wherein a dose of the PD-1 pathway inhibitor is administered to the subject once every three weeks, preferably up to three times.

79. A method according to any of claims 61 to 78, wherein a plurality of doses of radiotherapy are administered to the subject.

80. A method according to claim 79, wherein the radiotherapy is administered to the subject five times a week for five or six weeks.

81. A method according to claim 79 or 80, wherein the radiotherapy is administered to the subject in conventional fractionation of 2 Gy / day five times a week.

82. A method according to any of claims 61 to 81 , wherein the total radiation dose administered to the subject is 40-60 Gy.

83. A method according to any of claims 61 to 82, wherein the subject is a human subject.

84. A method according to any of claims 61 to 83, wherein the cancer is a cancer that is treatable by radiotherapy and / or surgery.

85. A method according to any of claims 61 to 84, wherein the cancer is a PD-L1 -positive, or a PD-L2-positive cancer.

86. A method according to any of claims 61 to 85, wherein the cancer is a breast cancer, skin cancer, lung cancer (for example NSCLC or SCLC), ovarian cancer, renal cancer (for example renal cell carcinoma), colon cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, bladder cancer, urothelial cancer, liver cancer, melanoma (for example, metastatic malignant melanoma), prostate cancer (for example hormone refractory prostate adenocarcinoma), head and neck cancer (for example, head and neck squamous cell carcinoma), cervical cancer, endometrial cancer, uterine cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma (for example, a B cell lymphoma or Hodgkin lymphoma), adrenal gland cancer, AIDS-associated cancer, alveolar soft part sarcoma, astrocytic tumor, bone cancer, brain and spinal cord cancer, metastatic brain tumor, carotid body tumor, chondrosarcoma, chordoma, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasia of the bone, gallbladder or bile duct cancer, gestational trophoblastic disease, germ cell tumor, haematological malignancy, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, lipoma / benign lipomatous tumor, liposarcoma / malignant lipomatous tumor, medulloblastoma, meningioma, Merkel cell carcinoma, multiple endocrine neoplasia, multiple myeloma, myelodysplasia syndrome, neuroblastoma, neuroendocrine tumor, papillary thyroid carcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, phaeochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematologic disorder, rhabdoid tumor, rhabdomysarcoma, sarcoma, soft-tissue sarcoma, squamous cell cancer, synovial sarcoma, mesothelioma, cutaneous squamous cell carcinoma, testicular cancer, thymic carcinoma, thymoma, and thyroid metastatic cancer.

87. A method according to any of claims 61 to 86, wherein the cancer is a sarcoma.

88. A method according to any of claims 61 to 87, wherein the cancer is a soft tissue sarcoma (STS).

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