WT1 was originally identified by complementary deoxyribonucleic acid (cDNA ) mapping to a region of chromosome 11p13. The WT1 cDNA encodes a protein of 575 amino acids (aa) in length, containing 4 Kruppel zinc fingers and exhibiting a complex pattern of alternative splicing, resulting in 4 different transcription factors (Haber et al, 1990; Hohenstein et al, 2016). Each WT1 protein isoform has different deoxyribonucleic acid (DNA) binding and transcriptional activities, and can positively or negatively regulate various genes involved in cellular proliferation, differentiation, apop tosis, organ development, and sex determination ( Haber et al, 1991). WT1 is normally expressed in tissues of the mesodermal origin during embryogenesis, including the kidney, gonads, heart, mesothelium, and spleen (Oji et al, 1999; Sharnhorst et al, 1999). In normal adult human tissues, WT1 expression is limited to very low levels in the nuclei of normal CD34+ hematopoietic stem cells, myoepithelial progenitor cells, renal podocytes, injured myocardium, and some cells in the testis and ovary ( Mundlos et al, 1993; Buckler et al, 1991; Fraizer et al, 1995). Although originally described as a tumor suppressor gene, the WT1 proteins appear to be involved in tumorigenesis as a bona fide oncogene ( Keilhoz et al, 2005; Lindstedt, et al, 2014). As WT1 is a transcription factor and is not expressed intact in the cell membrane, it has not been able to be targeted (druggable) with either small molecules or monoclonal antibodies (Toska et al, 2014). Nonetheless, the protein is processed by the proteasome in tumors and the derived peptides are presented in a major histocompatibility complex ( MHC)-dependent manner on the cell surface. Furthermore, WT1 peptide fragments when administered as direct SELLAS Life Sciences Group Galinpepimut -S Protocol : SLS17 -201/MK3475 -770 Protocol v1.1 (Amendment 1.0) 12 April 2019 Page 69 immunogens (vaccines) are also taken up by antigen -presenting cells (APCs) and processed by the immunoproteasome for MHC-dependent presentation (Jiagirdar et al, 2016; Asemissen et al, 2006; Müller et al, 2003). The above features render WT1 a highly attractive target for immunotherapy (Oka et al, 2002; Oka et al, 2007). WT1 was ranked as the top cancer antigen by a working group organized by the National Cancer Institute (NCI) in 2009 (Cheever, et al, 2009). The strong expression of WT1 protein in assorted cancers, coupled with its proposed mechanism of antigenicity and induction of immune responses (both CD4 and CD8 [Chapuis et al, 2013; Dao et al, 2016; Tyler et al, 2013; Tyler and Koehne, 2013; Fujiki et al, 2008; Krug et al; 2010]; after direct immunization against WT1 in patients with assorted cancers), makes it a rational candidate for the development of specific immunotherapies, such as peptide vaccines ( Van Driessche et al, 2012; Oka and Sugiyama, 2010; Oka et al 2009; Dao and Seheinbert, 2008). The WT1 antigen is one of the most widely expressed cancer antigens in multiple malignancies. It has been ranked by the NCI – National Institutes of Health (NIH) - as the top priority among cancer antigens for immunotherapy (Cheever MA, et al 2009). WT1 is overexpressed in numerous hematological malignancies, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and multiple myeloma (MM), as well as in many solid malignancies such as malignant pleural mesothelioma (MPM), gastrointestinal cancers, i.e., colorectal cancer (CRC), glioblastoma multiforme (GBM), triple-negative breast cancer (TNBC), ovarian cancer (OvC), small-cell lung cancer (SCLC); all in all WT1 is expressed in at least 50% of tumor pathology specimens in up to 25 assorted malignancies (Oji Y et al, 2003; Bejrananda T et al, 2010-2011; Inoue K, et al, 1997; Nakatsuka S-I et al, 2006; Provenzano E, et al, 2016; Narita M et al, 2010; Di Stasi A et al, 2015; Tyler EM and Koehne G. 2013; Babiak A et al, 2014; Menssen HDet al, 2000; Oji Yet al, 2016; Nakahara Y et al, 2004; Van Driessche Aet al, 2005). Despite WT1’s broad expression in assorted malignancies, due to its nature - an intracellular transcription factor localized in the cell nucleus, and as mentioned earlier - it has been ‘un- druggable’ via standard pharmacology appro aches, such as small molecules or surface-acting macromolecules (such as monoclonal antibodies). Thus, immunotherapy approaches, such as SELLAS Life Sciences Group Galinpepimut -S Protocol : SLS17 -201/MK3475 -770 Protocol v1.1 (Amendment 1.0) 12 April 2019 Page 70 direct immunizers (such as peptide-based vaccines) emerged as an attractive pathway to therapeutically target this key protein (Brayer and Pinilla-Ibarz, 2013; Schwartz J et al, 2003; Oka Y et al, 2006; Van Driessche A et al, 2012). Because WT1 protein is a self-antigen, breaking tolerance is a potential problem for an effective treatment. When developing potential treatment candidates, a strategy in use at Memorial Sloan-Kettering Cancer Center (MSKCC) to circumvent the poor immunogenicity and potential tolerance of tumor-associated peptides is to design synthetic analog peptides that will be more immunogenic. Such peptide analogues could generate an immune response that not only recognizes the immunizing epitopes, but also cross -reacts with the original native peptides; this is known as a heteroclitic response. By using computer prediction analysis, a large number of synthetic peptides derived from WT1 protein sequences were designed in which single or double aa substitutions were introduced into the peptides at key human leukocyte antigen (HLA)-A*A0201 binding positions (Pinilla-Ibarz et al, 2006; Gomez-Nunez M et al, 2006). Peptides predicted to bind with high affinity to HLA-A*A0201 molecules were directly assayed for their ability to stabilize major histocompatibility complex c lass I A0201 molecules on the surface of antigen-transporting-deficient T2 cell lines. The new synthetic peptides stabilized major histocompatibility complex class I A0201 molecules better than native sequences. Avidly binding peptides were then assayed in an antigen -specific T-cell expansion in vitro system for the ability to elicit HLA -restricted, peptide-specific cytotoxic T-lymphocyte (CTL) responses using purified T cells from healthy donors. The synthetic analog peptides generated more effective immune responses than the native peptides. In addition, CD8+ T-cells stimulated with the new synthetic peptides displayed heteroclitic features and cross reacted w ith the native WT1 peptides and also were able to mediate peptide-specific cytotoxicity. Importantly, T-cells stimulated with the new synthetic peptides cross-reacted with the native WT1 peptide sequence and were able to destroy HLA -matched CML blasts (Pinilla-Ibarz J et al, 2000;). This validates the expression and possibility of killing tumors in this system. Other groups have validated the same native sequence in use as a target (Mail änder et al, 2004). SELLAS Life Sciences Group Galinpepimut -S Protocol : SLS17 -201/MK3475 -770 Protocol v1.1 (Amendment 1.0) 12 April 2019 Page 71 Synthetic peptides with longer WT1 sequences were also designed by modifying previously identified WT1 peptide segments and adding fla nking aa segments. These peptides are capable of stimulating a CD4+ response, which is necessary for inducing long-term T-cell memory. They can induce a peptide-specific CD4+ response that can recognize WT1-positive tumor cells in multiple HLA -DRB1 settings. Using cross-priming experiments, it was shown that the WT1 peptide is presented on the surface of mesothelioma tumor cells and could be recognized by the T-cells stimulated by the individual WT1 -DR peptides (May et al, 2007). In order to broaden immunogenicity over a range of HLA subtypes, 4 WT1-derived peptides were chosen to combine into a mixture to be used as a direct immunizer . The current candidate product, now known as galinpepimut-S, contains 1 WT1 heteroclitic peptide to stimulate CD8 responses (WT1-A1), 2 longer WT1 native peptides to stimulate CD4 responses (WT1-427 long and WT1-331 long) and 1 longer heteroclitic peptide that could stimulate both CD4 and CD8 cells (WT1-122A1 long). Galinpepimut-S, along with the immunological adjuvant Montanide™ with granulocyte-macrophage colony-stimulating factor (GM-CSF) pre-stimulation was then taken into human trials , initially in AML and mesothelioma, and subsequently in other indications (multiple myeloma and ovarian cancer) . With regard to the use of GM-CSF as an immune adjuvant and to establish the paucity of any confounding immunologic and/or clinical effects thereof when co-administered with galinpepimut-S, the following information is of relevance: Firstly, GM -CSF has been recognized as an activator of the first step in adaptive immunity responses to antigens, especially peptide vaccines over the last 25 years (Caux et al , 1996). Pre-stimulation of host macrophages/monocytes and other antigen -presenting cells (APCs) is absolutely required for the induction immune responses against moieties of relatively low antigenic potential (such as tumor-associated antigens, or TAAs) presented through the MHC Class I and II systems (Mach et al, 2000; Olatunde et al, 2018). This is true for oligopeptide fragments of the WT1 protein, including those within the galinpepimut-S tetravalent mixture. In essence, in order for most peptide vaccines to induce immunogenicity – leading to CD8+ and/or CD4+ SELLAS Life Sciences Group Galinpepimut -S Protocol : SLS17 -201/MK3475 -770 Protocol v1.1 (Amendment 1.0) 12 April 2019 Page 72 lymphocyte activation, which is the prerequisite for the emergence of an immunologically mediated antitumor clinical effect, they need to be co -administered with an immune adjuvant. In a wide variety of such vaccines, including galinpepimut-S, one of the most commonly used (and more widely studied) adjuvant for this purpose is GM -CSF. Secondly, several clinical and immunobiological studies have shown lack of consistent effect of GM -CSF monotherapy, i.e., when this adjuvant is administered by itself and in the absence of another tumor-directed immunotherapy, with regard to GM -CSF’s ability to induce or maintain antigen-specific T- and B-cell immune responses and clinical effect in patients with variou s malignancies (O’Day et al, 2009; Greter et al, 2012; Lawson et al, 2015). Indeed, although GM-CSF monotherapy in cancer patients has been shown to exert various modulatory effects in the number/frequency and activation level of dendritic cells (DCs) and other immunocytes, none of these cell subpopulations have even been consistently shown to be specifically directed to/reactive against tumor -associated antigens. This is especially true when GM -CSF is used in small doses (such as that used in this study, 70 µg per administration - rather than the per-label dose of 250 µg per administration) (Parmiani et al, 2007). Of note, in an earlier phase 2b, double-blinded, randomized, controlled clinical study of galinpepimut -S (plus GM- CSF) versus GM-CSF alone given in the maintenance setting after frontline debulking multimodality therapy in patients with malignant pleural mesothelioma, no WT1 (antigen) - specific T-cell (CD8 or CD4) immune responses were observed in the control arm. Further, the clinical outcome (as measured by overall survival since randomization) in the control arm was comparable to that reported in patients who relapsed during follow up for the natural history of their disease (Zauderer et al, 2017). Therefore, when all the above is taken into consideration, GM-CSF alone appears to be inactive with regard to antigen -specific immunogenicity and antitumor effect. 1.1.1.2. Justification for Galinpepimut -S Dose A dose of 200 μg for each WT1 peptide within the galinpepimut -S mixture (total weight of all 4 peptides per inoculation being equal to 800 μg) was chosen because it is within the range of safe and active doses used in other WT1 -based treatments. Peptide vaccines have SELLAS Life Sciences Group Galinpepimut -S Protocol : SLS17 -201/MK3475 -770 Protocol v1.1 (Amendment 1.0) 12 April 2019 Page 73 generated immune and clinical responses within a wide range of doses (100 to 2000 μg injected) without clear evidence of dose response relationships. Higher doses have the theoretical possibility of stimulating lower affinity T -cell receptors on T cells and reducing the response. 1.1.2. Galinpepimut -S Clinical Studies1.1.2.1. Phase 1 Clinical Experience with Galinpepimut -S (Study 06 -085) The MSKCC conducted a single institution, open-label, single arm Phase 1 study (#06-085) in patients with AML, non-small cell lung cancer (NSCLC), or mesothelioma to determine the safety and immunogenicity of treatment with galinpepimut-S (Krug et al, 2010; Maslak et al, 2010; ClinicalTrials.gov identifier: NCT00398138 ). Patients with myelodysplastic syndrome (MDS) were eligible to enroll in this study; however, none were treated.