32 19.0 REFERENCES ............................................................................................................................. 33 DC Vaccine for Colorectal Cancer Page 5 of 34 Version 7.11-11-2021 CONFIDENTIAL This protocol is the property of the University of Pennsylvania. Do not disclose or use except as authorized in writing by the study spons or. 1.0 BACKGROUND AND RATIONALE 1.1 Overview Immunotherapy with checkpoint inhibitors is now widely accepted as a n effective therapeutic approach that offers, in some instances, durable complete remission for patients with various types of cancer such as melanoma, lung, and bladder cancer [1]. One unifying theme is the high mutational burden present in tumors analyzed from responding patients provi ding evidence that the im mune system is capable of recognizing genomic alterations (such as missense mutations) [1]. Emerging evidence confirms the activity of checkpoint inhibitors in patients with hypermutated GI malignancies such as MSI -H colorectal cancer (CRC) [2]. This present study aims to elicit T cell immunity to unique tumor encoded neoa ntigens using a dendritic cell (DC) vaccine pl atform for the purposes of identifying the genomic alterations that serve as target neoantigens in patients with CRC. The hope is this information will allow investigators to better design the next generation o f personalized immunotherapies which are specific for each patient’s cancer. 1.2 Colorectal Cancer (CRC) Statistics CRC continues to be a leading cause of death in North America and worldwide. I n 2018, the estimated number of new cases of CRC in the US is 140,250. The estimated number of deaths due to CRC in the US is 50,630 total (27,390 males and 23,240 females) [3]. The lifetime risk of developing invasive colorectal cancer in the US is approximately 4.5% (1/22 individuals). C RC is the fourth leading cause of death due to cancer among all age groups. In the tri -state area (Pennsylvania, New Jersey, Delaware), it is estimated that 3920 deaths due to CRC will occur in 2018[3] . Recent reports suggest that the incidence of CRC is rising in young er adults less than 50 years of age [4, 5]. 1.3 Colorectal Cancer (CRC) Genetics The molecular basis of CRC was initially outlined in 1990 by Fearon and Vogelstein[6] . A multistep model of genetic alterations including the accumulation of key driver mutations (such as KRAS) and loss of various tumor suppressor genes (such as p53) defined a conceptual framework to delineate the origi ns an d evolution of CRC. The original multihit model has been refined and extended to include inherited susceptibility factors, environmental influences an d tumor -host interactions, including the role of the immune system [7]. Recent advances now provide compelling evidence linking genetic instability with high mutational burden and recognition by the host immune system which is able to mediate tumor elimination under certain conditions. Despite the fact that most sporadic CRCs have a low mutational burden (1 -2 mutations/Mb), it is now appreciated that a distinct subgroup of patients, including individuals with a familial cancer predisposition , have hyper -mutated CRC frequently associated with mismatch repair ( MMR ) deficiency [8]. The original 1990 model of multistep tumorigenesis defined the clonal nature of adenoma transformation to malignancy followed by metastasis but did not outline the complexities of intratumor heterogeneity as revealed by next generation sequencing methodologies. Data from The Cancer Genome Atlas ( TCGA ) and related efforts have defined the 4 major consensus molecular subtypes (CMS) of c olorectal cancer as shown belo w[9]: DC Vaccine for Colorectal Cancer Page 6 of 34 Version 7.11-11-2021 CONFIDENTIAL This protocol is the property of the University of Pennsylvania. Do not disclose or use except as authorized in writing by the study spons or. It is now well established that ~15% of CRCs (CMS1) are hypermutated (>10 mutations/Mb) and associated w ith inherited cancer syndromes (most often, Lynch syndrome), CpG island methylator phenotype, and microsatellite instability. Interestingly, the CMS1 molecular subgroup of patient s is responsive to checkpoint inhibitor therapy. In 2017, both anti -PD-1 agents, pembrolizumab and nivolumab, received regulatory approval as monotherapy for treatment of relapsed/refractory MSI - H CRC that progressed after treatment with 5 -FU based chemot herapy[10]. The remaining 3 molecular subtypes [(CMS2, canonical) (CMS3, metabolic) and (CMS4, mesenchymal)] are typically low mutational burden (1 -2 mutations/Mb) malignancies and are not responsive to checkpoint inhibition with either an anti -PD-1 agent or anti- CTLA -4 antibody. CMS1 type CRCs are seen in both sporadic cases of CRC as well as in association with hereditary CRC predisposition syndromes . The most common inherited CRC syndrome is Lynch syndrome which accounts for 3% of all CRC and is characterized by a germline mutation in one of the four DNA mismatch repair genes including MSH2 , MLH1 , MSH6 , and PMS2 , or in EPCAM , which can lead to epigenetic silencing of MSH2 [8]. Current guidelines recommend universal testing of primary CRC specimens for MMR deficiency in all patients [11, 12] . Congenital mismatch repair deficiency (CMMRD) is a rare syndrome with inherited mutation in both alleles of a given MMR gene resulting in cancer predisposition at a younger age and increased risk for CMS1 type CRCs . Finally, a distinct familial syndrome is polymerase proofreading associated polyposis ( PPAP) which is a rare autosomal dominant syndrome involving a mutation in POLE or POLD1 and is characterized by ultra-hypermutated cancers (>100 mutations /Mb)[13]. Despite many CMS1 type CRCs being related to hereditary syndromes, the majority of CMS1 type CRCs are sporadic, with m icrosatellite instability (MSI) resulting from CpG island methylation of a DNA mismatch repair gene – typically, MLH1 – with loss of expression resulting in DNA mismatch repair deficiency. Approximately 12% of CRC patients exhibit a sporadic MSI -H phenotype resulting in the accumulation of mutatio ns most notably at repe titive DNA s equences know n as satellites[8] . By employing routine ( standard of care ) IHC methods to assess for MMR loss ( MLH1 /MSH2/MSH6/PMS2) in conjunction with molecular microsatellite testing, we are now able to universally screen all CRC patients in order to identify hypermutated CRC genomes. DC Vaccine for Colorectal Cancer Page 7 of 34 Version 7.11-11-2021 CONFIDENTIAL This protocol is the property of the University of Pennsylvania. Do not disclose or use except as authorized in writing by the study spons or. 1.4 Cancer Neoantigens In 2005, Galon and colleagues published a landmark report describing the immune landscape within prima ry CRCs and concluded that the presence of infiltrating memory CD8+ T cells correlated with improved overall survival [14]. A follow up report with different patient cohorts confirmed this finding one year later and advanced the concept that the type, density and location of CD8+ T cells (Immunoscore) in the primary tumor was a superior predictor of outcome compared to the widely accepted TNM classification [15]. Moreover, it further supported the notion that a type -1 biased (Th1) immune response correlated with improved outcomes. Despite this important finding, it has been difficult to translate this finding into new personalized therapeutic approaches for CRC, in part, because few target antigens are well defined. However, new universal testing methods to detect mismatch repair deficiency (by IHC) and the related microsatellite instability (by PCR) can easily identif y both somatic MMR deficient/MSI -H CRC as well as familial cancer syndromes (such as Lynch Syndrome). Characterization of mutational burden using NGS platforms have collectively simplified our ability to find target neoantigens in CRCs and thus, have opened the door to new therapeutic strategies [12]. Somatic gene mutations within cancer cells may be translated into peptides that are processed and presented on the surface of tumor cells. These mutated peptides can serve as foreign epitopes, or neoantigens, that may be recognized by the host immune system [16]. Neoantigen -specific T cell responses have been well documented in patients for whom immune checkpoint blockade therapy has been successful. Recent studies - including our own published work in melanoma - have demonstrated neoantigen -specific T cell responses can be elicited in cancer patients and interestingly, administration of anti- PD-1 antibody after neoantigen vaccination can result in complete melanoma regression with durable remissions [17-19]. Adoptive transfer of neoantigen - specific T cell products can also result in tumor regression in patients with melanoma as well as various GI cancers as published in case reports [16]. Thus, targeting cancer neoa ntigens has proven to be feasible and in certain instances, clinically effecti ve based on durable tumor regression. Based on new scientifi c advances related to immuno -oncology and unmet medical need for improved adjuvant therapy , there appears to be strong rationale to investigate hypermutated CRC as a new disease indication for neoantigen directed immunotherapy[10]. 1.5 Adjuvant Treatment for CRC: Current Standard A role for adjuvant treatment of high risk, surgically resected stage III CRC was established in 1990 with the publication of a randomized controlled clinical trial with 5 -fluorouracil (5-FU) and levamisole [20]. The NIH consensus panel supported the routine use of adjuvant chemotherapy for patients with stage III resected CRC but not stage II resected disease[21] . It was later shown that 5-fluorouracil/leucovorin was superior to 5- fluorouracil/levamisole and this was adopted as the standard of care by the mid -1990’s [21] . The Multicenter International Study of Oxaliplatin/5- FU/Leucovorin in the Adjuvant Treatment of Colon Cancer (MOSAIC) [22] was a landmar k trial which provided clear evidence in support of adding oxaliplatin to 5- FU/Leucovorin for stage III resected CRC with a hazard ratio for recurrence, 0.77; P=0.002. A second trial (NSABP C -07)[23] provided confirmation for the superiority of oxali platin/5 -FU/Leucovorin (FOLFOX) over 5 -FU/Leucovorin; however, high rates of neurotoxicity and diarrhea/dehydra tion related to oxaliplatin remain a concern. Recent non - inferior ity analysis of multiple trials presented at ASCO 2017 meeting Plenary session (LBA1) reviewed data from >13,000 patients and concluded that 3 months of adjuvant chemotherapy is sufficient for the specific subset of patients with low risk stage III disease (T3N1) and decreases the risk of neurotoxicity without necessarily compromising efficacy. It is important to note that DC Vaccine for Colorectal Cancer Page 8 of 34 Version 7.11-11-2021 CONFIDENTIAL This protocol is the property of the University of Pennsylvania. Do not disclose or use except as authorized in writing by the study spons or. only 20% of stage III CRC patients receive benefit from adjuvant chemotherapy and thus, new treatment options are needed. Data from mu ltiple randomized clinical trials evaluating the use of adjuvant chemotherapy , including FOLFOX, fail to convincingly show benefit for patients with stage II resected CRC . However, for high risk stage II patients such as those with T4 lesions, perforation or obstruction at presentation, poorly differentiated histology, lymphovascular invasion, perineural invasion or fewer than 12 lymph nodes examined during surgery, chemotherapy may be considered. An important exception to this is the MSI -H or MMR deficient stage II CRC patients. In this particular group, regardless of other high risk features, there is no established benefit to adjuvant chemotherapy and in fact, literature suggests that 5 -FU adjuvant therapy may indeed be detrimental in this subgr oup[24, 25]. 1.6 Dendritic Cells Antigen presentation to T cells in the context of class I and II major histocompa tibility molecules is critical f or priming neoantigen -specific CD8+ and CD4+ T cell responses, respectively. Antigen presentation is mediated by antigen presenting cells which include dendritic cells, B cells and macrophages. Dendritic cells (DC) are the most potent antigen presenting cells given their ability to express high levels of costimulatory molecules and secrete Th1 polarizing cytokines (e.g. IL -12) that are essential for the generation of cytotoxic T cell responses [24]. A variety of technical advances involving the isolation of human DC as well as an improved understanding of DC biology have led investigators to study autologous DC as adjuvant for peptide vaccination in cancer. Various clinical studies have demonstrated the safety and tolerability of DC immunization and serious adverse events related to DC vaccination are rare [26] . 1.7 Regulatory T cells Recent advances in immunology confirm the presence of a small population of circulating CD4+CD25+ T cells known as regulatory T cells (Treg) that function to suppress T cell immunity toward pathogens and cancer. In healthy adults, approximately 5% of peripheral blood CD4+ T cells are Treg based on co -expression of CD25 and the transcription factor FoxP3. Several randomized trials have confirmed that vaccines administered along with cyclophosphamide have enhanced immunogenicity . Low dose cyclophosphamide, which may be administered orally or intravenously, is an effective strategy to eliminate Treg in patients prior to vaccination [27]. 1.8 Organoid Cultures As part of participation on a separate tissue acquisition trial, subjects will have fresh tissue from both benign colonic mucosa as well as from t he resected colon cancer, collected at the time of routine care surgery and transported to the labs of Dr. Anil Rustgi and Dr. Chris Lengner for organoid culture establishment. Collection of tumor tissue samples will occur prior to participation on this i nterventional trial. For establishment of organoids from benign colonic tissue, healthy colonic epithelium will be mechanically fragmented and separated from underlying submucosal tissues, and subsequently intestinal crypts will be isolated as previously described [29]. Once isolated these crypts will be plated and grown in a mixture of Matrigel and Wnt -containing cell culture media. For establishment of organoids from neoplastic tissue, the tumor will first be mechanically fragmented and then chemically dissociated f rom surrounding tumor stroma. Further mechanical dissociation will be performed through a combination of pipetting and vortexing, and cells will be plated and DC Vaccine for Colorectal Cancer Page 9 of 34 Version 7.11-11-2021 CONFIDENTIAL This protocol is the property of the University of Pennsylvania. Do not disclose or use except as authorized in writing by the study spons or. grown in a mixture of Matrigel and Wnt -free cell culture media as previously described [30] After growth of organoid cultures from both benign and neoplastic tissue is established, these organoids will be passaged as needed, and will be cryopreserved to allow for future experimental use including morphologic a nd biochemical viability assays in the presence of subject’s post -DC vaccination T -cells. 1.9 Study Rationale Recent advances provide compelling evidence that the immune system plays a significant role in controlling cancer. Breakthrough treatments such as immune checkpoint inhibitors a nd CAR T cells demonstrate new therapeutic strategies which can translate into durable disease control rates, responses and even complete remissions in patients with advanced malignancies. For patients with stage II, MMR deficient CRC, no accepted adjuvant regimen exists. Although recurrence rates are low in this population, the development of therapies to further improve upon outcomes hold appeal. Patients with hypermutated CRC defined by the CMS1 subgroup represent a unique opportunity to evaluate personalized cancer vaccines which have be en shown in melanoma to elicit tumor- specific T cells and mediate complete regression of metastatic disease after administration of anti-PD-1 antibody in select cases [17, 18] . Moreover, personalized cancer vaccines are safe and well tolerated [17, 18, 19]. 1.10 Study Design This is a pilot study to assess the safety and tolerability , as well as the immune response rate, of mDC3 /8 vaccine in patients with colorectal cancer . The duration of a subject’s participation in this study will be approximately 12 months from receipt of the first mDC3/8 vaccine infusion. Approximately 12 subjects wil l be enrolled in this study. Additional secondary/exploratory objectives are described below. 2.0 OBJECTIVES 2.1 Primary Objective 1. To as sess the immune response rate to DC vaccination in subjects with hypermutated CRC. 2. To determine the safety of DC vaccination in subjects with surgically resected hypermutated CRC. 2.2 Secondary Objective 1. To determine the percentage of CD8+ cells in the primary tumor tissue. 2.3 Exploratory Objectives 1. To evaluate tumor and immune biomarkers and their association with treatment outcomes. 2. To isolate and characterize TIL from metastatic tumors that relapse after study vaccine treatment. DC Vaccine for Colorectal Cancer Page 10 of 34 Version 7.11-11-2021 CONFIDENTIAL This protocol is the property of the University of Pennsylvania. Do not disclose or use except as authorized in writing by the study spons or. 3.0 ELIGIBILITY CRITERIA Patients must fulfill the following eligibility requirements: 3.1 Inclusion Criteria 1. Pathologically -confirmed stage I and II hypermutated colorectal cancer (CRC). Hypermutated CRC is defined by one of the following categories: a. Microsatellite instability – high (MSI- H) by PCR. MSI -H is defined as two or more of the five markers for microsatellite instability being positive. These markers are: BAT25, BAT26, D2S123, D5S346 and D17S250. b. Mismatch repair deficient (MMRd) as defined by loss of expression of MMR protein(s) (MLH1, MSH2, MSH6, and/or PMS2) on MMR immunohistochemistry (IH C), which includes hereditary syndromes such as Lynch syndrome and congenital mismatch repair deficiency (CMMRD) as well as CRCs with somatic MMRd . c. Those arising in patients with polymerase proofreading associated polyposis (PPAP) with inherited mutations in either POLE or POLD1 . [Hypermutated malignancies including CRC typically harbor >10 mutations/Mb]. 2. Surgically resected disease 3. Male or f emale patients age ≥ 18 years 4. ECOG performance status 0 -1 5. Required initial laboratory values ( performed within 14 days prior to eligibility confirmation by physician- investigator ): a. WBC >3 THO/µL b. Hg ≥ 9.0 gm/dl c. Platelets >75 THO/µL d. Serum T otal Bilirubin ≤ 2.0 mg/dl ; unless the subject has known or suspected Gilbert’s syndrome for which ≤ 3 mg/dl is permitted . e. Serum Creatinine < 2.0 mg/dl 6. Subjects of reproductive potential must agree to use a medically accepted birth control method during the trial and for at least two months following the trial . Ple ase see Section 5.0 for additional details. 7. Provide written informed consent. 3.2 Exclusion Criteria 1. Prior malignancy within 3 years that, in opinion of the physician- investigator, would put subject at additional risk 2. Pregnant or nursing (lactating) women 3. Concurrent t reatment with systemic immunosuppressants including corticosteroids (e.g prednisone), calcineurin inhibitors (e.g tacrolimus, cyclosporine), antiproliferative agents (e.g mycophenolate mofetil, azathioprine) within 2 weeks of eligibility confirmation by physician - investigator . Local (inhaled or topical) steroids or replacement dose prednisone ( ≤10 mg daily) are permitted . 4.