Th17-inducing autologous dendritic cell vaccination promotes antigen-specific cellular and humoral immunity in ovarian cancer patients

In ovarian cancer (OC), IL-17-producing T cells (Th17s) predict improved survival, whereas regulatory T cells predict poorer survival. We previously developed a vaccine whereby patient-derived dendritic cells (DCs) are programmed to induce Th17 responses to the OC antigen folate receptor alpha (FRα). Here we report the results of a single-arm open-label phase I clinical trial designed to determine vaccine safety and tolerability (primary outcomes) and recurrence-free survival (secondary outcome). Immunogenicity is also evaluated. Recruitment is complete with a total of 19 Stage IIIC-IV OC patients in first remission after conventional therapy. DCs are generated using our Th17-inducing protocol and are pulsed with HLA class II epitopes from FRα. Mature antigen-loaded DCs are injected intradermally. All patients have completed study-related interventions. No grade 3 or higher adverse events are seen. Vaccination results in the development of Th1, Th17, and antibody responses to FRα in the majority of patients. Th1 and antibody responses are associated with prolonged recurrence-free survival. Antibody-dependent cell-mediated cytotoxic activity against FRα is also associated with prolonged RFS. Of 18 patients evaluable for efficacy, 39% (7/18) remain recurrence-free at the time of data censoring, with a median follow-up of 49.2 months. Thus, vaccination with Th17-inducing FRα-loaded DCs is safe, induces antigen-specific immunity, and is associated with prolonged remission.

: Generation of IFN- + T cell, IL-17 + T cell and humoral immunity to whole FRprotein is linked to generation of immunity to vaccine peptides. Shown in Panels A-E are correlation analyses comparing the magnitude of maximal peptide-specific IFN- + T cell response to the maximal FR IFN- + response. Each panel represents a unique peptide and each symbol represents a unique patient (n=18). Panels F-J and Panels K-O show the same for IL-17 + T cell and antibody responses, respectively. r = spearman correlation rho coefficient and unadjusted 2-sided P values indicate significant deviation from zero slope. Inset best-fit lines were calculated using non-linear least squares regression for data trend visualization.           Ovarian cancer is the most lethal gynecologic malignancy, with a 5-year survival rate of 45% due to the fact that it is rarely diagnosed while the cancer is still localized [1]. After initial surgical debulking, standard chemotherapeutic agents used include platinum/ taxane combinations. Nearly 80% of patients with advanced stage disease respond to these drugs [1]. However, at least 70% of patients with an initial complete clinical response to treatment will subsequently experience recurrent disease. While second-line treatments are available and widely used, once ovarian cancer recurs it is not generally considered curable. Maintenance chemotherapies after completion of initial postoperative chemotherapy are occasionally prescribed, but no regimen has been shown to extend overall survival or improve the cure rate. Novel adjuvant strategies to prevent or delay recurrence of ovarian cancer are desperately needed.

Role of the immune system in the clinical course of ovarian cancer
Many studies have demonstrated the importance of the immune system in ovarian cancer patient outcome. Notably, Zhang and colleagues demonstrated that CD3 T cell infiltration was positively associated with survival [2]. These investigators also found that patients with tumor-infiltrating T cells were more likely to be optimally debulked at surgery, suggesting that T cell infiltration may directly limit disease spread.
Despite strong evidence for anti-tumor immunity, it has become increasingly apparent that ovarian tumors avail themselves of multiple mechanisms of immune evasion, the most prominent of which is recruitment and infiltration of regulatory T cells that suppress anti-tumor immunity. Regulatory T cells (Treg) are recruited to ovarian tumors by the chemokine CCL22 (predominantly expressed by ovarian tumors), and the presence of Treg confers immune privilege and is associated with a poor prognosis and increased mortality [3]. Other investigators have corroborated these observations, showing that high expression of the forkhead box transcription factor Foxp3, which is preferentially expressed by CD4 + Treg, is an independent prognostic factor for reduced overall survival in ovarian cancer [4], and that a high CD8 + T cell/Treg ratio is associated with a more favorable prognosis for this disease [5]. Further mechanisms that contribute to the immunosuppressed state include expression of B7-H1, which can promote T cell anergy and apoptosis through engagement of PD-1 expressed by effector T cells [6,7] and expression of indoleamine 2,3-dioxygenase (IDO). Expression of both B7-H1 and IDO are associated with differentiation and recruitment of Treg [8][9][10], and clinical studies have shown that each of these mechanisms correlates independently with increased morbidity and mortality in ovarian cancer patients [11][12][13].

Th17-based immunotherapy and vaccination
In contrast with the evidence that Treg infiltration is associated with poor outcomes in ovarian cancer, a counterpoint is furnished by the recent observation that Th17 T cell infiltration correlates with more favorable clinical outcomes [14] (manuscript included in Section 8). Tumor-infiltrating Th17 cells were positively associated with effector cells and negatively associated with Treg infiltration [14], with the latter relationship arguably being founded on the known reciprocal regulation of Treg and Th17 differentiation [15,16]. These observations have led to the question of whether Th17 cells could be induced or expanded to therapeutic advantage, either by tumor vaccines or adoptive immunotherapy [17] (manuscript included in Section 8).
Current knowledge of the immunopathology of ovarian cancer presents a strong case in favor of Th17-based anti-tumor immunotherapy. This point is further supported by a recent report showing that human Th17 T cells are long-lived effector memory cells with the capacity to mediate effective antitumor immunity in collaboration with CD8 T cells. It was also found that Th17 cells were relatively resistant to apoptosis, and that apoptosis and persistence were regulated by high expression of HIF-1α, suggesting that Th17 cells may have a survival advantage in the hypoxic tumor microenvironment.

Dendritic cell vaccination in ovarian cancer
Dendritic cells (DCs) are myeloid-lineage immune cells and are primarily responsible for presenting antigens to T cells during the initiation of an adaptive immune response.
Although DC vaccination has been tested for the treatment of many other malignancies, clinical studies of DC vaccination in ovarian cancer have been limited. DC pulsed with killed autologous primary ovarian tumor cells induced antigen-specific T cells that secreted IFNγ upon stimulation with autologous tumor cells [18], suggesting that antigenpulsed DC may be a viable option for therapeutic vaccination against ovarian cancer. Various reports showing that DC loaded with tumor lysates, DC pulsed with acid-eluted peptides from ovarian cancer cells, DC fused with ovarian tumor cells, or DC loaded with ovarian tumor cells killed by oxidation could induce HLA class I-restricted CTL responses against autologous ovarian tumor cells [19][20][21][22] support this position. A limitation of these approaches is that the identity of the tumor antigens recognized by DC-stimulated CTL is not well defined, and it is not clear that T cell responses retain specificity for the tumor.
A phase I trial of autologous tumor antigen-loaded DC vaccination in 6 patients with ovarian cancer revealed no significant toxicity and 3 of 6 patients showed stable disease lasting 25 to 45 weeks [23]. Lymphoproliferative responses to tumor antigen were detected in 2 patients. Follow-up CT at 5 months after the last vaccination showed a partial response, and CT at 16 months showed greater than 50% remission of lymph node metastases. CA-125 levels were greatly reduced after the 1 st vaccination (from 640 U/mL to 60 U/mL) and remained at baseline 11 months after completion of vaccination. A clinical trial of MUC1 and HER2/neu peptide-pulsed DC vaccination in patients with advanced ovarian or breast cancer reported peptide-specific CTL responses in 5 of 10 patients, and also showed evidence of epitope spreading [24]. In one patient vaccinated with MUC1 peptides, carcinoembryonic antigen and MAGE3 peptide-specific T-cell responses were detected, and in a second patient, MUC1-specific T-cell responses were detected after seven vaccinations with HER2/neu peptide-pulsed DC.
The most durable clinical response to DC vaccination was described in a case report of a patient with recurrent metastatic ovarian cancer, who received 10 vaccinations of autologous DC loaded with mRNA encoding folate receptor-α [25].

1.5
The folate receptor alpha as a vaccine target antigen The tumor antigen targeted by the DC vaccine in this protocol is folate receptor alpha (FRα), a high affinity folate-binding protein that is overexpressed on 70-90% of ovarian tumors [26]. It is expressed at low levels in a small number of other tissues in the body where it functions to move folic acid from one compartment to another. For example, in the kidneys, FRα is responsible for retrieving folate from the urine prior to its excretion; in the central nervous system, FRα appears to concentrate folate in cerebrospinal fluid. It has recently been shown that FRα−expression is common in metastatic foci present at the time of diagnosis of ovarian cancer as well as on recurrent tumors, indicating that FRα is a good tumor target regardless of whether the patient is newly diagnosed or experiencing disease recurrence [27] (manuscript included in Section 8).

1.6
Pre-clinical human and mouse studies evaluating immunity to FR

Identification of FRα-derived peptide epitopes
Since the FRα is a self-antigen, there is likely to be some level of tolerance to the antigen. Thus, in order to target the protein in vaccine strategies, it was necessary to define epitopes (i.e. antigenic fragments) to which there remained a T cell repertoire. We hypothesized that such epitopes could be identified in patients with either ovarian or breast cancer who would have recognized the antigen during the clinical course of their disease and generated an immune response. To do this, the sequence of human FRα was screened as described [28] for peptides predicted to bind HLA class II molecules using RANKPEP. Fourteen peptides (15-18 amino acids in length) predicted to bind to 3 or more HLA DR molecules were selected for further testing (Figure 1) (manuscript included in Section 8).
The peptides were chosen based on their ability to bind to several different HLA class II antigen presenting molecules. HLA class II antigens, expressed on antigen presenting cells, present peptides to CD4 T cells. The ability of the peptides to bind to multiple HLA class II molecules makes it likely that each patient would have antigen presenting molecules that could bind to some of the peptides, so the vaccine should be able to increase immune responses in most people. The peptides in Figure 1 were synthesized and peripheral bloods from 30 patients and 18 normal healthy volunteers were assessed for immune responses to each epitope and the whole protein using T cell ELIspot assays and antibody ELISAs. ELIspot analysis identified four peptides (FR30, FR56, FR 113, and FR238) that generated responses in more patients than in healthy donor counterparts ( Figure 2). Overall, 70% of these patients showed FR-specific immunity to at least one epitope of FRα (Figure 3). This demonstrates that the ability to mount an immune response against FRα is intact and therefore should be able to be boosted. The same set of FRα peptides (see Table 1, Section 1.5) are currently being evaluated in a Phase I trial of FRα peptide vaccination (Mayo Clinic protocol MC1015, ClinicalTrails.gov Identifier NCT01606241, IND #14546).

Dendritic cell activation of Th17 responses
This clinical trial is based on the premise that DC vaccination designed to drive a tumor antigen-specific Th17 T cell response holds the potential to be of clinical benefit for patients with ovarian cancer. DC are remarkable for their plasticity in directing T cell differentiation and effector function, and thus the key to success may reside in our ability to educate DC to drive ovarian tumor antigen-specific Th17 responses.
Several studies have indicated that regulation of the p38 and ERK MAPK signal transduction pathways in DC plays a central role in direction of T cell differentiation. Inhibition of MEK 1/2 and ERK MAPK signaling promotes IL-12 production and Th1 T cell responses, whereas inhibition of p38 MAPK increases signal transduction through ERK 1/2 and blocks IL-12 production [29]. At face value, these observations suggest that inhibition of p38 MAPK signaling would be disadvantageous for DC-driven anti-tumor T cell responses, since this would abrogate Th1 responses. However, p38 inhibition promotes differentiation and survival of monocyte-derived DC [30], and p38 inhibition or MEK/ERK MAPK activation restores deficiencies in DC function in myeloma patients [31], suggesting that treatment of DC with pharmacological inhibitors of p38 signaling may confer benefit. Of particular significance, blockade of the p38 pathway can attenuate regulatory T cell induction by DC [32], whereas blockade of the ERK pathway suppresses DC-driven Th17 responses [33], suggesting that p38 blockade (which enhances ERK phosphorylation) may favor a switch from Treg induction to Th17 differentiation and expansion.

Preclinical studies
Treatment of ovarian tumor antigen-loaded, cytokine-matured DC with a combination of IL-15 and a p38 MAPK inhibitor affords synergy in antagonism of Treg induction and redirection toward Th17 responses that correlate with strong CD8 + CTL activation [34] (manuscript included in Section 8). Furthermore, DC vaccination of mice bearing advanced ovarian tumors has shown that ex vivo pharmacological inhibition of p38 signaling in vaccine DC yields an enhanced survival rate that correlates with increased Th17 frequencies.
Outcomes of IL-15/p38 MAPK inhibitor treatment of human monocyte-derived DC, relative to cytokine-matured DC: a. Diminished activation and expansion of Foxp3 + CD4 + Treg and strong activation of tumor antigen-specific Th17 responses. b. Potent activation of tumor antigen-specific CD8 + CTL responses. c. B7-H1 has been implicated in differentiation of adaptive Foxp3 + Treg [8], and p38 MAPK inhibition leads to loss of B7-H1 expression by DC, suggesting a possible mechanism for reduced recruitment and activation of CD4 + Foxp3 + Treg. d. Diminished CD4 + T cell expression of CTLA-4, a co-inhibitory ligand associated with Treg function.
Protocol Version Date: 17Apr2019 e. Diminished CD4 + T cell expression of PD-1, indicating the potential for reduced susceptibility to B7-H1 (PDL-1)-induced apoptosis or anergy in the tumor microenvironment. f. Diminished DC expression of CD80 and CD86, but conserved expression of ICOS-L, suggesting a pattern of costimulation that favors Th17 responses [35]. g. IDO expression by DC contributes to Treg responses [9,10]. IDO also inhibits Th17 responses [36], suggesting that IDO expression may play a key role in regulation of the Treg/Th17 balance. Inhibition of p38 signaling ablates IDO activity in DC. h. Multiplex and flow cytometric analyses show increased ERK phosphorylation following p38 inhibition, suggesting that signal transduction via the ERK MAPK pathway in DC is associated with recruitment of Th17 responses. i. In the ID8 mouse model of ovarian cancer, therapeutic vaccination with cytokine matured rAAV-SP17-transduced DC prolonged survival of tumor-bearing mice, but all animals succumbed to disease. In contrast, therapeutic vaccination with DC treated with a p38 MAPK inhibitor resulted in disease-free survival of >300 days in 19/20 vaccinated animals.
Collectively, these results support the proposal that treatment of DC with p38 MAPK inhibition plus IL-15 will drive ovarian tumor antigen-specific Th17 and CTL responses, and further suggest that this innovative approach may offer the potential for effective DC vaccination against ovarian cancer.

Clinical data to date
There are no available clinical research data to date on the investigational product.

1.9
Dose Rationale and Risk/Benefits

Dosage, dosage regimen and dosage period
FRα peptide-pulsed DC will be administered at a vaccine dose of 15 x 10 6 DC (allowable range as release criterion is 10-20 x 10 6 ). The vaccine volume will be 0.8 mL and will be administered intradermally in order to increase proximity of vaccination and local lymph node draining basins for stimulation of the immune response. Approximately 0.1 mL will be injected in a single site, with the total volume being divided into eight injections split between two areas. The same areas may be used for repeated vaccinations, although areas may be rotated from cycle to cycle. Alternatively, the vaccine dose may be administered via the 3M hollow Microneedle Transdermal system (hMTS). The entire vaccine dose is loaded into the hMTS device and the cells are subsequently delivered at one site. The useable injections sites are the same as the BD microinjection needle. Only individuals trained in the use of the 3M hMTS device can perform the injections.
Five DC vaccines will be administered at 21 day intervals, plus or minus 3 days to maximize patient convenience and protocol adherence. The dose and schedule is based on past experience of DC vaccine trials for gynecological malignancies [37].
Patients who tolerate treatment and do not have symptomatic recurrence of OC may continue therapy to include vaccination every 3 months from the completion of the 5 th vaccination for a period of up to 2 years, or until progressive/recurrent disease is confirmed. Duration of potential extension, and the option for such treatment, will be Protocol Version Date: 17Apr2019 patient specific as supply of additional vaccinations varies based on quantity produced at study initiation.

Rationale for selection of dose
A phase I dose escalation clinical trial of DC vaccination in patients with cervical cancer indicated optimal stimulation of tumor antigen-specific T cell responses with a dose of 1.5 x 10 7 DCs in injection-grade saline containing 20% heat-inactivated autologous serum, delivered s.c. and intradermal (intradermal preferred) at 14 day intervals [37].
Leukapheresis (10 liter volume) followed by CliniMACS isolation of CD14 + cells and high density DC culture in G-Rex flasks provided an optimal yield of 2.5 x10 8 mature DC from 10 9 CD14 + cells. This yield is sufficient for cryopreservation of 12 vials at 2 x10 7 DC/vial, which is the maximum number of DC vaccine treatments under this protocol (see Section 5.3).

Potential risks and benefits
Known toxicities associated with DC vaccination: • DC vaccination has not resulted in significant systemic toxicity in the clinical trials that have been performed. Of the adverse effects noted, mild symptoms, such as lowgrade fever and local reactions at the injection site, were most common.
• In a recently conducted phase I clinical trial of DC vaccination of patients with earlystage cervical cancer (BB-IND 11307), no adverse side effects were observed or reported by subjects following immunization beyond the immediate discomfort associated with injection. We noticed, however, local reactions (mild erythema, swelling/induration, pruritus) at the subcutaneous vaccination sites that increased with the number of vaccinations in most of the patients. We also observed a slight enlargement in the draining lymph node in the groin after DC injections in some of the patients. The patients were monitored during treatment with complete blood counts and serum chemistries that included liver and renal function tests and electrolytes. No alterations in liver and renal function were detected.
• One of the major concerns regarding DC vaccination with self tumor antigens is the possible induction of autoimmunity. Vitiligo has been seen in some melanoma patients, but no cases of severe autoimmune reactions have been reported. The target antigen in this trial, FRα, is expressed at low levels in some normal tissues.
• The possible risks of stimulation of Th17 responses through DC vaccination in ovarian cancer patients are not known. Although a number of studies have documented potent anti-tumor activity for Th17 T cells, other studies have reported pro-angiogenic and tumor-promoting properties ascribed to Th17 immune responses [38,39]. Th17 responses are pro-inflammatory, and may be associated with as yet unknown side effects. All subjects will be monitored closely for treatment-related adverse events and dose-limiting toxicities.
Known toxicities associated with FRα vaccination: Vaccination with FRα peptides with GM-CSF as an adjuvant is currently being tested in the clinical protocol MC1015. Thus far, 18 patients have initiated treatment, and 7 patients have completed planned study treatment. One patient had grade 4 sepsis, which was attributed as unrelated to treatment. One patient had a grade 3 injection site reaction manifesting as an ulceration. Grade 2 adverse events are as shown in Table 2:

2.36
Determine whether FRαDC vaccination is associated with changes in peripheral blood immune cell subsets.

2.37
Determine whether FRαDC vaccination leads to increases in plasma antibodies directed against OC-associated antigens, and whether changes in antibody levels are associated with recurrence-free survival.
Protocol Version Date: 17Apr2019

Patient Eligibility
Prior to discussing protocol entry with the patient, call the MCCC Registration Office to insure that a place on the protocol is open to the patient.

3.12
Histologically confirmed surgical diagnosis of stage IIIC or stage IV epithelial ovarian, fallopian tube, or primary peritoneal cancer. Patients with stage III cancer must have had peritoneal metastasis beyond pelvis more than 2 cm in greatest dimension and/or regional lymph node metastasis. NOTE: Histologic confirmation of the primary tumor is required. Eligible histologies include serous, endometrioid, clear cell, mucinous, transitional cell, undifferentiated, or mixed carcinoma.

3.13
Completion of cytoreductive surgery and has completed one (and only one) course of platinum-based chemotherapy (5-9 cycles) ≥ 4 but ≤20 weeks prior to registration.
NOTE: Cytoreductive surgery may have been prior to or after the first cycle of chemotherapy but must include hysterectomy and bilateral salpingooophorectomy, if the uterus and/or ovaries had not previously been removed.
NOTE: Patients may have had more than one chemotherapy regimen (ex: paclitaxel/ carboplatin switched to docetaxel/carboplatin due to allergy; weekly treatment switched to every 3 week treatment due to intolerance), but may not have received a separate course of treatment for recurrent OC.
NOTE: Patients may receive both neoadjuvant and adjuvant chemotherapy provided both regimens are platinum-based and total 9 or fewer chemotherapy cycles.
3.14 No evidence of disease at the time of registration, including no clinical concern for disease recurrence based on each of the following: • No evidence of disease by history and physical exam • CA125 within normal limits • CT abdomen/pelvis demonstrating no radiological evidence of disease performed after completion of chemotherapy ≤28 days before entering study 3.15 ECOG performance status 0 or 1 (Appendix I).

3.16
The following laboratory values obtained ≤28 days prior to registration.

3.24
Receiving any other investigational agent which would be considered as a treatment for the primary neoplasm.

3.25
Other active malignancy ≤3 years prior to registration. EXCEPTIONS: Non-melanotic skin cancer or carcinoma-in-situ of the cervix. NOTE: If there is a history or prior malignancy, they must not be receiving other specific treatment for their cancer.

3.26
History of myocardial infarction ≤6 months prior to registration, or congestive heart failure requiring use of ongoing maintenance therapy for life-threatening ventricular arrhythmias.

3.27
Epithelial ovarian cancer of low malignant potential (borderline tumor).

3.28
Treatment with chemotherapy, radiation therapy, or other immunotherapy ≤4 weeks prior to registration.
3.29a Immunosuppressive therapy (excluding topical steroids) for any other condition ≤4 weeks prior to registration.
3.29b Persistent fever (>24 hours) documented by repeated measurement ≤4 weeks prior to registration An optional correlative research component is part of this study, there will be an option to select if the patient is to be registered onto this component (see Section 17).
• Patient has/has not given permission to give her tissue sample for research testing.

6.3
Documentation of IRB approval must be on file in the Registration Office before an investigator may register any patients.
In addition to submitting initial IRB approval documents, ongoing IRB approval documentation must be on file (no less than annually) at the Registration Office If the necessary documentation is not submitted in advance of attempting patient registration, the registration will not be accepted and the patient may not be enrolled in the protocol until the situation is resolved. When the study has been permanently closed to patient enrollment, submission of annual IRB approvals to the Registration Office is no longer necessary.

6.4
Prior to accepting the registration, registration application will verify the following: • IRB approval at the registering institution • Patient eligibility • Existence of a signed consent form • Existence of a signed authorization for use and disclosure of protected health information 6.5 At the time of registration, the following will be recorded: • Patient has/has not given permission to store and use his/her sample(s) for future research of ovarian cancer at Mayo. • Patient has/has not given permission to store and use his/her sample(s) for future research to learn, prevent, or treat other health problems. • Patient has/has not given permission for MCCC to give his/her sample(s) to researchers at other institutions. 7.323 If two or three of the first three patients experiences DLT, the study will be temporarily closed until the data are reviewed by the study investigators and the Data Safety Monitoring Board.

7.33
Three additional patients will be treated with cycle 1 of the vaccine and observed for a minimum of 21 days, to assess toxicities, before new patients are treated.
7.321 If zero or one of the first six patients experiences DLT, the study will open to accrual for all remaining patients.
7.322 If two or more of the first six patients experiences DLT, the study will be temporarily closed until the data are reviewed by the study investigators and the Data Safety Monitoring Board.

7.34
Investigators are to contact the Study Chair as soon as any dose-limiting toxicity (DLT) occurs.

Antiemetics
Antiemetics may be used at the discretion of the attending physician.

Full supportive care
Patients should receive full supportive care while on this study. This includes blood product support, antibiotic treatment, and treatment of other newly diagnosed or concurrent medical conditions. All blood products and concomitant medications such as antidiarrheals, analgesics, and/or antiemetics received from the first day of study treatment administration until 30 days after the final dose will be recorded in the medical records.

9.3
Acute vaccine reactions 9.31 Fever: Fever may be treated symptomatically with acetaminophen 650-1000 mg by mouth every six hours as needed. Maximum total daily dose of acetaminophen is 4000 mg/24 hrs.

9.32
Myalgias/arthralgias: Myalgias and arthralgias may be treated symptomatically with acetaminophen 650-1000 mg by mouth every six hours as needed.

9.33
Injection site reactions: Erythema and induration are common injection site reactions that are expected to be self-limited. Injection site reactions should not be treated unless causing significant symptoms. If intense pruritis occurs, this should be treated with over-the-counter topical or antihistamines. If topical antihistamines are ineffective, over-the-counter oral anti-histamines and/or topical steroids may be used.

Immune-related adverse events
Immune-related adverse events are toxicities associated with vaccine treatment for which the pathophysiology is consistent with an immune mechanism. If dose-limiting immunerelated adverse events occur, these should be treated with prednisone 1 mg/kg, given once daily or in two divided doses per the discretion of the treating investigator. Prednisone should continue at the starting dose for at least two weeks and until symptoms improve to grade 1 or lower. At that point, prednisone may be tapered per the discretion of the treating investigator.

No ice on injection site
Ice should not be used on injection sites within 24 hours of vaccine administration.

Each CTCAE term in the current version is a unique representation of a specific event used for medical documentation and scientific analysis and is a single MedDRA
Lowest Level Term (LLT). Grade is an essential element of the Guidelines and, in general, relates to severity for the purposes of regulatory reporting to NCI. NOTE: A severe AE, as defined by the above grading scale, is NOT the same as serious AE which is defined in the table in Section 10.4.

Expected vs. Unexpected Events
• The determination of whether an AE is expected is based on agent-specific information provided in Section 15.0 of the protocol and the study specific consent form. • Unexpected AEs are those not listed in the agent-specific information provided in Section 15.0 of the protocol and the study specific consent form. NOTE: "Unexpected adverse experiences" means any adverse experience that is neither identified in nature, severity, or frequency of risk in the information provided for IRB review nor mentioned in the consent form.

10.3
Assessment of Attribution When assessing whether an adverse event is related to a medical treatment or procedure, the following attribution categories are utilized: Definite -The adverse event is clearly related to the agent(s). Probable -The adverse event is likely related to the agent(s).
Possible -The adverse event may be related to the agent(s). Unlikely -The adverse event is doubtfully related to the agent(s). Unrelated -The adverse event is clearly NOT related to the agent(s).
Events determined to be possibly, probably or definitely attributed to a medical treatment suggest there is evidence to indicate a causal relationship between the drug and the adverse event. Any death more than 30 days after the patient's last study treatment or procedure that is felt to be at least possibly treatment related must also be submitted as a Grade 5 AE, with a CTCAE type and attribution assigned.
Protocol Version Date: 17Apr2019

11.1
For the purposes of this study, patients should be re-evaluated for tumor recurrence with physical exam and laboratory testing every three months, including CA-125.
11.11 Imaging evaluation will take place at the discretion of the treating investigator if there is clinical or laboratory suspicion for ovarian cancer recurrence, including concerning clinical findings or increase in CA-125 >35 U/ml.

11.12
If a patient has a CA-125 >35 U/ml, then a second CA-125 should be obtained at least three weeks later.

11.2
At the time of reevaluation, patients will be classified in the following manner: 11.21 No evidence of disease (NED): Patients will be considered to have NED if both of the following conditions are met: 11.211 The patient has a CA-125 ≤35 U/mL and has no symptom that the treating investigator suspects to be due to disease recurrence. (If a single CA-125 level is >35 U/mL, the patient is still considered NED until a confirmatory CA-125 at least 3 weeks after the initial elevated CA-125 is also >35 U/mL.) 11.212 There is no evidence of disease on any imaging tests performed.
(Imaging is not required unless deemed clinically necessary by the treating investigator.) 11.22 Recurrent disease (PD): Patients will be considered to have PD if any of the following conditions are met: 11.221 The patient has a CA-125 >35 U/mL that has been confirmed by a second elevated CA-125 at least 3 weeks later.
11.222 There is evidence of disease on any imaging test performed.

13.1
Patients who are NED will continue treatment per protocol.

13.2
Patients who develop symptomatic PD while receiving therapy will go to the eventmonitoring phase.

13.3
Patients who develop PD but do not have any symptoms that the treating investigator believes are caused by EOC may continue treatment per protocol for as long as they remain asymptomatic and the treating investigator and patient believe there is benefit to continuing therapy.

13.4
Patients who go off protocol treatment for reasons other than PD will go to the eventmonitoring phase per Section 18.0.

13.5
If a patient fails to complete the first cycle of treatment for reasons other than toxicity, the patient will be regarded as non-evaluable and will be replaced.

13.6
A patient is deemed ineligible if after registration, it is determined that at the time of registration, the patient did not satisfy each and every eligibility criteria for study entry. The patient will go directly to the event-monitoring phase of the study (or off study, if applicable).
• If the patient received treatment, all data up until the point of confirmation of ineligibility must be submitted. Event monitoring will be required per Section 18.0 of the protocol. • If the patient never received treatment, on-study material must be submitted. Event monitoring will be required per Section 18.0 of the protocol.

13.7
A patient is deemed a major violation, if protocol requirements regarding treatment in cycle 1 of the initial therapy are severely violated that evaluability for primary end point is questionable. All data up until the point of confirmation of a major violation must be submitted. The patient will go directly to the event-monitoring phase of the study. The patient may continue treatment off-protocol at the discretion of the physician as long as there are no safety concerns, and the patient was properly registered. Event monitoring will be required per Section 18.0 of the protocol.

13.8
A patient is deemed a cancel if he/she is removed from the study for any reason before any study treatment is given. Collect 20 mL with no additive (two 10 mL red top tubes) and 180 mL with sodium heparin (eighteen 10 mL green top tubes or three 60 mL heparin syringes).
14.212 Prior to each cycle of treatment: Collect 5 mL with no additive (one 5 mL red top tubes) and 20 mL with sodium heparin (two 10 mL green top tubes). MCCC Addendum 6 Protocol Version Date: 17Apr2019 14.213 Three weeks after Cycles 5 and 12: Collect 20 mL with no additive (two 10 mL red top tubes) and 180 mL with sodium heparin (eighteen 10 mL green top tubes or three 60 mL heparin syringes).
14.214 At the time of disease recurrence due to PD (mandatory): Collect 20 mL with no additive (two 10 mL red top tubes) and 180 mL with sodium heparin (eighteen 10 mL green top tubes or three 60 mL heparin syringes).
14.22 Blood processing

No additive whole blood (red top tubes)
No mixing is required, but keep in upright position during transport.
14.222 Sodium heparin blood (green top tubes or sodium heparin syringes) Immediately mix at least 10 times by gentle inversion.
14.3 Shipping and handling 14.31 Kits will not be used for this study.
14.32 Send blood samples to Guggenheim 3-23, Mayo Clinic Rochester, Attn: Courtney Erskine via Mayo General Service.
14.4 Background and methodology

ELISpot Assays (enzyme-linked immunosorbent spot assays)
A 2-day ELISpot, which detects both activated and memory T cell effectors will be used to detect immunity to FRα as previously described [28,40] (manuscript for reference 28 included in Section 8). Furthermore, the ELIspot assay is able to measure the frequency of the responding T cells per unit of peripheral blood mononuclear cells (PBMCs). On day 1, 3 x10 5 PBMCs/well will be plated into 96-well plates in 3-well replicates in 200 µl of RPMI-1640 containing Lglutamine, penicillin, streptomycin, and 10% fetal calf serum (T-cell medium) in the presence or absence of 10 mcg/ml peptide antigen, 1 mcg/ml protein, or 1 mcg/ml tetanus vaccine. The cells will be incubated at 37°C for 42-46 hours followed by washing three times with phosphate-buffered saline (PBS) containing 0.05% Tween-20. The plate will be incubated for 2 hours at 37°C in PBS with 5 mcg/ml biotinylated anti-cytokine Ab, washed in PBS, and further incubated with 100 mcl/well avidin-horseradish peroxidase (HRP, Vector Laboratories, Burlingame, CA) for 1 hour at room temperature. The anti-cytokine and biotinylated anti-cytokine antibody pair will be obtained from Mabtech (Sweden). After 3 washes in PBS, the plate will be incubated with 100 mcl/well HRP-colorimetric substrate (Vector Laboratories) for 5-20 minutes, rinsed with cool tap water, and allowed to dry completely. The nitrocellulose plates will be read on an AID ELIspot reader (Cell Technology, Inc., Columbia MD, reader software v.3.1.1.). A positive response is defined as a frequency that is significantly (p < 0.05, two-tailed t test) greater than the mean of control noantigen wells and detectable (i.e., >1:100,000). A15-amino acid HLA-DR binding irrelevant peptide from Cyclin D1 will be used as a negative control peptide. Phytohemagglutinin (PHA) will be used as a positive control. The CEF viral peptide pool from the NIH AIDS Research and Reference Reagent Program will be used to evaluate non-specific increases in immunity [28]. The CEF Control Peptide Pool is a group of 32 peptides, 8-12 amino acids in length, with sequences derived from the human Cytomegalovirus, Epstein-Barr Virus and Influenza Virus. A vaccine-induced increase in FRα-specific T cell responses will be defined as (1) a 2-fold or greater increase in FRα-specific T cells at any point during treatment if there were detectable pre-treatment levels of FRαspecific T cells or (2) FRα-specific T cells at any point during treatment if pretreatment levels of FRα-specific T cells are non-detectable.
14.42 ELISAs (enzyme-linked immunosorbent assays) Antibodies have made outstanding surrogates for response to vaccine. Furthermore, the FRα vaccine in this protocol incorporates a known antibody recognition epitope of FRα. Antigen (10µg/well), peptide or whole protein (tetanus protein will be included as a control) will be prepared in 0.06M carbonate buffer and added to ELISA microtiter plates for 24 hours. Plates will be washed with PBS and blocked with 3% BSA-PBS. One hundred microliters of diluted sera (1:125 for peptide and 1:40 for tetanus toxoid in 1%BSA-PBS) will be added and the plates further incubated for 2 hr at RT followed by washing with PBS/0.1% Tween-20. A 1:2000 dilution of anti-IgG-HRP is then added to wells for 1 hour followed by washing and color development after adding 100 µl TMB (3,3',5,5' tetramethylbenzidine) substrate to the wells. Color development is stopped with 50 µl of a 0.1N HCl solution. For the standard curve, serial dilutions of human IgG will be added to separate wells. As a peptide control, a peptide derived from human collagen II, HII.71 (PPGLTGPAGEPGRQGSPGAD), will be used. Optical densities will be read on a Victor V multiplate reader and concentrations of antibody will be determined using the standard curve with Graphpad Prism (Graphpad Software). A vaccineinduced increase in FRα-specific antibody responses will be defined as (1) a 2fold or greater increase in FRα-specific antibody at any point during treatment if there were detectable pre-treatment levels of FRα-specific T cells or (2) FRαspecific antibodies at any point during treatment if pre-treatment levels of FRαspecific antibodies are non-detectable.
14.43 Peripheral blood immune cell subtype and ancillary studies Cancer patients have well-defined alterations in circulating immune cells and are characterized by systemic reductions in T cell numbers and functions as well as increases in inhibitory cells like immunosuppressive monocytes and regulatory T cells. These systemic changes in patient immunity have significant implications for response to immunotherapy. Therefore, we will perform peripheral blood immunophenotyping using flow cytometry on patient samples. We will use a ten color 8 tube flow cytometry panel that encompasses lymphocyte, monocyte, granulocyte subsets, regulatory T cells, immunosuppressive monocytes and myeloid derived suppressor cells. This analysis will generate a detailed picture of the patient's peripheral blood immunophenotype and how they respond to the vaccine. We will also collect plasma at selected collection points that may be used to measure changes in cytokines and growth factors. We may also collect and store immune cell subtypes (such as T cells and monocytes) for transcriptome analysis or other analyses (DNA sequencing) to identify changes in the behavior of the immune response during the course of therapy. The drug used in this study is patient's autologous dendritic cells (DC). Dendritic cells are cells manufactured in the laboratory from monocytes removed from patients' blood by leukapheresis. The dendritic cells need to acquire tumor antigens in order to stimulate anti-tumor immunity. Patients will be treated with DC exposed to peptides (small protein fragments) from the ovarian cancer-associated protein folate receptor alpha (FRα). DCs loaded with FRα peptides are referred to in this protocol as FRα DCs.

Formulation:
The drug is supplied as recently thawed cells prepared from patient material. Cells are manufactured and released by the Human Cellular Therapy Lab, Mayo Clinic Rochester. Before being released for use, cells will undergo testing for sterility and potency.

Preparation and storage:
Vaccine vials should be stored at less than -150ºC. The drug will be prepared and stored at the Human Cellular Therapy Lab, Mayo Clinic Rochester according to approved SOPs included in the IND. The drug will be delivered in a cooler on wet ice to the appropriate administration area by personnel from the Human Cell Therapy Lab.
15.14 Administration: The vaccine dose may be administered via the 3M hollow In the induction phase, the interval between FRαDC doses is 3 weeks ±3 days. In the maintenance phase, the interval between doses is 3 months ±1 week.

Number and location of intradermal unloaded DC doses (for DTH testing)
On Cycles 1, 5, and 12, patients will receive both FRαDCs and unloaded DCs, which serve as a control for DTH skin testing. Each unloaded DC dose will be administered in the same total volume (0.8 mL) as the FRαDC dose, and should be administered in 8 injections. The unloaded DCs are injected into 4 intradermal sites premarked at least 3 cm apart from each other, at each of two areas of the body. These should be the same 2 areas of the body that the FRαDC vaccine is given for that cycle, but on the contralateral side of the body.
15.143 Intradermal Injection using the BD microneedle  Only individuals trained in the use of the BD microneedle can perform the injections.  Clean the skin with alcohol and allow it to dry prior to injection.  Remove white cap from ID needle.  Holding the syringe in your dominant hand, hold the skin taut with your other hand.  Insert the needle perpendicular to the skin, in a short and quick movement.  Maintain a light pressure on the syringe and inject slowly pushing the plunger with your index finger.  Following injection, keep the needle inserted, remove the index finger, and wait a few seconds to prevent oozing.  Using the same needle repeat injections for each site. The syringe plunger rod will be marked with guidelines for each injection  Remove the needle from the skin and dispose the syringe into a sharps container.  Document administration of the product on the Injection Site Record form (see appendix II). 6. Hold 10 seconds to promote adhesion being careful not to press so hard as to actuate the injector. 7. To actuate the injector, place fingers or palm of hand on the spherical dome of the injector. Press down on the spherical dome quickly to actuate the injector. An audible click will be heard as the injector inserts the microneedle array into the skin. The infusion of the fluid begins immediately upon actuation. The injector will be held securely by the adhesive. 8. Remove hand from the injector. 9. The blue Progress Indicator becomes visible in the clear window during infusion and will advance across the window as fluid is delivered into the intradermal space. NOTE: Depending on the fluid viscosity and delivery location, the infusion may last from 1 to 10 minutes. Patients will be instructed to remain as still as possible. 10. The hMTS device injection is complete when the blue indicator has advanced across the full length of the viewing window. 11. After completion, wait at least 1 minute before removing the injector. 12. To remove the injector, peel off from the skin by placing thumb or finger onto the adhesive tabs and then slowly roll the device to the side to peel adhesive off the skin. NOTE: No post-delivery treatment is required at the injection site. NOTE: Transient blanching and erythema may be observed at the injection site. It is normal to see a bleb where the fluid resides until it is absorbed.

16.1
Study design: This is a single-arm pilot study designed to determin the safety and immunogenicity of folate receptor alpha peptide-loaded dendritic cell vaccination in patients with advanced stage epithelial ovarian cancer. The safety lead-in accrual schedule is based on a 3+3 phase I design.
16.11 Accrual and study duration: This pilot study may involve a minimum of 3 patients and maximum of 22 patients. It is expected that the monthly accrual will be 1.5 patients. Thus the accrual portion of the trial will require approximately 15 months. With the 2 planned stops and approximately 2 months required to assess each cohort, the trial will require 19 months to accrue and assess.
16.12 Accrual Schema: 16.121 Safety lead-in: • Three patients will be accrued and then the study will be temporarily closed to observe for 1 cycle. If there are at least 2 patients with DLTs the study team will decide if further study is desired and a plan will be formulated for the DSMB. • If there are 0 or 1 patients with DLTs, three more patients will be accrued and the study will be temporarily closed to observe for 1 cycle. If there are at least 2 patients with DLTs (out of the first 6) the study team will decide if further study is desired and a plan will be formulated for the DSMB. 16.122 Remaining accrual: If there are fewer than 2 DLTs (out of the first 6 evaluable patients), the final 16 patients will be accrued. 16.222 Overall Survival (OS) is defined as the number of days from study registration until death due to any cause. The Kaplan-Meier method will be used to estimate the distribution of OS.
16.23 Correlative Objectives: For continuous correlate data, change from baseline measures will be calculated to determine if levels have increased. Simple summary statistics (mean and 95% confidence intervals) will be used to assess. For categorical data (e.g. DTH skin reaction which is positive or negative) the percent of each category will be calculated along with a 95% confidence interval.

16.3
Adverse Event Stopping Rule: The principal investigator and the study statistician will review the study periodically (at least twice a year) to identify accrual, toxicity, and endpoint problems that might be developing. The study statistician will prepare a report containing accrual, adverse event, and efficacy data which will be submitted to the Mayo Clinic Cancer Center Data and Safety Monitoring Board (MCCC DSMB) every 6 months until all patients are off study treatment.
During the first 6 patients, if 2 or more have a DLT (as defined in section 7.3), enrollment will be suspended so that the adverse event data can be examined. A trial recommendation will be formulated and presented to the MCCC DSMB.
At any point in the enrollment process after 6 or more patients have been accrued, if more than 30% of these patients develop a DLT (as defined in section This study will be available to all eligible patients, regardless of race, gender, or ethnic group. Due to the site of disease, this study will accrue only female patients.
There is no information currently available regarding differential treatment effects in subsets defined by race or ethnicity, and there is no reason to expect such differences exist. Therefore, although the planned analyses will, as always, look for differences in treatment effect based on gender and racial groupings, the samples sizes are not increased in order to provide additional power for such subset analyses.
The geographic area that Mayo Clinic Rochester serves has a very small minority population. It is expected that only 5% of eligible patients are of minority background. Hispanic or Latino -a person of Cuban, Mexican, Puerto Rican, South or Central American, or other Spanish culture or origin, regardless of race. The term "Spanish origin" can also be used in addition to "Hispanic or Latino." Not Hispanic or Latino Racial Categories: American Indian or Alaskan Native -a person having origins in any of the original peoples of North, Central, or South America, and who maintains tribal affiliations or community attachment. Asian -a person having origins in any of the original peoples of the Far East, Southeast Asia, or the Indian subcontinent including, for example, Cambodia, China, India, Japan, Korea, Malaysia, Pakistan, the Philippine Islands, Thailand, and Vietnam. (Note: Individuals from the Philippine Islands have been recorded as Pacific Islanders in previous data collection strategies.) Black or African American -a person having origins in any of the black racial groups of Africa. Terms such as "Haitian" or "Negro" can be used in addition to "Black or African American." Native Hawaiian or other Pacific Islander -a person having origins in any of the original peoples of Hawaii, Guam, Samoa, or other Pacific Islands. White -a person having origins in any of the original peoples of Europe, the Middle East, or North Africa. As a correlative objective, FRα expression in the tumor will be examined as previously described [27]. FRα is expressed in both epithelial (breast, ovarian, uterine, testicular, colon, renal, etc.) and nonepithelial malignancies (myelogenous leukemias and sarcomas). Particular interest in therapeutic targeting of FRα has focused on ovarian cancer because 70-90% of nonmucinous tumors express the protein at high levels [41,42].
Samples will be examined for FRα expression by assessing slides made from a full paraffin block. Tissues will be stained with FBP343, a monoclonal IgG1 Protocol Version Date: 17Apr2019 antibody derived by immunization with human FRα purified from the KB nasopharyngeal carcinoma cell line.
Five-micron sections will be cut and placed on positively charged slides. After rehydration, tissues will be subjected to antigen retrieval and blocking of endogenous peroxidases prior to staining with 3.6 μg/ml FBP343 or a nonspecific isotype matched antibody as a negative control for 30 min. After washing the slides, signals will be detected using the mouse MACH3 system (Biocare Medical, Walnut Creek, CA). Slides will be counterstained with Modified Schmidt's Hematoxylin and permanently mounted.
Slides will then be archived using digital imaging performed using a Bliss "Virtual Microscopy" microscope and computer system (Bacus Laboratories, Lombard, IL). The staining intensity (strong, moderate, weak, or negative) and proportion of FRα-positive cells among the malignant cells will be scored independently on the digital images by two observers in Dr. Knutson's and Dr.
Kalli's group who are blinded to all clinical outcome data.