However, a randomized Phase 3 trial evaluating the CDX-110 did not demonstrate efficacy [7]. There are several agents currently under investigation that target the local immune response in solid tumors, particularly in melanoma, such as ipilimumab (blockade of cytotoxic T lymphocyte–associated protein 4 [CTLA-4]) and programmed cell death protein 1 (PD-1; PD-L1 blockade). Such agents are either FDA approved or being evaluated in various solid tumors with the most activity seen in [8]. There are many immunologic approaches to treatment of GBM. Gliomas can upregulate B7-H1 expression in circulating monocytes and tumor-infiltrative macrophages through modulation of 19 Abbreviated Title: GBM Pembro HSPPC Version Date: 03/26/2024 autocrine/paracrine interleukin 10 (IL-10) signaling, resulting in an immunosuppressive phenotype.[ 9] Glioma-associated cancer-initiating cells have been shown to express the costimulatory inhibitory molecule B7-H1, [ 10] which is a key factor in mediating immune resistance in glioma[11] and can induce T-cell apoptosis. [12]. Direct cell-to-cell contact experiments have shown that the glioma-associated cancer-initiating cells induced T-cell apoptosis in the setting of B7-H1 expression. [10] found that cancer- initiating cells markedly inhibited T-cell proliferation and activation, induced regulatory T cells, and triggered T-cell apoptosis that was mediated by B7-H1 and soluble galectin-3. Loss of tumor suppressor phosphotase and tensin homolog (PTEN) function increases B7-H1 expression and immunoresistance in glioma. [11] It also been shown that glioma-associated cancer-initiating cells induce immunosuppression, thereby allowing tumor cells to evade immunosurveillance. Yao et al. [13] found that B7-H1 was correlated with the malignancy grade of human astrocytic tumors, but preferentially in the non-dividing tumor cells. It was also upregulated at the growing edge of the tumors and had a negative correlation with tumor-infiltrating CD8 T cells. Tumor-infiltrating lymphocytes and PD-L1 expression are seen in the majority of GBM samples. [14, 15] Data show that lymphopenia induced by temozolomide (TMZ) can actually potentiate cancer immunotherapy, which may be due in part to cell death, which releases cellular contents that increase the immunoreactivity of the tumor environment. [16] 1.2.2 Intervention Background & Overview 1.2.2.1 Programmed Cell Death Protein 1 The importance of intact immune surveillance in controlling outgrowth of neoplastic transformation has been known for decades.[17] Accumulating evidence shows a correlation between tumor-infiltrating lymphocytes (TILs) in cancer tissue and favorable prognosis in various malignancies. [ 12] [18] [19, 20] [21]. In particular, the presence of CD8+ T-cells and the ratio of CD8+ effector T-cells / FoxP3+ regulatory T-cells seems to correlate with improved prognosis and long-term survival in many solid tumors. The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress immune control. The normal function of PD-1, expressed on the cell surface of activated T-cells under healthy conditions, is to down-modulate unwanted or excessive immune responses, including autoimmune reactions. PD-1 (encoded by the gene Pdcd1) is an Ig superfamily member related to CD28 and CTLA-4 which has been shown to negatively regulate antigen receptor signaling upon engagement of its ligands (PD-L1 and/or PD-L2). [22, 23] The structure of murine PD-1 has been resolved. [24] PD-1 and family members are type I transmembrane glycoproteins containing an Ig Variable-type (V-type) domain responsible for ligand binding and a cytoplasmic tail which is responsible for the binding of signaling molecules. The cytoplasmic tail of PD-1 contains 2 tyrosine-based signaling motifs, an immunoreceptor tyrosine-based inhibition motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Following T-cell stimulation, PD-1 recruits the tyrosine phosphatases SHP-1 and SHP-2 to the ITSM motif within its cytoplasmic tail, leading to the dephosphorylation of effector molecules such as CD3 ζ, PKCθ and ZAP70 which are involved in the CD3 T-cell signaling cascade [23, 25-27]. The 20 Abbreviated Title: GBM Pembro HSPPC Version Date: 03/26/2024 mechanism by which PD-1 down modulates T-cell responses is similar to, but distinct from that of CTLA-4 as both molecules regulate an overlapping set of signaling proteins[ 25] [26]. PD-1 was shown to be expressed on activated lymphocytes including peripheral CD4+ and CD8+ T- cells, B-cells, T regs and Natural Killer cells. [ 28] [29] Expression has also been shown during thymic development on CD4-CD8- (double negative) T-cells as well as subsets of macrophages and dendritic cells. [ 30] The ligands for PD-1 (PD-L1 and PD-L2) are constitutively expressed or can be induced in a variety of cell types, including non-hematopoietic tissues as well as in various tumors. [30] [ 31] [ 32, 33] Both ligands are type I transmembrane receptors containing both IgV- and IgC-like domains in the extracellular region and contain short cytoplasmic regions with no known signaling motifs. Binding of either PD-1 ligand to PD-1 inhibits T-cell activation triggered through the T-cell receptor. PD-L1 is expressed at low levels on various non- hematopoietic tissues, most notably on vascular endothelium, whereas PD-L2 protein is only detectably expressed on antigen-presenting cells found in lymphoid tissue or chronic inflammatory environments. PD-L2 is thought to control immune T-cell activation in lymphoid organs, whereas PD-L1 serves to dampen unwarranted T-cell function in peripheral tissues. [34] Although healthy organs express little (if any) PD-L1, a variety of cancers were demonstrated to express abundant levels of this T-cell inhibitor. PD-1 has been suggested to regulate tumor- specific T-cell expansion in subjects with melanoma. [35] This suggests that the PD- 1/PD-L1 pathway plays a critical role in tumor immune evasion and should be considered as an attractive target for therapeutic intervention. Pembrolizumab (previously known as SCH 900475) is a potent and highly selective humanized monoclonal antibody (mAb) of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. It is currently FDA approved for use in patients with unresectable or metastatic melanoma and disease progression following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor. It is also FDA approved for advanced (metastatic) non-small cell lung cancer (NSCLC) whose disease has progressed after other treatments and with tumors that express a protein called PD-L1 and for the treatment of patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) with disease progression on or after platinum-containing chemotherapy. 1.2.3 Rationale for the Pembrolizumab dose Selection of 200 mg as the appropriate dose for a switch to fixed dosing is based on simulation results indicating that 200 mg will provide exposures that are reasonably consistent with those obtained with 2 mg/kg dose and importantly will maintain individual patient exposures within the exposure range established in melanoma as associated with maximal clinical response. A population PK model, which characterized the influence of bodyweight and other patient covariates on exposure, has been developed using available data from 476 subjects from PN001. The distribution of exposures from the 200 mg fixed dose are predicted to considerably overlap those obtained with the 2 mg/kg dose, with some tendency for individual values to range slightly higher with the 200 mg fixed dose. The slight increase in PK variability predicted for the fixed dose relative to weight-based dosing is not expected to be clinically important given that the range of individual exposures is well contained within the range of exposures shown in the melanoma studies of 2 and 10 mg/kg to provide similar efficacy and safety. The population PK evaluation revealed that there was no significant impact of tumor burden on exposure. In addition, exposure was similar between the NSCLC 21 Abbreviated Title: GBM Pembro HSPPC Version Date: 03/26/2024 and melanoma indications. Therefore, there are no anticipated changes in exposure between different tumor types and indication settings. Available pharmacokinetic results in subjects with melanoma, NSCLC, and other solid tumor types support a lack of meaningful difference in pharmacokinetic exposures obtained at a given dose among tumor types. Population PK analysis has been performed and has confirmed the expectation that intrinsic factors do not affect exposure to Pembrolizumab to a clinically meaningful extent. Taken together, these data support the use of lower doses (with similar exposure to 2 mg/kg Q3W) in all solid tumor indications. 2 mg/kg Q3W is being evaluated in NSCLC in PN001, Cohort F30 and PN010, and 200 mg Q3W is being evaluated in head and neck cancer in PN012, which are expected to provide additional data supporting the dose selection. This trial will use Pembrolizumab 200 mg every three weeks to fit with treatment schedules currently used. 1.2.4 Heat Shock Protein-Peptide Complex-96 (HSPPC-96) Immunotherapy offers the promise of improving outcomes for patients with GBM by evoking specific immune responses that may produce a more sustained and less toxic effect than conventional therapy. Heat-shock proteins (HSPs), which function as intracellular chaperones, can be used to deliver a variety of tumor antigens to antigen presenting cells for immune stimulation. Heat Shock Protein-Peptide Complex-96 (HSPPC-96) consists of the heat shock protein glycoprotein-96 (HSP gp-96) and a wide array of chaperoned proteins, including autologous antigenic peptides. Heat shock proteins (HSP) are molecules that respond to cellular stress and counteract abnormal protein folding. They are known to modulate immune responses, especially the HSP gp-96. In a stressful environment, such as a tumor, HSPs are upregulated and highly expressed on tumor cells. This protects the tumor and leads to resistance to therapy. HSP expression is associated with cellular proliferation, apoptosis evasion, tissue invasion, metastasis, and angiogenesis. HSPPC-96 immunization works mechanistically by interacting with antigen presenting cells (APCs) via specific receptors, including CD91. [36, 37] highly specific nature of the interaction between HSPPC-96 and APCs is a significant advantage over other cancer vaccine approaches; and has been shown to facilitate robust CD4+ and CD8+ T-cell immune responses. There are multiple mechanisms by which the vaccine works. The first is the following: The autologous antigens in the vaccine are bound to the chaperone proteins (HSP) with noncovalent bonds. Macrophages and other transfer the antigens from HSP to antigen presenting cells (e.g., DCs) bind and take up the HSP-antigen complexes, via receptor- mediated endocytosis (with receptors such as CD91). The antigens are then expressed via MHC class I and II on antigen presenting cells (APCs), interacting with naïve T cells, which are activated into specific CD8 T and CD4 T cells. Another mechanism of the vaccine is an innate immunity pathway cells. HSPPC-96 Ag is internalized into the APC, which activates the APC. This leads to the following: 22 Abbreviated Title: GBM Pembro HSPPC Version Date: 03/26/2024 oActivated APCs express co-stimulatory molecules (CD80, CD86, and CD40) and express MHC class II, which interacts with T cells and produces tumor specific CD4 cells. oActivated APCs cause cytokine release (IL-1b, IL-12, IL-6, TNF-alph, GMCSF, chemokines, and nitric oxide). IL-12 stimulates NK cells, which enhance the antitumor activity of T cells. oThe interaction between HSP-antigen and APCs causes dendritic cell maturation, which stimulates NK cells. A final mechanism of the vaccine is that exposure to Gp96 leads to translocation of NFkB into the APC nucleus, which then regulates cancer cell growth, progression, and apoptosis. The advantage of the HSPPC-96 vaccine over others is the following: oMultifactorial mechanism: as described above, the vaccine works by multiple pathways to exert its effect, rather than 1 pathway. oThe vaccine is individualized and does not require identification of immunogenic antigens required by other vaccines. oMultiple antigens are used, which decreases the chance of immune evasion that occurs with a single antigen vaccine. Clinical grade HSPPC-96 can be easily purified from solid tumor and has been safely tested in hundreds of patients with newly diagnosed and recurrent melanoma and renal cell cancer. The toxicity encountered in renal cell carcinoma in prior trials is the following: injection site erythema (50%), injection site induration (48%), back pain (12%), headache (12%), fatigue (10%), nasopharyngitis (9%), incision site complication (9%), arthralgia (9%), hypertension (9%), urinary tract infection (8%), nausea (7%), diarrhea (6%), asthenia (6%), influenza (6%), constipation (5%), pyrexia (5%), and dizziness (5%). In melanoma, toxicity seen was pyrexia (8%), fatigue (6%), and nausea (5%). Between the trials there were 3 serious adverse events, 2 were thyroid dysfunction, and 1 was cellulitis. [ 38] [ 39] [ 11] The rate of successful HSPPC-96 vaccine manufacture is anticipated to be 80% or greater based on prior clinical trials. A prior phase 2 study of new diagnosed glioblastoma, vaccine was successfully manufactured in 84% (63/77) of attempted cases. The majority of manufacture failures were caused by insufficient number of vials available, below the required 4, for clinical use (9 cases). A single center single arm Phase I clinical trial and a multicenter single arm Phase II clinical trial for recurrent GBM patients treated with HSPPC-96 have been completed. Collectively these studies have shown that: 1) HSPPC-96 vaccine made from recurrent GBM tumor is safe and well tolerated, 2) HSPPC-96 vaccine extends survival in recurrent GBM patients over historical controls and 3) HSPPC-96 is immunogenic in recurrent GBM patients, inducing both an immunologic response locally and peripherally and via innate and adaptive immune mechanisms [39] and confers clinical efficacy in patients with newly diagnosed and recurrent GBM. Importantly, in patients with newly diagnosed GBM, HSPPC-96 vaccine in combination with standard of care temozolomide extended response. Specifically, in the phase I trial, testing was performed pre- and post-vaccine on all patients. Eleven of 12 patients were shown to have a significant immune response, which was measured by testing peripheral blood lymphocytes (PBLs). PBLs were tested for phenotype, reactivity to vaccine, IFN gamma production, and 23 Abbreviated Title: GBM Pembro HSPPC Version Date: 03/26/2024 proliferation. Vaccine was well tolerated with no serious adverse events and the only toxicity noted was injection site erythema or induration. The median progression survival in responders was 47 weeks, compared to 16 weeks in the non-responder. In the phase II trial, 33 patients were treated. Again, there were no serious adverse events attributed to vaccine. Toxicities reported were injection site reaction in 12 patients, and fatigue in 9 patients. Eleven patients were still alive at time of analysis, with median survival of 333 days for the evaluable population. Also, all patients studied showed immune response, based on CD8 IFN gamma production [9]. In a separate single arm phase II trial of newly diagnosed GBM patients who were treated with RT + TMZ + HSPPC-96, median PFS and overall survival were two to three times longer than expected based on historical controls (median PFS was 18 v 6-9 mos and median OS was 24 v 14-16 mos) of 24 months. Importantly, patients with low PDL1 expression on peripheral monocytes had longer survival than those with high PD-L1 expression, suggesting that an optimal group for HSPPC-96 use with a checkpoint inhibitor would be in the high PD-L1 expressing patients. Importantly, in data presented at ASCO 2015, in patients with less elevated PD-L1 expression on peripheral monocytes, treatment with HSPPC-96 was associated with an unexpected and impressively longer survival than those with high more elevated PD-L1 on circulating monocytes (mPFS of 27 v 6-9 mos and mOS 47 v 14-16; [38] ). Figure 1: Overall Survival by Monocyte PD-L1 1.2.5 Rationale for the Current Study The preclinical data about the immunologic aspects of gliomas and the data using vaccines, suggest that there is a strong rationale to evaluate these agents in GBM. While the blood brain 24 Abbreviated Title: GBM Pembro HSPPC Version Date: 03/26/2024 barrier (BBB) is often an issue for drug penetration, this would be less of an issue for checkpoint inhibitors as the effect is on trafficking T-Cells which can migrate peripherally to the brain. [ 15] Nonetheless, in the setting of contrast enhancement, the BBB is leaky, allowing drug penetration. Taken together, these data support the rationale that the combination of an autologous cancer vaccine and an anti-PD-1 inhibitor may synergistically result in an increase in tumor specific immune response with downregulation of tumor immunosuppression resulting in important clinical responses. There is pre-clinical and some clinical precedence for using an immune educating strategy with a release of tumoral immunosuppression. Several studies have shown a benefit of the combination over check point modulators alone or vaccine alone in several types of solid tumors. [ 19, 40] This study will be the first to evaluate this combination of vaccine (HSPPC-96) and PD-1 inhibition (Pembrolizumab) in newly diagnosed GBM with unmethylated MGMT promoter and will provide important data on immune-modulatory effect of this combination. This may be of particular value in patients with high peripheral PD-L1 expression, but also there also be value adding PD-1 to standard GBM therapy. We will be assessing OS-12 and other PFS and OS rates. A phase I study adding Pembrolizumab (200 mg) to RT+TMZ using standard starting doses was found to be safe and without a DLT.[41] The DLT window was from the start of RT until 4 weeks post Radiation. There were no grade 3 or 4 toxicities seen. Related grade 1 and 2 toxicities were anemia, lymphopenia, neutropenia, thrombocytopenia, fatigue, rash, and GI upset/poor PO intake. Pseudo-progression and inflammatory responses were not seen in in a phase I of RT + TMZ + Pembrolizumab. We do not believe there will be any added toxicity of adding HSPPC-96 to RT + TMZ + Pembrolizumab; while an increased inflammatory reaction might occur, these patients will all have achieved ≥ 80% resection. Tumor tissue will be analyzed to assess for MGMT, PDL-1 levels and T-cell infiltration as well as collection of peripheral markers of T-Cell activation and tryptophan metabolites. Patients who progress and will benefit from further surgery will have their tumor tissue collected and analyzed. 1.2.6 Rationale for the patient-reported outcomes: This study seeks to establish effective therapies. We hypothesize that using a combination of pembrolizumab, temozolomide and HSPPC-96 will result in improved survival. However, given the intensive nature of this regimen, it will be important to determine whether any determined survival benefit is associated with improvements in symptoms or whether a worsening of symptoms offsets the increase in survival. Precedence for measuring “non-therapeutic” endpoints exists in oncology research. For example, Gemcitabine was approved by the FDA partially as a consequence of the decrease in pain reported in pancreatic patients who were treated, not on the basis of survival improvement which was modest at best [42]. There have been efforts in neuro-oncology to evaluate secondary endpoints using validated instruments as an additional indicator of benefit. 25 Abbreviated Title: GBM Pembro HSPPC Version Date: 03/26/2024 The M.D. Anderson Symptom Inventory-Brain Tumor Module (MDASI-BT) allows the self- reporting of symptom severity and interference with daily activities. The MDASI-BT has demonstrated reliability and validity in the adult primary brain tumor patient population. [43] This tool represents a modification of the widely used and validated M.D. Anderson Symptom Inventory, with particular attention to symptoms common in patients with brain tumors. The availability of validated instruments provides an opportunity to prospectively assess the impact of treatment, both positive and negative, on patients.