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74 Background: CYAD-101 is a first-in-class, non-gene edited allogeneic CAR T-cell product that combines the broad breadth of tumor targeting of the NKG2D-based chimeric antigen receptor (CAR) with a peptide-based approach that controls graft versus host disease (GvHD). NKG2D binds eight ligands commonly over-expressed across many tumors while the co-expressed T-cell receptor (TCR) inhibitory (TIM) peptide interferes with signaling by the endogenous TCR. A bank of CYAD-101 cells was produced from a single donor and evaluated in the AlloSHRINK phase 1 study (NCT03692429) in patients with unresectable metastatic colorectal cancer (mCRC). Methods: Three CYAD-101 infusions, each administered following a FOLFOX standard cycle as preconditioning chemotherapy, were tested in a 3+3 dose-escalation study (dose-levels (DL): 108, 3x108 and 109 T-cells per infusion) in patients with relapsed/refractory mCRC who progressed after previous treatment with oxaliplatin-based chemotherapy, with or without irinotecan-based chemotherapy. Results: Fifteen patients (pts) were enrolled (3 pts at DL-1, 3 pts at DL-2, 9 pts at DL-3). No dose-limiting toxicity (DLT), Grade ≥ 3 related adverse events or GvHD were reported after any of the CYAD-101 infusions, thus confirming the overall good safety profile of CYAD-101 post FOLFOX. Encouraging anti-tumor activity was observed with 2 confirmed partial responses (PR), including one response in a KRAS mutated patient. In addition, 9 pts achieved stable disease (SD), with 7 SD lasting at least 3 months. The median progression-free survival in this heavily pre-treated population was 3.9 months (95% CI). Whilst engraftment of the CYAD-101 cells was observed after each infusion, the relative level of systemic cytokines appeared to be primarily modulated by cell dose with some suggestion that the magnitude of modulation might be associated with clinical response. Interestingly, preliminary analysis of the T-cell repertoire identified some evidence of TCR diversity after therapy in the patient showing the most durable partial response. Conclusions: These clinical results demonstrate the safety and tolerability of a fist-in-human non-gene edited allogeneic CAR T-cell treatment with early promising anti-tumor activity in advanced mCRC pts. Preliminary translational analysis present intriguing observations that the modulation of systemic cytokine levels may be associated with dose which is uncommon in CAR T-cell therapies reported to date while limited T-cell clonal diversification in the best responding patient underscores the likely central role of the adoptively transferred T-cell in driving therapeutic response in this particular patient. Extension cohort evaluating CYAD-101 following other preconditioning chemotherapy is expected to be initiated end 2020. Clinical trial information: NCT03692429.

3032 Background: In contrast to autologous CAR-T cell therapies, allogeneic donor-derived CAR-T cells can be banked and used in a timely fashion overcoming the critical time delay of just in time autologous cell manufacture. CYAD-101 is an allogeneic CAR-T that uses a non-gene edited peptide-based technology (TIM) to control graft versus host disease (GvHD) combined with a NKG2D-based CAR. Pre-clinical studies confirmed that CYAD-101 maintained CAR-directed anti-tumor activity in the absence of the induction of GvHD. Clinical grade CYAD-101 cells were produced for the phase 1 alloSHRINK trial (NCT03692429). Methods: A bank of clinical grade CYAD-101 cells was generated through two production runs using a single donor apheresis. Together, the bank generated > 53 billion CYAD-101 cells suitable for the entire dose escalation segment and short expansion phase of the trial (15 patients in total). Both runs showed high consistency with the CYAD-101 product generated composed mainly of CD4+ T cells (>85%) with a transduction level of > 92%, low relative expression of CD69/CD25 and largely absent expression levels of PD-1/LAG-3. The CYAD-101 cells were predominantly (>80%) CD45RA−/ CD62L−/ CD27− suggestive of an effector memory T cell population. Results: Upon co-culture with target K562 cells, CYAD-101 readily produced IFN-γ that was blocked by a NKG2D blocking antibody confirming specificity of the CAR. CYAD-101 cells showed in vitro cytotoxicity against tumor cells and produced an array of Th1 (IFN-γ, IL-2 and TGF-β) and Th2 (IL-4, IL-5) cytokines. Importantly, minimal IFN-γ was produced upon TCR stimulation while stimulation with a non-TCR mitogen (PMA + ionomycin) lead to high levels of IFN-γ. Together, these data show that clinical grade CYAD-101 cells were able to functionally respond through the CAR but showed minimal TCR-driven activation. Fifteen refractory metastatic CRC patients who had previously failed at least one line of oxaliplatin—containing therapy were treated with three doses of CYAD-101 cells given on Day 3 of three successive FOLFOX chemotherapy cycles. Updated clinical results continue to demonstrate an encouraging clinical activity (2 patients with partial response and 9 with stable disease) and the absence of GvHD in the context of CYAD-101 cell engraftment. Conclusions: These early clinical results demonstrate the safety and tolerability of a non-gene edited predominantly CD4+ CAR-T therapeutic approach. The initial observations of clinical activity in metastatic CRC patients warrants the continued development of this therapy. Clinical trial information: NCT03692429 .

Purpose Active immunization against the tumor-specific MAGE-A3 antigen is followed by a few but impressive and durable clinical responses. This randomized phase II trial evaluated two different immunostimulants combined with the MAGE-A3 protein to investigate whether a more robust and persistent immune response could be associated with increased clinical benefit. Patients and Methods Patients with MAGE-A3–positive stage III or IV M1a melanoma were randomly assigned to receive the MAGE-A3 protein combined either with AS02B or with AS15 immunostimulant. Clinical end points were toxicity and rates of objective clinical responses, progression-free survival (PFS), and overall survival (OS). Results Seventy-five patients were treated, with 36 eligible patients per arm. Both treatments were well tolerated. In the AS15 arm, four objective responses were observed (three complete responses and one partial response) versus one partial response in the AS02B arm. In the AS15 and AS02B arms, the PFS rates after 6 months were 25% and 14%, respectively; and the median OS times were 33 months and 19.9 months, respectively, with a median observation period of 48 months. Antibodies against MAGE-A3, found in all patients, showed three-fold higher titers in the AS15 arm. The anti–MAGE-A3 cellular response was also more pronounced in the AS15 arm. Conclusion In the MAGE-A3+AS15 arm, clinical activity was higher and the immune response more robust. Therefore, the AS15 immunostimulant was selected for combination with the MAGE-A3 protein in phase III trials.

Purpose The MAGE-A3 protein is expressed in approximately 35% of patients with resectable non–small-cell lung cancer (NSCLC). Several immunization approaches against the MAGE-A3 antigen have shown few, but often long-lasting, clinical responses in patients with metastatic melanoma. Patients and Methods A double-blind, randomized, placebo-controlled phase II study was performed assessing clinical activity, immunologic response, and safety following immunization with recombinant MAGE-A3 protein combined with an immunostimulant (13 doses over 27 months) in completely resected MAGE-A3–positive stage IB to II NSCLC. The primary end point was disease-free interval (DFI). Results Patients were randomly assigned to either MAGE-A3 immunotherapeutic (n = 122) or placebo (n = 60). After a median postresection period of 44 months, recurrence was observed in 35% of patients in the MAGE-A3 arm and 43% in the placebo arm. No statistically significant improvement in DFI (hazard ratio [HR], 0.75, 95% CI, 0.46 to 1.23; two-sided P = .254), disease-free survival (DFS; HR, 0.76; 95% CI, 0.48 to 1.21; P = .248), or overall survival (HR, 0.81; 95% CI, 0.47 to 1.40; P = .454) was observed. Corresponding analysis after a median of 70 months of follow-up revealed a similar trend for DFI and DFS. All patients receiving the active treatment showed a humoral immune response to the MAGE-A3 antigen, although no correlation was observed with outcome. No significant toxicity was observed. Conclusion In this early development study with a limited number of patients, postoperative MAGE-A3 immunization proved to be feasible with minimal toxicity. These results are being investigated further in a large phase III study.

TPS3093 Background: Chimeric Antigen Receptor (CAR)-T therapy has potentially serious limitations related to target antigen loss, toxicity due to pre-conditioning regimen, and lack of activity in many tumor types. To overcome these limitations, we have developed a novel CAR-T, called NKR-2, incorporating the full-length human natural killer receptor NKG2D fused with the human CD3 zeta signaling domain. When expressed in T-cells, the naturally-expressed DAP10 provides co-stimulatory signals to NKR-2 to produce cytokines and selectively target tumor cells upon recognition of up to 8 different stress-induced NKG2D ligands expressed in many solid and hematologic malignancies. In preclinical studies, NKR-2 demonstrated long-term anti-tumor activity towards a breadth of tumor indications, in the absence of pre-conditioning, whilst simultaneously targeting tumor cells and cells from the local tumor neo-vasculature and suppressive immune environment. In our recently completed First-in-Human Phase 1 study (NCT02203825) in hematologic cancers, a single administration of autologous NKR-2 was safe with initial signs of clinical benefit. Methods: Exploiting the multiple ligand targeting capability and unique mode of action of NKR-2, the THINK trial (THerapeutic Immunotherapy with NKR-2) is an open-label Phase I study that will assess the safety and clinical activity of multiple infusion NKR-2 treatment (every 2 weeks x 3 infusions) in relapse/refractory patients with metastatic or locally advanced CRC, urothelial carcinoma, TNBC, pancreatic cancer, recurrent epithelial ovarian and fallopian tube carcinoma, AML/MDS and MM, post standard treatment. The study contains two consecutive segments. The dose escalation segment will enroll 18 patients in two separate hematologic and solid malignancy arms, and will evaluate 3 dose levels of NKR-2 (3x108, 1x109 and 3x109 per injection) following a 3+3 design. The expansion segment will then enroll 96 additional patients in 7 separate cohorts for each indication with 3 steps of statistical analysis (overall futility, cohort futility and final evaluation). The study is open for recruitment in both EU and US. Clinical trial information: NCT03018405.

TPS3102Background: Canonical CAR-T cell constructs encode a single chain antibody variable fragment, costimulatory domain, and signaling domain of CD3 zeta. This restricts recognition to 1 tumor an...

7013 Background: Previous trials with the MAGE-A3 recombinant (rec) protein formulated with an immunostimulant have shown induction of specific immune responses and signals of clinical activity in different cancer diseases. In this phase I/II study (NCT 00455572), we evaluated the safety profile of the MAGE-A3 cancer immunotherapeutic (CI), formulated with the rec MAGE-A3 protein and the AS15 immunostimulant, and the induction of specific immune response with or without adjuvant chemotherapy (CT). Methods: MAGE-A3 CI was administered i.m. 8q3w in resected MAGE-A3+ stage IB-III NSCLC patients (pts). Three cohorts (C) were evaluated: Immunization with concurrent cisplatin plus vinorelbine (C1), sequentially after the same CT (C2) or with no CT (C3). The anti-MAGE-A3 humoral and cellular immune responses were evaluated by ELISA and intracellular cytokine staining (T cells producing both IFNg and TNF) respectively. Adverse Events (AEs) were graded according to CTCAE v. 3.0. Results: A total of 38/55 treated pts received the 8 doses schedule (15/19 in C1, 14/18 in C2, 9/18 in C3). Almost all pts reported at least one AE, mostly general constitutional disorders and administration site reactions. Six patients reported related grade 3 AEs. No related grade 4-5 AE or related SAE were reported. Immunogenicity results in the total treated population are reported in the table below. Conclusions: In this trial, patients in cohorts 2 and 3 correspond to the two populations enrolled in the Phase III MAGRIT trial evaluating the same MAGE-A3 CI. The phase I/II results suggest that this CI is well tolerated and induces in all treated pts specific antibodies against MAGE-A3 after 4 doses in presence or not of concurrent or sequential adjuvant CT. About 25% of the treated pts are considered as CD4 responders in presence or not of concurrent or sequential adjuvant CT. [Table: see text]

8535 Background: Active immunization towards MAGE-A3 tumor antigen is a potential new approach to treat cancer. Current data from pilot studies have shown that a strong and persistent immune respon...

7501 Background: A phase II randomized trial in 182 patients (pts) with completely resected stage IB or II MAGE-A3 (+) NSCLC comparing postoperative MAGE-A3 recombinant protein combined with an Adjuvant System to placebo (q3w x 5, followed by q3m x 8) showed a strong positive signal for activity (J Clin Oncol 25 Suppl 18:398S, 2007). We report here the analysis of gene expression profiling of tumors prior to treatment to identify a predictive signature that correlates with clinical activity of MAGE-A3 ASCI treatment. Methods: Microarray analysis (Affymetrix) was performed on 159 fresh-frozen tumor biopsies (MAGE-A3: 108; placebo: 51; stage IB: 109; stage II: 50). Clinical data on relapse are based on a median follow-up of 44 months. Results: Study of tumor samples from pts in the placebo arm led to identification of a gene signature associated with a high risk of postoperative relapse. The absence of this signature correlated with a very low relapse rate in stage IB pts: placebo 0%; MAGE-A3: 4%. In the MA...

9065 Background: The MAGE-A3 antigen, expressed in ±60% of metastatic melanomas, is a target of anti-tumor CD4+ and CD8+ T-cells. Previous data showed a few clinical responses of MAGE-A3 Antigen-Sp...

9045 Background: Gene expression profiling by microarrays was used to identify markers predictive of the clinical activity of the MAGE-A3 Antigen-Specific Cancer Immunotherapeutic (ASCI) recorded in a phase II study in metastatic melanoma. Methods: 75 patients with progressive, unresectable stage III or stage IV M1a MAGE-A3 (+) melanomas, were randomized as 1st-line therapy between immunization with MAGE-A3 protein combined with GSK proprietary Adjuvant Systems AS15 or AS02B. Gene expression profiling was performed on tumor biopsies taken prior to any immunization. Results: Initial analysis using supervised hierarchical clustering of 2 objective responders (OR) with 7 non-responders identified 2 gene clusters based on differential expression. The correlation of this gene expression signature (GS) and objective response was further confirmed on 22 patients. Interestingly, mixed response (MR) and stable disease > 6 months (SD) clustered with OR, suggesting a strong association between the identified signatu...

7554 Background: After complete resection, about 50% of patients with stages IB-II NSCLC disease die within 5 years. Adjuvant chemotherapy improves overall survival at the expense of substantial toxicity. Activity of MAGE-A3 immunotherapeutic (i.e. recombinant MAGE-A3 protein and a potent GlaxoSmithKline adjuvant) was previously demonstrated in metastatic melanoma. As about 35% of NSCLCs express MAGE-A3 antigen, post-operative MAGE-treatment may be a tumor-specific, well tolerated, and effective adjuvant therapy. Methods: Patients with completely resected, MAGE-A3 (+), stage pIB or pII were randomly assigned to postoperative MAGE-A3 or placebo (2:1), with 5 administration at 3-week intervals, followed by 8 administrations every 3 months. Randomization was stratified for stage (IB vs. II), histology (squamous vs. other), and lymph-node (LN) procedure (sampling vs. dissection). Primary endpoint was disease-free interval (DFI); other endpoints were safety, disease-free survival (DFS), and overall survival (OS). This exploratory Phase II study was designed to detect a clinically relevant HR with a 10% one-sided a. Results: 182 patients (122 stage IB, 60 stage II) from 59 centers in 14 countries were randomized over 2 years: Median age 63 (45–81); 87% male; 65% squamous cell carcinoma; 65% lymph-node dissection. After a median follow-up of 28 months, 67 recurrences and 45 deaths were recorded. Group comparisons of DFI, DFS and OS gave respectively a hazard ratio (HR) of 0.74 (95% CI 0.44–1.20, p=0.107), 0.73 (95% CI 0.45–1.16) and 0.66 (95% CI 0.36–1.20) in favor of the MAGE-A3 group. Overall, treatment was well tolerated, with excellent protocol compliance. Subset analysis also suggests that LN dissection may have an effect on survival. Conclusions: The final analysis of this randomized phase II study shows a positive trend for activity of MAGE-A3 treatement in NSCLC with a relative improvement of DFI and DFS of 27%. Further phase III evaluation is planned. This study also suggests that complete lymph-node dissection may have an effect on survival and should be confirmed prospectively. [Table: see text]

7019 Background: About half of the patients with completely resected early-stage NSCLC will relapse. Adjuvant chemotherapy reduces the number of relapses at the expense of substantial toxicity. Activity of MAGE-A3 vaccination (i.e. recombinant MAGE-A3 protein and a potent GSK immunological adjuvant) has been proven in advanced melanoma. As about 35% of NSCLCs express the MAGE-A3 antigen, post-operative MAGE-A3 vaccination may be a targeted, well tolerated, and effective adjuvant therapy. Methods: Patients with completely resected, MAGE-A3 positive (assessed by quantitative RT-PCR), stage pIB or pII were 2:1, double-blind, randomly assigned to postoperative MAGE-A3 vaccination or placebo. Vaccination was started ≥6 weeks after surgery, with 5 vaccinations at 3-week intervals, followed by 8 vaccinations every 3 months. Randomization was stratified for stage (IB vs. II), histology (squamous vs. other), and lymph node procedure (sampling vs. dissection). Other anti-cancer adjuvant therapy was not allowed. Primary endpoint was time-to-recurrence, other endpoints were recurrence rates at different times, and survival. A sample size of 180 patients (120 active, 60 placebo) was chosen, to achieve 48% power (α=10%) to detect a 10% difference, assuming a 40% recurrence rate at month 30 in the placebo group (log rank test). A thorough interim efficacy analysis by an independent statistician (blinded to investigators and sponsor) is planned for 18 months after completion of recruitment. Results: 35% of 1,089 biopsies from 59 sites were MAGE-A3 positive. In two years, 182 patients were randomized (121 stage IB, 61 stage II). Overall, treatment was well tolerated, with high protocol compliance. Data collection for the interim efficacy analysis was initiated on December 19th, 2005. Median follow-up was 21 months, with 62 recurrences recorded. Conclusions: This study confirms expression of MAGE-A3 antigen in 35% of early NSCLC cases, and demonstrates good tolerability of postoperative MAGE-A3 vaccination. Efficacy results of the interim analysis will be available in February 2006. [Table: see text]