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P145

Expanding Iovance’s tumor infiltrating lymphocytes (TIL) from core biopsies for adoptive T cell therapy using a 22-day manufacturing process

Michelle Abelson, PhD, Kenneth D'Arigo, Florangel Hilton, Maria Fardis, PhD, MBA, Cecile Chartier

Iovance Biotherapeutics, Inc., Tampa Bay, FL, United States Correspondence:Cecile Chartier ([email protected]) Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P145 Background

Iovance’s TIL products Lifileucel and LN-145 have demonstrated re- markable clinical activity in melanoma and cervical cancer utilizing Iovance’s proprietary 22-day manufacturing process and surgically resected tumor lesions ~ 1.5-cm diameter [1, 2]. Using a core needle biopsy procedure to obtain tumor samples could allow for greater convenience of collecting the tumor from patients [3]. We asked whether a streamlined manufacturing process could be implemented to produce therapeutically relevant TIL from multiple histologies starting with a core biopsy.

Methods

Core biopsies obtained from 4 melanoma and 3 pancreatic, 2 breast, 2 ovarian, and 1 lung tumors were processed in vitro, using a 22-day expansion method termed ‘Core process’. Core biopsy-derived TIL were assessed for expansion, phenotype (lineage, youth/differenti- ation, activation, and exhaustion markers), function (IFN-gamma and CD107a mobilization), and TCR repertoire.

Results

Iovance’s Core process successfully generated TIL products from all tested samples. One to 2 cores yielded more than 10e9 T cells for 10 of the 12 preparations. Phenotypic analyses revealed no significant differences in terms of T cell lineages and memory subsets, or ex- pression of activation, differentiation, and exhaustion markers when compared to Iovance’s current products. Core-derived TIL products responded to PMA and to anti-CD3 stimulations by inducing levels of CD107a mobilization and IFN-gamma secretion like those produced by TIL derived from excisional biopsies. Preliminary TCR sequencing data suggest that high-diversity products can be also be obtained from small samples, similar to what is obtained from TIL expansion.

Conclusions

This work demonstrates that the Iovance 22-day Core manufacturing method is highly robust and that it is feasible to expand TIL to thera- peutically relevant numbers from as little as 1 to 2 core biopsies from multiple histologies with this method. Resulting products were shown to be phenotypically comparable to, and as potent as, products gener- ated with Iovance_’s process from excisional biopsy. Iovance anticipates implementing this process in the clinic in the near future.

References

1. Jazaeri AA, Zsiros E, Amaria RN, Artz AS, Edwards RP, Robert Michael Wenham RM, et al. Safety and efficacy of adoptive cell transfer using autologous tumor infiltrating lymphocytes (LN-145) for treatment of recurrent, metastatic, or persistent cervical carcinoma. Clin Oncol. 2019;37:15:2538 (suppl).

2. Sarnaik A, Khushalani NI, Chesney JA, Kluger HM, Curti BD, et al. Safety and efficacy of cryopreserved autologous tumor infiltrating lymphocyte therapy (LN-144, lifileucel) in advanced metastatic melanoma patients who progressed on multiple prior therapies including anti-PD-1. J Clin Oncol. 2019;37:15:2518 (suppl).

3. Ullenhag GJ, Sadeghi AM, Carlsson B, Ahlström H, Mosavi F, Wagenius G, Tötterman TH, et al. Adoptive T-cell therapy for malignant melanoma pa- tients with TILs obtained by ultrasound-guided needle biopsy. Cancer Immunol Immunother. 2012;61:725–732.

P146

AUTO6NG: Next generation GD2-targeting CAR T-cell therapy with improved persistence and insensitivity to TGFb and checkpoint inhibition for relapsed/refractory neuroblastoma

Daniela Achkova, PhD1_{, Adrian Zarzoso}1_{, Yusuf Demir}1_{, Fernando} Gallardo1, Maria Stavrou1, Marco Della Peruta1, Saket Srivastava1, Mathew Robson1_{, Shimobi Onuoha}1_{, Simon Thomas}1_{, Shaun Cordoba}1_{, Martin} Pule1,2

1_{Autolus Ltd, London, United Kingdom;}2_{University College London} Cancer Institute, London, United Kingdom

Correspondence:Martin Pule ([email protected]) Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P146 Background

Neuroblastoma is the most common extracranial solid cancer in chil- dren with poor long-term survival in those with high-risk disease. A currently ongoing phase I clinical study of GD2-targeted CART for re- fractory/relapsed neuroblastoma (NCT02761915) shows activity against disseminated disease without inducing on target/off tumor toxicity. However, CART persistence was limited and clinical activity transient and incomplete.

Building on the GD2 CAR used in this study, we have developed a next generation T-cell product candidate termed AUTO6NG. The AUTO6NG product consists of 3 distinct populations of GD2-targeted CAR T-cells, produced by dual transduction of T-cells with two separate retroviral vectors. The first vector directs the expression of a GD2-targeting CAR, co-expressed with a constitutively signalling IL7 cytokine receptor (IL7R_CCR) (product A), while the second vector is a tri-cistronic retro- viral vector encoding the same GD2 CAR, co-expressed with dominant negative TGFbRII (dnTGFbRII) and truncated SHP2 (dSHP2) (product B). dSHP2 confers resistance to inhibitory signals such as those from PD1. Methods

Human T-cells were either dual transduced with both vectors yield- ing a mix of product A/B/A+B (AUTO6NG) or single transduced with each vector individually giving raise to product A or B. Both single and dual transduced CAR T-cells were extensively evaluated in vitro for redirected lysis, cytokine secretion, T-cell proliferation and survival and resistance to immunosuppressive pathways (including TGFb and PD1/PDL1 inhibition) in co-culture assays with GD2-positive and negative tumour cell lines. Additionally, anti-tumour activity of AUTO6NG was evaluated in vivo by intravenous administration in an established neuroblastoma xenograft model in NSG mice.

Results

AUTO6NG T-cells (product A/B/A+B) were highly potent in cytotox- icity assays against GD2 positive tumour cell lines with no differences

observed compared with single transduced CAR T-cells (product A or B). Expression of the IL7R_CCR in both AUTO6NG and product A con- ferred exogenous-cytokine-independent viability and homeostatic proliferation of modified T-cells, without causing autonomous T-cell growth. Furthermore, AUOTO6NG T-cells and product B but not product A proved resistant to both TGFb- and PD1/PDL1-mediated immunosuppression in vitro due to the presence of dnTGFbRII and dSHP2 in those genetically engineered CAR T-cells. Finally, intraven- ous delivery of AUTO6NG exhibited potent anti-tumour activity and extended survival in NSG mice with established tumour burden. Conclusions

These results demonstrate the feasibility, safety, and efficacy of AUTO6NG T-cells. The addition of IL7R_CCR, dnTGFbRII and dSHP2 modules to the AUTO6NG product augment its functions by extend- ing T-cell persistence and rendering modified T-cells resistant to TGFb- and PD1/PDL1-driven immune inhibition.

P147

Effect of chemotherapy on cellular kinetics of NKG2D-based CAR T- cells in metastatic colorectal cancer patients

Erik Marcelo Alcantar Orozco, Eytan Breman, MSc, Marie-Sophie Dheur, PhD, Fabian Borghese, PhD, Emilie Cerf, PhD, Nathalie Braun, Caroline Lonez, PhD, Anne Flament, Frederic Lehmann, MD

Celyad, Mont-Saint-Guibert, Belgium

Correspondence:Frederic Lehmann ([email protected]) Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P147 Background

Autologous and allogeneic Chimeric Antigen Receptor (CAR) T-cells are under thorough investigation to translate their success in B-cell malig- nancies to other types of cancer. Previous studies associated the anti- tumour effect of CAR T-cells to their long-term persistence. Most stud- ies use cyclophosphamide and fludarabine (CyFlu) preconditioning chemotherapy to facilitate CAR T-cell persistence. However, the effect of CyFlu preconditioning was rarely compared to other chemotherapies or to CAR T-cells alone. The THINK, SHRINK and ALLOSHRINK trials evaluate the safety and clinical activity of NKG2D receptor-based CAR T-cells in metastatic colorectal cancer (mCRC) patients. THINK and SHRINK utilize autologous CAR T-cells, whereas ALLOSHRINK utilizes allogeneic CAR T-cells. In THINK, CAR T-cells are injected without pre- conditioning chemotherapy or after CyFlu. In SHRINK and ALLOSHRINK, FOLFOX chemotherapy is given before CAR T-cell injections. Herein we present cellular kinetics results from these three trials.

Methods

Whole blood samples were drawn at various timepoints from pa- tients receiving at least one injection of CAR T-cells. Peripheral blood mononuclear cells (PBMCs) were isolated by ficoll gradient centrifu- gation at a central laboratory designated by the Sponsor. Genomic DNA was isolated using a commercially available kit. Engraftment of CAR T-cells was measured by digital droplet polymerase chain reac- tion (ddPCR) using transgene-specific primers and reported as trans- gene copies per microgram of genomic DNA. Long-term persistence of CAR T-cells was measured by calculating the area under the curve (AUC) using the linear trapezoidal rule.

Results

35 mCRC patients have been treated in THINK (14), SHRINK (9) and ALLOSHRINK (12). Preliminary results are available for 29 subjects. Cell kinetics for subjects having received one injection of autologous CAR T- cells show a seven-fold increase in mean peak levels of T-cell engraft- ment with CyFlu compared to FOLFOX. Mean AUC is four times higher with CyFlu compared to FOLFOX. Peak levels of engraftment and per- sistence observed with FOLFOX and without previous chemotherapy are similar. Additionally, allogeneic CAR T-cells exhibit a five-fold in- crease in mean AUC and a ten-fold increase in mean peak levels com- pared to autologous cells with the same prior chemotherapy regimen. Additional analyses will be presented during the congress.

Conclusions

Analyses of the initial 29 patients receiving either autologous or allo- geneic NKG2D-based CAR T-cells demonstrate that CyFlu enhances peak levels and persistence of adoptively transferred cells. FOLFOX

does not appear to influence engraftment or persistence of CAR T-cells. Allogeneic CAR T-cells show higher peaks and time-averaged persist- ence compared to autologous cells. Analysis of the results is ongoing. Ethics Approval

The studies referred to in this abstract were approved by all relevant ethical committees and authorities.

P148

High affinity NK cells expressing a PD-L1 chimeric antigen receptor demonstrate anti-tumor activity in head and neck cancer through multiple distinct mechanisms

Yevtte Robbins1_{, Jay Friedman, PhD}1_{, Sarah Greene}1_{, Kellsye Fabian,} PhD1, Michelle Padget1, John Lee, MD2, Patrick Soon-Shiong, MD2, Kayvan Niazi3_{, Lennie Sender}2_{, Laurent Boissel}2_{, Jeffrey Schlom, PhD}1_, James Hodge, PhD, MBA1, Clint Allen, MD1

1_{NIH, Bethesda, MD, United States;}2_{NantKwest, Culver City, CA, United} States;3Nantworks, Culver City, CA, United States;4NIH/NIDCD, Bethesda, MD, United States

Correspondence:Clint Allen ([email protected]) Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P148 Background

A significant portion of head and neck cancers (HNCs) harbor gen- omic alterations that render them insensitive to T cell detection. For these patients, natural killer (NK) cellular therapy may be an effective complementary treatment approach. We studied the anti-tumor ac- tivity of a novel, off the shelf, NK cellular therapy consisting of high affinity NK cells engineered to express a chimeric antigen receptor (CAR) targeting PD-L1 (PD-L1 t-haNKs).

Methods

Irradiated (15 Gy) PD-L1 t-haNK cells were assessed for direct cytotox- icity of five human and two murine HNC cell lines by real-time imped- ance analysis. PD-L1 knockout by CRISPR/Cas9 gene editing was performed in select cells to assess PD-L1-specific killing. Co-culture as- says with PD-L1 t-haNKs and murine or human peripheral and tumor infiltrating leukocytes were performed to determine selective elimin- ation of cells. Wild-type C57BL/6 (B6) or NSG mice were engrafted with parental or PD-L1 knockout murine or human tumors and assessed for tumor growth inhibition (TGI) following PD-L1 t-haNK treatment. Results

PD-L1 CAR expression on PD-L1 t-haNKs was verified. PD-L1 t-haNKs killed all human and murine HNC cell lines at low effector:target ra- tios. Killing of cells was significantly enhanced with increased PD-L1 expression following IFN-γ pre-treatment. Baseline killing was par- tially reversed and IFN-γ-enhanced killing was completely abrogated in PD-L1 knockout cells. Ex vivo co-culture of PD-L1 t-haNKs with per- ipheral and tumor infiltrating leukocytes from tumor bearing mice or with peripheral leukocytes from HNC patients revealed selective elim- ination of PD-L1 high macrophages and myeloid derived suppressor cells (MDSC) but not lymphocyte subsets. Treatment of B6 mice bear- ing murine oral cancers with PD-L1 t-haNKs in vivo resulted in≥50% reduction in PD-L1 high macrophages and MDSC but no reduction in lymphocytes. Treatment of NSG mice bearing parental human HNC or B6 mice bearing parental murine oral cancer resulted in significant TGI after PD-L1 t-haNK treatment. TGI was completely abrogated in mice bearing PD-L1 knockout tumors.

Conclusions

PD-L1 t-haNKs mediated potent PD-L1-specific cytotoxicity against HNC cells and selectively eliminate immunosuppressive macrophages and MDSC expressing high levels of PD-L1 from the periphery and tumor microenvironment. PD-L1 t-haNK monotherapy resulted in PD- L1-specific TGI in xenograft and syngeneic models. These data pro- vide the pre-clinical rationale for the clinical study of PD-L1 t-haNKs in solid tumors. Evidence that PD-L1 t-haNKs selectively eliminate im- munosuppressive macrophages and MDSC support the clinical study of PD-L1 t-haNKs as a monotherapy or in combination with treat- ments designed to activate T cell immunity.

Ethics Approval

The study was approved by the NIH Animal care and Use Committee, approval number 1464-18.

P149

Silencing PD-1 using self-delivering RNAi PH-762- to improve Iovance TIL effector function using Gen 2 manufacturing method Inbar Azoulay-Alfaguter, PhD1, Michelle Abelson, PhD1, Krit Ritthipichai, DVM, PhD1, Kenneth D’Arigo1, Florangel Hilton1, Marcus Machin, BS2, Dingxue Yan2_{, James Cardia}2_{, Maria Fardis, PhD, MBA}1_{, Cecile Chartier}1 1_{Iovance Biotherapeutics, Inc., Tampa, FL, United States;}2_Phio Pharmaceuticals, Tampa, FL, United States

Correspondence:Cecile Chartier ([email protected]) Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P149 Background

Adoptive T-cell transfer with tumor infiltrating lymphocytes (TIL) is an investigational immunotherapy for advanced solid cancers. Ongoing Phase II clinical trials of Iovance_’s lifileucel and LN-145 TIL products have demonstrated efficacy with ORRs of 38% and 44% in patients with melanoma and cervical cancer, respectively [1,2]. Anti-PD-1 therapy has been widely used as a first-line ther- apy in several types of cancer. TIL infusion products from the pa- tients previously treated with anti-PD-1 therapy still sustain PD-1 expression, especially the subset of tumor antigen-specific TIL [3]. Building on the therapeutic efficacy of PD-1 blockade, we rea- soned that intrinsic silencing of PD-1 in our TIL products, may provide similar benefits to systemic administration of anti-PD-1 therapy, while decreasing the side effects associated with sys- temic anti-PD-1 [3]. Self-delivering small interfering RNA (sd- rxRNA) is a chemically modified siRNA molecule, which has ability to penetrate cell types with high knockdown efficiency of specific target genes [4]. Furthermore, a knockdown approach yields a transient effect, which may prove a more favorable approach when compared with permanent genetic modification. Here, we tested the silencing efficiency of a PD-1-targeted sd-rxRNA, termed PH-762, in TIL and its effect on TIL phenotype and function.

Methods

TIL from melanoma, breast cancer, lung cancer, H&N cancer, and sarcoma were expanded ex vivo with Iovance’s proprietary 22-day process in the presence of PH-762. Resulting TIL products were assessed for PD-1 knockdown, cell expansion and viability, phenotype (T-cell lineage, differentiation, activa- tion, and exhaustion), and effector functions (IFN-gamma induction).

Results

Average silencing of the PD-1 levels was 85%. Sixteen of the 19 tumors tested demonstrated >80% silencing at the surface of PH-762-treated TIL relative to control sd-rxRNA-treated TIL. The remaining 3 samples had ~70% silencing efficiency. Expression of T-cell activation markers including 4-1BB and OX40 was sig- nificantly increased in TIL expanded with PH-762. Importantly, other inhibitory and exhaustion molecules remained unaffected, suggesting that compensatory mechanisms were not triggered by PD-1 silencing. Functionally, PD-1 knockdown TIL displayed elevated IFN-gamma secretion when co-cultured with autolo- gous tumor cells, indicating improved effector function upon specific T-cell re-stimulation.

Conclusions

sd-rxRNA-mediated silencing of PD-1 with PH-762 in TIL was highly efficient and generated TIL products with elevated effector function, providing a strong rationale for clinical testing.

Acknowledgements

PH-762 was kindly provided by Phio Pharmaceuticals. References

1. Sarnaik A. et al. Safety and efficacy of cryopreserved autologous tumor infiltrating lymphocyte therapy (LN-144, lifileucel) in advanced metastatic melanoma patients who progressed on multiple prior therapies including anti-PD-1. J Clin Oncol. 2019;37:2518-2518.

2. Jazaeri A A, et al. Safety and efficacy of adoptive cell transfer using autologous tumor infiltrating lymphocytes (LN-145) for treatment of

recurrent, metastatic, or persistent cervical carcinoma. J ClinOncol. 2019;37:2538-2538.

3. Gros A, et al. PD-1 identifies the patient-specific CD8(+) tumor- reactive repertoire infiltrating human tumors. J Clin Invest.

2014;124:2246-2259.

4. Ligtenberg M A, et al. Self-Delivering RNAi Targeting PD-1 Improves Tumor-Specific T Cell Functionality for Adoptive Cell Therapy of Malig- nant Melanoma. Mol Ther. 2018;26:1482-1493.

P150

1st-in-human CAR T clinical trial for metastatic breast cancers Cynthia Bamdad, PhD , Andrew Stewart, PhD, Pengyu Huang, PhD, Benoit Smagghe, PhD, Scott Moe, PhD, Tyler Swanson, Thomas Jeon, Danica Page, Ketan Mathavan, PhD, Trevor Grant, PhD, Rachel Herrup

Minerva Biotechnologies, Waltham, MA, United States

Correspondence:Cynthia Bamdad ([email protected]) Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P150 Background

Minerva will open a 1st-in-human CAR T clinical trial for meta- static breast cancers at the Fred Hutchinson Center September, 2019. huMNC2-CAR44 targets a novel form of MUC1; no thera- peutic that targets this form has ever been tested in humans. All previous, failed attempts to therapeutically target MUC1 have targeted the tandem repeat domains, which are cleaved and shed from the surface of cancer cells. Cleavage and shed- ding of the tandem repeat domain increases as tumor stage in- creases. huMNC2-CAR44 targets the truncated extra cellular domain of MUC1* (muk 1 star), also known as MUC1-C, which is the transmembrane cleavage product that remains after MUC1 is cleaved and the tandem repeat domain is shed from the can- cer cells. The MNC2 antibody, which is the targeting head of the CAR, cannot bind to full-length MUC1. It binds to an ectopic epitope that is only unmasked by cleavage and release of the MUC1 tandem repeat domain. MUC1* growth factor receptor is activated when onco-embryonic growth factor NME7AB dimer- izes its truncated extracellular domain. NME7AB and the huMNC2 antibody both compete for the same binding site, which is masked in full-length MUC1.

Methods

Monoclonal antibody MNC2 was selected because it recognizes a conformational epitope within MUC1* that is created by cleav- age by MMP9, which is overexpressed in breast cancers and is an indicator of poor prognosis. The luminal edge of some nor- mal tissues express a cleaved MUC1*-like form; however, on normal tissues, MUC1 is cleaved by a different cleavage enzyme, which alters the conformation of the truncated extra cellular domain and it is not recognized by the MNC2 antibody. Results

huMNC2-scFv recognizes 95% of breast cancers, across all subtypes, wherein the average percent staining for each tissue specimen is