Abstract: Background: Celularity, Inc. is developing human placental hematopoietic stem cells-derived, cryopreserved, off-the-shelf, ex-vivo expanded and allogenic natural killer (PNK) cells for various hematological malignancies and solid tumors. NK cells play a central role in antibody dependent cell mediated cytotoxicity (ADCC) through Fc receptor CD16 in monoclonal antibody mediated anti-tumor therapies. Two allelic forms of CD16 have been identified. The 158Val/Val form has shown to have higher IgG binding affinity compared to the 158Phe/Phe form.1 The high IgG binding allele are found in about 10-20% of the normal population.2,3 In addition, activation of NK cells induces CD16 shedding by matrix metalloprotease ADAM17 at 197Ser, thus limiting ADCC responses. A single mutation (Ser197Pro) prevents CD16 shedding and increases ADCC activity in NK cells.4 Since the antibody binding affinity and CD16 expression of PNK could vary with different donors, we hypothesize that expressing a high affinity (158Val) and proteinase cleavage resistant (197Pro) CD16 variant (CD16VP) augments anti-tumor ADCC activity. Methods: Lentivirus expressing CD16VP was used to transduce human placental CD34+ cells. After transduction, the cells were cultured in the presence of cytokines including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2, for 35 days to generate PNK-CD16VP cells. Non-transduced PNK cells (NT) served as a control. Expression of CD16VP was evaluated by activating cells with PMA/ionomycin to induce CD16 cleavage (CD16 shedding assay) followed by immunostaining with CD16 antibody and analyzed using flowcytometry. ADCC of PNK-CD16VP cells was assessed against Daratumumab (anti-CD38) or Rituximab (anti-CD20) opsonized lymphoma cell lines at various effector to target (E:T) ratios. IgG was used as ADCC control. In vivo anti-tumor activity was assessed in a Daudi disseminated Xenograft model in NSG mice. Luciferase-expressing Daudi cells (3x106) were intravenously (IV) administered at day 0, followed by PNK-CD16VP cells (10x106) IV at day 1 and day 3, and Daratumumab at day 3. Tumor burden in mice was monitored by Bioluminescence Imaging (BLI). Statistical differences between the groups were calculated using paired t-test using Prism. Results: Lentiviral transduction of CD16VP achieved high expression efficiency in multiple placental CD34+ donors. These cells expanded [7095 ± 2998 folds (n=8)] and differentiated into PNK cells ( & gt;90% CD56+CD3-) at day 35. PNK-CD16VP expressed 64.6 ± 10.3% (n=8) of CD16, while the NT expressed 12.1 ± 3.3% (n=8) CD16. PMA/ionomycin induced & gt;89% shedding of CD16 in NT cells, while significantly less ( & lt;11%) CD16 shedding was observed in PNK-CD16VP cells. These results indicated that CD16VP was expressed and maintained throughout the culture process. In vitro ADCC assay demonstrated improved anti-tumor activity of PNK-CD16VP cells over NT cells against Daratumumab or Rituximab opsonized lymphoma cell lines. At 10:1 E:T ratio PNK-CD16VP cells elicited higher cytotoxicity compared to NT: 47 ± 13% against Daratumumab opsonized Daudi cells versus 25 ± 5% (n=5; p & lt;0.05); 30 ± 13% against Daratumumab opsonized HS-Sultan cells versus 21 ± 14% (n=3; p & lt;0.05); 30 ± 7% against Daratumumab opsonized Sudhl6 cells versus 16 ± 10% (n=3; p & lt;0.05). Improved ADCC activities in PNK-CD16VP were also observed in other cell lines including Raji and Sudhl4 with Daratumumab and Rituximab antibodies. PNK-CD16VP were used to test anti-tumor ADCC in vivo using a disseminated Daudi Xenograft model. The preliminary data demonstrated that PNK-CD16VP combined with Daratumumab reduced BLI signal ( & gt;50%) compared to vehicle or Daratumumab alone at day 10 after treatment. This observation suggested that PNK-CD16VP demonstrated in vivo ADCC anti-tumor activity. Conclusions: In this study, we genetically modified PNK to express high affinity and cleavage resistant CD16 variant using lentivirus. The PNK-CD16VP cells demonstrated enhanced ADCC function against lymphoma cell lines in vitro and in vivo. Further development of PNK-CD16VP for immune-oncology therapeutics is warranted. References: Wu J et al. J Clin Invest. 1997;100(5):1059-1070.Sugita N et al. Clin Exp Immunol. 1999;117(2):350-354.Koene HR et al. Blood. 1997;90(3):1109-1114.Jing Y et al. PLoS One. 2015;10(3):e0121788. Disclosures Guo: Celularity, Inc.: Employment. Somanchi:Celularity Inc: Employment. Mathur:Celularity Inc: Employment. He:Celularity Inc: Employment. Ye:Celularity Inc: Employment. Difiglia:Celularity Inc: Employment. Rotondo:Celularity Inc: Employment. Rana:Celularity Inc: Employment. Ling:Celularity Inc: Employment. Edinger:Celularity Inc: Employment. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.

Abstract: Tumors often develop resistance to surveillance by endogenous immune cells, which include natural killer (NK) cells. Ex vivo activated and/or expanded NK cells demonstrate cytotoxicity against various tumor cells and are promising therapeutics for adoptive cancer immunotherapy. Genetic modification can further enhance NK effector cell activity or activation sensitization. Here, we evaluated the effect of the genetic deletion of ubiquitin ligase Casitas B-lineage lymphoma pro-oncogene-b ( CBLB ), a negative regulator of lymphocyte activity, on placental CD34 + cell-derived NK (PNK) cell cytotoxicity against tumor cells. Methods Using CRISPR/Cas9 technology, CBLB was knocked out in placenta-derived CD34 + hematopoietic stem cells, followed by differentiation into PNK cells. Cell expansion, phenotype and cytotoxicity against tumor cells were characterized in vitro. The antitumor efficacy of CBLB knockout (KO) PNK cells was tested in an acute myeloid leukemia (HL-60) tumor model in NOD- scid IL2R gamma null (NSG) mice. PNK cell persistence, biodistribution, proliferation, phenotype and antitumor activity were evaluated. Results 94% of CBLB KO efficacy was achieved using CRISPR/Cas9 gene editing technology. CBLB KO placental CD34 + cells differentiated into PNK cells with high cell yield and 〉 90% purity determined by CD56 + CD3 − cell identity. Ablation of CBLB did not impact cell proliferation, NK cell differentiation or phenotypical characteristics of PNK cells. When compared with the unmodified PNK control, CBLB KO PNK cells exhibited higher cytotoxicity against a range of liquid and solid tumor cell lines in vitro. On infusion into busulfan-conditioned NSG mice, CBLB KO PNK cells showed in vivo proliferation and maturation as evidenced by increased expression of CD16, killer Ig-like receptors and NKG2A over 3 weeks. Additionally, CBLB KO PNK cells showed greater antitumor activity in a disseminated HL60-luciferase mouse model compared with unmodified PNK cells. Conclusion CBLB ablation increased PNK cell effector function and proliferative capacity compared with non-modified PNK cells. These data suggest that targeting CBLB may offer therapeutic advantages via enhancing antitumor activities of NK cell therapies.

Abstract: Introduction: Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in ~20% of gastric cancer cases and correlated with poor outcome. Celularity is developing CYNK-101, an allogeneic, off-the-shelf human placental CD34+-derived NK cell product with genetically modified CD16 variant (CD16VP) for cancer treatment. CYNK-101 is designed to enhance the anti-tumor antibody dependent cellular cytotoxicity (ADCC) activity by expressing high-affinity and cleavage resistant CD16VP. Here we report the preclinical efficacy data of CYNK-101 plus Trastuzumab, an anti-HER2 monoclonal antibody, against HER2+ gastric cancer cells. Methods: Human placental CD34+ cells were transduced with lentivirus expressing CD16VP and cultured with cytokines to generate CYNK-101. ADCC activity of CYNK-101 plus Trastuzumab against HER2+ gastric cancer cells was measured by real-time xCELLigence, and cytokine secretion was quantified by Luminex. Ex vivo-CYNK-101 was isolated from NOD-scid IL2Rγnull immunodeficient (NSG) mice 13 days post CYNK-101 infusion. A subcutaneous NCI-N87 xenograft NSG-hIL15 transgenic mouse model was used for in vivo study. Results and conclusion: CYNK-101 was generated from multiple placental CD34+ donors (n=7) with & gt;90% CD56+CD3-and 74.1 ± 5.6% CD16 expression. While 4h PMAi treatment resulted in & gt;89% CD16 cleavage on non-transduced NK cells, & lt;11% cleavage from CYNK-101 demonstrated CD16 shedding resistance. At an effector to target (E:T) ratio of 1:1, CYNK-101 (n=7) showed enhanced lysis of NCI-N87 cells in the presence of Trastuzumab compared to IgG control, 59.6 ± 13.7% vs. 9.8 ± 3.2% at 4h, 97.1 ± 7.1% vs. 26.2 ± 16.1% at 24h, respectively (p & lt;0.001), and secreted increased GM-CSF, IFN-γ, and TNF-α at 24h. Ex vivo-CYNK-101 plus Trastuzumab exhibited enhanced cytotoxicity against NCI-N87 compared to IgG control (30.9% vs. 6.6% at 4h, 90.7% vs. 9.1% at 24h) at the E:T of 0.5:1, as well as increased production of GM-CSF, IFN-γ, and TNF-α at 24h. Compared to CYNK-101 pre-infusion, ex vivo-CYNK-101 showed not only a higher ADCC activity against NCI-N87, but also a more matured NK phenotype with increased expression of CD16, KIR, NKG2D, and CD94. In addition, CD16 shedding resistance was demonstrated on ex vivo-CYNK-101. CYNK-101 in vivo anti-tumor ADCC activity was assessed in a subcutaneous xenograft NCI-N87 mouse model. Significant tumor reduction was shown in the animals treated with CYNK-101 plus Trastuzumab compared to vehicle control, Trastuzumab or CYNK-101 alone (P & lt;0.0001, n=20 mice per group). In summary, our results demonstrated synergistic anti-tumor ADCC activities of CYNK-101 plus Trastuzumab against HER2+ gastric cancer cells in vitro, ex vivo and in vivo. Further development of CYNK-101 in combination with Trastuzumab for HER2+ gastric cancer immunotherapy is underway. Citation Format: Lin Kang, Shuyang He, Irene Raitman, Salvatore Rotondo, Joseph Gleason, Valentina Rousseva, Xuan Guo, Hemlata Rana, Qian Ye, Robert Hariri, Xiaokui Zhang. Potent immunotherapy of human placental CD34+-derived natural killer cells with high affinity and cleavage resistant CD16 (CYNK-101) plus Trastuzumab for HER2+ gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 58.

Abstract: CYNK-101, an allogeneic off-the-shelf human placental CD34+-derived natural killer (NK) cell product, is genetically modified to express a variant of CD16 (FcγRIII) via lentiviral vector transduction. When targeting tumors with monoclonal antibodies, NK cells are key effectors of antibody dependent cellular cytotoxicity (ADCC) following recognition of antibody Fc by CD16. CYNK-101 expresses the CD16 variant (CD16VP) which has a high-affinity due to a Valine at amino acid position 158 along with proteolytic cleavage resistance imparted by Proline at amino acid position 1971,2,3,4. We hypothesize that expressing CD16VP enhances anti-tumor activity of CYNK-101 in combination with monoclonal antibody therapy. Reported here are the in vitro and ex vivo results of evaluating CYNK-101 cytotoxicity against human epidermal growth factor receptor 2 (HER2)+ solid tumors in combination with Trastuzumab, an anti-HER2 monoclonal antibody. Human placental CD34+ cells were transduced with lentivirus expressing CD16VP, and cultured in the presence of cytokines, including TPO, SCF, Flt3L, IL-7, IL-15, and IL-2, to generate CYNK-101 cells. Lentiviral transduction with CD16VP achieved high expression efficiency in multiple placental CD34+ donors. These cells (n=7) expanded at 12041 ± 6394-fold and differentiated into CYNK-101 being & gt;90% CD56+CD3-. CYNK-101 expressed 74.1 ± 5.6% (n=7) of CD16 at the end of cultivation. Proteolytic cleavage resistance conferred by CD16VP in CYNK-101 was evaluated by activating NK cells with PMA/ionomycin (PMAi) to induce CD16 shedding. While 4h PMAi treatment resulted in & gt;89% CD16 cleavage on non-transduced NK cells, & lt;11% cleavage was observed on CYNK-101 cells, demonstrating CD16 shedding resistance. The in vitro anti-tumor activity of CYNK-101 against HER2+ solid tumor cell lines was assessed in combination with Trastuzumab. At effector to target (E:T) ratio of 0.6:1, CYNK-101 (n=7 donors) showed enhanced lysis of NCI-N87 gastric cancer cells in the presence of Trastuzumab compared to that of IgG control, 41.2 ± 11.1% vs. 5.3 ± 4.7% at 4h, 91.3 ± 15.0% vs. 7.5 ± 9.3% at 24h, respectively (p & lt;0.005). Increased production of GM-CSF, IFN-γ, and TNF-α was shown in CYNK-101 plus Trastuzumab against NCI-N87 at 24h compared to that of IgG control. Overall, the enhanced ADCC activity of CYNK-101 in combination with Trastuzumab has been demonstrated against both HER2+ gastric cancer cell lines, NCI-N87 and OE-19; and HER2+ breast cancer cell lines, AU565, BT-474, HCC-1954, SKBR-3, and ZR-75-30. To further evaluate ex vivo ADCC activity of CYNK-101, CYNK-101 cells were intravenously (IV) injected at 2x107 into busulfan-pretreated NOD-scid IL2Rγnull immunodeficient (NSG) mice at Day 0. Recombinant human IL-15 was intraperitoneally injected at Days 0, 2, 4, 6, 8, 10 and 12. At Day 13, CYNK-101 cells were isolated from mouse livers (ex vivo-CYNK-101) and assessed for phenotype and ADCC activity. Ex vivo-CYNK-101 exhibited enhanced cytotoxicity against NCI-N87 in combination with Trastuzumab compared to that of IgG control (30.9% vs. 6.6% at 4h, 90.7% vs. 9.1% at 24h) at the E:T ratio of 0.5:1, as well as increased cytokine productions of GM-CSF, IFN-γ, and TNF-α at 24h. Compared to pre-infusion CYNK-101, ex vivo-CYNK-101 showed not only a higher ADCC activity against NCI-N87, but also a more matured NK cell phenotype with increased expression of CD16, KIR, NKG2D, CD94, and CD11a. Besides, these ex vivo-CYNK-101 cells demonstrated CD16 shedding resistance post PMAi stimulation. In summary, our results demonstrated enhanced in vitro and ex vivo ADCC activities of CYNK-101 in combination with Trastuzumab against HER2+ solid tumors. CYNK-101 exhibited high resistance to CD16 shedding in vitro and ex vivo. Further maturation of CYNK-101 cells post IV infusion was accompanied by ADCC activity enhancement. Evaluation of in vivo ADCC activity of CYNK-101 in combination with Trastuzumab will include both subcutaneous and orthotopic gastric cancer models. Disclosures Kang: Celularity Inc.: Current Employment. Raitman:Celularity Inc.: Current Employment. Rotondo:Celularity Inc.: Current Employment. Gleason:Celularity Inc.: Current Employment, Current equity holder in private company. He:Celularity Inc.: Current Employment, Current equity holder in private company. Somanchi:Celularity Inc.: Current Employment, Current equity holder in private company. Rousseva:Celularity Inc.: Current Employment. Guo:Celularity Inc.: Current Employment. Rana:Celularity Inc.: Current Employment. DiFiglia:Celularity Inc.: Current Employment. Ye:Celularity Inc.: Current Employment, Patents & Royalties. Mahlakõiv:Celularity Inc.: Current Employment. Van Der Touw:Celularity Inc.: Current Employment. Hariri:Celularity Inc.: Current Employment, Current equity holder in private company. Zhang:Celularity Inc.: Current Employment, Current equity holder in private company.

Abstract: Natural Killer (NK) cells are key mediators of antibody dependent cellular cytotoxicity (ADCC) via the CD16 Fc receptor. NK cellular therapies can effectively be targeted to tumor antigens when combined with tumor specific antibodies. Celularity Inc. is developing human placental CD34 +-derived, cryopreserved, off-the-shelf, allogenic NK cells (CYNK-101) with a high IgG binding affinity and proteinase cleavage resistant CD16 variant (CD16VP) for cancer treatment. We hypothesize that expressing CD16VP augments anti-tumor ADCC activity. Reported here are the in vitro and in vivo results of evaluating CYNK-101 cytotoxicity against CD38 expressing multiple myeloma (MM) and lymphoma tumor cell lines when in combination with daratumumab, an anti-CD38 monoclonal antibody. Human placental CD34 + cells were transduced with lentivirus expressing CD16VP and cultured in the presence of cytokines to generate CYNK-101 cells. The in vitro cytotoxic activity of CYNK-101 against CD38 + MM (MOLP-8, LP-1, MM.1S) and lymphoma (Daudi) tumor cell lines, and normal B-cells was assessed in combination with daratumumab via flow cytometry based ADCC assays and cytokine secretion was assessed via multiplex Luminex analysis. In vivo ADCC activity of CYNK-101 was assessed using a disseminated B-cell lymphoma xenograft model in B-NDG-hIL15 mice. B-NDG-hIL15 mice lack T, B, and NK cells and are transgenic for human IL-15 to support CYNK-101 persistence and maturation. Luciferase expressing Daudi cells (3×10 6) were intravenously (IV) injected on Day 0 three days after preconditioning with a myeloablative dose of busulfan to allow for better tumor cell engraftment. CYNK-101 cells (1x10 7) and/or daratumumab (0.05 mg/kg) were IV injected on Days 7, 14 and 21. Tumor burden was assessed weekly by bioluminescence imaging (BLI) and the mice were followed for assessment of their survival (n=5 mice per group). In vitro ADCC studies indicate enhanced cytolysis of CYNK-101 in combination with daratumumab against both MM and lymphoma tumor cells compared to that of IgG control. At 24h at the effector to target (E:T) ratio of 5:1, CYNK-101 (n=5 donors) demonstrated a cytolysis of 87.6 ± 6.3% with daratumumab vs. 37.3 ± 9.5% with IgG control against MOLP-8 (p & lt;0.001), 73.9 ± 2.5% vs. 32.1 ± 7.2% against LP-1 (p & lt;0.001), 77.2 ± 11.5% vs. 67.4 ± 10.7% against MM.1S (p & lt;0.001), and 54.7 ± 24.0% vs. 4.3 ± 2.6% against Daudi (p & lt;0.01) tumor cells. Secretion of GM-CSF, IFN-γ, and TNF-α was increased in CYNK-101 co-cultured with the tumor cell lines in the presence of daratumumab for 24h (n=5 donors, p & lt;0.05). When cocultured with mixed LP-1 and CD38 + normal B-cells, CYNK-101 in combination with daratumumab displayed specific cytotoxicity against LP-1, while sparing CD38 + normal B-cells even at an E:T ratio up to 100:1, demonstrating that CYNK-101 can distinguish CD38 + tumor cells from CD38 + normal cells. Additionally, despite expression of CD38 on CYNK-101 there was no NK fratricide observed when CYNK-101 were in combination with daratumumab. In vivo studies in the lymphoma xenograft model revealed a significant decrease in tumor burden as evidenced from bioluminescence imaging at day 28 (1 week after last CYNK-101 injection) for mice that received CYNK-101 in combination with daratumumab compared to vehicle control (p & lt;0.001), CYNK-101 (p & lt;0.05) and daratumumab (p & lt;0.05). Furthermore, CYNK-101 in combination with daratumumab demonstrated an enhanced survival benefit with a median survival of 35 days versus a median survival of 28 days for the vehicle treated group (p & lt;0.005). In summary, our results demonstrate enhanced in vitro and in vivo ADCC activities of CYNK-101 in combination with daratumumab against CD38 + hematological tumors and warrant further development of this combination therapy for these cancers. Disclosures Raitman: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Gleason: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Rotondo: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. He: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Rousseva: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Guo: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Rana: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. van der Touw: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Ye: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Kang: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Hariri: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang: Celularity Inc.: Current equity holder in publicly-traded company, Ended employment in the past 24 months.

Abstract: Background: Natural killer (NK) cells exhibit anti-tumor activity in a non-antigen-specific manner without causing graft-versus-host disease. T cell and cord blood NK cells expressing chimeric antigen receptor (CAR) targeting CD19 have demonstrated remarkable clinical efficacies against B cell lymphomas (Maude et al, N Engl J Med 2018; Neelapu et al, N Engl J Med 2017; Liu et al, N Engl J Med 2020). Celularity has developed a platform for the expansion and differentiation of human placental CD34 + stem cells towards NK cells. The introduction of CD19 CAR enables generation of CAR19-CYNK cells that can be used as an off-the-shelf, cryopreserved, allogeneic cell therapy for CD19 + B cell malignancies. Reported here are the in vitro and in vivo results evaluating anti-tumor activity of CAR19-CYNK against CD19 + B cell malignancies. Methods: CAR19-CYNK cells were generated by retroviral transduction of human placental CD34 + cells with an anti-CD19 CAR (CD19scFv-CD28CD3ζ, Sorrento Therapeutics), followed by culture expansion in the presence of cytokines. CD19 CAR expression and phenotype of CAR19-CYNK cells were characterized by flow cytometry using the following surface markers: CD56, CD3, CD226, CD16, CD11a, CD94, NKG2D, NKp30, NKp44, NKp46. The in vitro anti-tumor activity of CAR19-CYNK against the B cell lymphoma cell lines, Daudi and Nalm-6, was assessed at various effector to target (E:T) ratios using a flow cytometry-based cytotoxicity assay and multiplex Luminex analysis for cytokine profiling. Non-transduced (NT) NK cells were used as control. In vivo efficacy of CAR19-CYNK was assessed using a disseminated B-cell lymphoma xenograft model in B-NDG-hIL15 mice. B-NDG-hIL15 mice lack T, B, and NK cells and are transgenic for human IL-15 to support CAR19-CYNK persistence and maturation. Luciferase expressing Daudi cells (3×10 6) were intravenously (IV) injected on Day 0 three days after the mice were preconditioned with a myeloablative dose of busulfan to allow for better tumor cell engraftment. CAR19-CYNK cells (1x10 7) were IV injected on Day 7. Tumor burden was assessed weekly by bioluminescence imaging (BLI) and the mice were followed for assessment of their survival (n=5 mice per group). Results: Placental CD34 + cells were genetically modified using a retroviral vector and achieved an average of 29.2% ± 12.4% (range 17.5% to 50.1%; n=5 donor lots) CD19 CAR expression on CAR19-CYNK cells at the end of 35-day culture. The average fold expansion of CAR19-CYNK was 6186 ± 2847 with the range of 2692 to 10626 (n=5 donor lots). Post-thaw evaluation of CAR19-CYNK (n=5 donor lots) revealed 93.8 ± 3.9% of CD56 +CD3 - NK cells, and transduction of CD19 CAR on CYNK did not significantly alter NK cell phenotype based on various activation and lineage markers (CD226, CD16, CD11a, CD94, NKG2D, NKp30, NKp44, NKp46). CAR19-CYNK displayed enhanced in vitro cytotoxicity against lymphoma cell lines, Daudi and Nalm-6, compared to that of NT NK cells. At the E:T ratio of 10:1, CAR19-CYNK (n=5 donor lots) elicited significant increased cytotoxicity against Nalm-6 compared to that of NT NK cells, with 75.9 ± 14.8% vs. 0.00 ± 0.00% at 24h (p & lt;0.005). Under the same condition, CAR19-CYNK (n=4 donor lots) showed higher cytotoxicity against Daudi compared to that of NT NK cells with 23.6 ± 18.9% vs. 4.9 ± 4.0%. When cocultured with tumor cell lines, CAR19-CYNK showed increased secretion of the proinflammatory cytokines GM-CSF (p & lt;0.05 for both Nalm-6 and Daudi), IFN-g (p & lt;0.05 for Nalm-6), and TNF-a compared to that of NT NK cells at an E:T ratio of 1:1 for 24h. To evaluate the in vivo efficacy of CAR19-CYNK, a disseminated Daudi xenograft B-NDG-hIL15 model was used. CAR19-CYNK treated mice demonstrated a significant survival benefit with a median survival of 39 days versus a median survival of 28 days for the vehicle treated group (p & lt;0.05). Conclusions: In summary, we have successfully established a process for generating CAR19-CYNK cells from human placental CD34 + cells. CAR19-CYNK demonstrated enhanced in vitro cytotoxicity against CD19 + B cell malignancies and in vivo survival benefit in a disseminated lymphoma xenograft B-NDG-hIL15 model. Further development of CAR19-CYNK for CD19 + B cell malignancies is warranted. Disclosures Gergues: Celularity Inc: Current Employment, Current equity holder in publicly-traded company. Raitman: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Gleason: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Rousseva: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. He: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Van Der Touw: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Ye: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Kang: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Pai: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Guo: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Ji: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Hariri: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang: Celularity Inc.: Current equity holder in publicly-traded company, Ended employment in the past 24 months.

Abstract: Background Ischemic cardiovascular diseases are the major causes of death in the world. Recently, mesenchymal or tissue progenitor cell derived exosomes are being investigated as an emerging therapeutic agent for treating cardiovascular diseases due to their potentials in restoring the damaged vasculature via promoting angiogenesis. Exosome therapy has the advantages in low immunogenicity and stability features comparing to cells 1. Tissue and organ derived exosomes could have a broader functionality than those derived from a homogeneous cell population as they contain multiple cell types. Placenta is a large and highly vascularized organ and placenta exosomes are known to play essential roles during pregnancy in supporting fetus development 2. Placenta is abundant with endothelial cells, epithelial cells, cytotrophoblast, syncytiotrophoblasts and stem cells including both hematopoietic and non-hematopoietic stem cells. It is perceivable that exosomes isolated from a placenta possess the functions of the cells in a placenta. Materials and Methods Full-term human placentas were obtained under the full consent of healthy donors. Placenta tissues were cultured in serum-free DMEM medium supplemented with antibiotics. After culturing 8 to 16 hours, supernatant was harvested, medium was changed every 8 to 12 hours for up to two days. Placenta derived exosomes (pExo) were isolated through sequential centrifugation of the supernatant of the placenta culture and final preparation were suspended in PBS and stored at -80oC. Quantification of pExo was performed with Bicinchoninic Acid protein assay kit. pExo size was determined using NanoSight. MILLIPLEX-MAP human cytokine/chemokine-PX41 was used to analyze the cytokine composition of pExo. In vitro cell migration assays were performed with seeding 1x10e5/mL human umbilical vessel endothelial cells (HUVEC) in basal media (BM) on the top chamber of an 8-um transwell on a 24-well plate with 500uL BM with or without pExo in the bottom chamber. After 6-24 hours of culture, transwells were observed under an inverted microscope after staining. In vitro cell proliferation assay was performed by seeding HUVEC at 2-4x10e3 cells/96-well with a water-soluble tetrazolium salt-based cell viability assay. To evaluate the pro-angiogenic activity of pExo in vivo, a hind limb ischemia (HLI) model in db/db diabetic mice was used. After surgical induction of HLI on Day 0, pExo from two different donors or vehicle (n=15 each group) was injected via i.v. at 100ug/mice or 100uL on Days 1, 6 and 11. The blood flow through hind paws was measured by Speckle Doppler. Results Utilizing the established cultivation and isolation methods, we achieved the average yield of pExo at 327±91 mg per placenta (n=10). pExo has an average size of 118±15 nm (n=10) as determined with nano-particle tracking analysis, consistent with consensus exosome size ranging 50nm to 200nm 1. Flow cytometry and Western blot analyses confirmed that pExo exhibits characteristic markers known for exosomes including CD9, CD63 and CD81. pExo contained abundant IL-8, HGF, FGF2, PDGF-BB, RANTES, MCP-1 and GM-CSF which are known to play roles in supporting angiogenesis and chemotaxis. In co-culture assays, pExo promoted the proliferation of HUVECs by 149±13% (n=5) comparing to basal medium controls. pExo also demonstated chemotactic activities comparable to that of complete HUVEC growth medium in stimulating the migration of HUVECs across membrane of transwell. Chlorpromazine and Pitstop2 abolished pExo-induced transwell-migration of HUVECs, suggesting that HUVECs may uptake pExo through endocytosis. In the HLI study, pExo treated mice showed significantly higher (50% to 250%) blood flow on Days 14, 28 and 35 comparing to the vehicle group. Histology analysis showed pExo treated mice had 40% to 140% higher density of CD34+ capillary on Day 35, suggesting a significant increase of angiogenesis in the pExo treated groups comparing to the vehicle group. Conclusion: In summary, pExo contains proangiogenic cytokines and chemokines and demonstrates the promising pro-angiogenic activities in vitro and in HLI mice model. These results support further development of pExo as a potential therapeutic agent for the treatment of cardiosvascular diseases. References: Phinney and Pittenger (2017). Stem Cells. 35:851 Sarker et al., (2014). Journal of Translational Medicine. 12:204 Disclosures Ye: Celularity Inc.: Current Employment, Patents & Royalties. He:Celularity Inc.: Current Employment, Current equity holder in private company. Gleason:Celularity Inc.: Current Employment, Current equity holder in private company. Stout:Celularity: Current Employment, Current equity holder in private company. Shah:Celularity: Current Employment. Somanchi:Celularity Inc.: Current Employment, Current equity holder in private company. Zhang:Celularity Inc.: Current Employment, Current equity holder in private company. Hariri:Celularity Inc.: Current Employment, Current equity holder in private company.