scholarly journals Non-Viral Sleeping Beauty Transposon Engineered CD19-CAR-NK Cells Show a Safe Genomic Integration Profile and High Antileukemic Efficiency

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2797-2797
Author(s):  
Tobias Bexte ◽  
Lacramioara Botezatu ◽  
Csaba Miskey ◽  
Julia Campe ◽  
Lisa Marie Reindl ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Nk Cells ◽  
Nk Cell ◽  
Sleeping Beauty ◽  
Feeder Cell ◽  
Genomic Integration ◽  
Vector Integration ◽  
Sleeping Beauty Transposon

Abstract Background: Natural Killer (NK) cells are known for their high intrinsic cytotoxic capacity. Recently, we and others showed that virally transduced NK cells equipped with a synthetic chimeric antigen receptor (CAR) targeting CD19 induced enhanced killing of acute lymphoblastic leukemia (ALL) cells. Here, we demonstrate for the first time that primary NK cells can be engineered using the non-viral Sleeping Beauty (SB) transposon/transposase system to stably express a CD19-CAR with a safe genomic integration profile and high anti-leukemic efficiency in vitro and in vivo. Methods: Primary NK cells were isolated from PBMCs from healthy donors. SB transposons vectorized as minicircles (MC), which encode either a Venus fluorescent protein or a CD19-CAR together with truncated EGFR (tEGFR) as a marker, were introduced in combination with the hyperactive SB100X transposase into primary NK cells via nucleofection. The genetically engineered NK cells were expanded using IL-15 cytokine stimulation under feeder-cell free conditions. Vector integration sites were mapped by analyzing the genomic region around each insertion site in genomic DNA from long-term cultivated gene-modified NK cells, engineered ether by lentiviral (LV) or SB-based technology. Stable gene delivery and biological activity were monitored by flow cytometry and cytotoxicity of CD19-CAR NK cells against CD19-positive ALL and CD19-negative cell lines. Results: Applying a protocol optimized with respect to nucleofection pulses, time points and plasmid ratios, primary NK cells showed long-lasting Venus expression (up to 50%) upon SB-mediated gene delivery with similar viability as non-treated (NT) NK cells during feeder-cell free ex-vivo expansion using IL-15. Likewise, SB transposon-engineered CD19-CAR NK cells displayed high viability, durable transgene expression (Fig 1 A), and no significant change in the NK cell phenotype profile. Next, we assessed vector integration into genomic safe harbors (GSH). GSH are defined as regions of human chromosomes that fulfill the following five criteria: not ultraconserved, >300 kb away from miRNA genes, >50 kb away from transcriptional start sites (TSS), >300 kb away from genes involved in cancer and outside transcription units. CD19-CAR NK cells generated using SB100X showed a significantly higher frequency of vector integration into GSH compared to LV-transduced CAR-NK cells and a significantly more-close to random nucleotide frequency (computer-generated random positions in the genome map to GSHs; random 43.68%; LV 14.78%, SB100X 23.99%; p<0.05) (Fig 1 B). MC.CD19-CAR NK cells generated with the SB platform demonstrated significantly higher cytotoxicity compared to NT NK cells against CD19-positive Sup-B15 ALL cells after long-term cultivation for two to three weeks and no loss of natural intrinsic cytotoxicity. After 4-hour co-culture, significantly enhanced specific tumor cell lysis was found for MC.CD19-CAR NK cells vs NT NK cells at all effector to target cell ratios (E:T) tested (E:T 20:1 83.88% vs 43.13%; E:T 10:1 75.18% vs 31.32%; E:T 5:1 67.38 vs 32.22%; E:T 1:1 42.54 vs 10.19%; p<0.05) (Fig 1 C). With regard to intrinsic natural cytotoxicity of NK cells, no significant decrease in cell killing was overserved for SB-gene-modified CD19-CAR NK cells compared to NT NK cells against CD19-negative K562 cells (E:T 5:1 83%; p<0.05) (Fig 1 D). Significantly enhanced antitumor potential of SB-generated CD19-CAR NK cells was confirmed in a systemic CD19-positive lymphoma xenograft model (NSG-Nalm-6/Luc) in vivo. After injection of 0.5x10 6 tumor cells per mouse and lymphoma engraftment, animals were treated with a single dose of 10x10 6 SB-modified CD19-CAR NK cells pooled from three different donors with a mean tEGFR/CAR expression of 34%. MC.CD19-CAR NK cell therapy resulted in rapid lymphoma eradication in all treated mice (n=4; p<0.05), whereas mice receiving similar amounts of NT NK cells showed progressive lymphoma growth comparable to untreated control mice (Fig 1 E-F). Conclusion: Taken together, the Sleeping Beauty transposon system represents an innovative gene therapy approach for non-viral engineering of safe, highly functional and relatively cost-efficient CAR-NK cells that may not only be suitable for ALL therapy but also for a broad range of other applications in cancer therapy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals 188 Development of WU-NK-101, a feeder cell-free expanded allogeneic memory NK cell product with potent anti-tumor activity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A200-A200
Author(s):  
Ryan Sullivan ◽  
Mary Mathyer ◽  
Jennifer Govero ◽  
John Dean ◽  
Andrew Martens ◽  
...  
Keyword(s):  
Nk Cells ◽  
Large Scale ◽  
Nk Cell ◽  
Feeder Cell ◽  
Free Expansion ◽  
Anti Tumor Activity ◽  
Memory Nk Cells

BackgroundAllogeneic Natural Killer (NK) cells are emerging as a safe and effective modality for the treatment of cancer, overcoming several limitations associated with adoptive T cell therapies. Cytokine induced memory-NK cells offer several advantages over conventional NK cells, including enhanced functional persistence, efficacy, and metabolic fitness. Additionally, unlike iPSC and cord blood derived NK cells, they do not require engineering to enable functionality. Here we describe the use of WU-PRIME, a GMP-grade fusion protein complex to generate memory NK cells, and WU-EXPAND, a feeder cell free expansion system to expand memory-NK cells and create WU-NK-101. Further cryopreservation enables the large-scale, off-the-shelf manufacture of memory NK for cancer immunotherapy, with high anti-tumor activity.MethodsNK cells derived from healthy donor leukopheresate were either activated with WU-PRIME and then expanded with WU-EXPAND to form WU-NK-101 or immediately expanded with WU- EXPAND as controls and then cryopreserved. We compared NK cell expansion as well as post- thaw NK cell functionality as assessed by cytokine secretion and short-term and long-term anti- tumor functionality, long-term persistence in NSG mice, as well as anti-tumor activity in vivo.ResultsNK cells activated with WU-PRIME followed by WU-EXPAND (WU-NK-101), expand robustly in large-scale reactions, over 250-fold in 14 days. The cells maintain durable expression of CD25 after expansion, as well as several other hallmarks of the memory-NK phenotype as assessed by mass cytometry. As compared to cells expanded with WU-EXPAND only, WU-NK-101 cells have improved in vitro activity against K562 cells, as well as AML cell lines (TF-1, THP-1, and HL-60). Notably, this functionality is maintained long-term upon repeated challenge. In vivo, WU-NK-101 cells, compared to expanded NK cells have improved in vivo persistence (figure 1; 50,290 v. 9,623, p<0.0001). In vivo anti-tumor activity was also assessed in leukemia models, where Memory NK cells demonstrate superior anti-tumor activity compared to expanded NK cells.Abstract 188 Figure 1NK cell persistence in tumor-bearing mice. 10e6 cryopreserved NK cells were injected into K562 tumor-bearing mice, and supported with 50,000IU human IL-2 every other day. After 9 days, blood was harvested by cheek bleed and assessed for NK cells (hCD45+, CD56+, CD3) in the blood by flow cytometry.ConclusionsThe data demonstrate that WU-NK-101 generated using a feeder cell-free expansion system has a memory phenotype and improved in vitro and in vivo anti-tumor activity compared to conventional NK cells. This activation and expansion platform will enable the development and clinical translation of multiple allogeneic NK cell therapies.

scholarly journals Long-Term PEDF Release in Rat Iris and Retinal Epithelial Cells after Sleeping Beauty Transposon-Mediated Gene Delivery

2017 ◽  
Vol 9 ◽  
pp. 1-11 ◽  
Author(s):  
Laura Garcia-Garcia ◽  
Sergio Recalde ◽  
Maria Hernandez ◽  
Jaione Bezunartea ◽  
Juan Roberto Rodriguez-Madoz ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Epithelial Cells ◽  
Sleeping Beauty ◽  
Sleeping Beauty Transposon ◽  
Rat Iris

Pancreatic Cancer Induced by In Vivo Electroporation-Enhanced Sleeping Beauty Transposon Gene Delivery System in Mouse

Pancreas ◽  
2014 ◽  
Vol 43 (4) ◽  
pp. 614-618 ◽  
Author(s):  
June-Shine Park ◽  
Kyung-Min Lim ◽  
Sung Goo Park ◽  
Sun Young Jung ◽  
Hyun-Ji Choi ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Gene Delivery ◽  
Delivery System ◽  
Sleeping Beauty ◽  
Gene Delivery System ◽  
In Vivo Electroporation ◽  
Sleeping Beauty Transposon

scholarly journals 181. Quantitative Evaluation of Long Term Gene Expression In Vivo after Delivery of Polyethylenimine-Conjugated Sleeping Beauty Transposon DNA

2006 ◽  
Vol 13 ◽  
pp. S70
Author(s):  
Kelly M. Podetz-Pedersen ◽  
Jason B. Bell ◽  
Terry W. Steele ◽  
Joel L. Frandsen ◽  
Thomas W. Shier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quantitative Evaluation ◽  
Sleeping Beauty ◽  
Sleeping Beauty Transposon

scholarly journals Helper-Independent sleeping beauty Transposon–Transposase vectors for efficient nonviral gene delivery and persistent gene expression in vivo

2003 ◽  
Vol 8 (4) ◽  
pp. 654-665 ◽  
Author(s):  
Jacob Giehm Mikkelsen ◽  
Stephen R Yant ◽  
Leonard Meuse ◽  
Zan Huang ◽  
Hui Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Delivery ◽  
Sleeping Beauty ◽  
Nonviral Gene Delivery ◽  
Sleeping Beauty Transposon

Real-Time In Vivo Biodistribution of Multipotent Adult Progenitor Cells (MAPC): Role of the Immune System in MAPC Resistance in Non-Transplanted and Bone Marrow Transplanted Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 507-507
Author(s):  
Jakub Tolar ◽  
Scott Bell ◽  
Ron McElmurry ◽  
Lily Xia ◽  
R. Scott McIvor ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Nk Cells ◽  
Nk Cell ◽  
Sleeping Beauty ◽  
Whole Body ◽  
Term Survival ◽  
Hematopoietic Stem

Abstract MAPC are non-hematopoietic stem cells derived from adult BM with the potential for a wide differentiation pattern in vitro and in vivo. MAPCs are MHC class I and thus may be a target of natural killer (NK) cell mediated elimination in the syngeneic setting. To determine whether MAPC are susceptible targets for NK mediated killing, splenocytes from poly I:C (an inducer of NK activity) treated C57BL/6 mice were mixed with Yac-1 (H2a; a NK sensitive target) or MAPC (from C57BL/6J-rosa26) in a chromium release assay. Effector:target ratios indicated that MAPC were susceptible to NK lysis albeit less so than Yac-1 cells. To assess in vivo immune responses to MAPC, we infused MAPC into mice with various degrees of T-, B-, and NK- cell immune competence. To follow biodistribution of MAPC in live animals with whole body imaging (WBI), we labeled MAPC with red fluorescent protein DsRed2 and luciferase, using Sleeping Beauty transposons. MAPC (106) were co-nucleofected (Amaxa) with 5mcg of each pT/CAGGS-DsRed2 and pT/CAGGS-Luciferase and an SB transposase-encoding plasmid (p/CMV-HSB2) at a 1:50 ratio. Selected double transgenic MAPC (MAPC DL) clones were euploid, and maintained their characteristic trilineage differentiation. MAPC DL (106) were injected IV into cohorts (n=5–6) of adult C57BL/6 (B6), Rag2−/− (T- and B-cell deficient) and B6 Rag2/IL-2Rgc (T-, B- and NK deficient mice). Additional cohorts of B6 and Rag2−/− were given anti-NK1.1 mAb 2x/wk to deplete NK cells. In B6 mice, MAPC DL were detected on d4 but not d14 or d30. In Rag2−/− mice, MAPC DL were detected throughout the 30d period. NK depletion did not substantially increase MAPC DL number in B6 mice. However, in Rag2/IL-2Rgc mice MAPC DL were persistent and in 50% of mice they increased in number from d4‡d30. Post-mortem analysis revealed MAPC DL cells in all but B6 wild type mice: Rag2/IL-2Rgc ≥ Rag2−/− with NK depletion&gt;&gt; Rag2−/−. These data suggest that endogenous NK cells and T cells resist MAPC DL. Interestingly, in vitro studies indicate that MAPCs suppress an allogeneic mixed lymphocyte reaction (MLR) culture. Therefore, the T cell resistance to MAPC may be due to an immune response generated to the multiple foreign reporter proteins expressed by these cells. Since MAPCs may be useful as cellular therapies for the treatment of regimen-related toxicity, studies were performed in which B10.BR mice were lethally irradiated (TBI) and given B6 BM ± MAPC DL (106). MAPC DL were seen in the chest, abdomen, face, and paws on d4, d7, d10 and d28 at high numbers suggesting that TBI conditioning overcomes both NK and T cell mediated resistance resuting in a widespread homing/migration of MAPC. These data are the first to illustrate the immune responses to MAPCs and indicate that TBI conditioning may be advantageous in the long-term survival and widespread homing of MAPCs.

Assessment of antitumor function of NK cells expanded with exosomes from K562.mb21 cells.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 132-132 ◽  
Author(s):  
Jeremiah Oyer ◽  
Sarah B. Gitto ◽  
Sara Khederzadeh ◽  
Kari Shaver ◽  
Dean A. Lee ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Expansion ◽  
Nk Cell ◽  
Ex Vivo ◽  
K562 Cells ◽  
In Vitro Cytotoxicity ◽  
Feeder Cell ◽  
Nsg Mice

132 Background: NK cells can kill malignant cells to provide innate immunity against tumors. Due to their low abundance in blood, a focus is to expand NK cells ex vivo having enhanced anti-tumor cytotoxicity to be used as a treatment. Our group has pioneered a cell-free method using plasma membrane (PM) particles derived from K562 cells expressing 41BBL and membrane-bound IL-21 (K562.mb21) which were developed for NK cell expansion. Compared to feeder cell based methods for NK cell expansion, PM21-particles improve safety and allow for potential wide-spread dissemination, and also allows direct in vivo use. Exosomes, vesicles naturally secreted by cells, may yet be another novel feeder cell free way for NK cell expansion and may have further advantageous therapeutic dimensions. Methods: EX21-exosomes and PM21-particles were prepared from K562.mb21 cells and characterized by Nanosight and Western blot analysis. CD3-depleted PBMCs were cultured with EX21 for 14 days, NK cell amounts were monitored and media changed every 2-3 days. In vitro cytotoxicity against K562 cells were comparatively assessed for EX21-NK cells and PM21-NK cells. In vivo anti-tumor efficacy of EX21- and PM21-NK cells was assessed in NSG mice implanted ip with SKOV3_luc ovarian tumor cells (1 x 106 cells seeded for 4 days). SKOV3-bearing mice were treated with vehicle, or two doses of EX21-NK cells or PM21-NK cells (1 x 107, in 5 day intervals), and with or without in vivo administration of EX21 (10 µg, 3x/week) or PM21-particles (600 µg, 3x/week). All groups were injected ip with IL-2 (10 KU, 3x/week). Survival analysis was performed with a Log-rank (Mantel-Cox) test. Results: NK cells cultured with EX21 expanded 530 fold (344-710) over 14 days compared to 735 fold (667-802) in presence of PM21-particles. Treatment of SKOV3 engrafted NSG mice with NK cells, expanded with either EX21 or with PM21, allowed significant ( < 0.0001) increase in survival compared to untreated animals (41-44 vs 29 days post treatment). Ip delivery of EX21 to SKOV3 bearing mice had no effect on survival in either untreated control or EX21-NK cell treated groups. Conclusions: EX21 efficiently expands NK cells and EX21-NK cells have equal anti-tumor effect as PM21-NK cells, both in vitro and in vivo.

scholarly journals The IL-12– and IL-23–Dependent NK-Cell Response is Essential for Protective Immunity Against SecondaryToxoplasma GondiiInfection

2019 ◽  
Author(s):  
Daria L. Ivanova ◽  
Tiffany M. Mundhenke ◽  
Jason P. Gigley
Keyword(s):  
Nk Cells ◽  
Cell Fate ◽  
Cell Response ◽  
Nk Cell ◽  
Secondary Infection ◽  
Protective Role ◽  
Parasite Infection ◽  
A Cell

AbstractNatural Killer (NK) cells can develop memory-like features and contribute to long-term immunity in mice and humans. NK cells are critical for protection against acuteT. gondiiinfection. However, whether they contribute to long-term immunity in response to this parasite is unknown. We used a vaccine challenge model of parasite infection to address this question and to define the mechanism by which NK cells are activated during secondary parasite infection. We found NK cells were required for control of secondary infection. NK cells increased in number at the infection site, became cytotoxic and produced IFNγ. Adoptive transfer and NK-cell fate mapping revealed thatT. gondii–experienced NK cells were not intrinsically different from naïve NK cells with respect to their long-term persistence and ability to protect. Thus, they did not develop memory-like characteristics. Instead, a cell-extrinsic mechanism may control protective NK-cell responses during secondary infection. To test the involvement of a cell-extrinsic mechanism, we used anti-IL-12p70 and IL-12p35-/-mice and found that the secondary NK-cell response was not fully dependent on IL-12. IL-23 depletion with anti-IL-23p19in vivosignificantly reduced the secondary NK-cell response, suggesting that both IL-12 and IL-23 were involved. Anti-IL-12p40 treatment, which blocks both IL-12 and IL-23, eliminated the protective secondary NK-cell response, supporting this hypothesis. Our results define a previously unknown protective role for NK cells during secondaryT. gondiiinfection that is dependent on IL-12 and IL-23.

scholarly journals Preclinical Development of Inducible MyD88/CD40 (iMC)-Enhanced Chimeric Antigen Receptor Natural Killer (GoCAR-NK) Cells to Target BCMA+ Tumors

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 39-40
Author(s):  
Xiaomei Wang ◽  
MyLinh T Duong ◽  
Alan D. Guerrero ◽  
Aruna Mahendravada ◽  
Kelly L Sharp ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cytokine Production ◽  
Nk Cell ◽  
Target Cells ◽  
Publicly Traded ◽  
Nsg Mice ◽  
Co Activation ◽  
Tumor Cytotoxicity

Background: Natural Killer (NK) cells possess potent innate anti-tumor cytotoxicity that can be augmented and focused by engineering with chimeric antigen receptors (CARs). Because NK cells do not express T cell receptors that can direct alloreactivity, they have potential as an off-the-shelf (OTS) cell therapy for the treatment of cancer. We recently demonstrated that a drug-inducible co-activation molecule (inducible MyD88/CD40; iMC) synergizes with transgenic IL-15 to boost CAR-NK cell proliferation, survival and anti-tumor cytotoxic effects (Blood Adv.4:1950 [2020]). Here, we describe the pre-clinical development of an OTS iMC/IL-15-enhanced CAR-NK cell platform targeting B cell maturation antigen (BCMA) for the treatment of multiple myeloma. Methods: NK cells were isolated from peripheral blood mononuclear cells by CD56+ selection, activated with IL-15 and microparticles conjugated with IL-21 and 4-1BB ligand. Activated NK cells were transduced with retrovirus encoding an optimized iMC and IL-15-expressing BCMA CAR construct (iMC-BCMA.z-IL15) where iMC signaling could be activated by exposure to rimiducid (Rim), a small molecule dimerizing ligand. Anti-tumor cytotoxicity and cytokine production was assessed using co-culture assays with control or modified CAR-NK cells against BCMA-expressing myeloma cells (NCIH929, RPMI8226, MM1S, U266 and NALM-6-BCMA). Additional experiments were performed with BCMA-edited cell lines (CRISPR/Cas9) to evaluate the innate cytotoxic potential of GoCAR-NK cells. In vivo anti-tumor efficacy and NK cell expansion was measured using immunodeficient NSG mice engrafted with 1.5 x 106 NCIH929-GFPffluc, MM1S-GFPffluc or THP1-GFPffluc cells followed by i.v. treatment with up to 1 x 107 BCMA GoCAR-NK cells. Tumor and NK cells were tracked via bioluminescence imaging. Results: Following IL-15 and IL-21/4-1BBL microparticle stimulation, NK cells were efficiently transduced (40-70%) and exhibited rapid ex vivo expansion (200-fold in 13 days). iMC-BCMA.CAR-IL15-modified NK cells exhibited potent cytotoxicity against BCMA+target cells compared with mock-transduced NK cells (MM1S, 58±4% versus 17±2%; Nalm-6-BCMA, 61±2% versus 19±6%) after 24 hours. Long-term (7 day) co-culture assays revealed the effect of iMC/IL-15 enhancement on NK cell potency, proliferation and cytokine production where iMC-BCMA.z-IL15-modified NK cells stimulated with Rim showed a &gt;70% increase in tumor-specific killing compared to cells without iMC activation. Further, rimiducid-induced activation led to NK cell persistence and proliferation, 8.1±4.0-fold expansion compared to the start of the coculture. In comparison, there was an 80% reduction mock transduced NK cells or GoCAR-NK cells in cocultures without rimiducid. Induced-MC signaling also drove production of cytokines such as TNF-α, IFN-g (6.6X stimulation with 1 nM Rim relative to no drug), GMCSF, IP-10, and IL-13. In addition, activation of the iMC co-activation protein in combination with IL-15 secretion prevented NK cell exhaustion and led to retained functional activity of the modified GoCAR-NK cells for over 4-weeks in culture. In contrast, unmodified NK cells or modified GoCAR-NK cells without Rim exposure became functionally deficient. Of interest, a comparison of NK and T cells modified with the iMC/IL-15 BCMA CAR construct indicated that CAR-NK cells display more rapid target killing, which is further augmented by iMC-mediated cell signaling in the presence of Rim. Furthermore, GoCAR-NK cells were capable of lysis of BCMA-null target cells due to their innate anti-tumor activity. In vivo efficacy studies showed that neither iMC activation nor IL-15 secretion alone were sufficient to support CAR-NK cell engraftment in NSG mice but, in combination, they resulted in CAR-NK cell expansion and persistence. iMC/IL-15-enhanced BCMA GoCAR-NK cells proliferation was associated with improved control of tumor outgrowth in mice challenged with BCMA+ myeloma cells. Summary: These results indicate that the synergistic activity of iMC signaling combined with transgenic IL-15 production can enhance BCMA-specific CAR-NK cytotoxicity, cytokine production, long-term proliferation and persistence and may improve overall anti-tumor efficacy of a potential OTS cell therapy for the treatment of myeloma. Disclosures Wang: Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Duong:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Guerrero:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Mahendravada:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Sharp:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Brandt:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Gagliardi:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Foster:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Bayle:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

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