The authors would like to thank Dr. Carl June (University of Pennsylvania) for help generating and providing aAPC clone #4 and Dr. Perry Hackett (University of Minnesota) for help with the SB system.
Grant support: Cancer Center Core Grant (CA16672); RO1 (CA124782, CA120956, CA141303); R33 (CA116127); P01 (CA148600); SPORE (CA136411); Albert J Ward Foundation; Burroughs Wellcome Fund; Gillson Longenbaugh Foundation; Cancer Prevention and Research Institute of Texas; CLL Global Research Foundation; Department of Defense; Estate of Noelan L. Bibler; Harry T. Mangurian, Jr., Fund for Leukemia Immunotherapy; Institute of Personalized Cancer Therapy; Leukemia and Lymphoma Society; Lymphoma Research Foundation; MDACC's Sister Institution Network Fund; Miller Foundation; Mr. Herb Simons; Mr. and Mrs. Joe H. Scales; Mr. Thomas Scott; National Foundation for Cancer Research; Pediatric Cancer Research Foundation; Production Assistance for Cellular Therapies (PACT); William Lawrence and Blanche Hughes Children's Foundation.

Day 0 or Before
1. Isolation of Mononuclear Cells (MNC) from PB and UCB
<ol>
 <li>Dilute PB with equal volume, and UCB with four volumes of PBS-EDTA.</li>
 <li>Slowly layer diluted blood (25 ml) onto Ficoll (12 ml) in a 50 ml centrifuge tube(s) and centrifuge at 400 x g for 30 - 40 min (no brake).</li>
 <li>Collect and transfer the mononuclear cell fraction (interface) using a transfer pipette to a fresh 50 ml centrifuge tube.</li>
 <li>Bring up the volume to 50 ml with PBS-EDTA and centrifuge at 450 x g for 10 min.</li>
 <li>Aspirate the supernatant, gently re-suspend the cell pellet(s) in 50 ml of Complete Culture Media (CCM) and, centrifuge at 400 x g for 10 min.</li>
 <li>Gently re-suspend and pool the cell pellets in CCM and perform a cell count using Trypan blue exclusion method (Cellometer, PBMC program).</li>
 <li>MNC can now be used for electroporation (Nucleofection) or cryopreserved for future use.</li>
</ol>
2. Preparation of T Cells for Electroporation on Day 0
<ol>
 <li>If using cryopreserved MNC, quickly thaw sufficient cells for a full scale electroporation (2 x 108 adding ~20% to account for cell loss during centrifugation and 2 hr incubation) in 37 &deg;C water bath. If using freshly isolated MNC proceed to step 2.3.</li>
 <li>Gently re-suspend and transfer cells to an appropriately sized centrifuge tube(s) contained pre-warmed Complete Phenol-Free RPMI culture media (PF-RPMI) and centrifuge at 200 x g for 10 min (no brake), aspirate supernatant.</li>
 <li>Re-suspend the MNC in PF-RPMI, perform a cell count (Cellometer) and transfer cells to an appropriately sized cell culture vessel(s) at a concentration of 106 cells/ml.</li>
 <li>Incubate in a humidified 37 &deg;C/5% CO2 incubator for 2 hr &plusmn; 30 min.</li>
 <li>Transfer the MNC to sterile centrifuge tube(s), spin at 200 x g for 5 min (no brakes), aspirate the supernatant and gently re-suspend and combine the cell pellet(s) in PF-RPMI.</li>
 <li>Perform a cell count (Cellometer) and calculate the volume of the cell suspension required (2 x 108 MNC).</li>
 <li>Transfer the calculated volume to a sterile 50 ml centrifuge tube and spin at 200 x g for 10 min (no brake).</li>
 <li>Aspirate the supernatant so that no residual media remains and gently re-suspend by tapping side of tube.</li>
</ol>
3. Electroporation (Nucleofection) of MNC (Full Scale Process Using 10 Cuvettes) on Day 0
<ol>
 <li>Pre-incubate a sterile 12-well plate with 10 wells containing 4 ml of warm PF-RPMI in a humidified 37 &deg;C/5% CO2 incubator.</li>
 <li>Prepare and pre-warm the Lonza Nucleofector Solution Human T cell kit (reconstituted per manufacturer's instructions, <a href="http://www.lonza.com" target="_blank">www.lonza.com</a>) to ambient temperature in a Biosafety Cabinet (BSC).</li>
 <li>Prepare Nucleofector solution/DNA master mix by adding 100 &mu;l of supplemented Nucleofector solution, 15 &mu;g of transposon (supercoiled DNA plasmid designated as CD19RCD28/pSBSO) and 5 &mu;g of transposase (supercoiled DNA plasmid designated as pCMV-SB11) per reaction/cuvette.</li>
 <li>Disperse the cell pellet (from step 2.8) by gently tapping the side of the centrifuge tube and re-suspend in Nucleofection solution/DNA master mix (Final cell concentration: 2 x 107 cells/100 &mu;l).</li>
 <li>Carefully transfer 100 &mu;l of the cell suspension (from step 3.4) to each of ten (10) Lonza Nucleofection cuvettes, being careful to avoid bubbles.</li>
 <li>Tap the cuvette once, and electroporate using program U-014 (for unstimulated T cells).</li>
 <li>Transfer the cuvettes and the 12-well plate (Step 3.1) to the BSC.</li>
 <li>Harvest the electroporated cells from each cuvette using an Amaxa fine tip transfer pipette, by adding ~500 &mu;l of the pre-warmed culture medium from the corresponding well (12-well plate prepared in Step 3.1) and return the plate to a humidified 37 &deg;C/5% CO2 incubator for 2 hr &plusmn; 30 min.</li>
 <li>Following the 2-hr incubation, harvest and transfer the cells from all wells to a sterile centrifuge tube.</li>
 <li>Wash cells by centrifugation at 140 x g for 8 min, ambient temperature, no brake and aspirate and discard the supernatant so that no residual medium covers the cell pellet.</li>
 <li>Disperse the cell pellet by gently tapping the side of the centrifuge tube and gently re-suspend in CCM to achieve a single cell suspension.</li>
 <li>Perform cell count and adjust cell concentration to 106 cells/ml in CCM.</li>
 <li>Transfer cell suspension to cell culture flask(s) and place in incubator overnight.</li>
 <li>Process steps 2.1 to 3.8 were repeated for control: EGFP-transfected cells (5 x 106 cells/cuvette with 5 &mu;g Amaxa control EGFP supercoiled plasmid, pmaxGFP).</li>
</ol>
Day 1 of 1st and Subsequent Stimulation Cycles
4. Analysis of CAR Expression by Flow Cytometry on Day 1
<ol>
 <li>Harvest the electroporated cells and perform a cell count using Trypan blue exclusion method (Hemocytometer).</li>
 <li>Stain cells (1 to 2 x 106) with antibody specific for CD3, CD4, CD8, and human IgG Fc&gamma; (as a measurement of CAR expression).</li>
 <li>Acquire cells on FACS Calibur and analyze the data using FCS Express software to calculate expression of CAR.
 <ol>
 <li>Calculate CAR+ cells in culture by the formula: 
 (No. of Total viable cells) x (% CAR+ cells) = No. of CAR+ cells</li>
 </ol>
 </li>
</ol>
5. Preparation of aAPC (clone #4) on Day 1. aAPC (clone #4) were Derived from K562 Cells (Parental Line Obtained from American Type Culture Collection) to Co-express Desired T Cell Co-stimulatory Molecule
<ol>
 <li>Thaw an aliquot of frozen 100 Gy irradiated aAPC in a 37 &deg;C water bath.</li>
 <li>Cells are washed twice by centrifugation at 400 x g, 10 min in CCM and counted using Cellometer (Trypan blue exclusion).</li>
 <li>Calculate number of viable aAPC required for stimulation: 
 (No. of CAR+ cells) x 2 = No. of irradiated aAPC required</li>
</ol>
6. aAPC-mediated Stimulation of CAR+ T Cells on Day 1 Beginning of 1st and Subsequent Stimulation Cycles
<ol>
 <li>Mix electroporated cells (expressing CAR) and &gamma;-irradiated aAPC (clone #4) in a sterile container at a ratio of 1:2 (CAR+ cell: viable aAPC) in CCM.
 <ol>
 <li>Note the aAPC ratio is adjusted for the expression of CAR based on flow cytometry the day after electroporation.</li>
 </ol>
 </li>
 <li>Add IL-21 (30 ng/ml) to the cell suspension.</li>
 <li>Aliquot in T-75 cm2 flask(s) and/or Vue Life Culture bags at a concentration of 106 cells/ml and return to the incubator.</li>
</ol>
Days 3, 5
7. Continued Culture of CAR+ T Cells
<ol>
 <li>Perform a half-media change, replenish IL-21, and maintain T cells at a concentration of 106 cells/ml.</li>
</ol>
Day 7
8. End of First aAPC-mediated Stimulation Cycle
<ol>
 <li>Harvest cells, count and stain for CD3, CD4, CD8, and Fc&gamma; (CAR) and proceed to step 10.1.</li>
</ol>
9. Depletion of CD56+ Cells (Usually between 7 and 14 days after electroporation)
<ol>
 <li>Perform a CD56 depletion using paramagnetic beads if CD56+CD3neg lymphocytes &ge; 10%.</li>
</ol>
Stimulation Cycles #2, #3, &amp; #4 Corresponding to Days 8 &rarr; 14, Days 15 &rarr; 21, &amp; Days 22 &rarr; 28
10. Recursive Addition of aAPC to Propagate T Cells to Clinically-sufficient Numbers
<ol>
 <li>Repeat the stimulation process (4 times) as described in steps 4.3-8.1.</li>
 <li>Add IL-2 (50U/ml) to the cultures beginning on Days 7, 14 and 21, &amp; then at each media change (three times a week, on a Monday-Wednesday-Friday schedule).</li>
 <li>Cryopreserve (archive) excess T cells as needed.
 <ol>
 <li>T cells frozen using controlled rate freezer.</li>
 </ol>
 </li>
</ol>
Day 28
11. End of Last aAPC-mediated Stimulation Cycle: Harvest T Cells
<ol>
 <li>Cryopreserve T cells for release testing and infusion.</li>
</ol>

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B., Dotti, G., Brentjens, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CARs+on+track+in+the+clinic.">CARs on track in the clinic.</a> Mol. Ther. 19, 432-438 (2011).</li><li>Aronovich, E. L., McIvor, R. S., Hackett, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sleeping+Beauty+transposon+system:+a+non-viral+vector+for+gene+therapy.">The Sleeping Beauty transposon system: a non-viral vector for gene therapy.</a> Hum. Mol Genet. 20, R14-R20 (2011).</li><li>Hackett, P. B., Largaespada, D. A., Cooper, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transposon+and+transposase+system+for+human+application.">A transposon and transposase system for human application.</a> Mol. 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No conflicts of interest declared.

Transposon and transposase systems, such as from piggyBac12,19 and SB18,20-22, are non-viral approaches to gene therapy that are an alternative to viral-mediated transduction of clinical grade CAR+ T cells. The SB was chosen as the gene transfer system based on its potential for human gene therapy1,6,23. We developed the dual technologies of SB transposition (to introduce a CAR) and recursive addition of &gamma;-irradiated aAPC (to retrieve genetically modified T cells stably expressing a CAR) to serve as platform technologies in the manufacture of TAA-specific T cells in compliance with cGMP for Phase I/II trials (Figure 4). After 28 days (four 7-day stimulation cycles) of co-culture on &gamma;-irradiated aAPC, we are typically able to generate at least ~1010 genetically modified T cells suitable for human applications. As needed, additional stimulation cycles can be undertaken to generate larger numbers of genetically modified T cells. Furthermore, if less CAR+ T cells are needed, the approach to electroporation and propagation can be scaled back employing fewer cuvettes and carrying forward just a sub-set of the numerically expanded T cells for subsequent rounds of proliferation on aAPC at the beginning of each Stimulation cycle. The majority of the electroporated and propagated T cells harvested for infusion stably express the CAR. The outgrowth of CD4+ and CD8+ T cells expressing our 2nd generation CAR include cells with a memory/naive phenotype and exhibit three hallmarks of re-directed specificity. Firstly, the genetically modified T cells specifically lyse CD19+ targets, secondly, produce IFN-&gamma; in response to CD19+ stimulator cells and thirdly, proliferate in response to CD19+ feeder cells, all in a CAR-dependent manner13,18. Our approach to integrate a CAR transgene by electro-transfer of non-viral DNA plasmids from the SB system can be undertaken in quiescent primary T cells derived from PB and UCB. We and others have genetically modified K562 cells to serve as aAPC to efficiently propagate clinically-sufficient numbers of T cells for infusion24,25. The aAPC and tissue culture environment (e.g. the addition of IL-21) have been have been modified to generate patient- and donor-derived CD19-specific T cells for infusion after hematopoietic stem-cell transplantation (Table 1)13,18. We can produce CAR+ T cells from PB simply obtained by venipuncture which avoids the cost, discomfort, and inconvenience of obtaining MNC from PB by apheresis. The ability to derive large numbers of CAR+ T cells from small numbers of MNC is particularly appealing for infusing T cells after allogeneic UCB transplantation. The small size and anonymity of the neonatal donor precludes re-accessing this individual at a later time point and only limited numbers of harvested MNC are available as starting material for T cell manufacture to avoid interfering with hematopoiesis. Further advances to the manufacturing process are currently underway to include a high throughput electroporation device coupled with a fully closed WAVE bioreactor to minimize handling. In aggregate, the SB and aAPC are appealing platforms to generate CD19-specific CAR+ T cells that can be adapted to generate large numbers of genetically modified T cells that can recognize alternative cell-surface TAAs in compliance with cGMP.

<table border="1">
 <tbody>
 <tr>
 <td>Reagents</td>
 <td>Vendor</td>
 <td>Catalogue number</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>DNA plasmid expressing GFP (pmaxGFP)</td>
 <td>Lonza</td>
 <td>VPA-1002 (kit)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Sigma</td>
 <td>D8537</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CliniMACS PBS-EDTA</td>
 <td>Miltenyi Biotec</td>
 <td>70026</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HyQ RPMI-1640</td>
 <td>Thermo Scientific</td>
 <td>SH30096.01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phenol Free RPMI-1640</td>
 <td>Thermo Scientific</td>
 <td>SH30605.01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hyclone Fetal Bovine Serum</td>
 <td>Thermo Scientific</td>
 <td>SH3007003HI</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GlutaMAX (100x)</td>
 <td>Gibco</td>
 <td>3505-061</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ficoll-Paque</td>
 <td>GE Healthcare Biosciences</td>
 <td>17-1440-02</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Recombinant human IL-21</td>
 <td>PeproTech</td>
 <td>AF-200-21</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Recombinant human IL-2 (Proleukin)</td>
 <td>Novartis</td>
 <td>NDC 65483-116-07</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Human T cell Kit (Nucleofector Solution)</td>
 <td>Lonza</td>
 <td>VPA-1002</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CliniMACS CD56 Reagent</td>
 <td>Miltenyi</td>
 <td>70206</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FITC-conjugated to mouse mAb specific for human CD3</td>
 <td>BD Biosciences</td>
 <td>349201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>APC-conjugated to mouse mAb specific for human CD4</td>
 <td>BD Biosciences</td>
 <td>340443</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PerCPCy5.5-conjugated to mouse mAb specific for human CD8</td>
 <td>BD Biosciences</td>
 <td>341051</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PE-conjugated to F(ab')2 fragment of goat antibody specific for human Fc&gamma;</td>
 <td>Invitrogen</td>
 <td>H10104</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium Azide</td>
 <td>Sigma</td>
 <td>S2002</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Disposables</td>
 </tr>
 <tr>
 <td>12-well plate</td>
 <td>Corning</td>
 <td>3513</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>T-75 cm2 flask</td>
 <td>Corning</td>
 <td>430641</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Culture bags</td>
 <td>Vue Life</td>
 <td>290C; 750C</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 ml centrifuge tube</td>
 <td>BD Biosciences</td>
 <td>352098</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Amaxa Nuceleofector II</td>
 <td>Lonza</td>
 <td>AAB-1001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cellometer</td>
 <td>Nexcelom Bioscience</td>
 <td>Cellometer Vision</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sorvall Legend RT Centrifuge</td>
 <td>Sorvall</td>
 <td>75004377</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BD FACS Calibur</td>
 <td>BD Biosciences</td>
 <td>342975</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Controlled rate freezer</td>
 <td>Planer</td>
 <td>Kryo 750</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Irradiator</td>
 <td>CIS bio International</td>
 <td>IBL-437 C#09433</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>
 <table border="1">
 <tbody>
 <tr>
 <td colspan="4">Expression of Molecules (Endogenous and Introduced)</td>
 </tr>
 <tr>
 <td>Cell Line</td>
 <td>Common Name</td>
 <td>Cell Type</td>
 <td>Molecules Expressed</td>
 </tr>
 <tr>
 <td>CJKT64.86.41BBL.GFP-IL15.CD19</td>
 <td>Clone 4</td>
 <td>K562</td>
 <td>CD86, CD64, CD137L, CD19, GFP co-expressed with membrane bound IL-15</td>
 </tr>
 </tbody>
 </table>
 Complete Culture Media (CCM) (stored at 4 &deg;C for up to 30 days) 
 HyQ RPMI-1640/Phenol-free RPMI-1640 
 10% FBS 
 1% GlutaMAX
 Flow Cytometry Buffer (stored at 4 &deg;C for up to 30 days) 
 PBS 
 2% FBS 
 0.1% Sodium Azide</td>
 </tr>
 </tbody>
</table>

clinical application of sleeping beauty and artificial antigen presenting cells to genetically modify t cells from peripheral and umbilical cord blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

We report that electro-transfer of DNA plasmids and propagation of T cells on &gamma;-irradiated aAPC can be used to generate clinically-appealing numbers of T cells derived from PB and UCB for human applications. These genetically modified T cells express an introduced CAR that recognizes the TAA CD19, independent of major histocompatibility complex. The SB-derived DNA plasmids to express the (i) transposon, a 2nd generation CAR (CD19RCD28) that signals through CD28 and CD3-&epsilon;14, and (ii) transposase, SB1115, have been previously described13,16,17. The plasmids used in the current study were produced commercially by Waisman Clinical Biomanufacturing Facility (Madison, WI). The aAPC (clone #4), derived from K562 cells (parental line obtained from American Type Culture Collection), co-express desired T cell co-stimulatory molecules (each introduced molecule at 390% on cell surface of aAPC), as previously described12. Here we show that CD19-specific T cells could be generated from mononuclear cells (MNC) derived from PB or UCB using SB transposition to introduce the CAR followed by addition of aAPC to numerically expand the T cells in a CAR-dependent manner (Figures 1, 4)13,18. Ten cuvettes (2x107 MNC/cuvette) are electroporated for each recipient using 15 &mu;g of DNA plasmid (CD19RCD28/pSBSO) coding for transposon (CAR) and 5 &mu;g of DNA plasmid (pCMV-SB11) coding for transposase (SB11). The number of cuvettes can be reduced if MNC are limiting or scaled back for laboratory work. The day of electroporation is defined as "Day 0" of Stimulation cycle #1. As controls for flow cytometry and culture conditions, autologous T cells are mock electroporated (without DNA plasmid) and numerically expanded on &gamma;-irradiated aAPC (clone #4) that had been pre-loaded with OKT3 to cross-link CD3 to sustain T cell proliferation. We routinely assess the efficiency of electrotransfer and viability of the T cells the day after electroporation (Figure 2B). The expression of EGFP from control DNA plasmid (designated pmaxGFP) and CAR at this initial time point reflects protein expression from the integrated and episomal plasmid. Typically, the day after electroporation we measure EGFP expression at ~60% and CAR expression at ~40% (Figure 2A) with T cell viability between 40-50%. Recursive additions of &gamma;-irradiated aAPC in the presence of soluble recombinant human IL-2 and IL-21 retrieve T cells stably expressing CAR (CD19RCD28). CD3negCD56+ NK cells are depleted from the culture using CD56-specific paramagnetic beads if the percentage of these NK cells is &ge;10% and especially if the percentage of CAR expressed on the T cells is low. This depletion prevents the rapid overgrowth of NK cells which interferes with the ability of aAPC to sustain the proliferation of CAR+ T cells. On occasion, depletion of NK cells from CAR+ T cells is undertaken during the last two stimulation cycles, but this introduces a loss of desired cells due to co-expression of CD56 on some CAR+ T cells. The T cells were grown in a functionally closed system using Vue Life culture bags past Day 14. A subset of the genetically modified and propagated T cells are typically cryopreserved at Day 14 or Day 21 (end of Stimulation cycles #2 or #3) of co-culture on aAPC to serve as a source of archived material for future analyses and to be thawed if unanticipated problems subsequently occur during the manufacturing process. T cells are typically harvested on or about Day 28 of culture (Figure 3) that routinely express &gt;90% CAR and are &gt;80% viable (Figure 2C, D). We have previously shown that, after four weeks of co-culture on aAPC the average fold-expansion of CD3+ T cells is 19,800&plusmn;11,313 with CAR+ expression being 90%&plusmn;7.5 13. These T cells are cryopreserved and undergo in-process and release testing that informs on the safety and therapeutic potential of the manufactured product. Release testing is undertaken in compliance with clinical laboratory improvement amendments (CLIA) to generate a certificate of analysis prior to infusion into recipients on clinical trials.
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50070/50070fig1highres.jpg" src="/files/ftp_upload/50070/50070fig1.jpg" /> 
 Figure 1. Steps outlining the process to electroporate and propagate CAR+ T cells from PB and UCB. <a href="https://www.jove.com/files/ftp_upload/50070/50070fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" fo:content-width="4.5in" fo:src="/files/ftp_upload/50070/50070fig2highres.jpg" src="/files/ftp_upload/50070/50070fig2.jpg" /> 
 Figure 2. Characterization of genetically modified T cells from PB. (A) Expression of EGFP at Day 0 of first stimulation cycle to assess the efficiency of gene transfer. Expression of CD19-specific CAR (CD19RCD28) as assessed by flow cytometry on CD3+, CD8+ and CD4+ T cells at (B) approximately 24 hr after electroporation and (C) 28 days after co-culture on aAPC. Similar expression of CAR was observed with UCB-derived T cells. (D) Kinetics of CAR expression. <a href="https://www.jove.com/files/ftp_upload/50070/50070fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" fo:content-width="4.5in" fo:src="/files/ftp_upload/50070/50070fig3highres.jpg" src="/files/ftp_upload/50070/50070fig3.jpg" /> 
 Figure 3. Propagation of PB-derived CAR+ T cells. Rate of numeric expansion of CD3+ and CAR+ T cells derived from PB by repeated co-culture on &gamma;-irradiated aAPC in presence of recombinant human soluble IL-2 and IL-21. Upward arrows indicate the additions of &gamma;-irradiated aAPC that mark the beginning of each Stimulation cycle. UCB-derived CAR+ T cells exhibit similar rates of numeric expansion.
<img alt="Figure 4" fo:content-width="6in" fo:src="/files/ftp_upload/50070/50070fig4highres.jpg" src="/files/ftp_upload/50070/50070fig4.jpg" /> 
 Figure 4. Schematic of the manufacturing process using SB and aAPC systems to genetically modify and propagate CAR+ T cells derived from PB and UCB. CD19-specific CAR+ T cells were generated by electro-transfer of SB-derived supercoiled DNA plasmids and subsequent co-culture on K562-derived aAPC (clone #4) in the presence of recombinant human soluble IL-2 and IL-21. <a href="https://www.jove.com/files/ftp_upload/50070/50070fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td>T-cell source</td>
 <td>Transposon*</td>
 <td>Transposase</td>
 <td>T-cell infusion</td>
 <td>IRB #</td>
 <td>NIH-OBA #</td>
 <td>IND #</td>
 </tr>
 <tr>
 <td>Autologous (patient-derived)**</td>
 <td>CD19RCD28</td>
 <td>SB11</td>
 <td>After autologous hematopoietic stem-cell transplantation</td>
 <td>2007-0635</td>
 <td>0804-922</td>
 <td>14193</td>
 </tr>
 <tr>
 <td>Allogeneic (donor-derived)</td>
 <td>CD19RCD28</td>
 <td>SB11</td>
 <td>After allogeneic hematopoietic stem-cell transplantation</td>
 <td>2009-0525</td>
 <td>0910-1003</td>
 <td>14577</td>
 </tr>
 <tr>
 <td>Allogeneic (donor-derived)</td>
 <td>CD19RCD28</td>
 <td>SB11</td>
 <td>After allogeneic umbilical cord blood transplantation</td>
 <td>2010-0835</td>
 <td>1001-1022</td>
 <td>14739</td>
 </tr>
 </tbody>
</table>
Table 1. Clinical trials under the auspices of the FDA at MDACC to infuse CD19-specific CAR+ T-cells propagated on aAPC. *T-cells are rendered specific for CD19 through enforced expression of SB transposon coding for a 2nd generation CAR, designated CD19RCD28, that signals through CD28 and CD3-z. **Trial described in reference # 7

Watch this Scientific Journal Video about Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood at JoVE

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

clinical-application-sleeping-beauty-artificial-antigen-presenting

Research

JoVE Journal

Immunology and Infection

18.2K Views.  U.T. MD Anderson Cancer Center. T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Video: Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34+ cells. A method for CD34+ cell isolation and megakaryocyte differentiation is described here.

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...