"Cancer Regression in Patients After Transfer of Genetically Engineered Lymphocytes".

Richard A. Morgan ¹, Mark E. Dudley ¹, John R. Wunderlich ¹, Marybeth S. Hughes ¹, James C. Yang ¹, Richard M. Sherry ¹, Richard E. Royal ¹, Suzanne L. Topalian ¹, Udai S. Kammula ¹, Nicholas P. Restifo ¹, Zhili Zheng ¹, Azam Nahvi ¹, Christiaan R. de Vries ¹, Linda J. Rogers-Freezer ¹, Sharon A. Mavroukakis ¹, Steven A. Rosenberg ^1, *

¹ Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, USA.

* To whom correspondence should be addressed.
Steven A. Rosenberg , E-mail: SAR@mail.nih.gov

Using adoptive transfer of lymphocytes given after host immunodepletion it is possible to mediate objective cancer regression in patients with metastatic melanoma. However, the generation of tumor-specific T cells in this mode of immunotherapy is often limiting. Using a retrovirus encoding a T cell receptor, we report here the ability to specifically confer tumor recognition by autologous lymphocytes from peripheral blood. Adoptive transfer of these transduced cells in fifteen patients resulted in durable engraftment at levels exceeding ten percent of peripheral blood lymphocytes for at least two months post infusion. We observed high sustained levels of circulating, engineered cells at one year post-infusion in two patients, that both demonstrated objective regression of metastatic melanoma lesions. This study suggests the therapeutic potential of genetically engineered cells for the biologic therapy of cancer.

In the last two decades, fundamental advances in immunology have opened new opportunities for the
development of cellular-based therapies for the treatment of cancer (1, 2). After ex vivo expansion, transfer and clonal repopulation in patients following lymphodepleting conditioning, autologous tumor infiltrating lymphocytes (TIL) have been found to mediate objective cancer regression in a measurable proportion of patients with metastatic melanoma (3–5). A limitation of this approach is the requirement that patients have pre-existing tumor reactive cells that can be expanded ex vivo. In addition, in many cancer patients, especially those with cancers other than melanoma, it is difficult to identify these tumor reactive lymphocytes. To overcome this limitation, we set out to develop an approach to cancer immunotherapy based on the genetic modification of normal peripheral blood lymphocytes (PBL).

Tumor associated antigens (TAA) are recognized by the T cell receptor (TCR) on the T lymphocyte surface, which is composed of the TCR alpha and beta-chains (6). The genes encoding TCR specific for a variety of TAA have now been cloned and these include the TCR recognizing the MART-1 and gp100 melanoma/melanocyte differentiation antigens, the NY-ESO-1 cancer-testis antigen present on many common epithelial cancers and an epitope from the p53 molecule, expressed on the surface of approximately 50% of cancers of common epithelial origin (7–12). In each case, these antigens were detected by the TCR when they were presented as peptides by molecules encoded by the major
histocompatibility complex protein HLA-A2. In vitro transcribed RNA from four TAA reactive TCRs (recognizing MART-1: 27-35, gp100:209-217, NY-ESO-1:157-165, and p53:264-272) were electroporated into CD8+ PBL, which were then co-cultured with peptide pulsed T2 cells. These transfected cells produced large amounts of interferon-g , upon stimulation with their respective peptides (Fig. 1A),

Fig. 1A. Transduction and analysis of TCR engineered cells.

Fig. 1. Transduction and analysis of TCR engineered cells.

(A) CD8 + human lymphocytes were electroporated with RNA encoding control (GFP) or cloned TCRs reactive with HLA-A2 restricted epitopes from human tumor antigens MART-1, gp100, NY-ESO-1, and p53. Effector T-cells were co-cultured with T2 cells pulsed with 1 µM of the indicated peptide (values are interferon-g , pg/ml). 

and were able to recognize HLA-A2 matched tumors including melanoma, lung and breast cancer (table S1). Furthermore, transduction with these TCR encoding retroviral vectors converted normal PBL to cells capable of specifically recognizing and destroying both fresh and cultured cells from multiple common cancers (e.g., breast, lung, esophagus, sarcomas and liver) in vitro (9–12).

To test the ability of genetically engineered PBL derived from patients with melanoma to recognize and destroy tumor cells in vivo, we transduced PBL with the genes encoding the alpha and beta chains of the anti-MART-1 TCR. These genes were cloned from a TIL clone obtained from a cancer patient who demonstrated a near complete regression of metastatic melanoma following adoptive TIL transfer (5). A retroviral vector was constructed and optimized to express the MART-1 TCR alpha and beta chains (Fig. 1B and SOM).

Fig. 1B: Diagram of recombinant retroviral vector MSGV1AIB used to engineer human lymphocytes.

Fig. 1B: Diagram of recombinant retroviral vector MSGV1AIB used to engineer human lymphocytes.
 

Gene transfer, assessed by staining for the specific Vb12 protein in this TCR, resulted in 30% staining of CD8+ cells (Fig. 1C) as compared with approximately 1% for control cultures (gene transfer was about equally divided between CD4 and CD8 cells).

Fig. 1C: Transduced (Td) lymphocytes were analyzed five days post-transduction for the expression of
Vb12 and MART-1 tetramer in CD8+ cells in comparison to untransduced (UnTd) cells. Numbers are the percent positive cells in that region.

Fig. 1C: Transduced (Td) lymphocytes were analyzed five days post-transduction for the expression of
Vb12 and MART-1 tetramer in CD8+ cells in comparison to untransduced (UnTd) cells. Numbers are the percent positive cells in that region.

Fifteen percent of the transduced CD8+ cells bound the MART-1 peptide specific HLA-A*0201 tetramer (Fig. 1C; data on all patients are shown in table S2). The TCR transduced cells were biologically active, as demonstrated by the specific secretion of interferon-g following co-culture with both MART-1 peptide pulsed cells and HLA-A2 positive melanoma cell lines (Fig. 1D).

Fig. 1D: TCR vector-engineered cells from patient 6 (TCR) were co-cultured with MART-1 peptide pulsed T2 cells, HLA-A2- melanoma line (Mel 888), or HLA-A2+ melanoma line (Mel 526) and the amount of interferon-g produced determined.

Fig. 1D: TCR vector-engineered cells from patient 6 (TCR) were co-cultured with MART-1 peptide pulsed T2 cells, HLA-A2- melanoma line (Mel 888), or HLA-A2+ melanoma line (Mel 526) and the amount of interferon-g produced determined. Control effectors were untransduced cells (PBL) and MART-1 reactive TIL JKF6 (JKF6). 

To test the in vivo efficacy of these MART-1 TCR engineered T cells, 17 HLA-A*0201 patients with
progressive metastatic melanoma (Table 1) were selected for treatment. All patients were refractory to prior therapy with IL-2.

Fig. 1E: Anti-melanoma properties of genetically engineered lymphocytes were determined for all patients
prior to infusion. The production of interferon-g (pg/ml) following co-culture with peptide pulsed T2 cells (Peptide Reactivity) and anti-melanoma activity (Tumor Reactivity) for HLA-A2 + lines (526, 624) and HLA-A2- lines (888,938). Values demonstrating specific release of cytokine are in bold.

Patients received adoptive cell transfer (ACT) with MART-1 TCR transduced autologous PBL at a time of
maximum lymphodepletion (SOM). An initial cohort of three patients was treated with cells following an extended culture period of 19 days, at which point they had cell doubling times ranging from 8.7 to 11.9 days (Table 1, cohort 1; patients 1, 2a, 3). In these patients, less than 10% of the transduced cells
persisted across the time points tested during the first 30 days post-infusion and 2% or less persisted beyond 50 days (Fig. 2A). These first three patients showed no delay in the progression of disease.

Fig. 2A-D. Persistence of gene marked cells.

Cohort 1:                                                                     Cohort 2:

Fig. 2A-D. Persistence of gene marked cells. DNA extracted from PBMC was subjected to real-time quantitative PCR to determine the percent of vector-transduced cells in patient circulation at various times post-infusion. Each line represents data from a separate patient. (A), cohort 1; (B),cohort 2; (C), cohort 3. (D) Mean value of the % of gene marked cells for all patients in each cohort at the given time interval post-treatment. 

(E) The percentage of CD8+/ Vb12+ cells in the intermediate gate (see SOM) for patients in cohorts 2 and 3 are shown. 

(F) The percentage of CD8+/MART-1 tetramer+ cells was determined for patients in cohorts 2 and 3 at the times shown. Pre-treatment values for each patient are plotted as day 0 post-infusion.

These first three patients showed no delay in the progression of disease.

In an effort to administer gene-modified lymphocytes that were in their active growth phase, the culture conditions were modified (SOM) to limit the ex vivo culture period to between 6 and 9 days after stimulation of cells with anti-CD3 antibody (Table 1; cohort 2, doubling times two days or less). In a further cohort, larger numbers of actively dividing cells for ACT were generated by performing a second rapid expansion protocol (13) after 8-9 days (Table 1, cohort 3; doubling time 0.9 to 3.3 days). In contrast to the lack of cell persistence seen in cohort 1 (Fig. 2A), patients in cohorts 2 and 3 (Fig. 2, B to D), all exhibited persistence of the transduced cells at greater than 9% at one and four weeks post-treatment (range 9%-56%).

All eight patients providing samples at greater than 50 days exhibited persistence at greater than 17%, and this was durable in the seven patients over a monitoring period of over 90 days. One patient (patient 14) had > 60% of circulating lymphocytes positive for the gene marked cells (Fig. 2C).

In 14 patient samples tested at one month post-transfer, quantitative RT-PCR assays revealed the presence of vector derived RNA confirming that gene expression continued (table S3). All but one of 15 patients analyzed had increased levels of CD8+/Vb12 cells at one week post-treatment and 11 of 15 were higher at one month compared to pretreatment levels (Fig. 2E) All 13 patients examined had increased
MART-1 tetramer-binding cells post-treatment (Fig. 2F), and 11 of 14 had increased number of elispot positive cells (table S4).

There was, however, a discordance between the mean persistence of transduced cells at one month in cohorts 2 and 3 as measured by PCR (mean 26%), compared to the measurement of Vb12 expressing cells (8.1%) and of MART-1 tetramer-binding cells (0.8%). This discordance may in part be due to mispairing of the introduced TCR chains with the endogenous chain as well as the different sensitivities of the assays. The reduced expression of the transgene in the persisting cells at one month and later may also be a function of the described decrease (14) in the transcription of retrovirally inserted trangenes and the decline in metabolic activity during the conversion of activated cells to memory cells. This decrease in expression of the retroviral transgene would be expected to impact more heavily on the measurement of tetramers which relies on the aggregation of multiple receptors than on the detection of Vb12 directly
by antibody.

Most importantly, two patients demonstrated a sustained objective regression of their metastatic melanoma assessed by standard RECIST criteria (15).

Patient 4, a 52 year old male, had received prior treatment with alpha interferon, lymph node dissection, experimental vaccine, and high-dose IL-2. He then developed progressive disease in the liver (4.4 X 3.3 cm) and axilla (1.3 X 1.2 cm). Following treatment in the current protocol he experienced complete regression of the axillary mass, and an 89% reduction of the liver mass (Fig. 3, A and B) at which time it was removed.

Fig. 3A-D. Cancer regression in two patients.

Fig. 3. Cancer regression in two patients. (A) CT images of patient 4 liver metastasis; pre-treatment, one month and 10 months post-treatment with TCR engineered T-cells. (B) Size of liver and axillary tumors and tempo of regression of tumor sites in patient 4 (treatment time = day 0). He remains clinically disease free at 19 months after treatment.

(C) CT images of patient 14 hilar lymph node metastasis; pre-treatment, beginning of treatment (Day 0), and two months and 12 months post-treatment. (D) Size of tumor and tempo of regression in patient 14.

Following treatment in the current protocol he underwent regression of the hilar mass and is now clinically disease free 18 months later (Fig. 3, C and D). Thus, two patients with rapidly progressive metastatic melanoma showed full clinical regression of disease after transfer of genetically engineered
autologous PBL.

In responding patients 4 and 14, gene marked cells in the circulation (assumed to be 1% of total body lymphocytes) expanded 1400 fold and 30 fold respectively compared to the infusion cell number. At one year post-infusion, both responding patients had sustained high levels (between 20%-70%) of circulating gene-transduced cells (Fig. 3E).

(E) Quantitation of gene marked cells in patients 4 and 14 PBMC was determined by real-time
quantitative PCR. Day of infusion (Inf.) indicated by arrow. (F) The percentage of CD8+/Vb12 +cells in the intermediate gate (see SOM) in the circulation of patients 4 and 14.

http://www.sciencemag.org/cgi/content/full/1129003/DC1

Materials and Methods
Supporting Tables S1 to S5
Supporting References

Supplemental On-line Material:

Methods

Patient Peripheral Blood Mononuclear Cells (PBMC), and cell lines.

All of the Peripheral Blood Lymphocytes (PBL) used in this study were cryopreserved PBMC obtained by leukapheresis of metastatic melanoma patients treated at the Surgery Branch, National Cancer Institute, NIH, Bethesda, MD. T2 is a lymphoblastoid cell line deficient in TAP function, whose HLA class I proteins can be easily loaded with exogenous peptides (1). Melanoma lines 938mel, 888mel, 624mel, and 526mel were generated at the Surgery Branch from resected tumor lesions. Other cell lines used were non-small cell lung cancer line H2087 (ATCC/CRL-5922 ), breast cancer line MDA 453 (ATCC/HTB-131), the
human lymphoid cell line Sup T1 (ATCC CRL-1942), and PG13 gibbon ape leukemia virus packaging cell line (ATCC CRL 10, 686). All the cell lines described above were maintained in R10 (RPMI1640, Invitrogen Corp, Grand Isle, NY) supplemented with 10% FCS (Biofluids, Inc., Gaithersburg, MD). Culture medium (CM) for established human T lymphocyte lines was RPMI1640 with 0.05mM mercaptoethanol, 300 IU/ml interleukin-2 (IL-2) (Chiron Corp. Emerville, CA) plus 10% human AB serum (Valley Biomedical Winchester, VA).

Patient Treatment

This human gene therapy protocol was reviewed and approved by the National Institutes of Health institutional biosafety committee, National Cancer Institute institutional review board, recombinant DNA advisory committee of the Office of Biotechnology Activities, Office of the Director, National Institutes of Health, and the Center for Biologics Evaluation and Research of the Food and Drug Administration (all, Bethesda, MD). Patients eligible for treatment on this protocol were older than 18 years, HLA-A0201+, HIV infection, hepatitis B and C negative with a life expectancy greater than 3 months with good performance status. All patients signed institutional review board-approved consents and had their pathology confirmed by pathologists at the Clinical Center, National Institutes of Health in Bethesda,
MD. All patients had measurable disease on computed tomography scan, magnetic resonance imaging or by physical exam. Tumor response was determined by RECIST criteria (2). Patients were required to have progressed after prior interleukin-2 therapy before enrollment and did not have tumor infiltrating lymphocyte cultures available for treatment.

PBMC were isolated by leukapheresis, se parated by centrifugation on a ficoll-hypaque cushion, washed in HBSS, then resuspended at a concentration of 1 X 10⁶ /ml in T-cell culture medium AIM-V (Invitrogen Corp, Gr and Isle, NY) with 300IU/ml IL-2, 100U/ml penicillin, 100 µg/ml streptomycin, 1.25 µg/ml amphotericin, 10 µg/ml ciprofoloxicin, and 5% human AB serum supplemented with 50ng/ml OK T3. After 2 days of culture, cells were collected, resuspended in fresh T cell culture medium without OKT3. A retroviral vector (MSGV1AIB) was constructed (3) and optimized to express the MART-1 TCR alpha and beta chains using an Murine Stem Cell Virus (MSCV) long terminal repeat (LTR) (known to be
relatively resistant to in vivo silencing), incorporating the splicing and optimized start codon of the MFG-design of vectors, and the use of a highly efficient internal ribosome entry site (IRES) derived from the human polio virus (4). After selection of high-titer producer cell clones a clinical grade retroviral vector supernatant was produced and used in a solid-phase transduction protocol that resulted in highly efficient gene transfer without the use of any selection method. The transduction of up to 5 X 10⁸ cells was performed by overnight culture on Retronectin (CH-296, GMP grade Retronectin purchased from Takara Bio. Inc, Japan) coated, MSGV1AIB vector-preloaded six well tissue culture plates, using 6 ml vector
and up to 5 X 10⁶ cells per well. In cohorts 1 and 3, PBL were transferred the following day to a second set of pre-coated Retronectin/retroviral vector tissue culture plates. Patients in cohort 2, received cells with only one cycle of transduction. Two days after the last transduction, the PBL cultures were assayed for the presence of Vb12 TCR protein by FACS analysis and for activity by cytokine release assay.

An initial cohort of three patients was treated with cells following an extended culture period of 19 days at which point they had cell doubling times ranging from 8.7 to 11.9 days (Table 1, cohort 1; patients 1, 2a, 3). In an effort to administer gene-modified lymphocytes that were in their active growth phase, we modified the culture conditions to limit the ex vivo culture time to 6 to 9 days after stimulation of cells with OKT-3. This modification was based on prior animal and human data indicating that the in vivo proliferation and persistence of the transferred cells was highly correlated with their ability to mediate cancer regression (5-9). Thus patient lymphocytes were s timulated with OKT-3, transduced and administered either on days 6, 7, or 8 at which time they had doubling times of two days or less (Table 1, cohort 2; 11 patients). To generate larger numbers of actively dividing cells for ACT, we cultured transduced cells for 17-18 days, exposing a portion of the culture to an OKT-3 based rapid expansion protocol (10) after 8-9 days (Table 1, cohort 3; 4 patients).

The doubling time of the cells in cohort 3 was 0.9 to 3.3 days. The mean number of cells infused in cohort 2 was 6.2 X 10⁹ and in cohort 3 was 45.3 X 10⁹ . All patients received nonmyeloablative lymphodepleting chemotherapy, consisting of 2 days of cyclophosphamide (60mg/kg) and 5 days of fludarabine (25mg/m² ). One to four days after the final dose of chemotherapy, patients received MSGV1AIB (anti-MART-1 TCR)
transduced lymphocytes by intravenous bolus infusion over thirty minutes. Patients then received high dose intravenous IL-2 (720,000 IU/kg every eight hours to tolerance or a maximum of 12 doses), and MART-1:27-36( 27L) peptide vaccine (1 mg peptide, manufactured by the National Cancer Institute through a contract with Ben Venue Laboratories) emulsified in Montanide® ISA-51 (Seppic, Inc., Fairchild, NJ) by intramuscular injection on days 1, 2, 3, 4, 5, 12, and 19 after cell infusion.

Electroporation of in vitro transcribed mRNA.

mRNA encoding the GFP was prepared from pGEM4Z/EGFP/A64 (kindly provided by Dr. Eli Gilboa, Duke University Medical Center. Durham, NC). mRNA encoding TCR a and b chains of MART-1, gp100, NY-ESO-1, and p53 were prepared from PCR products made using gene specific primer pairs containing the T7 RNA polymerase promoter sequence (11). mMASSAGE mMACHINE High Yield Capped RNA transcription Kit (Ambion Inc. Austin TX )was utilized to generate in vitro transcribed (IVT) RNA. The IVT RNA was purified using an RNeasy Mini Kit (Qiagen, Valencia , CA) and purified RNA was resuspended in RNase free water at 1-0.5 mg/ml. For the stimulation of the T cells, human PBLs were
stimulated with IL-2 (300 IU/ml) plus 50 ng/ ml OKT3 for 7 days, and enriched for CD8+ cells before electroporation. The stimulated CD8+ PBLs subjected to electroporation following resuspension in OPTI-MEM (Invitrogen ) medium at the final concentration of 25X10⁶ /ml. Cells and cuvettes were pre-chilled by putting them on ice for >5 min. For electroporation 0.2 ml of the cells were mixed with 2 µg/1X10⁶ T cells of IVT RNA and electroporated in a 2 mm cuvette (Harvard Apparatus BTX, Holliston, MA), using ECM830 Electro Square Wave Porator (Harvard Apparatus BTX, Holliston, MA) at 500V/500 µs. Immediately after electroporation, the cells were transferred to fresh CM with 300 IU/ml IL-2
and incubated at 37 °C until use.

FACS analysis

Cell surface expression of TCRVb12, TCRVb8, CD3, CD4, and CD8 molecules on PBL were measured using FITC, PE or APC-conjugated antibodies according to the manufacturer’s instructions. Anti-TCR Vb8 and Vb12 were purchased from Immunotech (Westbrook, ME) and the other antibodies were from Becton Dickinson (San Jose, CA). MART-1 specific TCR were stained with a tetrameric HLA-A2/MART-1:27-36(27L) – fluorochrome conjugate (iTAg MHC Tetramer, Beckman Coulter, Fullerton, CA) according to the manufacturer’s recommendations. Cells we restained in a FACS buffer made of PBS (BioWhitaker, Walkersville, MD) and 1% FC S. Prior to staining of peripheral blood
lymphocytes, Fc receptors were blocked with normal mouse IgG (Caltag Labs, Burlingame, CA). Immunofluorescence, was measured using a FACscan or FACScaliber flow cytometer (Becton Dickinson). A combination of forward angle light scatter and propidium iodide staining was used to gate out dead cells. Approximately 1 x 10⁵ events were acquired. Staining was analyzed using Cell Quest (Becton Dickson) or FlowJo (Treestar, Ashland OR) software. The Vb12 intermediate cells were identified based on the Vb12 profile of the preinfusion cells using markers set to exclude both CD8 + Vb12 high and CD8 + Vb12 negative events. Other markers were set by comparison to fluorochrome-conjugated isotype control
antibodies or HLA-A2 tetramer containing an irrelevant control peptide.

Cytokine release assays

PBL cultures were tested for specificity and tu mor reactivity in cytokine release assays. For these assays, 1x10⁵ responder cells and 1x10⁵ stimulator cells (T2 or human tumor lines) were incubated for 18 to 24 h in a 0.2-ml culture vol ume in individual wells of 96-well plates. T2 cells were pulsed with peptide (1 µ g/ml) in medium for 2 h at 37°C, followed by washing (three times) before initiation of co-cultures. Ex vivo lymphocyte activity was measured after overnight culture in T cell medium supplemented with 300 IU/ml IL-2. Cytokine secretion was measured in culture supernatants diluted as to be in the linear range of the assay using commercially available ELISA kits (IFN-g , Endogen, Cambridge, MA) according to the
manufacturer’s recommendations.

Analysis of gene marked cells.

The persistence of the gene-marked cells was determined from patient PB MC. Genomic DNA was isolated using QIAa mp® DNA Blood Midi Kit (Qiagen, Valencia, CA) according to the manufacture’s instruction. 100ng of each DNA was used for the Real-time quantitative PCR assay (TaqMan, Applied Biosys tems Inc, Foster City, CA). Total RNA was isolated from PBL using RNeasy® Mini Kit (Qiagen). 1µg of total RNA was used in the first strand of cDNA synthesis reaction using ThermoScript™ RT-PCR System (Invitrogen, Carlsbad, CA) using random hexamers and diluted 10-fold with RNA-free water after the reaction. One tenth of the diluted reaction mix was used later for the real-time quantitative PCR (TaqMan). All PCR reactions were performed using an ABI 7500 Fast Real-time PCR System instrument (Applied Biosystems Inc,). The TaqMan gene specific assay was designed by ABI Assays-by-Design SM software (Applied Biosytems Inc.,) using as target, the sequence between TCRa and IRES genes. The assay primers/probe were:
5’-GCCGGGTTTAATCTGCTCATG-3’ (AIBAI-forward primer);
5’-GCCGGGTTTAATCTGCTCATG -3’ (AIBAI-reverse primer), and 5’-FAM-TCCAGCTGAACTAGAACTA-3 ’ (AIBAI- FAM labeled probe).
The reference standard curve was established using the genomic DNA from a MSGV1AIB transduced SupT1 cell clone by mixing known ratios of genomic DNA into untransduced SupT1 cell DNA. TaqMan b-actin control reagents kit (Applied Biosytems Inc.,) was used to normalize reactions to input RNA/DNA amounts.

Supplemental References

1. R. D. Salter, D. N. Howell, P. Cresswell, Immunogenetics 21, 235 (1985).

2. P. Therasse et al. , JNatlCancerInst 92, 205 (Feb 2, 2000).

3. M. S. Hughes et al. , HumGeneTher 16, 457 (Apr, 2005).

4. R. A. Morgan et al. , Nucleic Acids Res 20, 1293 (Mar 25, 1992).

5. J. Zhou et al. , J Immunol 175, 7046 (Nov 15, 2005).

6. J. Zhou, M. E. Dudley, S. A. Rosenberg, P. F. Robbins, J Immunother 28, 53 (Jan-Feb, 2005).

7. P. F. Robbins et al. , J Immunol 173, 7125 (Dec 15, 2004).

8. J. Huang et al. , J Immunother 28, 258 (May-Jun, 2005).

9. L. Gattinoni et al. , J E x p Med 202, 907 (Oct 3, 2005).

10. S. R. Riddell, P. D. Greenberg, J Immunol Methods 128, 189 (Apr 17, 1990).

11. Y. Zhao et al. , Mol Ther 13, 151 (Jan, 2006).

                                          Effector TCR RNA

Tumor                 GFP            MART-1          gp100          NY-ESO-1          p53
________________________________ _____________________________
938mel                23                68                     35                47                        38
526mel                61            1146                 317               163                   306
H2087                 62              224                     96               116                 >1654
MDA 435S-A2   96              175                   125              342                    127
MDA 435S       130                65                      73                 75                      65
________________________________ _____________________________
CD8+ PBLs were electroporated with IVT GFP RNA or tumor antigen specific TCRs (MART-1, gp100, NY-ESO-1, p53). Two hours post electroporation, the cells were co-cultured with tumor cell lines and after overnight co-culture, the supernatants were collected and subjected to ELISA detection of interferon-g secretion (in pg/ml). The known phenotypes of these tumor cell line were; melanoma 938m el (HLA-A2-/MART-1+/gp100+), melanoma 526mel (HLA-A2+/MAR T-1+/gp100+/p53+), non-small cell
lung cancer H2087 (HLA-A2+/NY-ESO-1-/p53+ ), breast cancer MDA 453S-A2 (HLA-A2+/NY-ESO-1+/p53-), and breast cancer MDA 453S (HLA-A2-/NY-ESO-1+/p53-).
Values demonstrating specific release of cytokine are (underlined) in bold.

                                                 Patient Number
                1    2a  3    4      5   6    7    8   9   10  11 12  13  2b  14  15   16  17  Ave
Vb12
(%CD4)  58  63  21  n/d  18  36  45  38 43  52  51 66  43  66  58  47  18   41  45
Tetramer
(%CD4)    8  15    1  n/d    2   10  15   8  15  21  22 33  15  15  7     4   10   7   12

Vb12
(%CD8)   49  65  63  24   17   32  23 29  43  42  43 59  44  72  51  51  36  24   42
Tetramer
(%CD8)   16  28   3   23     3   10   6   5   13  15  19 30  16   33  17  19  11 15   17

Samples were taken from transduced T lymphocytes cultures at or near time of infusion. FACS analysis for CD4, CD8, Vb12, and MART-1 Tetramer (M27L) was as described in methods. n/d, not determined.; Ave, average value.

                                                       Patient Number

                 4      5      6       7         8        9       10    11      12    13      14    15    16   17
Infusion   5.8   2.2   14.5   10.7   21.5   20.8   4.0   10.3   9.6   11.2   4.0   5.1   24   11.1

Post
Infusion   1.2   0.8    3.5     1.9    0.6      2.8     0.9    1.4    0.6    2.9    2.8   1.1   1.9   2.2

Amount of TCR vector-derived RNA normalized to cellular actin mRNA. Using RNA derived from a stable transduced human T cell line (Sup T1) as reference; total RNA from infused T cells or patient PBMC post-infusion were subjected to Q-RT PCR and then normalized to the amount of actin mRNA (values are vector/1 x 10⁶ actin). Samples were 2-5 weeks post-infusion, with exception of patients 8, 9, and 11, which were 2 months, 3 months, and 2 weeks respectively.

                                             Patient Number
                 1         2a      3       4         5         6         8         9       13     2b      14       15       16       17
Infusion   1523   1783   215   4527   1367   1203   1352   460   3380 4877  2023   2938   1393   190

Pre
treatment     22       21       1       14         0         1         0       0         0       0      20         1         6       0

Post
infusion         13       45       0       41         7         1     215     20     700      13    530      35       92   108

Number of interferon-g positive Elispots/100,000 cells. Post-infusion values derived from PBL taken at one to four weeks post-infusion and rested overnight in medium (without cytokine) prior to assay.

Patient 4:

                                         Melanoma Cell Line

                                    A2-                              A2+                            Peptide pulsed T2 cells

                              888           938            526            624        None       gp100             MART-1
                None   A1,24         A1,24        A2,3          A2,3                   1.0µM 10 µM   1.0 µM  0.1µM
Controls
None             0          0                0                0               0               0           0         0              0           0
AK 1700-3 181       66                9              37             10             46         36        32           35         32
JKF6              0       22              28         10250     10680              1         0   >16910  >15430    7770
JR6C12          0       41                0           4190      4640               0     8000           0        8520      143

Patient 4
Pre-treatment 130  204           117               98           133          521        421       422         462      520
Day 7                40    65            44             131          165          104         94   >1255   >1480     851
Day 8                87   110           75                 92            78          122        190     957         893     537
Day 18              42     27           23                 43            55          184           63      108         160       113

Anti-melanoma properties of genetically engineered lymphocytes determined for patient 4 PBL following overnight culture in IL-2 ( 300 IU/ml). The production of interferon-g (pg/ml) following co-culture with peptide pulsed T2 cells (peptide reactivity) and anti-melanoma activity (tumor reactivity) for HLA-A2 +
lines (526, 624) and HLA-A2- lines (888, 938). Controls included; non-peptide reactive TIL AK 1700-3, MART-1 reactive TIL JKF6, and gp100 reactive TIL JR6C12. JR6C12 recognition of 1.0 µM MART-1 peptide was not reproducible in repeated assays. Values demonstrating specific release of cytokine are in
underline bold.

Patient 14:
                                                       Melanoma Cell Line

                                           A2-                         A2+                                   Peptide pulsed T2 cells
                                   888          938          526          624           None            gp100               MART-1
                      None   A1,24       A1,24      A2,3         A2,3                      1.0 µM   10 µM   1.0 µM  0.1 µM
Controls
None                    0        0             43            0              0                0            23          70            0         18
AK 1700-3         35      53             33          21            26               52           43          94           73       105
JKF6                  15      11            66       6750      9160               37         337   >2168  >2168   >1828
JR6C12               0        0              0        4410      3760                0      >1510          23          15         61

Patient 14
Pre-treatment  368    410          352          284          344             639          645        874        614       471
Day 5                 66      53            67          110         170            134            96       918       716     634
Day 7                 69      52            45            41             55              33            57       192       221       109
Day 10               79      91              0            80             46              33            52       245       214       108
Day 27               52      76            35            33             55              67          103       282       232       158
Day 88               26      50            31              0             52              75            41           52        43          31
Day 257           105      52            93            53             46            111            83         597     492       399

Anti-melanoma properties of genetically engineered lymphocytes determined for patient 14 PBL following overnight culture in IL-2 ( 300 IU/ml). The production of interferon-g (pg/ml) following co-culture with peptide pulsed T2 cells (peptide reactivity) and anti-melanoma activity (tumor reactivity) for HLA-A2 +
lines (526, 624) and HLA-A2- lines (888, 938). Controls included; non-peptide reactive TIL AK 1700-3, MART-1 reactive TIL JKF6, and gp100 reactive TIL JR6C12. Values demonstrating specific release of cytokine are in underlined bold.

References and Notes

1. S. A. Rosenberg, Immunity 10, 281 (1999).

2. J. H. Blattman, P. D. Greenberg, Science v. 305, no. 5681, pp. 200-205 (July 9, 2004).. http://www.sciencemag.org/cgi/content/abstract/305/5681/200

3. S. A. Rosenberg, P. Spiess, R. Lafreniere, Science 233, 1318 (1986).

4. M. E. Dudley et al., J. Clin. Oncol. v. 23, no. 10, pp. 2346-2357 (April, 2005).
"Adoptive Cell Transfer Therapy Following Non-Myeloablative but Lymphodepleting Chemotherapy for the Treatment of Patients With Refractory Metastatic Melanoma".
http://www.jco.org/cgi/content/abstract/23/10/2346

5. M. E. Dudley et al., Science 298, 850 (2002).

6. M. Krogsgaard, M. M. Davis, Nat. Immunol. 6, 239 (2005).

7. T. N. Schumacher, Nat. Rev. Immunol. 2, 512 (2002).

8. M. Sadelain, I. Riviere, R. Brentjens, Nat. Rev. Cancer 3, 35 (2003).

9. Y. Zhao et al., J. Immunol. 174, 4415 (2005).

10. R. A. Morgan et al., J. Immunol. 171, 3287 (2003).

11. M. S. Hughes et al., Hum. Gene Ther. 16, 457 (2005).

12. C. J. Cohen et al., J. Immunol. 175, 5799 (2005).

13. S. R. Riddell, P. D. Greenberg, J. Immunol. Methods 128, 189 (1990).

14. D. B. Kohn et al., Nat. Med. 4, 775 (1998).

15. P. Therasse et al., J. Natl. Cancer Inst. 92, 205 (2000).

16. X. Zhu et al., J. Immunol. 176, 3223 (2006).

17. L. Yang, D. Baltimore, Proc. Natl. Acad. Sci. USA 102, 4518 (2005).

18. L. Gattinoni et al., J. Exp. Med. 202, 907 (2005).

19. W.W. Overwijk et al., J. Exp. Med. 198, 569 (2003).

20. The authors would like to acknowledge the expert help in the care of these patients provided by the Surgery Branch Immunotherapy Fellows, Juan Gea-Banacloche for valuable advice concerning the management of infectious complications, nurses on the 3NW and Surgical ICU wards in the Clinical Center, NIH, as well as Arnold Mixon and Shawn Farid for FACS analysis. Thanks also to Dr. Ken Cornetta and the National Gene Vector Laboratory for production of the clinical grade retroviral vector. This work was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer
Institute, National Institutes of Health.

This exciting pilot study by Richard Morgan, Mark Dudley, John Wunderlich, Marybeth Hughes, James Yang, Richard Sherry, Richard Royal, Suzanne Topalian, Udai Kammula, Nicholas Restifo, Zhili Zheng, Azam Nahvi, Christiaan de Vries, Linda Rogers-Freezer, Sharon Mavroukakis, and Steven A. Rosenberg uses immune RNA transduction applied into engineered retroviruses to activate the patient's own T-lymphocytes against distantly-spread malignant melanomas. RNA species are often immunogenic, and may be present in many immune responses.

a. Kern DH, Chow N, and Pilch YH, "Lymphocyte populations participating in cellular antitumor immune responses mediated by immune RNA", J. Natl. Cancer Inst. 60, 335 (1978).

b. Coughlin CM, Vance BA, Grupp SA, and Vonderheide RH, "RNA-transfected CD40-activated B cells induce functional T cell responses against viral and tumor antigen targets: implications for pediatric immunotherapy", Blood, 15 March 2004, Vol. 103, No. 6, pp. 2046-2054.

c. Liao X, Li Y, Bonini C, Nair S, Gilboa E, Greenberg PD, and Yee C, "Transfection of RNA Encoding Tumor Antigens Following Maturation of Dendritic Cells Leads to Prolonged Presentation of Antigen and the Generation of High-Affinity Tumor-Reactive Cytotoxic T Lymphocytes".

d. Frenster JH, "Phytohemagglutinin-Activated Autochthonous Lymphocytes for Systemic Immunotherapy of Human Neoplasms".

a. Kern DH, Chow N, and Pilch YH, "Lymphocyte populations participating in cellular antitumor immune responses mediated by immune RNA", J. Natl. Cancer Inst. 60, 335 (1978).

b. Coughlin CM, Vance BA, Grupp SA, and Vonderheide RH, "RNA-transfected CD40-activated B cells induce functional T cell responses against viral and tumor antigen targets: implications for pediatric immunotherapy", Blood, 15 March 2004, Vol. 103, No. 6, pp. 2046-2054.

c. Liao X, Li Y, Bonini C, Nair S, Gilboa E, Greenberg PD, and Yee C, "Transfection of RNA Encoding Tumor Antigens Following Maturation of Dendritic Cells Leads to Prolonged Presentation of Antigen and the Generation of High-Affinity Tumor-Reactive Cytotoxic T Lymphocytes".

d. Frenster JH, "Phytohemagglutinin-Activated Autochthonous Lymphocytes for Systemic Immunotherapy of Human Neoplasms".

Links to RNA and Biological Causality:

A Brief History of  Activator RNA:

Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to RNA-Induced Embryogenesis:
Links to RNA and Biological Causality:
Links to Reprogramming and Neoplasia:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".