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Clinical Trial
. 2009 Jul 16;114(3):535-46.
doi: 10.1182/blood-2009-03-211714. Epub 2009 May 18.

VSports在线直播 - Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen

Laura A Johnson  1 , Richard A Morgan, Mark E Dudley, Lydie Cassard, James C Yang, Marybeth S Hughes, Udai S Kammula, Richard E Royal, Richard M Sherry, John R Wunderlich, Chyi-Chia R Lee, Nicholas P Restifo, Susan L Schwarz, Alexandria P Cogdill, Rachel J Bishop, Hung Kim, Carmen C Brewer, Susan F Rudy, Carter VanWaes, Jeremy L Davis, Aarti Mathur, Robert T Ripley, Debbie A Nathan, Carolyn M Laurencot, Steven A Rosenberg
Affiliations
Clinical Trial

Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen

Laura A Johnson et al. Blood. .

Abstract

Gene therapy of human cancer using genetically engineered lymphocytes is dependent on the identification of highly reactive T-cell receptors (TCRs) with antitumor activity. We immunized transgenic mice and also conducted high-throughput screening of human lymphocytes to generate TCRs highly reactive to melanoma/melanocyte antigens. Genes encoding these TCRs were engineered into retroviral vectors and used to transduce autologous peripheral lymphocytes administered to 36 patients with metastatic melanoma. Transduced patient lymphocytes were CD45RA(-) and CD45RO(+) after ex vivo expansion VSports手机版. After infusion, the persisting cells displayed a CD45RA(+) and CD45RO(-) phenotype. Gene-engineered cells persisted at high levels in the blood of all patients 1 month after treatment, responding patients with higher ex vivo antitumor reactivity than nonresponders. Objective cancer regressions were seen in 30% and 19% of patients who received the human or mouse TCR, respectively. However, patients exhibited destruction of normal melanocytes in the skin, eye, and ear, and sometimes required local steroid administration to treat uveitis and hearing loss. Thus, T cells expressing highly reactive TCRs mediate cancer regression in humans and target rare cognate-antigen-containing cells throughout the body, a finding with important implications for the gene therapy of cancer. This trial was registered at www. ClinicalTrials. gov as NCI-07-C-0174 and NCI-07-C-0175. .

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Figures

Figure 1
Figure 1
Tumor-reactive DMF5 or gp100(154) alpha and beta TCR chain RNA electroporated into PBLs confer high reactivity to melanoma tumor antigens. (A) Ten-day anti-CD3–stimulated donor PBLs were electroporated with in vitro–transcribed RNA encoding paired DMF4, DMF5, or gp100(154) TCR alpha and beta chains, or GFP control. Cells were cocultured for 18 hours with T2 cells pulsed with peptide, HLA-A*02+ melanomas mel624+ or mel526+, or HLA-A*02− melanomas mel888− or mel938−. IFN-γ in the supernatant was detected by ELISA. (B) Structure of the MSGV-based γ-retroviral vectors DMF4 and gp100(154), incorporating an IRES and DMF5 with a furin 2A ribosomal skip sequence, allowing for dual gene expression.
Figure 2
Figure 2
DMF5 and gp100(154) TCR retroviral constructs conferred greater antitumor reactivity to donor PBLs than the original DMF4 receptor. (A) Donor PBLs were stimulated with anti-CD3 mAb OKT-3 and separated into CD4 and CD8 populations before retroviral transduction with DMF4, DMF5, or gp100(154) TCR constructs. TCR expression was analyzed 7 days later by tetramer staining and flow cytometry. (B) Donor PBLs transduced with retroviral TCR constructs were cocultured with T2 cells pulsed with MART-1:27-35 or gp100:154-162 peptide, and IFN-γ secretion was measured by ELISA. (C) Transduced PBLs were cocultured with mel624+ melanomas, and tumor target lysis was evaluated by 51Cr-release assay. Cells did not lyse HLA-mismatched tumors (data not shown).
Figure 3
Figure 3
TCR-transduced cells from responding patients persisted and showed antitumor activity ex vivo. Blood samples were taken from patients' cells before and after TCR-transduced cell infusion. PBMCs were evaluated for persistence of infused cells in peripheral blood after treatment by specific tetramer staining and were also used directly in coculture assays with mel624 tumors (MART1+, gp100+, HLA-A2+). Antitumor activity was evaluated by IFN-γ and IL-2 ELISPOT, and also by intracellular staining for IFN-γ production. (A) Persistence and activity of DMF5 patient treatment cells before, 2 weeks after, and 1 month after infusion. Responding (PR) and nonresponding (NR) patients are represented by solid and broken lines, respectively. (B) Comparison of PBMCs from patients treated with either DMF5 or gp100(154) TCR-transduced cells. Tetramer staining and ELISPOT analysis of IFN-γ and IL-2 production from nonresponding (NR) and responding (PR) patients at 1 month after treatment. All samples had less than 10 ELISPOTs (per 100 000 PBMCs) and less than 1% IFN-γ–positive cells, respectively, against the HLA-A*02− mel888 tumor (data not shown). Patients with objective clinical responses (PR) had higher numbers of antitumor IFN-γ (P = .02) and IL-2 (P = .02) secreting cells than nonresponders (NR).
Figure 4
Figure 4
Phenotype of patient treatment cells before and after infusion. DMF5 patient PBMCs were stained by tetramers for TCR-recognizing MART1:27-35, and by mAb for CD3 and the activation and differentiation markers CD27, CD28, CD45RA, and CD45RO. Cell phenotype was evaluated by flow cytometry, gated on CD3+ cells before treatment, and on CD3+tetramer+ cells for infusion (Rx) and 1 month after treatment (1 mo) samples. Error bars indicate mean ± SEM. Responding patients (PR) are represented by solid symbols, and nonresponding patients (NR) by open symbols.
Figure 5
Figure 5
Patients treated with the highly reactive DMF5 or gp100(154), but not DMF4 TCR, had increased serum IFN-γ levels after treatment. Patient IFN-γ serum levels were measured daily before, during, and after cell infusion by ELISA. (A) Serum IFN-γ levels increased from baseline to a peak at 3 to 6 days after cell infusion (3 representative DMF5 patients are shown). (B) Peak IFN-γ levels in serum after treatment for patients treated with DMF5, gp100(154), and DMF4 TCR-transduced cells. Only patients treated with the highly reactive DMF5 or gp100(154) TCR demonstrated increased IFN-γ in serum (P = .001 and P < .001, respectively, compared with P = .09 for DMF4 TCR).
Figure 6
Figure 6
Tissue trafficking and target cell destruction in patients after infusion of TCR-transduced cells. (A) Biopsy of inflamed skin from DMF5 patient 2, day 5 after treatment, demonstrating spongiotic vesicles and necrotic/dyskeratotic keratinocytes (arrows), stained with hematoxylin and eosin, and immunohistochemically stained for CD8+ cells or the Melan-A antibody recognizing MART-1 antigen. (Bottom left) Positive control staining for the anti-MART-1 Melan-A antibody showing normal epidermal melanocytes and a subcutaneous melanoma deposit (original magnification ×20). (B) One week after treatment, biopsies from gp100(154) patient with preexistent patchy vitiligo, demonstrating CD8+ cellular infiltrate into the epidermis of pigmented, but not vitiliginous skin (original magnification ×20). (C) Slit-lamp ophthalmologic evaluation of DMF5 patient 5 eye, 2 weeks after treatment, demonstrating cloudy cellular anterior chamber infiltrate (above) and (below) with induced iris dilation 6 months after TCR treatment and steroid eye drop administration, demonstrating posterior synechiae (asymptomatic). (D) Cells present in ocular fluid from panel C (top), analyzed directly by staining and flow cytometry. (E) Audiologic examination of DMF5 patient 2 before TCR treatment, day 13 after treatment showing hearing loss, and 11 months after treatment, showing hearing recovery after intratympanic steroid treatment.
Figure 7
Figure 7
Highly reactive transferred cells traffic to and destroy melanoma tumors in patients. (A) Sequential biopsies of subcutaneous tumors from DMF5 patient 4 before (d0) and after treatment, stained with hematoxylin and eosin (left), or anti-CD8 (right). Original magnification ×20. (B) DMF5 patient 4 thigh covered with multiple subcutaneous melanoma lesions before treatment, and after partial tumor regression 5 weeks after treatment. (C) Flow cytometry of TILs grown from DMF5 patient 4 subcutaneous tumor resected 4 weeks after treatment. (D-F) Before treatment and after treatment CT scans of (D) DMF5 patient 2 lungs, (E) DMF5 patient 8 brain (top) and axilla (bottom), and (F) gp100(154) patient 14 lungs (top) and liver (bottom). Arrows represent location of melanoma metastases.
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References

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