Transferred melanoma-specific CD8+ T cells persist, mediate tumor regression, and acquire central memory phenotype Aude G. Chapuisa, John A. Thompsonb, Kim A. Margolinb, Rebecca Rodmyrea, Ivy P. Laia, Kaye Dowdya, Erik A. Farrara, Shailender Bhatiab, Daniel E. Sabathc, Jianhong Caoa, Yongqing Lia, and Cassian Yeea,1 a Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; bGeneral Oncology and Hematology, Seattle Cancer Care Alliance and University of Washington, Seattle, WA 98109; and cDepartment of Laboratory Medicine, University of Washington, Seattle, WA 98195
Edited by Tak W. Mak, The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute at Princess Margaret Hospital, University Health Network, Toronto, ON, Canada, and approved February 3, 2012 (received for review August 30, 2011)
Adoptively transferred tumor-specific T cells offer the potential for non–cross-resistant therapy and long-term immunoprotection. Strategies to enhance in vivo persistence of transferred T cells can lead to improved antitumor efficacy. However, the extrinsic (patient conditioning) and intrinsic (effector cell) factors contributing to long-term in vivo persistence are not well-defined. As a means to enhance persistence of infused T cells in vivo and limit toxicity, 11 patients with refractory, progressive metastatic melanoma received cyclophosphamide alone as conditioning before the infusion of peripheral blood mononuclear cell-derived, antigen-specific, CD8+ cytotoxic T-lymphocyte (CTL) clones followed by low-dose or high-dose IL-2. No life-threatening toxicities occurred with low-dose IL-2. Five of 10 evaluable patients had stable disease at 8 wk, and 1 of 11 had a complete remission that continued for longer than 3 y. On-target autoimmune events with the early appearance of skin rashes were observed in patients with stable disease or complete remission at 4 wk or longer. In vivo tracking revealed that the conditioning regimen provided a favorable milieu that enabled CTL proliferation early after transfer and localization to nonvascular compartments, such as skin and lymph nodes. CTL clones, on infusion, were characterized by an effector memory phenotype, and CTL that persisted long term acquired phenotypic and/or functional qualities of central memory type CTLs in vivo. The use of a T-cell product composed of a clonal population of antigen-specific CTLs afforded the opportunity to demonstrate phenotypic and/or functional conversion to a central memory type with the potential for sustained clinical benefit. adoptive transfer
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trategies aimed at increasing the number and quality of autologous T cells targeting melanoma-associated antigens have been effective in reducing tumor burden in a limited number of patients, and clinical responses have been correlated with the duration of in vivo persistence of transferred T cells (1–4). Adoptive transfer of ex vivo-expanded melanoma-specific cytotoxic T-lymphocyte (CTL) clones can increase the in vivo frequency of melanoma-reactive T cells in a setting in which the elicited response can be tracked and the transferred cells characterized (5, 6). Improving the benefit of infused T cells has involved either extrinsic modulation of the host environment or intrinsic manipulation of the infused product to promote ultimate in vivo survival of T cells (7). Nonmyeloablative chemotherapy conditioning regimens [highdose (HD) cyclophosphamide (CY) and fludarabine] or ablative doses of total body irradiation (TBI; 1,200 rad) administered before T-cell infusions have been shown to facilitate the in vivo engraftment and expansion of adoptively transferred cells at the cost of serious and potentially life-threatening toxicities (8). We hypothesized that administration of HD CY given alone may induce a transient lymphopenia sufficient to allow engraftment and persistence of transferred T cells without incurring prolonged immunosuppression (8–10). Concurrent HD IL-2 (600,000– 4592–4597 | PNAS | March 20, 2012 | vol. 109 | no. 12
720,000 U/kg thrice daily) may contribute to increased toxicity (8), and although low-dose (LD) IL-2 (250,000 U/m2 s.c. twice daily) can enhance T-cell survival by several days, it is not sufficient to prolong the clinical efficacy of adoptively transferred cells significantly (3). We sought to determine in a phase I/II clinical trial whether HD CY (4 g/m2) followed by LD or HD IL-2 was sufficient to promote survival of melanoma-specific T-cell clones and induce measurable clinical responses. Previous studies in mice and nonhuman primates have shown that infusion of T cells derived from a central memory (Tcm) population exhibit greater replicative potential in response to antigen and prolonged in vivo persistence compared with those derived from an effector memory (Tem) source (11, 12), and may likely be poised to eliminate tumor more effectively. However, it remains unclear if cytotoxic T cells can be established from a Tcm pool in patients with high tumor burden, wherein the majority of endogenous circulating T cells are likely Tem; thus, T-cell clones generated from these patients in vitro are likely to be Tem or naive T cell-derived. In this study, particular attention was given to the analysis of the phenotype and function of T-cell clones that had prolonged in vivo survival because studies in the nonhuman primate model demonstrated that after in vitro expansion, T cells derived from Tcm or Tem share a Tem phenotype but their behavior is ultimately representative of their source, reverting to their original phenotype in vivo (11). Here, we show that conversion from a Tem to Tcm phenotype occurred in the setting of minimal, nonablative, lymphodepleting conditioning in two patients, wherein long-term in vivo persistence of transferred CTLs was observed. Furthermore, in this limited number study, HD CY alone accompanied by LD IL-2 postinfusion allowed for a transient expansion of the transferred cells, on-target autoimmune skin toxicity, disease stabilization in at least half of the patients at 8 wk, and a continuous complete response in one patient. Results Adoptive Transfer of Melanoma-Specific CTL Clones Preceded by HD CY and Followed by LD IL-2 Is Well-Tolerated and Safe. Eleven
patients with bulky metastatic melanoma who had failed previous therapy (Table 1) received CTL clones specific for Mart1/A2, Tyr/A2, gp100/A2, gp100/A3, and Tyr/B44. Characteristics of the infused melanoma-specific CTL clones are described in Fig. S1, Tables S1, S2, and the treatment plan in Fig. S2. Of the patients who received LD IL-2 following CY and T cells (cohort 1), all
Author contributions: C.Y. designed research; A.G.C., J.A.T., K.A.M., R.R., I.P.L., K.D., E.A.F., S.B., D.E.S., J.C., Y.L., and C.Y. performed research; A.G.C., J.A.T., K.A.M., R.R., and C.Y. analyzed data; and A.G.C. and C.Y. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
To whom correspondence should be addressed. E-mail:
[email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1113748109/-/DCSupplemental.
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Table 1. Patient characteristics Patient no.
Age, y, (gender)
Previous treatments
Disease site
LD IL-2 1 2
57 (M) 38 (F)
Surgery, radiation, HD IL-2, anti–CTLA-4 Surgery, radiation, carboplatin, and paclitaxel
3 4* 5 6
65 (M) 60 (M) 68 (M) 40 (F)
Surgery, radiation, MEK inhibitor Ocular enucleation, surgery, Mel48 vaccine Surgery, radiation, gamma-knife, temozolomide Surgery, radiation, anti–CTLA-4, peptide-based vaccine
7 8 HD IL-2 9 10* 11*
61 (F) 50 (M)
Surgery, radiation, bevacizumab, paclitaxel Surgery, radiation, IFN-α, HD IL-2, paclitaxel
Mesenteric LN, scapular, s.c. Cervical, supraclavicular LN, chest wall, breast, pulmonary nodes Lung, mediastinal and mesenteric LN Kidney, pancreas, liver, muscle Mediastinal LN, pulmonary nodes Mediastinal, supraclavicular, mammary chain, periportal, portacaval nodes. Right and left kidneys, adrenal, liver Pulmonary, inguinal, s.c.
75 (M) 35 (M) 55 (M)
HD IL-2 Brachytherapy, lipiodol, 90Y microsphere liver embolization Surgery, radiation, cisplatin, vinblastine, dacarbazine, IFN-α HD IL-2
Liver, peritoneum, bone Liver, mesenteric LN, sacrum Liver, mesenteric LN, sacrum
F, female; LN, lymph node; M, male. *Choroidal melanoma.
Assessment of Clinical Responses. Clinical response to treatment was not the primary end point of this study because of the small number of patients enrolled. However, 1 of 10 evaluable patients achieved confirmed complete remission (CR) and 5 of 10 patients experienced stable disease (SD) at 8 wk (Table 2). Patient 1 presented with a sizable abdominal lymph node that regressed entirely by 4 wk (Fig. 1A), and the patient remains in confirmed CR >36 mo after CTL infusion without further intervention. In patient 6, there was a 42% decrease in all target lesions 19 wk after infusion (a growing paraspinal lesion was surgically removed
before imaging was performed). Except for patient 1 (CR), all patients eventually progressed by 12–19 wk. Patients who experienced stabilization or regression of disease ≥4 wk after infusions experienced skin rashes within 7 d of CTL infusion. Patient 1’s rash (Fig. 1B) was biopsied at day 5, and the T-cell receptor-γ (TCR-γ) rearrangement found in DNA extracted from the skin biopsy was identical to the infused CTL clone (Fig. 1C), demonstrating that infused CTLs were present in the skin and were the likely cause of the inflammatory rash. Although not all rashes were biopsied, these results suggest that infused CTLs have the potential to migrate to the skin consistent with our previous findings (13). Melanoma-Specific CTL Clones Isolated from Patients with Metastatic Melanoma Can Persist in Vivo Following Transfer. Tracking of the
infused cells in vivo was performed by clone-specific TCR Vβ quantitative PCR and, where possible, corresponding multimer stains (3) (Fig. 2). These methods have different limits of detection (1 of 105 cells for quantitative TCR-specific PCR and 0.1% for multimer stains), and although they have previously been individually used to quantitate in vivo frequencies of infused CTL clones (1, 14), they were compared for patient 1 (Fig. 2A). The frequency of multimer-positive CTLs reached 70.4%, 2.2%, and 1.5% compared with 86.9%, 2.6%, and 1.4% by TCR Vβ-specific PCR at days 4, 56, and 508, respectively, thus yielding near-coincident results for frequencies ≥0.1% of CD8+ T cells and validating the use of either method for tracking purposes.
Table 2. Assessment of clinical response On study Patient no. 1 2 3 4 5 6 7 8 9 10 11
Follow-up
Cohort
T-cell specificity
HLA type
4 wk
8 wk
1 1 1 1 1 1 1 1 2 2 2
Mart1 Tyr gp100 gp100 Tyr Tyr Tyr Mart1 Mart1 Mart1 Mart1
A2 B44 A2 A3 A2 A2 B44 A2 A2 A2 A2
CR (−100%) ND SD (+0.6%) SD (+0.6%) SD (+10%) SD (−7%) SD (+6.3%) PD (+37%) SD (+13%) SD (−9.1%) PD (+36%)
CR (−100%) SD (−13%) ND* SD (+8%) PD (new) SD (−2%) PD (+28%) PD (+47%) SD (+15%) SD (−4.6%) PD (+31%)
12 wk
16 wk
19 wk
24 wk
Rash
PD (NT)
CR (−100%) PD (new)
Y Y Y Y Y Y Y N Y Y N
CR (−100%) PD* (+52%) PD (+24%) NE† PD (+61%) PD (+64%) SD (+9%)
PD (new) PD (+21%) PD (+51%)
N, no; ND, not done; new, new metastasis; NT, nontarget lesion progression; Y, yes. *Patient initiated alternate treatment modality. † Not evaluable: 42% reduction in all lesions except growing paraspinal mass surgically removed before scans.
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experienced transient (0.1% in vivo by multimer stains (patient 1) and/or TCR PCR (patients 1 and 2) (Fig. 2A). Patient 2 developed enlargement of a lymph node in the neck 51 d after Tcell infusion. The node was excised for diagnostic purposes. Tumor microinvasion with an associated lymphocytic infiltrate was identified within connective tissue (Fig. 3B, Left), and expression of tyrosinase was confirmed on the melanoma tumor cells (Fig. 3B, Right). Specific IFN-γ secretion in response to peptide-pulsed autologous monocytes was detected by ELISpot 4594 | www.pnas.org/cgi/doi/10.1073/pnas.1113748109
Fig. 2. In vivo persistence of melanoma-specific CTL clones. (A) TCR copies per 100 CD8+ T-cell DNA equivalents (Left, x axis) and percentage of multimer-positive (%Mult.) CD8+ T cells (Right, x axis) in PBMCs collected 7 d (±2 d) before and after infusions for four of eight patients in cohort 1 whose clones showed persistence (≥48 d). Inset dot plots above the graph for patient 1 show percentages of multimer-positive CD8+ T cells at days 4, 56, and 508. (B) Same analysis performed for four of eight patients in cohort 1 whose clones demonstrated persistence for 90%) entered the cell cycle early after transfer (day 7). By day 14, the number of detectable CTLs in the peripheral blood contracted and either persisted in a Ki-67− quiescent state (patient 1) or had disappeared from peripheral blood (patients 9, 10, and 11) (Fig. 5A). Localization of transferred CTLs to nonvascular compartments (i.e., lymph node, tumor sites) cannot be excluded. Proliferation of infused cells was distinct from the recovery of endogenous CD8+ T cells after lymphodepletion because the peak percentage of replicating endogenous cells occurred later (day 14) and involved a lower fraction of total CD8+ T cells (mean of 51%). Plasma IL-15 levels (Fig. 5B, Upper) were increased in patients postinfusion. Significantly higher levels were observed in patients who had received HD IL-2 (Fig. 5B, Lower). However, there was no correlation between peak levels or cumulative levels (area under the curve) of IL-15 and clone persistence. Similarly, plasma levels of other cytokines tested in vivo (Fig. S3) did not correlate with clone persistence or survival. Regulatory T-cell (T-reg) dynamics were also monitored because murine and human studies suggested a role for CY in reducing the circulating T-reg populations (19, 20). After a transient decrease in absolute numbers of T-regs coinciding with lymphodepletion induced by CY, both percentages and absolute numbers of T-regs peaked 14 d after infusions; they then declined and reached pretreatment levels at 21 and 49 d, respectively (Fig. S4 A and B). Ki-67 expression by T-regs was significantly higher before and after treatment compared with CD4+ T-effector cells, suggesting a replicative advantage of Tregs compared with CD4+ T-effector cells (Fig. S4C). Overall, we observed that CY lymphodepletion followed by LD or HD IL-2 fostered an environment that enabled infused cells to proliferate early after transfer. However, neither early proliferation detected in the peripheral blood or plasma IL-15 nor the dynamics of T-regs translated into long-term peripheral in vivo persistence.
studies in nonhuman primates have demonstrated that cells derived from Tcm, differentiated to Tem ex vivo, can reacquire inherent features shared with Tcm in vivo (11). In this study, analysis of the transferred CTLs in two patients (patients 1 and 2) demonstrated long-term persistence, up-regulation of phenotypic markers associated with Tcm, and/or acquisition of helper independence (antigen-driven IL-2 production), suggesting that these melanoma-specific CTL clones may have originated from a Tcm population. Although the infused CTLs were not genetically marked, the established specific Vβ clonal identity argues almost unequivocally in favor of phenotypic reexpression of markers shared with Tcm and against the outgrowth of a Tcm subset in the infused population as might be conceivable when infusing polyclonal T cells. For patient 1, CTL persistence coincided with tumor regression by 4 wk and continuous remission for 3+ years. Patient 2 demonstrated extended in vivo persistence of the transferred CTLs but eventually progressed by 19 wk. Studies performed by the Surgery Branch at the National Cancer Institute (NCI) have also demonstrated the significant impact of lymphodepletion conditioning on in vivo persistence of adoptively transferred tumor-infiltrating lymphocytes (TILs); in these seminal studies, CR rates as high as 16% were observed using myeloablative regimens combining HD CY, fludarabine, and 12-Gy TBI, followed by HD IL-2 postinfusion (8, 9, 21). However, there are marked differences in the configuration and toxicity profile of these NCI trials: A surgical procedure was required to acquire TILs, TILs were propagated in vitro with high doses of IL-2, and the regimens led to acute morbidity and potentially life-threatening toxicities, thereby limiting the pool of eligible patients. In the present study, preinfusion conditioning with a regimen of HD CY alone when used in conjunction with postinfusion LD IL-2 is safe and well-tolerated and represents a clinically feasible alternative with a broader patient eligibility profile. HD CY alone achieved a transient lymphodepleted condition that induced measurable circulating levels of the “prosurvival” cytokine IL-15 consistent with previous studies (8, 10) and enabled transferred cells to undergo early cell division as demonstrated by Ki-67 expression (22). On-target toxicities (skin rashes) appeared within 1 wk of infusion in patients who achieved SD or CR at ≥4 wk, suggesting that the environment fostered by HD CY enabled transferred T cells to reach peripheral tissue. In our study, the administration of HD IL-2 after T-cell infusion yielded unacceptable toxicities and added uncertain benefit. The use of CTL clones in this study provided very strong evidence that transferred CTLs that persist in vivo (a reported predictor of response) acquire characteristics of Tcm. In contrast to a previous study using a transferred polyclonal T-cell product, the use of CTL clones eliminates the possibility of an outgrowth of a preexisting Tcm subpopulation (4). However, the selection of persistent T-cell clones used for patients 1 and 2 was stochastic. A means to generate Tcm-like effector cells for adoptive therapy prospectively would be desirable. More precise control over a Tcell product with a Tcm phenotype may be achieved through the use of in vitro cytokine modulation (23, 24), Tcm selection and TCR gene transfer, or infusion of a polyclonal T-cell population with an increased statistical probability of comprising a Tcm population. Such Tcm-derived effectors become highly responsive to homeostatic cytokines, such as IL-15, which is up-regulated in vivo following CY lymphodepletion. In future studies, it is
Fig. 4. Adoptively transferred melanoma-specific CTLs show phenotypic and functional characteristics shared with CD8+ Tcm in vivo. (A) Patient (Pt)
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1: Expression of CD45RO, CD27, CD28, CD127 CD62L, and CCR7 (bold lines) on the HLA*0201-Mart1-specific CTL clone preinfusion (Pre-inf.; Left), and on days 6, 34, and 508 after transfer (Right). MFIs are shown for surface markers with >1% positivity compared with isotype controls (gray areas). (B) Expression of CD27 (y axis), CD62L (x axis) (Upper), and CCR7 (Lower) gated on multimer-positive CTLs 508 d after transfer. Expression of CD28; secretion of TNF-α and IL-2 within IFN-γ+ cells on infused clones; and PBMCs preinfusion and on days 20, 56, and 508 (C; patient 1) and days 20, 56, and 117 (D; patient 2).
Chapuis et al.
Fig. 5. CY conditioning followed by exogenous IL-2 fosters a favorable environment for replication of infused CTLs. (A) Intranuclear Ki-67 expression on clones (Left) and CD8+ multimer-positive or -negative cells within PBMCs of patients 1, 9, 10, and 11 (rows) before CY and on days 0, 7, 14, 28, 56, and 508 (patient 1 only) after T-cell infusion (columns). Multimer (y axis) and Ki-67 (x axis). (B) IL-15 plasma levels (pg/mL) (y axis) plotted over time (x axis). Gray lines represent individual patients, and filled circles show mean and SD. Patients in cohort 1 (Upper) and cohort 2 (Lower).
Methods
static melanoma (Table 1), and had an historical estimated survival of less than 40% at 1 y (25). Detailed descriptions of the materials and methods used, including selection of targets, treatment plan, cytotoxicity assays, T-cell tracking assays, and flow cytometry, are provided in SI Methods.
All clinical investigations were conducted according to the Declaration of Helsinki principles. Protocol 2140 was approved by the Fred Hutchinson Cancer Research Center Institutional Review Board and the Food and Drug Administration. All patients provided written informed consent, had meta-
ACKNOWLEDGMENTS. This work was supported by National Institutes of Health/National Cancer Institute Grant R21 CA128283. A.G.C. was supported by the Oncology Fellowship T32 Grant of the University of Washington Medical Center. C.Y. is supported by a Burroughs Wellcome Fund Translational Scientist Award TS60637.
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conceivable that the combination of adoptively transferred Tcm effectors, together with a source of LD exogenous cytokines, may eliminate the requirement for a conditioning regimen altogether while providing long-term antitumor control.
