Phase I Trial of Adoptive Cell Transfer with Mixed-Profile Type-I/Type-II Allogeneic T Cells for Metastatic Breast Cancer

Abstract

PURPOSE

Metastatic breast cancer (MBC) response to allogeneic lymphocytes requires donor T-cell engraftment and is limited by graft-versus-host disease (GVHD). In mice, Type-II-polarized T cells promote engraftment and modulate GVHD whereas Type-I-polarized T cells mediate more potent graft-versus-tumor (GVT) effects. This Phase-I translational study evaluated adoptive transfer of ex-vivo-costimulated Type-I/Type-II (T1/T2) donor T cells with T-cell-depleted (TCD) allogeneic stem-cell transplantation (AlloSCT) for MBC.

EXPERIMENTAL DESIGN

Patients had received anthracycline, taxane and antibody therapies, been treated for metastatic disease and an HLA-identical-sibling donor. Donor lymphocytes were costimulated ex vivo with anti-CD3/anti-CD28 antibody-coated magnetic beads in IL-2/IL-4-supplemented media. Patients received reduced-intensity conditioning, donor stem cells and T1/T2 cells, and monitoring for toxicity, engraftment, GVHD and tumor response; results were compared with historical controls, identically treated except for T1/T2-product infusions.

RESULTS

Mixed Type-I/Type-II CD4⁺-T cells predominated in T1/T2 products. Nine patients received T1/T2 cells at Dose-Level 1 (5×10⁶ cells/kg). T-cell donor chimerism reached 100% by a median of 28 days. Seven (78%) developed acute GVHD. At Day +28, five patients had partial responses (56%) and none had MBC progression; thereafter, two patients had continued responses. Donor-T-cell engraftment and tumor responses appeared faster than in historical controls, but GVHD rates were similar and responders progressed early, often following treatment of acute GVHD.

CONCLUSION

Allogeneic T1/T2 cells were safely infused with TCD-AlloSCT, appeared to promote donor engraftment, and may have contributed to transient early tumor responses.

INTRODUCTION

Metastatic breast cancer (MBC) remains incurable while novel therapeutic targets and investigational agents are identified at an unprecedented pace, with little progress on survival after first-line treatment failure.(1) Several immunotherapeutic approaches have activity against breast cancer, including cellular therapies;(2) synergy with cytotoxic agents might improve treatment outcomes.(3) We have previously reported a clinically relevant graft-versus-tumor (GVT) effect after allogeneic stem cell transplantation (AlloSCT) in MBC, attributable to allogeneic lymphocytes.(4) While prior reports suggested possible graft-versus-MBC,(5–7) our study design distinguished immune-mediated responses from chemotherapy effects with reduced-intensity conditioning (RIC) and T-cell depleted (TCD) stem-cell allografts with delayed donor lymphocyte infusions (DLI). Recipients underwent induction for targeted lymphocyte depletion (TLD) prior to RIC to prevent rejection of the TCD-allograft during the expected delay in donor T-cell engraftment.(8) Breast cancer responses coincided with full-donor T-cell chimerism and were strongly associated with development of acute GVHD; unfortunately, tumor progression often rapidly followed systemic treatment of GVHD.(4) Subsequently, other reports substantiated these observations.(9–11)

While TCD reduces acute GVHD, it may delay donor engraftment and increase risk of graft rejection and relapse.(12–14) Delayed DLI offsets these negative consequences but carries risk of acute GVHD.(15) Both graft rejection and GVHD are predominantly mediated through Type-I immune responses.(16) In murine models, we have shown that ex-vivo polarized donor CD4⁺-Th2 and CD8⁺-Tc2 cells exert beneficial effects on donor engraftment and GVHD.(17, 18) Further, while donor Tc1-cell-mediated tumor cytotoxicity is more potent, donor Tc2 cells exert significant antitumor effects with less coincident GVHD.(18–22) Ex-vivo expansion of lymphocytes from healthy donors via CD3/CD28 costimulation generates activated, Type-I polarized CD4⁺- and CD8⁺-T cells;(23) addition of IL-4 yields T cells with mixed features of Type-I/Type-II polarization.(24) We hypothesized that mixed Type-I/Type-II polarized (“T1/T2”) donor T cells given at the time of CD34⁺ stem-cell enriched allograft infusion might facilitate rapid donor engraftment and promote balanced inflammatory conditions in vivo without eliminating the GVHD protection afforded by TCD. As observed in our murine models, Type-II effects might attenuate acute GVHD while permitting early GVT contributions by both Type-I and Type-II donor lymphocytes. Earlier immune antitumor responses with less high-grade acute GVHD could increase the clinical benefit of allogeneic lymphocyte therapy for MBC. Large numbers of donor T cells costimulated/expanded ex vivo with anti-CD3/anti-CD28 antibody-coated magnetic beads have been safely administered to patients, including Type-II-activated CD4⁺-T cells(24, 25) and activated DLI.(23)

We initiated a Phase I clinical study to evaluate donor T1/T2 cell therapy (BB-IND 11720; NCT00082953). Primary aims were to determine the feasibility and safety of administering ex-vivo-generated donor T1/T2 cells. Secondary aims were to assess the effects of T1/T2 cells on donor T-cell engraftment and tumor response through comparisons with historical control patients treated on a previous trial (NCT00020176). Both trials used the identical AlloSCT regimen except donor T1/T2 cell infusion.(4)

MATERIALS AND METHODS

Patients

Subjects had an eligible, HLA-identical (6/6 antigen) sibling donor and MBC with measurable disease for which they had received a taxane, an anthracycline, appropriate targeted therapy (e.g., hormonal agents and/or trastuzumab for receptor-positive and/or HER2-overexpressing tumor) and at least one cytotoxic drug for metastatic disease. Eligibility allowed treated central nervous system (CNS) metastases that had not progressed for at least four weeks. The National Cancer Institute Institutional Review Board approved this study in 2004; all patients and donors provided informed, written consent. The trial permitted accrual of up to 36 recipient-donor pairs and treatment of three patient-cohorts with escalating T1/T2 cell-doses.

Cell Processing

Donor T1/T2 Cells

Donor peripheral blood mononuclear cells (PBMC) and plasma were collected from 5–15 liters of whole blood processed (Fenwal CS 3000 Plus^® blood cell separator, Baxter Healthcare Corporation, Deerfield, IL or COBE Spectra^®, Gambro BCT, Inc, Lakewood, CO). A portion of the fresh collection was ACK-lysed, incubated with anti-CD3/anti-CD28 antibody-coated magnetic beads at a 3:1 bead/cell ratio (courtesy of Carl June, University of Pennsylvania, BB-IND 6675), and T-cell enriched (MPC™-1, Dynal^®, Invitrogen Corporation, Carlsbad, CA). Bead-bound cells were seeded in gas-permeable cell culture bags (Lifecell^®, Baxter) in Type-II culture media (X-VIVO 20^®, Cambrex, Walkersville, MD, with 5% heat-inactivated, filtered autologous (donor) plasma, recombinant human IL-4 (rhIL-4), 1000 IU/ml, BB-IND 4348, and rhIL-2, 20 IU/ml, Chiron Therapeutics, Emeryville, CA). Cells were cultured at 37°C in 5–7% CO₂, humidified incubators for 12 days. Type-II media was added on days 2 and 4 (10X) and as needed (1X) to maintain a concentration of 0.5×10⁶ cells/ml through at least day 8 of culture. Release criteria included attainment of 5×10⁶ CD3⁺ cells/kg recipient weight and standard sterility assays. T1/T2-cell products were cryopreserved in 5×10⁶ CD3⁺ cells/kg-aliquots in Plasmalyte A^® (Baxter) with 4% human serum albumin, 5% dimethyl sulfoxide (Research Industries, Salt Lake City, UT) and 6% pentastarch (B. Braun, Irvine, CA) and stored in liquid nitrogen under Good Manufacturing Practice conditions.

Peripheral Blood Stem Cells (PBSC)

Following PBMC collection, donors underwent PBSC mobilization with filgrastim (8 µg/kg, subcutaneous injection, b.i.d.) and collection with daily apheresis starting on Day 5 (Fenwal CS 3000 Plus^®) until at least 10×10⁶ CD34⁺ cells/kg were collected. Donor cells were subjected to a 4-to-5-log T-cell depletion ex vivo, using one of two methods: a combined positive selection of CD34⁺ cells and negative selection of T cells, using CD2-, CD6- and CD7-targeted antibodies;(8) or, following modification of institutional cell-processing procedures, a positive selection of CD34⁺ cells (CliniMACS^® CD34 Reagent and Immunomagnetic Selection System, Miltenyi Biotec, Auburn, CA). A lymphocyte add-back to 1×10⁵ CD3⁺ cells/kg was performed to minimize T-cell dose variability and allografts were cryopreserved as described.

Study Design

Recipients underwent AlloSCT as previously described (Figure 1).(26) Briefly, patients received one or two 21-day cycles of TLD followed by restaging and a peripheral-blood CD4⁺-T cell count. Absent disease progression, patients with circulating CD4⁺ count greater than 50 cells/µl received a second cycle of TLD. After the second cycle, or after Cycle One if restaging demonstrated disease progression, patients proceeded to RIC and donor-cell infusions, irrespective of the CD4⁺-T cell count. Cyclosporine prophylaxis was begun on Day -1. On Day 0, donor PBSC were infused, followed one hour later by T1/T2 cells. Filgrastim (10 µg/kg/day) was given from Day 0 through neutrophil recovery and cyclosporine was given from Day -1 through Day +28 (200–300 µg/L) then, in the absence of acute GVHD, rapidly tapered off over two weeks. Patients were monitored for toxicity in-hospital through neutrophil recovery then as outpatients biweekly through Day +100 and monthly through Day +180.

Figure 1. Study Design.

Abbreviations: CD4: CD4⁺-T cell; µl: microliter; mg/m²: milligram per square meter; µg/kg: micrograms per kilogram; HLA: human leukocyte antigen; DLI: donor lymphocyte infusion.

Donor chimerism was assessed with PCR-based analysis of short tandem repeats (STR) using commercially available probes (Perkin-Elmer Cetus, Irvine, CA). Bone marrow chimerism was assessed at Transplant Days +28 and +98, and PBMC chimerism, including CD3⁺ and CD14⁺CD15⁺ mononuclear cell subsets, was assessed every two weeks through Day +98, and at four, five and six months post-transplant. The severity of acute and chronic GVHD was graded according to Keystone Consensus Criteria(27) and the International Bone Marrow Transplant Registry Consensus Criteria(28), respectively. (During the course of the trial, NIH Consensus Criteria for scoring chronic GVHD(29) were adopted in our institution and are reported when available). Tumor response was assessed with computed tomographic (CT) scanning after each cycle of TLD and at four and six weeks after Day 0, then monthly through six months. Serial CT measurements were made by independent study radiologists (CC, AD) and responses were recorded according to Response Evaluation Criteria In Solid Tumors (RECIST).(30) Responses of historical controls were retrospectively assigned according to RECIST to permit comparisons.

Characterization of Donor T1/T2 Products and Recipient PBL

Donor T1/T2 cells and recipient PBL populations were evaluated by flow cytometry to quantify CD3⁺ lymphocytes and CD4⁺- and CD8⁺-T cell subsets. To assess Type-I and Type-II polarization, T1/T2 product CD4⁺- and CD8⁺-T cell subsets were assessed with intracellular flow cytometry for nuclear expression of transcription factors T-bet and GATA-3 and for IFNγ, TNFα, FoxP3, IL-2, IL-4, IL-10 and IL-13. Cytolytic potential was assessed with intracellular flow cytometry of T-cell subsets for perforin and granzyme expression. Two weeks following donor PBSC and T1/T2-product infusions, recipient PBL were assessed for secretion of IFNγ,IL-2, IL-4 and IL-10 (MACS® Cytokine Secretion Assay, Miltenyi Biotec, Auburn, CA).

Statistical Methods

This Phase-I trial tested feasibility and safety of donor T1/T2 cells. The primary safety endpoint was development of Grade II–IV acute GVHD through Day +42, coinciding with planned discontinuation of cyclosporine. Secondary endpoints included time to full-donor T-cell engraftment, tumor response and progression-free and overall survival, and included comparisons with historical controls who underwent AlloSCT for MBC without T1/T2 cells. Three dose-levels were planned, treating up to 12 subjects per cohort; accrual to the subsequent cohort was permitted if no more than six patients in a given cohort developed Grade II–IV acute GVHD by Day +42. An early stopping rule for dose-limiting toxicity specified termination if greater than one-third of patients in a cohort experienced unresponsive Grade III acute GVHD or if there was any Grade IV acute GVHD and/or treatment-related death.

Comparisons between T1/T2 recipients and historical controls included rates of Grade II–IV acute GVHD using Fisher’s exact test; time to full-donor T-cell chimerism using an exact Wilcoxon rank sum test; and ordered-response categories using the Cochran-Armitage test for trend.

RESULTS

Patient Characteristics

Eleven patients with MBC and sibling-donors were enrolled on study (Table 1) at Dose-Level 1 (5×10⁶ T1/T2 cells/kg). Recipient median age was 45 years (range, 37–57) and median duration of MBC was 15 months (range, 5–58). All cancers demonstrated one or more adverse prognostic factors, including tumors lacking estrogen and progesterone receptors (ER⁻/PR⁻, n=4, of which 2 were ER⁻/PR⁻/HER2⁻), over-expression of HER2 (n=5), visceral metastases (n=9, liver and/or lung) and prior brain metastases (n=3). Patients had received a median of four chemotherapy regimens for metastatic disease (range, 2–7).

Table 1.

Recipient Characteristics

		Breast Cancer Diagnosis				Metastatic Disease
Patient No.	Age (Yr)	ER	PR	HER2	Duration (Months)	Duration (Months)	# Chemo Regimens	Brain	Liver	Lung	Chest Wall	Bone	BM
2	41	+	+	−	58	34	3	x	x		x	x	x
4*	42	+	+	−	127	58			x			x	x
6*	37	−	−	−	22	10					x		x
8	46	+	−	+	29	13	2			x	x	x
10	47	−	−	+	30	15	6			x	x
12	56	+	+	+	71	71	4		x	x	x	x
14	57	+	+	+	67	11	4		x	x		x
16	35	−	−	+	29	10	4				x
18	42	−	−	−	30	5	4			x
20	48	+	+	−	61	42	7	x	x	x		x	x
22	45	+	+	−	79	29	6	x
Median	45				58	15	4

Abbreviations/Definitions: ER: estrogen receptor expression (immunohistochemistry, IHC); PR: progesterone receptor expression (IHC); HER2: HER2 receptor overexpression (IHC or Her2 fluorescence in-situ hybridization); Breast Cancer Diagnosis/Duration: months from diagnosis to study enrollment; Metastatic Breast Cancer/Duration: months from diagnosis of metastatic disease to study enrollment; Metastatic Breast Cancer/Chemo Regimens: number of chemotherapy regimens received since diagnosis of metastatic disease.

Characterization of Donor T1/T2 Products

Ten donors underwent steady-state lymphocyte collection; T1/T2 products were generated from each. T1/T2 products were predominantly comprised of CD3⁺ T cells (median, 97%, range, 77–100), with a median CD4/CD8 ratio of 3.41 (range, 1.3–22.7 Table 2). As shown in Figure 2, approximately 40% of expanded CD4⁺-T cells expressed IL-2 and IL-13 while less than 10% expressed IFNγ and IL-10; none expressed TNFα. Less than 10% of CD8⁺-T cells expressed IL-2, IFNγ and IL-13, rare cells expressed IL-4 or IL-10, and none expressed TNFα. Intracellular expression of polarizing transcription factors followed a similar pattern. One-quarter of CD4⁺-T cells expressed T-bet and only rare GATA-3⁺ cells; FoxP3 expression was similar to T-bet. Within CD8⁺-T cells, less than 10% expressed T-bet and GATA-3 expression was essentially absent. Examination of intracellular cytolytic protein expression showed that 40–50% of CD4⁺-T-cells expressed both perforin and granzyme, while only granzyme was expressed in significant numbers of CD8⁺-T cells.

Table 2.

Donor Cell Products

	Allografts		T1/T2 Cells		DLI
Patient No.	CD34 (106)	CD3 (105)	Dose (106)	4/8 Ratio	#1 (1×106)	#2 (5×106)	#3 (1×107)
2	7.29	1	5	1.9	-	-	-
4*	-	-	-	1.3	-	-	-
6*	-	-	-	-	-	-	-
8	6.24	1	5	3.8	42	69	-
10	5.48	10*	5	1.4	43	69	97
12	7.50	0.14*	5	4.2	74	-	-
14	6.41	1	5	22.7	-	-	-
16	6.32	1	5	5.2	68	103	-
18	7.92	1	5	2.0	-	-	-
20	10.94	1	5	3.0	-	-	-
22	10.54	1	5	5.7	-	-	-
Median	7.29	1	5	3.41	56	69	97

Abbreviations/Definitions: Allograft/CD34: donor stem cell dose per kilogram recipient weight; Allograft/CD3: donor T-cell dose after lymphocyte add-back per kilogram recipient weight (*note: the doses for Patient Nos. 10 and 12 reflect errors in the lymphocyte add-back); T1/T2 cells/Dose: donor T1/T2 cell dose per kilogram recipient weight; T1/T2 Cells/4/8 Ratio: ratio of CD4⁺ to CD8⁺-T cells in donor T1/T2 cell products; DLI: cell dose as CD3⁺ cells per kilogram recipient weight and day of administration relative to Day 0 (donor stem cell and T1/T2 infusions).

Figure 2. Functional Phenotype of Donor T1/T2 Products and Post-infusion Recipient PBL at Two Weeks.

T1/T2 products were evaluated for CD4⁺ and CD8⁺ subsets and, samples permitting, for transcription factor and cytokine expression by cell-surface and intracellular flow cytometry, respectively. Two weeks after donor PBSC and T1/T2 cell infusions, recipient PBL CD4⁺ and CD8⁺-T cells were assessed for secretion of Type-I and Type-II cytokines using MACS® Cytokine Secretion Assay detection kits.

Treatment

Patient Number 6 (PN6) voluntarily came off study prior to evaluation of Cycle 1 of TLD. In the remaining 10 patients, TLD achieved a median 95% reduction in circulating CD4⁺-T cell counts (range, 26–98), from 491 CD4⁺-T cells/µl at baseline (range, 60–960) to 34 CD4⁺-T cells/µl after TLD (range, 9–80). PN4 was removed from study prior to RIC due to rapid disease progression resulting in hepatic failure. Nine patients completed RIC and received donor stem cells and T1/T2 cells. Five patients developed acute GVHD prior to completion of cyclosporine taper; four patients completed cyclosporine taper and received DLI for disease progression per protocol.

Toxicity

GVHD

Following administration of T1/T2 cells, no infusion reactions, organ-specific toxicities or hyperacute GVHD were observed. At hematologic recovery from RIC, three patients developed engraftment syndrome requiring steroid treatment (Table 3). Grade II–IV acute GVHD developed in seven patients (77.9%), five occurring on or before Day +42; six patients required systemic steroid treatment and all responded to therapy. Chronic GVHD developed in 3 of 8 evaluable patients (37.5%). In historical controls, 9 of 18 evaluable patients (50%) developed Grade II–IV acute GVHD (p=0.23; excludes PN4, who died on Day +2), of which three occurred on or before Day +42. Chronic GVHD developed in 8 of 15 evaluable patients (53.3%), of which four were “late-acute” GVHD coincident with establishment of full-donor T-cell chimerism after Day +100 (Supplemental Data).

Table 3.

Clinical Outcomes

	100% DONOR		STEROIDS		GVHD				RESPONSE			MORTALITY
Patient No.	T Cell (Day)	Myeloid (Day)	Day	Indication	Acute			Chronic Stage	TLD	Day +28	TTP (Days)	Survival (Days)	Cause of Death
					Onset	Skin/Liver/GI	Grade	IBMTR/NIH
2	28	14	5	ES	20	3/0/1	II	No	PR	PR	98	108	MBC
8	28	28	71	Palliative	44	0/0/2	III	N/A	SD	SD	42	90	MBC
10	98	14	-	-	-	0/0/0	O	No	SD	PR	42	116	MBC
12	70	14	10	ES	80	3/0/0	II	No	SD	SD	70	101	MBC
14	28	14	46	GVHD	42	2/0/1	II	Ext/Mod	SD	PR	70	540	MBC
16	14	14	100	Palliative	-	0/0/0	O	No	SD	SD	42	117	MBC
18	28	14	16	GVHD	14	3/0/2	III	Ext/Mod	SD	SD	126	410	MBC
20	14	14	9	ES	41	2/0/2	III	Ext/Mod	SD	PR	70	223	MBC
22	14	14	18	GVHD	14	4/0/0	IV	No	SD	PR	70	152	Sepsis
Median	28	14	17		41						70	117

Abbreviations/Definitions: 100% Donor: first day following donor stem cell and T1/T2 cell infusions that sustained full-donor chimerism (greater than 90%) was observed in whole-blood, column-separated T-cell (CD3⁺) and myeloid (CD14⁺CD15⁺) cell fractions (MACS) Cell Separation Columns, Miltenyi, Auburn, CA); Steroids/Day and Indication: initial systemic steroid administration; GVHD/Acute: day of onset, maximum organ-specific staging and grade of acute GVHD; GVHD/Chronic/Stage: both IBMTR/NCI Consensus Criteria provided; Response: RECIST responses after induction chemotherapy (TLD) and Day +28 post-transplant; TTP: first progression from Day 0; Survival: from Day 0.

Treatment-Related Mortality (TRM)

There was no TRM attributable to the T1/T2 cells. A single treatment-related death (sepsis) occurred on Day +152 (11.1%); there was one death from disease progression prior to Day +100 (Table 3). In historical controls there were four treatment-related deaths (21.1%) and one death due to disease progression prior to Day +100 (Supplemental Data).(4, 31)

Engraftment

All patients engrafted, with rapid hematologic recovery and full-donor chimerism (neutrophils > 5000 cells/µl: median, 10 days, range, 9–11; platelets > 50,000/µl: median, 12 days, range, 8– 48, one patient remained transfusion-dependent). The median time to full-donor engraftment was 28 days in circulating T cells (range, 14–84); in historical controls, the median time to full-donor T-cell engraftment was 77 days (range, 14–365; p = 0.051, exact two-tailed Wilcoxon rank-sum test). Donor chimerism in circulating myeloid cells reached 100% in a median of 14 days in T1/T2 recipients and in historical controls (ranges, 14–28 and 14–98, respectively).(4, 8, 31)

In-Vivo Effects of Donor T1/T2 Cell Infusion

At Day +14 after T1/T2 cell infusion, circulating CD4⁺ and CD8⁺-T cells expressed a mixed Type-I/Type-II cytokine profile, with relatively balanced frequencies of T cells secreting IFNγ, IL-2, IL-4, and IL-10 (Figure 2). An increase in eosinophils, associated with Type-II immune responses and antitumor activity,(32–34) was observed in 7 of 9 patients (Figure 3); absolute eosinophil counts exceeded the upper limit of normal (ULN) by Day +42 in all patients who had not received systemic steroids. In some, the rise in eosinophils coincided with tumor response and acute GVHD, as illustrated by the clinical course of PN14 (Figure 3). Clinical evaluation of a pleural effusion on Day +42 (PN12, pleural tumor nodules) permitted analysis of the bloody exudate, in which 44% of the cells were lymphocytes and 58% of those were CD3⁺ (75% CD4⁺ and 20% CD8⁺). By STR analysis, 95% of the CD3⁺ fraction was donor-derived (relative to 89% in the peripheral blood).

Figure 3. In-vivo Effects of Donor T1/T2 Cell Infusion.

A. Percent-change in sum-of-product tumor measurements from baseline (per RECIST) for T1/T2 cell recipients (top) and historical controls (bottom). Black bars denote patients with a tumor response at Day +28; gray bars denote patients with stable or progressive disease at Day +28. The day relative to donor T1/T2 cell infusion is shown on the horizontal axis for both graphs (middle). B. Eosinophilia, a Type-II inflammatory response, was observed in most patients. The relative percent-change in absolute eosinophil counts (AEC) from baseline at peak are shown for each patient using the left vertical axis: black columns identify patients who received steroids on or before Day +42, gray columns represent patients who did not. Using the right vertical axis, each patient’s peak AEC is shown (♦); the horizontal axis identifies the Patient Number and day of peak relative to donor T1/T2 cell infusion. The horizontal dotted line indicates the upper limit of normal (ULN) for the AEC. C. In PN14, the rise in eosinophils coincided with early tumor response and the peak with onset of acute GVHD (Day +42). The line graph depicts absolute eosinophil counts; the bar graph shows percent-change in sum-of-product tumor measurements from baseline at each time-point; the ULN is shown by the horizontal dotted line; the vertical dashed line indicates when systemic steroids were initiated (Day +46).

Efficacy

Following pre-transplant TLD, one patient had a partial response (PR) and the others had stable disease (SD). By Day +28 post-infusion, five patients had achieved PR, four had SD, and none had progressive disease (PD). In 17 evaluable historical controls, the Day +28 responses included five PR (four in whom MBC had responded to pre-transplant chemotherapy), eight SD, and 4 PD (trend toward higher response rate in T1/T2 cell recipients: p=0.14, two-tailed Cochran-Armitage test for trend). At Day +42, two T1/T2-cell recipients’ tumors had responded further. The median time-to-progression was 70 days from AlloSCT (range, 42–126, Table 3).

DISCUSSION

While allogeneic lymphocytes effectively mediate MBC responses, clinical benefit is limited GVHD, which is closely associated with donor engraftment and GVT response.(4, 8, 9, 11) This first-in-human Phase I trial of cytokine-polarized allogeneic CD4⁺/CD8⁺-T cell transfer established the feasibility of generating mixed-profile donor T cells, which were well tolerated with respect to infusion-related toxicity. Evaluation of this cell product on the same platform employed in our earlier MBC trial (host immune depletion, RIC and TCD-allografts) allowed us to explore T1/T2 efficacy with respect to engraftment, GVHD prevention and antitumor activity.

As expected, costimulated donor T1/T2 cells were comprised predominantly of activated CD4⁺-T cells;(35) nonetheless, there was also significant expansion of CD8⁺-T cells. Both CD4⁺ and CD8⁺ subsets of the T1/T2 products expressed the Type-I transcription factor T-bet, whereas very low frequencies of T cells expressed the Type-II transcription factor GATA-3. Cytokine expression in CD4⁺ cells was more consistent with a mixed Th1/Th2 product, with both IL-2 and IL-13 production and minimal production of other Type-I and Type-II cytokines. A smaller proportion of activated CD8⁺-T cells produced cytokines, with similar a similar mixed Type-I/Type-II pattern. We previously reported observations made while this study was ongoing that purified CD4⁺- T cells costimulated with Type-II cytokines ex vivo demonstrated a similar mixed Th1/Th2 cytokine phenotype.(24, 25) Recipients of T1/T2 cells also produced a mixed pattern of Type-I and Type-II cytokines at Day +14, suggesting that the infused cell product may have influenced T-cell cytokine production in vivo. Frequent eosinophilia after T1/T2 cell infusion further suggested in-vivo Type-II effects.

Relative to historical controls,(8) T1/T2-cell recipients appeared to have faster donor T-cell engraftment. T1/T2 recipients also appeared to have better disease control than historical controls by Day +28. While it is not possible to precisely delineate immunologic versus chemotherapeutic contributions to earlier tumor responses observed in T1/T2 recipients, it is noteworthy that these patients had fewer tumor responses to induction chemotherapy (TLD) than historical controls. It seems plausible that the rapid engraftment observed T1/T2-cell recipients expedited donor-T-cell infiltration into tumor tissue and contributed to earlier antitumor effects. Our study design to evaluate T1/T2 cells provided a rigorous test for whether T1/T2 cells could separate therapeutic from toxic effects of alloreactivity. The combination of CD34-selection and cyclosporine is a standard regimen to prevent acute GVHD; early cyclosporine taper was used in an aggressive maneuver to elicit GVT responses, exploiting cyclosporine’s differential potency in suppression of Tregs and activated effector T cells, with rapid tapering to unleash alloreactivity,(36, 37) At Dose-Level 1, T1/T2 cells did not reduce the incidence of acute GVHD relative to historical controls, nor improve opportunities to use of DLI for antitumor effect. Indeed, the onset of GVHD was prior to cyclosporine taper in one-third of patients, with GVHD treatment rapidly followed by cancer progression.(4) The timing of T1/T2 administration (Day 0) may have contributed to the rate of acute GVHD,(24) when lymphopenia from the preparative regimen may have its greatest effect on proliferation and rapid turnover of effector T-cell populations. As postulated in lymphopenia-induced autoimmunity,(38) even polarized donor lymphocytes infused on Day 0 may undergo rapid expansion and give rise to tissue-antigen directed effector populations and promote GVHD.

Study enrollment was terminated when seven patients treated at Dose-Level 1 developed acute GVHD, thereby precluding our ability to evaluate whether higher doses of T1/T2 cells might result in enhanced GVT effects. The patients on this study had very advanced breast cancer with metastatic disease progression on a median of four prior chemotherapy regimens; in addition, most patients carried multiple adverse prognostic factors. Furthermore, the current patient cohort appeared to be more chemotherapy-refractory relative to our previous trial; that is, we observed a lower rate of response to pre-transplant induction chemotherapy. It is important to note that this combination of adverse disease characteristics is infrequently represented in CIBMTR-registered trials of AlloSCT for MBC.(10) Therefore, it is possible that donor T1/T2 cells might mediate improved tumor control in patients with disease characteristics previously associated with GVT responses, including: chemosensitive disease, lower tumor burden and/or bone-only metastases.(9, 11) It is also possible that the current approach, whereby T-cell therapy is delayed relative to conventional transplant approaches that do not involve a prolonged course of induction chemotherapy may have proven disadvantageous in the current patient population. Nonetheless, it seems unlikely that theT1/T2 cells we evaluated would sufficiently address the significant limitations of allogeneic immunotherapy of MBC.

Successful allogeneic immunotherapy for MBC will require a robust immune response and precise discrimination between the therapeutic GVT effect and GVHD toxicity. For allogeneic approaches to improve treatment outcomes in MBC, novel strategies to modulate alloreactivity are essential. The counter-regulatory potential of the Type-II immune response appears promising in preclinical murine models. In an ongoing trial in patients with high-risk hematologic malignancies, we are evaluating the clinical potential of using rapamycin to stabilize Type II function in ex-vivo polarized T cells.(39–41) If early promise is sustained in promoting engraftment and early tumor response in high-risk hematologic malignancies, it would be reasonable to test rapamycin-enhanced Type-II donor T cells in MBC.

Harnessing intrinsic regulatory mechanisms of T-cell immune responses may provide sufficient separation of therapeutic (GVT) from pathologic (GVHD) effects to extend the curative potential of allogeneic cell therapy in hematologic malignancies to individuals with metastatic breast cancer. Ultimately useful approaches must be applicable in a broad range of clinical venues; while complex study designs may be useful in early-phase trials such as this, the translation of effective cell therapies will require studied modification to increase clinical utility.

TRANSLATIONAL RELEVANCE.

We conducted a second-generation clinical trial to further harness allogeneic lymphocyte-mediated graft-versus-breast cancer effects. In our initial trial, metastatic breast cancer (MBC) responses required donor T-cell engraftment and were limited clinically by graft-versus-host disease (GVHD). Separation of graft-versus-tumor (GVT) effects from GVHD is therefore essential for allogeneic lymphocyte therapy of MBC. In murine models, Type-I cytokine-polarized T cells (T1) mediate potent GVT effects and severe GVHD whereas Type-II polarized cells (T2) mediate modest GVT effects and moderate GVHD. In this phase I trial, we infused ex-vivo manufactured T cells of mixed T1/T2 cytokine phenotype. At Dose-Level 1, T1/T2 cells promoted donor engraftment and may have contributed to earlier GVT effects; however, T1/T2 dose escalation was not possible due to GVHD. More robust methods to balance GVHD and GVT effects will be required to benefit patients with metastatic breast cancer.