Targeted T-cell Therapy in Stage IV Breast Cancer: A Phase I Clinical Trial

Abstract

Purpose: This study reports a phase I immunotherapy trial in 23 women with metastatic breast cancer consisting of eight infusions of anti-CD3 × anti-HER2 bispecific antibody (HER2Bi) armed anti-CD3-activated T cells (ATC) in combination with low-dose IL-2 and granulocyte-macrophage colony-stimulating factor to determine safety, maximum tolerated dose (MTD), technical feasibility, T-cell trafficking, immune responses, time to progression, and overall survival (OS).

Experimental design: ATC were expanded from leukapheresis product using IL2 and anti-CD3 monoclonal antibody and armed with HER2Bi. In 3+3 dose escalation design, groups of 3 patients received 5, 10, 20, or 40 × 10(9) armed ATC (aATC) per infusion.

Results: There were no dose-limiting toxicities and the MTD was not defined. It was technically feasible to grow 160 × 10(9) ATC from a single leukapheresis. aATC persisted in the blood for weeks and trafficked to tumors. Infusions of aATC induced anti-breast cancer responses and increases in immunokines. At 14.5 weeks after enrollment, 13 of 22 (59.1%) evaluable patients had stable disease and 9 of 22 (40.9%) had progressive disease. The median OS was 36.2 months for all patients, 57.4 months for HER2 3+ patients, and 27.4 months for HER2 0-2+ patients.

Conclusions: Targeting HER2(+) and HER2(-) tumors with aATC infusions induced antitumor responses, increases in Th1 cytokines, and IL12 serum levels that suggest that aATC infusions vaccinated patients against their own tumors. These results provide a strong rationale for conducting phase II trials.

Conflict of interest statement

Conflict of Interest

L.G.L is co-founder of Transtarget Inc. and A.T., Z.A., F.J.C., R.D.L., D.S.D., N.K., A.M., W.C., Q.L. and R.R. have no conflicts of interest.

©2015 American Association for Cancer Research.

Figures

Figure 1

Treatment schema shows leukapheresis to obtain T cells for expansion. HER2Bi armed ATC (aATC) were administered twice weekly for four consecutive weeks. All patients received SQ IL-2 (300,000 IU/m2/day) and GM-CSF (250 µg/m2/twice weekly), beginning 3 days before the first aATC infusion and ending 1 week after the last aATC infusion. Immune testing was performed at indicated time points after aATC infusions. 1B) Shows a PET/CT of a partial responder in the HER2-negative group who had 2 well-defined liver metastases (2.5 × 1.7 cm and 2.5 × 1.3 cm as shown in Before). Reimaging after IT showed regression of the two lesions after 6 months (After). 1C) Left panel shows K-M curve for entire group (All), HER2 (3+) positive and HER2 (0–2+) negative patients. One patient with an unknown HER2 status was analyzed with the HER2 negative group. Right panel shows K-M curve for time to progression for entire group, HER2 (3+) positive and HER2 (0–2+) negative groups.

Figure 2

A) Shows the bar graph of cytotoxicity mediated by fresh PBMC of individual patients (n=12) against SK-BR-3 and their corresponding HER2Bi armed ATC doses, OS and TTP. B) Upper panel shows the enhanced cytotoxicity by PBMC directed at SK-BR-3 and K562 at pre IT, mid IT (infusion #4 or #5) and post IT (4 weeks after completion of IT). Lower panel shows T cell IFN-γ EliSpots directed at SK-BR-3 and K562 targets at pre IT, mid IT and post IT.

Figure 3

Shows profile of serum cytokines. Analysis of serum samples (n=13) at pre-immunotherapy (PreIT), after aATC infusions #4 (midIT), and post IT (4 weeks after completion of IT). Armed ATC infusions (IT) show increase in IFN-γ, IL-2, IL-12, TNF-α, GM-CSF, and IL-10 levels during aATC infusions (Mid IT). Bottom right graph shows the mean ratio of Th1/Th2=[IL-2+IFNγ]/[IL-4+IL-10] at pre-, mid- and postIT time points.

Figure 4

Left panel shows the proportion of circulating CD3+ cells and IgG2a+ cells in sequential samples from 1 patient stained with anti-human CD3 and anti-mouse IgG2a+ to quantitate the proportion of HER2Bi armed ATC in the PBMC by flow cytometry. Right panel shows the proportion of circulating PBL from four patients that were stained with anti-mouse IgG2a and quantitated by flow cytometry at preinfusion #1 (Inf #1), preinfusion #5 (Inf #5), preinfusion #8 (Inf #8), and 1 week after the 8th infusion (1w post). B) Left panel shows HER2Bi-armed ATC localized to the bone marrow, lung, liver, and spleen within 4 h of injection. By 24 hours, armed ATC had cleared the lungs but persisted in the bone marrow, liver and spleen for up to 96 hours (4 days) post-infusion. Blood was sampled and counted to determine the clearance of armed ATC from the blood. Right panel shows the clearance of 111In labeled aATC. Heparinized blood (1ml aliquots) were drawn at 0, 0.7, 1, 6, 8, 27 and 96 hrs post infusion and counted for gamma irradiation. Results are presented as % radioactivity in serum (cpm) of injected dose. C) Left panel shows the sternal tumor biopsy 1 week (Left panel) and 1 month (Right panel) post IT showing poorly differentiated mammary ductal carcinoma. Composite immunofluorescent staining detected HER2Bi bispecific antibody using anti-mouse IgG-FITC overlaying IHC for detection of T cells using anti-CD3. HER2Bi detection co-localized with T cells infiltrating connective tissue surrounding nests of tumor cells. D) Shows the HAMA response before each aATC infusion and post infusions at indicated time points. HAMA antibody response (n=11) could not be detected more than 10ng/ml at any time point.