Supplementary Video 2 from Combining Adoptive Cellular and Immunocytokine Therapies to Improve Treatment of B-Lineage Malignancy

Supporting Information from Optimizing Radiolabeled Engineered Anti-p185^HER2 Antibody Fragments for <i>In vivo</i> Imaging

PDF file - 64K, Boxplots of cellular proliferation rates for different treatment groups calculated by applying ADC data to a logistic model of tumor growth. Boxplots for (A) control, (B) CRLX101 and (C) CPT-11 animals between day 0/2, day 0/4, day 2/4, and day 4/7 are shown.

Supplementary Video 2 from Combining Adoptive Cellular and Immunocytokine Therapies to Improve Treatment of B-Lineage Malignancy

Supplementary Video 1 from Combining Adoptive Cellular and Immunocytokine Therapies to Improve Treatment of B-Lineage Malignancy

Supporting Information from Optimizing Radiolabeled Engineered Anti-p185HER2 Antibody Fragments for In vivo Imaging

PDF file - 74K, P-values of histological comparisons of CRLX101 and CPT-11 cohorts with controls on different days. Statistically significant values are highlighted (p < 0.01, *, p < 0.05, ).

PDF file - 112K, Quantitative histological assessment of CRLX101 and CPT-11 response. Tumors treated with CRLX101 (5 mg/kg), CPT-11 (100 mg/kg) or saline were harvested for histology on days 0, 2, 4, and 7. Tumor samples were subsequently sectioned and stained with (A) Cleaved caspase-3 (co-stained with hematoxylin), (B) TUNEL (co-stained with hematoxylin), (C) Ki-67 (co-stained with hematoxylin) and (D) H&E. Three fields of view from each tumor slide were analyzed to obtain measurements of positive cleaved caspase-3 and TUNEL staining, percentage of Ki-67 expression and cellular density (as demonstrated by hematoxylin staining). Statistically significant values, as compared to the control cohort for each time point, are highlighted (p < 0.01, *, p < 0.05, ). Error bars denote standard error.

OBJECTIVESReport the feasibility, toxicities, and long-term results of a Phase I/II trial of 90Y-labeled anticarcinoembryonic antigen (anti-CEA) (cT84.66) radioimmunotherapy (RIT), gemcitabine, and hepatic arterial infusion (HAI) of fluorodeoxyuridine (FUdR) after maximal hepatic resection of metastatic colorectal cancer to the liver.METHODSPatients with metastatic colorectal cancer to the liver postresection or ablation to minimum disease were eligible. Each cohort received HAI of FUdR for 14 days on a dose escalation schedule. The maximum HAI FUdR dose level planned was 0.2 mg/kg/day, which is the standard dose for HAI FUdR alone. On day 9, 90Y-cT84.66 anti-CEA at 16.6 mCi/m2 as an i.v. bolus infusion and on days 9-11 i.v. gemcitabine at 105 mg/m2 were given. Patients could receive up to three cycles every 6 weeks of protocol therapy. Four additional cycles of HAI FUdR were allowed after RIT.RESULTSSixteen patients were treated on this study. A maximum tolerated dose of 0.20 mg/kg/day of HAI FUdR combined with RIT at 16.6 mCi/m2 and gemcitabine at 105 mg/m2 was achieved with only 1 patient experiencing grade 3 reversible toxicity (mucositis). After surgery, 10 patients had no evidence of visible disease and remained without evidence of disease after completion of protocol therapy. The remaining 6 patients demonstrated radiological visible disease after surgery and after protocol therapy 2 patients had a CR, 1 patient had PR, 2 had stable disease, and 1 had progression. With a median follow-up of 41.8 months (18.7-114.6), median progression free survival was 9.6 months. Two patients demonstrated long-term disease control out to 45+ and 113+ months.CONCLUSIONThis study demonstrates the safety, feasibility, and potential utility of HAI FUdR, RIT, and systemic gemcitabine. The trimodality approach does not have higher hematologic toxicities than seen in prior RIT-alone studies. Future efforts evaluating RIT in colorectal cancer should integrate RIT with systemic and regional therapies in the minimal tumor burden setting.

The goal of this study was to characterize the relationship between tumor uptake of 64Cu-DOTA-trastuzumab as measured by PET/CT and standard, immunohistochemistry (IHC)-based, histopathologic classification of human epidermal growth factor receptor 2 (HER2) status in women with metastatic breast cancer (MBC). Methods: Women with biopsy-confirmed MBC and not given trastuzumab for 2 mo or more underwent complete staging, including 18F-FDG PET/CT. Patients were classified as HER2-positive (HER2+) or -negative (HER2−) based on fluorescence in situ hybridization (FISH)–supplemented immunohistochemistry of biopsied tumor tissue. Eighteen patients underwent 64Cu-DOTA-trastuzumab injection, preceded in 16 cases by trastuzumab infusion (45 mg). PET/CT was performed 21–25 (day 1) and 47–49 (day 2) h after 64Cu-DOTA-trastuzumab injection. Radiolabel uptake in prominent lesions was measured as SUVmax. Average intrapatient SUVmax (pt) was compared between HER2+ and HER2− patients. Results: Eleven women were HER2+ (8 immunohistochemistry 3+; 3 immunohistochemistry 2+/FISH amplified), whereas 7 were HER2− (3 immunohistochemistry 2+/FISH nonamplified; 4 immunohistochemistry 1+). Median pt for day 1 and day 2 was 6.6 and 6.8 g/mL for HER 2+ and 3.7 and 4.3 g/mL for HER2− patients (P < 0.005 either day). The distributions of pt overlapped between the 2 groups, and interpatient variability was greater for HER2+ than HER2− disease (P < 0.005 and 0.001, respectively, on days 1 and 2). Conclusion: By 1 d after injection, uptake of 64Cu-DOTA-trastuzumab in MBC is strongly associated with patient HER2 status and is indicative of binding to HER2. The variability within and among HER2+ patients, as well as the overlap between the HER2+ and HER2− groups, suggests a role for 64Cu-DOTA-trastuzumab PET/CT in optimizing treatments that include trastuzumab.

Background: DI-Leu16-IL2 is a recombinant fusion immunocytokine composed of interleukin-2 and a CD20-targeting monoclonal antibody that maintains the activities of both antibody and cytokine components but is also involved in tumor targeting, engagement of the immune system, and induction of an anti-cancer vaccine effect.

Relapse remains a major cause of treatment failure in patients with relapsed/refractory B-NHL undergoing RIC allo-HCT, especially in chemoresistant or PET positive disease. Zevalin® offers the advantage of targeted radiation delivery to disease sites and is an effective therapy for B-cell NHL. We hypothesized that adding Zevalin® to Fludarabine and melphalan (Flu/Mel) may improve disease control in PET positive and chemoresistant disease thus reducing the risk of relapse post allo-HCT. Patients received Zevalin® 0.4mCi/kg on day -14, flu 25 mg/m2 daily x 5 days on days -9 to -5 and mel 140 mg/m2 on day -4. Rituximab level was measured on Day -22 and -15 and if the level was ≥ 10 μg/ml, Rituximab was not given prior to Zevalin® to enhance biodistribution. All pts received Tacrolimus/sirolimus for GVHD prophylaxis; methotrexate was added for mismatched URD. Between 10/2007 and 4/2014, 41patients were treated. Median age was 55 (range 27-67), M: F =23:18, median # of regimen =4 (range 2-9). Two had prior auto-HCT. The median time from dx to HCT was 23 months (range 5-127). Histology: DLBCL (including transformed) =16 (39%), MCL =9 (22%), low-grade, FL and MZ= 9 (27%) and SLL/CLL=5 (16%). Disease status at HCT: 1CR=7, Relapse= 12, ≥2CR=7, primary refractory =15. Nineteen (46%) had chemoresistant. Twenty-seven (66%) had PET positive at HCT. The donors were matched siblings 20 (49%) and URD 21 (51%). All patients except one early death engrafted. There were 20 deaths from disease progression (6), infection (6), GVHD (1) and multi-organ failure (5) and unknown (2). TRM at day +100 and at 2 year was 5% and 20%, respectively. Nine patients relapsed at a median of 6 months (range 1.4-60) post HCT. The overall incidence grade II-IV aGVHD was 44%. Among 27 with PET positive, 19 became PET negative at day +100 while 8 remained PET positive and 5 subsequently relapsed. Twenty-one patients are alive at a median follow-up of 52 months (range 16-82) for surviving patients. The 3 yrs OS and PFS for all patients was 58% (95% CI, 41-71%) and 55% (95% CI, 39-69%), respectively. The probability of relapse at 1 and 2 years were both 20% (95% CI, 9-33%). Univariate analysis using the following variables: age, disease status, # of regimen, histology (DLBCL/Transformed vs. other), donor type, chemoresistant, and PET status identified histology as the only factor predict for OS (p=0.0087) and PFS (0.0047), while disease status at HCT (p=<.0001) and histology (p=<.0001) predict for relapse. OS based on histology is shown in figure 1. This study demonstrates that adding Zevalin® to Flu/Mel leads to improve outcome in chemoresistant and PET positive B-NHL undergoing allo-HCT and suggest that pre-transplant PET positive does not preclude successful allo-HCT especially in low-grade B-NHL. However, this approach was not effective in DLBCL and transformed lymphoma thus novel approach with new target agents should be explored.

Natural killer (NK) cells play a vital antitumor role as part of the innate immune system. Efficacy of adoptive transfer of NK cells depends on their ability to recognize and target tumors. We investigated whether low dose focused ultrasound with microbubbles (ldbFUS) could facilitate the targeting and accumulation of NK cells in a mouse xenograft of human colorectal adenocarcinoma (carcinoembryonic antigen (CEA)-expressing LS-174T implanted in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice) in the presence of an anti-CEA immunocytokine (ICK), hT84.66/M5A-IL-2 (M5A-IL-2). Human NK cells were labeled with an FDA-approved ultra-small superparamagnetic iron oxide particle, ferumoxytol. Simultaneous with the intravenous injection of microbubbles, focused ultrasound was applied to the tumor. In vivo longitudinal magnetic resonance imaging (MRI) identified enhanced accumulation of NK cells in the ensonified tumor, which was validated by endpoint histology. Significant accumulation of NK cells was observed up to 24 hrs at the tumor site when ensonified with 0.50 MPa peak acoustic pressure ldbFUS, whereas tumors treated with at 0.25 MPa showed no detectable NK cell accumulation. These clinically translatable results show that ldbFUS of the tumor mass can potentiate tumor homing of NK cells that can be evaluated non-invasively using MRI.

DI-Leu16-IL2 immunocytokine is a recombinant fusion protein composed of interleukin 2 (IL2) and a CD20 targeting monoclonal antibody. Pre-clinical studies have shown it maintains the activities of both antibody and cytokine components but is also involved in tumor targeting, engagement of the immune system and induction of an anti-cancer vaccine effect. In a SCID mouse model,DI-Leu16-IL2 is more effective than the individual components (IL-2 and CD20) given either alone or in combination (Gillies SD; 2005). DI-Leu16-IL2 administered intravenously to 8 relapsed/refractory Non-Hodgkin's Lymphoma (NHL) patients at a maximum dose of dose of 0.5 mg/m2 resulted in 1 complete, 1 possible partial response and 4 patients with stable disease (Nakamura R; 2013).

Transformation from indolent non-Hodgkin lymphoma (NHL) to diffuse large B cell lymphoma (DLBCL) has historically been associated with a poor prognosis. A small series of autologous stem cell transplantation (ASCT) studies using conventional conditioning regimens has demonstrated durable progression-free survival (PFS) rates ranging from 25% to 47%, but data in the rituximab era are lacking. Here we report the results of a multicenter retrospective trial evaluating ASCT in patients with transformed lymphoma using the Z-BEAM conditioning regimen, which combines yttrium-90-labeled ibritumomab tiuxetan (Zevalin) with high-dose BEAM (carmustine, etoposide, cytarabine, melphalan) chemotherapy. Sixty-three patients from 4 institutions were treated between 2003 and 2011. Histological confirmation of transformation was required and defined as a diagnosis of DLBCL in patients with either a prior history or concomitant diagnosis of low-grade B cell NHL. Median patient age at ASCT was 59.5 years, median number of prior regimens was 2, and all patients were exposed to rituximab. Disease status at ASCT was as follows: first complete remission (CR) (n = 30), first partial remission (n = 11), first relapse (n = 14), and at least second CR (n = 8). The median time from diagnosis of histological transformation to ASCT was 7.5 months (range, 2.8 to 116). Two-year nonrelapse mortality was 0%. Median follow-up for living patients was 28 months (range, 5 to 103). Two-year PFS was 68% (95% confidence interval, 58% to 75%), and overall survival was 90% (95% confidence interval, 80% to 95%). In conclusion, the Z-BEAM conditioning regimen for ASCT is well tolerated by patients with transformed lymphoma and demonstrates encouraging clinical outcomes. (C) 2014 American Society for Blood and Marrow Transplantation.

Objective: Systemic radiation therapy may require the clinical use of relatively high levels (tens of millicuries) of energetic pure, beta-emitting radionuclides such as P, sgsr and goy. To deliver such amounts of radioactivity to the patient presents a unique radiation hazard to the staff in nuclear pharmacy, radiation oncology and nuclear medicine due to the brake, or bremsstrahlung, radiation produced in the injection volume and syringe. Methods: We designed a portable shield and automated delivery system which can be operated remotely to perform intravenous or intraperitoneal administration of a given amount of radioactivity and in a fixed period of time. Results: By encasing a 60-cc syringe in 1 .4 em of acrylic and covering that layer with 6 mm of lead, we could reduce the exposure rate to approximately 0.12 mR hr1 mci1 at 5 em from the side of the device containing a goy source. Corresponding exposure rates were 0.027 and 0.001 mR hr1 mci-1, at 20 and 100 em respectively, from the side of the injection syringe. For a 50-mCi clinical injection, we estimated exposure rates of approximately 6 mR hr1 at 5 em from a goy source enclosed in this prefabricated, portable syringe holder. Conclusions: We recommend that clinics, using highenergy beta emitters for radiotherapy, fabricate acrylic and lead holders for syringes used in the protocols to reduce their staffs' exposure.

Women with human epidermal growth factor receptor 2 (HER2)–positive breast cancer are candidates for treatment with the anti-HER2 antibody trastuzumab. Assessment of HER2 status in recurrent disease is usually made by core needle biopsy of a single lesion, which may not represent the larger tumor mass or other sites of disease. Our long-range goal is to develop PET of radiolabeled trastuzumab for systemically assessing tumor HER2 expression and identifying appropriate use of anti-HER2 therapies. The purpose of this study was to evaluate PET/CT of 64Cu-DOTA-trastuzumab for detecting and measuring tumor uptake of trastuzumab in patients with HER2-positive metastatic breast cancer. Methods: Eight women with biopsy-confirmed HER2-positive metastatic breast cancer and no anti-HER2 therapy for 4 mo or longer underwent complete staging, including 18F-FDG PET/CT. For 6 of the 8 patients, 64Cu-DOTA-trastuzumab injection (364–512 MBq, 5 mg of trastuzumab) was preceded by trastuzumab infusion (45 mg). PET/CT (PET scan duration 1 h) was performed 21–25 (day 1) and 47–49 (day 2) h after 64Cu-DOTA-trastuzumab injection. Scan fields of view were chosen on the basis of 18F-FDG PET/CT. Tumor detection sensitivity and uptake analyses were limited to lesions identifiable on CT; lesions visualized relative to adjacent tissue on PET were considered PET-positive. Radiolabel uptake in prominent lesions was measured as maximum single-voxel standardized uptake value (SUVmax). Results: Liver uptake of 64Cu was reduced approximately 75% with the 45-mg trastuzumab predose, without significant effect on tumor uptake. The study included 89 CT-positive lesions. Detection sensitivity was 77%, 89%, and 93% for day 1, day 2, and 18F-FDG, respectively. On average, tumor uptake was similar for 64Cu-DOTA-trastuzumab and 18F-FDG (SUVmax and range, 8.1 and 3.0–22.5 for day 1 [n = 48]; 8.9 and 0.9–28.9 for day 2 [n = 38]; 9.7 and 3.3–25.4 for 18F-FDG [n = 56]), but same-lesion SUVmax was not correlated between the 2 radiotracers. No toxicities were observed, and estimated radiation dose from 64Cu-DOTA-trastuzumab was similar to 18F-FDG. Conclusion:64Cu-DOTA-trastuzumab visualizes HER2-positive metastatic breast cancer with high sensitivity and is effective in surveying disseminated disease. A 45-mg trastuzumab predose provides a 64Cu-DOTA-trastuzumab biodistribution favorable for tumor imaging. 64Cu-DOTA-trastuzumab PET/CT warrants further evaluation for assessing tumor HER2 expression and individualizing treatments that include trastuzumab.

TPS11121 Background: We have developed 64Cu-DOTA-trastuzumab for PET imaging of HER2-positive breast cancer. We have determined that administering trastuzumab (45 mg) prior to 64Cu-DOTA-trastuzumab sharply reduces liver uptake of the radiotracer. We are now testing whether tumor uptake of 64Cu-DOTA-trastuzumab correlates with variable IHC staining in women with advanced breast cancer. Methods: Eligibility criteria included biopsy confirmation of metastatic disease that was HER2 1+, 2+, or 3+ by IHC, no anti-HER therapy within the prior 4 mo, and at least 1 non-hepatic site of metastasis > 20 mm outside the biopsy site. Staging workup included 18F-FDG PET-CT. Patients received 45 mg of cold trastuzumab prior to 64Cu-DOTA-trastuzumab. PET-CT scans were obtained at 21-25 h (Day 1) and 47-48 h (Day 2) over axial fields of view chosen in reference to 18F-FDG. Uptake in prominent lesions was measured in terms of maximum single-voxel SUV (SUVmax). Lesions identified on CT and judged to have image intensity > adjacent tissue by an expert radiologist were considered positive on PET. Results: Fourteen women (median age 56, range 35-75 y) have undergone imaging. HER2 status by IHC was 3+ in 9 pts., 2+ in 5 and 1+ in 1. Two women with IHC 2+ disease were FISH+. In the patients considered clinically HER2 positive (IHC 3+ or 2+, FISH+), 64Cu-DOTA-trastuzumab sensitivity was 75 and 90%, respectively, on Days 1 and 2, compared with 94% for 18F-FDG. Tumor uptake of 64Cu-DOTA-trastuzumab was also readily visualized in HER2-negative patients (measured detection sensitivity 90%). There were no false positive findings with 64Cu-DOTA-trastuzumab. Lesion uptake of 64Cu-DOTA-trastuzumab was higher in HER2+ than in HER2- patients (SUVmaxmean ± sem: Day 1 8.9±0.6 vs 4.3±0.2; Day 2 9.9±0.8 vs 5.4±0.3, p < 0.001). Conclusions:64Cu-DOTA-trastuzumab PET visualizes HER2 1+, 2+ and 3+ metastatic breast cancer with high sensitivity and specificity. Tumor uptake of 64Cu-DOTA-trastuzumab-PET in IHC 1+ and 2+ patients implies possible benefit from anti-HER2 therapies for individuals whose cancers are currently considered HER2 negative. Research Support: DOD1024511. Clinical trial information: NCT01093612.

Cancer radiotherapy may be immunogenic, but it is unclear why its immunogenic effects are rarely sufficient to prevent tumor recurrence. Here, we report a novel Toll-like receptor 9 (TLR9)-dependent mechanism that initiates tumor regrowth after local radiotherapy. Systemic inhibition of TLR9, but not TLR4, delayed tumor recurrence in mouse models of B16 melanoma, MB49 bladder cancer, and CT26 colon cancer after localized high-dose tumor irradiation. Soluble factors in the microenvironment of regressing tumors triggered TLR9 signaling in freshly recruited myeloid cells appearing within four days of radiotherapy. The tumorigenic effects of TLR9 depended on MyD88/NF-κB-mediated upregulation of interleukin (IL)-6 expression, which in turn resulted in downstream activation of Jak/STAT3 signaling in myeloid cells. In comparing global gene expression in wild-type, TLR9-, or STAT3-deficient myeloid cells derived from irradiated tumors, we identified a unique set of TLR9/STAT3-regulated genes involved in tumor-promoting inflammation and revascularization. Blocking STAT3 function by two myeloid-specific genetic strategies corrected TLR9-mediated cancer recurrence after radiotherapy. Our results suggest that combining localized tumor irradiation with myeloid cell-specific inhibition of TLR9/STAT3 signaling may help eliminate radioresistant cancers.

Abstract Background: We have utilized 64Cu-DOTA-trastuzumab with PET imaging to assess the in vivo expression of HER2 in women with advanced breast cancer. We have demonstrated that a preadministered dose of trastuzumab 45 mg prior to injection of 64Cu-DOTA-trastuzumab resulted in a 75% decrease in the hepatic uptake of Cu-64, resulting in improved image quality. Methods: Patients with biopsy confirmation of recurrent disease located outside the breast and axilla considered for study. Complete staging workup included CT of the chest, abdomen, and pelvis, bone scintigraphy and 18F FDG PET. At least 1 non-hepatic site of metastasis that was > 2 cm separate from the biopsy site was also required. HER2 status was assessed by both IHC and FISH. Index lesions were identified on CT imaging. After the first two patients, all patients received a cold dose of 45mg of trastuzumab immediately prior to 64Cu-DOTA-trastuzumab to decrease liver uptake. 64Cu-DOTA-trastuzumab PET imaging was performed at 24 and 48 hours. Uptake on 64Cu-DOTA-trastuzumab was correlated with HER2 status by IHC and FISH. Results: Fifteen women have undergone 64Cu-DOTA-trastuzumab PET imaging and quantitative image analysis. 10 patients were HER2+ (7 IHC 3+, 3 IHC 2+/FISH+), 3 patients were IHC 2+/FISH-, and 2 patients were IHC 1+. Tumor uptake by 64Cu-DOTA-trastuzumab PET max SUV was higher in HER2+ positive than HER2- patients (1.9-fold higher on day1, p<0.02, and 1.7-fold higher on day2, p<0.05). However, the lowest max SUV was in a HER2+ patient (HER2 2+/FISH+), demonstrating considerable heterogeneity. Conclusion: 64Cu-DOTA-trastuzumab PET correlates with HER2+ status. However, due to high within and between patient variability, 64Cu-DOTA-trastuzumab PET imaging could potentially enrich for HER2+ patients that respond to HER2-targeted therapy, and could also suggest some HER2- patients that may benefit from HER2-targeted therapy. This hypothesis needs to be further explored in patients undergoing HER2-targeted therapy. This work was supported by the Department of Defense grant # BC095002. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-03-05.