Low-dose targeted radionuclide therapy synergizes with CAR T cells and enhances tumor response

Ionizing radiation has garnered considerable attention as a combination partner for immunotherapy due to its potential immunostimulatory effects. In contrast to the more commonly used external beam radiation, we explored the feasibility of combining chimeric antigen receptor (CAR) T cell therapy with targeted radionuclide therapy (TRT), which is achieved by delivering β-emitting 177Lu-DOTATATE to tumor via tumor-infiltrating CAR T cells that express somatostatin receptor 2 (SSTR2). We hypothesized that the delivery of radiation to tumors could synergize with CAR T therapy, resulting in enhanced antitumor immunity and tumor response. To determine the optimal dosage and timing of 177Lu-DOTATATE treatment, we measured CAR T cell infiltration and expansion in tumors longitudinally through positron emission tomography (PET) using a SSTR2-specific positron-emitting radiotracer,18F-NOTA-Octreotide. In animals receiving CAR T cells and a low–dose (2.5 Gy) of TRT following the administration of 177Lu-DOTATATE, we observed a rapid regression of large subcutaneous tumors, which coincided with a dramatic increase in serum proinflammatory cytokines. Tumor burden was also reduced when a higher radiation dose (6 Gy) was delivered to the tumor. However, this higher dose led to cell death in both the tumor and CAR T cells. Our study suggests that there may exist an optimum range of TRT dosage that can enhance T cell activity and sensitize tumor cells to T cell killing, which may result in more durable tumor control compared to a higher radiation dose

Novel Strategy for Selection of Monoclonal Antibodies Against Highly Conserved Antigens: Phage Library Panning Against Ephrin-B2 Displayed on Yeast

Ephrin-B2 is predominately expressed in endothelium of arterial origin, involved in developmental angiogenesis and neovasculature formation through its interaction with EphB4. Despite its importance in physiology and pathological conditions, it has been challenging to produce monoclonal antibodies against ephrin-B2 due to its high conservation in sequence throughout human and rodents. Using a novel approach for antibody selection by panning a phage library of human antibody against antigens displayed in yeast, we have isolated high affinity antibodies against ephrin-B2. The function of one high affinity binder (named as ‘EC8’) was manifested in its ability to inhibit ephrin-B2 interaction with EphB4, to cross-react with murine ephrin-B2, and to induce internalization into ephrin-B2 expressing cells. EC8 was also compatible with immunoprecipitation and detection of ephrin-B2 expression in the tissue after standard chemical fixation procedure. Consistent with previous reports on ephrin-B2 induction in some epithelial tumors and tumor-associated vasculatures, EC8 specifically detected ephrin-B2 in tumors as well as the vasculature within and outside of the tumors. We envision that monoclonal antibody developed in this study may be used as a reagent to probe ephrin-B2 distribution in normal as well as in pathological conditions and to antagonize ephrin-B2 interaction with EphB4 for basic science and therapeuti

Detection of ephrin-B2 expression in human tissue arrays.

<p>(A&B) Immunostaining of ephrin-B2 expression in human tumor tissue arrays using EC8. Control denotes immunostaining without EC8 as a primary antibody. Tumor and stromal cells were indicated with arrowhead and arrow, respectively. Scale bar = 20 µm. PAC = Papillary Adenocarcinoma; AC = Adenocarcinoma; BR = Bronchus; AL = Alveoli; SC = Squamous Cell Carcinoma; SAC = Serous Adenocarcinoma. IDC = Nonspecific Infiltrating Duct Carcinoma. (C) Immunofluorescence staining on human colon tumor tissue demonstrating that EC8 (red) detects ephrin-B2 expressions in both cancer cells and tumor-associated vasculature (green). Blow up views of the two areas indicated with dashed box are shown in the right panel. Scale bar = 100 µm.</p

Selection, validation, and sequence of ephrin-B2-specific human single-chain antibodies.

<p>(A) A schematic diagram of phage panning against antigens expressed in yeast display system <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030680#pone.0030680-Boder1" target="_blank">[34]</a>. (B) Immunofluorescence flow cytometry measurements of protein and phage binding to yeast cells. Surface-displayed ephrin-B2 was detected by the binding of anti-Myc antibody (‘Myc’) as well as recombinant human EphB4-Fc (‘EphB4’) to yeast cells (top panel). Progressive enrichment of phage clones from first three rounds of panning (denoted as ‘1 st’, ‘2 nd’ and ‘3 rd’) was detected by antibody against His tag (bottom panel). Histograms drawn in shaded area and solid lines indicate antibody binding to uninduced and induced yeast cells, respectively. The percentage of phage clones with positive binding is indicated. (C) SDS-PAGE of scFv-EB1 (lane ‘1’) and scFv-EC8 (lane ‘2’). (D) Ephrin-B2 specific scFv binding to irrelevant yeast cells, yeast cells with expression of ephrin-B2 ectodomain, 293T cells, and 293T cells with transient expression of full-length ephrin-B2. Shown are the histograms of cells labeled with secondary antibody with (solid line) and without (shaded area) ephrin-B2 specific scFv as primary antibody. (E) Sequence alignment of scFv-EA6, scFv-EB1, and scFv-EC8. Complementarity determining regions (CDR), the beginning of immunoglobulin variable heavy (VH) and variable light (VL) chain domains, and the linker connecting VH and VL are noted. <b>*</b> indicates amino acids differ between scFvs.</p

Detection of ephrin-B2 expression in human cancer cell lines and tumor xenograft in mice.

<p>(A) Flow cytometry measurements of EC8 binding to COLO205 and HCT116 cells (solid line) in comparison to the isotype control (shaded area). CHO cells with no ephrin-B2 expression were also included for comparison. (B) RT-PCR detection of ephrin-B2 expression in different cell lines. (C) Immunostaining of ephrin-B2 on human colon cancer xenografted in mice. Control denotes immunostaining without EC8 as a primary antibody. Tumor and stromal cells were indicated with arrowhead and arrow, respectively. Circle indicates murine endothelium stained with EC8. Scale bar = 20 µm.</p

Supplementary Material for: Comprehensive, Individualized, Person-Centered Management of Community-Residing Persons with Moderate-to-Severe Alzheimer Disease: A Randomized Controlled Trial

<p><b><i>Background/Aims: </i></b>The aim was to examine added benefits of a Comprehensive, Individualized, Person-Centered Management (CI-PCM) program to memantine treatment. <b><i>Methods: </i></b>This was a 28-week, clinician-blinded, randomized, controlled, parallel-group study, with a similar study population, similar eligibility criteria, and a similar design to the memantine pivotal trial of Reisberg et al. [N Engl J Med 2003;348:1333-1341]. Twenty eligible community-residing Alzheimer disease (AD) subject-caregiver dyads were randomized to the CI-PCM program (n = 10) or to usual community care (n = 10). Primary outcomes were the New York University Clinician's Interview-Based Impression of Change Plus Caregiver Input (NYU-CIBIC-Plus), assessed by one clinician set, and an activities of daily living inventory, assessed by a separate clinician set at baseline and at weeks 4, 12, and 28. <b><i>Results: </i></b>Primary outcomes showed significant benefits of the CI-PCM program at all post-baseline evaluations. Improvement on the NYU-CIBIC-Plus in the management group at 28 weeks was 2.9 points over the comparator group. The memantine 2003 trial showed an improvement of 0.3 points on this global measure in memantine-treated versus placebo-randomized subjects at 28 weeks. Hence, globally, the management program intervention benefits were 967% greater than memantine treatment alone. <b><i>Conclusion: </i></b>These results are approximately 10 times those usually observed with both nonpharmacological and pharmacological treatments and indicate substantial benefits with the management program for advanced AD persons.</p