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Supplementary data Table S1. Ex vivo biodistribution (24 h p.i.) of antibody fragments in SCID mice bearing 38C13-huCD20, blocked or CD20-negative 38C13 subcutaneous allografts. Data are presented as mean (%ID/g) with standard deviation ({plus minus} SEM). Table S2. Ex vivo biodistribution (24 h p.i.) of GA101 cys-diabody and cys-minibody fragments in human CD20 transgenic mice (huCD20TM). Data are presented as mean (%ID/g) with standard deviation ({plus minus} SEM). Figure S1 Biochemical characterization. (A) SDS-PAGE analysis of purified rituximab-based cys-diabody (RxcDb, 52 kDa) and cys-minibody (RxcMb, 82 kDa) shows correctly assembled dimers under non-reducing conditions (NR) and monomeric fragments under reducing conditions. (B) Size exclusion chromatography of engineered antibody fragments in comparison to full-length rituximab IgG. Rx cMb eluted as a single peak corresponding to the expected molecular weight. Rx cDb eluted slightly later, corresponding to the 52 kDa dimer but also showed a smaller second peak of higher molecular weight. Figure S2. (A) Schematic representation of site-specific conjugation to the C-terminal cys-tag of cys-diabody and cys-minibody antibody fragments, respectively. (B) Reduction and conjugation of Alexa Fluor 488 Maleimide (using the example of the cys-diabody) results in monomeric fragments migrating under non-reducing conditions in SDS-PAGE shown as Coomassie stain (left panel) and fluorescence (right panel). (C) Size exclusion chromatography analysis comparing unconjugated (black) and mal-DFo conjugated (grey) antibody fragments (using Superdex 75 column for cys-diabodies and Superdex 200 column for cys-minibodies. Figure S3. Obinutuzumab (GA101)-based antibody fragments retain type II binding characteristics. Figure S4. Immunoreactive fractions of radiolabeled antibody fragments were determined by cell binding studies using CD20 positive Ramos cells and CD20 negative Bc-1 cells. Shown is one representative plot for 124I-GAcDb. Immunoreactive fractions for obinutuzumab (GA1010)-based fragments ranged from 65-85 %. Figure S5 89Zr-GAcMb immunoPET imaging of huCD20TM. Figure S6 Immunohistochemical staining of human CD20 expression in murine 38C13 B-cell lymphoma allografts.

Purpose: The inability to visualize cancer during prostatectomy contributes to positive margins, cancer recurrence, and surgical side effects. A molecularly targeted fluorescent probe offers the potential for real-time intraoperative imaging. The goal of this study was to develop a probe for image-guided prostate cancer surgery.Experimental Design: An antibody fragment (cys-diabody, cDb) against prostate stem cell antigen (PSCA) was conjugated to a far-red fluorophore, Cy5. The integrity and binding of the probe to PSCA was confirmed by gel electrophoresis, size exclusion, and flow cytometry, respectively. Subcutaneous models of PSCA-expressing xenografts were used to assess the biodistribution and in vivo kinetics, whereas an invasive intramuscular model was utilized to explore the performance of Cy5-cDb–mediated fluorescence guidance in representative surgical scenarios. Finally, a prospective, randomized study comparing surgical resection with and without fluorescent guidance was performed to determine whether this probe could reduce the incidence of positive margins.Results: Cy5-cDb demonstrated excellent purity, stability, and specific binding to PSCA. In vivo imaging showed maximal signal-to-background ratios at 6 hours. In mice carrying PSCA+ and negative (−) dual xenografts, the mean fluorescence ratio of PSCA+/− tumors was 4.4:1. In surgical resection experiments, residual tumors Conclusions: Fluorescently labeled cDb enables real-time in vivo imaging of prostate cancer xenografts in mice, and facilitates more complete tumor removal than conventional white light surgery alone. Clin Cancer Res; 22(6); 1403–12. ©2015 AACR.See related commentary by van Leeuwen and van der Poel, p. 1304

Supplemental Figure 1: Transfection of 22rv1 with FLuc-IRES-mCherry and 22rv1_PSCA with Fluc-IRES-GFP and FACS sorting created FLuc expressing PSCA positive and negative cell lines. Supplemental Figure 2: H&E Staining of 22rv1 and 22rv1_PSCA intratibial xenografts shows mixed osteolytic (blue dashed arrows) and osteoblastic (solid red arrows) type tumor formation. Supplemental Figure 3: Comparison of optical imaging, 18F-Fluoride Bone Scan, and A11 immunoPET in mice bearing 22rv1 intratibial xenograft 6 weeks post-xenograftimplantation. Supplemental Figure 4: Comparison of optical imaging, 18F-Fluoride Bone Scan, and A11 immunoPET in a mouse bearing a 22rv1_PSCA intratibial xenograft at 2 and 6 weeks postxenograft- implantation. Supplemental Figure 5: Quantification of tumor necrosis using ImageJ based color Segmentation. Supplemental Figure 6: H&E staining of LAPC-9 tumors treated with either MDV-3100 or vehicle control. Supplemental Table 1: 44 hour biodistribution of mice bearing 22rv1 and 22rv1_PSCA intratibial xenografts. Histologic analysis of control and MDV-3100 treated tumors.

Figure S1. Biochemical characterization of Cy5 conjugated anti-PSCA cys-Diabody. Figure S2. Validation of in vivo tumor detection using endogenously PSCA-expressing line LAPC-9 Figure S3. Determining the optimal time point for in vivo Cy5-cDb imaging. Figure S4. Cy5-cDb enabled fluorescence-guided surgical resection of PSCA-expressing tumors. Figure S5. LAPC-9 intramuscular tumor extent was not readily detectable under white light (A-B), but was clearly visible under fluorescence guidance (C). Figure S6. Intraoperative white light and florescent images of 22Rv1 intramuscular xenografts utilizing anti-PSCA Cy5-cDb. Figure S7. Additional H&E histopathology of tumor margins from the prospective, randomized trial.

Purpose: The B-cell antigen CD20 provides a target for antibody-based positron emission tomography (immunoPET). We engineered antibody fragments targeting human CD20 and studied their potential as immunoPET tracers in transgenic mice (huCD20TM) and in a murine lymphoma model expressing human CD20.Experimental Design: Anti-CD20 cys-diabody (cDb) and cys-minibody (cMb) based on rituximab and obinutuzumab (GA101) were radioiodinated and used for immunoPET imaging of a murine lymphoma model. Pairwise comparison of obinutuzumab-based antibody fragments labeled with residualizing (89Zr) versus non-residualizing (124I) radionuclides by region of interest analysis of serial PET images was conducted both in the murine lymphoma model and in huCD20TM to assess antigen modulation in vivo.Results:124I-GAcDb and 124I-GAcMb produced high-contrast immunoPET images of B-cell lymphoma and outperformed the respective rituximab-based tracers. ImmunoPET imaging of huCD20TM showed specific uptake in lymphoid tissues. The use of the radiometal 89Zr as alternative label for GAcDb and GAcMb yielded greater target-specific uptake and retention compared with 124I-labeled tracers. Pairwise comparison of 89Zr- and 124I-labeled GAcDb and GAcMb allowed assessment of in vivo internalization of CD20/antibody complexes and revealed that CD20 internalization differs between malignant and endogenous B cells.Conclusions: These obinutuzumab-based PET tracers have the ability to noninvasively and quantitatively monitor CD20-expression and have revealed insights into CD20 internalization upon antibody binding in vivo. Because they are based on a humanized mAb they have the potential for direct clinical translation and could improve patient selection for targeted therapy, dosimetry prior to radioimmunotherapy, and prediction of response to therapy. Clin Cancer Res; 23(23); 7242–52. ©2017 AACR.

Regulatory T cells (Tregs) play an important role in maintaining immune homeostasis by preventing excessive inflammation in normal tissues. In cancer, Tregs hamper antitumor immunosurveillance and may be a resistance mechanism to anti-PD-1 therapies. In preclinical cancer models, Treg depletion enhances antitumor cytotoxic immune responses, but systemic and persistent Treg removal can elicit immunopathology. Therapeutic strategies that selectively deplete highly suppressive activated intratumoral Tregs may avoid immune-related toxicities and amplify the activity of antitumor CD8 T cells during PD-1 blockade. CD30 is a member of the TNF receptor superfamily and is expressed by a subset of activated lymphocytes and various lymphomas. Here, transcriptomics and flow cytometry data from tumor and peripherally derived Tregs demonstrated CD30 is enriched on intratumoral Tregs. Among peripheral Tregs, CD30 expression was associated with an activated effector phenotype (FoxP3hiCD45RA-, fraction II). Further, treatment of dissociated NSCLC tumor spheroids with anti-PD-1 resulted in upregulated CD30 expression by Tregs but not intratumoral CD4 or CD8 T cells, which is consistent with Treg reactivation and supports CD30 as an activated Treg target. In vitro, CD30 expression on Tregs was dependent on signaling through the TCR and was significantly amplified by IL-2 and IL-15, suggesting CD30 may identify a subpopulation of activated antigen-specific Tregs. Brentuximab vedotin (BV) is an antibody-drug conjugate approved for clinical use in multiple types of lymphomas including cHL and PTCL. BV consists of a CD30-directed monoclonal antibody conjugated to the highly potent microtubule-disrupting agent monomethyl auristatin E (MMAE). The activity of BV in lymphomas is thought to primarily result from tumor-directed intracellular MMAE release leading to apoptosis, though multiple immunomodulatory effects of BV have also been suggested. We have demonstrated through in vitro and in vivo preclinical model systems of human T cell activation that BV can selectively deplete CD30-expressing Tregs, amplifying cytotoxic CD8 T cell expansion. These and Treg CD30 expression data motivated the clinical exploration of BV plus pembrolizumab in metastatic PD-1 refractory melanoma and NSCLC (NCT04609566). Consistent with preclinical data, preliminary biomarker analysis from these studies showed a small but significant decrease in Tregs (CD4+CD25+CD127low/-) in peripheral blood after treatment with BV plus pembrolizumab. Further, limited on-treatment biopsies showed a trend toward increased intratumoral CD8:Treg (FoxP3) ratio in some patients receiving combination therapy. These data support the ongoing evaluation of BV in combination with PD-1 inhibitors in solid tumors and a potential additional mechanism of action for BV. Citation Format: Ryan A. Heiser, Bryan M. Grogan, Reice D. James, Michelle L. Ulrich, Jason D. Berndt, Brian P. O'Connor, Shyra J. Gardai, Hailing Lu, Scott M. Knowles. CD30 is a marker of activated effector regulatory T cells in solid tumors providing clinical rationale for the combination of brentuximab vedotin and PD-1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3253.

Background: Racial inequities for patients with heart failure (HF) have been widely documented. HF patients who receive cardiology care during a hospital admission have better outcomes. It is unknown whether there are differences in admission to a cardiology or general medicine service by race. This study examined the relationship between race and admission service, and its effect on 30-day readmission and mortality Methods: We performed a retrospective cohort study from September 2008 to November 2017 at a single large urban academic referral center of all patients self-referred to the emergency department and admitted to either the cardiology or general medicine service with a principal diagnosis of HF, who self-identified as white, black, or Latinx. We used multivariable generalized estimating equation models to assess the relationship between race and admission to the cardiology service. We used Cox regression to assess the association between race, admission service, and 30-day readmission and mortality. Results: Among 1967 unique patients (66.7% white, 23.6% black, and 9.7% Latinx), black and Latinx patients had lower rates of admission to the cardiology service than white patients (adjusted rate ratio, 0.91; 95% CI, 0.84–0.98, for black; adjusted rate ratio, 0.83; 95% CI, 0.72–0.97 for Latinx). Female sex and age >75 years were also independently associated with lower rates of admission to the cardiology service. Admission to the cardiology service was independently associated with decreased readmission within 30 days, independent of race. Conclusions: Black and Latinx patients were less likely to be admitted to cardiology for HF care. This inequity may, in part, drive racial inequities in HF outcomes.