Abstract The rapidly advancing field of cancer immunotherapy is currently limited by the scarcity noninvasive and quantitative technologies capable monitoring presence abundance CD8+ T cells other immune cell subsets. In this study, we describe generation 89Zr-desferrioxamine–labeled anti-CD8 cys-diabody (89Zr-malDFO-169 cDb) for immuno-PET tracking endogenous cells. We demonstrate that a sensitive tool detecting changes in systemic tumor-infiltrating CD8 expression preclinical syngeneic...

Significance Anti-CD8 immuno-PET imaging agents provide the potential to monitor localization, migration, and expansion of CD8-expressing cells noninvasively in vivo. Shown here is successful generation functional anti-CD8 based on engineered antibodies for use a variety preclinical disease immunotherapeutic models.

The proliferation and trafficking of T lymphocytes in immune responses are crucial events determining inflammatory responses. To study whole-body lymphocyte dynamics noninvasively vivo, we generated anti-CD4 -CD8 cys-diabodies (cDbs) derived from the parental antibody hybridomas GK1.5 2.43, respectively, for (89)Zr-immuno-PET detection helper cytotoxic cell populations.Anti-CD4 cDbs were engineered, produced via mammalian expression, purified using immobilized metal affinity chromatography,...

Inflammatory bowel diseases (IBDs) in humans are characterized part by aberrant CD4-positive (CD4+) T-cell responses. Currently, identification of foci inflammation within the gut requires invasive procedures such as colonoscopy and biopsy. Molecular imaging with antibody fragment probes could be used to noninvasively monitor cell subsets causing intestinal inflammation. Here, GK1.5 cys-diabody (cDb), an antimouse CD4 derived from hybridoma, was a PET probe for CD4+ T cells dextran sulfate...

The therapeutic efficacy of recombinant antibodies such as single-chain Fv fragments and small bispecific or bifunctional molecules is often limited by rapid elimination from the circulation because their size. Here, we have investigated effects N-glycosylation on activity pharmacokinetics a diabody (scDb CEACD3) developed for retargeting cytotoxic T cells to CEA-expressing tumor cells. We could show that introduction sequons into flanking linker C-terminal extension results in production...

Fusion of small recombinant antibody fragments to an albumin-binding domain (ABD) from streptococcal protein G strongly extends their plasma half-life. This ABD binds with nanomolar affinity human (HSA) and mouse serum albumin (MSA). It was speculated that increase in should lead a further In the present study, we analyzed effects valency on pharmacokinetic properties bispecific single-chain diabody (scDb), applied previously investigate various half-life extension strategies. The scDb is...

Prostate stem cell antigen (PSCA) is expressed on the surface in 83%–100% of local prostate cancers and 87%–100% cancer bone metastases. In this study, we sought to develop immunoPET agents using ¹²⁴I- ⁸⁹Zr-labeled anti-PSCA A11 minibodies (scFv-C_H3 dimer, 80 kDa) evaluate their use for quantitative imaging cancer. <b>Methods:</b> minibody was alternatively labeled with or ⁸⁹Zr-desferrioxamine injected into mice bearing either matched 22Rv1...

Tumor necrosis factor (TNF) signals through two membrane receptors, TNFR1 and TNFR2, is known to be the major pathogenic mediator of chronic acute inflammatory diseases. Present clinical intervention based on neutralization ligand TNF. Selective inhibition TNF receptor 1 (TNFR1) provides an alternative opportunity neutralize pro-inflammatory activity while maintaining advantageous immunological responses mediated by including immune regulation, tissue homeostasis neuroprotection. We recently...

Purpose: The B-cell antigen CD20 provides a target for antibody-based positron emission tomography (immunoPET). We engineered antibody fragments targeting human and studied their potential as immunoPET tracers in transgenic mice (huCD20TM) murine lymphoma model expressing CD20.Experimental Design: Anti-CD20 cys-diabody (cDb) cys-minibody (cMb) based on rituximab obinutuzumab (GA101) were radioiodinated used imaging of model. Pairwise comparison obinutuzumab-based labeled with residualizing...

Abstract Purpose: The inability to visualize cancer during prostatectomy contributes positive margins, recurrence, and surgical side effects. A molecularly targeted fluorescent probe offers the potential for real-time intraoperative imaging. goal of this study was develop a image-guided prostate surgery. Experimental Design: An antibody fragment (cys-diabody, cDb) against stem cell antigen (PSCA) conjugated far-red fluorophore, Cy5. integrity binding PSCA confirmed by gel electrophoresis,...

Pancreatic cancer has a high mortality rate due to late diagnosis and the tendency invade surrounding tissues metastasize at an early stage. A molecular imaging agent that enables both presurgery antigen-specific PET (immuno-PET) intraoperative near-infrared fluorescence (NIRF) guidance might benefit of pancreatic cancer, staging, surgical resection, which remains only curative treatment. <b>Methods:</b> We developed dual-labeled probe based on A2 cys-diabody (A2cDb) targeting cell-surface...

Inadequate diagnostic methods for prostate cancer lead to over-and undertreatment, and the inability intraoperatively visualize positive margins may limit success of surgical resection.Prostate visualization could be improved by combining complementary modalities immuno-positron emission tomography (immunoPET) preoperative disease detection, fluorescence imaging-guided surgery (FIGS) real-time intraoperative tumor margin identification.Here, we report on evaluation dual-labeled humanized...

Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence visualization of superficial tissue layers image-guided surgery. <b>Methods:</b> We developed a universal linker (DML) that facilitates site-specific conjugation to cysteine residue–bearing antibody fragment, introduction commercially available fluorescent dye (via an amine-reactive prosthetic group), rapid efficient...

Epithelial membrane protein-2 (EMP2) is upregulated in a number of tumors and therefore remains promising target for mAb-based therapy. In the current study, image-guided therapy an anti-EMP2 mAb was evaluated by PET both syngeneic immunodeficient cancer models expressing different levels EMP2 to enable better understanding its tumor uptake off accumulation clearance. The therapeutic efficacy initially high- low-expressing tumors, reduced load high EMP2-expressing 4T1 HEC-1-A tumors. To...

Activated leukocyte cell adhesion molecule (ALCAM) is an immunoglobulin superfamily that aberrantly expressed in a wide variety of human tumors, including melanoma, prostate cancer, breast colorectal carcinoma, bladder cancer and pancreatic adenocarcinoma. This spectrum malignancies makes ALCAM prospective pan-cancer immunoPET target to aid detection diagnosis multiple malignancies. In this study, we assess site-specific versus non-site-specific conjugation strategies for (64)Cu-DOTA...

The inability to intraoperatively distinguish primary tumor, as well lymphatic spread, increases the probability of positive surgical margins, tumor recurrence, and toxicity. goal this study was develop a tumor-specific optical probe for real-time fluorescence-guided surgery.A humanized antibody fragment against PSCA (A11 minibody, A11 Mb) conjugated with near-infrared fluorophore, IRDye800CW. integrity binding were confirmed by gel electrophoresis, size-exclusion chromatography, flow...

Abstract Purpose: Prostate stem cell antigen (PSCA) is highly expressed in local prostate cancers and cancer bone metastases its expression correlates with androgen receptor activation a poor prognosis. In this study, we investigate the potential clinical applications of immunoPET anti-PSCA A11 minibody, an antibody fragment optimized for use as imaging agent. We compare minibody to 18F-Fluoride PET scans detecting tumors evaluate ability image tumor response deprivation. Experimental...

Abstract MET, the receptor of hepatocyte growth factor, plays important roles in tumorigenesis and drug resistance numerous cancers, including non–small cell lung cancer (NSCLC). As increasing numbers MET inhibitors are being developed for clinical applications, antibody fragment–based immunopositron emission tomography (immunoPET) has potential to rapidly quantify vivo expression levels response evaluation patient stratification these targeted therapies. Here, fully human single-chain...

Phage display libraries of human single-chain variable fragments (scFvs) are a reliable source fully antibodies for scientific and clinical applications. Frequently, scFvs form the basis larger, bivalent formats to increase valency avidity. A small versatile antibody fragment is diabody, cross-paired scFv dimer (∼55 kDa). However, generation diabodies from selected requires decreasing length interdomain linker, typically by overlap PCR. To simplify this process, we designed two linkers with...

Abstract Introduction: Human prostate stem cell antigen (hPSCA) is a surface protein that overexpressed in pancreatic cancer but minimally expressed normal tissues, making it ideal as therapeutic marker for targeted radiopharmaceutical therapy (TRT). Humanized anti-PSCA A2 scFv-Fc2 antibody was engineered with double mutation (A2DM, t1/2 12 h) the FcRn binding site rapid clearance through liver and reduced bone marrow toxicity to improve index. The potential of A2DM investigated imaging...

The 22nd Annual Antibody Engineering and 9th Therapeutics international conferences, the 2011 Meeting of Society, organized by IBC Life Sciences with contributions from Society two Scientific Advisory Boards, were held December 5–8, in San Diego, CA. meeting drew ~800 participants who attended sessions on a wide variety topics relevant to antibody research development. As preview main events, pre-conference workshop 4, focused antibodies as probes structure. Conference comprised eight...

The 23rd Annual Antibody Engineering, 10th Therapeutics international conferences, and the 2012 Meeting of Society, organized by IBC Life Sciences with contributions from Society two Scientific Advisory Boards, were held December 3–6, in San Diego, CA. meeting drew over 800 participants who attended sessions on a wide variety topics relevant to antibody research development. As prelude main events, pre-conference workshop 2, focused intellectual property issues that impact engineering....