BACKGROUND:Dogs spontaneously develop prostate carcinoma (PC) and share prostate gland anatomy, physiology, and size to men. Over the last 15 years, we have developed and refined a canine model of focal PC to evaluate therapeutic-diagnostic (theranostic) interventions. A comprehensive description of the pathology and synthesis of the various studies has not been performed. The goal of this manuscript was to describe the canine model tumor pathology within the framework of its methodological development to help guide future translational PC research. METHODS:In published and unpublished studies, we previously inoculated prostate glands of immunosuppressed, intact beagle dogs (n = 56) with a canine PC cell line (Ace-1) transduced with human or canine genes for targeted theranostics. Gross tumor assessment and histology were performed in all cases. Molecular tumor and microenvironmental pathology was investigated using digital image analysis, immunohistochemistry, laser-capture microdissection, and quantitative real-time PCR. RESULTS:The model reliably (85.7% engraftment rate) formed prostatic tumors resembling intermediate and high-grade localized PC, with poorly differentiated morphology, stromal invasion, and peripheral growth. Soft tissue metastasis occurred in 13/48 (27.1%) dogs. Most dogs formed multifocal prostatic tumors with occasional tumors outside the prostate gland. Tumor location influenced growth behavior and the microenvironment. Allografts were histologically classified as intraglandular intraprostatic, invasive intraprostatic, capsular, or extraprostatic. Compared to intraprostatic tumors, capsular/extraprostatic tumors had increased proliferation (Ki-67 index), epithelial-to-mesenchymal transition, and microenvironmental alterations that included increased collagenous stroma, fibroplasia, and reduced immune cell infiltration. CONCLUSIONS:The canine model of PC captured important pathologic features of men undergoing curative-intent therapy alongside model- and species-specific characteristics of interest to researchers. Beyond defining pathology, the results highlighted applications of the canine model in studying the tumor microenvironment and advancing preclinical, anti-cancer strategies in a large animal species.

Cancer patients benefit from significantly higher survival rates if tumors are detected at early stages and prior to metastasis. Positron emission tomography (PET), computed tomography (CT), Single-Photon Emission Computed Tomography (SPECT), and other imaging techniques allow for noninvasive diagnosis of various tumors in relatively short periods. Targeted delivery of radiation is also an important approach in cancer therapy. Due to the dynamic nature of RNA, RNA nanoparticles demonstrate spontaneous tumor targeting, resulting in rapid accumulation in tumors without the use of targeting ligands. Incorporating tumor-targeting ligands on RNA nanoparticles generates enhanced tumor accumulation and targeting. Here a unique technology to specifically label three-way junction (3WJ) RNA nanoparticles is reported to carry radioisotopes or other imaging markers for imaging. Two RNA nanoparticles were constructed to target prostate specific membrane antigen (PSMA) via a PSMA RNA aptamer or conjugated tert-Butyl-DCL (DCL). The spontaneous cancer homing resulted in the detection of tumors with high sensitivity in mouse models, which can be applied to any cancer subtype at an early stage. Tumor accumulation occurred due to the motile and deformable nature of RNA nanoparticles, allowing for passage of the high concentration of leaky vasculature in the tumor environment. Furthermore, RNA nanoparticles conjugated with a NOTA radioisotope chelator were incubated with 68Ga in a pH- and temperature-controlled environment to prevent 68Ga non-specific interactions with the negatively charged phosphodiester backbone of RNA. The low pH during 68Ga3+ conjugation neutralized the negative charge of the phosphate backbone on the RNA, ensuring only specific radioisotope chelation to NOTA. To prove the concept of the proposed system, 68Ga-labeled 3WJ was tested in a prostate cancer animal model by PET/CT. The 68Ga-SF5 3WJ accumulated in and identified prostate cancer tumors with high sensitivity, resolution, specificity, and reliability. The proof-of-concept study reported in this paper is an important step in the direction of developing novel radiotherapeutic agents for various cancers. The radioisotope- or fluorophore-labelled nanoparticles were excreted from the body quickly, thus reducing the chance of toxicity and side effects. This molecular imaging platform, based on RNA nanoparticles, shows great promise in early diagnosis, staging, and precise treatment of any tumor subtype.

Chronic lymphocytic leukemia (CLL) is a disease characterized by the accumulation of mature CD19+CD5+CD23+ B cells in the bloodstream and in lymphoid organs. It usually affects people over 70 years of age, which limits the options for treatments. The disease is typically well-managed, but to date is still incurable. Hence, the need for novel therapeutic strategies remains. Nurse-like cells (NLCs) are major components of the microenvironment for CLL, supporting tumor cell survival, proliferation, and even drug resistance. They are of myeloid lineage, guided toward differentiating into their tumor-supportive role by the CLL cells themselves. As such, they are analogous to tumor-associated macrophages and represent a major therapeutic target. Previously, it was found that a mushroom extract, Active Hexose-Correlated Compound (AHCC), promoted the death of acute myeloid leukemia cells while preserving normal monocytes. Given these findings, it was asked whether AHCC might have a similar effect on the abnormally differentiated myeloid-lineage NLCs in CLL. CLL-patient PBMCs were treated with AHCC, and it was found that AHCC treatment showed a direct toxic effect against isolated CLL cells. In addition, it significantly reduced the number of tumor-supportive NLCs and altered their phenotype. The effects of AHCC were then tested in the Eµ-TCL1 mouse model of CLL and the MllPTD/WT Flt3ITD/WT model of AML. Results showed that AHCC not only reduced tumor load and increased survival in the CLL and AML models, but it also enhanced antitumor antibody treatment in the CLL model. These results suggest that AHCC has direct and indirect effects against CLL and that it may be of benefit when combined with existing treatments.

Overactivation of immune responses is a hallmark of autoimmune disease pathogenesis. This includes the heightened production of inflammatory cytokines such as Tumor Necrosis Factor α (TNFα), and the secretion of autoantibodies such as isotypes of rheumatoid factor (RF) and anticitrullinated protein antibody (ACPA). Fcγ receptors (FcγR) expressed on the surface of myeloid cells bind Immunoglobulin G (IgG) immune complexes. Recognition of autoantigen-antibody complexes by FcγR induces an inflammatory phenotype that results in tissue damage and further escalation of the inflammatory response. Bromodomain and extra-terminal protein (BET) inhibition is associated with reduced immune responses, making the BET family a potential therapeutic target for autoimmune diseases such as rheumatoid arthritis (RA). In this paper, we examined the BET inhibitor PLX51107 and its effect on regulating FcγR expression and function in RA. PLX51107 significantly downregulated expression of FcγRIIa, FcγRIIb, FcγRIIIa, and the common γ-chain, FcϵR1-γ, in both healthy donor and RA patient monocytes. Consistent with this, PLX51107 treatment attenuated signaling events downstream of FcγR activation. This was accompanied by a significant decrease in phagocytosis and TNFα production. Finally, in a collagen-induced arthritis model, PLX51107-treatment reduced FcγR expression in vivo accompanied by a significant reduction in footpad swelling. These results suggest that BET inhibition is a novel therapeutic approach that requires further exploration as a treatment for patients with RA.

Target-specific biomolecules, monoclonal antibodies (mAb), proteins, and protein fragments are known to have high specificity and affinity for receptors associated with tumors and other pathological conditions. However, the large biomolecules have relatively intermediate to long circulation half-lives (>day) and tumor localization times. Combining superior target specificity of mAbs and high sensitivity and resolution of the PET (Positron Emission Tomography) imaging technique has created a paradigm-shifting imaging modality, ImmunoPET. In addition to metallic PET radionuclides, 124I is an attractive radionuclide for radiolabeling of mAbs as potential immunoPET imaging pharmaceuticals due to its physical properties (decay characteristics and half-life), easy and routine production by cyclotrons, and well-established methodologies for radioiodination. The objective of this report is to provide a comprehensive review of the physical properties of iodine and iodine radionuclides, production processes of 124I, various 124I-labeling methodologies for large biomolecules, mAbs, and the development of 124I-labeled immunoPET imaging pharmaceuticals for various cancer targets in preclinical and clinical environments. A summary of several production processes, including 123Te(d,n)124I, 124Te(d,2n)124I, 121Sb(α,n)124I, 123Sb(α,3n)124I, 123Sb(3He,2n)124I, natSb(α, xn)124I, natSb(3He,n)124I reactions, a detailed overview of the 124Te(p,n)124I reaction (including target selection, preparation, processing, and recovery of 124I), and a fully automated process that can be scaled up for GMP (Good Manufacturing Practices) production of large quantities of 124I is provided. Direct, using inorganic and organic oxidizing agents and enzyme catalysis, and indirect, using prosthetic groups, 124I-labeling techniques have been discussed. Significant research has been conducted, in more than the last two decades, in the development of 124I-labeled immunoPET imaging pharmaceuticals for target-specific cancer detection. Details of preclinical and clinical evaluations of the potential 124I-labeled immunoPET imaging pharmaceuticals are described here.

Several radioisotopes of iodine (123I, 124I, 125I, and 131I) are available for medical use. One of them can be used, depending on the application, for radioiodine labeling of New Chemical Entities (NCEs) and biomolecules (peptides, proteins, protein fragments, monoclonal antibodies, etc.) for the development of novel imaging and therapeutic pharmaceuticals. Direct, using inorganic and organic oxidizing agents and enzyme catalysts, and indirect, using prosthetic groups, radioiodine-labeling methods have been used routinely in the past. In this report, a comprehensive review of the physical properties of various iodine radionuclides, their medical applications, and a summary of various radioiodine labeling reagents and methods for NCEs and biomolecules are provided.

Nuclear medicine was recognized as a potential medical field a long time ago when 131I was used in thyroid cancer patients [...].

Radioiodine labeling of peptides and proteins is routinely performed by using various oxidizing agents such as Chloramine T, Iodobeads, and Iodogen reagent and radioactive iodide (I−), although some other oxidizing agents were also investigated. The main objective of the present study was to develop and test a novel reagent, inorganic monochloramine (NH2Cl), for radioiodine labeling of new chemical entities and biomolecules which is cost-effective, easy to make and handle, and is selective to label amino acids, peptides, and proteins. The data presented in this report demonstrate that the yields of the non-radioactive iodine labeling reactions using monochloramine are >70% for an amino acid (tyrosine) and a cyclic peptide (cyclo Arg-Gly-Asp-d-Tyr-Lys, cRGDyK). No evidence of the formation of N-chloro derivatives in cRGDyK was observed, suggesting that the reagent is selective in iodinating the tyrosine residue in the biomolecules. The method was successfully translated into radioiodine labeling of amino acid, a peptide, and a protein, Bovine Serum Albumin (BSA).

2063 Objectives: The learning objectives are: 1. Introduction of longer distance drone Aircraft Systems (UAS) and its current use in healthcare 2. A review of the fundamental legal and regulatory issues for the use of drones / UAS 3. Needs for efficient longer range distribution of 4. How would a comprehensive approach look like 5. Current developments for a demonstration project in Kenia but designed for general applicability. Methods: As distribution of imaging pharmaceuticals is highly time critical and Unmanned Aircraft Systems (UAS) are now in place even for delivery of non-essential goods to residential locations and we had previously introduced the concept, we now report on the current state of the art for a country wide delivery concept in Kenia that is being considered for the transport of PET radiopharmaceuticals. Zipline is a drone logistic company specializing in longer range deliveries and has established commercial feasibility and reliability of its operations. New concepts of radiopharmaceutical handling as well as dose management at the receiving side have to be part of such a logistic approach and are enabling to utilize this technology. Synergistically, we had previously demonstrated a low dose / table time optimized approach for whole body FDG PET/CT imaging enabling innovative approaches including advanced reconstruction. Results: After our previous introduction of the concept of drone transport of imaging pharmaceuticals, we continued to develop a technical design requirement approach in order to facilitate and optimize the process for preparation and handling of the radioactive tracer doses as well as the associated regulatory as well as organizational requirements. While today’s clinical PET/CT operation typically rely on multiple deliveries per day, long distance drone operation will be most efficient by single delivery and efficient use of the delivered pharmaceuticals. This required also the development and validation of count density oriented PET imaging protocols with higher dose and shorter acquisition early after delivery and lower dose longer acquisitions at later time points. While radiopharmaceuticals have been transported in the air for decades, drone transport has different challenges and opportunities. We have identified and conceptualized solutions which will be reviewed. Conclusions: The need for rapid and efficient transportation even of molecular imaging pharmaceuticals has become more obvious in the re-recent years and even accelerated during the Covid pandemic. In this presentation we introduce the conceptual approaches as well as the current development effort in Kenya to the regional distribution of tracers from a single cyclotron facility as a demonstration project that could be adapted to other environments. Research Support: This work is supported by the Wright Center of Innovation development fund.

Gallium-68 (68Ga) is an important radionuclide due to its successful use in the clinic. Currently, 68Ga is produced and supplied by using germanium-68/Gallium-68 (68Ge/68Ga) generator systems in the preclinical and clinical environments. Until recently, the supply of 68Ge/68Ga generators, specifically the good manufacturing practices (GMP) grade, had a long lead time (up to 18 months). This led researchers to investigate the cyclotron production of 68Ga by using solid and liquid targets. This report summarizes an overview of the chemistry, coordination chemistry, and radiochemistry of Ga and the current status of manufacturing and supply of 68Ga radionuclide.

Validated analytical methods must be used for accurate and precise determination of identity, purity, strength, and quality of any pharmaceutical dose intended for human administration. Consequently, all analytical methods must be validated per USP <1225> and the ICH Q2R1 guidelines to meet the cGMP requirements of the FDA and EMA. A reversed-phase high-performance liquid chromatography analytical method, with <10 min run time, was developed for the analysis of [11C]nicotine. The analytical method was found to be specific, accurate, suitable, robust, precise, and linear for [11C]nicotine, nicotine, and (±) nornicotine in the concentration ranges studied. The LOD and LOQ values were also established.

1097 Objectives: The objective of the present work was (1) to measure and compare in vitro stability of 68Ga-labeled PSMA targeting conjugates of acyclic and macrocyclic chelators in mouse serum at 37oC, and (2) to correlate stability data with in-vivo tumor uptake in 22Rv1 xenografted mice1. The knowledge of mouse serum stability is critical for the understanding of the efficacy of a potential imaging pharmaceutical. Methods: Glu-Urea-Lys (GUL) derivatives of acyclic and macrocyclic chelators have been reported as prostate-specific membrane antigen (PSMA) agents. In the present work, GUL was conjugated with two chelators, NOTA and DOTA, via a thiourea linker and designated as NOTA-GUL and DOTA-GUL, respectively. The well-known radioligand PSMA-11, NOTA-GUL, and DOTA-GUL were labeled with 68Ga (37-100 MBq) using a standard radiolabeling technique developed in our laboratories. 68GaCl3 was produced using a Gallia Pharm generator from Eckert & Ziegler. The 68Ga-labeled PSMA conjugates were analyzed and mouse serum stability was monitored by using a reversed-phase High-Performance Liquid Chromatography (RP-HPLC) method. In a typical stability study, a 68Ga-labeled conjugate sample was incubated with mouse serum for 4 hours at 37 °C and the sample was injected onto a RP-HPLC column at a predefined time intervals. Progress of the degradation of the 68Ga-labeled PSMA conjugate in mouse serum was monitored by monitoring area of the main peak with time. Control experiments were performed in each case by monitoring degradation of 68Ga-labeled NOTA-GUL, DOTA-GUL, and PSMA-11 in Phosphate Buffer Saline (PBS). Results: Efficiency of 68Ga-labeling of NOTA-GUL, DOTA-GUL, and PSMA-11 was achieved >96% with > 99% purity. No degradation of 68Ga-labeled PSMA-11 and NOTA-GUL was observed after 4 h incubation in mouse serum. On the contrary, degradation (mainly demetalation) of 68Ga-labelded DOTA-GUL was seen even after 1 h incubation and reaching up to 8% after 4 h incubation. Consistent with the human serum stability study,1 mouse serum stability of 68Ga-labeled PSMA conjugates followed the order: PSMA-11~NOTA-GUL >DOTA-GUL. A similar trend was observed in the in-vivo tumor uptake (%ID/g) in 22Rv1 xenografted mice 1 h post-injection,i.e 6.5, 5.4, and 4.66 for PSMA-11, NOTA-GUL, and DOTA-GUL, respectively. Interestingly, in the present work 68Ga-labeled DOTA-GUL is less inert than the 68Ga-labeled PSMA-11 in mouse serum. Conclusions: In summary, the in-vitro mouse serum stability of the three of 68Ga-Labeled PSMA targeting conjugates of acyclic and macrocyclic chelators follow the order PSMA-11~ NOTA-GUL >DOTA-GUL which reflects in the in-vivo tumor uptake in preclinical mice model.1Ref. Moon S-H, Hong MK, Kim YJ et al. Bioorg Med Chem 2018; 26: 2501-2507.

The prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) are identified as important targets on prostate cancer. Receptor-targeting radiolabeled imaging pharmaceuticals with high affinity and specificity are useful in studying and monitoring biological processes and responses. Two potential imaging pharmaceuticals, AMBA agonist (where AMBA = DO3A-CH2CO-G-[4-aminobenzyl]- Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) and RM1 antagonist (where RM1 = DO3A-CH2CO-G-[4-aminobenzyl]-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2), have demonstrated high binding affinity (IC50) to GRP receptors and high tumor uptake. Antagonists, despite the poor tumor cell internalization properties, can show clearer images and pharmacokinetic profiles by virtue of their higher tumor uptake in animal models compared to agonists. For characterization, development, and translation of a potential imaging pharmaceutical into the clinic, it must be evaluated in a series of tests, including in vitro cell binding assays, in vitro buffer and serum stability studies, the biodistribution of the radiolabeled material, and finally imaging studies in preclinical animal models. Data related to acetate buffer, mouse, canine, and human sera stability of 177Lu-labeled RM1 are presented here and compared with the acetate buffer and sera stability data of AMBA agonist. The samples of 177Lu-labeled RM1 with a high radioconcentration degrade faster than low-radioconcentration samples upon storage at 2-8 °C. Addition of stabilizers, ascorbic acid and gentisic acid, improve the stability of 177Lu-labeled RM1 significantly with gentisic acid being more efficient than ascorbic acid as a stabilizer. The degradation kinetics of 177Lu-labeled AMBA and RM1 in sera follow the order (fastest to slowest): mouse > canine > human sera. Finally, 177Lu-labeled RM1 antagonist is slower to degrade in mouse, canine, and human sera than 177Lu-labeled AMBA agonist, further suggesting that an antagonist is a more promising candidate than agonist for the positron emission tomography (PET) imaging and therapy of prostate cancer patients.

1614 Objectives: The objective of the present work was to study in vitro serum stability of two GRPR-targeting probes based on DO3A conjugates, one being agonist (AMBA) and another being antagonist (RM26), in mouse, canine, and human serum at 37oC. Methods: Two targeting ligands that recognize GRPR receptors were synthesized, purified, and characterized by MALDI mass spectral and HPLC analysis. These are: DO3A-t-BBN (AMBA, Agonist) where DO3A is DO3A-CH2CO, t-BBN = G-[4-aminobenzoyl]-QWAVGHLM-NH2; andDO3A-RM26 (Antagonist); RM26 = G-[4-aminiobenzoyl]-F(d)QWAVGHLµL-NH2 with F(d)-Phenyl alanine with configuration d, and amino acid abbreviation µ being 3S, 4S-4-amino-3-hydroxy-6-methylheptanoic acid. Mouse and human serum (MP Biomedicals), canine serum (Immunoreagents, Inc.), and 177LuCl3 (MURR) were used in this study. Both probes were radiolabeled with 177Lu and a known amount of a 177Lu-labeled probe was mixed with the serum, incubated at 37oC, and the degradation, in triplicate, was monitored with time by a Reversed-Phase (RP) HPLC method. The method involved using an Agilent HPLC which is interfaced with a Lablogic Radioisotope Detector (RID) and a Laura software. Results: The GRPR-targeting ligands were purified by a semi-prep RP-HPLC method and characterized by MALDI mass spectral analysis. A 177Lu labeling and an RP-HPLC analytical method for monitoring degradation of the GRPR-targeting probes in mouse, human and canine serum were developed. Serum stability of 177Lu-AMBA and 177Lu(DO3A-RM26) in mouse, canine, and human, respectively, was monitored by the reduction in the main radioactivity HPLC peak of the corresponding probe. Half-lives of in vitro serum degradation for each probe were calculated, by fitting the degradation data to a first-order kinetics model. The calculated half-lives (in h, n=3) were 3.1 [1], 16.4+0.6; 10.1+0.5, 19.0+0.8; and 38.8 [1], 141+5 in mouse, canine, and human serum for 177Lu-AMBA and 177Lu(DO3A-RM26), respectively. In general, the antagonist was found to be more stable than the agonist in mouse, canine, and human serum and the serum stability followed the order human>canine>mouse. Conclusions: The study demonstrates that the serum stability of antagonist, 177Lu(DO3A- RM26), is greater than agonist, 177Lu-AMBA. [1] Lantry LE, Cappelletti E, Maddalena, ME et al. 177Lu-AMBA: Synthesis and Characterization of a Selective 177Lu-Labeled GRP-R Agonist for Systemic Radiotherapy of Prostate Cancer. J Nucl Med. 2006; 47: 1144-1152.

The magnitude of antibody responses varies across the individual proteins that constitute any given microorganism, both in the context of natural infection and vaccination with attenuated or inactivated pathogens. The protein-specific factors underlying this variability are poorly understood. In 267 individuals exposed to intense seasonal malaria, we examined the relationship between immunoglobulin G (IgG) responses to 861 Plasmodium falciparum proteins and specific features of these proteins, including their subcellular location, relative abundance, degree of polymorphism, and whether they are predicted to have human orthologs. We found that IgG reactivity was significantly higher to extracellular and plasma membrane proteins and also correlated positively with both protein abundance and degree of protein polymorphism. Conversely, IgG reactivity was significantly lower to proteins predicted to have human orthologs. These findings provide insight into protein-specific factors that are associated with variability in the magnitude of antibody responses to natural P. falciparum infection-data that could inform vaccine strategies to optimize antibody-mediated immunity as well as the selection of antigens for sero-diagnostic purposes.

The clinical applications of positron emission tomography (PET) imaging pharmaceuticals have increased tremendously over the past several years since the approval of 18fluorine-fluorodeoxyglucose (18F-FDG) by the Food and Drug Administration (FDA). Numerous 18F-labeled target-specific potential imaging pharmaceuticals, based on small and large molecules, have been evaluated in preclinical and clinical settings. 18F-labeling of organic moieties involves the introduction of the radioisotope by C-18F bond formation via a nucleophilic or an electrophilic substitution reaction. However, biomolecules, such as peptides, proteins, and oligonucleotides, cannot be radiolabeled via a C-18F bond formation as these reactions involve harsh conditions, including organic solvents, high temperature, and nonphysiological conditions. Several approaches, including 18F-labeled prosthetic groups, silicon, boron, and aluminum fluoride acceptor chemistry, and click chemistry have been developed, in the past, for 18F labeling of biomolecules. Linear and macrocyclic polyaminocarboxylates and their analogs and derivatives form thermodynamically stable and kinetically inert aluminum chelates. Hence, macrocyclic polyaminocarboxylates have been used for conjugation with biomolecules, such as folate, peptides, affibodies, and protein fragments, followed by 18F-AlF chelation, and evaluation of their targeting abilities in preclinical and clinical environments. The goal of this report is to provide an overview of the 18F radiochemistry and 18F-labeling methodologies for small molecules and target-specific biomolecules, a comprehensive review of coordination chemistry of Al3+, 18F-AlF labeling of peptide and protein conjugates, and evaluation of 18F-labeled biomolecule conjugates as potential imaging pharmaceuticals.

A novel, rapid, and automated loop method for the synthesis of [11C]nicotine was developed and optimized. The method involves, a reaction of the precursor, (+) nornicotine or (-) nornicotine, with a gas-phase produced [11C]CH3I in an 800 µL loop at 75 °C for 5 min followed by a semi-preparatory Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) purification. The optimized synthesis and purification process was complete in < 30 min and produced [11C]nicotine with > 99.9% Radiochemical Purity (RCP), no [11C]CH3I, no (+) nornicotine, 105 mCi/µmole specific activity, 7.0 – 7.2 pH, and 16.6% ethanol. The current method can be optimized, to reduce the ethanol content (<10%), and can be translated to a cGMP production of [11C]nicotine for human clinical trials.

Antibody (Ab) fragments have great clinical potential as cancer therapeutics and diagnostics. Their small size allows for fast clearance from blood, low immunoreactivity, better tumor penetration, and simpler engineering and production. The smallest fragment derived from a full-length IgG that retains binding to its antigen, the single-chain variable fragment (scF(V)), is engineered by fusing the variable light and variable heavy domains with a peptide linker. Along with switching the domain orientation, altering the length and amino acid sequence of the linker can significantly affect scF(V) binding, stability, quaternary structure, and other biophysical properties. Comprehensive studies of these attributes in a single scaffold have not been reported, making design and optimization of Ab fragments challenging. Here, we constructed libraries of 3E8, an Ab specific to tumor-associated glycoprotein 72 (TAG-72), a mucinous glycoprotein overexpressed in 80% of adenocarcinomas. We cloned, expressed, and characterized scF(V) s, diabodies, and higher-order multimer constructs with varying linker compositions, linker lengths, and domain orientations. These constructs dramatically differed in their oligomeric states and stabilities, not only because of linker and orientation but also related to the purification method. For example, protein L-purified constructs tended to have broader distributions and higher oligomeric states than has been reported previously. From this library, we selected an optimal construct, 3E8.G(4)S, for biodistribution and pharmacokinetic studies and in vivo xenograft mouse PET imaging. These studies revealed significant tumor targeting of 3E8.G(4)S with a tumor-to-background ratio of 29:1. These analyses validated 3E8.G(4)S as a fast, accurate, and specific tumor-imaging agent.