Dual-inhibitory domain iCARs improve the efficiency of the AND-NOT gate CAR T strategy.

CAR (chimeric antigen receptor) T cell therapy has shown clinical success in treating hematological malignancies, but its treatment of solid tumors has been limited. One major challenge is on-target, off-tumor toxicity, where CAR T cells also damage normal tissues that express the targeted antigen. To reduce this detrimental side-effect, Boolean-logic gates like AND-NOT gates have utilized an inhibitory CAR (iCAR) to specifically curb CAR T cell activity at selected nonmalignant tissue sites. However, the strategy seems inefficient, requiring high levels of iCAR and its target antigen for inhibition. Using a TROP2-targeting iCAR with a single PD1 inhibitory domain to inhibit a CEACAM5-targeting CAR (CEACAR), we observed that the inefficiency was due to a kinetic delay in iCAR inhibition of cytotoxicity. To improve iCAR efficiency, we modified three features of the iCAR-the avidity, the affinity, and the intracellular signaling domains. Increasing the avidity but not the affinity of the iCAR led to significant reductions in the delay. iCARs containing twelve different inhibitory signaling domains were screened for improved inhibition, and three domains (BTLA, LAIR-1, and SIGLEC-9) each suppressed CAR T function but did not enhance inhibitory kinetics. When inhibitory domains of LAIR-1 or SIGLEC-9 were combined with PD-1 into a single dual-inhibitory domain iCAR (DiCARs) and tested with the CEACAR, inhibition efficiency improved as evidenced by a significant reduction in the inhibitory delay. These data indicate that a delicate balance between CAR and iCAR signaling strength and kinetics must be achieved to regulate AND-NOT gate CAR T cell selectivity.

First-in-man study of the PSMA Minibody IR800-IAB2M for molecularly targeted intraoperative fluorescence guidance during radical prostatectomy.

PURPOSE: Prostate-specific membrane antigen (PSMA) is increasingly used to image prostate cancer in clinical practice. We sought to develop and test a humanised PSMA minibody IAB2M conjugated to the fluorophore IRDye 800CW-NHS ester in men undergoing robot-assisted laparoscopic radical prostatectomy (RARP) to image prostate cancer cells during surgery. METHODS: The minibody was evaluated pre-clinically using PSMA positive/negative xenograft models, following which 23 men undergoing RARP between 2018 and 2020 received between 2.5 mg and 20 mg of IR800-IAB2M intravenously, at intervals between 24 h and 17 days prior to surgery. At every step of the procedure, the prostate, pelvic lymph node chains and extra-prostatic surrounding tissue were imaged with a dual Near-infrared (NIR) and white light optical platform for fluorescence in vivo and ex vivo. Histopathological evaluation of intraoperative and postoperative microscopic fluorescence imaging was undertaken for verification. RESULTS: Twenty-three patients were evaluated to optimise both the dose of the reagent and the interval between injection and surgery and secure the best possible specificity of fluorescence images. Six cases are presented in detail as exemplars. Overall sensitivity and specificity in detecting non-lymph-node extra-prostatic cancer tissue were 100% and 65%, and 64% and 64% respectively for lymph node positivity. There were no side-effects associated with administration of the reagent. CONCLUSION: Intraoperative imaging of prostate cancer tissue is feasible and safe using IR800-IAB2M. Further evaluation is underway to assess the benefit of using the technique in improving completion of surgical excision during RARP. REGISTRATION: ISCRCTN10046036: https://www.isrctn.com/ISRCTN10046036 .

Artificial intelligence in immunotherapy PET/SPECT imaging.

OBJECTIVE: Immunotherapy has dramatically altered the therapeutic landscape for oncology, but more research is needed to identify patients who are likely to achieve durable clinical benefit and those who may develop unacceptable side effects. We investigated the role of artificial intelligence in PET/SPECT-guided approaches for immunotherapy-treated patients. METHODS: We performed a scoping review of MEDLINE, CENTRAL, and Embase databases using key terms related to immunotherapy, PET/SPECT imaging, and AI/radiomics through October 12, 2022. RESULTS: Of the 217 studies identified in our literature search, 24 relevant articles were selected. The median (interquartile range) sample size of included patient cohorts was 63 (157). Primary tumors of interest were lung (n = 14/24, 58.3%), lymphoma (n = 4/24, 16.7%), or melanoma (n = 4/24, 16.7%). A total of 28 treatment regimens were employed, including anti-PD-(L)1 (n = 13/28, 46.4%) and anti-CTLA-4 (n = 4/28, 14.3%) monoclonal antibodies. Predictive models were built from imaging features using univariate radiomics (n = 7/24, 29.2%), radiomics (n = 12/24, 50.0%), or deep learning (n = 5/24, 20.8%) and were most often used to prognosticate (n = 6/24, 25.0%) or describe tumor phenotype (n = 5/24, 20.8%). Eighteen studies (75.0%) performed AI model validation. CONCLUSION: Preliminary results suggest broad potential for the application of AI-guided immunotherapy management after further validation of models on large, prospective, multicenter cohorts. CLINICAL RELEVANCE STATEMENT: This scoping review describes how artificial intelligence models are built to make predictions based on medical imaging and explores their application specifically in the PET and SPECT examination of immunotherapy-treated cancers. KEY POINTS: • Immunotherapy has drastically altered the cancer treatment landscape but is known to precipitate response patterns that are not accurately accounted for by traditional imaging methods. • There is an unmet need for better tools to not only facilitate in-treatment evaluation but also to predict, a priori, which patients are likely to achieve a good response with a certain treatment as well as those who are likely to develop side effects. • Artificial intelligence applied to PET/SPECT imaging of immunotherapy-treated patients is mainly used to make predictions about prognosis or tumor phenotype and is built from baseline, pre-treatment images. Further testing is required before a true transition to clinical application can be realized.

In vivo NIR-II structured-illumination light-sheet microscopy.

Noninvasive optical imaging with deep tissue penetration depth and high spatiotemporal resolution is important to longitudinally studying the biology at the single-cell level in live mammals, but has been challenging due to light scattering. Here, we developed near-infrared II (NIR-II) (1,000 to 1,700 nm) structured-illumination light-sheet microscopy (NIR-II SIM) with ultralong excitation and emission wavelengths up to ∼1,540 and ∼1,700 nm, respectively, suppressing light scattering to afford large volumetric three-dimensional (3D) imaging of tissues with deep-axial penetration depths. Integrating structured illumination into NIR-II light-sheet microscopy further diminished background and improved spatial resolution by approximately twofold. In vivo oblique NIR-II SIM was performed noninvasively for 3D volumetric multiplexed molecular imaging of the CT26 tumor microenvironment in mice, longitudinally mapping out CD4, CD8, and OX40 at the single-cell level in response to immunotherapy by cytosine-phosphate-guanine (CpG), a Toll-like receptor 9 (TLR-9) agonist combined with OX40 antibody treatment. NIR-II SIM affords an additional tool for noninvasive volumetric molecular imaging of immune cells in live mammals.

Harnessing imaging tools to guide immunotherapy trials: summary from the National Cancer Institute Cancer Imaging Steering Committee workshop.

As the immuno-oncology field continues the rapid growth witnessed over the past decade, optimising patient outcomes requires an evolution in the current response-assessment guidelines for phase 2 and 3 immunotherapy clinical trials and clinical care. Additionally, investigational tools-including image analysis of standard-of-care scans (such as CT, magnetic resonance, and PET) with analytics, such as radiomics, functional magnetic resonance agents, and novel molecular-imaging PET agents-offer promising advancements for assessment of immunotherapy. To document current challenges and opportunities and identify next steps in immunotherapy diagnostic imaging, the National Cancer Institute Clinical Imaging Steering Committee convened a meeting with diverse representation among imaging experts and oncologists to generate a comprehensive review of the state of the field.

Pre-conditioning modifies the TME to enhance solid tumor CAR T cell efficacy and endogenous protective immunity.

Chimeric antigen receptor (CAR) T cell therapy has led to impressive clinical responses in patients with hematological malignancies; however, its effectiveness in patients with solid tumors has been limited. While CAR T cells for the treatment of advanced prostate and pancreas cancer, including those targeting prostate stem cell antigen (PSCA), are being clinically evaluated and are anticipated to show bioactivity, their safety and the impact of the immunosuppressive tumor microenvironment (TME) have not been faithfully explored preclinically. Using a novel human PSCA knockin (hPSCA-KI) immunocompetent mouse model, we evaluated the safety and therapeutic efficacy of PSCA-CAR T cells. We demonstrated that cyclophosphamide (Cy) pre-conditioning significantly modified the immunosuppressive TME and was required to uncover the efficacy of PSCA-CAR T cells in metastatic prostate and pancreas cancer models, with no observed toxicities in normal tissues with endogenous expression of PSCA. This combination dampened the immunosuppressive TME, generated pro-inflammatory myeloid and T cell signatures in tumors, and enhanced the recruitment of antigen-presenting cells, as well as endogenous and adoptively transferred T cells, resulting in long-term anti-tumor immunity.

Cross-Link-Functionalized Nanoparticles for Rapid Excretion in Nanotheranostic Applications.

Most NIR-IIb fluorophores are nanoparticle-based probes with long retention (≈1 month or longer) in the body. Here, we applied a novel cross-linked coating to functionalize core/shell lead sulfide/cadmium sulfide quantum dots (PbS/CdS QDs) emitting at ≈1600 nm. The coating was comprised of an amphiphilic polymer followed by three crosslinked amphiphilic polymeric layers (P3 coating), imparting high biocompatibility and >90 % excretion of QDs within 2 weeks of intravenous administration. The P3 -QDs were conjugated to an engineered anti-CD8 diabody (Cys-diabody) for in vivo molecular imaging of CD8+ cytotoxic T lymphocytes (CTLs) in response to anti-PD-L1 therapy. Two-plex molecular imaging in combination with down-conversion Er nanoparticles (ErNPs) was performed for real-time in vivo monitoring of PD-L1 positive tumor cells and CTLs with cellular resolution by non-invasive NIR-IIb light sheet microscopy. Imaging of angiogenesis in the tumor microenvironment and of lymph nodes deep in the body with a signal-to-background ratio of up to ≈170 was also achieved using P3 -QDs.

18F-labeled anti-human CD20 cys-diabody for same-day immunoPET in a model of aggressive B cell lymphoma in human CD20 transgenic mice.

PURPOSE: Metabolic imaging using [18F]FDG is the current standard for clinical PET; however, some malignancies (e.g., indolent lymphomas) show low avidity for FDG. The majority of B cell lymphomas express CD20, making it a valuable target both for antibody-based therapy and imaging. We previously developed PET tracers based on the humanised anti-CD20 antibody obinutuzumab (GA101). Preclinical studies showed that the smallest bivalent fragment, the cys-diabody (GAcDb, 54.5 kDa) with a peak uptake at 1-2 h post-injection and a biological half-life of 2-5 h, is compatible with short-lived positron emitters such as fluorine-18 (18F, t1/2 110 min), enabling same-day imaging. METHODS: GAcDb was radiolabeled using amine-reactive N-succinimidyl 4-[18F]-fluorobenzoate ([18F]SFB), or thiol-reactive N-[2-(4-[18F]-fluorobenzamido)ethyl]maleimide ([18F]FBEM) for site-specific conjugation to C-terminal cysteine residues. Both tracers were used for immunoPET imaging of the B cell compartment in human CD20 transgenic mice (hCD20TM). [18F]FB-GAcDb immunoPET was further evaluated in a disseminated lymphoma (A20-hCD20) syngeneic for hCD20TM and compared to [18F]FDG PET. Tracer uptake was confirmed by ex vivo biodistribution. RESULTS: The GAcDb was successfully 18F-radiolabeled using two different conjugation methods resulting in similar specific activities and without impairing immunoreactivity. Both tracers ([18F]FB-GAcDb and [18F]FBEM-GAcDb) specifically target human CD20-expressing B cells in transgenic mice. Fast blood clearance results in high contrast PET images as early as 1 h post injection enabling same-day imaging. [18F]FB-GAcDb immunoPET detects disseminated lymphoma disease in the context of normal tissue expression of hCD20, with comparable sensitivity as [18F]FDG PET but with added specificity for the therapeutic target. CONCLUSIONS: [18F]FB-GAcDb and [18F]FBEM-GAcDb could monitor normal B cells and B cell malignancies non-invasively and quantitatively in vivo. In contrast to [18F]FDG PET, immunoPET provides not only information about the extent of disease but also about presence and localisation of the therapeutic target.

Aligning physics and physiology: Engineering antibodies for radionuclide delivery.

The exquisite specificity of antibodies and antibody fragments renders them excellent agents for targeted delivery of radionuclides. Radiolabeled antibodies and fragments have been successfully used for molecular imaging and radioimmunotherapy (RIT) of cell surface targets in oncology and immunology. Protein engineering has been used for antibody humanization essential for clinical applications, as well as optimization of important characteristics including pharmacokinetics, biodistribution, and clearance. Although intact antibodies have high potential as imaging and therapeutic agents, challenges include long circulation time in blood, which leads to later imaging time points post-injection and higher blood absorbed dose that may be disadvantageous for RIT. Using engineered fragments may address these challenges, as size reduction and removal of Fc function decreases serum half-life. Radiolabeled fragments and pretargeting strategies can result in high contrast images within hours to days, and a reduction of RIT toxicity in normal tissues. Additionally, fragments can be engineered to direct hepatic or renal clearance, which may be chosen based on the application and disease setting. This review discusses aligning the physical properties of radionuclides (positron, gamma, beta, alpha, and Auger emitters) with antibodies and fragments and highlights recent advances of engineered antibodies and fragments in preclinical and clinical development for imaging and therapy.

Engineered antibody fragments for immuno-PET imaging of endogenous CD8+ T cells in vivo.

The noninvasive detection and quantification of CD8(+) T cells in vivo are important for both the detection and staging of CD8(+) lymphomas and for the monitoring of successful cancer immunotherapies, such as adoptive cell transfer and antibody-based immunotherapeutics. Here, antibody fragments are constructed to target murine CD8 to obtain rapid, high-contrast immuno-positron emission tomography (immuno-PET) images for the detection of CD8 expression in vivo. The variable regions of two anti-murine CD8-depleting antibodies (clones 2.43 and YTS169.4.2.1) were sequenced and reformatted into minibody (Mb) fragments (scFv-CH3). After production and purification, the Mbs retained their antigen specificity and bound primary CD8(+) T cells from the thymus, spleen, lymph nodes, and peripheral blood. Importantly, engineering of the parental antibodies into Mbs abolished the ability to deplete CD8(+) T cells in vivo. The Mbs were subsequently conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid for (64)Cu radiolabeling. The radiotracers were injected i.v. into antigen-positive, antigen-negative, immunodeficient, antigen-blocked, and antigen-depleted mice to evaluate specificity of uptake in lymphoid tissues by immuno-PET imaging and ex vivo biodistribution. Both (64)Cu-radiolabeled Mbs produced high-contrast immuno-PET images 4 h postinjection and showed specific uptake in the spleen and lymph nodes of antigen-positive mice.

Engineered antibodies for molecular imaging of cancer.

Antibody technology has transformed drug development, providing robust approaches to producing highly targeted and active therapeutics that can routinely be advanced through clinical evaluation and registration. In parallel, there is an emerging need to access similarly targeted agents for diagnostic purposes, including non-invasive imaging in preclinical models and patients. Antibody engineering enables modification of key properties (immunogenicity, valency, biological inertness, pharmacokinetics, clearance route, site-specific conjugation) in order to produce targeting agents optimized for molecular imaging. Expanded availability of positron-emitting radionuclides has led to a resurgence of interest and applications of immunoPET (immuno-positron emission tomography). Molecular imaging using engineered antibodies and fragments provides a general approach for assessing cell surface phenotype in vivo and stands to play an increasingly important role in cancer diagnosis, treatment selection, and monitoring of molecularly targeted therapeutics.

Noninvasive Imaging of PSMA in prostate tumors with (89)Zr-Labeled huJ591 engineered antibody fragments: the faster alternatives.

Engineered antibody fragments offer faster delivery with retained tumor specificity and rapid clearance from nontumor tissues. Here, we demonstrate that positron emission tomography (PET) based detection of prostate specific membrane antigen (PSMA) in prostatic tumor models using engineered bivalent antibodies built on single chain fragments (scFv) derived from the intact antibody, huJ591, offers similar tumor delineating properties but with the advantage of rapid targeting and imaging. (89)Zr-radiolabeled huJ591 scFv (dimeric scFv-CH3; (89)Zr-Mb) and cysteine diabodies (dimeric scFv; (89)Zr-Cys-Db) demonstrated internalization and similar Kds (∼2 nM) compared to (89)Zr-huJ591 in PSMA(+) cells. Tissue distribution assays established the specificities of both (89)Zr-Mb and (89)Zr-Cys-Db for PSMA(+) xenografts (6.2 ± 2.5% ID/g and 10.2 ± 3.4% ID/g at 12 h p.i. respectively), while minimal accumulation in PSMA(-) tumors was observed. From the PET images, (89)Zr-Mb and (89)Zr-Cys-Db exhibited faster blood clearance than the parent huJ591 while tumor-to-muscle ratios for all probes show comparable values across all time points. Ex vivo autoradiography and histology assessed the distribution of the probes within the tumor. Imaging PSMA-expressing prostate tumors with smaller antibody fragments offers rapid tumor accumulation and accelerated clearance; hence, shortened wait periods between tracer administration and high-contrast tumor imaging and lower dose-related toxicity are potentially realized.

Integrating Artificial Intelligence and PET Imaging for Drug Discovery: A Paradigm Shift in Immunotherapy.

The integration of artificial intelligence (AI) and positron emission tomography (PET) imaging has the potential to become a powerful tool in drug discovery. This review aims to provide an overview of the current state of research and highlight the potential for this alliance to advance pharmaceutical innovation by accelerating the development and deployment of novel therapeutics. We previously performed a scoping review of three databases (Embase, MEDLINE, and CENTRAL), identifying 87 studies published between 2018 and 2022 relevant to medical imaging (e.g., CT, PET, MRI), immunotherapy, artificial intelligence, and radiomics. Herein, we reexamine the previously identified studies, performing a subgroup analysis on articles specifically utilizing AI and PET imaging for drug discovery purposes in immunotherapy-treated oncology patients. Of the 87 original studies identified, 15 met our updated search criteria. In these studies, radiomics features were primarily extracted from PET/CT images in combination (n = 9, 60.0%) rather than PET imaging alone (n = 6, 40.0%), and patient cohorts were mostly recruited retrospectively and from single institutions (n = 10, 66.7%). AI models were used primarily for prognostication (n = 6, 40.0%) or for assisting in tumor phenotyping (n = 4, 26.7%). About half of the studies stress-tested their models using validation sets (n = 4, 26.7%) or both validation sets and test sets (n = 4, 26.7%), while the remaining six studies (40.0%) either performed no validation at all or used less stringent methods such as cross-validation on the training set. Overall, the integration of AI and PET imaging represents a paradigm shift in drug discovery, offering new avenues for more efficient development of therapeutics. By leveraging AI algorithms and PET imaging analysis, researchers could gain deeper insights into disease mechanisms, identify new drug targets, or optimize treatment regimens. However, further research is needed to validate these findings and address challenges such as data standardization and algorithm robustness.

89Zr-ImmunoPET for the Specific Detection of EMP2-Positive Tumors.

Epithelial membrane protein-2 (EMP2) is upregulated in a number of tumors and therefore remains a promising target for mAb-based therapy. In the current study, image-guided therapy for an anti-EMP2 mAb was evaluated by PET in both syngeneic and immunodeficient cancer models expressing different levels of EMP2 to enable a better understanding of its tumor uptake and off target accumulation and clearance. The therapeutic efficacy of the anti-EMP2 mAb was initially evaluated in high- and low-expressing tumors, and the mAb reduced tumor load for the high EMP2-expressing 4T1 and HEC-1-A tumors. To create an imaging agent, the anti-EMP2 mAb was conjugated to p-SCN-Bn-deferoxamine (DFO) and radiolabeled with 89Zr. Tumor targeting and tissue biodistribution were evaluated in syngeneic tumor models (4T1, CT26, and Panc02) and human tumor xenograft models (Ramos, HEC-1-A, and U87MG/EMP2). PET imaging revealed radioactive accumulation in EMP2-positive tumors within 24 hours after injection, and the signal was retained for 5 days. High specific uptake was observed in tumors with high EMP2 expression (4T1, CT26, HEC-1-A, and U87MG/EMP2), with less accumulation in tumors with low EMP2 expression (Panc02 and Ramos). Biodistribution at 5 days after injection revealed that the tumor uptake ranged from 2 to approximately 16%ID/cc. The results show that anti-EMP2 mAbs exhibit EMP2-dependent tumor uptake with low off-target accumulation in preclinical cancer models. The development of improved anti-EMP2 Ab fragments may be useful to track EMP2-positive tumors for subsequent therapeutic interventions.

Yttrium-90 Anti-CD25-BEAM-Conditioning for Autologous Hematopoietic Cell Transplantation in Peripheral T Cell Lymphoma.

Peripheral T cell lymphomas (PTCL) have a poor prognosis with current treatments. High-dose chemotherapy followed by autologous hematopoietic cell transplant (AHCT) is used as a consolidation strategy after achieving clinical remission with first-line therapy, as well as in chemosensitive relapse if allogeneic transplant is not an option. CD25 is a targetable protein often highly expressed in PTCL. In this phase 1 clinical trial, we tested the addition of beta-emitting 90Y-labeled chimeric anti-CD25 basiliximab (aTac) to BEAM (carmustine, etoposide, cytarabine, melphalan) as conditioning for AHCT in patients with PTCL. Twenty-three AHCT-eligible patients were enrolled, and 20 received therapeutic 90Y-aTac-BEAM AHCT. Radiation doses of 0.4, 0.5 and 0.6 mCi/kg were tested. With no observed dose-limiting toxicities, 0.6 mCi/kg was deemed the recommended phase 2 dose. The most prevalent adverse effect, grade 2 mucositis, was experienced by 80% of patients. As of this report, 6 (30%) of the treated patients had died, 5 due to progressive disease and 1 due to multiple organ failure [median time of death 17 mo (range: 9-21 mo)] post-AHCT. Median follow-up was 24 mo (range: 9-26 mo) overall and 24 mo (range: 13-26 mo) for surviving patients. For patients who received therapeutic 90Y-aTac-BEAM AHCT, the 2-year progression-free and overall survival were 59% (95% CI: 34-77%) and 68% (95% CI: 42-84%), respectively. 90Y-aTac-BEAM appears to be safe as an AHCT conditioning regimen for PTCL, with no increased toxicity over the toxicities historically seen with BEAM alone in this patient population. This trial was registered at www.clinicaltrials.gov as # NCT02342782.

Anti-carcinoembryonic antigen single-chain variable fragment antibody variants bind mouse and human neonatal Fc receptor with different affinities that reveal distinct cross-species differences in serum half-life.

Serum half-life of IgG is controlled by the neonatal Fc receptor (FcRn) that interacts with the IgG Fc region and may be increased or decreased as a function of altered FcRn binding. Preclinical evaluations of modified IgGs are frequently carried out in mice, but such IgGs may bind differently to mouse and human FcRn (mFcRn and hFcRn). Here, we report a detailed characterization of a matched set of mouse-human chimeric T84.66 scFv-Fc variants with specificity for the tumor carcinoembryonic antigen and mutations in the FcRn-binding site. Binding to soluble mFcRn and hFcRn was measured using in vitro assays, and the results were compared with blood clearance in vivo in normal (mFcRn bearing) and hFcRn transgenic mice. All variants bound better to mFcRn than to hFcRn. The loss of affinity varied among the mutants, however, and also the hierarchy of binding differed depending on the receptor. The mutations had no major impact on binding to the classical Fcγ receptors. Importantly, the trend of blood clearance in both strains of mice correlated with the hierarchy of binding obtained using soluble FcRn. Consequently, in vitro interaction analysis of engineered IgGs regarding their cross-species FcRn binding ability provides information for prediction of in vivo pharmacokinetics.

Endocytosis and intracellular trafficking properties of transferrin-conjugated block copolypeptide vesicles.

Block polypeptides are an emerging class of materials that have the potential to be used in many biomedical applications, including the field of drug delivery. We have previously developed a negatively charged block copolypeptide, poly(L-glutamate)60-b-poly(L-leucine)20 (E60L20), which forms spherical vesicles in aqueous solution. Since these vesicles are negatively charged, they are minimally toxic toward cells. However, the negative charge also inhibits these vesicles from effectively being internalized by cells, which can be problematic as many therapeutics have intracellular targets. To overcome this limitation of the E60L20 vesicles, transferrin (Tf) was conjugated onto the vesicle surface, since the receptor for Tf is overexpressed on cancer cells. The enhanced uptake of the Tf-conjugated vesicle was verified through confocal microscopy. Furthermore, endocytosis and immunostaining experiments confirmed that the Tf conjugated on the vesicle surface plays a critical role in the internalization and subsequent intracellular trafficking behavior of the vesicles.

An engineered anti-CA19-9 cys-diabody for positron emission tomography imaging of pancreatic cancer and targeting of polymerized liposomal nanoparticles.

BACKGROUND: Antibody-based therapeutics is a rapidly growing field. Small engineered antibody fragments demonstrate similar antigen affinity compared with the parental antibody but have a shorter serum half-life and possess the ability to be conjugated to nanoparticles. The goal of this study was to engineer an anti-carbohydrate antigen 19-9 (CA19-9) cys-diabody fragment in hopes of targeting nanoparticles to pancreatic cancer. METHODS: The anti-CA19-9 cys-diabody was created by engineering a C-terminal cysteine residue into the DNA single-chain Fv construct of the anti-CA19-9 diabody and expressed in NS0 cells. Maleimide chemistry was used to conjugate the cys-diabody to polymerized liposomal nanoparticles (PLNs) through the cysteine residues. Flow cytometry was used to evaluate targeting of cys-diabody and cys-diabody-PLN conjugate to human pancreatic cancer cell lines. The cys-diabody was radiolabeled with a positron emitter ((124)I) and evaluated in a mouse model of CA19-9-positive and CA19-9-negative xenografts with micro-positron emission tomography/micro-computed tomography at successive time intervals after injection. Percentage of injected dose per gram of radioactivity was measured in blood and tumor to provide objective confirmation of the micro-positron emission tomographic images. RESULTS: Tumor xenograft imaging of the anti-CA19-9 cys-diabody demonstrated an average tumor-to-blood ratio of 3.0 and positive-to-negative tumor ratio of 7.4. Successful conjugation of the cys-diabody to PLNs was indicated by flow cytometry showing specific binding of cys-diabody-PLN conjugate to human pancreatic cancer cells in vitro. CONCLUSIONS: Our results show that the anti-CA19-9 cys-diabody targets pancreatic cancer providing specific molecular imaging in tumor xenograft models. Furthermore, the cys-diabody-PLN conjugate demonstrates target-specific binding of human pancreatic cancer cells with the potential to deliver targeted treatment.

Development of 18F-Labeled hydrophilic trans-cyclooctene as a bioorthogonal tool for PET probe construction.

We report the development of a hydrophilic 18F-labeled a-TCO derivative [18F]3 (log P = 0.28) through a readily available precursor and a single-step radiofluorination reaction (RCY up to 52%). We demonstrated that [18F]3 can be used to construct not only multiple small molecule/peptide-based PET agents, but protein/diabody-based imaging probes in parallel.

First-In-Human Pilot PET Immunoimaging Study of 64Cu-Anti-Carcinoembryonic Antigen Monoclonal Antibody (hT84.66-M5A) in Patients with Carcinoembryonic Antigen-Producing Cancers.

Background: PET imaging using radiolabeled immunoconstructs shows promise in cancer detection and in assessing tumor response to therapies. The authors report the first-in-human pilot study evaluating M5A, a humanized anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb), radiolabeled with 64Cu in patients with CEA-expressing malignancies. The purpose of this pilot study was to identify the preferred patient population for further evaluation of this agent in an expanded trial. Methods: Patients with CEA-expressing primary or metastatic cancer received 64Cu-DOTA-hT84.66-M5A with imaging performed at 1 and 2 days postinfusion. 64Cu-DOTA-hT84.66-M5A PET scan findings were correlated with CT, MRI, and/or FDG PET scans and with histopathologic findings from planned surgery or biopsy performed postscan. Results: Twenty patients received 64Cu-DOTA-hT84.66-M5A. Twelve patients demonstrated positive images, which were confirmed in 10 patients as tumor by standard-of-care (SOC) imaging, biopsy, or surgical findings. Four of the 8 patients with negative imaging were confirmed as true negative, with the remaining 4 patients having disease demonstrated by SOC imaging or surgery. All 5 patients with locally advanced rectal cancer underwent planned biopsy or surgery after 64Cu-DOTA-hT84.66-M5A imaging (4 patients imaged 6-8 weeks after completing neoadjuvant chemotherapy and radiation therapy) and demonstrated a high concordance between biopsy findings and 64Cu-DOTA-hT84.66-M5A PET scan results. Three patients demonstrated positive uptake at the primary site later confirmed by biopsy and at surgery as residual disease. Two patients with negative scans each demonstrated complete pathologic response. In 5 patients with medullary thyroid cancer, 64Cu-DOTA-hT84.66-M5A identified disease not seen on initial CT scans in 3 patients, later confirmed to be disease by subsequent surgery or MRI. Conclusions: 64Cu-DOTA-hT84.66-M5A demonstrates promise in tumor detection, particularly in patients with locally advanced rectal cancer and medullary thyroid cancer. A successor trial in locally advanced rectal cancer has been initiated to further evaluate this agent's ability to define tumor extent before and assess disease response after neoadjuvant chemotherapy and radiotherapy. clinical trial.gov (NCT02293954).