Enhancing brain entry and therapeutic activity of chimeric antigen receptor T cells with intra-arterial NEO100 in a mouse model of CNS lymphoma.

OBJECTIVE: Malignancies of the CNS are difficult to treat because the blood-brain barrier (BBB) prevents most therapeutics from reaching the intracranial lesions at sufficiently high concentrations. This also applies to chimeric antigen receptor (CAR) T cells, for which systemic delivery is inferior to direct intratumoral or intraventricular injection of the cells. The authors previously reported on a novel approach to safely and reversibly open the BBB of mice by applying intra-arterial (IA) injections of NEO100, a pharmaceutical-grade version of the natural monoterpene perillyl alcohol. The authors hypothesized that this method would enable enhanced brain entry and therapeutic activity of intravenously delivered CAR T cells, which the authors tested in a mouse model of CNS lymphoma. METHODS: Human Raji lymphoma cells were implanted into the brains of immune-deficient mice. After tumor uptake was confirmed with bioluminescent imaging, 0.3% NEO100 was injected intra-arterially, which was followed by intravenous (IV) delivery of CD19-targeted CAR T cells. After this single intervention, tumor growth was monitored with imaging, long-term survival of mice was recorded, and select mice were euthanized to analyze the distribution of CAR T cells in brain tissue. RESULTS: Intravenously injected CAR T cells could be readily detected in brain tumor areas after IA injection of NEO100 but not after IA injection of the vehicle (without NEO100). Although all untreated control animals died within 3 weeks, all mice that received IA NEO100 followed by IV CAR T cells survived and thrived for 200 days, when the experiment was terminated. Of the mice that received IV CAR T cells without prior IA NEO100, 3 died within 3 weeks and 2 survived long-term. CONCLUSIONS: BBB opening by IA NEO100 facilitates brain entry of intravenously delivered CD19 CAR T cells. The long-term survival of all mice with CNS lymphoma, along with the disappearance of the tumor as determined with imaging, suggests that this one-time therapeutic intervention was curative. BBB opening by IA NEO100 may offer a novel option to increase brain access by CAR T cells.

Brain Disposition of Antibody-Based Therapeutics: Dogma, Approaches and Perspectives.

Due to their high specificity, monoclonal antibodies have been widely investigated for their application in drug delivery to the central nervous system (CNS) for the treatment of neurological diseases such as stroke, Alzheimer's, and Parkinson's disease. Research in the past few decades has revealed that one of the biggest challenges in the development of antibodies for drug delivery to the CNS is the presence of blood-brain barrier (BBB), which acts to restrict drug delivery and contributes to the limited uptake (0.1-0.2% of injected dose) of circulating antibodies into the brain. This article reviews the various methods currently used for antibody delivery to the CNS at the preclinical stage of development and the underlying mechanisms of BBB penetration. It also describes efforts to improve or modulate the physicochemical and biochemical properties of antibodies (e.g., charge, Fc receptor binding affinity, and target affinity), to adapt their pharmacokinetics (PK), and to influence their distribution and disposition into the brain. Finally, a distinction is made between approaches that seek to modify BBB permeability and those that use a physiological approach or antibody engineering to increase uptake in the CNS. Although there are currently inherent difficulties in developing safe and efficacious antibodies that will cross the BBB, the future prospects of brain-targeted delivery of antibody-based agents are believed to be excellent.

Spatio-temporal biodistribution of 89Zr-oxine labeled huLym-1-A-BB3z-CAR T-cells by PET imaging in a preclinical tumor model.

Quantitative in vivo monitoring of cell biodistribution offers assessment of treatment efficacy in real-time and can provide guidance for further optimization of chimeric antigen receptor (CAR) modified cell therapy. We evaluated the utility of a non-invasive, serial 89Zr-oxine PET imaging to assess optimal dosing for huLym-1-A-BB3z-CAR T-cell directed to Lym-1-positive Raji lymphoma xenograft in NOD Scid-IL2Rgammanull (NSG) mice. In vitro experiments showed no detrimental effects in cell health and function following 89Zr-oxine labeling. In vivo experiments employed simultaneous PET/MRI of Raji-bearing NSG mice on day 0 (3 h), 1, 2, and 5 after intravenous administration of low (1.87 ± 0.04 × 106 cells), middle (7.14 ± 0.45 × 106 cells), or high (16.83 ± 0.41 × 106 cells) cell dose. Biodistribution (%ID/g) in regions of interests defined over T1-weighted MRI, such as blood, bone, brain, liver, lungs, spleen, and tumor, were analyzed from PET images. Escalating doses of CAR T-cells resulted in dose-dependent %ID/g biodistributions in all regions. Middle and High dose groups showed significantly higher tumor %ID/g compared to Low dose group on day 2. Tumor-to-blood ratios showed the enhanced extravascular tumor uptake by day 2 in the Low dose group, while the Middle dose showed significant tumor accumulation starting on day 1 up to day 5. From these data obtained over time, it is apparent that intravenously administered CAR T-cells become trapped in the lung for 3-5 h and then migrate to the liver and spleen for up to 2-3 days. This surprising biodistribution data may be responsible for the inactivation of these cells before targeting solid tumors. Ex vivo biodistributions confirmed in vivo PET-derived biodistributions. According to these studies, we conclude that in vivo serial PET imaging with 89Zr-oxine labeled CAR T-cells provides real-time monitoring of biodistributions crucial for interpreting efficacy and guiding treatment in patient care.

Advances in Immuno-PET for the Detection of Cancer and Assessment of Response to Therapy.

BACKGROUND: Monoclonal antibodies (mAbs) against tumor-associated antigens have been shown to target tumors with specificity and selectivity; therefore, it was hypothesized that cancer could be treated with mAbs without side effects. In the early 1980s, clinical studies demonstrated that tumors could be visualized using radiolabeled mAbs. However, with the introduction of positron emission tomography (PET) with 18F-fluorodeoxyglucose (18F-FDG), antibody-based imaging became less important because of its limited diagnostic accuracy. During the last two decades, a revival of imaging with radiolabeled mAbs has taken place, specifically PET with longer half-life isotopes. Development of immune checkpoints as targets for immunotherapy has opened opportunities for the development of a wide variety of antibodies, such as anti-CTLA-4, anti-PD-L1, and anti-PD1. Thus, imaging with these antibodies radiolabeled with 89Zr or another long-half-life PET isotope, known as immuno-PET, has become mainstream. OBJECTIVE: This study aimed to review the rapid development of immuno-PET for the detection of cancer and assessment of therapeutic response combining surgery, radiation, chemotherapy, and/or immunotherapy. This review includes reports on the radiolabeling, imaging and clinical utility of 89Zr-, 64Cu- and 124I-labeled mAbs. RESULTS: More than 120 research and review articles on immuno-PET were reviewed. CONCLUSION: Many mAbs have been developed and used for the treatment of cancer; however, a limited number of antibodies have been radiolabeled for immuno-PET. While much progress has been made with the therapeutic applications of mAbs, immuno-PET for diagnosis and treatment assessment needs more research. Improved chelating agents and extensive imaging studies are needed to refine immuno-PET for the diagnosis of cancers and assessment of response to therapy.

Principles of N-Linked Glycosylation Variations of IgG-Based Therapeutics: Pharmacokinetic and Functional Considerations.

The development of recombinant therapeutic proteins has been a major revolution in modern medicine. Therapeutic-based monoclonal antibodies (mAbs) are growing rapidly, providing a potential class of human pharmaceuticals that can improve the management of cancer, autoimmune diseases, and other conditions. Most mAbs are typically of the immunoglobulin G (IgG) subclass, and they are glycosylated at the conserved asparagine position 297 (Asn-297) in the CH2 domain of the Fc region. Post-translational modifications here account for the observed high heterogeneity of glycoforms that may or not impact the stability, pharmacokinetics (PK), efficacy, and immunogenicity of mAbs. These modifications are also critical for the Fc receptor binding, and consequently, key antibody effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Moreover, mAbs produced in non-human cells express oligosaccharides that are not normally found in serum IgGs might lead to immunogenicity issues when administered to patients. This review summarizes our understanding of the terminal sugar residues, such as mannose, sialic acids, fucose, or galactose, which influence therapeutic mAbs either positively or negatively in this regard. This review also discusses mannosylation, which has significant undesirable effects on the PK of glycoproteins, causing a decreased mAbs' half-life. Moreover, terminal galactose residues can enhance CDC activities and Fc-C1q interactions, and core fucose can decrease ADCC and Fc-FcγRs binding. To optimize the therapeutic use of mAbs, glycoengineering strategies are used to reduce glyco-heterogeneity of mAbs, increase their safety profile, and improve the therapeutic efficacy of these important reagents.

A Humanized Lym-1 CAR with Novel DAP10/DAP12 Signaling Domains Demonstrates Reduced Tonic Signaling and Increased Antitumor Activity in B-Cell Lymphoma Models.

PURPOSE: The murine Lym-1 mAb targets a discontinuous epitope (Lym-1 epitope) on several subtypes of HLA-DR, which is upregulated in a majority of human B-cell lymphomas and leukemias. Unlike CD19, the Lym-1 epitope does not downregulate upon crosslinking, which may provide an advantage as a target for CAR T-cell therapy. Lym-1 CAR T cells with a conventional 4-1BB and CD3ζ (BB3z) signaling domain exhibited impaired ex vivo expansion. This study aimed to identify the underlying mechanisms and develop strategies to overcome this effect. EXPERIMENTAL DESIGN: A functional humanized Lym-1 antibody (huLym-1-B) was identified and its scFv form was used for CAR design. To overcome observed impaired expansion in vitro, a huLym-1-B CAR using DAP10 and DAP12 (DAP) signaling domains was evaluated for ex vivo expansion and in vivo function. RESULTS: Impaired expansion in huLym-1-B-BB3z CAR T cells was shown to be due to ligand-dependent suboptimal CAR signaling caused by interaction of the CAR binding domain and the surface of human T cells. Using the novel DAP signaling domain construct, the effects of suboptimal CAR signaling were overcome to produce huLym-1-B CAR T cells with improved expansion ex vivo and function in vivo. In addition, the Lym-1 epitope does not significantly downregulate in response to huLym-1-B-DAP CAR T cells both ex vivo and in vivo. CONCLUSIONS: DAP intracellular domains can serve as signaling motifs for CAR, and this new construct enables nonimpaired production of huLym-1-B CAR T cells with potent in vivo antitumor efficacy.

Biodistribution and efficacy of an anti-TENB2 antibody-drug conjugate in a patient-derived model of prostate cancer.

TENB2, a transmembrane proteoglycan protein, is a promising target for antibody drug conjugate (ADC) therapy due to overexpression in human prostate tumors and rapid internalization. We previously characterized how predosing with parental anti-TENB2 monoclonal antibody (mAb) at 1 mg/kg in a patient-derived LuCap77 explant model with high (3+) TENB2 expression could (i) block target-mediated intestinal uptake of tracer (& 0.1 mg/kg) levels of radiolabeled anti-TENB2-monomethyl auristatin E ADC while preserving tumor uptake, and (ii) maintain efficacy relative to ADC alone. Here, we systematically revisit this strategy to evaluate the effects of predosing on tumor uptake and efficacy in LuCap96.1, a low TENB2-expressing (1+) patient-derived model that is more responsive to ADC therapy than LuCap77. Importantly, rather than using tracer (& 0.1 mg/kg) levels, radiolabeled ADC tumor uptake was assessed at 1 mg/kg - one of the doses evaluated in the tumor growth inhibition study - in an effort to bridge tissue distribution (PK) with efficacy (PD). Predosing with mAb up to 1 mg/kg had no effect on efficacy. These findings warrant further investigations to determine whether predosing prior to ADC therapy might improve therapeutic index by preventing ADC disposition and possible toxicological liabilities in antigen-expressing healthy tissues.

Lym-1 Chimeric Antigen Receptor T Cells Exhibit Potent Anti-Tumor Effects against B-Cell Lymphoma.

T cells expressing chimeric antigen receptors (CARs) recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR) on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgammanull (NSG) mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluorescent protein (Raji/Luc-GFP) in NSG mice with similar or better reactivity than CD19 CAR T cells. Lym-1 CAR transduction of T cells is a promising immunotherapy for patients with Lym-1 epitope positive B-cell malignancies.

Unraveling the Effect of Immunogenicity on the PK/PD, Efficacy, and Safety of Therapeutic Proteins.

Biologics have emerged as a powerful and diverse class of molecular and cell-based therapies that are capable of replacing enzymes, editing genomes, targeting tumors, and more. As this complex array of tools arises a distinct set of challenges is rarely encountered in the development of small molecule therapies. Biotherapeutics tend to be big, bulky, polar molecules comprised of protein and/or nucleic acids. Compared to their small molecule counterparts, they are fragile, labile, and heterogeneous. Their biodistribution is often limited by hydrophobic barriers which often restrict their administration to either intravenous or subcutaneous entry routes. Additionally, their potential for immunogenicity has proven to be a challenge to developing safe and reliably efficacious drugs. Our discussion will emphasize immunogenicity in the context of therapeutic proteins, a well-known class of biologics. We set out to describe what is known and unknown about the mechanisms underlying the interplay between antigenicity and immune response and their effect on the safety, efficacy, pharmacokinetics, and pharmacodynamics of these therapeutic agents.

Systemic delivery of chTNT-3/CpG immunoconjugates for immunotherapy in murine solid tumor models.

CpG oligodeoxynucleotides (CpG) potently activate the immune system by mimicking microbial DNA. Conjugation of CpG to chTNT-3, an antibody targeting the necrotic centers of tumors, enabled CpG to accumulate in tumors after systemic delivery, where it can activate the immune system in the presence of tumor antigens. CpG chemically conjugated to chTNT-3 (chTNT-3/CpG) were compared to free CpG in their ability to stimulate the immune system in vitro and reduce tumor burden in vivo. In subcutaneous Colon 26 adenocarcinoma and B16-F10 melanoma models in BALB/c and C57BL/6 mice, respectively, chTNT-3/CpG, free CpG, or several different control constructs were administered systemically. Intraperitoneal injections of chTNT-3/CpG delayed tumor growth and improved survival and were comparable to intratumorally administered CpG. Compared to saline-treated mice, chTNT-3/CpG-treated mice had smaller average tumor volumes by as much as 72% in Colon 26-bearing mice and 79% in B16-bearing mice. Systemically delivered free CpG and CpG conjugated to an isotype control antibody did not reduce tumor burden or improve survival. In this study, chTNT-3/CpG retained immunostimulatory activity of the CpG moiety and enabled delivery to tumors. Because systemically administered CpG rapidly clear the body and do not accumulate into tumors, chTNT-3/CpG provide a solution to the limitations observed in preclinical and clinical trials.

Key factors influencing ADME properties of therapeutic proteins: A need for ADME characterization in drug discovery and development.

Protein therapeutics represent a diverse array of biologics including antibodies, fusion proteins, and therapeutic replacement enzymes. Since their inception, they have revolutionized the treatment of a wide range of diseases including respiratory, vascular, autoimmune, inflammatory, infectious, and neurodegenerative diseases, as well as cancer. While in vivo pharmacokinetic, pharmacodynamic, and efficacy studies are routinely carried out for protein therapeutics, studies that identify key factors governing their absorption, distribution, metabolism, and excretion (ADME) properties have not been fully investigated. Thorough characterization and in-depth study of their ADME properties are critical in order to support drug discovery and development processes for the production of safer and more effective biotherapeutics. In this review, we discuss the main factors affecting the ADME characteristics of these large macromolecular therapies. We also give an overview of the current tools, technologies, and approaches available to investigate key factors that influence the ADME of recombinant biotherapeutic drugs, and demonstrate how ADME studies will facilitate their future development.

Evaluating the Use of Antibody Variable Region (Fv) Charge as a Risk Assessment Tool for Predicting Typical Cynomolgus Monkey Pharmacokinetics.

The pharmacokinetic (PK) behavior of monoclonal antibodies in cynomolgus monkeys (cynos) is generally translatable to that in humans. Unfortunately, about 39% of the antibodies evaluated for PKs in cynos have fast nonspecific (or non-target-mediated) clearance (in-house data). An empirical model relating variable region (Fv) charge and hydrophobicity to cyno nonspecific clearance was developed to gauge the risk an antibody would have for fast nonspecific clearance in the monkey. The purpose of this study was to evaluate the predictability of this empirical model on cyno nonspecific clearance with antibodies specifically engineered to have either high or low Fv charge. These amino acid changes were made in the Fv region of two test antibodies, humAb4D5-8 and anti-lymphotoxin α. The humAb4D5-8 has a typical nonspecific clearance in cynos, and by making it more positively charged, the antibody acquires fast nonspecific clearance, and making it less positively charged did not impact its clearance. Anti-lymphotoxin α has fast nonspecific clearance in cynos, and making it more positively charged caused it to clear even faster, whereas making it less positively charged caused it to clear slower and within the typical range. These trends in clearance were also observed in two other preclinical species, mice and rats. The effect of modifying Fv charge on subcutaneous bioavailability was also examined, and in general bioavailability was inversely related to the direction of the Fv charge change. Thus, modifying Fv charge appears to impact antibody PKs, and the changes tended to correlate with those predicted by the empirical model.

A novel in vitro method to model the fate of subcutaneously administered biopharmaceuticals and associated formulation components.

Subcutaneous (SC) injection is becoming a more common route for the administration of biopharmaceuticals. Currently, there is no reliable in vitro method that can be used to anticipate the in vivo performance of a biopharmaceutical formulation intended for SC injection. Nor is there an animal model that can predict in vivo outcomes such as bioavailability in humans. We address this unmet need by the development of a novel in vitro system, termed Scissor (Subcutaneous Injection Site Simulator). The system models environmental changes that a biopharmaceutical could experience as it transitions from conditions of a drug product formulation to the homeostatic state of the hypodermis following SC injection. Scissor uses a dialysis-based injection chamber, which can incorporate various concentrations and combinations of acellular extracellular matrix (ECM) components that may affect the release of a biopharmaceutical from the SC injection site. This chamber is immersed in a container of a bicarbonate-based physiological buffer that mimics the SC injection site and the infinite sink of the body. Such an arrangement allows for real-time monitoring of the biopharmaceutical within the injection chamber, and can be used to characterize physicochemical changes of the drug and its interactions with ECM components. Movement of a biopharmaceutical from the injection chamber to the infinite sink compartment simulates the drug migration from the injection site and uptake by the blood and/or lymph capillaries. Here, we present an initial evaluation of the Scissor system using the ECM element hyaluronic acid and test formulations of insulin and four different monoclonal antibodies. Our findings suggest that Scissor can provide a tractable method to examine the potential fate of a biopharmaceutical formulation after its SC injection in humans and that this approach may provide a reliable and representative alternative to animal testing for the initial screening of SC formulations.

Dose dependent pharmacokinetics, tissue distribution, and anti-tumor efficacy of a humanized monoclonal antibody against DLL4 in mice.

Delta-like-4 ligand (DLL4) plays an important role in vascular development and is widely expressed on the vasculature of normal and tumor tissues. Anti-DLL4 is a humanized IgG1 monoclonal antibody against DLL4. The purpose of these studies was to characterize the pharmacokinetics (PK), tissue distribution, and anti-tumor efficacy of anti-DLL4 in mice over a range of doses. PK and tissue distribution of anti-DLL4 were determined in athymic nude mice after administration of single intravenous (IV) doses. In the tissue distribution study, radiolabeled anti-DLL4 (mixture of (125)Iodide and (111)Indium) was administered in the presence of increasing amounts of unlabeled anti-DLL4. Dose ranging anti-DLL4 anti-tumor efficacy was evaluated in athymic nude mice bearing MV522 human lung tumor xenografts. Anti-DLL4 had nonlinear PK in mice with rapid serum clearance at low doses and slower clearance at higher doses suggesting the involvement of target mediated clearance. Consistent with the PK data, anti-DLL4 was shown to specifically distribute to several normal tissues known to express DLL4 including the lung and liver. Maximal efficacy in the xenograft model was seen at doses ≥ 10 mg/kg when tissue sinks were presumably saturated, consistent with the PK and tissue distribution profiles. These findings highlight the importance of mechanistic understanding of antibody disposition to enable dosing strategies for maximizing efficacy.

Comparative physiology of mice and rats: radiometric measurement of vascular parameters in rodent tissues.

A solid understanding of physiology is beneficial in optimizing drug delivery and in the development of clinically predictive models of drug disposition kinetics. Although an abundance of data exists in the literature, it is often confounded by the use of various experimental methods and a lack of consensus in values from different sources. To help address this deficiency, we sought to directly compare three important vascular parameters at the tissue level using the same experimental approach in both mice and rats. Interstitial volume, vascular volume, and blood flow were radiometrically measured in selected harvested tissues of both species by extracellular marker infusion, red blood cell labeling, and rubidium chloride bolus distribution, respectively. The latter two parameters were further compared by whole-body autoradiographic imaging. An overall good interspecies agreement was observed for interstitial volume and blood flow on a weight-normalized basis in most tissues. In contrast, the measured vascular volumes of most rat tissues were higher than for mouse. Mice and rats, the two most commonly utilized rodent species in translational drug development, should not be considered as interchangeable in terms of vascular volume per gram of tissue. This will be particularly critical in biodistribution studies of drugs, as the amount of drug in the residual blood of tissues is often not negligible, especially for biologic drugs (e.g., antibodies) having long circulation half-lives. Physiologically based models of drug pharmacokinetics and/or pharmacodynamics also rely on accurate knowledge of biological parameters in tissues. For tissue parameters with poor interspecies agreement, the significance and possible drivers are discussed.

Quantitative cumulative biodistribution of antibodies in mice: effect of modulating binding affinity to the neonatal Fc receptor.

The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn's role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn's role in antibody PK and catabolism at the tissue level.

Enhanced tumor retention of a radiohalogen label for site-specific modification of antibodies.

A known limitation of iodine radionuclides for labeling and biological tracking of receptor targeted proteins is the tendency of iodotyrosine to rapidly diffuse from cells following endocytosis and lysosomal degradation. In contrast, radiometal-chelate complexes such as indium-111-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (In-111-DOTA) accumulate within target cells due to the residualizing properties of the polar, charged metal-chelate-amino acid adduct. Iodine radionuclides boast a diversity of nuclear properties and chemical means for incorporation, prompting efforts to covalently link radioiodine with residualizing molecules. Herein, we describe the Ugi-assisted synthesis of [I-125]HIP-DOTA, a 4-hydroxy-3-iodophenyl (HIP) derivative of DOTA, and demonstration of its residualizing properties in a murine xenograft model. Overall, this study displays the power of multicomponent synthesis to yield a versatile radioactive probe for antibodies across multiple therapeutic areas with potential applications in both preclinical biodistribution studies and clinical radioimmunotherapies.

Cytoreductive chemotherapy improves the biodistribution of antibodies directed against tumor necrosis in murine solid tumor models.

Current strategies in cancer treatment employ combinations of different treatment modalities, which include chemotherapy, radiotherapy, immunotherapy, and surgery. Consistent with that approach, the present study demonstrates how chemotherapeutic agents can potentiate the delivery of radiolabeled, necrosis-targeting antibodies (chTNT-3, NHS76) to tumor. All chemotherapeutics in this study (5-fluorouracil, etoposide, vinblastine, paclitaxel, and doxorubicin) resulted in statistically significant increases in tumor uptake of radiolabeled antibodies and their F(ab')2 fragments compared to no pretreatment with chemotherapy. Labeled antibodies were administered at various time points following a single dose of chemotherapy in multiple tumor models, and the biodistribution of the antibodies was determined by measuring radioactivity in harvested tissues. MicroPET/CT was also done to demonstrate clinical relevancy of using chemotherapy pretreatment to increase antibody uptake. Results of biodistribution and imaging data reveal specific time frames following chemotherapy when necrosis-targeting antibodies are best delivered, either for imaging or radiotherapy. Thus, the present work offers the prospect of using cytoreductive chemotherapy to increase tumor accumulation of select therapeutic antibodies, especially when combined with other forms of immunotherapy, for the successful treatment of solid tumors.