CAR T Cells Targeting MISIIR for the Treatment of Ovarian Cancer and Other Gynecologic Malignancies.

The prognosis of patients diagnosed with advanced ovarian or endometrial cancer remains poor, and effective therapeutic strategies are limited. The Müllerian inhibiting substance type 2 receptor (MISIIR) is a transforming growth factor ß (TGF-ß) receptor family member, overexpressed by most ovarian and endometrial cancers while absent in most normal tissues. Restricted tissue expression, coupled with an understanding that MISIIR ligation transmits apoptotic signals to cancer cells, makes MISIIR an attractive target for tumor-directed therapeutics. However, the development of clinical MISIIR-targeted agents has been challenging. Prompted by the responses achieved in patients with blood malignancies using chimeric antigen receptor (CAR) T cell therapy, we hypothesized that MISIIR targeting may be achieved using a CAR T cell approach. Herein, we describe the development and evaluation of a CAR that targets MISIIR. T cells expressing the MISIIR-specific CAR demonstrated antigen-specific reactivity in vitro and eliminated MISIIR-overexpressing tumors in vivo. MISIIR CAR T cells also recognized a panel of human ovarian and endometrial cancer cell lines, and they lysed a battery of patient-derived tumor specimens in vitro, without mediating cytotoxicity of a panel of normal primary human cells. In conclusion, these results indicate that MISIIR targeting for the treatment of ovarian cancer and other gynecologic malignancies is achievable using CAR technology.

Therapeutic Efficacy of a Family of pHLIP-MMAF Conjugates in Cancer Cells and Mouse Models.

The targeting of therapeutics specifically to diseased tissue is crucial for the development of successful cancer treatments. The approach here is based on the pH(low) insertion peptide (pHLIP) for the delivery of a potent mitotic inhibitor monomethyl auristatin F (MMAF). We investigated six pHLIP variants conjugated to MMAF to compare their efficacy in vitro against cultured cancer cells. While all pHLIP-MMAF conjugates exhibit potent pH- and concentration-dependent killing, their cytotoxicity profiles are remarkably different. We also show that the lead conjugate exhibits significant therapeutic efficacy in mouse models without overt toxicities. This study confirms pHLIP-monomethyl auristatin conjugates as possible new therapeutic options for cancer treatment and supports their further development.

Compounds identified by virtual docking to a tetrameric EGFR extracellular domain can modulate Grb2 internalization.

BACKGROUND: Overexpression or mutation of the epidermal growth factor receptor (EGFR) potently enhances the growth of many solid tumors. Tumor cells frequently display resistance to mechanistically-distinct EGFR-directed therapeutic agents, making it valuable to develop therapeutics that work by additional mechanisms. Current EGFR-targeting therapeutics include antibodies targeting the extracellular domains, and small molecules inhibiting the intracellular kinase domain. Recent studies have identified a novel prone extracellular tetrameric EGFR configuration, which we identify as a potential target for drug discovery. METHODS: Our focus is on the prone EGFR tetramer, which contains a novel protein-protein interface involving extracellular domain III. This EGFR tetramer is computationally targeted for stabilization by small molecule ligand binding. This study performed virtual screening of a Life Chemicals, Inc. small molecule library of 345,232 drug-like compounds against a molecular dynamics simulation of protein-protein interfaces distinct to the novel tetramer. One hundred nine chemically diverse candidate molecules were selected and evaluated using a cell-based high-content imaging screen that directly assessed induced internalization of the EGFR effector protein Grb2. Positive hits were further evaluated for influence on phosphorylation of EGFR and its effector ERK1/2. RESULTS: Fourteen hit compounds affected internalization of Grb2, an adaptor responsive to EGFR activation. Most hits had limited effect on cell viability, and minimally influenced EGFR and ERK1/2 phosphorylation. Docked hit compound poses generally include Arg270 or neighboring residues, which are also involved in binding the effective therapeutic cetuximab, guiding further chemical optimization. CONCLUSIONS: These data suggest that the EGFR tetrameric configuration offers a novel cancer drug target.

Inhibition of cancer cell proliferation and breast tumor targeting of pHLIP-monomethyl auristatin E conjugates.

Localized delivery is vital for the successful development of novel and effective therapeutics for the treatment of cancer. The targeting and delivery described herein is based on the pH (low) insertion peptide (pHLIP), a unique delivery peptide that can selectively target tumors in mice and translocate and release cargo molecules intracellularly based solely on the low extracellular pH intrinsic to cancer cells. In this study, we investigate the efficacy of pHLIP to target and deliver the highly potent and clinically validated microtubule inhibitor monomethyl auristatin E (MMAE) to cancer cells and breast tumors. We show that pHLIP-MMAE conjugates induce a potent cytotoxic effect (>90% inhibition of cell growth) in a concentration- and pH-dependent manner after only 2 h incubation without any apparent disruption of the plasma membrane. pHLIP-MMAE conjugates exhibit between an 11- and 144-fold higher antiproliferative effect at low pH than that at physiological pH and a pronounced pH-dependent cytotoxicity as compared to that of free drug. Furthermore, we demonstrate that a pHLIP-MMAE drug conjugate effectively targets triple-negative breast tumor xenografts in mice. These results indicate that pHLIP-based auristatin conjugates may have an enhanced therapeutic window as compared to that of free drug, providing a targeting mechanism to attenuate systemic toxicity.

Identification of inhibitory scFv antibodies targeting fibroblast activation protein utilizing phage display functional screens.

Fibroblast activation protein (FAP) is a serine protease selectively expressed on tumor stromal fibroblasts in epithelial carcinomas and is important in cancer growth, adhesion, and metastases. As FAP enzymatic activity is a potent therapeutic target, we aimed to identify inhibitory antibodies. Using a competitive inhibition strategy, we used phage display techniques to identify 53 single-chain variable fragments (scFvs) after three rounds of panning against FAP. These scFvs were expressed and characterized for binding to FAP by surface plasmon resonance and flow cytometry. Functional assessment of these antibodies yielded an inhibitory scFv antibody, named E3, which could attenuate 35% of FAP cleavage of the fluorescent substrate Ala-Pro-7-amido-4-trifluoromethylcoumarin compared with nonfunctional scFv control. Furthermore, a mutant E3 scFv was identified by yeast affinity maturation. It had higher affinity (4-fold) and enhanced inhibitory effect on FAP enzyme activity (3-fold) than E3. The application of both inhibitory anti-FAP scFvs significantly affected the formation of 3-dimensional FAP-positive cell matrix, as demonstrated by reducing the fibronectin fiber orientation from 41.18% (negative antibody control) to 34.06% (E3) and 36.15% (mutant E3), respectively. Thus, we have identified and affinity-maturated the first scFv antibody capable of inhibiting FAP function. This scFv antibody has the potential to disrupt the role of FAP in tumor invasion and metastasis.

IL-38 blockade induces anti-tumor immunity by abrogating tumor-mediated suppression of early immune activation.

Immune checkpoint inhibitors that overcome T cell suppressive mechanisms in tumors have revolutionized the treatment of cancer but are only efficacious in a small subset of patients. Targeting suppressive mechanisms acting on innate immune cells could significantly improve the incidence of clinical response by facilitating a multi-lineage response against the tumor involving both adaptive and innate immune systems. Here, we show that intra-tumoral interleukin (IL)-38 expression is a feature of a large frequency of head and neck, lung and cervical squamous cancers and correlates with reduced immune cell numbers. We generated IMM20324, an antibody that binds human and mouse IL-38 proteins and inhibits the binding of IL-38 to its putative receptors, interleukin 1 receptor accessory protein-like 1 (IL1RAPL) and IL-36R. In vivo, IMM20324 demonstrated a good safety profile, delayed tumor growth in a subset of mice in an EMT6 syngeneic model of breast cancer, and significantly inhibited tumor expansion in a B16.F10 melanoma model. Notably, IMM20324 treatment resulted in the prevention of tumor growth following re-implantation of tumor cells, indicating the induction of immunological memory. Furthermore, exposure of IMM20324 correlated with decreased tumor volume and increased levels of intra-tumoral chemokines. Together, our data suggest that IL-38 is expressed in a high frequency of cancer patients and allows tumor cells to suppress anti-tumor immunity. Blockade of IL-38 activity using IMM20324 can re-activate immunostimulatory mechanisms in the tumor microenvironment leading to immune infiltration, the generation of tumor-specific memory and abrogation of tumor growth.

Impact of expression system on the function of the C6.5 diabody PET radiotracer.

The ability of engineered antibodies to rapidly and selectively target tumors that express their target antigen makes them well suited for use as radioimaging tracers. The combination of molecular size and bivalent nature makes diabody molecules a particularly promising structure for use as radiotracers for diagnostic imaging. Previous data have demonstrated that the anti-HER2 C6.5 diabody (C6.5db) is an effective radiotracer in preclinical models of HER2-positive cancer. The aim of this study was to evaluate the impact on radiotracer performance, associated with expressing the C6.5db in the Pichia pastoris (P-C6.5db) system as compared to Escherichia coli (E. C6.5db). Glycosylation of P-C6.5db led to faster blood clearance and lower overall tumor uptake than seen with E. coli-produced C6.5db. However, P-C6.5db achieved high tumor/background ratios that are critical for effective imaging. Dosimetry measurements determined in this study for both (124)I-P-C6.5db and (124)I-E-C6.5db suggest that they are equivalent to other radiotracers currently being administered to patients.

Protein-intrinsic and signaling network-based sources of resistance to EGFR- and ErbB family-targeted therapies in head and neck cancer.

Agents targeting EGFR and related ErbB family proteins are valuable therapies for the treatment of many cancers. For some tumor types, including squamous cell carcinomas of the head and neck (SCCHN), antibodies targeting EGFR were the first protein-directed agents to show clinical benefit, and remain a standard component of clinical strategies for management of the disease. Nevertheless, many patients display either intrinsic or acquired resistance to these drugs; hence, major research goals are to better understand the underlying causes of resistance, and to develop new therapeutic strategies that boost the impact of EGFR/ErbB inhibitors. In this review, we first summarize current standard use of EGFR inhibitors in the context of SCCHN, and described new agents targeting EGFR currently moving through pre-clinical and clinical development. We then discuss how changes in other transmembrane receptors, including IGF1R, c-Met, and TGF-ß, can confer resistance to EGFR-targeted inhibitors, and discuss new agents targeting these proteins. Moving downstream, we discuss critical EGFR-dependent effectors, including PLC-γ; PI3K and PTEN; SHC, GRB2, and RAS and the STAT proteins, as factors in resistance to EGFR-directed inhibitors and as alternative targets of therapeutic inhibition. We summarize alternative sources of resistance among cellular changes that target EGFR itself, through regulation of ligand availability, post-translational modification of EGFR, availability of EGFR partners for hetero-dimerization and control of EGFR intracellular trafficking for recycling versus degradation. Finally, we discuss new strategies to identify effective therapeutic combinations involving EGFR-targeted inhibitors, in the context of new system level data becoming available for analysis of individual tumors.

IMM-BCP-01, a patient-derived anti-SARS-CoV-2 antibody cocktail, is active across variants of concern including Omicron BA.1 and BA.2.

Monoclonal antibodies are an efficacious therapy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, rapid viral mutagenesis led to escape from most of these therapies, outlining the need for an antibody cocktail with a broad neutralizing potency. Using an unbiased interrogation of the memory B cell repertoire of patients with convalescent COVID-19, we identified human antibodies with broad antiviral activity in vitro and efficacy in vivo against all tested SARS-CoV-2 variants of concern, including Delta and Omicron BA.1 and BA.2. Here, we describe an antibody cocktail, IMM-BCP-01, that consists of three patient-derived broadly neutralizing antibodies directed at nonoverlapping surfaces on the SARS-CoV-2 Spike protein. Two antibodies, IMM20184 and IMM20190, directly blocked Spike binding to the ACE2 receptor. Binding of the third antibody, IMM20253, to its cryptic epitope on the outer surface of RBD altered the conformation of the Spike Trimer, promoting the release of Spike monomers. These antibodies decreased Omicron SARS-CoV-2 infection in the lungs of Syrian golden hamsters in vivo and potently induced antiviral effector response in vitro, including phagocytosis, ADCC, and complement pathway activation. Our preclinical data demonstrated that the three-antibody cocktail IMM-BCP-01 could be a promising means for preventing or treating infection of SARS-CoV-2 variants of concern, including Omicron BA.1 and BA.2, in susceptible individuals.

Highly sensitive detection of HER2 extracellular domain in the serum of breast cancer patients by piezoelectric microcantilevers.

Rapid and sensitive detection of serum tumor biomarkers are needed to monitor cancer patients for disease progression. Highly sensitive piezoelectric microcantilever sensors (PEMS) offer an attractive tool for biomarker detection; however, their utility in the complex environment encountered in serum has yet to be determined. As a proof of concept, we have functionalized PEMS with antibodies that specifically bind to HER2, a biomarker (antigen) that is commonly overexpressed in the blood of breast cancer patients. The function and sensitivity of these anti-HER2 PEMS biosensors was initially assessed using recombinant HER2 spiked into human serum. Their ability to detect native HER2 present in the serum of breast cancer patients was then determined. We have found that the anti-HER2 PEMS were able to accurately detect both recombinant and naturally occurring HER2 at clinically relevant levels (>2 ng/mL). This indicates that PEMS-based biosensors provide a potentially effective tool for biomarker detection.

Unbiased interrogation of memory B cells from convalescent COVID-19 patients reveals a broad antiviral humoral response targeting SARS-CoV-2 antigens beyond the spike protein.

Patients who recover from SARS-CoV-2 infections produce antibodies and antigen-specific T cells against multiple viral proteins. Here, an unbiased interrogation of the anti-viral memory B cell repertoire of convalescent patients has been performed by generating large, stable hybridoma libraries and screening thousands of monoclonal antibodies to identify specific, high-affinity immunoglobulins (Igs) directed at distinct viral components. As expected, a significant number of antibodies were directed at the Spike (S) protein, a majority of which recognized the full-length protein. These full-length Spike specific antibodies included a group of somatically hypermutated IgMs. Further, all but one of the six COVID-19 convalescent patients produced class-switched antibodies to a soluble form of the receptor-binding domain (RBD) of S protein. Functional properties of anti-Spike antibodies were confirmed in a pseudovirus neutralization assay. Importantly, more than half of all of the antibodies generated were directed at non-S viral proteins, including structural nucleocapsid (N) and membrane (M) proteins, as well as auxiliary open reading frame-encoded (ORF) proteins. The antibodies were generally characterized as having variable levels of somatic hypermutations (SHM) in all Ig classes and sub-types, and a diversity of VL and VH gene usage. These findings demonstrated that an unbiased, function-based approach towards interrogating the COVID-19 patient memory B cell response may have distinct advantages relative to genomics-based approaches when identifying highly effective anti-viral antibodies directed at SARS-CoV-2.

Effective treatment of established human breast tumor xenografts in immunodeficient mice with a single dose of the alpha-emitting radioisotope astatine-211 conjugated to anti-HER2/neu diabodies.

PURPOSE: Successful radioimmunotherapy strategies depend on selecting radioisotopes with physical properties complementary to the biological properties of the targeting vehicle. Small, engineered antitumor antibody fragments are capable of rapid, highly specific tumor targeting in immunodeficient mouse models. We hypothesized that the C6.5 diabody, a noncovalent anti-HER2 single-chain Fv dimer, would be an ideal radioisotope carrier for the radioimmunotherapy of established tumors using the short-lived alpha-emitting radioisotope (211)At. EXPERIMENTAL DESIGN: Immunodeficient nude mice bearing established HER2/neu-positive MDA-MB-361/DYT2 tumors treated with N-succinimidyl N-(4-[(211)At]astatophenethyl)succinamate ((211)At-SAPS)-C6.5 diabody. Additional cohorts of mice were treated with (211)At-SAPS T84.66 diabody targeting the carcinoembryonic antigen or (211)At-SAPS on a diabody specific for the Müllerian inhibiting substance type II receptor, which is minimally expressed on this tumor cell line. RESULTS: A single i.v. injection of (211)At-SAPS C6.5 diabody led to a 30-day delay in tumor growth when a 20 muCi dose was administered and a 57-day delay in tumor growth (60% tumor-free after 1 year) when a 45 muCi dose was used. Treatment of mice bearing the same tumors with (211)At-SAPS T84.66 diabody at the same doses led to a delay in tumor growth, but no complete responses, likely due to substantially lower expression of this antigen on the MDA-MB-361/DYT2 tumors. In contrast, a dose of 20 muCi of (211)At-SAPS on the anti-Müllerian-inhibiting substance type II receptor diabody did not affect tumor growth rate, demonstrating specificity of the therapeutic effect. CONCLUSIONS: These findings indicate that diabody molecules can be effective agents for targeted radioimmunotherapy of solid tumors using powerful, short-lived alpha-emitting radioisotopes.

Quantitative immuno-positron emission tomography imaging of HER2-positive tumor xenografts with an iodine-124 labeled anti-HER2 diabody.

Positron emission tomography (PET) provides an effective means of both diagnosing/staging several types of cancer and evaluating efficacy of treatment. To date, the only U.S. Food and Drug Administration-approved radiotracer for oncologic PET is (18)F-fluoro-deoxyglucose, which measures glucose accumulation as a surrogate for malignant activity. Engineered antibody fragments have been developed with the appropriate targeting specificity and systemic elimination properties predicted to allow for effective imaging of cancer based on expression of tumor associated antigens. We evaluated a small engineered antibody fragment specific for the HER2 receptor tyrosine kinase (C6.5 diabody) for its ability to function as a PET radiotracer when labeled with iodine-124. Our studies revealed HER2-dependent imaging of mouse tumor xenografts with a time-dependent increase in tumor-to-background signal over the course of the experiments. Radioiodination via an indirect method attenuated uptake of radioiodine in tissues that express the Na/I symporter without affecting the ability to image the tumor xenografts. In addition, we validated a method for using a clinical PET/computed tomography scanner to quantify tumor uptake in small-animal model systems; quantitation of the tumor targeting by PET correlated with traditional necropsy-based analysis at all time points analyzed. Thus, diabodies may represent an effective molecular structure for development of novel PET radiotracers.

Development of engineered antibodies specific for the Müllerian inhibiting substance type II receptor: a promising candidate for targeted therapy of ovarian cancer.

The Müllerian inhibiting substance type II receptor (MISIIR) is involved in Müllerian duct regression as part of the development of the male reproductive system. In adult females, MISIIR is present on ovarian surface epithelium and is frequently expressed on human epithelial ovarian cancer cells. Müllerian inhibiting substance has been found to be capable of inhibiting the growth of primary human ovarian cancer cells derived from ascites and ovarian cancer cell lines. This suggested to us that MISIIR could be an attractive target for antibody-based tumor targeting and growth inhibition strategies. Here, we describe the production of recombinant human MISIIR extracellular domain-human immunoglobulin Fc domain fusion proteins and their use as targets for the selection of MISIIR-specific human single-chain variable fragments (scFv) molecules from a human nonimmune scFv phage display library. The binding kinetics of the resulting anti-MISIIR scFv clones were characterized and two were employed as the basis for the construction of bivalent scFv:Fc antibody-based molecules. Both bound specifically to human ovarian carcinoma cells in flow cytometry assays and cross-reacted with mouse MISIIR. These results indicate that antibody-based constructs may provide a highly specific means of targeting MISIIR on human ovarian carcinoma cells for the purpose of diagnosing and treating this disease.

Cerenkov Luminescence Imaging as a Modality to Evaluate Antibody-Based PET Radiotracers.

Antibodies, and engineered antibody fragments, labeled with radioisotopes are being developed as radiotracers for the detection and phenotyping of diseases such as cancer. The development of antibody-based radiotracers requires extensive characterization of their in vitro and in vivo properties, including their ability to target tumors in an antigen-selective manner. In this study, we investigated the use of Cerenkov luminescence imaging (CLI) as compared with PET as a modality for evaluating the in vivo behavior of antibody-based radiotracers. METHODS: The anti-prostate-specific membrane antigen (PSMA) huJ591 antibody (IgG; 150 kDa) and its minibody (Mb; 80 kDa) format were functionalized with the chelator 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) and radiolabeled with the positron-emitting radionuclide 64Cu (half-life, 12.7 h). Immunoreactive preparations of the radiolabeled antibodies were injected into NCr nu/nu mice harboring PSMA-positive CWR22Rv1 and PSMA-negative PC-3 tumor xenografts. Tumor targeting was evaluated by both PET and CLI. RESULTS: 64Cu-NODAGA-PSMA-IgG and 64Cu-NODAGA-PSMA-Mb retained the ability to bind cell surface PSMA, and both radiotracers exhibited selective uptake into PSMA-positive tumors. Under the experimental conditions used, PSMA-selective uptake of 64Cu-NODAGA-PSMA-IgG and 64Cu-NODAGA-PSMA-Mb was observed by CLI as early as 3 h after injection, with tumor-to-background ratios peaking at 24 (IgG) and 16 (Mb) h after injection. Targeting data generated by CLI correlated with that generated by PET and necropsy. CONCLUSION: CLI provided a rapid and simple assessment of the targeting specificity and pharmacokinetics of the antibody-based PET radiotracers that correlated well with the behavior observed by standard PET imaging. Moreover, CLI provided clear discrimination between uptake kinetics of an intact IgG and its small-molecular-weight derivative Mb. These data support the use of CLI for the evaluation of radiotracer performance.

Isolation of scFvs to in vitro produced extracellular domains of EGFR family members.

The members of the epidermal growth factor receptor (EGFR) family are over expressed in a variety of malignancies and are frequently linked to aggressive disease and a poor prognosis. Although clinically effective monoclonal antibodies (MAbs) have been developed to target HER2 and EGFR, the remaining two family members, HER3 and HER4, have not been the subject of significant efforts. In this paper, we have taken the initial steps required to generate antibodies with potential clinically utility that target the members of the EGFR family. The genes for the extracellular domains (ECDs) of all four members of the EGFR family were cloned and used to stably transfect 293 (HEK) cells. Milligram quantities of each ECD were produced and characterized. The HER3, HER4, and EGFR ECDs were then employed as targets for the selection of antibodies from naïve human scFv (single-chain Fv) phage display libraries. Six unique scFv clones were isolated that bound specifically to HER3, 13 unique clones were isolated with specificity for HER4 and 52 unique anti-EGFR clones were isolated. These scFvs provide a valuable and potentially clinically relevant panel of agents to target the members of the EGFR family.

Oligoclonal antibodies to target the ErbB family.

INTRODUCTION: Use of mAbs to inhibit signaling through the ErbB receptor tyrosine kinase family has proven to be an effective strategy for treating ErbB-driven cancers. Advances in the field of antibody engineering and manufacturing now allow us to more effectively mimic the natural immune response by generating oligoclonal mixtures of antibodies against desired targets of interest. AREAS COVERED: In this review, we examine the literature describing the development of oligoclonal mixtures of antibodies against ErbB family members and the impact of those mixtures on preclinical and clinical efficacy. EXPERT OPINION: Oligoclonal antibodies, facilitated by the improved antibody engineering and manufacturing techniques, hold the promise of improving patient outcomes. Through the use of empirical methods, oligoclonal mixtures with enhanced capacity to block signaling through ErbB family members can be identified. The intrinsic mechanisms associated with each of the component mAbs provide an opportunity to block signaling via multiple mechanisms of action. In addition, combinations of antibodies targeting multiple ErbB family members provide the capacity to down-regulate signaling through multiple components of this critical pathway.

Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the VL framework using a structure-guided approach.

Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a VH and VL domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in VH and VL domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the VL domain to one that best pairs with the existing VH. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the VL domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability.

Quantitation of small-animal (124)I activity distributions using a clinical PET/CT scanner.

UNLABELLED: Time-dependent PET imaging can be an important tool in the assessment of radiotracer performance in murine models. We have performed a quantitative analysis of PET images of (124)I, acquired on a clinical PET system using a small-animal phantom. We then compared the recovered activity concentrations with the known activity concentration in the phantom spheres. The recovery coefficients found from the phantom data were applied to in vivo (124)I anti-HER2/neu C6.5 diabody PET data and compared with necropsy biodistribution data from the same tumor-bearing immunodeficient mouse. METHODS: The small-animal phantom consisted of a 4 x 8 cm water-filled acrylic cylinder with hollow spheres filled with water ranging in volume from 0.0625 to 1.0 mL and activity concentration of 27 +/- 2 kBq/mL. The background activity concentrations varied from 0 to 0.05 to 0.10 of the spheres. Data were acquired at 0, 5, and 10 cm from the scanner longitudinal axis. Recovery coefficients were theoretically calculated for spheres of different volume, background-to-target concentrations, and distance from the scanner's longitudinal axis. The theoretic recovery coefficients were applied to the maximum sphere activity concentration measured from the PET images, thus obtaining a recovered activity concentration to be compared with the known activity concentration of the spheres. RESULTS: The mean recovered activity concentration for the phantom spheres was 25 +/- 2 kBq/mL. The (124)I diabody PET image of a mouse with a tumor xenograft was then analyzed using the techniques described. The tumor percentage injected dose per gram estimated from the murine PET image (4.8 +/- 0.4) compared well with those obtained from necropsy studies (5.1). CONCLUSION: This study indicates the feasibility of performing quantitative imaging on murine (124)I antibody fragment PET images using a large-bore clinical scanner, which enables high-throughput studies to evaluate the performance of PET tracers in a timely and cost-effective manner by imaging multiple animals simultaneously. Tracers deemed promising by this screening method can then be further evaluated using traditional necropsy studies. Our group is currently conducting time-dependent (124)I diabody PET and necropsy comparative studies with larger numbers of mice.

Combining anti-ERBB3 antibodies specific for domain I and domain III enhances the anti-tumor activity over the individual monoclonal antibodies.

BACKGROUND: Inappropriate signaling through the epidermal growth factor receptor family (EGFR1/ERBB1, ERBB2/HER2, ERBB3/HER3, and ERBB4/HER4) of receptor tyrosine kinases leads to unregulated activation of multiple downstream signaling pathways that are linked to cancer formation and progression. In particular, ERBB3 plays a critical role in linking ERBB signaling to the phosphoinositide 3-kinase and Akt signaling pathway and increased levels of ERBB3-dependent signaling is also increasingly recognized as a mechanism for acquired resistance to ERBB-targeted therapies. METHODS: We had previously reported the isolation of a panel of anti-ERBB3 single-chain Fv antibodies through use of phage-display technology. In the current study scFv specific for domain I (F4) and domain III (A5) were converted into human IgG1 formats and analyzed for efficacy. RESULTS: Treatment of cells with an oligoclonal mixture of the A5/F4 IgGs appeared more effective at blocking both ligand-induced and ligand-independent signaling through ERBB3 than either single IgG alone. This correlated with improved ability to inhibit the cell growth both as a single agent and in combination with other ERBB-targeted therapies. Treatment of NCI-N87 tumor xenografts with the A5/F4 oligoclonal led to a statistically significant decrease in tumor growth rate that was further enhanced in combination with trastuzumab. CONCLUSION: These results suggest that an oligoclonal antibody mixture may be a more effective approach to downregulate ERBB3-dependent signaling.