Epithelial cell-expressed type II IL-4 receptor mediates eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic, food-driven allergic disease, characterized by eosinophil-rich inflammation in the esophagus. The histopathological and clinical features of EoE have been attributed to overproduction of the type 2 cytokines IL-4 and IL-13, which mediate profound alterations in the esophageal epithelium and neutralizing of their shared receptor component (IL-4Rα) with a human antibody drug (dupilumab) demonstrates clinical efficacy. Yet, the relative contribution of IL-4 and IL-13 and whether the type II IL-4 receptor (comprised of the IL-4Rα chain in association with IL-13Rα1) mediates this effect has not been determined. METHODS: Experimental EoE was induced in WT, Il13ra1-/- , and Krt14Cre /Il13ra1fl/fl mice by skin-sensitized using 4-ethoxymethylene-2-phenyl-2-oxazolin (OXA) followed by intraesophageal challenges. Esophageal histopathology was determined histologically. RNA was extracted and sequenced for transcriptome analysis and compared with human EoE RNAseq data. RESULTS: Induction of experimental EoE in mice lacking Il13ra1 and in vivo IL-13 antibody-based neutralization experiments blocked antigen-induced esophageal epithelial and lamina propria thickening, basal cell proliferation, eosinophilia, and tissue remodeling. In vivo targeted deletion of Il13ra1 in esophageal epithelial cells rendered mice protected from experimental EoE. Single-cell RNA sequencing analysis of human EoE biopsies revealed predominant expression of IL-13Rα1 in epithelial cells and that EoE signature genes correlated with IL-13 expression compared with IL-4. CONCLUSIONS: We demonstrate a definitive role for IL-13 signaling via IL-13Rα1 in EoE. These data provide mechanistic insights into the mode of action of current therapies in EoE and highlight the type II IL-4R as a future therapeutic target.

Autoantibodies to Annexin A2 and cerebral thrombosis: Insights from a mouse model.

INTRODUCTION: Antiphospholipid syndrome (APS) is an autoimmune disorder manifested by thromboembolic events, recurrent spontaneous abortions and elevated titers of circulating antiphospholipid antibodies. In addition, the presence of antiphospholipid antibodies seems to confer a fivefold higher risk for stroke or transient ischemic attack. Although the major antigen of APS is ß2 glycoprotein I, it is now well established that antiphospholipid antibodies are heterogeneous and bind to various targets. Recently, antibodies to Annexin A2 (ANXA2) have been reported in APS. This is of special interest since data indicated ANXA2 as a key player in fibrinolysis. Therefore, in the present study we assessed whether anti-ANXA2 antibodies play a pathological role in thrombosis associated disease. MATERIALS AND METHODS: Mice were induced to produce anti-ANXA2 antibodies by immunization with ANXA2 (iANXA2) and control mice were immunized with adjuvant only. A middle cerebral artery occlusion stroke model was applied to the mice. The outcome of stroke severity was assessed and compared between the two groups. RESULTS: Our results indicate that antibodies to ANXA2 lead to a more severe stroke as demonstrated by a significant larger stroke infarct volume (iANXA2 133.9 ± 3.3 mm3 and control 113.7 ± 7.4 mm3; p = 0.017) and a more severe neurological outcome (iANXA2 2.2 ± 0.2, and control 1.5 ± 0.18; p = 0.03). CONCLUSIONS: This study supports the hypothesis that auto-antibodies to ANXA2 are an independent risk factor for cerebral thrombosis. Consequently, we propose screening for anti-ANXA2 antibodies should be more widely used and patients that exhibit the manifestations of APS should be closely monitored by physicians.

Unravelling plasmidome distribution and interaction with its hosting microbiome.

Horizontal gene transfer via plasmids plays a pivotal role in microbial evolution. The forces that shape plasmidomes functionality and distribution in natural environments are insufficiently understood. Here, we present a comparative study of plasmidomes across adjacent microbial environments present in different individual rumen microbiomes. Our findings show that the rumen plasmidome displays enormous unknown functional potential currently unannotated in available databases. Nevertheless, this unknown functionality is conserved and shared with published rat gut plasmidome data. Moreover, the rumen plasmidome is highly diverse compared with the microbiome that hosts these plasmids, across both similar and different rumen habitats. Our analysis demonstrates that its structure is shaped more by stochasticity than selection. Nevertheless, the plasmidome is an active partner in its intricate relationship with the host microbiome with both interacting with and responding to their environment.

In vivo anti-MUC1+ tumor activity and sequences of high-affinity anti-MUC1-SEA antibodies.

Cleavage of the MUC1 glycoprotein yields two subunits, an extracellular alpha-subunit bound to a smaller transmembrane beta-subunit. Monoclonal antibodies (mAbs) directed against the MUC1 alpha-beta junction comprising the SEA domain, a stable cell-surface moiety, were generated. Sequencing of all seven anti-SEA domain mAbs showed that they clustered into four groups and sequences of all groups are presented here. mAb DMB5F3 with picomolar affinity for the MUC1 SEA target was selected for further evaluation. Immunohistochemical staining of a series of malignancies with DMB5F3 including lung, prostate, breast, colon, and pancreatic carcinomas revealed qualitative and qualitative differences between MUC1 expression on normal versus malignant cells: DMB5F3 strongly stained malignant cells in a near-circumferential pattern, whereas MUC1 in normal pancreatic and breast tissue showed only weak apical positivity of ductal/acinar cells. Humanized chimeric DMB5F3 linked to ZZ-PE38 (ZZ IgG-binding protein fused to Pseudomonas exotoxin) induced vigorous cytotoxicity of MUC1+ malignant cells in vitro. The intensity of cell killing correlated with the level of MUC1 expression by the target cell, suggesting a MUC1 expression threshold for cell killing. MUC1+ Colo357 pancreatic cancer cells xenotransplanted into nude and SCID mice models were treated with the chDMB5F3:ZZ-PE38 immunocomplex. In both transplant models, chDMB5F3:ZZ-PE38 exhibited significant in vivo anti-tumor activity, suppressing up to 90% of tumor volume in the SCID model compared with concomitant controls. The efficacy of chDMB5F3:ZZ-PE38 immunotoxin in mediating tumor killing both in vitro and in vivo strongly suggests a clinical role for anti-MUC1 SEA antibody in the treatment of MUC1-expressing malignancies.

Immune checkpoint inhibitor combinations: Current efforts and important aspects for success.

Immune checkpoint inhibitors (ICI) have emerged as a remarkable treatment option for diverse cancer types. Currently, ICIs are approved for an expanding array of cancer indications. However, the majority of patients still do not demonstrate a durable long-term response following ICI therapy. In addition, many patients receiving ICI therapy develop immune-related adverse events (irAEs) affecting a wide variety of organs. To increase the percentage of patients who benefit from ICI therapy and to reduce the occurrence of irAEs, there is an ongoing effort to combine current ICIs with novel checkpoints inhibitors or other therapeutic approaches to achieve a synergistic effect which is larger than the sum of its parts. In this review we highlight the essential factors for more effective ICI combinations. We describe how the design of these strategies should be driven by the tumor's immunological context. We analyze current combination strategies and describe how they can be improved to unleash the immune system's full anti-cancer potential as well as convert immunologically "cold" tumors into "hot" ones. We examine the efforts to combine current ICIs (PD-1 and CTLA-4) with novel checkpoints (TIM-3, LAG-3, VISTA, TIGIT and others), immunotherapies (CAR-T cells and Cancer Vaccines) and delivery strategies (bispecific antibodies and other delivery platforms). Importantly, we outline how can one optimally combine ICIs with traditional pillars of cancer therapy such as radiation therapy (RT) and chemotherapy. We discuss the considerations regarding successful combination with RT and chemotherapy; these include fractionation schemes and selection of chemotherapeutics which can both directly eradicate cancer cells as well as increase the infiltration of immune cells into tumors. Finally, we critically assess these approaches and attempt to establish their strengths and weaknesses based on pre-clinical and clinical data.

Universal Biofactor-Releasing Scaffold Enabling in Vivo Reloading.

The supply of growth factors to engineered tissues is essential for many physiological processes. These processes include the proper organization of the cells into functioning tissues, maintenance of their viability, vasculogenesis, proliferation, and differentiation. Systems to efficiently control the release of growth factors were previously incorporated into tissue engineering scaffolds to affect cells. However, because the initial concentration of the factors in these systems is finite, their ability to provide a long-term physiological effect is limited. Here, we report on a new reloadable system in which 3D fibrous scaffolds conjugated with an anti His-tag antibody enable the retention and controlled release of any His-tag-modified proteinaceous growth factor. The scaffolds can be reloaded in vitro or in vivo with any His-tagged biomolecule at any time according to the physiological need. We show the ability of the scaffolds to release angiogenic factors in a static cell culture or under flow in a microfluidics device and effect on endothelial cells. We also demonstrate the potential of the system to be sequentially reloaded in vivo with various factors, and as a proof of concept, we provide evidence for the efficient in vivo vascularization of scaffolds after reloading with tagged VEGF.

Novel immune check point inhibiting antibodies in cancer therapy-Opportunities and challenges.

Drug resistance of tumor cells to chemotherapy is limiting the therapeutic efficacy of most anticancer drugs and represents a major obstacle in medical oncology. However, treatment of various human malignancies with biologics, mostly monoclonal antibodies (mAbs), is not limited by such chemoresistance mechanisms. However, other resistance or evasion mechanisms limit the efficacy to anticancer therapeutic mAbs that engage tumor-associated antigens on the surface of the malignant cells. Immune checkpoint blocking monoclonal antibodies are heralded as a promising therapeutic approach in clinical oncology. These mAbs do not directly attack the malignant cells as most anticancer mAbs; rather, they enhance the anti-tumor response of the immune system by targeting immune regulatory pathways. Three mAbs targeting immune checkpoint molecules are currently used in the clinic and new mAbs that target other potential inhibitory targets are being actively investigated. This therapeutic approach, while proving as highly beneficial for many patients, is prone to toxicities and side effects of an autoimmune nature. Defining suitable management algorithms and biomarkers that predict therapeutic effects and adverse toxicity are required to provide survival benefit for larger numbers of cancer patients. Overcoming these challenges, along with opportunities for new agents and combinatorial strategies are the main focus of immune checkpoint blockade research today.

Sustained secretion of anti-tumor necrosis factor α monoclonal antibody from ex vivo genetically engineered dermal tissue demonstrates therapeutic activity in mouse model of rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a symmetric inflammatory polyarthritis associated with high concentrations of pro-inflammatory, cytokines including tumor necrosis factor (TNF)-α. Adalimumab is a monoclonal antibody (mAb) that binds TNF-α, and is widely used to treat RA. Despite its proven clinical efficacy, adalimumab and other therapeutic mAbs have disadvantages, including the requirement for repeated bolus injections and the appearance of treatment limiting anti-drug antibodies. To address these issues, we have developed an innovative ex vivo gene therapy approach, termed transduced autologous restorative gene therapy (TARGT), to produce and secrete adalimumab for the treatment of RA. METHODS: Helper-dependent (HD) adenovirus vector containing adalimumab light and heavy chain coding sequences was used to transduce microdermal tissues and cells of human and mouse origin ex vivo, rendering sustained secretion of active adalimumab. The genetically engineered tissues were subsequently implanted in a mouse model of RA. RESULTS: Transduced human microdermal tissues implanted in SCID mice demonstrated 49 days of secretion of active adalimumab in the blood, at levels of tens of microgram per milliliter. In addition, transduced autologous dermal cells were implanted in the RA mouse model and demonstrated statistically significant amelioration in RA symptoms compared to naïve cell implantation and were similar to recombinant adalimumab bolus injections. CONCLUSIONS: The results of the present study report microdermal tissues engineered to secrete active adalimumab as a proof of concept for sustained secretion of antibody from the novel ex vivo gene therapy TARGT platform. This technology may now be applied to a range of antibodies for the therapy of other diseases.

Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes.

Nonhuman proteins have valuable therapeutic properties, but their efficacy is limited by neutralizing antibodies. Recombinant immunotoxins (RITs) are potent anticancer agents that have produced many complete remissions in leukemia, but immunogenicity limits the number of doses that can be given to patients with normal immune systems. Using human cells, we identified eight helper T-cell epitopes in PE38, a portion of the bacterial protein Pseudomonas exotoxin A which consists of the toxin moiety of the RIT, and used this information to make LMB-T18 in which three epitopes were deleted and five others diminished by point mutations in key residues. LMB-T18 has high cytotoxic and antitumor activity and is very resistant to thermal denaturation. The new immunotoxin has a 93% decrease in T-cell epitopes and should have improved efficacy in patients because more treatment cycles can be given. Furthermore, the deimmunized toxin can be used to make RITs targeting other antigens, and the approach we describe can be used to deimmunize other therapeutically useful nonhuman proteins.

Cross-immunogenicity: antibodies to infliximab in Remicade-treated patients with IBD similarly recognise the biosimilar Remsima.

OBJECTIVE: The cross-immunogenicity of the recently approved infliximab-biosimilar Remsima (CT-P13) with the originator drug Remicade is still unknown. DESIGN: Sera of patients with IBD with or without measurable anti-Remicade antibodies to infliximab (ATI) were tested for their cross-reactivity to two batches of Remsima. Experiments were repeated after deglycosylation of Remicade/Remsima, IgG purification, excipients' dialysis and monomer purification by size exclusion chromatography. Anti-Remicade antibodies were tested for their functional inhibition of TNF-α binding by Remsima/Remicade by competition assay. Cross-reactivity of anti-adalimumab antibodies with Remicade/Remsima was also investigated. RESULTS: 125 patients' and controls' sera were tested (median age 31 years, IQR 24.5-39.5). All 56 anti-Remicade ATI-negative controls (14 healthy individuals, 42 patients with IBD) were also negative for anti-Remsima ATI. All 69 positive anti-Remicade IBD sera were cross-reactive with Remsima. ATI titres against Remicade or Remsima were strongly correlated (r values between 0.92 and 0.99, p<0.001 for all experiments, Spearman's correlation test). The background ELISA signal for Remsima was slightly higher compared with Remicade in negative controls (1.25±0.6 µg/mL vs 0.76±0.5 µg/mL, respectively, p<0.001), and persisted after deglycosylation, dialysis or protein size filtration, but abolished by IgG purification and significantly diminished by monomer purification. Anti-Remicade ATIs of patients with IBD (n=10) exerted similar functional inhibition on Remsima or Remicade TNF-α binding capacity (p=NS for all inhibition curve points). Antibodies-to-adalimumab in adalimumab-treated patients with IBD (n=7) did not cross-react with either Remicade or Remsima. CONCLUSIONS: Anti-Remicade antibodies in patients with IBD recognise and functionally inhibit Remsima to a similar degree, suggesting similar immunogenicity and shared immunodominant epitopes on these two infliximab agents. In contrast, anti-adalimumab antibodies do not cross-react with Remsima or Remicade.

Antibody-targeted drugs and drug resistance--challenges and solutions.

Antibody-based therapy of various human malignancies has shown efficacy in the past 30 years and is now one of the most successful and leading strategies for targeted treatment of patients harboring hematological malignancies and solid tumors. Antibody-drug conjugates (ADCs) aim to take advantage of the affinity and specificity of monoclonal antibodies (mAbs) to selectively deliver potent cytotoxic drugs to antigen-expressing tumor cells. Key parameters for ADC include choosing the optimal components of the ADC (the antibody, the linker and the cytotoxic drug) and selecting the suitable cell-surface target antigen. Building on the success of recent FDA approval of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla), ADCs are currently a class of drugs with a robust pipeline with clinical applications that are rapidly expanding. The more ADCs are being evaluated in preclinical models and clinical trials, the clearer are becoming the parameters and the challenges required for their therapeutic success. This rapidly growing knowledge and clinical experience are revealing novel modalities and mechanisms of resistance to ADCs, hence offering plausible solutions to such challenges. Here, we review the key parameters for designing a powerful ADC, focusing on how ADCs are addressing the challenge of multiple drug resistance (MDR) and its rational overcoming.

Insights into the bovine rumen plasmidome.

Plasmids are self-replicating genetic elements capable of mobilization between different hosts. Plasmids often serve as mediators of lateral gene transfer, a process considered to be a strong and sculpting evolutionary force in microbial environments. Our aim was to characterize the overall plasmid population in the environment of the bovine rumen, which houses a complex and dense microbiota that holds enormous significance for humans. We developed a procedure for the isolation of total rumen plasmid DNA, termed rumen plasmidome, and subjected it to deep sequencing using the Illumina paired-end protocol and analysis using public and custom-made bioinformatics tools. A large number of plasmidome contigs aligned with plasmids of rumen bacteria isolated from different locations and at various time points, suggesting that not only the bacterial taxa, but also their plasmids, are defined by the ecological niche. The bacterial phylum distribution of the plasmidome was different from that of the rumen bacterial taxa. Nevertheless, both shared a dominance of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. Evidently, the rumen plasmidome is of a highly mosaic nature that can cross phyla. Interestingly, when we compared the functional profile of the rumen plasmidome to two plasmid databases and two recently published rumen metagenomes, it became apparent that the rumen plasmidome codes for functions, which are enriched in the rumen ecological niche and could confer advantages to their hosts, suggesting that the functional profiles of mobile genetic elements are associated with their environment, as has been previously implied for viruses.

Identification and elimination of an immunodominant T-cell epitope in recombinant immunotoxins based on Pseudomonas exotoxin A.

Recombinant immunotoxins (RITs) are chimeric proteins that are being developed for cancer treatment. We have produced RITs that contain PE38, a portion of the bacterial protein Pseudomonas exotoxin A. Because the toxin is bacterial, it often induces neutralizing antibodies, which limit the number of treatment cycles and the effectiveness of the therapy. Because T cells are essential for antibody responses to proteins, we adopted an assay to map the CD4(+) T-cell epitopes in PE38. We incubated peripheral blood mononuclear cells with an immunotoxin to stimulate T-cell expansion, followed by exposure to overlapping peptide fragments of PE38 and an IL-2 ELISpot assay to measure responses. Our observation of T-cell responses in 50 of 50 individuals correlates with the frequency of antibody formation in patients with normal immune systems. We found a single, highly immunodominant epitope in 46% (23/50) of the donors. The immunodominant epitope is DRB1-restricted and was observed in subjects with different HLA alleles, indicating promiscuity. We identified two amino acids that, when deleted or mutated to alanine, eliminated the immunodominant epitope, and we used this information to construct mutant RITs that are highly cytotoxic and do not stimulate T-cell responses in many donors.

Behavioral and neural effects of intra-striatal infusion of anti-streptococcal antibodies in rats.

Group A ß-hemolytic streptococcal (GAS) infection is associated with a spectrum of neuropsychiatric disorders. The leading hypothesis regarding this association proposes that a GAS infection induces the production of auto-antibodies, which cross-react with neuronal determinants in the brain through the process of molecular mimicry. We have recently shown that exposure of rats to GAS antigen leads to the production of anti-neuronal antibodies concomitant with the development of behavioral alterations. The present study tested the causal role of the antibodies by assessing the behavior of naïve rats following passive transfer of purified antibodies from GAS-exposed rats. Immunoglobulin G (IgG) purified from the sera of GAS-exposed rats was infused directly into the striatum of naïve rats over a 21-day period. Their behavior in the induced-grooming, marble burying, food manipulation and beam walking assays was compared to that of naïve rats infused with IgG purified from adjuvant-exposed rats as well as of naïve rats. The pattern of in vivo antibody deposition in rat brain was evaluated using immunofluorescence and colocalization. Infusion of IgG from GAS-exposed rats to naïve rats led to behavioral and motor alterations partially mimicking those seen in GAS-exposed rats. IgG from GAS-exposed rats reacted with D1 and D2 dopamine receptors and 5HT-2A and 5HT-2C serotonin receptors in vitro. In vivo, IgG deposits in the striatum of infused rats colocalized with specific brain proteins such as dopamine receptors, the serotonin transporter and other neuronal proteins. Our results demonstrate the potential pathogenic role of autoantibodies produced following exposure to GAS in the induction of behavioral and motor alterations, and support a causal role for autoantibodies in GAS-related neuropsychiatric disorders.

On the limited recognition of inorganic surfaces by short peptides compared with antibodies.

The vast potential applications of biomolecules that bind inorganic surfaces led mostly to the isolation of short peptides that target selectively specific materials. The demonstrated differential affinity toward certain surfaces created the impression that the recognition capacity of short peptides may match that of rigid biomolecules. In the following, we challenge this view by comparing the capacity of antibody molecules to discriminate between the (100) and (111A) facets of a gallium arsenide semiconductor crystal with the capacity of short peptides to do the same. Applying selection from several peptide and single chain phage display libraries, we find a number of antibody molecules that bind preferentially a given crystal facet but fail to isolate, in dozens of attempts, a single peptide capable of such recognition. The experiments underscore the importance of rigidity to the recognition of inorganic flat targets and therefore set limitations on potential applications of short peptides in biomimetics.

Antibody Isolation from Human Synthetic Libraries of Single-Chain Antibodies and Analysis Using NGS.

Antibody libraries came into existence 30 years ago when the accumulating sequence data of immunoglobulin genes and the advent of PCR technology made it possible to clone antibody gene repertoires. Phage display (most common) and additional display and screening technologies were applied to pan out desired binding specificities from antibody libraries. As other antibody discovery tools, phage display is not an off-the-shelf technology and not offered as a kit but rather requires experience and expertise for making it indeed very useful.Next-generation sequencing (NGS) coupled with bioinformatics is a powerful tool for analyzing large amount of DNA sequence output of the panning. Here, we demonstrate how NGS analysis of phage biopanning (phage-Seq) of complex antibody libraries can facilitate the antibody discovery process and provide insights regarding the biopanning process (see Fig. 1).

Lipid nanoparticles-loaded with toxin mRNA represents a new strategy for the treatment of solid tumors.

Background and rationale: Cancer therapy have evolved remarkably over the past decade, providing new strategies to inhibit cancer cell growth using immune modulation, with or without gene therapy. Specifically, suicide gene therapies and immunotoxins have been investigated for the treatment of tumors by direct cancer cell cytotoxicity. Recent advances in mRNA delivery also demonstrated the potential of mRNA-based vaccines and immune-modulators for cancer therapeutics by utilizing nanocarriers for mRNA delivery. Methods: We designed a bacterial toxin-encoding modified mRNA, delivered by lipid nanoparticles into a B16-melanoma mouse model. Results: We showed that local administration of LNPs entrapping a modified mRNA that encodes for a bacterial toxin, induced significant anti-tumor effects and improved overall survival of treated mice. Conclusions: We propose mmRNA-loaded LNPs as a new class of anti-tumoral, toxin-based therapy.

Reciprocal interactions between innate immune cells and astrocytes facilitate neuroinflammation and brain metastasis via lipocalin-2.

Brain metastasis still encompass very grim prognosis and therefore understanding the underlying mechanisms is an urgent need toward developing better therapeutic strategies. We uncover the intricate interactions between recruited innate immune cells and resident astrocytes in the brain metastatic niche that facilitate metastasis of melanoma and breast cancer. We show that granulocyte-derived lipocalin-2 (LCN2) induces inflammatory activation of astrocytes, leading to myeloid cell recruitment to the brain. LCN2 is central to inducing neuroinflammation as its genetic targeting or bone-marrow transplantation from LCN2-/- mice was sufficient to attenuate neuroinflammation and inhibit brain metastasis. Moreover, high LCN2 levels in patient blood and brain metastases in multiple cancer types were strongly associated with disease progression and poor survival. Our findings uncover a previously unknown mechanism, establishing a central role for the reciprocal interactions between granulocytes and astrocytes in promoting brain metastasis and implicate LCN2 as a prognostic marker and potential therapeutic target.

An immunoconjugate of anti-CD24 and Pseudomonas exotoxin selectively kills human colorectal tumors in mice.

BACKGROUND & AIMS: Effective and selective treatment options are needed for patients with colorectal cancer (CRC). The CD24 mucin-like glycoprotein is overexpressed in CRCs; monoclonal antibodies (mAbs) against CD24 inhibit tumor cell growth in vitro and in vivo. Based on the tumor-specific expression of CD24, we investigated the potential of anti-CD24 SWA11 mAb, to deliver a cytotoxic agent into CRC cells. METHODS: We conjugated SWA11 to a Pseudomonas exotoxin derivative (PE38) via an Fc-binding ZZ domain from Staphylococcal protein A (which binds the Fc domain of mouse IgG2a immunoglobulins) to generate the immunotoxin SWA11-ZZ-PE38; IgG-ZZ-PE38 was used as control. Human HT-29 and COLO320 (CD24-positive) and HCT116 (CD24-negative) CRC cell lines were assayed for immunotoxin binding, cytotoxicity, viability, and apoptosis. Toxicity and antitumor efficacy were tested in mice. RESULTS: The immunotoxin preserved the affinity and specificity of SWA11, bound and selectively killed CD24-expressing CRC cells via apoptosis. IC(50) values ranged from 20 to 50 ng/mL-several orders of magnitude lower than that of the mAb alone. The immunotoxins were not toxic to mice at the maximum dose of 0.75 mg/kg. Growth of HT-29 xenograft tumors was significantly reduced in mice given SWA11-ZZ-PE38 (by 78%) compared to untreated mice. CONCLUSIONS: Anti-CD24 SWA11 mAb can deliver a PE exotoxin derivative to CRC cells and cause them to undergo apoptosis, without toxicity to normal tissues. This immunotoxin might be developed as a therapeutic treatment for patients with CRC.

Selection of antibodies from synthetic antibody libraries.

More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.