Antibody Therapeutics - IBC Life Sciences

IBC’s 23rd Annual International Conference 

Co-Located with IBC’s 10th Annual 

Poster Presentations Featured in the Exhibit Hall 

Student Posters 

S1 Site-Specific UV Crosslinking of Peptides and Functional Moieties to Antibodies via Conserved Nathan Alves University of Notre Dame 

Nucleotide Binding Sites 

Described here is a robust method for covalently attaching peptides and small molecules to antibodies through a highly conserved binding domain located on the antibody variable fragment (Fv). Based on antibody 

crystal structure overlays and taking into account antibody symmetry there are at least two conserved nucleotide binding sites (NBS) per antibody. An in silico screening of small molecules was carried out to select 

for a high affinity nucleotide analog to target the NBS, monovalent Kd = 1-10 micromolar, experimentally validated based on various IgG and IgE antibodies tested. A stable covalent bond is formed through a photochemical 

reaction in a UV energy dependent manner providing for a unique, homogenous insertion of a target molecule selectively to the antibody light chains. Using this method we have been able to develop an 

immunosensor that maintains an exceedingly high level of antibody activity, including Fc recognition, through the oriented immobilization of antibodies onto surfaces. This method of crosslinking also has implications 

in the development of next generation pharmaceutical antibodies by providing a site specific insertion of function moieties such as: chemotherapeutics, cell penetrating peptides, targeting sequences (bispecific 

antibodies), imaging molecules, and facilitates oriented immobilization of antibodies onto various nanoparticle drug delivery platforms. 

S2 Development and Optimization of an E. coli-based Display Platform for Selection of Affinity Proteins Ken Andersson Royal Institute of Technology 

(KTH) 

Displaying proteins and peptides libraries on the surface of Gram-negative E. coli is potentially very favorable due to the high transformation frequency of this host organism. In contrast to phages, bacteria can be 

subjected to high-throughput screening using FACS for isolation of proteins with new properties. However, display of libraries based on larger proteins (e.g. antibody fragments) on the outer membrane of E. coli has 

proven to be challenging. Autotransporters is a class of surface proteins in Gram-negative bacteria that enable efficient translocation of recombinant proteins to the outer membrane. Previous studies have shown 

promising results with high surface expression levels of large recombinant proteins without decreasing cell viability. We are currently working on development of an E. coli-based display platform for expression of 

protein libraries intended for combinatorial protein engineering applications. In this project, we are using the AIDA-I protein (Adhesin Involved in Diffuse Adherence) for membrane anchoring of recombinant proteins, 

which is a naturally occurring autotransporter in E. coli. The development and investigation includes analysis of different promoters, expression conditions, strains as well as engineering of the expression cassette. We 

will present our most recent results from this work. 

S3 Site-specific Antibody-Drug Conjugates and Bispecific Antibodies using Unnatural Amino Acids Jun Axup The Scripps Research Institute 

Antibody conjugates are becoming of increased interest for the targeted treatment of cancer and other diseases. Traditionally, protein conjugation uses nonselective cysteine or lysine chemistries that often lead to 

heterogeneous products and makes optimization of biological, physical, and pharmacological properties challenging. We demonstrate the use of genetically encoded unnatural amino acids with orthogonal chemical 

reactivity to synthesize homogeneous antibody-drug conjugates and bispecific antibodies with precise control of conjugation site and stoichiometry. para-Acetylphenylalanine (pAcPhe) was site-specifically incorporated 

into an anti-Her2 antibody and conjugated to small molecule toxin auristatin or anti-CD3 antibody, which recruits cytotoxic T cells. The resulting conjugates demonstrated excellent pharmacokinetics, potent in vitro 

cytotoxicity against Her2-positive cancer cells, and tumor regression in rodent xenograft models. The synthesis and characterization of homogeneous antibody-drug conjugates and bispecific antibody conjugates with 

medicinal chemistry-like control over macromolecular structure should facilitate the optimization of antibody conjugates for a host of therapeutic uses. 

S4 Developing a Quantitative Model of Antibody Effector Function Austin Boesch Dartmouth College 

Monoclonal antibodies represent the fastest growing class of anti-cancer therapeutics. Strong genetic evidence indicates that in vivo, these antibodies act as molecular beacons to recruit innate immune cells to 

recognize malignant cells as pathogenic. The ability of an antibody to efficiently recruit innate immune cells depends on complex interactions between the pathogenic target, the antibody, and an effector cell. Here we 

present a quantitative model of natural killer cell mediated antibody dependent cellular cytotoxicity (ADCC), and examine the impact of core fucosylation, FcgR3A occupancy, serum IgG competition and mutations in 

the Fc binding domain. The results indicate that FcgR3A site occupancy can predict the percentage of cells killed on an ADCC dose response curve. In addition, it appears ADCC is initiated past a threshold of FgR3A 

site occupancy on the natural killer cell. This provides insight into the mechanism of the innate immune response and provides powerful evidence that the effector function of antibodies should be a key consideration in 

the development and design of this promising class of cancer therapeutics. 

S5 Towards a Comprehensive Map of B Cell Epitopes in VACV Antigens: Parallel Application of Multiple Thomas Kaever La Jolla Institute for Allergy & 

Epitope Mapping Approaches 

Immunology 

B cell responses to the smallpox vaccine are crucial, yet their epitopes recognized in humans remain poorly understood. We have set out to systematically discover and characterize B cell epitopes in vaccinia virus (VACV) 

antigens implicated as targets of neutralizing responses. We found that the application of different epitope discovery techniques (X-ray crystallography, linear peptide recognition by ELISA and foot printing by deuterium 

exchange) was necessary to fully characterize the epitopes recognized by a panel of antibodies recognizing the D8 antigen. Here we describe the application of two additional techniques, namely viral mutagenesis and site 

directed mutagenesis. Viral mutagenesis was applied to create a VACV mutant (pp7) which successfully evades recognition by the anti-H3 monoclonal antibody (mAb) JH4. Sequencing of pp7 revealed a point mutation at 

residue 112 of the H3 gene (glutamic acid [E] to lysine [K]). Site directed mutagenesis confirmed a significantly decreased binding affinity of JH4 to the mutated E112A H3 protein. Overall, our parallel epitope mapping 

studies reveal significant differences in the success of different mapping techniques for different antibodies, and highlight the need to compare and contrast the information gained by different methodologies. 

S6 The Ankyrin-repeat Protein, AnkGAG1D4, Specifically Targets HIV-1 capsid and Shows an Anti-HIV-1 Wannisa 

The Scripps Research Institute 

Effect in CD4+ T Cells 

Khamaikawin 

Anti-HIV gene delivery has been proposed as an alternative approach for multi-drug resistant salvage therapy as a less toxic, long-term replacement for anti-retroviral drug treatments. Designed ankyrin repeat proteins have 

been shown to be a robust and versatile scaffold for binding proteins. Recently, we developed and selected AnkGAG1D4, an ankyrin-repeat protein, which targets the HIV capsid and disrupts viral capsid assembly. We now 

demonstrate that AnkGAG1D4 can be expressed from integrated lentiviral vectors in CD4+ T cells and upon viral challenge the spread in tissue culture was attenuated up to 13-fold as compared to infection of control, CD4+ T 

cells. Moreover, stable expression of AnkGAG1D4 in primary CD4+ T cells and T cell lines did not show evidence of detectable cellular toxicity. The AnkGAG1D4 expressing cells showed similar growth patterns as control, nonexpressing 

AnkGAG1D4 cells. These studies support notion that ankyrin repeat proteins targeting viral protein may be applicable for use in hematopoietic stem and T cells. Furthermore, ankyrin-mediated capsid targeting may be 

less prone to resistance development. Our studies provide proof of concept for the continued development of ankyrin-repeat proteins as potential intracellular therapeutic agents against a variety of pathogens. 

S7 Novel Human Antibody Fragments Against EpCAM and c-MET Show Great Potential for Therapeutic Keyu LI UCLA 

and Diagnostic Applications in Targeted Therapy of Lung Cancer 

While EGFR targeted therapies show great potential in some non-small cell lung cancer patients, their limited response rate calls for better prediction and evaluation of drug resistance. Antibodies against important 

biomarkers like EpCAM and c-MET are very valuable for drug resistance evaluation and therapeutic applications. We successfully identified several novel human antibodies against EpCAM and c-MET from human 

scFv phage display libraries. The selected scFv clones were then re-formatted in to cys-diabodies or scFv-Fcs and expressed in bacteria or CHO cells. By flow cytometry using Hcc827 cells, we confirmed these antibody 

fragments have high affinities, ranging from 0.3nM to 9nM. These antibodies were then characterized and studied for therapeutic and diagnostic applications. One of our EpCAM antibodies, the D10 scFv-Fc, shows 

better performance than commercial antibody in a magneto-nanosensor assay that can detect serum markers with extremely high sensitivity. Some of our anti-c-MET cys-diabodies show therapeutic effects on erlotinib 

resistant cell lines with c-MET amplification, while other cells without c-MET amplification are not affected. In conclusion, these novel human antibodies with high affinity and low immunogenicity can greatly help the 

study of drug resistance in EGFR targeted therapies, and improve the diagnosis and treatment of cancer patients 

S8 An Alternative Approach for the Production of Recombinant Antibodies Based on Next Generation Javier Valdes-Aleman National Institute of Public 

Sequencing Data 

Health (Mexico) 

High-throughput sequencing of the antibody repertoire has enabled a more thorough analysis of Ig diversity, which has led to an improved antibody discovery process. An adequate analysis allows the identification 

of presumably antigen-specific antibodies. Gene synthesis is commonly used for the production of recombinant antibodies; however, it could be bypassed to generate lower-cost antibody production technologies. 

METHODS: We developed a generic workflow for the analysis, identification and recovery of potentially antigen-specific antibody sequences. Several on-line bioinformatic tools were assembled to generate a pipeline 

for the VH analysis. An alternative gene-synthesis-free strategy for the recovery of antibodies was coupled to allow experimental assessment of in silico results. RESULTS: Potentially antigen-specific clonal groups were 

identified and their sequences were efficiently recovered. Antibody production and experimental specificity evaluation remain to be completed shortly. CONCLUSION: A proper analysis if the Ig repertoire allows the 

identification of antigen-specific sequences and the gene-synthesis-free strategy reduces cost and increases the experimental capacity. *Some components of the presented work are going through a patenting process; 

therefore, undisclosed information should remain confidential. 

1 See You Next Year • December 9-12, 2013 • Hyatt Regency Huntington Beach • Huntington Beach, CA 

December 2-6, 2012 

Hilton San Diego Bayfront Hotel • San Diego, CA 

Antibody Therapeutics


S9 Inhibitory Variants Derived from Albumin-binding Domain Targeting Human IL-23 Receptor Lucie Vankova Institute of Biotechnology, Academy 

of Sciences of the Czech Republic 

Interleukin-23 (IL-23), a cytokine of p19 and p40 subunits, has been recently described to play a pivotal role in the development of chronic autoimmune diseases. As an alternative to antibody-based drugs, we engineer 

novel protein binders suppressing IL-23-mediated signaling. Library of three-helix bundle variants of albumin-binding domain (ABD) from streptococcal protein G (Ahmad et al. 2012) was used to identify binders 

of extracellular human IL-23R via ribosome display selection. After selection campaigns we identified group of clones (REX binders) that inhibit binding of p19 proteins and hIL-23 to a refolded IL-23R or soluble IL- 

23R-IgG chimera using direct competition ELISA. The strongest competitors REX009, REX125 and REX128 were confirmed to recognize hIL-23R-Fc chimera using surface plasmon resonance and their Kd values were 

estimated to be in sub- to nanomolar range. Thermal stability of the binders was verified by fluorescence-based thermal-shift assay. We further demonstrate that REX variants bind to K-562 and THP-1 human cell lines 

and this binding correlates with IL-23R cell-surface expression. As binding of the REX clones to THP-1 cells can be substantially diminished by a high dose of p19 protein we conclude that we identified novel IL-23Rbinding 

inhibitors that might be useful in designing novel anti-inflammatory tools. 

S10 Development of a Visual Immunoassay Utilizing Circularly Permutated Proteins to Detect 

Sara Zunnoon Khan University of Guelph 

Environmental Contaminants 

Recently, a fusion protein composed of antibody variable fragments and circularly permutated beta-lactamase protein was created by Kojima et al. (Bioconjugate Chem. 22: 633), with antigen specificities against 

osteocalcin and imidacloprid. The enzyme was split, circularly permutated, and fused to the variable heavy and light chain fragments, thereby ensuring inactivity until binding of the target antigen to the antibody fragments 

triggered enzyme activation. Upon activation, the beta-lactamase hydrolyzed a colorimetric reaction producing a detectable signal, indicating antigen presence. Based on this design, fusion proteins composed of single-chain 

variable antibody fragments specific for benzo[a]pyrene (B[a]P; Anal. Bioanal. Chem. 402: 499) and 2,4-dichlorophenoxyacetic acid (2,4-D; Vet. Med. – Czech 48: 237), with the superfolder variant of green fluorescent 

protein (sf-GFP; Nat. Biotechnol. 24(1): 79) were designed and expressed in E.coli for the development of a visual detection method. Relying on a similar mechanism to that used by the osteocalcin/imidacloprid detector, 

it is predicted that the split, circularly permutated sf-GFP will not fluoresce until activated by the binding of B[a]P/2,4-D to the split antibody variable fragments, subsequently resulting in a detectable fluorescent signal. 

Extraction and purification data have successfully confirmed the expression of fusion protein (~55 kDa) in E.coli periplasm, and immunoassays involving the fusion protein are under way. 

Advances in B Cell Cloning and Antibody-Production Cell Selection 

A1 Antigen-specific Plasma/plasmablast Cells Identification From a Variety of Animals for the Production Nobuyuki Kurosawa University of Toyama 

of Monoclonal Antibodies 

Nobuyuki Kurosawa , Megumi Yoshioka, Rika Fujimoto and Masaharu Isobe Direct cloning of cognate pairs of immunoglobulin genes from single antigen-specific plasma/plasmablast cells (ASPCs) using PCR has 

attracted attention as an alternative method for generating monoclonal antibodies (mAbs) from immunized animals. Although the use of ASPCs is best suited for the isolation of high affinity mAbs, the application 

of this method for species other than humans and mice is limited because the current plasma/plasmablast cell (PC) isolation protocols rely on a small number of identified PC-specific markers combined with the 

absence of one or more B-cell differentiation antigens. We propose a simple method to identify ASPCs from a variety of animals for the production of mAbs. By staining lymph node cells with an antibody against 

immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, we achieved PC identification from humans, mice, rats, rabbits and guinea pigs with 70-90% purity, representing a 48-223-fold enrichment 

of PCs compared with the original lymphocyte populations. Furthermore, by using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, ASPCs were sorted from the rest of the cell 

population by fluorescence-activated cell sorting. This method can greatly contribute to the isolation of high affinity mAbs from a variety of animals. 

A2 A Robust and Rapid System for High-throughput Generation of Recombinant Monoclonal Antibodies Masaharu Isobe University of Toayma 

from a Variety of Species 

Masaharu Isobe, Megumi Yoshioka, Rika Fujimoto and Nobuyuki Kurosawa Molecular cloning and expression of polymerase chain reaction (PCR)-amplified immunoglobulin variable (V) genes from single, isolated 

primary B cells provide powerful tools for the generation of recombinant monoclonal antibodies (mAbs). To achieve rapid and scalable automation for the generation of mAbs from a large numbers of single cells, 

we previously proposed a high-throughput single-cell-based immunoglobulin gene cloning method by developing a non-contact magnetic power transmission system (MAGrahd) for single-cell-based cDNA synthesis 

and a target-selective joint PCR (TS-jPCR) for the construction of the IgH and IgL gene expression units. To further improve the efficiency in isolation of antigen specific mAbs, we incorporated a new technique named 

endoplasmic reticulum (ER)-based identification of antigen-specific antibody-producing cells (ERIAA) for isolation of antigen-specific plasma cells. Our approach of utilizing ERIAA and high-throughput cloning enables 

the rapid generation of antigen-specific mAbs from a variety of animals as short as four days. We verified the advantage of our technology by isolating hundreds of human insulin-specific mAbs from guinea pigs. Our 

methods will greatly contribute to the isolation of high performance mAbs needed for sensitive diagnostics and therapeutic purposes. 

A3 A Novel Human Fusion Partner Cells, SPYMEG, for Development of Fully Human Monoclonal Kenichiro Ono Medical & Biological 

Antibodies and Therapeutic Antibody Candidates Generated from SPYMEG. 

Laboratories, Co., Ltd. 

We have developed a novel fusion partner cell, SPYMEG, to establish hybridomas producing fully human monoclonal antibody (HuMAb) from human B cells. Various procedures obtaining fully HuMab have been 

developed. Previously, the mouse myelomas have been used to establish hybridoma cells producing HuMAb, however, the establishment of a hybridoma has not been succeeded. A famous human fusion partner cell, 

Karpus, has been reported that it is hard to handle. In contrast, the advantages of SPYMEG in human antibody development are: a) easy to culture, b) easy to fuse with lymphocytes by PEG, c) easy to select on HAT, 

d)high efficiency on fusion and, e)stable production of antibody. In this presentation, we report that functional HuMAbs neutralizing all dengue virus serotypes (DENV-1 to -4) were established by SPYMEG cell using 

lymphocytes from patients with acute phase of the secondary infection of dengue in Thailand. Moreover, we successflly isolated influenza A group-specific broadly neutralizing HuMAbs from patients and influenza 

B type-specific broadly neutralizing HuMAbs from vaccinated healthy donors. All of which are therapeutic candidates and we found SPYMEG is very useful tool to establish native HuMAbs against tropical infectious 

diseases. This study is a part of JST/JICA SATREPS project. 

A4 Method for Robust and High Through-put Rabbit mAb Generation Pernille Gry Larsen Novo Nordisk A/S 

The capability to generate high affinity mAbs, recognising a wide repertoire of mechanistically relevant epitopes, sets the competitive edge in therapeutic mAb development. Rabbits have traditionally been regarded as 

an efficient source for generating Abs with high affinity and large diversity and therefore, the ability to generate rabbit mAbs provides an attractive technology supplementing other mAb generation platforms. Moreover, 

rabbits are evolutionary distant from mice, and implementation of a platform to generate rabbit mAbs inherently increases the possibility to generate surrogate mAbs for studies in mouse models. Here we describe an 

in-house developed method for robust and high through-put rabbit mAb generation. In short, the approach exploits the capability of primary B cells to proliferate and differentiate to secrete high amounts of IgG in 

vitro. It is based on fluorescence-activated cell sorting of antigen-specific monoclonal B cells, which are propagated in vitro in the presence of B cell stimulatory factors (recombinant rabbit BAFF, recombinant rabbit 

IL2, supernatant from PMA-activated rabbit splenocytes, and irradiated EL4.B5 cells). We have successfully optimized the protocol to proliferate and differentiate single B cells into high IgG-producing monoclonal 

cultures allowing both for efficient screening of antibody binding and functionality, as well as automated cloning of the antibodies selected from the screening. 

A5 Harvesting the Immune Repertoires from Antigen-Specific Bone-marrow Plasma Cells. Rapid 

Ana Paula Galvao AnaptysBio, Inc. 

Generation and Selection of Humanized Antibodies Without Making Hybridomas 

Da Silva 

Galvao Da Silva, A. P.; Brown, M.; Tomlinson G.; Spasojevic, V; McConnell, A.; Hare, E.; King, D.; Kehry, M. The generation of humanized antibodies from mouse monoclonal antibodies is a well established procedure, but 

requires a number of time consuming steps. These include the need to generate a panel of murine hybridomas, select antibodies with suitable properties, clone, sequence, generate CDR-grafted antibodies and undertake 

additional antibody engineering to generate optimal humanized antibodies with similar activity to the murine parent. In an alternative approach we have used direct harvesting of an immune repertoire through recovery of 

rearranged CDR3 sequences from antigen-specific bone marrow plasma cells (BMPC), followed by direct cloning as a library of CDR3 sequences into human IgG1 with otherwise germline sequence V-regions. The resulting 

library, constructed using four human VH-genes (IgHV1-69, IgHV3-23, IgHV4-59, and IgHV5-51) was displayed on the surface of HEK293 cells in intact IgG format, and antigen positive cells recovered via flow cytometry with 

fluorescently labeled antigen. The resulting humanized antibodies are then affinity matured via in vitro somatic hypermutation, initiated by the coexpression of activation-induced cytidine deaminase (AID). 

Antibodies in a Complex Environment: Targeting to Directly Affect Cellular Processes 

B1 Technology To Optimize Antibodies For Targeting Therapeutics Mark Federspiel Mayo Clinic 

Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery 

and optimization of high affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage and yeast display that 

would over-come the protein translation limitations of microorganisms and thereby improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a 

large-scale mam-malian expression system for clinical production. We will present data that demonstrate the replication and pol-ypeptide display characteristics of a eukaryotic retrovirus, Avian Leukosis Virus (ALV), 

may offer a robust, eu-karyotic version of bacteriophage display. The binding affinity of a model scFv was optimized improving affinity from micromolar to picomolar levels using ALV display. We believe ALV display 

provides an extension to anti-body display on microorganisms and offers both virus and cell display platforms in a eukaryotic expression sys-tem. ALV display enables an improvement in the diversity of properly 

processed and functional antibody vari-ants that can be screened and affinity optimized to improve promising antibody candidates. 

B2 Engineering of Bispecific Fynomer-antibody Fusions (FynomAbs) for Therapeutic Applications Ulrich Wuellner Covagen AG 

Fynomers are small binding proteins derived from the human Fyn SH3 domain. They can be selected from large combinatorial libraries against a variety of target proteins. Their small size and expression profile offer 

the possibility to create bispecific antibody Fynomer fusion proteins (FynomAbs) with superior properties compared to the unmodified antibody. At the same time, FynomAbs maintain the advantageous drug-like 

properties of antibodies such as excellent stability and long half-life. We will demonstrate the versatility of our FynomAb technology by showing the impressive results of two case studies with two different FynomAbs. 

The first FynomAb comprises an anti-IL-17A Fynomer fused to an anti-TNF antibody and the second an anti-HER2 Fynomer fused to an anti-HER2 antibody (bi-paratopic). Both FynomAbs show very favorable 

physicochemical properties and excellent in vitro and in vivo bioactivity in complex disease models like inflammation and cancer. The results show that FynomAbs represent an attractive new class of bispecific drug 

candidates for preclinical and clinical development. 

www.IBCLifeSciences.com/antibodyeng 2


B3 Site-selective Antibody Labeling Without Engineering: Answering Unmet Needs in Antibody 

Brian Agnew Molecular Probes-Life 

Labeling Applications 

Technologies 

We have developed a modular copperless click chemistry-mediated method for the site-specific enzymatic labeling of essentially any antibody on the heavy chain N-linked glycans. While standard antibody labeling 

techniques can be cumbersome and tedious, with variable reproducibility and low yields, the described approach results in highly reproducible labeling with built-in robustness. The site-selective approach prevents 

disruption of the antigen binding domain that often occurs with standard amine or thiol labeling methods. This is especially important when labeling monoclonal antibodies with vulnerable amino acids in or 

around the antigen binding domain. Additionally, this modular “Lego-like” labeling system allows for the simple attachment of numerous detection molecules including fluorescent proteins, nanoparticles, biotin, 

fluorescent dyes, and other small molecule payloads. The enzymatic labeling approach involves the use of a mutant beta-Gal-T1 enzyme, Gal-T(Y289L), that is substrate-permissive and specifically labels terminal 

N-acetylglucosamine (GlcNAc) residues on N-linked antibody sugars with azide-modified N-acetylgalactosamine (GalNAz). Once labeled, the azide-tagged antibodies are reacted with dibenzocyclooctyne (DIBO)functionalized 

molecules in a copper-free click reaction. We demonstrate here the site-specific labeling of azide-tagged antibodies with fluorescent probes and other small molecule payloads with remarkable 

reproducibility. Absolute site-specificity of the labeling method is demonstrated using endoglycosidase treatments of labeled antibodies combined with fluorescence gel electrophoresis quantitation. Site-selective 

labeling with easy and robust labeling site characterization will allow for accelerated regulatory approval when required. 

B4 Lung Endothelial Retargeting Via Anti-ACE mAbs Sergei Danilov University of Illinois at 

Chicago 

Monoclonal antibodies to angiotensin I-converting enzyme (ACE) accumulates extremely specifically and efficiently into the lung capillaries. Lung endothelial targeting via anti-ACE mAbs still is the most effective and 

most specific approach in comparison with any other anti-endothelial mAbs . This effect was demonstrated on all animals to which ACE our mAbs were cross-reacted: mAb 9B9-rat, hamster, cat, monkey, human; 

i2H5-monkey; 1A2 (and several others)-rat), 4B10.5 (and several others)-mouse. We demonstrated not only effective and specific delivery of the reporter genes into lung endothelium in vivo via anti-ACE mAbs but 

also a therapeutic effects of the delivery of the gene: eNOS synthase for attenuation of spontaneous hypertension in SHR rats and BMPR2-for attenuation of pulmonary hypertension in rat animal models. Conjugates 

of catalase (CAT) with anti-ACE mAb 9B9 effectively protect i) isolated perfused lungs from hydrogen-peroxide induced lung injury; ii) lungs from ischemia-reperfusion injury in vivo. Therefore the CAT-9B9 conjugate 

might increase dramatically the preservation of lung transplant during transportation. The scFv 9B9 was recently cloned and isolated , which allows us to start genetic engineering of the surfaces of viral particles and 

therapeutic cells (somatic and stem cells) and preparation of fusions with therapeutic proteins for therapy of pulmonary diseases. 

B5 Reducing the Immunogenicity of Recombinant Immunotoxin by Identifying and Silencing Human B Masanori Onda NCI/NIH 

Cell Epitopes 

Recombinant immunotoxns (RITs) are hybrid proteins used to treat cancer. They are composed of a truncated Pseudomonas exotoxin A, which kills the cell and an Fv that delivers the toxin to the cancer cells. Our 

clinical studies show that in many patients RITs induce neutralizing antibodies and limit the number of doses that can be given. We previously identified seven major mouse B-cell epitopes in the toxin, and were able to 

silence them using point mutations, yet retained full antitumor activity. Here we show that we have identified and silenced human B-cell epitopes in the RIT HA22. We obtained B cells from the patients with antibodies 

to RITs, isolated Fvs and constructed a phage-display library containing Fvs that bind to RITs. We then used alanine scanning mutagenesis to locate the epitopes. We found that human and mouse epitopes frequently 

overlap but are not identical. Many mutations that modify mouse epitopes did not affect human epitopes and vice versa. We used this information to construct a new immunotoxin, HA22-LR-LO10, with low reactivity 

with human sera, yet excellent cytotoxic and antitumor activity. The toxin portion of this RIT can be used with Fvs targeting other cancer antigens and is suitable for clinical development. 

B6 Detergent-solubilized Cell Lysates Facilitate in Vitro Antibody Engineering Against Membrane Proteins Ben Tillotson UW-Madison 

Membrane proteins, especially membrane receptors, represent a valuable class of drug targets due to their unique role in cell signal transduction and intracellular transport. Here we show that antigen preparations in 

the form of detergent-solubilized cell lysates render membrane proteins (MPs) compatible with in vitro antibody engineering techniques. To this end, affinity maturation of an anti-human transferrin receptor (hTfR) 

single chain antibody (scFv) was carried out via yeast surface display, using detergent solubilized mammalian cell lysates as the sole antigen source. Selective biotinylation of plasma membrane proteins enabled a 

competitive dissociation rate screen that identified anti-hTfR scFvs with up to 4-fold improved dissociation rates on the surface of yeast. Importantly, although the lysates contained a complex mixture of proteins, the 

engineered scFvs retained TfR binding specificity. When secreted as soluble proteins, mutant scFvs bound to cell-surface TfR with up to 7-fold improvements in equilibrium affinity. The lysate-based framework provides 

a flexible, quantitative, and potentially high-throughput platform for generation and optimization of antibodies against membrane proteins. 

B7 Production of Elicitor Protein Cross-reactive Monoclonal Antibodies Through Immunization with a Jillian Tarling University of Guelph 

Common Structural Motif 

Nep-1-like proteins (NLP) are pore-forming virulence factors secreted by bacteria, oomycetes, and fungi, triggering immune responses and necrosis in dicotyledonous plants. All NLPs possess the NPP1-domain, a 

conserved motif of residues (GHRHDWE), which contributes three amino acids to a cation-binding cavity crucial for NLP function. Based on its functional significance, the NPP1-domain was chosen as an antigenic 

target for antibody development. Mice were immunized with a GHRHDWE-keyhole limpet hemocyanin conjugate and IgM-secreting hybridoma clones were produced. The IgMs were analyzed for their ability to bind 

four NLP entities: NLP-Pya (or PaNie, from Pythium aphanidermatum), NLP-Fo (or Nep1, from Fusarium oxysporum), NLP-Bh (from Bacillus halodurans), and NLP-Ssclr (from Sclerotina sclerotiorum). Antibodies with 

high affinity and cross-reactivity were propagated in single-chain variable fragment (scFv) format and characterized with respect to their affinity constants. Thus, by immunizing mice with a conserved structural motif, 

monoclonal antibodies were produced that are specific for multiple members of a virulence protein family. Since the NPP1-domain has functional significance to the NLP family, antibodies able to bind this motif may 

be capable of inhibiting NLP pore-forming ability; this would negate the pathogenic significance of these NLPs and possibly help mitigate plant disease caused by pathogens that produce them. 

B8 The SIMPLE Antibody Platform and Best-in-Class c-Met Antibodies Hans de Haard Utrecht University 

arGEN-X’s SIMPLE Antibody platform has many outstanding qualities as a source of fully human antibodies with therapeutic potential. Relative to other technologies, it excels in delivering functionally diverse 

antibodies against complex targets, in particular cell surface receptors and highly conserved proteins. Exploiting the active immunization of outbred llamas, the SIMPLE Antibody approach taps into a more 

comprehensive range of target epitopes than antibodies derived from non-immune or inbred rodent systems. Our platform generates fully human antibodies with intrinsically high affinities and potencies requiring 

no in vitro affinity engineering. Based on V gene sequences as expressed in B cells, SIMPLE Antibody products have outstanding physicochemical properties, consistent manufacturability and allow a seamless 

transition from discovery into development. Exciting progress in our c-Met program is presented, highlighting how functional antibody diversity allows identification of antibodies with best-in-class characteristics 

for the immunotherapy of solid tumors. c-Met is a compelling yet challenging oncology target (tumors of lung, stomach, prostate, brain,…). C-Met is a receptor tyrosine kinase and the natural ligand is HGF/scatter. 

Dimerization of c-Met induced by HGF leads to signaling & activation. Through aberrant signaling, c-Met can play a key role in cancer development (survival/proliferation/mobility). The challenge for a therapeutic 

antibody is to antagonize the receptor without inducing agonistic activity. 

Antibody Engineering: Libraries, Expression, Characterization 

C1 Selection of Human-derived Antibodies against Targets Expressed on Live Cells with dsDNA Display Yan Chen X-BODY Biosciences 

and Massively Parallel Next Generation Sequencing Analysis 

Diverse human libraries of antibody VH and VL domains were generated from B cells of normal human donors. Selections were performed against HER2 expressed on the surface of live cells with a proprietary dsDNA 

display methodology. Epitope binning and functional selection was employed on live cell selections to select for VH domains recognizing broad epitopes and with desired functional properties. Selection pools were 

subjected to next generation sequencing and analyzed by tabulating the frequency of the numerous hits. The Individual VH domains were purified and shown to bind HER2 expressed on breast cancer cells. As predicted 

from the next generation sequence analysis, one VH domain targeting HER2 bound to an epitope different from either Herceptin or Pertuzumab; further, this domain was found to synergize with Herceptin and 

Pertuzumab activity in cell proliferation assays. This platform has broad utility for directly screening cells to find antibodies that interact with challenging cell surface targets such as integral membrane proteins. 

C2 Proteome-wide Epitope Survey of Antibodies Using Ultra-dense Peptide Arrays Björn Forsström KTH Biotechnology 

Antibodies are of tremendous importance in all areas of bioscience. A frequent concern is the possible presence of cross-reactivity to off-target epitopes. Here, we have studied the binding of both monoclonal 

and polyclonal antibodies using ultra-dense planar microarrays involving 2.1 million in situ synthesized tiled peptide sequences representing the entire open reading frame (ORF)-space of the human proteome, 

complemented with precise mapping of epitopes based on full amino acid substitution scans of all residues in the epitope region. The analysis suggest that the linear epitopes are relatively short, confined to five to 

seven residues, resulting in off-target binding to peptides comprising sub-elements of the cognate epitope sequence. Interestingly, subsequent analysis using an array of recombinant proteins suggests that these linear 

epitopes have a strict conformational component. The results show the usefulness of proteome-wide epitope survey (PWES) showing a path forward for high-throughput analysis of antibody interactions. 

C3 Improved Panning Output and Antibody Fragment Production by Co-expression with the Peptidyl Raphael Levy XOMA Corporation 

Prolyl Isomerase, FkpA, in the Cytoplasm of Escherichia coli 

Low expression or low functional activity of antibody fragments in the periplasm of E. coli can often be attributed to protein folding or aggregation issues. Since antibody fragments displayed on phage are also 

secreted into the periplasm of E. coli, improper expression can also impact the selection of antibody candidates from phage libraries. We have developed a novel approach to enhance the secretion of properly folded 

antibody fragments into the periplasm. This involves co-expressing of antibody fragments with the peptidyl prolyl cis-trans isomerase, FkpA, without its signal sequence (cytFkpA) which consequently is expressed in 

the E. coli cytosol. Expression of cytFkpA in the bacterial cytoplasm considerably improved the periplasmic secretion of functional fragments. The effects of cytFkpA co-expression were even more pronounced than the 

effects of co-expressing Fab fragments with the native, periplasmic FkpA. Importantly, in the presence of cytFkpA, both the number of unique clones selected from Fab and scFv naïve phage libraries, as well as their 

functional expression levels and diversity, were significantly increased. 

C4 Development of High Capacity Magnetic Protein A Beads and Protein G Beads for Rapid Antibody Nidhi Nath Promega Corporation 

Purification from Small Sample Volume 

We have developed high capacity magnetic Protein A and magnetic Protein G beads by combining two novel technologies. First, is the use of magnetic beads based on macroporous cellulose that provides high surface 

area for high capacity and low non-specific binding surface for high purity. Second, is the immobilization of Protein A and Protein G on the magnetic bead in a covalent and oriented fashion using HaloTag Technology. 

Oriented immobilization of the proteins has been shown to improve the functionality of the proteins and may contribute to the high capacity of these beads. Magnetic Protein A Beads and Magnetic Protein G Beads 

allow purification of polyclonal and monoclonal antibodies from serum, ascites fluid and cell media. These beads offer several advantages for antibody purification including: a) High capacity of up to 25mg of Human 

IgG per ml of settled beads; b) quick and easy handling of 1-96 samples in parallel; c) easy handling of 20 microliters to 30ml of samples; d) higher reproducibility for purification from small samples and e) ability to 

process samples manually or on robotic platforms. We offer several examples highlighting these advantages. 

3 See You Next Year • December 9-12, 2013 • Hyatt Regency Huntington Beach • Huntington Beach, CA


C5 Optimization of Transcriptional Regulatory Elements for an Improved Expression of Antibodies in the Alexandre Fontayne LFB Biotechnologies 

EMABling Plateform 

The EMABling® platform has been developed to express functionally improved monoclonal antibodies (mAbs) with low fucose content in the glycan moiety that results in increased antibody dependent cell-mediated 

cytotoxicity (ADCC). For industrial antibody production, the increased productivity might be obtained by the optimization of a number of variables including expression vector design. In this study, transcription 

regulation through modification of promoters, enhancers, 5’UTR and introns have been sequentially evaluated in transient transfection in YB2/0 cell line based on the quantification of the IgG light chain molecule. 

Optimal conditions were defined then confirmed in stable pools for the production of the entire IgGs. As results, the new combination composed of CKD9 promoter, SV40ie enhancer, eIF4G1 5’UTR and EF1_ intron 

allows a 6 folds improvement of the antibody titer in comparison with the former expression construct composed of LTR RSV promoter and pCineo derived intron. 

C6 Application of Eukaryotic Cell-free in Vitro Protein Expression Platform on scFv Antibody 

Hsu Chuan-Lung Development Center for 

Library Screening 

Biothechnology 

Rabbit reticulocyte lysate (RRL) translation systems are used for characterizing protein products from RNA transcripts and investigating transcriptional and translational control. It has been reported that RRL can 

provide an alternative approach for the development of antibody based biotherapeutics and biological detection by coupling single chain variable fragment library screening technology.Here, we demonstrate that 

the home-made RRL has been prepared for in vitro protein expression and further optimized the protocols in previously references and technical manuals for the construction of the rabbit reticulocyte lysate in vitro 

translation system. The RRL activities are quantified by measuring the fluorescent optical absorption by the addition of the capped luciferase transcripts in RRL. The results display that the quality of RRL prepared 

from day 8th after acetylphenylhydrazine-induced hemolytic anemia have the optimal ribosomal translation activity for protein expression in the presence of 79mM KCl and 0.5mM MgOAc. The translation activity of 

our home-made RRL is nearly equal to that of the commercial products. In our current study, we further combined the single chain display technology with the cell-free ribosomal protein expression system to produce 

antigen-specific scFv library for developing in vitro scFv library screening method. The scFv construction of anti-IL20 and anti- Tn7E were constructed for in vitro ribosomal display, and then mRNA–ribosome–antibody 

complexes were produced by our home-made rabbit reticulocyte lysate system have been confirmed. 

C7 ADAPT: An Integrated Antibody Display Platform that Facilitates Antibody Discovery Hoa Giang XOMA 

Phage display of antibody fragments is an extremely successful tool for isolating potential therapeutic agents. The main factors in the success of XOMA’s antibody libraries are the ability to construct large libraries 

(over 1011 cfu), high heavy and light chain diversity, ease of display and expression in E. coli, and versatility of panning against a wide range of antigens. As with most systems there are some limitations in that certain 

antibody fragments show decreased antigen binding and function when reformatted to IgGs. Combining the use of phage display with eukaryotic display systems increases the chances of identifying a greater number 

of functional antibodies in a short time by utilizing rapid reformatting and direct testing as full IgGs. Furthermore, these eukaryotic display systems allow the utilization of FACS to accelerate antibody discovery by 

eliminating poorly functioning antibodies, such as weak binders with high expression. Here we show the versatility of an integrated system using both phage and eukaryotic display. 

C8 Technology for Exceedingly Large Synthetic Antibody Library for Therapeutic Antibody Optimization Lian Gao University of Houston 

Antibody libraries are typically obtained from pathogen stimulation of a host system, such as mouse or disease human B-cells. The pool of antibodies is used to isolate antigen-specific antibodies after a long tedious 

process. The obtained antibodies suffer from problems of low affinity of binding, thus poor specificity and selectivity. Various mutagenesis methods have been introduced to expand the diversity of antibody pools 

by introducing DNA sequence variations for antibody genes. We have developed an efficient work flow for creating synthetic monoclonal antibodies with precisely controlled CDR oligonucleotides and thus designed 

antigen binding sequences. This allowed the use of the abundant sequence information of antibodies with known high binding affinity and specificity. We overcome the difficulties that the exceedingly large designed 

CDR oligonucleotides would be expensive and slow to obtain by introducing a new generation of microchip based parallel synthesis technology. Which allow parallel synthesis of millions of oligonucleotide of designed 

sequences on a single microchip. i.e., traditionally, a single oligo nucleotide is made at one location of a microchip. In the second generation microchip for oligonucletoide technology, multiple oligonucleotides (upto 

1,000 different sequences of designed context) are synthesized at one location. The resultant CDR oligonucleotides are incorporated into mAb antibody expression plasmid for construction of antibodylibrary reaching 

diversity in tens of millions. The method has been used to construct anti ERBB2 (HER2) libraries. The screening experiments generated new antibodies of improved properties compared to herceptaine, which is 

clinically prescribed therapeutic antibody for treatment of HER2 expression breast cancers. The efficient generation of rationally designed antibody libraries allowed possibilities of improving the effectiveness and the 

biosafety of the existing antibody therapeutics in short mount of time. 

C9 Retained Display - a Combined Cellular and Polyvalent Lambdoid Display Platform, Capable of MathewBeasley Affinity Biosciences 

Extremely Fast Library Screening 

Affinity bio has developed ReD (Retained Display), an antibody selection platform that combines the parallel screening capability of lambdoid-phage display, with the quantifiable clone selection of FACS. Our 

proprietary, fully human, single-chain Ab scaffold library is expressed from a lambdoid-phage vector that is deficient for bacterial lysis. Upon phage induction, the single-chain Ab is loaded at high valency onto 

the phage capsid. The bacterial cell may then be either enzymatically lysed to release the phage for conventional panning or the cell, with encapsulated phage, may be permeabilised for diffusion and binding of a 

fluorescently labelled protein target for detection by FACS. Recovery of FACS-positive clones is easily achieved with the addition of a lysin to the cells and infection of fresh bacteria. ReD offers high capture frequencies 

of low-abundance clones and the capability for a seamless switch between phage panning to cellular FACS without sub-cloning. 

C10 Antibody Library Display on a Mammalian Virus Vector: Combining the Advantages of Both Phage Ernest Smith Vaccinex, inc 

and Yeast Display Into One Technology 

Utilizing a vaccinia virus based library technology we have developed an antibody discovery platform that enables efficient expression of a library of fully human antibodies on the surface of vaccinia virus; an enveloped 

mammalian virus. Similar in concept to phage display, conditions are utilized where each vaccinia virion will express a single antibody specificity on its surface. Various panning and magnetic bead based methods have 

been developed to allow screening of a library of vaccinia-MAb virions and selection of recombinant vaccinia virus encoding specific antibodies. Upon infection of mammalian cells the antibody is not only incorporated 

into newly produced virus, it is also displayed on the surface of the host cell. Similar to methods utilized in yeast display, the cells displaying vaccinia encoded antibody can be selected using a combination or magnetic 

beads and cell sorting, and the virus encoding the specific antibody readily recovered and analyzed. This technology allows for rapid high efficient selection of vaccinia-MAb virions in a cell free panning system, and then 

to incorporate the cell based screening for high specificity and selection of optimal antibodies. This technology can be employed for de novo antibody selection, affinity improvement of existing human antibodies, or 

for the robust conversion of a non-human antibody into a panel of fully human antibodies. In all applications, there is a built in selection for antibodies that are efficiently expressed in mammalian cells. 

C11 Antibody Stability Engineering by Deep Sequencing of Phage-displayed Antibody Libraries and High An-Suei Yang Academia Sinica 

Throughput Physical Measurements 

Single chain antibody fragments (scFv) are important components in protein therapeutics. However, unlike intact antibodies, single chain antibody fragments are frequently unstable in physiological conditions. Next 

generation sequencing (NGS) has extensively expanded the viable antibody VH and VL domain sequence database, providing informatics basis for scFv stability engineering. In this work, groups of consecutive or 

noncontiguous residues (4~9 residues) in the framework regions of a scFv template were randomized in phage-displayed antibody libraries and tens of thousands sequences for functional scFv variants were derived with 

NGS. The stability of hundreds of randomly selected functional scFv variants were also assessed quantitatively with high throughput thermal inactivation measurement, so that the amino acid preference statistics in the 

scFv framework structure were understood in terms of the protein stability measurements. The NGS information in conjunction with the high throughput thermal stability measurements enabled a novel platform for 

scFv stability engineering, for which the outcomes are superior to those based on the conventional consensus sequence approach built on natural antibody sequence databases. The knowledge gained from this work is 

expected to further the understanding of sequence-structure relationships in antibody variable domains and to enhance the basis for rational antibody stability engineering. 

C12 MapEng, a New Platform to Aid in the Structural Analysis and Engineering of Biobetter Therapeutics Bob DuBridge Full Spectrum Genetics 

Genetic analysis of point mutations has been a valuable tool in elucidating protein structure/function relationships. With the increased efficiency of gene synthesis to create point mutants and the dramatic increases in DNA 

sequencing technology for identifying these changes, we designed a new method to analyze the effects of hundreds of point mutants on protein binding in parallel. In this system small phage libraries containing all possible 

single amino acid substitutions in the CDRs of an antibody combining site are separated via equilibrium binding, the results of these binding reactions are then quantitatively assessed using a next generation sequencing 

platform. The output of this system aids in the design of biobetter molecules with a variety of engineered traits, not just limited to affinity maturation. It also allows the creation of detailed structural models of protein 

binding sites. We have used this system to assess the effects on affinity of all possible point mutants in and around heavy chain CDR3 of Avastin. In this experiment the effects of 500 individual point mutations on antibody 

binding were analyzed in parallel. Up, down and neutral mutations were discovered or reconfirmed published results. These results and their implications for creating biobetter therapeutics will be discussed. 

Antibody Refinement for Therapeutic Development 

D1 A Novel Approach to Detect and Visualize Aggregation-prone Residues in Immunoglobulins Johannes Maier Chemical Computing Group 

The success in applying therapeutic proteins as drugs in the past twenty years made immunoglobulins one of the most attractive targets for protein engineering. One of the most challenging obstacles in using 

antibodies as therapeutics is their tendency to form aggregations when stored at high concentrations that often is associated with significant loss in efficacy. Here, we demonstrate a novel method capable of identifying 

aggregation-prone residues in silico that can be used to examine a series of mutants to elucidate or compare the critical hydrophobic interactions within antibodies. The approach determines the hydrophobic surface 

patches based on Poisson-Boltzmann as well as Accessible Surface Area (ASA) calculations without using very long and computationally expensive molecular dynamics simulations. This SAR-type approach to protein 

mutations might help to generate novel protein therapeutics with lower aggregation propensities without compromising their specificity. 

D2 Introducing Camelid Single Domain Antibody Fragments On Label Free Platforms For Antibody Pim Hermans BAC BV 

Quantitation And Characterization 

Since more and more antibody based therapeutics are lacking a regular protein A binding site it becomes a challenge to find a protein A equivalent for primary capture of antibody formats for detection and 

characterization. For this reason a diverse set of camelid single domain antibody fragments has been developed directed towards a unique panel of antibody sub-domains (e.g. CH2, CH3 on Fc and CL_ / _ , CH1 on 

Fab) further providing isotype, subclass and species selectivity. This set of camelid based affinity ligands enables the set-up of analytical tools for virtually any antibody format. Due to the small size (± 14 kDa) and 

rigid structure of camelid affinity ligands, functionality is retained upon immobilization onto sensor surfaces resulting in high signal to noise ratio’s. Good compatibility is found for a variety of label free platforms like 

Biacore, Octet and IBIS and examples will be presented on detection and quantitation of different antibody formats. The concept of multiplex analysis will also be exemplified using the IBIS MX96 platform that allows 

specific - and simultaneous detection of different antibody isotypes in complex mixtures like human serum samples. 



D3 Antibody Engineering to Improve Manufacturability Arnold Horwitz XOMA 

Antibody expression variation in CHO cells is well established. While developing CHO-K1 cells expressing human antibodies, we encountered a very poor-expressing antibody which also displayed elevated levels of high 

molecular weight aggregate. Using transient CHO-K1 expression, we identified light chain as the source of the manufacturability issues. While other antibodies achieved optimal expression at 1:1 or 2:1 LC:HC ratios, 

this antibody required a 6:1 LC to HC ratio for maximal expression. A “swap” experiment with heavy and light chains from a highly expressed antibody confirmed light chain as the cause for poor expression. Light chain 

shuffling was performed to select antibodies which retained binding affinity. Evaluation by transient CHO-K1 expression of antibodies containing the new light chains yielded candidates with high expression at 1:1 or 

2:1 LC to HC ratios. Expression in permanently transfected CHO-K1 cells of antibodies containing the original heavy chain with new light chains confirmed the initial transient expression results. In addition, antibodies 

with the new light chains contained very low aggregate levels. These results demonstrate that antibody manufacturability issues can be due to light chain and that pairing heavy chains with alternate light chains can 

improve antibody expression and product quality while maintaining affinity. 

D4 Use of Dip and Read Anti-Human Fab-CH1 Biosensors for Rapid Quantitation and Kinetic Analysis of Dominic Andrada ForteBio - A Division of 

Human IgG Fab Fragments 

Pall Life Sciences 

Specific detection, quantitation and kinetic analysis of human IgG Fab or F(ab’)2 molecules can be performed using the Anti-human Fab-CH1 biosensors. The biosensor is capable of distinguishing the Fab/F(ab’)2 

from contaminating free light and heavy chains. The sensor can be used in crude samples allowing for the specific capture of the human antibody fragment without the need for purification. Data from both 

quantitation and kinetic applications is presented below. 

D5 Rapid and Robust Process Scale Manufacturing of F(ab’)2 Fragments Fredrik Olsson Genovis 

The production of F(ab’)2 fragment is commonly performed at various stages during monoclonal antibody development. These fragments are used in applications such as histology, imaging, characterization, crystallization 

and more. The most widely used process for creating these bivalent fragments is based on enzymatic digestion using pepsin. However, the pepsin enzymatic process is commonly accompanied by several problems such as low 

yield, over digestion, need for optimization and loss of immune reactivity due to low pH. IdeS, a cysteine protease from the pathogenic microorganism Streptococcus pyogenes, has recently gained interest in applications of 

characterization of mAb:s. We have investigated the potential of using this enzyme immobilized on agarose for continuous production of F(ab’)2 and Fc fragments. Based on immobilized IdeS a rapid, robust and efficient 

process of producing F(ab’)2 fragments has been set up that circumvents all of the problems usually associated with pepsin fragmentation. Our data also indicates that the process is highly versatile and scalable. 

D6 A Novel Bioluminescent Assay to Measure ADCC & CDC with the use of Automation Sumant Dhawan Cell Technology, Inc. 

The classical “gold standard” ADCC/CDC assay incorporates 51Cr. The 51Cr method is less sensitive, time consuming and involves the use and eventual costly disposal of radioactive material. We describe the use of a 

highly sensitive, non-radioactive luminescent chemistry to simplify the assay process and improve the data quality. The ADCC reaction involves recruitment of NK cells by the test antibody leads to lysis of the target cell. This 

causes release of Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) from the lysed cell. ATP production ensues, which is coupled to a luciferase/luciferin reaction producing a luminescent signal that is measured by Cell 

Technology’s aCella-TOXTM kit. With this method, no target cell preparation time is necessary. The procedure can be completed in half the time of the original procedure without any complicated and expensive regulatory 

permissions. Experimental data using control antibodies, including Rituximab, as well as fresh and cryopreserved NK cells are shown to prove the validity of the method. Results of antibody-mediated complement dependent 

cytotoxicity (CDC) and Cell-mediated cytotoxicity (CMC) are also presented. The assay is easily automated in 96-well format and can be conducted using a semi-automated assay system. 

Bispecific Antibody Engineering 

E1 Cytotoxic Enhancement of a Bispecific Diabody by Format Conversion to Tandem Single-chain Ryutaro Asano Tohoku University 

Variable Fragment 

Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these 

formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) that target epidermal growth 

factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon 

resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format 

in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed between hEx3-Db and hEx3-taFv with surface plasmon 

resonance spectroscopy. Furthermore, hEx3-taFv inhibited cancer growth in mice more effectively than did hEx3-Db. Our results show that converting the format of small bispecific antibodies can improve their 

function. 

E2 AlbuCORE – Structure-Guided Engineering and Design of a Multi-Valent Albumin Scaffold Paula Asano Zymeworks Inc. 

Albumin is the most abundant protein in the bloodstream and is critical in the regulation of plasma osmotic pressure and in the native transport of a variety of metabolites. Albumin has a long circulatory half-life 

of 19 days and can accumulate in tumor interstitium by binding to SPARC proteins following gp60-mediated endothelial transcytosis. Albumin is an attractive antibody-alternative vehicle for carrying and delivering 

therapeutic and diagnostic agents with albumin protein fusions designed to reduce immunogenicity and improve pharmacokinetics and efficacy compared to the proteins alone. A drawback is that fusions are limited 

to the amino- and carboxy-termini of albumin and cannot be customized for increased valencies and/or spatial separation and orientation depending on target geometries. The AlbuCORE scaffold is an engineered 

human serum albumin where the natural protein sequence is spliced and reconstituted to form a quasi-native molecule to which other active protein domains can be fused at the two natural, as well as the two newly 

formed termini. We will be showcasing the pharmacological and biophysical characteristics of this antibody-alternative multi-valent scaffold as we pursue internal and external drug development opportunities. 

E3 Azymetric Scaffold – Optimized Fc Heterodimer for Improved Purity and Stability in the 

David Poon Zymeworks Inc 

Development of Bispecific Antibodies 

Naturally occurring IgG antibodies, and the majority of therapeutic monoclonal antibodies, are bivalent and monospecific binders of their target antigens derived from the homodimeric assembly of heavy chains. The 

chains can be engineered and expressed recombinantly to form heterodimeric scaffolds that can be utilized in the design of bispecific antibody therapeutics. These dual antigen-binding moieties can take advantage 

of novel therapeutic mechanisms of action while retaining traditional IgG-like PK properties. Key challenges addressed in this engineering approach are: 1) Purity – the different heavy chains must associate and 

preferentially form obligate heterodimers as any contaminant homodimers will pose significant downstream purification and/or mechanistic challenges, and 2) Stability – the resultant antibodies must possess stability 

profiles similar to the wild-type antibody as this can directly impact the scale-up and manufacturability of the therapeutic. Structure guided computational modeling and simulation approaches were used to engineer 

the CH3 domain of IgG1 antibodies such that highly pure and stable heterodimeric antibodies were produced. We have evaluated and will showcase the biophysical properties, manufacturability, pharmacokinetics, 

and pharmacological potential of Azymetric candidates as we progress in the internal and external development of best-in-class antibody therapeutics for multiple indications. 

E4 Validation of MeMo®, a Transgenic Mouse Platform to Generate Fully Human, Functionally Potent Rob Roovers Merus BV 

Bispecific Antibodies 

Merus has developed and validated a powerful discovery engine for human bispecific antibodies. The MeMo® transgenic mouse is engineered to generate antibody diversity based on a common light chain (cLC) and 

diversified heavy chains. MeMo® features normal B cell development and, upon immunization, comparable serum antibody titers to those of wild type mice. Using small cohorts of MeMo® mice and straightforward 

immunizations with EGFR and cMet receptor tyrosine kinases, large and diverse panels of cLC antibodies against both targets were obtained from phage display libraries constructed from immunized mice. For EGFR, 

antibodies against all four sub-domains of the receptor were obtained, with affinities as low as pM and many functionally inhibited the receptor. Similarly, a diverse panel of anti-cMet antibodies recognising relevant 

epitopes was obtained, as many of the clones competed for binding to cMet with clinically-relevant antibodies. These antibodies serve as building blocks for the generation of bispecific therapeutic cLC IgG. Using a 

proprietary CH3 dimerisation technology, two different heavy chains and the cLC are expressed in a single cell and efficiently assemble into full length bispecific antibodies: Biclonics. This technology was used to 

screen 1000’s of Her1 x Her3 bispecifics and several highly potent bispecific antibodies were identified. 

E5 A Novel Fc-containing anti-CD19 x Anti-CD3 Bispecific Depletes B Cells in Cynomolgus Monkeys Gregory Moore Xencor, Inc. 

With a Single Intravenous Dose 

We have generated a potent anti-CD19 x anti-CD3 bispecific that contains variable regions cross-reactive for cynomolgus monkey CD19 and CD3 and an Fc region that allows for favorable pharmacokinetics. The 

bispecific mediates lysis of B lymphoma lines in the presence of either human or cyno T cells, and causes upregulation of CD25 and CD69 on the surface of T cells. In contrast, it produces no T cell activation in the 

absence of CD19-expressing target cells, a critical feature for avoiding toxicity. We have found that the bispecific is capable of rapid and sustained depletion of B cells in cynomolgus monkeys, with less than 5% of 

the blood B cell population remaining one day after infusion. The bispecific was developed using a novel bispecifics platform employing a heterodimeric Fc region that allows for efficient heterodimer purification and 

production. The antibody Fc provides structural and functional benefits including a large size that precludes renal filtration; binding to various activating and inhibitory Fc receptors (including FcRn, which prolongs 

half-life) and affinity for proteins A and G to facilitate large scale purification 



Diagnostic Antibody Engineering 

F1 A PSMA Specific Diabody for SPECT Imaging of Advanced Prostate Cancer Florian Kampmeier King’s College London 

Purpose: Primary prostate cancer is well manageable, however there is currently no effective tool to determine nodal status, detect metastases and low volume recurrent disease and to assess response to treatment. 

Prostate specific membrane antigen (PSMA) is a specific and well established marker for prostate carcinoma and is investigated in a number of clinical trials as a target for radioimmunotherapy and more recently ADC 

therapy. In this study a diabody derived from the monoclonal antibody J591 is developed as a SPECT tracer for the detection of PSMA in prostate cancer. Experimental: The diabody with a stabilizing C-terminal cysteine 

was expressed in 293T cells and purified by IMAC/SEC. Specificity and affinity were determined in cell binding studies. For SPECT imaging, the diabody was site specifically labelled with Tc99m via the C-terminal His 

tag and evaluated in a subcutaneous prostate carcinoma xenograft model. Results: J591c diabody binds to PSMA expressing cells with low nanomolar affinity (0.82+/-0.53 nM). Imaging studies showed a peak uptake 

of 4.9+/-1.6 %ID/cm3 at 8h post injection and a PSMA+ to PSMA- tumour ratio of 2.1. The Tc99m labelled J591 diabody allows SPECT imaging of PSMA within a short time frame. Feasibility of LN and metastasis 

imaging should be investigated. 

F2 Comparison of a Cys-Minibody and a Cys-Diabody for Anti-CD20 ImmunoPET Imaging Kirsten Zettlitz Crump Institute for Molecular Imaging 

The CD20 cell surface molecule is expressed at high levels on most B-cell lymphomas and is a useful target for antibody-based therapy. We developed, optimized and evaluated novel antibody-based (GA101) PET 

tracers as an efficacious diagnostic tool to assess antigen presence and expression, tumor localization and also for therapy management and follow-up. GA101 is a humanized, non-internalizing type II anti-CD20 mAb 

that binds CD20 in a different orientation from type I antibodies (e.g. rituximab) and is not internalized. Based on GA101 we engineered antibody fragments differing in size and with pharmacokinetics optimized for 

PET imaging. Both fragments, a cys-diabody (CDb, 55 kDa) and a cys-minibody (CMb, 83 kDa), retain cell-binding characteristics (type II epitope, stoichiometry, apparent affinity) of the parental IgG but lack Fcmediated 

functions and FcRn recycling. GA101_CMb and GA101_CDb were radioiodinated with 124I and evaluated in mice bearing CD20-positive subcutaneous tumors. MicroPET images at 4, 8, and 24 h show high 

activity in the CD20-positive tumor with low background for both fragments. Specific uptake in the CD20–positive tumor as determined by ex vivo biodistribution at 24 h was higher for GA101_CMb (9.16 ± 1.9 %ID/g) 

while GA101_CDb reached a higher ratio of positive tumor:blood (12.78 ± 3.74). 

F3 Engineered Anti-murine CD8 Minibody Fragment for Cu-64 ImmunoPET Imaging of 

Richard Tavare UCLA 

CD8 Expression in Vivo 

As a means to non-invasively detect CD8 expression in vivo, the variable regions of the rat anti-mouse CD8 IgG2b antibody (clone 2.43) has been sequenced from the hybridoma for reformatting to minibodies and 

subsequent Cu-64 ImmunoPET imaging. The ~80 kDa minibody is a divalent construct that contains the rat anti-mouse CD8 scFv followed by the mouse IgG2a hinge and CH3 domain. Engineering to the minibody 

format serves two purposes: firstly, the minibody lacks the full Fc fragment and will, therefore, not deplete CD8+ cells in vivo, and secondly the minibody has optimal pharmacokinetics for radionuclide imaging because 

the lack of a full Fc domain decreases the blood half-life. After SCN-NOTA conjugation and Cu-64 radiolableing, PET imaging and subsequent ex vivo biodistribution was acquired for 4 different groups of mice four 

hours post-intravenous injection: wild type C57BL/6, NOD scid gamma mice, C57BL/6 mice blocked with 4 mg/kg cold minibody, and C57BL/6 mice treated with an anti-CD8 depleting antibody. The radiolabeled 

2.43 minibody showed specific uptake in the spleen and lymph nodes of the wild type C57BL/6 mice and 4- to 5-fold reduced uptake in the spleen and lymph nodes of NSG SCID mice, blocked C57BL/6 mice, and 

depleted C57BL/6 mice. 

F4 Anti-CA-15.3 IgY Antibodies as a Potential Diagnostic Tools Renata Grzywa Wroclaw University of 

Technology 

Efficiency of anticancer therapy depends not only on well-aimed treatment strategies but also on early disease diagnosis. The development of new diagnostic methods relies on selection of specific tumor markers. One 

such biomarker associated mainly with breast cancer is CA 15-3. This serum-based glycoprotein is a soluble form of the transmembrane MUC-1 protein. Elevated level of CA 15-3 are associated with breast cancer as 

well as other adenocarcinomas including lung, ovarian and pancreatic cancer. In this study, we obtained and characterized chicken anti-CA 15-3 IgY antibody. The antigens used for hen immunization included the 

full protein in addition to two epitopes administered as conjugates with KLH protein. Immunochemical characterization of IgY antibodies isolated from egg yolks confirmed their high specificity towards all injected 

antigens. In addition, some of the isolated antibodies demonstrated cross-reactivity between specific epitopes and full-length CA 15-3 protein. In conclusion, the polyclonal specificity and low cost of production of IgY 

antibodies makes them promising candidates for use in future diagnostic immunoassays. 

F5 Generation and Application of PSA-Specific IgY Antibodies Agnieszka Lupicka Wroclaw University Of Technology 

According to the American Cancer Society prostate cancer is the second leading cause of cancer death in men. Early diagnosis reduces the risk of metastasis and thus increases the chances of successful treatment. 

Prostate specific antigen (PSA) is a member of the serine protease family produced by the prostate gland. The physiological function of PSA is primarily associated with liquefying of the coagulum necessary for sperm 

liberation. Elevated levels of PSA are associated with the development of prostate tumors, thus PSA is considered to be a useful biomarker for diagnostic purposes. The aim of our studies was the generation of avian 

antibodies (IgY) directed against prostate specific antigen. The advantages of using IgY technology include ease of antibody isolation, low production cost, and stability in vivo. Moreover, IgY antibodies do not react 

with human complement or rheumatoid factor, making them ideal for diagnostic purposes. In order to generate PSA-specific IgY antibodies we immunized hens either with native protein or with selected, highly 

immunogenic peptidyl epitopes. Peptidyl PSA fragments used for immunization were synthesized using the SPP method and conjugated to carrier protein. The isolated specific antibodies demonstrated high specificity 

of antigen recognition (including native PSA recognition by hapten-derived IgY antibodies). 

F6 Development of HIV-1 gp120-specific IgY Antibodies Marcin Sieńczyk Wroclaw University of 

Technology 

Although extensive research into the development of HIV-1 aspartyl protease inhibitors led to discovery and use of nine clinically approved drugs, the number of HIV infections continues to rise. The WHO statistics 

indicate that in 2011 more than 2.5 million new infections were noted and 34.2 million people are living with HIV globally. Lack of an effective vaccine, and the high mutation rate of HIV contribute to the spread of 

this epidemic. Thus, there is a significant need to develop cost-efficient, reliable and widely affordable HIV diagnostic tools. Located on the surface of HIV particle, the gp120 protein is crucial for virus entry into the cell 

via CD4/CCR5 binding. We focused our attention on the development of gp120-specific antibodies that were elicited in hens. In order to generate specific IgY antibodies we immunized with either native gp120 protein 

(HIV-1 IIIB) or its polypeptide fragment. Here we report the first example of gp120-specific IgY antibodies that may be used to diagnose HIV-1 infection. Our isolated IgY antibodies showed high specificity of binding to 

gp120. The major advantages of this technique include ease of isolation (the IgY antibodies can be purified from egg yolk), high yield and low cost of production. 

F7 Generation of Antibodies Specific to Staphylococcus Aureus Adhesion Proteins Map and Efb Maciej Walczak Wroclaw University Of Technology 

Effective treatment of the increasing number of Staphylococcus aureus infections, especially infections caused by antibiotic-resistant strains replies upon rapid and reliable diagnostic tools. The most common current 

diagnostic methods are based on microbiological techniques (Gram staining, isolation, culturing on mannitol containing medium). Further species differentiation is then performed via detection of specific enzymes 

such as catalase, coagulase, or DNAse. These approaches are limited by their time-consuming character and, in the case of some Staphylococcus species, low sensitivity for specific enzyme expression. Here we 

report the application of two constitutively expressed S.aureus adhesion proteins – Map and Efb which were used for development of specific antibodies. Map (EAP, p70) allows bacteria adhesion to the extracellular 

structures which facilitates tissues colonization and spread of the infection. Efb protein is able to bind fibrinogen, and disrupts immune response by inhibiting complement activation through C3b binding, and 

preventing B-cell stimulation. Efb and Map proteins as well as their polypeptide fragments were used to generate specific IgY antibodies. These antibodies represent an interesting alternative to classic mammalian IgG, 

and may be used to develop novel diagnostics as well as therapies to support conventional antibiotic treatment. The biochemical evaluation of the generated IgY antibodies will be discussed. 

F8 Monitoring Therapeutic Monoclonal Antibody Concentration: Development of Reagents for Detecting Francisco Ylera AbD Serotec 

Free and Total Drug 

In the bloodstream, therapeutic antibodies against targets such as TNF_ exist in free or bound forms. As bio-analytical methods for determining drug concentration are often based on ligand-binding assays using the 

drug target as capture agent, only the free drug concentration can be measured. However, drug target concentration is known to increase in disease state and also may also accumulate upon antibody treatment, which 

in turn leads to an underestimation of total drug concentration, when only free drug is measured. Here we present the development of reagents for measuring total drug and drug-target complexes. Using the antibody 

drugs Remicade® (Infliximab), Humira® (Adalimumab) and an in house drug candidate as examples, , we developed recombinant antibodies using the HuCAL® PLATINUM library that bind specifically to either the 

free drug or to the drug complexed with its target. The in vitro phage display approach for reagent generation enabled antibody-target complexes to be used as antigens and simultaneous subtraction of unwanted 

specificities. Such antibodies are extremely difficult to develop using traditional animal immunization methods. The development and use of these recombinant antibodies in immunoassays to measure total drug or 

drug-target complexes in biological matrices like human serum is shown. 

F9 Ultra-Quenchbodies: Antibody Probes that Fluoresce Upon Antigen Binding Hiroshi Ueda The University of Tokyo 

Recently we reported “Quenchbody”, which is a site-specifically labeled scFv whose fluorescence is initially quenched, but de-quenched by the antigen binding. Investigation of the enhancement mechanism of the 

carboxytetramethylrhodamine (TAMRA)-labeled anti-osteocalcin scFv showed that this antigen-dependency was due to the quenching and its antigen-dependent release by semi-conserved Trp residues near the VH/ 

VL interface [1]. Here we describe an extension of this approach to more natural Fab fragments. Using a cell-free translation-mediated position-specific protein labeling system, we made Fab fragments that have 

an N-terminal TAMRA in either H or L chain, or both. Surprisingly, the Fab with one dye in H chain showed an antigen-dependent fluorescence enhancement up to 9.6-fold, which surpassed that of TAMRA-scFv. 

Furthermore, the Fab with two dyes showed 26-fold enhancement, probably due to H-dimer formation between the dyes. Also, when the H and L chains were labeled with different dyes that cause FRET between 

them, fluorescence enhancement up to 50-fold was observed. Similar enhancements were seen for other Fabs recognizing haptens to proteins. Due to its versatility, stability and practical responses, these “Ultra- 

Quenchbodies” are expected to have a range of applications, including validation of novel therapy targets. Reference: Abe et al., JACS 133 (2011) 17386-94. 



Therapeutic Antibody Development, Receptor Agonists/Antagonists & Target Validation 

G1 Human scFvs For Cholera Treatment Viktoriya Dubrovskaya The Scripps Research Institute 

Cholera, is an infectious gastroenteritis caused by enterotoxin-producing strains of the bacterium Vibrio cholerae and is generally associated with consumption of raw shellfish and raw vegetables. Massive liquid loss 

occurs after the cholera toxin first attaches to the gut epithelia via its B subunit (CTB), and then upon cell entry of its A subunit affects the electrolyte balance by activation of adenyl cyclase and if not treated may cause 

patient’s death within 3 h. To date there is no currently approved vaccine treatment for cholera available, and public health measures, such as adequate sewage treatment and a reliable source of safe drinking water, 

are considered the most effective way of combating the disease. In this work a phage display library of human scFvs was generated from VH and VL gene repertoires of peripheral blood lymphocytes of 44 donors. The 

library was enriched against CTB and specific anti-CTB scFv have been selected. We have demonstrated that certain antibody fragments compete effectively with CTB for binding to GM1-containing liposomes. We have 

also shown that these antibody fragments effectively block binding of CTB to fixed and non-fixed membrane of Caco2 gut epithelial cells. 

G2 Engineering Agonists and Antagonists to Complex Membrane Targets: Peptide and Domain Grafting Eric Tam Innovative Targeting Solutions 

of Antibody CDRs Utilizing Mammalian Display Libraries. 

Inc 

Although antibodies have been generated against numerous epitopes, there are important clinically relevant receptor classes, including GPCRs and ion channels, which have historically been difficult to target. Peptides 

show promise against these challenging targets but they often have limitations as therapeutics. We report the use of a mammalian display system to graft peptides with reactivity to complex membrane antigens into the 

CDRs of the full length human IgG scaffold. Characterization of a library of greater than 100 million peptide variants generated in the context of all the human heavy chain variable and human light chain variable gene 

segments is presented. The advantages of diversifying both the length and composition of peptide flanking sequences as well as strategies to isolate rare variants with the desired properties are described. 

G3 Synthesis of GPCR by Wheat Cell-free Protein Synthesis System; Application for Functional Analysis Hiroyuki Takeda Ehime university 

and Monoclonal Antibody Production 

Membrane proteins account for one third of human genome, and are important player in in signal transduction and metabolism. More than half of drug target are membrane protein. However infrastructure for their 

functional analysis, such as expression and purification system or antibody production method, is not established enough. Recently we reported a membrane protein synthesis method, in which liposome was added 

into wheat cell-free synthesis system (Nozawa, et al., 2011). Membrane protein is embedded on the lipid layer of liposome during synthesis. Membrane proteins with various numbers of transmembrane domains are 

successfully synthesized. Membrane proteins prepared by the present method are applicable for variety of functional analysis. Using a GPCR, DRD1 as a model, we developed several application of proteoliposome 

prepared by the present method, such as ligand binding assay, in vivo G protein activation assay and antibody preparation. DRD1 proteoliposome was prepared by the present method and immunized to mice. Among 

800 clones of hybridomas produced, dozens of monoclonal antibodies bound to the antigen specifically. These antibodies were applicable for ELISA, Western blotting, immunoprecipitation, and immunostaining. 

These results demonstrated that proteoliposome prepared by wheat cell-free system was an advantageous materials for antibody production of membrane proteins. 

G4 A Novel Hexameric TNF-alpha Binding Molecule, EnbCS6hFc, Exhibits Potent Therapeutic Effect on Ya-Ping Lai Industrial Technology 

Arthritis Mouse Model 

Research Institute 

Tumor necrosis factor-alpha (TNF-alpha) plays an important role in the pathology of rheumatoid arthritis. A novel hexameric TNF-alpha-binding molecule, EnbCS6hFc, consisting of the extracellular domain of the 

TNF receptor II, a self-trimerizing collagen-like peptide and a human Fc domain was produced recombinantly to explore the therapeutic potential in rheumatoid arthritis. Both EnbCS6hFc and the bivalent counterpart, 

etanercept, exhibited similar potency in neutralizing soluble form TNF-alpha. However, EnbCS6hFc showed a 72.6-fold stronger binding strength to the transmembrane TNF-alpha than etanercept as determined by 

competitive binding assays using stable cell line expressing the non-cleavable transmembrane TNF-alpha. The pharmacokinetics of EnbCS6hFc in mouse was similar to etanercept. Possibly due to the avidity effect of 

multivalance of EnbCS6hFc to the transmembrane TNF-alpha, EnbCS6hFc is superior to etanercept in treating collagen antibody-induced arthritis mouse model. 

G5 GPCR Antibody Discovery Directly on the Cell Surface Cory Bentley Fabrus, Inc 

Fabrus, Inc. is developing functional therapeutic monoclonal antibodies to challenging cell surface targets. Therapeutic targets embedded in the cell membrane, such as GPCRs and ion channels, cannot easily be 

isolated and maintain native structure. Fabrus circumvents this challenge by screening antibodies against cell-surface targets directly on living cells with a spatially addressed IgG library. Fabrus has generated an IgG 

library with a single synthetic germline antibody per microtiter well. The Fabrus Library allows for a non-competitive evaluation of each library constituent, bringing small molecule screening approaches to biologic 

therapeutic discovery. This shift in therapeutic antibody discovery has generated specific library hits against GPCR-expressing target cells as identified by multiplexed high-throughput flow cytometry. Hits were against 

Family A and Family B GPCRs. Initial characterization of these hits, based on ligand-dependent modulation antibody binding to the cell surface, suggested that various hits had agonist or antagonist activity. After 

affinity and functional maturation, a Family B GPCR hits demonstrates positive modulatory activity and multiple Family A GPCR hits demonstrate inhibitory activity. These approaches give Fabrus access to targets 

previously intractable for antibody therapeutics discovery. 

G6 RAGE is a New Target for Antibody-based Therapy Estelle Leclerc North Dakota State University 

The Receptor for Advanced Glycation Endproducts is a pattern recognition Receptor that is found activated in a large number of human pathologies such as Alzheimer’s Disease, cancer and in complications of 

diabetes. In several mouse models of RAGE mediated pathologies, blocking RAGE by anti-RAGE antibodies has been shown to significantly reduce RAGE dependent deleterious effects. Activation of RAGE occurs 

following the interaction of RAGE ligands with the extracellular domains (V, C1 and C2) of the receptor. Despite numerous in vitro studies on RAGE/ligand interaction, the role of each extracellular domain of RAGE in 

in vivo ligand recognition is not fully understood. We have generated a pool of monoclonal antibodies against the distinct domains of RAGE (V, C1, C2) that will help to understand the mechanisms of RAGE activation 

by its ligands in cells. Moreover, these antibodies could be used as diagnostic reagents and potential therapeutic agents. 

G7 Targeting TGF-alpha for Antibody Therapy against Cetuximab Resistant Cancer. Yoshiro Kishi Medical & Biological 

Laboratories, Co., Ltd. 

Transforming Growth Factor alpha (TGF-alpha) is a member of the epidermal growth factor family. TGF-alpha is co-expressed with it receptor called Epidermal Growth Factor Receptor (EGFR) in various cancer cells. 

TGF-alpha and EGFR pathway is involved in both cancer growth and stroma development in an autocrine fashion. Therefore, we hypothesized that neutralization of TGF-alpha by specific monoclonal antibody blocks 

autocrine cancer growth, and prevents angiogenesis in stroma as well. We have generated a panel of mouse monoclonal anti-human TGF-alpha antibodies, which bind to both soluble and membrane-bound form 

of TGF-alpha. Those antibodies demonstrated inhibitory activities in TGF-alpha induced EGFR phosphorylation and on in vitro tumor cell growth. Furthermore, these antibodies revealed potent in vivo anti-tumor 

activities over various human cancer xenograft models including Cetuximab resistant cancers with K-ras gene mutation. In addition, some of them inhibited TGF-alpha induced angiogenesis in vivo. These results 

suggested that TGF-alpha neutralizing antibody could be a novel therapeutic candidate against Cetuximab resistant cancers. In poster session, we will discuss the mechanism of action of anti-TGF-alpha antibodies 

inhibiting development of tumors harboring a K-ras gene mutation. 

G8 Isolating Species-specific Antibodies Against Bacteria: a Novel Tool for Metagenomics Sandeep Kumar Institution: Los Alamos 

National Laboratory 

Metagenomics is a nascent field addressing the complexity and constitution of microbiomes in environmental samples, as well as their interaction with the host, for example, those in the human gut. Current 

methodologies employ shotgun sequencing and 16S sequence surveys of the whole genomes and tend to be biased towards over-represented communities in the microbiome. As an alternative approach, individual 

or similar species in a complex microbiome can be isolated in a high throughput fashion, or the commonest species depleted, by species-specific antibodies against the species. Whole genome sequencing following 

such sample pre-processing would lead to a broader coverage in the metagenomics studies. We have applied this approach to a representative community of bacteria found in human gut and we show the feasibility 

of selecting antibodies against individual species or the whole population in the community using phage display technique. Further, we show the application of high throughput sequencing of the CDR3 regions of 

polyclonal single-chain antibodies as a tool for identifying unique antibodies that are specific to individual bacterial species. Once isolated, species-specific antibodies will be used in combination with Fluorescenceactivated 

cell sorting (FACS) to evaluate their ability to enrich or deplete a complex sample for a targeted bacterial species 

G9 Isolation of Salmonella Serovar-specific Single-Domain Antibodies. Agnieszka Nowacka National Research Council 

Canada 

Worldwide, the consumption of Salmonella-contaminated food and water is extremely common. Each year, it results in thousands of infections leading to food poisoning, increased health problems and health costs, 

and in some cases, death. Three of the most common Salmonella serovars known to be involved in infection are Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella 

enterica serovar Newport. In this proof of concept study, we have isolated Salmonella flagellin protein antigens, which will provide serovar specificity due to the sequence variability present in their domain 3 (D3) 

composition, among each of the different serovars. In turn, when we isolate single-domain antibodies against our antigens using a phage display library, the antibodies that will be isolated should therefore be serovarspecific 

as well. Immunoassays will be carried out to test the affinity and cross-reactivity of the isolated antibodies. This work will provide the basis for the potential future development of an immuno-paper dipstick 

system, which can be used for detection purposes in order to help reduce the spread of Salmonella infection. 

G10 Effect of Basigin on Lapatinib Resistance of Gastric Cancer Cells Kristine Kim Kangwon National University 

Lapatinib is a dual inhibitor for EGFR and HER2 that has shown promising in-vitro results treating inhibition of HER2+ cancer cells. However, some tumors either do not respond or develop resistance to the agent. 

Using an integrated proteomic approach, we have identified a panel of significantly up-regulated plasma membrane proteins including CD44, CD133, EpCAM, CD151, basigin, and HER3 in the lapatinib-resistant 

gastric cancer cells compared to its parental cells. Expression levels of selected proteins were further validated using immunoblot assays. In the present study, we explored the role of basigin mediating the lapatinib drug 

resistance. We tested the effects of a basigin monoclonal antibody on the growth of the lapatinib resistance cells and observed significant inhibition of cell growth, suggesting basigin is an interesting target molecule 

involved in the lapatinib drug resistance. Here we present a systematic approach for the identification of a panel of differentially expressed plasma membrane proteins in the drug resistant cells. We also demonstrate 

that neutralizing basigin sensitizes the anti-cancer drug resistant cells. 



G11 Cloning of Primate FcR - A Cellular Tool for Late Phase Discovery and Species Selection Lauren Stephens Cambridge University 

The safety assessment of therapeutic monoclonal antibodies is often carried out in non-human primates (NHP). Many therapeutic targets are present on both NHP and human cells and this can be established using 

in vitro binding assays prior to in vivo testing. However antibodies interact with not only their cognate antigen through the variable region but cellular effector mechanism via Fc:FcR binding. Currently there is no 

established method to measure the ability of human antibody to interact with NHP FcR directly. We present work in which cell lines have been transfected with Fc_RIIIa from different NHP species and compared with 

human FcR in their ability to bind human antibody. Using a panel of human IgG subclasses we have shown that NHP receptors generally had a higher affinity for human Fc regions, similar to the human 158V allele 

and that the immunoglobulin binding hierarchy was comparable (G3>G1>G4>G2). A panel of clinical IgG1 antibodies (Alemtuzumab, Bevacizumab, Trastuzumab, Ritxuimab and Ofatumumab) were tested and 

interestingly there were notable differences in the EC50 values between human and NHP for some antibodies such as Bevacizumab even though all the mAb were of the same subclass. 

G12 FcRn Molecules and Accompanying Assays Sune Justesen University of Copenhagen, Denmark 

The human neonatal Fc receptor (FcRn, or Brambell factor) is structurally related to MHC class I molecules; it consists of an MHC class I-like heavy chain associated with the MHC class I light chain, beta-2microglobulin 

(?2m). In contrast to MHC class I molecules, however, it does not bind peptide; instead, it binds IgG and albumin in a pH dependent way, showing strong interaction at low pH and no interaction at 

physiological pH. Thus, upon cellular internalization and the ensuing endosome acidification, the FcRn binds internalized IgG and albumin and returns them to the cell surface, where they are released into the neutral 

extracellular environment. In effect, the FcRn rescues IgG and albumin from lysosomal degradation thereby explaining the unusual long half-life of these proteins compared to other plasma proteins. Exploiting FcRn 

mediated rescue has emerged as a potentially generalizable approach to improve the pharmacokinetics of therapeutics. In parallel, blocking of the FcRn mediated rescue of antibodies may pose a way systemic removal 

of pathogenic and autoreactive antibodies. Hence identification of novel or modified FcRn ligands could have significant clinical relevance. The ability to measure FcRn interactions is a prerequisite for any study and 

manipulation of FcRn mediated rescue. Here, we have used bacterially expressed and refolded soluble human FcRn to establish high-throughput homogenous assays capable of measuring the interaction of nearly any 

compound to the albumin or IgG binding site of FcRn. We demonstrate how the assay have been used to measure the affinity of, a panel of HSA mutants and therapeutic antibodies. Additionally the FcRn molecules 

have been used to screen a small molecule library using cutting edge technology. The screening identified a small molecule capable of inhibiting the interaction between IgG and FcRn. To our knowledge this is the first 

example of a small molecule FcRn antagonist. 

G13 A Series of Bispecific Antibody Formats Evaluated for Pretargeting: Comparison of Blood Clearance Paul Yazaki City of Hope Beckman 

and Tumor Uptake. 

Research Institute 

A series of anti-tumor/anti-chelate bispecific antibody formats were developed for pretargeted radioimmunotherapy. Based on the anti-carcinoembryonic antigen humanized hT84.66-M5A monoclonal antibody and 

the anti-DOTA C8.2.5 scFv antibody fragment, this cognate series of bispecific antibodies were radioiodinated to determine their tumor targeting, biodistribution and pharmacokinetic properties in a mouse xenograft 

tumor model. The in vivo biodistribution studies showed that all of the bispecific antibodies exhibited specific high tumor uptake but the tumor targeting was approximately one-half of the parental anti-CEA mAb due 

to faster blood clearance. Serum stability and FcRn studies showed no apparent reason for the faster blood clearance. A dual radiolabel biodistribution study revealed the 111In-DOTA bispecific antibody had increased 

liver and spleen uptake, not seen for the 125I-version due to metabolism and release of the radioiodine from the cells. This data suggests increased clearance of the antibody fusion formats by the mononuclear 

phagocyte system. Importantly, a pretargeted study showed specific tumor uptake of 177Lu-DOTA and a tumor:blood ratio of 199:1. This pretargeted radiotherapeutic and substantial reduction in the radioactive 

exposure to the bone marrow should enhance the therapeutic potential of RIT. 

Therapeutic Development of Early Stage Antibodies 

H1 CRB0017: A Novel Therapeutic Antibody to Treat Osteoarthritis Michela Visintin Rottapharm Biotech Srl 

Aggrecan cleavage by the disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 4 (ADAMTS-4) and 5 (ADAMTS-5) is crucial for the breakdown of cartilage matrix during osteoarthritis (OA), 

a degenerative joint disease that leads to the progressive destruction of articular structures. To date, several class of small-molecular inhibitors of ADAMTS-4 and ADAMTS-5 have been identified, but due to the high 

homology among the ADAMTS family that share common catalytic and structural features with other metalloproteases members, designing selectivity has been very challenging. In order to develop a specific inhibitor 

that lacks off-target side effect, we used the two-hybrid based IAC technology to isolate a panel of monoclonal antibodies (mAbs) against ADAMTS-5. mAb CRB0017 was identified with low nanomolar affinity 

constant and high specificity for ADAMTS-5. CRB0017 showed potent inhibition of biological function of ADAMTS-5 in two relevant OA models. CRB0017 administered intraarticularly twice in three months in the 

STR/ort mouse could modify the course of OA by delaying cartilage breakdown. Similar results were obtained testing the mAb in the medial meniscal tear rat model. The procedure of blind scoring of the histological 

samples clearly showed a dose dependent effect of the antibody in reducing the severity of the OA pathology in these animal models. 

H2 Human mAb Binding to Human and Mouse L1CAM for the Treatment of Cancers Hyo Jeong Hong Kangwon National University 

L1 cell adhesion molecule (L1CAM) was first described as a neural cell adhesion molecule and functions in the development of the nervous system by mediating homophilic and heterophilic binding at the cell surface. 

L1CAM is also expressed at low levels in other cell types such as lymphoid and myelomonocytic cells, kidney tubule epithelial cells, and intestinal crypt cells. L1CAM is highly expressed in several types of cancers and 

its expression correlates with tumor progression and metastasis. To evaluate whether anti-L1CAM antibody could serve as an anticancer agent, we developed a human mAb that specifically bind to human and mouse 

L1CAM with a high-affinity (KD = ~ 0.2 nM) and also constructed a humanized antibody that binds to only human L1CAM with a moderate affinity (KD = ~ 2.0 nM). Animal study showed that the human mAb greatly 

inhibited the growth of tumor in human bile duct carcinoma xenograft nude mice model, while the humanized antibody showed lower antitumor activity compared with the human antibody. The animals treated with 

each antibody 3 times a week for 4 weeks did not exhibit any symptoms. The results suggest that the human mAb has potential as an anticancer agent for the treatment of cancers expressing L1CAM and also will have 

advantaghdges over the mAb binding to only human L1CAM in pre-clinical studies. 

H3 Prophylactic and Therapeutic Antibody Against Herpes Simplex Virus Infection in Wild-type and Jiann-Shiun Lai Development Center for 

Immunodeficient Mice 

Biotechnology 

Herpes simplex virus type 1 (HSV-1) and type 1 (HSV-2) are common human pathogens that include primary and recurrent infections of mucous membranes (genital and orofacial herpes), neonatal HSV infection, 

visceral HSV infections in immunocompromised hosts, HSV encephalitis, and an association with erythema multiforme. However, infections in the eye, central nervous system, and brain can be life-threatening. Several 

approaches are currently available for treating HSV infection, including antiviral medication and vaccine. However, these approaches do not prevent or cure HSV infection and only reduce viral reproduction or alleviate 

complications associated with HSV infection. In this study we used scFv phage displayed human libraries to screen specific antibodies against HSV. We have identified three glycoprotein D (gD)-specific monoclonal 

antibodies (E317, E425, Y571), but not glycoprotein B, which show equal potency of HSV-1 and HSV-2-neutralizing activity in vitro and demonstrated that MAb E317 had prophylactic and therapeutic efficacy in SCID 

mice challenged with lethal dose of HSV-1. We, furthermore demonstrate that MAb E317 appeared to be more effective than acyclovir (ACV) suitable as therapeutic tools in clinical applications. When SCID mice were 

treated intraperitoneally with a single dosage of 15 mg per kg of E317 at 24 hours after HSV-1 challenge, the antibody protected 100% of mice from death compared to 0% survival rate with ten consecutive days of 

ACV treatment. These studies define the complexity of epitopes on gD of HSV-1 and HSV-2 that is recognized by highly protective antibodies with therapeutic potential. 

H4 Streamlining Antibody Development Using Large Scale, CHO Transient Gene Expression (TGE) Karen Donato MaxCyte 

Followed by Rapid Production of CHO Stable Pools 

Antibody production for early stage antibody development activities is commonly conducted using transiently transfected HEK cells to produce adequate quantities of antibody for characterization, while later stage 

screening and biomanufacturing rely on CHO-based stable cell lines. Migration from HEK to CHO cell backgrounds can lead to manufacturing challenges and changes in post translational modifications that can alter 

the antibody’s therapeutic potential. MaxCyte’s proprietary electroporation technology can greatly shorten the time line of antibody development by enabling large scale transient gene expression (TGE) directly within 

CHO cells. Data will be presented showing rapid gram-scale antibody production via transient transfection using the MaxCyte STX®. CHOS cells can be transfected with >95% transfection efficiency and cell viability. 

CHOS cells transfected in bulk via flow EP with a human IgG1 plasmid can be seeded at high density post EP, generating titers > 400 mg/L and enabling >1 gram of protein from 1g/L in 6 weeks. 

H5 Anti-PSMA x anti-CD3 Bispecific Antibody Redirects T cell Cytotoxicity in Castrate-resistant Prostate Jane Gross Emergent BioSolutions Inc 

Cancer Models 

Authors Toddy Sewell, Gabriela Hernandez-Hoyos, Ruth A. Chenault, Jennifer Wiens, John Kumer, Sateesh Natarajan, Catherine J. McMahan, Jane A. Gross, Paul A. Algate, and John W. Blankenship. Emergent Product 

Development Seattle, LLC. Seattle, WA, USA. Title: anti-PSMA x anti-CD3 bispecific antibody redirects T cell cytotoxicity in castrate-resistant prostate cancer models Abstract: Background: Prostate cancer is the most 

common cancer in men. Although screening, radiation, surgery and hormone ablation therapy have greatly improved the detection and treatment of early stage prostate cancer, few options exist to treat metastatic, 

castrate-resistant prostate cancer (CRPC). We have developed a therapeutic approach that uses a bispecific SCORPIONTM (multi-specific protein therapeutic) molecule that redirects T cell cytotoxicity against cells 

expressing a common prostate cancer antigen, PSMA (Prostate Specific Membrane Antigen). SCORPION molecules are antibody-like therapeutics containing two sets of binding domains linked to immunoglobulin 

Fc domains, which extends the half-life of the molecule in vivo. Materials and Methods: Cytotoxic activity was examined in vitro by treating PSMA(+) cell lines (LNCaP, C4-2B, MDA-PCa-2b) and PSMA(-) cell lines 

(DU-145, BxPC-3) with SCORPION molecules in the presence of purified human T cells or human peripheral blood mononuclear cells (PBMCs). Cytotoxic activity was determined by chromium release assays or by 

incorporation of a label such as propidium iodide or 7-aminoactinomycin D. CFSE-labeled T cells were assessed for activation and proliferation in a similar setting using multi-color flow cytometry. To evaluate serum 

stability, SCORPION proteins were incubated in mouse serum and monitored for functional activity. Results: SCORPION molecules targeting PSMA and CD3 efficiently redirected T cell cytotoxicity against PSMA(+) 

cell lines in vitro at low picomolar concentrations. Cytotoxic activity was target dependent and required the presence of T cells. T cells were activated and proliferated in response to the SCORPION therapeutic in the 

presence of PSMA(+) target cells; no proliferation was observed in response to PSMA(-) cells. SCORPION proteins were stable to freeze/thaw in mouse serum and showed stable functional activity in mouse serum 

when incubated at 37 °C for up to one week. Conclusions: These studies show that anti-PSMA x anti-CD3 SCORPION therapeutics may be efficient agents for redirecting T cell cytotoxicity and show promise as 

potential therapeutics in CRPC. 



H6 Preclinical Characterization of ARGX-110, Targeting CD70 as an Immune Checkpoint Inhibitor and Peter Ulrichts arGEN-X 

Proliferation Blocker 

In healthy individuals, expression of CD70 is limited to activated T- and B-cells and mature dendritic cells. In many autoimmune indications, chronic expression of CD70 on T-cells has been demonstrated. In 

addition, overexpression of CD70 is documented in a variety of solid and hematological tumors, where it is thought to play a role in evasion of immune surveillance and tumor proliferation and survival. ARGX-110 

is a defucosylated, human IgG1 monoclonal antibody that selectively targets and neutralizes CD70, the ligand of CD27. Detailed biochemical and functional characterization was carried out to support its clinical 

development as therapy for cancer and autoimmune diseases. ARGX-110 binds human and cynomolgus CD70 with picomolar affinity and inhibits receptor-ligand interaction with high potency as measured in 

bioassays. The therapeutic potential of ARGX-110 has been demonstrated in a Burkitt lymphoma xenograft model and in whole blood ex vivo depletion assays. When administered weekly with 5 doses of 30 mg/kg in 

cynomolgus monkeys, the antibody showed no signs of toxicity and had no effect on T-, B- and NK cell numbers. Moreover, the predicted half-life of ARGX-110 in humans is around 23 days. Initiation of a Phase I study 

in patients with CD70+ malignancies is planned for early in 2013. 

H7 A Bispecific SCORPION Molecule that Targets CD86 and Delivers an Immunosuppressive Cytokine, Phillip Tan Emergent Product 

IL10, is a Potent Inhibitor of Antigen Presenting Cells 

Development Seattle 

SCORPION (multi-specific protein therapeutic) molecules contain two binding moieties directed to either soluble and/or cell surface targets. The binding moieties are separated by an immunoglobulin Fc domain 

which can retain effector function including a long in vivo serum half life. SCORPION molecules possess process development characteristics potentially compatible with both clinical and commercial development. 

In this example, a SCORPION molecule is used to target and block a co-stimulation receptor, CD86 and delivers an immunosuppressive cytokine, IL10, to antigen presenting cells. By targeting multiple pathways, a 

SCORPION molecule can effectively inhibit antigen presenting cells, thereby preventing T cells stimulation, and may have potential application in autoimmunity and inflammatory diseases. We will present pre-clinical 

data of our SCORPION molecule showing high levels of in vitro/in vivo efficacy at low doses. 

H8 Engineering bs-scFv Based Immune Modulatory Antibodies Jimson D’Souza Fox Chase Cancer Center 

Jimson D’Souza, Heidi Simmons, Tatiana Pazina, Carl June, Gregory P. Adams, Matthew K. Robinson Antibody-based therapies employed against cancer typically function by either altering signal transduction and/or 

by focusing elements of the host immune response against the tumor cells. Bispecific antibodies that bind bivalently to a tumor antigen and an antigen present on the surface of immune effector cells have long been 

used clinically to directly focus the cytotoxic effect of the patient’s immune system. CD8 positive T cells represent a promising subset of immune effector cells for immunotherapy-based approaches. We hypothesize 

that targeting the activating T cell receptor ICOS or inhibitory receptors CTLA4 and PD1, in the context of a bs-scFv, represents promising immunomodulatory platform for inducing cytotoxic T cell-mediated antitumor 

response as well as a long-term memory response. To date we have expressed and purified the target antigens and used them for isolation of scFv antibodies against ICOS and PD-1 from a phage display library. 

Likewise, efforts are underway, to isolate antibodies against CTLA-4. Future work will focus on studying the effects of these antibodies on proliferation of immune cells in vitro and in vivo. 

H9 Glycoengineered Defucosylated Anti-CD20 Antibodies Activate Neutrophils Mediated Phagocytosis Josee Golay Ospedali Riuniti 

More Efficiently than Rituximab 

GA101 (obinutuzumab) is a glycoengineered defucosylated anti-CD20 antibody with higher affinity for CD16A and stronger antibody dependent cellular cytotoxicity activity (ADCC) by NK cells. Polymorphonuclear 

neutrophils (PMN) express the homologous GPI-anchored CD16B molecule which also shows enhanced affinity for glycoengineered antibodies. We have investigated the functional activity of human PMN in response 

GA101 or defucosylated rituximab (G2), compared to the parent rituximab (RTX) antibody. We show that GA101 activated PMN more efficiently than RTX, leading to 50% increased CD11b expression and 70% 

downmodulation of CD62L. This activation was not accompanied by generation of reactive oxygen species (ROS), or by ADCC. In contrast, activation of purified PMN through CD16B led to up to 45% phagocytosis of 

opsonised targets and was followed by PMN death. Furthermore significant phagocytosis (15% above background) could be observed in whole blood only in presence of defucosylated and not unmodified antibodies, 

similarly to what is observed for NK cell activation and ADCC. We conclude that phagocytosis of opsonised targets by PMN is an additional mechanism of action for defucosylated IgG1 antibodies. These data may 

offer an explanation for the increased neutropenia observed in patients treated with GA101, an effect therefore likely related to its anti-lymphoma activity. This study was in part supported by Roche Glycart 

H10 Development of Novel anti-IFN-gamma Humanized Antibody Maria Larina Institute of Bioorganic Chemistry 

Russian Academy of Sciences 

Interferon-gamma (IFN-gamma) plays important role in the generation and maintenance of Th1 lineage and promotes cellular immunity through the activation of macrophages, NK-cells and effector T-cells. Excessive 

release of IFN-gamma appears to be associated with a number of pathological processes. Thus using neutralizing antibodies to IFN-gamma might be considered as an approach for the treatment of several autoimmune 

diseases. The aim of present work was the development of anti- IFN-gamma humanized antibody. F1 is high affinity monoclonal murine antibody (Ab) to IFN-gamma possessing neutralizing activity. Humanization 

of F1 Ab was performed using the approach of sequential framework (FR) grafting. Human germline V gene segments were used as a source of human FR sequences. Comparison of the FR1 and FR4 grafting vs. full 

FR grafting in heavy and light chain showed that differences only in second and third heavy chain frameworks affect F1 Ab binding properties. The humanized version of F1 Ab obtained as a result of full FR grafting 

exhibited relative affinity 5-fold lower than the chimeric Ab. Substitution of heavy chain residues 69 and 71 with murine counterparts resulted in affinity and neutralizing activity determined in inhibition of HLA-DR 

induction assay similar to the chimeric Ab. 

H11 Comprehensive Optimization of a Single Chain Variable Domain Antibody Fragment as a Targeting Kathy Zhang Merrimack Pharmaceutical 

Module of Cytotoxic Nanoparticle. 

Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies. This application of antibody modules requires careful engineering as multiple 

parameters such as rate of internalization, colloidal, serum; lipid phase and chemical denaturation stability need to be optimized in addition to mouse cross-reactivity, expression levels and antigen-binding affinity. In 

this poster we present a case study of engineering of a single chain variable domain (scFv) of an antibody used to target liposome-based cytotoxic nanoparticles. We investigated the impact of orientation of VH and 

VL domains, length and composition of the linker, amino acid make-up of the framework and complementarity determining regions (CDRs) on the molecular properties of scFv. Additionally, we have explored the 

contributions of individual and grouped tyrosine residues in the CDRs to binding affinity and stability. Our studies demonstrated that comprehensive strategy was required to discover a ScFv with desired characteristics. 

This highlights the importance of high-throughput engineering, screening and characterization approaches for discovery of optimal antibody targeting modules for cytotoxic nanoparticles.

Antibody Therapeutics - IBC Life Sciences

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