Common light chain chickens produce human antibodies of high affinity and broad epitope coverage for the engineering of bispecifics.

Bispecific antibodies are an important and growing segment in antibody therapeutics, particularly in the immuno-oncology space. Manufacturing of a bispecific antibody with two different heavy chains is greatly simplified if the light chains can be the same for both arms of the antibody. Here, we introduce a strain of common light chain chickens, called OmniClic®, that produces antibody repertoires largely devoid of light chain diversity. The antibody repertoire in these chickens is composed of diverse human heavy chain variable regions capable of high-affinity antigen-specific binding and broad epitope diversity when paired with the germline human kappa light chain. OmniClic birds can be used in immunization campaigns for discovery of human heavy chains to different targets. Subsequent pairing of the heavy chain with a germline human kappa light chain serves to facilitate bispecific antibody production by increasing the efficiency of correct pairing. Abbreviations: AID: activation-induced cytidine deaminase; bsAb: bispecific antibody; CDR: complementarity-determining region; CL: light chain constant region; CmLC: common light chain; D: diversity region; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; Fc: fragment crystallizable; FcRn: neonatal Fc receptor; FR: framework region; GEM: gel-encapsulated microenvironment; Ig: immunoglobulin; IMGT: the international ImMunoGeneTics information system®; J: joining region; KO: knockout; mAb: monoclonal antibody; NGS: next-generation sequencing; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PGC: primordial germ cell; PGRN: progranulin; TCR: T cell receptor; V: variable region; VK: kappa light chain variable region; VL: light chain variable region; VH: heavy chain variable region.

Expression of human lambda expands the repertoire of OmniChickens.

Most of the approved monoclonal antibodies used in the clinic were initially discovered in mice. However, many targets of therapeutic interest are highly conserved proteins that do not elicit a robust immune response in mice. There is a need for non-mammalian antibody discovery platforms which would allow researchers to access epitopes that are not recognized in mammalian hosts. Recently, we introduced the OmniChicken®, a transgenic animal carrying human VH3-23 and VK3-15 at its immunoglobulin loci. Here, we describe a new version of the OmniChicken which carries VH3-23 and either VL1-44 or VL3-19 at its heavy and light chain loci, respectively. The Vλ-expressing birds showed normal B and T populations in the periphery. A panel of monoclonal antibodies demonstrated comparable epitope coverage of a model antigen compared to both wild-type and Vκ-expressing OmniChickens. Kinetic analysis identified binders in the picomolar range. The Vλ-expressing bird increases the antibody diversity available in the OmniChicken platform, further enabling discovery of therapeutic leads.

V(D)J Rearrangement Is Dispensable for Producing CDR-H3 Sequence Diversity in a Gene Converting Species.

An important characteristic of chickens is that the antibody repertoire is based on a single framework, with diversity found mainly in the CDRs of the light and heavy chain variable regions. Despite this apparent limitation in the antibody repertoire, high-affinity antibodies can be raised to a wide variety of targets, including those that are highly conserved. Transgenic chickens have previously been generated that express a humanized antibody repertoire, with a single framework that incorporates diversity by the process of gene conversion, as in wild-type chickens. Here, we compare the sequences and antibodies that are generated purely by gene conversion/somatic hypermutation of a pre-rearranged heavy chain, with the diversity obtained by V(D)J rearrangement followed by gene conversion and somatic hypermutation. In a gene converting species, CDR-H3 lengths are more variable with V(D)J rearrangement, but similar levels of amino acid diversity are obtainable with gene conversion/somatic hypermutation alone.

Chickens with humanized immunoglobulin genes generate antibodies with high affinity and broad epitope coverage to conserved targets.

Transgenic animal platforms for the discovery of human monoclonal antibodies have been developed in mice, rats, rabbits and cows. The immune response to human proteins is limited in these animals by their tolerance to mammalian-conserved epitopes. To expand the range of epitopes that are accessible, we have chosen an animal host that is less phylogenetically related to humans. Specifically, we generated transgenic chickens expressing antibodies from immunoglobulin heavy and light chain loci containing human variable regions and chicken constant regions. From these birds, paired human light and heavy chain variable regions are recovered and cloned as fully human recombinant antibodies. The human antibody-expressing chickens exhibit normal B cell development and raise immune responses to conserved human proteins that are not immunogenic in mice. Fully human monoclonal antibodies can be recovered with sub-nanomolar affinities. Binning data of antibodies to a human protein show epitope coverage similar to wild type chickens, which we previously showed is broader than that produced from rodent immunizations.

Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells.

The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds.

No Evidence of Antibodies against GAD65 and Other Specific Antigens in Children with Autism.

BACKGROUND: The presence of autoantibodies has been proposed as evidence for a role of autoimmunity in autism. This report investigates the prevalence of autoantibodies in children with autism using the luciferase immunoprecipitation systems (LIPS) immunoassay technology. A panel of autoantibody targets against several known and candidate neurological autoantigens, autoimmune-associated autoantigens and viruses was employed. METHODS: Serological analysis was performed on typically developing children (n = 55), developmentally delayed children without autism (n = 24) and children diagnosed with autism (n=104). Autoantibodies were measured against glutamic acid decarboxylase-65 (GAD65), a CNS autoantigen proposed to be associated with autism and against Ro52, glial fibrillary acidic protein, tyrosine hydroxylase, aquaporin-4, and gamma-enolase, the mouse mammary tumor virus and the xenotropic murine leukemia virus. Antibody levels and seropositivity prevalence were analyzed for statistically significant differences between the three groups. RESULTS: The majority of the children (98%) were seronegative for all targets in the antigen panel. No GAD65 seropositive children were detected in the cohort. Several low level seropositive sera against several of the protein targets were identified in isolated children in each of the three groups, but there was no difference in prevalence. CONCLUSION: Using this panel of antigens and a sensitive, robust assay, no evidence of unusual immunoreactivity was detected in children with autism, providing evidence against a role of autoimmunity against several previously implicated proteins in autism spectrum disorder pathogenesis. GENERAL SIGNIFICANCE: The idea that autoantibodies represent an underlying cause or are biomarkers for autism pathophysiology is not supported by this report.

Lateral Flow Immunoassay.

Lateral flow immunoassays (LFIAs) are a staple in the field of rapid diagnostics. These small handheld devices require no specialized training or equipment to operate, and generate a result within minutes of sample application. They are an ideal format for many types of home test kits, for emergency responders and for food manufacturers and producers looking for a quick evaluation of a given sample. LFIAs rely on high quality monoclonal antibodies that recognize the analyte of interest. As monoclonal antibody technology becomes more accessible to smaller laboratories, there has been increased interest in developing LFIA prototypes for potential commercial manufacture. In this chapter, the basics of designing and building an LFIA prototype are described.

Detection of shiga toxins by lateral flow assay.

Shiga toxin-producing Escherichia coli (STEC) produce shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA) for the rapid detection of Stxs in <10 min using a pair of monoclonal antibodies that bind epitopes common to Stx1 and six Stx2 variants. This LFA provides a rapid and sensitive test for the detection of Stxs directly from STEC culture supernatants or at risk food samples with a 0.1 ng/mL limit of detection (LOD) for Stx2a. This Stx LFA is applicable for use in the rapid evaluation of Stx production from cultured E. coli strains or as a tool to augment current methods as part of food safety testing.

Altered antibody profiles against common infectious agents in chronic disease.

Despite the important diagnostic value of evaluating antibody responses to individual human pathogens, antibody profiles against multiple infectious agents have not been used to explore health and disease mainly for technical reasons.  We hypothesized that the interplay between infection and chronic disease might be revealed by profiling antibodies against multiple agents. Here, the levels of antibodies against a panel of 13 common infectious agents were evaluated with the quantitative Luciferase Immunoprecipitation Systems (LIPS) in patients from three disease cohorts including those with pathogenic anti-interferon-γ autoantibodies (IFN-γ AAB), HIV and Sjögren's syndrome (SjS) to determine if their antibody profiles differed from control subjects.  The IFN-γ AAB patients compared to controls demonstrated statistically higher levels of antibodies against VZV (p=0.0003), EBV (p=0.002), CMV (p=0.003), and C. albicans (p=0.03), but lower antibody levels against poliovirus (p=0.04). Comparison of HIV patients with blood donor controls revealed that the patients had higher levels of antibodies against CMV (p=0.0008), HSV-2 (p=0.0008), EBV (p=0.001), and C. albicans (p=0.01), but showed decreased levels of antibodies against coxsackievirus B4 (p=0.0008), poliovirus (p=0.0005),   and HHV-6B (p=0.002). Lastly, SjS patients had higher levels of anti-EBV antibodies (p=0.03), but lower antibody levels against several enteroviruses including a newly identified picornavirus, HCoSV-A (p=0.004), coxsackievirus B4 (p=0.04), and poliovirus (p=0.02). For the IFN-γ AAB and HIV cohorts, principal component analysis revealed unique antibody clusters that showed the potential to discriminate patients from controls.  The results suggest that antibody profiles against these and likely other common infectious agents may yield insight into the interplay between exposure to infectious agents, dysbiosis, adaptive immunity and disease activity.

Rapid and selective detection of botulinum neurotoxin serotype-A and -B with a single immunochromatographic test strip.

Botulinum neurotoxins (BoNT) are the most potent toxins known. Produced by Clostridium botulinum, BoNTs are classified into seven, antigenically distinct serotypes, designated A-G. The toxin acts to inhibit acetylcholine release, resulting in paralysis and death. Naturally occurring foodborne disease is most often the result of improper canning of foods, while wound botulism, associated with injection drug users, is on the rise. Because of its potency, BoNTs have also been identified as targets for use by bioterrorists. The 'gold standard' of detection of BoNTs is the mouse bioassay, an expensive and time consuming test that requires specialized equipment and trained personnel. There is a need for a rapid, sensitive diagnostic for BoNTs that could be used by minimally trained personnel in the event of a foodborne outbreak or a bioterrorist threat. Here, we describe the use of a single lateral flow device (LFD) that can detect and distinguish between BoNT/A and B, two of the four serotypes that are known to intoxicate humans and together represent >80% of naturally occurring illness. The device could detect as little as 5 ng/mL of purified BoNT/A and 10 ng/mL of BoNT/B in 2% and 1% milk, respectively. In undiluted apple juice, 25 ng/mL of BoNT/A and 10 ng/mL of BoNT/B could be detected. No cross reactivity between BoNT/A and B antibodies was observed. The LFD described here is easy to use, requires no specialized training or equipment, and can identify and distinguish between BoNT/A and /B serotypes. These attributes make this rapid diagnostic device a potentially valuable tool in the fields of food safety and homeland security.

Two major autoantibody clusters in systemic lupus erythematosus.

Systemic lupus erythematosus is a chronic autoimmune disease of complex clinical presentation and etiology and is likely influenced by numerous genetic and environmental factors. While a large number of susceptibility genes have been identified, the production of antibodies against a distinct subset of nuclear proteins remains a primary distinguishing characteristic in disease diagnosis. However, the utility of autoantibody biomarkers for disease sub-classification and grouping remains elusive, in part, because of the difficulty in large scale profiling using a uniform, quantitative platform. In the present study serological profiles of several known SLE antigens, including Sm-D3, RNP-A, RNP-70k, Ro52, Ro60, and La, as well as other cytokine and neuronal antigens were obtained using the luciferase immunoprecipitation systems (LIPS) approach. The resulting autoantibody profiles revealed that 88% of a pilot cohort and 98% of a second independent cohort segregated into one of two distinct clusters defined by autoantibodies against Sm/anti-RNP or Ro/La autoantigens, proteins often involved in RNA binding activities. The Sm/RNP cluster was associated with a higher prevalence of serositis in comparison to the Ro/La cluster (P = 0.0022). However, from the available clinical information, no other clinical characteristics were associated with either cluster. In contrast, evaluation of autoantibodies on an individual basis revealed an association between anti-Sm (P = 0.006), RNP-A (P = 0.018) and RNP-70k (P = 0.010) autoantibodies and mucocutaneous symptoms and between anti-RNP-70k and musculoskeletal manifestations (P = 0.059). Serologically active, but clinically quiescent disease also had a higher prevalence of anti-IFN-α autoantibodies. Based on our findings that most SLE patients belong to either a Sm/RNP or Ro/La autoantigen cluster, these results suggest the possibility that alterations in RNA-RNA-binding protein interactions may play a critical role in triggering and/or the pathogenesis of SLE.

Serological studies confirm the novel astrovirus HMOAstV-C as a highly prevalent human infectious agent.

Molecular identification of a microbe is the first step in determining its prevalence of infection and pathogenic potential. Detection of specific adaptive immune responses can provide insights into whether a microbe is a human infectious agent and its epidemiology. Here we characterized human anti-IgG antibody responses by luciferase immunoprecipitation systems (LIPS) against two protein fragments derived from the capsid protein of the novel HMOAstV-C astrovirus. While antibodies to the N-terminal fragment were not informative, the C-terminal capsid fragment of HMOAstV-C showed a high frequency of immunoreactivity with serum from healthy blood donors. In contrast, a similar C-terminal capsid fragment from the related HMOAstV-A astrovirus failed to show immunoreactivity. Detailed analysis of adult serum from the United Sates using a standardized threshold demonstrated HMOAstV-C seropositivity in approximately 65% of the samples. Evaluation of serum samples from different pediatric age groups revealed that the prevalence of antibodies in 6-12 month, 1-2 year, 2-5 year and 5-10 year olds was 20%, 23%, 32% and 36%, respectively, indicating rising seroprevalence with age. Additionally, 50% (11/22) of the 0-6 month old children showed anti-HMOAstV-C antibody responses, likely reflecting maternal antibodies. Together these results document human humoral responses to HMOAstV-C and validate LIPS as a facile and effective approach for identifying humoral responses to novel infectious agents.

Antibody profiling of Borrelia burgdorferi infection in horses.

Infection with Borrelia burgdorferi is common in horses and ponies from the New England and mid-Atlantic regions of the United States. Here, we evaluated luciferase immunoprecipitation systems (LIPS) for profiling antibody responses against three different antigenic targets for the diagnosis of equine B. burgdorferi infection. LIPS testing of horse serum samples suspected of Lyme infection revealed that approximately 75% of the horse samples (114/159) were seropositive against the synthetic VOVO antigen, comprising repeated immunodominant C6 epitopes as well as OspC immunodominant epitopes. A comparison of VOVO and immunofluorescence assays (IFA) showed that 51% of the samples were positive in both assays (VOVO(+)/IFA(+)), 13% were VOVO(-)/IFA(+), 21% were VOVO(+)/IFA(-), and 15% were negative in both. To further understand humoral responses to B. burgdorferi and reconcile the diagnostic differences between IFA and VOVO, two additional B. burgdorferi LIPS tests were performed with DbpA and DbpB. Robust seropositive antibody responses against DbpA and/or DbpB were detected in 98% (79/81) of the VOVO(+)/IFA(+) and 93% (50/54) of the discrepant samples. Additionally, some of the samples negative by both VOVO and IFA showed immunoreactivity against DbpA and/or DbpB. Overall, 94% of the suspected horse samples were seropositive by LIPS, and heat map analysis revealed that seropositive samples often were immunoreactive with at least two of the three antigens. These results suggest that LIPS tests employing multiple recombinant antigens offer a promising approach for the evaluation of antibody responses in Lyme disease.

Emerging tactical strategies for fighting the war on cancer based on the genetic landscape.

Although it is well-established that cancer is driven by genetic mutations resulting in the acquisition of onco-genes and the loss of tumor suppressors, until recently many of the genomic details remained obscure. As a result of recent high-throughput DNA sequencing, basic insights into the spectrum of protein coding mutations in many cancers are now known. These findings provide an unprecedented framework of understanding and present new avenues for diagnosis, treatment, and prevention of cancer. In this article we discuss several high impact areas of global sequencing projects including developing drugs that specifically target cancer cells, creating personalized tools for better treatment and monitoring, and developing pre-symptomatic diagnostic tests. Capitalizing on these and other advances represent a new turning point in the war on cancer.

Searching for biomarkers: humoral response profiling with luciferase immunoprecipitation systems.

B-cell-mediated humoral responses are triggered in many human diseases, including autoimmune diseases, cancer, and neurologic and infectious diseases. However, the full exploitation of the information contained within a patient's antibody repertoire for diagnosis, monitoring and even disease prediction has been limited due to the poor diagnostic performance of many immunoassay formats. We have developed luciferase immunoprecipitation systems (LIPS) that harnesses light-emitting proteins to generate high-definition antibody profiles that are optimal for both diagnostics and biomarker discovery. Here, we describe the results and implications from a range of LIPS-antibody profiling studies performed in our laboratory. These include highly sensitive diagnostics for domestic and global pathogens, insights into infection-related diseases, discovery of new biomarkers for human diseases, subcategorization of symptoms and identification of pathogenic autoantibodies against self-proteins. These investigations highlight the types of humoral response profiles associated with different diseases, provide new information related to disease pathogenesis and offer a framework for incorporating LIPS antibody profiling into global health initiatives and disease monitoring.

LIPS arrays for simultaneous detection of antibodies against partial and whole proteomes of HCV, HIV and EBV.

For many infectious agents, the detection of antibodies is critical for diagnosing, monitoring and understanding vaccine responses. To facilitate the highly quantitative and simultaneous analysis of antibodies against multiple proteins from infectious agents, we have developed Luciferase Immunoprecipitation Systems (LIPS) arrays. By configuring microtiter plates with multiple antigens and testing control and infected serum samples at one time in solution, LIPS arrays provided highly reproducible antibody titers to panels of antigens with a wide dynamic range of detection. While all serum samples showed similar positive and negative immunoreactivity with internal control antigens derived from Influenza and Renilla luciferase-alone protein, respectively, antibody titers to many HCV and HIV antigens were generally 10 to over 400-fold higher in the infected versus uninfected samples. Additional screening of 18 proteins from the EBV proteome with serum samples from healthy EBV-infected individuals showed statistically significant antibody titers to 50% of the proteins tested. Antibody titers for the different EBV antigens in the healthy EBV-infected individuals were markedly heterogeneous highlighting the complexity of host humoral responses. These results suggest that LIPS arrays offer a highly discriminating platform for simultaneously profiling a wide spectrum of antibodies associated with many infectious agents.

Recombinant expression of the AChR-alpha1 subunit for the detection of conformation-dependent epitopes in Myasthenia Gravis.

Detection of autoantibodies associated with neurological disease typically involves immunoprecipitation of radioactively labeled native proteins. We explored whether single receptor subunits, fused to Renilla luciferase (Ruc), could detect patient autoantibodies in Luciferase Immunoprecipitation Systems. Myasthenia Gravis patient sera were tested for conformational autoantibodies to only the α1-subunit of the nicotinic acetylcholine receptor (AChR). Using a panel of 10 AChR-α1 fragments, AChR-α1-Δ5-Ruc demonstrated the highest immunoreactivity with a conformational-specific antibody and the highest sensitivity in a pilot cohort. Testing a larger cohort with AChR-α1-Δ5-Ruc demonstrated 21% sensitivity and 97% specificity. A point mutation within Ruc increased the diagnostic performance of AChR-α1-Δ5 (32% sensitivity, 97% specificity). The (125)I-α-bungarotoxin multi-subunit AChR assay demonstrated 63% sensitivity and 97% specificity. These findings highlight the difficulty in detecting Myasthenia Gravis conformational epitopes across assay formats and lay the foundation for detecting autoantibodies to defined recombinant chains of the AChR and potentially other neurotransmitter receptors.

Rapid induction of autoantibodies during ARDS and septic shock.

BACKGROUND: Little is known about the induction of humoral responses directed against human autoantigens during acute inflammation. We utilized a highly sensitive antibody profiling technology to study autoantibodies in patients with acute respiratory distress syndrome (ARDS) and severe sepsis, conditions characterized by intensive immune activation leading to multiple organ dysfunction. METHODS: Using Luciferase Immunoprecipitation Systems (LIPS), a cohort of control, ARDS and sepsis patients were tested for antibodies to a panel of autoantigens. Autoantibody titers greater than the mean plus 3 SD of the 24 control samples were used to identify seropositive samples. Available longitudinal samples from different seropositive ARDS and sepsis patient samples, starting from within the first two days after admission to the intensive care, were then analyzed for changes in autoantibody over time. RESULTS: From screening patient plasma, 57% of ARDS and 46% of septic patients without ARDS demonstrated at least one statistically significant elevated autoantibody compared to the controls. Frequent high titer antibodies were detected against a spectrum of autoantigens including potassium channel regulator, gastric ATPase, glutamic decarboxylase-65 and several cytokines. Analysis of serial samples revealed that several seropositive patients had low autoantibodies at early time points that often rose precipitously and peaked between days 7-14. Further, the use of therapeutic doses of corticosteroids did not diminish the rise in autoantibody titers. In some cases, the patient autoantibody titers remained elevated through the last serum sample collected. CONCLUSION: The rapid induction of autoantibodies in ARDS and severe sepsis suggests that ongoing systemic inflammation and associated tissue destruction mediate the break in tolerance against these self proteins.

Proteome-wide anti-hepatitis C virus (HCV) and anti-HIV antibody profiling for predicting and monitoring the response to HCV therapy in HIV-coinfected patients.

We quantified antibody responses to the hepatitis C virus (HCV) proteome that are associated with sustained virologic response (SVR) in human immunodeficiency virus (HIV)/HCV-coinfected patients treated with pegylated interferon and ribavirin. Analysis of pre- and posttreatment samples revealed significant decreases in the combined anti-core, anti-E1, and anti-NS4 HCV antibody titers in those with SVRs but not in those who experienced relapse or who did not respond. Furthermore, anti-HIV p24 antibody titers inversely correlated with treatment response. These results suggest that profiling anti-HCV antibody is useful for monitoring HCV therapy, especially in discriminating between those who experience relapse and those who have SVRs at 48 weeks.

Anti-cytokine autoantibodies are associated with opportunistic infection in patients with thymic neoplasia.

Patients with thymic malignancy have high rates of autoimmunity leading to a variety of autoimmune diseases, most commonly myasthenia gravis caused by anti-acetylcholine receptor autoantibodies. High rates of autoantibodies to cytokines have also been described, although prevalence, spectrum, and functionality of these anti-cytokine autoantibodies are poorly defined. To better understand the presence and function of anti-cytokine autoantibodies, we created a luciferase immunoprecipitation system panel to search for autoantibodies against 39 different cytokines and examined plasma from controls (n = 30) and patients with thymic neoplasia (n = 17). In this screen, our patients showed statistically elevated, but highly heterogeneous immunoreactivity against 16 of the 39 cytokines. Some patients showed autoantibodies to multiple cytokines. Functional testing proved that autoantibodies directed against interferon-α, interferon-ß, interleukin-1α (IL-1α), IL-12p35, IL-12p40, and IL-17A had biologic blocking activity in vitro. All patients with opportunistic infection showed multiple anti-cytokine autoantibodies (range 3-11), suggesting that anti-cytokine autoantibodies may be important in the pathogenesis of opportunistic infections in patients with thymic malignancy. This study was registered at http://clinicaltrials.gov as NCT00001355.