Toya Nath Baral

Differential tumor-targeting abilities of three single-domain antibody formats

The large molecular size of antibody drugs is considered one major factor preventing them from becoming more efficient therapeutics. Variable regions of heavy chain antibodies (HCAbs), or single-domain antibodies (sdAbs), are ideal building blocks for smaller antibodies due to their molecular size and enhanced stability. In the search for better antibody formats for in vivo imaging and/or therapy of cancer, three types of sdAb-based molecules directed against epidermal growth factor receptor (EGFR) were constructed, characterized and tested. Eleven sdAbs were isolated from a phage display library constructed from the sdAb repertoire of a llama immunized with a variant of EGFR. A pentameric sdAb, or pentabody, V2C-EG2 was constructed by fusing one of the sdAbs, EG2, to a pentamerization protein domain. A chimeric HCAb (cHCAb), EG2-hFc, was constructed by fusing EG2 to the fragment crystallizable (Fc) of human IgG1. Whereas EG2 and V2C-EG2 localized mainly in the kidneys after i.v. injection, EG2-hFc exhibited excellent tumor accumulation, and this was largely attributed to its long serum half life, which is comparable to that of IgGs. The moderate size (approximately 80 kDa) and intact human Fc make HCAbs a unique antibody format which may outperform whole IgGs as imaging and therapeutic reagents.

Transient expression and purification of chimeric heavy chain antibodies

Monoclonal antibodies have been successfully engineered as approved therapeutics. However, their large size is considered a major factor preventing them from having a more efficient tissue penetration. As the first step to establish a possibly more efficient antibody platform, we present here transient expression, purification and characterization of six chimeric heavy chain antibodies (cHCAbs), or fusion of camelid single domain antibodies (sdAbs) to human fragment crystallizable (Fc). All six HCAbs have a MW of approximately 80 kDa, expressed well in a HEK293 expression system and have G0, G1 and G2 types of glycosylation. The transient expression also provided a very fast way to generate high milligram to low gram amount of proteins for in vitro assays and preliminary animal studies.

Immunobiology of African trypanosomes: need of alternative interventions.

Trypanosomiasis is one of the major parasitic diseases for which control is still far from reality. The vaccination approaches by using dominant surface proteins have not been successful, mainly due to antigenic variation of the parasite surface coat. On the other hand, the chemotherapeutic drugs in current use for the treatment of this disease are toxic and problems of resistance are increasing (see Kennedy (2004) and Legros et al. (2002)). Therefore, alternative approaches in both treatment and vaccination against trypanosomiasis are needed at this time. To be able to design and develop such alternatives, the biology of this parasite and the host response against the pathogen need to be studied. These two aspects of this disease with few examples of alternative approaches are discussed here.

Isolation of functional single domain antibody by whole cell immunization: implications for cancer treatment.

Carcinoembryonic antigen related cell adhesion molecule (CEACAM) 6 is over-expressed in different types of cancer cells. In addition, it has also been implicated in some infectious diseases. Targeting this molecule by an antibody might have applications in diverse tumor models. Single domain antibody (sdAb) is becoming very useful format in antibody engineering as potential tools for treating acute and chronic disease conditions such as cancer for both diagnostic as well as therapeutic application. Generally, sdAbs with good affinity are isolated from an immune library. Discovery of a new target antigen would require a new immunization with purified antigen which is not always easy. In this study, we have isolated, by phage display, an sdAb against CEACAM6 with an affinity of 5 nM from a llama immunized with cancer cells. The antibody has good biophysical properties, and it binds to the cells expressing the target antigen. Furthermore, it reduces cancer cells proliferation in vitro and shows an excellent tumor targeting in vivo. This sdAb could be useful in diagnosis as well as therapy of CEACAM6 expressing tumors. Finally, we envisage it would be feasible to isolate good sdAbs against other interesting tumor associated antigens from this library. Therefore, this immunization method could be a general strategy for isolating sdAbs against any surface antigen without immunizing the animal with the antigen of interest each time.

Single‐Domain Antibodies and Their Utility

Engineered monoclonal antibody fragments have gained market attention due to their versatility and tailor‐made potential and are now considered to be an important part of future immunobiotherapeutics. Single‐domain antibodies (sdAbs), also known as nanobodies, are derived from VHHs [variable domains (V) of heavy‐chain‐only antibodies (HCAb)] of camelid heavy‐chain antibodies. These nature‐made sdAbs are well suited for various applications due to their favorable characteristics such as small size, ease of genetic manipulation, high affinity and solubility, overall stability, resistance to harsh conditions (e.g., low pH, high temperature), and low immunogenicity. Most importantly, sdAbs have the feature of penetrating into cavities and recognizing hidden epitopes normally inaccessible to conventional antibodies, mainly due to their protruding CDR3/H3 loops. In this unit, we will present and discuss comprehensive and step‐by‐step protocols routinely practiced in our laboratory for isolating sdAbs from immunized llamas (or other members of the Camelidae family) against target antigens using phage‐display technology. Expression, purification, and characterization of the isolated sdAbs will then be described, followed by presentation of several examples of applications of sdAbs previously characterized in our laboratory and elsewhere. Curr. Protoc. Immunol. 103:2.17.1‐2.17.57. ©2013 by John Wiley & Sons, Inc.

Single domain antibody against carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits proliferation, migration, invasion and angiogenesis of pancreatic cancer cells

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is over-expressed in pancreatic cancer cells, and it is associated with the progression of pancreatic cancer. We tested a single domain antibody (sdAb) targeting CEACAM6, 2A3, which was isolated previously from a llama immune library, and an Fc conjugated version of this sdAb, to determine how they affect the pancreatic cancer cell line BxPC3. We also compared the effects of the antibodies to gemcitabine. Gemcitabine and 2A3 slowed down cancer cell proliferation. However, only 2A3 retarded cancer cell invasion, angiogenesis within the cancer mass and BxPC3 cell MMP-9 activity, three features important for tumour growth and metastasis. The IC50s for 2A3, 2A3-Fc and gemcitabine were determined as 6.5μM, 8μM and 12nM, respectively. While the 2A3 antibody inhibited MMP-9 activity by 33% compared to non-treated control cells, gemcitabine failed to inhibit MMP-9 activity. Moreover, 2A3 and 2A3-Fc inhibited invasion of BxPC3 by 73% compared to non-treated cells. When conditioned media that were produced using 2A3- or 2A3-Fc-treated BxPC3 cells were used in a capillary formation assay, the capillary length was reduced by 21% and 49%, respectively. Therefore 2A3 is an ideal candidate for treating tumours that over-express CEACAM6.

Crystal Structure of a Human Single Domain Antibody Dimer Formed through V(H)-V(H) Non-Covalent Interactions.

Single-domain antibodies (sdAbs) derived from human VH are considered to be less soluble and prone to aggregate which makes it difficult to determine the crystal structures. In this study, we isolated and characterized two anti-human epidermal growth factor receptor-2 (HER2) sdAbs, Gr3 and Gr6, from a synthetic human VH phage display library. Size exclusion chromatography and surface plasmon resonance analyses demonstrated that Gr3 is a monomer, but that Gr6 is a strict dimer. To understand this different molecular behavior, we solved the crystal structure of Gr6 to 1.6 Å resolution. The crystal structure revealed that the homodimer assembly of Gr6 closely mimics the VH-VL heterodimer of immunoglobulin variable domains and the dimerization interface is dominated by hydrophobic interactions.

Expression of Single-Domain Antibodies in Bacterial Systems

In this chapter we describe in detail the current protocols that are used to express single-domain antibodies in bacteria. Bacteria are among the most common expression systems for expressing recombinant proteins. We present different approaches for carrying out periplasmic and cytoplasmic expression, as well as small-scale and large-scale expression. In addition, we discuss the advantages and possible drawbacks of each protocol. We present data related to expression vectors, expression conditions, methods of protein extraction and purification, and yield and purity analysis of sdAbs. We also highlight important points that need to be considered before sdAbs that have been expressed in bacteria are used either in vitro or in vivo.

A Novel Affinity Tag, ABTAG, and Its Application to the Affinity Screening of Single-Domain Antibodies Selected by Phage Display

ABTAG is a camelid single-domain antibody (sdAb) that binds to bovine serum albumin (BSA) with low picomolar affinity. In surface plasmon resonance (SPR) analyses using BSA surfaces, bound ABTAG can be completely dissociated from the BSA surfaces at low pH, over multiple cycles, without any reduction in the capacity of the BSA surfaces to bind ABTAG. A moderate throughput, SPR-based, antibody screening assay exploiting the unique features of ABTAG is described. Anti-carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) sdAbs were isolated from a phage-displayed sdAb library derived from the heavy chain antibody repertoire of a llama immunized with CEACAM6. Following one or two rounds of panning, enriched clones were expressed as ABTAG fusions in microtiter plate cultures. The sdAb-ABTAG fusions from culture supernatants were captured on BSA surfaces and CEACAM6 antigen was then bound to the captured molecules. The SPR screening method gives a read-out of relative expression levels of the fusion proteins and kinetic and affinity constants for CEACAM6 binding by the captured molecules. The library was also panned and screened by conventional methods and positive clones were subcloned and expressed for SPR analysis. Compared to conventional panning and screening, the SPR-based ABTAG method yielded a considerably higher diversity of binders, some with affinities that were three orders of magnitude higher affinity than those identified by conventional panning.