Biparatopic single-domain antibodies against Axl achieve ultra-high affinity through intramolecular engagement.

Overexpression of Axl, a TAM-family receptor tyrosine kinase, plays key roles in the formation, growth, and spread of tumors as well as resistance to targeted therapies and chemotherapies. We identified novel llama VHs against human Axl using multiple complementary phage display selection strategies and characterized a subset of high-affinity VHs. The VHs targeted multiple sites in Ig-like domains 1 and 2 of the Axl extracellular domain, including an immunodominant epitope overlapping the site of Gas6 interaction and two additional non-Gas6 competitive epitopes recognized by murine monoclonal antibodies.

Characterization of murine CEACAM1 reveals low expression on CD8 T cells and no tumor growth modulating activity by anti-CEACAM1 mAb CC1.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) has been reported to mediate both tumorigenic and anti-tumor effects . Blockade of the CEACAM1 signaling pathway has recently been implicated as a novel mechanism for cancer immunotherapy. CC1, a mouse anti-CEACAM1 monoclonal antibody (mAb), has been widely used as a pharmacological tool in preclinical studies to inform on CEACAM1 pathway biology although limited data are available on its CEACAM1 blocking characteristics or pharmacodynamic-pharmacokinetic profiles.

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.

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.

Low-temperature approach to highly emissive copper indium sulfide colloidal nanocrystals and their bioimaging applications.

We report our newly developed low-temperature synthesis of colloidal photoluminescent (PL) CuInS2 nanocrystals (NCs) and their in vitro and in vivo imaging applications. With diphenylphosphine sulphide (SDPP) as a S precursor made from elemental S and diphenylphosphine, this is a noninjection based approach in 1-dodecanethiol (DDT) with excellent synthetic reproducibility and large-scale capability. For a typical synthesis with copper iodide (CuI) as a Cu source and indium acetate (In(OAc)3) as an In source, the growth temperature was as low as 160 °C and the feed molar ratios were 1Cu-to-1In-to-4S.

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.

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.

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 V(H) 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 V(H) phage display library. Size exclusion chromatography and surface plasmon resonance analyses demonstrated that Gr3 is a monomer, but that Gr6 is a strict dimer.

Single-domain antibody bioconjugated near-IR quantum dots for targeted cellular imaging of pancreatic cancer.

Successful targeted imaging of BxPC3 human pancreatic cancer cells is feasible with near-IR CdTeSe/CdS quantum dots (QDs) functionalized with single-domain antibody (sdAb) 2A3. For specific targeting, sdAbs are superior to conventional antibodies, especially in terms of stability, aggregation, and production cost. The bright CdTeSe/CdS QDs were synthesized to emit in the diagnostic window of 650-900 nm with a narrow emission band.

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.

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.

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).

Parallel selection of multiple anti-infectome Nanobodies without access to purified antigens.

A strategy was developed to isolate Nanobodies, camelid-derived single-domain antibody fragments, against the parasite infectome without a priori knowledge of the antigens nor having access to the purified antigens. From a dromedary, infected with T. evansi, we cloned a pool of Nanobodies and selected after phage display 16 different Nanobodies specific for a single antigen, i.

A glycosylphosphatidylinositol-based treatment alleviates trypanosomiasis-associated immunopathology.

The GPI-anchored trypanosome variant surface glycoprotein (VSG) triggers macrophages to produce TNF, involved in trypanosomiasis-associated inflammation and the clinical manifestation of sleeping sickness. Aiming at inhibiting immunopathology during experimental Trypanosoma brucei infections, a VSG-derived GPI-based treatment approach was developed. To achieve this, mice were exposed to the GPI before an infectious trypanosome challenge.

Tumor necrosis factor (TNF) receptor-1 (TNFp55) signal transduction and macrophage-derived soluble TNF are crucial for nitric oxide-mediated Trypanosoma congolense parasite killing.

Control of Trypanosoma congolense infections requires an early cell-mediated immune response. To unravel the role of tumor necrosis factor (TNF) in this process, 6 different T. congolense strains were used in 6 different gene-deficient mouse models that included TNF(-/-), TNF receptor-1 (TNFp55)(-/-), and TNF receptor-2 (TNFp75)(-/-) mice, 2 cell type-specific TNF(-/-) mice, as well as TNF-knock-in mice that expressed only membrane-bound TNF.

Control of Trypanosoma evansi infection is IgM mediated and does not require a type I inflammatory response.

Very recent reports have documented that Trypanosoma evansi, the etiological agent of the livestock disease "surra," can cause human trypanosomiasis. In contrast to trypanosomes causing human African trypanosomiasis, T. evansi has a wide geographic distribution and host range, yet information about the immunobiological aspects of T.

Interferon-gamma and nitric oxide in combination with antibodies are key protective host immune factors during trypanosoma congolense Tc13 Infections.

The control of chronic Trypanosoma congolense trypanosomiasis was analyzed using several gene-deficient mouse strains. First, interferon (IFN)-gamma receptor (IFN-gamma-R)-deficient mice were used to show that IFN- gamma -mediated immune activation is crucial for parasitemia control. Second, infections in major histocompatibility complex (MHC) class II-deficient mice indicate that this molecule is needed for initiation of IFN- gamma and subsequent tumor necrosis factor (TNF) production.

Experimental therapy of African trypanosomiasis with a nanobody-conjugated human trypanolytic factor.

High systemic drug toxicity and increasing prevalence of drug resistance hampers efficient treatment of human African trypanosomiasis (HAT). Hence, development of new highly specific trypanocidal drugs is necessary. Normal human serum (NHS) contains apolipoprotein L-I (apoL-I), which lyses African trypanosomes except resistant forms such as Trypanosoma brucei rhodesiense.