PD-L1 immunohistochemistry correlates only moderately with patient survival and response to PD-(L)1 treatment. Heterogeneity of tumor PD-L1 expression might limit the predictive value of small ...biopsies. Here we show that tumor PD-L1 and PD-1 expression can be quantified non-invasively using PET-CT in patients with non-small-cell lung cancer. Whole body PD-(L)1 PET-CT reveals significant tumor tracer uptake heterogeneity both between patients, as well as within patients between different tumor lesions.
AdnectinsTM are a new family of therapeutic proteins based on the 10th fibronectin type III domain, and designed to bind with high affinity and specificity to therapeutically relevant targets. ...Adnectins share with antibody variable domains a beta-sheet sandwich fold with diversified loops, but differ from antibodies in primary sequence and have a simpler, single-domain structure without disulfide bonds. As a consequence, Adnectins bind targets with affinity and specificity as high as those of antibodies, but are easier to manipulate genetically and compatible with bacterial expression systems. Adnectins that bind macromolecular targets with nanomolar and picomolar affinity have been selected using in vitro evolution methods, including mRNA display, phage display and yeast display. CT-322, a PEGylated, anti-angiogenic Adnectin that binds vascular endothelial growth factor (VEGF) receptor 2 and blocks its interaction with VEGF A, C and D, is being evaluated in Phase II clinical trials for efficacy in several oncology indications.
This report describes a new cell-surface display system, the Secretion and Capture Technology (SECANT™) platform, which relies on in vivo biotinylation of the protein of interest followed by its ...capture on the avidinated surface of the parent cell. Cell sorting techniques are then used to isolate clones that display target-binding protein. A distinguishing feature of this method is its ability to display complex proteins, such as full-length immunoglobulin G (IgG) antibodies, on living cells. In this proof-of-concept study, Saccharomyces cerevisiae cells that displayed Herceptin IgG were isolated from a 10 000-fold excess of cells that displayed a lysozyme-binding antibody.
In-vitro display technologies combine two important advantages for identifying and optimizing ligands by evolutionary strategies. First, by obviating the need to transform cells in order to generate ...and select libraries, they allow a much higher library diversity. Second, by including PCR as an integral step in the procedure, they make PCR-based mutagenesis strategies convenient. The resulting iteration between diversification and selection allows true Darwinian protein evolution to occur in vitro.
We describe two such selection methods, ribosome display and mRNA display. In ribosome display, the translated protein remains connected to the ribosome and to its encoding mRNA; the resulting ternary complex is used for selection. In mRNA display, mRNA is first translated and then covalently bonded to the protein it encodes, using puromycin as an adaptor molecule. The covalent mRNA–protein adduct is purified from the ribosome and used for selection. Successful examples of high-affinity, specific target-binding molecules selected by in-vitro display methods include peptides, antibodies, enzymes, and engineered scaffolds, such as fibronectin type III domains and synthetic ankyrins, which can mimic antibody function.
The tenth human fibronectin type three domain (10Fn3) is a small (10 kDa), extremely stable and soluble protein with an immunoglobulin-like fold, but without cysteine residues. Selections from ...10Fn3-based libraries of proteins with randomized loops have yielded high-affinity, target-specific antibody mimics. However, little is known about the biophysical properties of such antibody mimics, which will determine their suitability for in vitro and medical applications. We characterized target binding and biophysical properties of two related 10Fn3-based antibody mimics that bind vascular endothelial growth factor receptor two (VEGF-R2). The first antibody mimic, which has a dissociation constant (Kd) of 13 nM, is highly stable melting temperature (Tm) = 62°C and soluble, whereas the second, which binds VEGF-R2 with 40× higher affinity, is less stable (Tm < 40°C) and relatively insoluble. We used our understanding of these two 10Fn3 derivatives and of wild-type 10Fn3 structure to engineer the next generation of antibody mimics, which have an improved combination of high affinity (Kd = 0.59 nM), stability (Tm = 53°C) and solubility. Our findings illustrate that 10Fn3-based antibody mimics can be engineered for favorable biophysical properties even when 20% of the wild-type 10Fn3 sequence is mutated in order to satisfy target-binding requirements.
The 10th human fibronectin type III domain (
10Fn3) is one of several protein scaffolds used to design and select families of proteins that bind with high affinity and specificity to macromolecular ...targets. To date, the highest affinity
10Fn3 variants have been selected by mRNA display of libraries generated by randomizing all three complementarity-determining region -like loops of the
10Fn3 scaffold. The sub-nanomolar affinities of such antibody mimics have been attributed to the extremely large size of the library accessible by mRNA display (10
12 unique sequences).
Here we describe the selection and affinity maturation of
10Fn3-based antibody mimics with dissociation constants as low as 350 pM selected from significantly smaller libraries (10
7–10
9 different sequences), which were constructed by randomizing only 14
10Fn3 residues. The finding that two adjacent loops in human
10Fn3 provide a large enough variable surface area to select high-affinity antibody mimics is significant because a smaller deviation from wild-type
10Fn3 sequence is associated with a higher stability of selected antibody mimics. Our results also demonstrate the utility of an affinity-maturation strategy that led to a 340-fold improvement in affinity by maximizing sampling of sequence space close to the original selected antibody mimic.
A striking feature of the highest affinity antibody mimics selected against lysozyme is a pair of cysteines on adjacent loops, in positions 28 and 77, which are critical for the affinity of the
10Fn3 variant for its target and are close enough to form a disulfide bond. The selection of this cysteine pair is structurally analogous to the natural evolution of disulfide bonds found in new antigen receptors of cartilaginous fish and in camelid heavy-chain variable domains. We propose that future library designs incorporating such an interloop disulfide will further facilitate the selection of high-affinity, highly stable antibody mimics from libraries accessible to phage and yeast surface display methods.
Hepatitis C virus NS3/4A protease Kwong, A D; Kim, J L; Rao, G ...
Antiviral research,
02/1999, Letnik:
41, Številka:
1
Journal Article
Recenzirano
Despite an urgent medical need, a broadly effective anti-viral therapy for the treatment of infections with hepatitis C viruses (HCVs) has yet to be developed. One of the approaches to anti-HCV drug ...discovery is the design and development of specific small molecule drugs to inhibit the proteolytic processing of the HCV polyprotein. This proteolytic processing is catalyzed by a chymotrypsin-like serine protease which is located in the N-terminal region of non-structural protein 3 (NS3). This protease domain forms a tight, non-covalent complex with NS4A, a 54 amino acid activator of NS3 protease. The C-terminal two-thirds of the NS3 protein contain a helicase and a nucleic acid-stimulated nucleoside triphosphatase (NTPase) activities which are probably involved in viral replication. This review will focus on the structure and function of the serine protease activity of NS3/4A and the development of inhibitors of this activity.
Smartbombs and cloaking devices Wagner, Richard W; Lipovsek, Dasa
Nature biotechnology,
03/1999, Letnik:
17, Številka:
3
Journal Article
Recenzirano
Ten years ago, the prospect of using "magic bullets" to combat disease caused much excitement in the biomedical community. One lead candidate for such therapeutics was an engineered immunotoxin, ...which consisted of a homing device (the antibody) and a poison lethal to the targeted cell (the bullet). Although they worked miracles for nude mice implanted with deadly cancers, immunotoxins suffered setbacks in the clinic, including a low therapeutic index and a neutralizing immune response. Not to be outmaneuvered, in this issue Suzuki et al. describe a new approach to the immunotoxin dilemma by engineering one of the body's own toxins, ribonuclease (RNase), to potentially thwart the antigenic effect. In the work reported in this issue, Suzuki et al. created new cytotoxic agents by modifying two human ribonucleases of the RNase A superfamily into forms insensitive to RI and thus toxic to cells. The idea was to cloak the RNase so that RI could not bind to it, while leaving the active site of the enzyme undisturbed. The crystal structure of an RNase A-RI complex shows the side chain of Ser89 in bovine RNase A to be in close contact with RI, but relatively far from the active site. Consequently, the authors introduced a cysteine residue into position 89 of human RNase A to make conjugation possible at that position. The resulting free thiol was then used to attach organic thiols or other proteins to the mutant RNase. None of the conjugation partners tested interfered with RNase folding or enzymatic activity; on the other hand, all protein conjugation partners were found to abolish the sensitivity of the RNase to RI. Similar results were obtained when human eosinophil-derived neurotoxin (EDN), an RNase related to RNase A, was conjugated through a cysteine in an analogous position.
Hepatitis C virus NS3/4A protease Kwong, Ann D; Kim, Joseph L; Rao, Govinda ...
Antiviral Research,
12/1998, Letnik:
40, Številka:
1
Book Review, Journal Article
Recenzirano
Despite an urgent medical need, a broadly effective anti-viral therapy for the treatment of infections with hepatitis C viruses (HCVs) has yet to be developed. One of the approaches to anti-HCV drug ...discovery is the design and development of specific small molecule drugs to inhibit the proteolytic processing of the HCV polyprotein. This proteolytic processing is catalyzed by a chymotrypsin-like serine protease which is located in the N-terminal region of non-structural protein 3 (NS3). This protease domain forms a tight, non-covalent complex with NS4A, a 54 amino acid activator of NS3 protease. The C-terminal two-thirds of the NS3 protein contain a helicase and a nucleic acid-stimulated nucleoside triphosphatase (NTPase) activities which are probably involved in viral replication. This review will focus on the structure and function of the serine protease activity of NS3/4A and the development of inhibitors of this activity.
PDF
