The role of small molecules in cell and gene therapy.

Cell and gene therapies have achieved impressive results in the treatment of rare genetic diseases using gene corrected stem cells and haematological cancers using chimeric antigen receptor T cells. However, these two fields face significant challenges such as demonstrating long-term efficacy and safety, and achieving cost-effective, scalable manufacturing processes. The use of small molecules is a key approach to overcome these barriers and can benefit cell and gene therapies at multiple stages of their lifecycle.

The Landscape of Early Clinical Gene Therapies outside of Oncology.

The field of cell and gene therapy (GT) is expanding rapidly and there is undoubtedly a wave of enthusiasm and anticipation for what these treatments could achieve next. Here we assessed the worldwide landscape of GT assets currently in early clinical development (clinical trial phase 1/2 or about to enter clinical trial). We included all gene therapies, i.

Current Status of Gene Engineering Cell Therapeutics.

manipulations of autologous patient's cells or gene-engineered cell therapeutics have allowed the development of cell and gene therapy approaches to treat otherwise incurable diseases. These modalities of personalized medicine have already shown great promises including product commercialization for some rare diseases. The transfer of a chimeric antigen receptor or T cell receptor genes into autologous T cells has led to very promising outcomes for some cancers, and particularly for hematological malignancies.

Delivering advanced therapies: the big pharma approach.

After two decades of focused development and some recent clinical successes, cell and gene therapy (CGT) is emerging as a promising approach to personalized medicines. Genetically engineered cells as a medical modality are poised to stand alongside or in combination with small molecule and biopharmaceutical approaches to bring new therapies to patients globally. Big pharma can have a vital role in industrializing CGT by focusing on diseases with high unmet medical need and compelling genetic evidence.

Creation of the large and highly functional synthetic repertoire of human VH and Vκ domain antibodies.

This protocol describes a method for creation of a highly diverse and functional synthetic phage-displayed repertoire of fully human domain antibodies (dAbs). The repertoire is based on two human frameworks (one VH and one Vκ) that express well in bacteria and are frequently used in human antibodies. To achieve this, we first build dAb libraries, containing full synthetic diversity at key positions in the complementarity-determining regions (CDRs).

Anti-serum albumin domain antibodies in the development of highly potent, efficacious and long-acting interferon.

Serum albumin-binding domain antibodies (AlbudAbs) have previously been shown to greatly extend the serum half-life of the interleukin-1 receptor antagonist IL-1ra. We have subsequently extended this approach to look at the in vitro activity, in vivo efficacy and pharmacokinetics of an agonist molecule, interferon (IFN)-alpha2b, fused to an AlbudAb. Here we describe this molecule and show that in this format AlbudAb half-life extension technology displays significant advantages in comparison with other methods of half-life extension, in particular genetic fusion to serum albumin.

Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs.

We have used phage display to isolate a range of human domain antibodies (dAbs) that bind to mouse, rat and/or human serum albumin (SA) and can be expressed at very high levels in bacterial, yeast or mammalian cell culture. In contrast to non-SA-binding dAbs, which have terminal half-lives of less than 45 min, the half-lives of these 12 kDa 'AlbudAbs' can match the half-life of SA itself. To demonstrate the use of AlbudAbs for extending the half-lives of therapeutic drugs, we created a fusion of the interleukin-1 receptor antagonist (IL-1ra) with an AlbudAb.

A technology platform for the fast production of monoclonal recombinant antibodies against plant proteins and peptides.

The application of recombinant antibodies in plant biology research is limited because plant researchers have minimal access to high-quality phage display libraries. Therefore, we constructed a library of 1.3 x 10(10) clones displaying human single-chain variable fragments (scFvs) that is available to the academic community.

Selection of optical biosensors from chemisynthetic antibody libraries.

We describe a method for creating antibodies with a fluorescent reporter integrated into the antigen-binding site. A reporter molecule was chemically linked to a hypervariable loop of an antibody repertoire displayed on phage, and this repertoire was selected for antigen binding. In one selected antibody, the fluorescence of the probe responded quantitatively to antigen binding.

Aggregation-resistant domain antibodies selected on phage by heat denaturation.

We describe a method for selecting aggregation-resistant proteins by heat denaturation. This is illustrated with antibody heavy chain variable domains (dAbs), which are prone to aggregate. The dAbs were displayed multivalently at the infective tip of filamentous bacteriophage, and heated transiently to induce unfolding and to promote aggregation of the dAbs.

Crystal structure of HEL4, a soluble, refoldable human V(H) single domain with a germ-line scaffold.

The antigen binding site of antibodies usually comprises associated heavy (V(H)) and light (V(L)) chain variable domains, but in camels and llamas, the binding site frequently comprises the heavy chain variable domain only (referred to as V(HH)). In contrast to reported human V(H) domains, V(HH) domains are well expressed from bacteria and yeast, are readily purified in soluble form and refold reversibly after heat-denaturation. These desirable properties have been attributed to highly conserved substitutions of the hydrophobic residues of V(H) domains, which normally interact with complementary V(L) domains.

Domain antibodies: proteins for therapy.

Occurring naturally in "heavy chain" immunoglobulins from camels, and now produced in fully human form, domain antibodies (dAbs) are the smallest known antigen-binding fragments of antibodies, ranging from 11 kDa to 15 kDa. dAbs are the robust variable regions of the heavy and light chains of immunoglobulins (VH and VL respectively). They are highly expressed in microbial cell culture, show favourable biophysical properties including solubility and temperature stability, and are well suited to selection and affinity maturation by in vitro selection systems such as phage display.

Polyethylene glycol-derivatized cysteine-substitution variants of recombinant staphylokinase for single-bolus treatment of acute myocardial infarction.

Background: Thrombolytic therapy of acute myocardial infarction (AMI) is evolving toward bolus administration. Derivatization of proteins with polyethylene glycol (PEG) may reduce their clearance.

Methods And Results: A staphylokinase (SakSTAR) variant with 12 amino acid substitutions to reduce its antigenicity, SakSTAR (K35A, E65Q, K74R, E80A, D82A, T90A, E99D, T101S, E108A, K109A, K130T, K135R), and with Ser in position 3 mutated into Cys (code SY161), was derivatized with maleimide-PEG with M:(r) of 5,000 (P5), 10,000 (P10), or 20,000 (P20).

High-density mutagenesis by combined DNA shuffling and phage display to assign essential amino acid residues in protein-protein interactions: application to study structure-function of plasminogen activation inhibitor 1 (PAI-I).

The identification of specific amino acid residues involved in protein-protein interaction is fundamental to understanding structure-function relationships. Supported by mathematical calculations, we designed a high-density mutagenesis procedure for the generation of a mutant library of which a limited number of random clones would suffice to exactly localize amino acid residues essential for a particular protein-protein interaction. This goal was achieved experimentally by consecutive cycles of DNA shuffling, under error prone conditions, each followed by exposure of the target protein on the surface of phages to screen and select for correctly folded, functional mutants.

Pharmacokinetic and thrombolytic properties of cysteine-linked polyethylene glycol derivatives of staphylokinase.

Recombinant staphylokinase (SakSTAR) variants obtained by site-directed substitution with cysteine, in the core (lysine 96 [Lys96], Lys102, Lys109, and/or Lys135) or the NH(2)-terminal region that is released during activation of SakSTAR (serine 2 [Ser2] and/or Ser3), were derivatized with thiol-specific (ortho-pyridyl-disulfide or maleimide) polyethylene glycol (PEG) molecules with molecular weights of 5,000 (P5), 10,000 (P10), or 20,000 (P20). The specific activities and thrombolytic potencies in human plasma were unaltered for most variants derivatized with PEG (PEGylates), but maleimide PEG derivatives had a better temperature stability profile. In hamsters, SakSTAR was cleared at 2.

Guiding a docking mode by phage display: selection of correlated mutations at the staphylokinase-plasmin interface.

During co-evolution of interacting proteins, functionally disruptive mutations on one side of the interface may be compensated by local amino acid changes on the other to restore binding affinity. This information can be useful for geometry-based docking approaches by reducing the translational and rotational space available to the proteins. Here, we demonstrate that correlated mutations at a protein-protein interface can be rapidly identified by selecting a phage-displayed library of a randomly mutated component of the complex for complementation of mutations that decreased binding in the interacting partner.

Structural and functional basis of plasminogen activation by staphylokinase.

Staphylokinase (Sak), a 15.5-kDa bacterial protein, forms a complex with human plasmin, which in turn activates other plasminogen molecules to plasmin. Three recombinant DNA-based approaches, (i) site directed substitution with alanine, (ii) search for proximity relationships at the complex interface, and (iii) active-site accessibility to protease inhibitors have been used to deduce a coherent docking model of the crystal structure of Sak on the homology-based model of microplasmin (microPli), the serine protease domain of plasmin.

High resolution mapping of the B cell epitopes of staphylokinase in humans using negative selection of a phage-displayed antigen library.

Staphylokinase (Sak), a 16-kDa protein secreted by Staphylococcus aureus, induces fibrin-specific thrombolysis in patients with thrombotic disorders. However, Sak also elicits high titers of neutralizing Abs that persist for several months and preclude its repeated use in humans. To identify the antigenic determinants of Sak recognized by humans, a phage-displayed library of Sak variants was selected for mutants that escape binding to an affinity matrix derivatized with patient-specific polyclonal anti-Sak Abs.

Arginine 719 in human plasminogen mediates formation of the staphylokinase:plasmin activator complex.

Staphylokinase (Sak), a 16-kDa bacterial protein, forms a 1:1 stoichiometric complex with the serine proteinase domain of human plasmin, which in turn converts other plasminogen molecules into plasmin. To identify amino acid residues critical for generating the Sak:plasmin activator complex, alanine-scanning mutagenesis was performed on phage-displayed micro-plasminogen (microPlg). Substitution of Arg719 with Ala [microPlg(R719A)] disrupted complex formation, although the sensitivity of phage-displayed microPlg(R719A) to activation by urokinase and the amidolytic activity of the micro-plasmin derivative [microPli(R719A)] remained unaffected.

Epitope mapping by negative selection of randomized antigen libraries displayed on filamentous phage.

Since most antibodies directed against protein antigens recognize epitopes composed of several discontinuous segments of the polypeptide chain, attempts to delineate the amino acids constituting these epitopes with the use of linear peptides have generally been unsuccessful. Here, a method is described based on error-prone PCR, phage display and negative selection, whereby amino acid residues constituting the functional epitope are identified in the context of the native protein. First a library of randomized antigen variants containing most single, double and triple amino acid mutants generated by single nucleotide substitutions is produced by error-prone PCR amplification of the DNA sequence encoding the protein antigen.

Screening panels of monoclonal antibodies using phage-displayed antigen.

A procedure is described to screen panels of hybridomas or purified monoclonal antibodies using antigen displayed on the surface of filamentous bacteriophage. In this system, samples containing murine monoclonal antibodies are incubated with phage-displayed antigen in microtiter plates coated with rabbit anti-mouse IgG, and bound antibody-phage complex is detected with horseradish peroxidase-sheep anti-phage M13 conjugate. The assay has been validated with a panel of 16 monoclonal antibodies directed against human plasminogen, using phage-displayed miniplasmin-(ogen) (amino acids Ala444 through Asn791 comprising kringle 5 and the proteinase domain of plasminogen) or microplasminogen (amino acids Ala543 through Asn791 comprising the proteinase domain).

Enzymatic properties of phage-displayed fragments of human plasminogen.

Two low-molecular-mass forms of human plasminogen, plasminogen-(543-791)-peptide (micro-plasminogen), comprising the serine protease domain, and plasminogen-(444-791)-peptide (mini-plasminogen), which in addition contains kringle 5, were displayed on filamentous phage by fusion to the N-terminus of the minor coat protein pIII, to levels of 0.5 molecules micro-plasminogen-pIII/phage particle and 0.1 molecules mini-plasminogen-pIII/phage particle.

Recombinant staphylokinase variants with altered immunoreactivity. III: Species variability of antibody binding patterns.

Background: The "charged cluster-to-alanine" substitution variants SakSTAR(K35A,E38A,K74A,E75A,R77A) and SakSTAR(K74A,E75A,R77A,E80A,D82A), previously identified as SakSTAR.M38 and SakSTAR.M89, respectively, induce less antibody formation in patients than wild-type recombinant staphylokinase (SakSTAR), but their specific activities are reduced by 50%.

LambdaZLG6: a phage lambda vector for high-efficiency cloning and surface expression of cDNA libraries on filamentous phage.

We describe a vector, lambdaZLG6, combining the high efficiency of cDNA library cloning in bacteriophage lambda with filamentous phage display of cDNA-encoded products. The cDNAs are expressed as fusions to the 3' end of M13 gene VI. The lambdaZLG library is converted to a pZLG6-cDNA phagemid library by in vivo mass excision.

Anticoagulant repertoire of the hookworm Ancylostoma caninum.

Hookworms are hematophagous nematodes that infect a wide range of mammalian hosts, including humans. There has been speculation for nearly a century as to the identity of the anticoagulant substances) used by these organisms to subvert host hemostasis. Using molecular cloning, we describe a family of potent small protein (75-84 amino acids) anticoagulants from the hookworm Ancylostoma caninum termed AcAP (A.