Alastair David Griffiths Lawson

Rings in Drugs

We have analyzed the rings, ring systems, and frameworks in drugs listed in the FDA Orange Book to understand the frequency, timelines, molecular property space, and the application of these rings in different therapeutic areas and target classes. This analysis shows that there are only 351 ring systems and 1197 frameworks in drugs that came onto the market before 2013. Furthermore, on average six new ring systems enter drug space each year and approximately 28% of new drugs contain a new ring system. Moreover, it is very unusual for a drug to contain more than one new ring system and the majority of the most frequently used ring systems (83%) were first used in drugs developed prior to 1983. These observations give insight into the chemical novelty of drugs and potentially efficient ways to assess compound libraries and develop compounds from hit identification to lead optimization and beyond.

Activation of resting human primary T cells with chimeric receptors: costimulation from CD28, inducible costimulator, CD134, and CD137 in series with signals from the TCR zeta chain.

Chimeric receptors that include CD28 signaling in series with TCRζ in the same receptor have been demonstrated to activate prestimulated human primary T cells more efficiently than a receptor providing TCRζ signaling alone. We examined whether this type of receptor can also activate resting human primary T cells, and whether molecules other than CD28 could be included in a single chimeric receptor in series with TCRζ to mediate the activation of resting human primary T cells. Human CD33-specific chimeric receptors were generated with CD28, inducible costimulator, CD134, or CD137 signaling regions in series with TCRζ signaling region and transfected by electroporation into resting human primary T cells. Their ability to mediate Ag-specific activation was analyzed in comparison with a receptor providing TCRζ signaling alone. Inclusion of any of the costimulatory signaling regions in series with TCRζ enhanced the level of specific Ag-induced IL-2, IFN-γ, TNF-α, and GM-CSF cytokine production and enabled resting primary T cells to survive and proliferate in response to Ag in the absence of any exogenous factors. Inclusion of CD28, inducible costimulator, or CD134 enhanced TCRζ-mediated, Ag-specific target cell lysis. Chimeric receptors providing B7 and TNFR family costimulatory signals in series with TCRζ in the same receptor can confer self-sufficient clonal expansion and enhanced effector function to resting human T cells. This type of chimeric receptor may now be used to discover the most potent combination of costimulatory signals that will improve current immunotherapeutic strategies.

The Loss of Telomerase Activity in Highly Differentiated CD8+CD28−CD27− T Cells Is Associated with Decreased Akt (Ser473) Phosphorylation

The enzyme telomerase is essential for maintaining the replicative capacity of memory T cells. Although CD28 costimulatory signals can up-regulate telomerase activity, human CD8+ T cells lose CD28 expression after repeated activation. Nevertheless, telomerase is still inducible in CD8+CD28− T cells. To identify alternative costimulatory pathways that may be involved, we introduced chimeric receptors containing the signaling domains of CD28, CD27, CD137, CD134, and ICOS in series with the CD3 zeta (ζ) chain into primary human CD8+ T cells. Although CD3 ζ-chain signals alone were ineffective, triggering of all the other constructs induced proliferation and telomerase activity. However, not all CD8+CD28− T cells could up-regulate this enzyme. The further fractionation of CD8+CD28− T cells into CD8+CD28− CD27+ and CD8+CD28−CD27− subsets showed that the latter had significantly shorter telomeres and extremely poor telomerase activity. The restoration of CD28 signaling in CD8+CD28−CD27− T cells could not reverse the low telomerase activity that was not due to decreased expression of human telomerase reverse transcriptase, the enzyme catalytic subunit. Instead, the defect was associated with decreased phosphorylation of the kinase Akt, that phosphorylates human telomerase reverse transcriptase to induce telomerase activity. Furthermore, the defective Akt phosphorylation in these cells was specific for the Ser473 but not the Thr308 phosphorylation site of this molecule. Telomerase down-regulation in highly differentiated CD8+CD28−CD27− T cells marks their inexorable progress toward a replicative end stage after activation. This limits the ability of memory CD8+ T cells to be maintained by continuous proliferation in vivo.

Cytokine Induced Killer cells for cell therapy of acute myeloid leukemia: improvement of their immune activity by expression of CD33-specific chimeric receptors

Background Cytokine-induced killer cells are ex vivo-expanded cells with potent antitumor activity. The infusion of cytokine-induced killer cells in patients with acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplant is well tolerated, but limited clinical responses have been observed. To improve their effector functions against acute myeloid leukemia, we genetically modified cytokine-induced killer cells with chimeric receptors specific for the CD33 myeloid antigen. Design and Methods SFG-retroviral vectors coding for anti-CD33-ζ and anti-CD33-CD28-OX40-ζ chimeric receptors were used to transduce cytokine-induced killer cells. Transduced cells were characterized in vitro for their ability to lyse leukemic targets (4-hour 51chromium-release and 6-day co-cultures assays on human stromal mesenchymal cells), to proliferate (3H-thymidine-incorporation assay) and to secrete cytokines (flow cytomix assay) after contact with acute myeloid leukemia cells. Their activity against normal CD34+ hematopoietic progenitor cells was evaluated by analyzing the colony-forming unit capacity after co-incubation. Results Cytokine-induced killer cells were efficiently transduced with the anti-CD33 chimeric receptors, maintaining their native phenotype and functions and acquiring potent cytotoxicity (up to 80% lysis after 4-hour incubation) against different acute myeloid leukemia targets, as also confirmed in long-term killing experiments. Moreover, introduction of the anti-CD33 chimeric receptors was accompanied by prominent CD33-specific proliferative activity, with the release of high levels of immunostimulatory cytokines. The presence of CD28-OX40 in chimeric receptor endodomain was associated with a significant amelioration of the anti-leukemic activity of cytokine-induced killer cells. Importantly, even though the cytokine-induced killer cells transduced with anti-CD33 chimeric receptors showed toxicity against normal hematopoietic CD34+ progenitor cells, residual clonogenic activity was preserved. Conclusions Our results indicate that anti-CD33 chimeric receptors strongly enhance anti-leukemic cytokine-induced killer cell functions, suggesting that cytokine-induced killer cells transduced with these molecules might represent a promising optimized tool for acute myeloid leukemia immunotherapy.

Antibody-targeted chemotherapy of B-cell lymphoma using calicheamicin conjugated to murine or humanized antibody against CD22

Antibody-targeted chemotherapy with immunoconjugates of calicheamicin is a clinically validated strategy in cancer therapy. This study describes the selection of a murine anti-CD22 mAb, m5/44, as a targeting agent, its conjugation to a derivative of calicheamicin (CalichDM) via either acid-labile or acid-stable linkers, the antitumor activity of CalichDM conjugated to m5/44, and its subsequent humanization by CDR grafting. Murine IgG1 mAb m5/44 was selected based on its subnanomolar affinity for CD22 and ability to be internalized into B cells. CalichDM conjugated to m5/44 caused potent growth inhibition of CD22+ human B-cell lymphomas (BCLs) in vitro. The conjugate of m5/44 with an acid-labile linker was more potent than an acid-stable conjugate, a nonbinding conjugate with a similar acid-labile linker, or unconjugated CalichDMH in inhibiting BCL growth. CalichDM conjugated to m5/44 caused regression of established BCL xenografts in nude mice. In contrast, both unconjugated m5/44 and a nonbinding conjugate were ineffective against these xenografts. Based on the potent antitumor activity of m5/44-CalichDM conjugates, m5/44 was humanized by CDR grafting to create g5/44, an IgG4 anti-CD22 antibody. Both m5/44 and g5/44 bound CD22 with subnanomolar affinity. Competitive blocking with previously characterized murine anti-CD22 mAbs suggested that g5/44 recognizes epitope A located within the first N-terminal Ig-like domain of human CD22. Antitumor efficacy of CalichDM conjugated to g5/44 against BCL xenografts was more potent than its murine counterpart. Based on these results, a calicheamicin conjugate of g5/44, CMC-544, was selected for further development as a targeted chemotherapeutic agent for the treatment of B-cell malignancies.

Blockade of colony stimulating factor‐1 (CSF‐1) leads to inhibition of DSS‐induced colitis

Background Intestinal inflammation associated with inflammatory bowel disease (IBD) is typically characterized by an inflammatory cell infiltrate and pro‐inflammatory cytokine production. Of particular interest, the frequency of colony stimulating factor‐1 (CSF‐1)‐expressing cells is increased in active lesions. In this study, we have investigated the role of CSF‐1 in mucosal inflammation, using a murine model of colitis induced by dextran sulfate sodium (DSS). Methods A neutralizing anti‐CSF‐1 antibody was administered to Balb/c mice that received DSS in their drinking water. Signs of colitis, such as clinical disease score, cellular infiltrate, and cytokine production, were assessed. Results Administration of a neutralizing anti‐CSF‐1 antibody significantly inhibited DSS‐induced colitis. Clinical symptoms, such as weight loss and the appearance of diarrhea or fecal blood, were reduced by CSF‐1 blockade; histologic scores were also improved. The cellular infiltrate of macrophages and T cells was inhibited and a trend toward reduced production of pro‐inflammatory cytokines was noted. Conclusions This is the first study to demonstrate that CSF‐1 plays an important role in mediating intestinal mucosal inflammation and therefore may prove to be an attractive therapeutic target for intestinal diseases such as inflammatory bowel disease. (Inflamm Bowel Dis 2006)

Co-expression of human protein disulphide isomerase (PDI) can increase the yield of an antibody Fab′ fragment expressed in Escherichia coli

Secretion to the periplasm of Escherichia coli enables production of many eukaryotic extracellular proteins in a soluble form. The complex disulphide bond arrangement of such proteins is probably a major factor in determining the low yield of correctly folded product observed in many cases. Here we show that co‐expression of human protein disulphide isomerase increased the yield of a monoclonal antibody Gab′ fragment in the periplasm of E. coli.

Drug Binding Assays do not Reveal Specific Binding of Lacosamide to Collapsin Response Mediator Protein 2 (CRMP-2)

Aims: Lacosamide (LCM; SPM 927, Vimpat®) is an antiepileptic drug (AED) used as adjunctive treatment for adults with partial‐onset seizures. LCM has a different mode of action from traditional sodium channel blocking AEDs in that it selectively enhances slow inactivation of sodium channels without affecting fast inactivation. Initial investigations suggested that LCM might have an additional mode of action by binding to the collapsin response mediator protein 2 (CRMP‐2), which is further investigated here. Methods: LCM binding to native and cloned human CRMP‐2 was determined using radioligand binding experiments and surface plasmon resonance measurements. Results: No specific binding of [3H]LCM (free concentration 100–1450 nM) to isolated or membrane bound human CRMP‐2 expressed in mammalian cell systems and bacteria was observed. Surface plasmon resonance analysis also showed that LCM, over a concentration range of 0.39–100 μM, does not specifically bind to human CRMP‐2. Conclusion: The diverse drug binding methods employed here are well suited to detect specific binding of LCM to CRMP‐2 in the micromolar range, yet the results obtained were all negative. Results of this study suggest that LCM does not specifically bind to CRMP‐2.

High-Throughput Screening for High Affinity Antibodies

UCB Selected Lymphocyte Antibody Method (SLAM) is a rapid and efficient process for the generation of high-quality monoclonal antibodies, in which variable region gene sequences are recovered directly from specific, single B cells. Monoclonal antibody generation has been limited in the past by the relatively low efficiency of the hybridoma process. UCB SLAM process is well suited to high-throughput screening and has been extensively automated at UCB. If necessary, in excess of 1 times 10 9 B cells can be screened in a campaign, to discover a rare therapeutic antibody candidate, which meets the stringent selection criteria. Primary screening for antigen binders, on purified or cell expressed antigen, is performed using a homogeneous fluorescence assay format. Supernatants from positive wells are consolidated to allow further secondary screening and selection of antibodies with desired characteristics. Individual, specific B cells are identified using a fluorescence based method and isolated using a micromanipulator. The antibody variable region genes are cloned from DNA extracted from the single B cell. The genes are sequenced then prepared for transient expression to confirm activity. Antibodies with affinities (K D) in the sub 10 pM range against a range of therapeutic targets are routinely recovered using this process.

The discovery, engineering and characterisation of a highly potent anti-human IL-13 fab fragment designed for administration by inhalation.

We describe the discovery, engineering and characterisation of a highly potent anti-human interleukin (IL)-13 Fab fragment designed for administration by inhalation. The lead candidate molecule was generated via a novel antibody discovery process, and the selected IgG variable region genes were successfully humanised and reformatted as a human IgG γ1 Fab fragment. Evaluation of the biophysical properties of a selection of humanised Fab fragments in a number of assays allowed us to select the molecule with the optimal stability profile. The resulting lead candidate, CA652.g2 Fab, was shown to have comparable activity to the parental IgG molecule in a range of in vitro assays and was highly stable. Following nebulisation using a mesh nebuliser, CA652.g2 Fab retained full binding affinity, functional neutralisation potency and structural integrity. Epitope mapping using solution nuclear magnetic resonance confirmed that the antibody bound to the region of human IL-13 implicated in the interaction with IL-13Rα1 and IL-13Rα2. The work described here resulted in the discovery and design of CA652.g2 human γ1 Fab, a highly stable and potent anti-IL-13 molecule suitable for delivery via inhalation.