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Molecularly engineered antibodies with fit-for-purpose properties will differentiate next generation antibody therapeutics from traditional IgG1 scaffolds. One requirement for engineering the most appropriate properties for a particular therapeutic area is an understanding of the intricacies of the target microenvironment in which the antibody is expected to function. Our group and others have demonstrated that proteases secreted by invasive tumors and pathological microorganisms are capable of cleaving human IgG1, the most commonly adopted isotype among monoclonal antibody therapeutics.
Specific cleavage in the lower hinge of IgG1 results in a loss of Fc-mediated cell-killing functions without a concomitant loss of antigen binding capability or circulating antibody half-life. Proteolytic cleavage in the hinge region by tumor-associated or microbial proteases is postulated as a means of evading host immune responses, and antibodies engineered with potent cell-killing functions that are also resistant to hinge proteolysis are of interest. Mutation of the lower hinge region of an IgG1 resulted in protease resistance but also resulted in a profound loss of Fc-mediated cell-killing functions. In the present study, we demonstrate that specific mutations of the C H2 domain in conjunction with lower hinge mutations can restore and sometimes enhance cell-killing functions while still retaining protease resistance. By identifying mutations that can restore either complement- or Fcγ receptor-mediated functions on a protease-resistant scaffold, we were able to generate a novel protease-resistant platform with selective cell-killing functionality. Introduction The mechanism of action for several therapeutic monoclonal antibodies (mAbs) is thought to be due in part to Fc-mediated effector functions (, ).
The most common human immunoglobulin isotype used for therapeutic intervention is IgG1 , which contains two Fab arms linked to a single Fc domain by a flexible hinge region. The Fc domain of IgG1 can bind to FcγRs expressed on immune effector cells and mediate target cell destruction by cellular means, including antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP).
Antigen-engaged mAbs can also recruit serum components of the immune system and mediate target cell destruction by initiating the complement cascade (–). The interactions of both immune cell FcγRs and the C1q component of complement require key amino acid recognition motifs in the lower hinge and proximal C H2 region of human IgG (, –). Our group and others have shown that the human IgG1 subclass is susceptible to limited proteolysis in the hinge region by a number of physiologically relevant proteases associated with microbial infections ( e.g. GluV8 of Staphylococcus aureus and IdeS of Streptococcus pyogenes) and invasive cancers ( e.g.
The matrix metalloproteinases (MMPs)) (–). Cleavage within the lower hinge of IgG1 occurs in a two-step process where first one heavy chain is cleaved, resulting in a singly cleaved intermediate (, ). Cleavage of the second heavy chain separates the Fc from the Fab arms, resulting in an Fc fragment and an F(ab′) 2 fragment. Previous studies have indicated that the singly cleaved intermediate is the dominant cleavage product generated on the cell surface and that single cleavage results in abrogated binding to FcγRs (, ) and a loss of complement-dependent cytotoxicity (CDC). Accordingly, the singly cleaved intermediate displays a profound loss of function in terms of cell killing both in vitro and in vivo (, ). However, the singly cleaved intermediate retains antigen binding capabilities as well as the long circulating half-life of the intact IgG1 counterpart.
For these reasons, our group and others have hypothesized that antibody cleavage by tumor-associated and microbial proteases can potentially function as an immune evasion mechanism (for reviews, see Refs. Previously, we have shown that the human IgG2 subclass is resistant to cleavage by a number of physiologically relevant proteases, including MMP-3, MMP-7, MMP-12, and MMP-13 as well as GluV8. Sequence alignments comparing the lower hinge of IgG1 and IgG2 suggested that the resistance to cleavage may be due to amino acid differences between IgG1 and IgG2.
However, the IgG2 subclass has very weak binding to FcγRIIIa and thus low to undetectable ADCC capacity. The IgG2 subclass also has greatly reduced CDC activity compared with IgG1. Several groups have exchanged the lower hinge/proximal C H2 of IgG1 and IgG2 (, ) and characterized the loss of function associated with this domain exchange. Armour et al. demonstrated that introduction of the lower hinge/proximal C H2 of IgG2 into IgG1 resulted in a profound loss of function and proposed that these substitutions could serve as a silent Fc platform. Shields et al.
introduced the lower hinge/proximal C H2 of IgG2 into IgG1 and showed a greater than 20-fold loss of binding to FcγRs. Therefore, additional efforts would be required to generate protease-resistant variants containing an IgG2 lower hinge/proximal C H2 region that retain Fc-dependent cell-killing functions. Antibody engineering has long been recognized as a means to improve mAb-based therapies (, ), particularly with regard to antibodies that target tumor antigens (, ).
Efforts to engineer anti-tumor mAbs in the Fc domain are often directed toward increasing the cell killing capacity of the mAb by augmenting binding to FcγRs (, –) or the C1q component of complement (, ). One common method to augment cell-killing functions is to mutate amino acids in the C H2 region and screen for variants with increased binding to C1q or FcγRs (, ). A number of published reports have also documented efforts to engineer the hinge region to improve effector function or increase antibody stability (, ).
In this study, we demonstrate that mutation of the lower hinge of IgG1 confers protease resistance but also results in the loss of Fc-mediated cell-killing functions. We show that specific mutations incorporated into the C H2 region of engineered mAbs with a protease-resistant lower hinge cannot only restore functional activities to IgG1 but in some cases substantially enhance Fc effector functions. Furthermore, we show that cleaved IgGs are detected within the tumor microenvironment of human head and neck squamous cell carcinoma, highlighting the need for a protease-resistant platform for diseases characterized by the presence of IgG-cleaving proteases. Antibodies The V-region cDNA sequences of the anti-CD20 variants were the same as those used in rituximab (VL GenBank TM accession number and VH GenBank accession number ), and the heavy and light chains were engineered onto IgG subclasses and variants by molecular cloning. Transient transfection and expression in 293T and/or CHO cells were performed with standard procedures at Janssen Research and Development, LLC.
MAbs were purified using protein A columns and underwent in-house quality controls for 95% purity prior to further experimental analyses. The complementarity-determining region sequences of the humanized, complementarity-determining region-grafted anti-CD142 mAb were derived from the murine anti-human CD142 mAb TF8-5G9, which originated at the Scripps Research Institute and has been described previously (, ). Protease Digestions Protease digestions were performed at pH 7.5 at 37 °C for 24 h in PBS for Ides and GluV8 or in Tris-buffered saline with 5 m m CaCl 2 for the MMP reactions. Anti-CD142 antibodies were used at a concentration of 0.5 mg/ml, and protease concentrations of 10% molar ratio for MMP-3 (Janssen Research and Development, LLC) and MMP-12 (Enzo Life Sciences), 20% molar ratio for GluV8 (Biocentrum), and 1% (w/w) for IdeS (Genovis) were used. Kinetic digests were performed with a 10% molar ratio for MMP-3 and 0.1% (w/w) IdeS and were quenched at the indicated time points using a final concentration of 10 m m EDTA for the MMP-3 digest and 10 m m iodoacetamide for the IdeS digest.
The percentage of intact IgG remaining was calculated as done previously. Immunohistochemistry All immunohistochemistry was performed by QualTek Molecular Laboratories (Newtown, PA). Four-micrometer sections were dewaxed through four changes of xylene (5 min each) followed by a graded alcohol series to distilled water. Steam heat-induced epitope recovery was used for 20 min in the capillary gap in the upper chamber of a Black and Decker steamer. Sections were incubated with primary rabbit polyclonal anti-hinge detection antibodies (125 ng/ml) directed against three cleavage sites in the hinge that were described previously. An anti-rabbit biotinylated secondary antibody was applied followed by avidin-biotin complex-HRP. Secondary antibodies were detected with 3,3′-diaminobenzidine chromogen.
Positive staining was indicated by the presence of a brown chromogen reaction product. Sections were counterstained with hematoxylin for 1 min. Slides were analyzed under a microscope using a 40× objective. ADCC The ADCC assays were performed as described previously with several modifications. ADCC assays were performed with increasing anti-CD20 IgG1 antibody variant concentrations. Briefly, human PBMCs purified from leukopaks were used as effector cells, and WIL2-S cells were used as targets in a 50:1 ratio. The WIL2-S cells were labeled with 2,2′:6′,2″-terpyridine-6,6″-dicarboxylate reagent (PerkinElmer Life Sciences) for 30 min, washed twice, and resuspended in RPMI 1640 medium supplemented with GlutaMAX, 10% heat-inactivated FBS, 0.1 m m nonessential amino acids, and 1 m m sodium pyruvate (Invitrogen).
0.5 × 10 6 PBMCs, 1 × 10 4 labeled WIL2-S, and antibody at the indicated concentration were combined in 200 μl total in U-shaped 96-well plates, centrifuged for 2 min at 200 × g, and incubated at 37 °C for 2 h. At the end of the assay, plates were centrifuged again at 200 × g for 5 min, and 20 μl of supernatant were mixed with 200 μl of DELPHIA europium-based reagent. The fluorescence signal was measured using an Envision 2101 Multilabel Reader (PerkinElmer Life Sciences).
The percentage of lysis was calculated as (Experimental release − Spontaneous release)/(Maximal release − Spontaneous release) × 100. Data were log-transformed and fit to a sigmoidal dose-response curve using GraphPad Prism v5. CDC CDC assays were performed as described previously with WIL2-S cells as the target. A total of 50 μl of 0.05 × 10 6 cells was added to the wells of 96-well U-bottom plates in RPMI 1640 medium, 10% heat-inactivated FBS, 0.1 m m nonessential amino acids, and 1 m m sodium pyruvate. An additional 50 μl of medium was added with and without mAbs, and plates were incubated at room temperature for 1 h.
After incubation, a total of 50 μl of a 10% rabbit complement (Invitrogen) solution was added, and plates were incubated at 37 °C for 20 min. Plates were centrifuged again at 200 × g for 5 min, and 50 μl of supernatant were mixed with 50 μl of the lactate dehydrogenase cytotoxicity detection kit (Roche Applied Science).
Plates were incubated for 15 min at room temperature and then analyzed on a SpectraMax M5 (Molecular Devices, Sunnyvale, CA) at 490 nm. Data were normalized to maximal cytotoxicity with Triton X-100 (Sigma) and minimal control containing only cells and complement in the absence of mAb. Data were log-transformed and fit to a sigmoidal dose-response model using GraphPad Prism v5. Twenty-four-hour ADCP Human PBMCs were isolated from leukopaks (Biologics Specialty) using Ficoll gradient centrifugation.
CD14 pos monocytes were purified from PBMCs by negative depletion using a CD14 isolation kit that did not deplete CD16 pos monocytes (Stem Cell Technologies). Purified monocytes were plated at 0.1 × 10 6 cells/cm 2 in X-VIVO-10 medium (Lonza) containing 10% FBS. Macrophages were differentiated from monocytes by the addition of 25 ng/ml macrophage colony-stimulating factor (R&D Systems) for 7 days. IFNγ (50 ng/ml; R&D Systems) was added for the final 24 h of differentiation.
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The target cells for the assay were GFP-expressing MDA-MB-231 cells. Isolated macrophages were incubated in a 37 °C incubator with GFP-expressing MDA-MB-231 cells at a ratio of four macrophages (0.1 × 10 6 cells/well) to one MDA-MB-231 cell (25,000 cells/well) for 24 h with wild-type anti-CD142 or protease-resistant variants of anti-CD142 in 96-well U-bottom plates. The final volume of medium (DMEM + 10% FBS) used for the assay was 200 μl.
At the end of 24 h, plates were centrifuged at 300 × g for 5 min, and the cells were removed from the 96-well plates using Accutase (Sigma). Macrophages were identified with anti-CD11b (clone ICRF44) and anti-CD14 (clone M5E2) antibodies (both from BD Biosciences) coupled to Alexa Fluor 647 (Invitrogen), and then cells were acquired on an LSRFortessa flow cytometer (BD Biosciences). The data were analyzed using FloJo software (Tree Star). The percentage (%) of cell killing was determined by measuring the reduction in GFP fluorescence resulting from its degradation in the lysosomes after internalization using the following equation: Percentage of tumor cells killed = ((Percentage of GFP pos, CD11b neg, CD14 neg cells in no mAb control) − (Percentage of GFP pos, CD11b neg, CD14 neg cells in the presence of mAb))/(Percentage of GFP pos, CD11b neg, CD14 neg cells in no mAb control) × 100. Cell Binding Assays Target cells were plated at 0.2 × 10 6 in 100 μl of FACS staining buffer (BD Bioscience) with the indicated antibody concentrations. Cells were incubated for 30 min at 4 °C, washed two times with PBS, and then incubated with 100 μl of a 1:10 dilution of anti-κ-phycoerythrin (Biolegend) in FACS buffer for 30 min at 4 °C.
Cells were washed two times and resuspended in PBS. Acquisition was performed on a FACSCalibur (BD Biosciences), and analysis was performed using FlowJo (TreeStar).
Data were fitted to a sigmoidal dose-response curve using GraphPad Prism v5. ELISA for Antigen Binding Nunc-Immuno MaxiSorp plates were coated overnight with 10 μg/ml streptavidin (Invitrogen) at 4 °C. At room temperature, plates were blocked for 1 h with 3% BSA, PBS and then coated with biotinylated CD142 in 3% BSA, PBS for an additional hour. Antibody variants were added at the indicated concentration for 1 h. The plate was washed three times with 0.15 m NaCl, 0.02% Tween 20 and then treated with anti-κ-HRP (Millipore) at a 1:5000 dilution for 1 h. Plates were washed three times, 3,3′,5,5′-tetramethylbenzidine reagent (Fitzgerald Industries International) was added for 5 min, and the reaction was stopped with 3 m HCl.
The absorbance was read at 450 nm. Data were log-transformed and fitted to a sigmoidal dose-response curve using GraphPad Prism v5. In Vitro Competition FcRn Binding Analysis A competitive binding assay was used to assess relative affinities of different antibody samples to in-house recombinant human FcRn-His 6 (transmembrane and cytoplasmic domains of FcRn were replaced with a polyhistidine affinity tag).
Ninety-six-well copper-coated plates (Thermo Scientific) were used to capture FcRn-His 6 at 5 μg/ml in PBS after which plates were washed with 0.15 m NaCl, 0.02% Tween 20, pH ∼5 and then incubated with blocking reagent (0.05 m MES, 0.025% BSA, 0.001% Tween 20, pH 6.0, 10% ChemiBLOCKER (Millipore)). Plates were washed as above, and then serial dilutions of competitor test antibody in blocking reagent were added to the plate in the presence of a fixed 1 μg/ml concentration of an indicator antibody (a biotinylated human IgG1 monoclonal antibody).
Plates were incubated at room temperature for 1 h, washed three times as above, and then incubated with a 1:10,000 dilution of streptavidin-HRP (Jackson ImmunoResearch Laboratories) at room temperature for 30 min. Plates were washed five times as above, and bound streptavidin-HRP was detected by adding 3,3′,5,5′-tetramethylbenzidine peroxidase substrate with Stable Stop (Fitzgerald Industries International) and incubating for 4 min. Color development was stopped by addition of 0.5 m HCl. Optical densities were determined with a SpectraMax Plus384 plate reader (Molecular Devices) at 450-nm wavelength. Data were fitted to a sigmoidal dose-response curve using GraphPad Prism v5. B Cell Depletion in Cynomolgus Monkeys The B cell depletion studies were performed by Huntingdon Life Sciences (East Millstone, NJ). Four cynomolgus monkeys per group were injected with saline or rituximab variants at 1 mg/kg.
At the indicated time points, blood was collected and analyzed by flow cytometry. Cell events within a lymphocyte scatter were further subdivided into T lymphocytes (CD3 pos, CD20 neg), natural killer lymphocytes (CD3 neg, CD159a pos), or B lymphocytes (CD3 neg, CD19 pos). Antibodies to CD3 (SP34), CD20 (2H7), CD16 (3G8), and CD40 (5C3) and corresponding isotype control antibodies were purchased from BD Biosciences. Antibodies to CD159a (Z199), CD14 (RMO52), and CD19 (J3-119) were purchased from Beckman Coulter. Individual antibodies or appropriate antibody mixtures were added to 12 × 75-mm FACS tubes.
Blood samples were mixed before use ( e.g. Gentle inversion or rolling). Fifty microliters of blood and 50 μl of heat-inactivated FBS (Sigma) were added to each of the tubes. Sample tubes were briefly vortexed after all additions, protected from light, and incubated at ambient temperature for a minimum of 10 min. Red blood cells were then lysed, and samples were fixed using the TQ Prep Workstation TM (Coulter). Samples were stored at 2–8 °C protected from light until analysis.
All sample acquisition took place on an FC500 flow cytometer (Coulter). FlowCount beads (Coulter) were utilized to determine cell concentrations.
Data analysis was performed using FCS Express (v3.0). Detection of Cleaved IgGs in Human Squamous Cell Carcinoma Based on the observation that a number of physiologically relevant proteases associated with invasive cancers can cleave human IgGs in vitro, we assessed the presence of cleaved IgGs in human tumor tissue. We had previously generated anti-hinge antibodies that bind to cleaved IgGs but do not react with the intact IgG counterpart (, ). The anti-hinge antibodies were used for immunohistochemical detection of cleaved IgGs in human head and neck squamous cell carcinoma (HNSCC). As shown in A ( left panel), cleaved IgGs were detected in HNSCC with an enrichment of detection at the tumor/stromal interface.
To demonstrate that the staining was specific for cleaved IgGs, we added a pool of hinge-cleaved F(ab′) 2 fragments (generated with MMP-3, GluV8, and IdeS) to block the antigen-binding arm of the anti-hinge antibodies ( A, right panel). Inclusion of pooled F(ab′) 2 fragments effectively blocked detection of cleaved IgGs, confirming the specificity of the detection reagent. Positive staining for cleaved IgGs was observed in 19 of 51 (37%) individual HNSCC cases ( B). It was not unexpected that cleavage was not seen in all of the sections due to the heterogeneity of human tumor samples ( e.g. Total levels of EGF receptor vary widely in human tumor samples and are not uniform among different individuals ). This finding indicated that human IgGs were subject to proteolytic cleavage in the lower hinge region within the tumor microenvironment, especially at the expanding edge of the tumor where protease expression is known to be heightened. Mutation of the Lower Hinge of IgG1 Confers Protease Resistance We next sought to determine whether we could mutate the lower hinge/proximal C H2 region of human IgG1 to confer protease resistance.
Initially, three variants were generated for this purpose. The first variant contained the IgG1 to IgG2 domain exchange E233P/L234V/L235A with Gly 236 deleted (EU numbering ); this substitution was designated 2h. To compensate for the loss of function associated with such a domain swap (, ), additional variants were generated with select mutations in the C H2 region. The variant 2h-DE contained the IgG2 lower hinge and the C H2 mutations S239D/I332E, which were previously shown to enhance FcγR binding to IgG1. The variant 2h-AA contained the IgG2 lower hinge and the C H2 mutations K326A/E333A, which were previously shown to enhance IgG1 CDC activity. To assess protease susceptibility of the newly generated variants, mAbs were incubated with proteases capable of cleaving human IgG1.
Similar to previous studies, IgG1 was cleaved by MMP-3, MMP-12, GluV8, and IdeS to varying degrees after a 24-h incubation (, A and B). IgG2, 2h, 2h-DE, and 2h-AA were all resistant to cleavage by MMP-3 and MMP-12. The variant 2h-DE was uniquely resistant to the Group A streptococcal protease IdeS but had increased susceptibility to S. Aureus protease GluV8 compared with IgG2, 2h, and 2h-AA.
These results indicated that lower hinge mutations augmented protease resistance and that incorporation of mutations into the C H2 influenced protease resistance as well. Select Protease-resistant Variants Have Restored CDC or Enhanced ADCC We probed the ability of the protease-resistant variants to mediate Fc-dependent immune effector functions using cell-based assays. For CDC activity, we opsonized WIL2-S lymphoma cells with anti-CD20 antibody variants containing the V-region of rituximab. IgG1 mediated CDC activity, whereas the IgG2 did not, and as expected, the 2h mutation resulted in a loss of CDC activity (, left). However, the K326A/E333A mutations (variant 2h-AA) were able to restore CDC activity albeit at a reduced level relative to IgG1.
The incorporation of the S239D/I332E mutations (variant 2h-DE) did not restore CDC activity. Mutation of specific amino acids can restore cell-killing functions to the 2h protease-resistant backbone. Concentration-dependent CDC ( A) and ADCC ( B) activities against WIL2-S target cells using anti-CD20 IgG1 ( black circles), IgG2 ( green squares).
ADCC was assessed with anti-CD20 variants using peripheral blood mononuclear effector cells allotyped to be heterozygous for the Fcγ RIIIa 158 V/F polymorphism and WIL2-S target cells. IgG1 mediated ADCC activity, whereas IgG2 and the variants 2h and 2h-AA did not (, right). The 2h-DE variant had enhanced ADCC activity with an ∼11-fold average increase in potency as compared with IgG1. Thus, this engineering strategy resulted in a novel methodology to select for specific cell-killing functions that are at times enhanced.
Protease-resistant Variants That Elicit Both CDC and Enhanced ADCC To develop a protease-resistant platform that could mediate Fc effector functions more broadly ( i.e. Both ADCC and CDC), we combined the CDC-restoring K326A/E333A mutations with either of the ADCC-restoring mutations, S239D or I332E, resulting in variants that were designated 2h S239D/K326A/E333A (2h-DAA) or 2h K326A/I332E/E333A (2h-AEA). Twenty-four-hour proteolytic digests confirmed that both 2h-DAA and 2h-AEA maintained protease resistance to MMP-3, MMP-12, and GluV8 compared with IgG1 ( A). The 2h-AEA variant showed greater protease resistance to IdeS compared with IgG1 and 2h-DAA, suggesting that the I332E mutation on the 2h backbone influenced protease resistance to IdeS. Protease resistance is maintained in variants with combinations of ADCC- and CDC-restoring mutations on the 2h protease-resistant backbone on the anti-CD142 V-region. A, IgG1 ( white), 2h-DAA ( gray), and 2h-AEA ( black) were incubated with MMP-3, MMP-12.
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We next assessed IgG proteolysis in kinetic digest assays comparing IgG1 with the protease-resistant variants 2h-DAA and 2h-AEA. MMP-3 cleavage of IgG1 was detected within 15 min, and no intact IgG1 was detected at the 24-h time point, whereas both 2h-DAA and 2h-AEA were resistant to MMP-3 cleavage throughout the 24-h assay ( B).
Because IdeS rapidly cleaves human IgG1 (, ), kinetic IdeS digests were performed with shorter time points at an enzyme concentration of 0.1% (w/w). Approximately 60% intact IgG1 was detected after 1 min, and complete loss of intact IgG occurred by 30 min. In contrast, the variant 2h-AEA maintained nearly complete resistance to IdeS throughout the assay. The 2h-DAA variant displayed some loss of intact IgG after 2 h with ∼90% intact mAb remaining ( C). These results confirmed that the combination of lower hinge mutations with I332E imparted the greatest level of resistance to IdeS. Next, we characterized the Fc effector functions of the all the protease-resistant variants.
The protease-resistant variants 2h-DAA and 2h-AEA displayed CDC activity at potencies comparable with the 2h-AA variant ( A and ). Either the S239D or the I332E mutation alone was capable of restoring ADCC activity to the 2h backbone with each variant demonstrating higher potency than IgG1 although to levels lesser than the 2h-DE variant ( B and ). The ADCC activities for both the 2h-DAA and 2h-AEA variants were ∼4-fold higher than IgG1 with heterozygous Fcγ RIIIa 158 V/F PBMC donors. Human PBMC donors that were homozygous for the high affinity Fcγ RIIIa 158 V/V polymorphism displayed similar relative differences in ADCC enhancements between the different Fc variants ( C).
ADCC was further enhanced by the protease-resistant variants compared with IgG1 when human PBMC donors that were homozygous for the low affinity Fcγ RIIIa 158 F/F polymorphism were used as effector cells ( D). The 2h-DE variant displayed an ∼30-fold average increase in potency compared with IgG1, whereas the 2h-DAA and 2h-AEA variants displayed average increases of ∼23- and ∼10-fold, respectively, among the two different donors tested.
Therefore, three protease-resistant variants demonstrated increased ADCC potency compared with IgG1, and the increased potency was most apparent using low affinity Fcγ RIIIa 158 F/F polymorphism PBMC donors. Antibody-dependent Macrophage Killing of Human Tumor Cell Lines We recently demonstrated that tumor-associated macrophages display potent antitumor activity in the presence of an anti-tumor mAb.
Therefore, we wanted to assess the function of the protease-resistant variants against mAb-opsonized tumor cells using macrophage effector cells. Fc-dependent effector functions of macrophages against mAb-opsonized tumor cells are often assessed by ADCP.
This is typically accomplished by co-incubating macrophages with mAb-opsonized tumor cells and assessing internalization of the tumor cells within macrophages over a time frame typically no longer than 4 h (, –). Our group previously developed a flow cytometry and microscopy-based ADCP assay where the target tumor cells (MDA-MB-231) expressed GFP to obviate the need to label the tumor cells with a dye (, ). Macrophage internalization of MDA-MB-231 cells opsonized with a tumor targeting anti-CD142 mAb was assessed by either flow cytometry or fluorescence microscopy (, ). We had previously noted that the GFP fluorescence within macrophages was punctate, whereas the GFP fluorescence of non-internalized tumor cells was uniform (, ).
Because the process of ADCP results in internalization and the subsequent lysosomal destruction of the tumor cell and GFP fluorescence is dependent on the structural integrity of the GFP protein, we hypothesized that the punctate GFP signal was due to breakdown of the tumor target cell within the macrophage. To test the ability of macrophages to kill anti-CD142 mAb-opsonized, GFP-expressing MDA-MB-231 tumor cells, we extended a traditional ADCP assay to 24 h. In this assay, we could observe complete destruction of the tumor cell both by flow cytometry and immunofluorescence.
This was accomplished by incubating effector macrophages in the presence of anti-CD142-opsonized, GFP-expressing MDA-MB-231 target cells and assessing the loss of GFP detection after 24 h (, A and B). The variants 2h-DE, 2h-DAA, and 2h-AEA displayed tumor cell killing comparable with IgG1. The tumor cell killing of the IgG2 was slightly reduced, whereas the variants 2h and 2h-AA had the lowest level of tumor cell killing ( C). These results demonstrated that the variants with ADCC-restoring mutations were capable of killing MDA-MB-231 tumor cells in the 24-h ADCP assay. Protease-resistant Variant Fab Arm Function, FcRn Binding, and Biophysical Property Assessment To test that the protease-resistant mutations did not affect Fab arm function, we assessed the ability of the variants to bind to antigen in both plate-based and cell-based assays. The results indicated that variants engineered onto two different V-regions bound identically to their respective targets (, A and B). We next assessed the ability of the variants to bind to the neonatal Fc receptor, FcRn, which is thought to contribute to the long circulating half-life of IgGs.
Fc binding to FcRn occurs proximal to the junction of the C H2 and C H3 regions , a region that is distal from the mutations present in all of the protease-resistant variants. All of the variants displayed FcRn binding comparable with IgG1 ( C). These results demonstrate that the protease-resistant mutations do not affect the ability of the mAbs to engage either antigen or FcRn.
Protease-resistant variant binding to antigen and FcRn is not affected. A, the variants 2h, 2h-AA, 2h-DE, 2h-DAA, and 2h-AEA containing the anti-CD142 V-region bound similarly to IgG1 on a plate-bound ELISA assay using CD142 antigen. Shown is a representative. We next assessed several biophysical properties of select variants (IgG1, IgG2, 2h-DE, 2h-DAA, and 2h-AEA). Cross-interaction chromatography was used to assess protein-protein interactions, and the results indicated that all of the assessed variants displayed minimal protein-protein interactions (data not shown). Differential scanning calorimetry was performed to assess the thermal stability of the variants. The results indicated varying degrees of thermal stability in the C H2 region with variants containing the I332E mutation having the lowest T m (55.6 °C for 2h-DE and 60.7 °C for 2h-AEA).
Of the variants tested, 2h-DAA decreased ∼7 °C compared with IgG1 (64.9 and 72.0 °C, respectively). Finally, the expression titers for each of the variants were comparable with the parent IgG1 mAb (data not shown). Protease-resistant Variants Mediate B Cell Depletion in Cynomolgus Monkeys Because of sequence differences between mouse and human FcγRs, the in vivo function of mAbs engineered for binding to human FcγRs are often assessed in non-human primates (, ) whose FcγRs are more homologous to the human counterparts. Because many anti-human CD20 mAbs are cross-reactive with cynomolgus CD20 expressed on B cells, the cytotoxic potential of engineered anti-CD20 variants is often assessed in cynomolgus monkey B cell depletion studies. A previous B cell depletion study in cynomolgus monkeys using an Fc silent anti-CD20 variant did not result in depletion of B cells in vivo , further supporting that this model is appropriate for the assessment of Fc-dependent effector functions of anti-CD20 mAbs. An anti-CD20 mAb was not used for B cell detection because murine-derived anti-CD20 mAbs, including rituximab, a murine/human chimeric antibody, often bind to a common epitope on the large extracellular loop of CD20 and can either fully or partially mask CD20. Therefore, we used an anti-CD19 antibody (clone J3-119) to detect cynomolgus monkey B cells.
A single dose intravenous injection of a 1.0 mg/kg concentration of anti-CD20 IgG1 or the variants 2h-DE or 2h-DAA resulted in nearly complete loss of detection of B cells by the 6-h time point ( and ). Depletion was sustained through day 7 of the study with B cell recovery starting at day 14 of the study. These results indicated that the protease-resistant variants 2h-DE and 2h-DAA were capable of depleting cynomolgus monkey B cells in vivo comparably with IgG1 at the 1.0 mg/kg dose.
. 1999. US. 1h 55min.
PG. Directed by: Michael Hoffman.
Cast: Kevin Kline, Michelle Pfeiffer, Rupert Everett, Stanley Tucci, Calista Flockhart, Anna Friel, Christian Bale, Dominic West, David Strathairn, Sophie Marceau, Roger Rees, Max Wright, Gregory Jbara, Bill Irwin, Sam Rockwell, Bernard Hill, John Sessions Not since Max Reinhardt's 1934 version has any film sought to pay loyal tribute to this classic tale of misbegotten romance and fairy magic. Hoffman, cautious that the text's classical Greek setting might distance his audience, has relocated to Tuscany at the turn of the century and draws upon those changing. 1999. US. 1h 27min. U.
Directed by: Tim Hill. Written by: Jerry Juhl, Joey Mazzarino, Ken Kaufman. Cast: Dave Goelz, Steve Whitmire, Bill Barretta, Frank Oz, Jeffrey Tambor, F Murray Abraham, David Arquette, Josh Charles, Kathy Griffin, Hollywood Hogan, Pat Hingle, Ray Liotta, Andie McDowell For their sixth big screen adventure, the focus of our Muppet attentions is Gonzo, that blue, hooked nosed thing. No one is really sure what Gonzo is, so when he gets a message which he believes is from space, the race is on to make contact with his extraterrestrial brethren.
Muppets From Space captures the spirit of. 1999. US. 1h 38min. 12.
Directed by: Gil Junger. Written by: Karen McCullah Lutz, Kirsten Smith. Cast: Julia Stiles, Heath Ledger, Joseph Gordon-Levitt, Larisa Oleynik, David Krumholtz, Andrew Keegan, Larry Miller It could have been horrible. But this high school-set reworking of Shakespeare's The Taming of the Shrew is not only faithful to its source, but is a funny, charming and enjoyable film in its own right.
Purists may be aghast at the hijacking of such a literary jewel, but films like this offer easy access to great stories. 1998. US. 18.
Directed by: Todd Solondz. Written by: Todd Solondz.
Cast: Jane Adams, Philip Seymour Hoffman, Jon Lovitz, Dylan Baker, Lara Flynn Boyle, Justin Elven, Cynthia Stevenson, Lila Glantzman-Leib, Rufus Read, Gerry Becker, Louise Lasser, Ben Gazzara, Camryn Manheim, Arthur J Nascarella, Molly Shannon, Ann Harada Three sisters, two small boys, one psychologist and a phone-harassment specialist. Out of these unlikely elements Solondz has wrought pure cinematic gold, which veers from belly laughter one moment to stark pathos in another.
What a sad coincidence that this movie was released the same day - Charlie day - the terrible slaughter in the deep heart of Paris occurred, where twelve people working for Charlie Hebdo satyric magazine were killed. Coincidence because the lead character of this outstanding crime film has Charlie as a nickmane. Yes, sad coincidence. Well, let's talk at last about this, I repeat, outstanding crime movie, inspired from actual events that shocked France during nearly ten long years. Horrible sex and torture crimes committed by the famous Guy Georges.
Everything here is very well made, shown in a meticulous, accurate way, explaining everything to the audiences. Acting is here at his top, and editing too. I know there always will be some folks to make critical comments. I respect that. Nothing is perfect. Don't miss it.
In the memory of Charlie.
John Bodkin Adams in the 1940s Born ( 1899-01-21)21 January 1899, Ireland Died 4 July 1983 ( 1983-07-04) (aged 84), England Occupation Known for Suspected serial killer Criminal charge Fraud Criminal penalty Removed from Medical Register in 1957, reinstated in 1961 John Bodkin Adams (21 January 1899 – 4 July 1983) was a, convicted and suspected. Between 1946 and 1956, more than 160 of his patients died in suspicious circumstances. Of these, 132 left him money or items in their. He was tried and for the murder of one patient in 1957. Another count of murder was withdrawn by the prosecution in what was later described as 'an abuse of process' by the presiding judge, causing questions to be asked in about the prosecution's handling of events. The trial was featured in headlines around the world and was described at the time as 'one of the greatest murder trials of all time' and 'murder trial of the century'.
It was also described at the time as 'unique' because, in the words of the judge, 'the act of murder' had 'to be proved by expert evidence.' The trial had several important legal ramifications.
It established the doctrine of, whereby a doctor giving treatment with the aim of relieving pain may, as an unintentional result, shorten life. Secondly, because of the publicity surrounding Adams's committal hearing, the law was changed to allow defendants to ask for such hearings to be held in private. Finally, though a defendant had never been required to, the judge underlined in his summing-up that no prejudice should be attached by the jury to Adams not doing so. Adams was found guilty in a subsequent trial of 13 offences of, lying on forms, and failing to keep a dangerous drugs register.
He was removed from the in 1957 and reinstated in 1961 after two failed applications. 's files on the case were initially closed to the public for 75 years, and would have remained so until 2033. Following a request by historian Pamela Cullen, special permission was granted in 2003 to reopen the files. Contents. Early years Adams was born and raised in into a deeply religious family of, an austere sect of which he remained a member for his entire life.
His father, Samuel, was a preacher in the local congregation and by profession was a watchmaker. He also had a passionate interest in cars, which he would pass on to John. In 1896 Samuel was 39 years old when he married Ellen Bodkin, aged 30, in,. John was their first son, followed by a brother, William Samuel, in 1903. In 1914 Adams's father died of a. Four years later William died in the. After attending for several years, Adams at at the age of 17.
There he was seen as a 'plodder' and 'lone wolf' by his lecturers and, partly because of an illness (probably ), he missed a year of studies. He graduated in 1921, having failed to qualify for honours. In 1921 surgeon offered Adams a position as assistant. He spent a year there but did not prove a success. On Short's advice, Adams applied for a job as a in a Christian practice in,. Eastbourne.
Kent Lodge, where Adams lived from 1929 to 1983 Adams arrived in Eastbourne in 1922, where he lived with his mother and his cousin, Sarah Florence Henry. In 1929, he borrowed 2,000 (equivalent to £104,247 at 2011 prices ) from a patient, William Mawhood, and bought an 18-room house called Kent Lodge, in Trinity Trees (then known as Seaside Road ), a select address.
Adams would frequently invite himself to the Mawhoods' residence at meal time, even bringing his mother and cousin. He also began charging items to their accounts at local stores without their permission. Mrs Mawhood would later describe Adams to the police as 'a real scrounger'. When Mr Mawhood died in 1949, Adams visited his widow uninvited and took a 22-carat gold pen from her bedroom dressing table, saying he wanted 'something of her husband's'. He never visited her again.
Gossip regarding Adams's unconventional methods had started by the mid-1930s. In 1935, Adams inherited £7,385 from a patient, Matilda Whitton; her whole estate amounted to £11,465, equivalent to £430,931 and £669,007 respectively at 2011 values. The was contested by her relatives but upheld in court, though a giving Adams's mother £100 was overturned. Adams then began receiving 'anonymous postcards' about him 'bumping off' patients, as he admitted in a newspaper interview in 1957. These were received at a rate of three or four a year until, and then commenced again in 1945. Adams stayed in Eastbourne throughout the war, and was 'furious' at not being deemed desirable by other doctors to be selected for a 'pool system' where GPs would treat the patients of colleagues who had been called up. In 1941, he gained a diploma in and worked in a local hospital one day a week, where he acquired a reputation as a bungler.
He would fall asleep during operations, eat cakes, count money, and even mix up the gas tubes, leading to patients waking up. In 1943, his mother died, and in 1952 his cousin Sarah developed.
Adams gave her an injection half an hour before she died and according to historian Pamela Cullen, this is the only 'case where it can be considered that the doctor was '. Adams's career was very successful, and by 1956 'he was probably the wealthiest GP in England'. He attended some of the most famous and influential people in the region, including and medal winner, society painter, industrialist Sir, the, Eastbourne's Richard Walker and a host of businessmen. After years of rumours, and Adams's having been mentioned in at least 132 wills of his patients, on 23 July 1956 Eastbourne police received an anonymous call about a death. It was from, the music hall performer, whose friend had died unexpectedly while being treated by Adams.
Police investigation The investigation was taken over from Eastbourne police on 17 August 1956 by two officers from the 's Murder Squad. The senior officer, of Scotland Yard, was known for having solved the in 1953. He was assisted. Hannam was in the unusual position that, instead of having to find a suspect for a known crime, he had a known suspect in Adams but needed to link him to more serious crimes than, making false statements and mishandling drugs. Devlin suggests that Hannam became fixated on the idea that Adams had murdered many elderly patients for legacies, regarding his receiving a legacy as grounds for suspicion, although Adams was generally only a minor beneficiary. The investigation decided to focus on cases from 1946 to 1956 only. Of the 310 death certificates examined by, 163 were deemed to be suspicious.
The police took numerous statements from nurses that had treated Adams' patients. Some were generally favourable to him, but others claimed Adams had given patients 'special injections' of substances Adams refused to describe to the nurses. The statements also claimed that his habit was to ask the nurses to leave the room before injections were given and that he would also isolate patients from their relatives, hindering contact between them. However, during the trial, the assertions of Mrs Morrell's nurses that they did not know what Adams was injecting or that he did not give these in front of them were disproved by the contents of their own note-books.
Obstruction On 24 August, in an 'extraordinary move' the (BMA) sent a letter to all doctors in Eastbourne reminding them of 'Professional Secrecy' ( i.e., ) if interviewed by the police. Hannam was not impressed, especially since any information gleaned would relate to dead patients. He, and the, Sir (who prosecuted all cases of poisoning), wrote to the BMA secretary, Dr Macrae, 'to try to get him to remove the ban'. The impasse continued until on 8 November Manningham-Buller met with Macrae to convince him of the importance of the case. During this meeting, in a highly unusual move, he passed Hannam's confidential 187-page report on Adams to Macrae. Macrae took the report to the President of the BMA and returned it the next day. In all likelihood, he also copied it and passed it on to the defence.
Convinced of the seriousness of the accusations, Macrae dropped his opposition to doctors talking to the police. In the end two Eastbourne doctors gave evidence to the police.
On 28 November 1956, opposition MPs and gave notice of two questions to be asked in the regarding the affair, one asking what 'reports the Attorney-General has sent' to the (GMC) in the 'past six months'. Manningham-Buller replied that he had 'had no communications' with the GMC, but only with an officer of it. He did not mention the report. Instead, he instigated an investigation into a, later concluding that Hannam himself had passed information regarding the meeting with Macrae to a journalist, probably of the. Meeting Hannam On 1 October 1956 Hannam bumped into Adams and Adams asked 'You are finding all these rumours untrue, aren't you?' Hannam mentioned a Adams had forged: 'That was very wrong.
I have had God's forgiveness for it', Adams replied. Hannam brought up the deaths of Adams' patients and his receipt of legacies from them. Adams answered: 'A lot of those were instead of fees, I don't want money. What use is it? I paid £1100 last year' Hannam later mentioned, 'Mr Hullett left you £500'.
Adams replied, 'Now, now, he was a life-long friend. I even thought it would be more than it was.' Finally, when asked why he had stated untruthfully on forms that he was not to inherit from the deceased, Adams said: Oh, that wasn't done wickedly, God knows it wasn't. We always want cremations to go off smoothly for the dear relatives. If I said I knew I was getting money under the Will they might get suspicious and I like cremations and burials to go smoothly. There was nothing suspicious really.
It was not deceitful. Search On 24 November, Hannam, Hewett and the head of Eastbourne, Pugh, searched Adams' house with a issued (in Pugh's name) under the Dangerous Drugs Act 1951. When told they were looking for, ', and the like', Adams was surprised: 'Oh, that group. You will find none here.
I haven't any. I very seldom ever use them', he said.
When Hannam asked for Adams' Dangerous Drugs Register – the record of those ordered and used – Adams responded: 'I don't know what you mean. I keep no register.' He had not kept one since 1949. When shown a list of dangerous drugs he had prescribed Morrell, and asked who administered them, Adams said, 'I did nearly all.
Perhaps the nurses gave some but mostly me' – contradicting what the nurses' notebooks would show during his trial. Hannam then observed, 'Doctor, you prescribed for her 75 – 1/6 grains heroin tablets the day before she died.' Adams replied, 'Poor soul, she was in terrible agony. It was all used. I used them myself. Do you think it is too much?'
Adams opened a cupboard for the police: amongst medicine bottles were 'chocolates – slabs stuck – butter, margarine, sugar'. While the officers inspected it Adams walked to another cupboard and slipped two objects into his jacket pocket. Hannam and Pugh challenged him and Adams showed them two bottles of morphine; one he said was for Annie Sharpe, a patient and major witness who had died nine days earlier under his care; the other said 'Mr Soden'. He had died on 17 September 1956 but pharmacy records later showed Soden had never been prescribed morphine. Adams was later (after his main trial in 1957) convicted of obstructing the search, concealing the bottles and for failing to keep a Dangerous Drugs register.
Later at the police station, Adams told Hannam: Easing the passing of a dying person isn't all that wicked. She Morrell wanted to die. That can't be murder.
It is impossible to accuse a doctor. In the basement of Adams's house, the police found, 'a lot of unused china and silverware. In one room there were 20 new motor car tyres still in their wrappings and several new motor car leaf springs. Wines and spirits were stored in quantity.' Hallworth reports that Adams was stockpiling in case of another. On the second floor, 'one room was given over to an : six guns in a glass-fronted display case, several automatic pistols'.
He had permits for these. Another room was used 'wholly for photographic equipment. A dozen very expensive cameras in leather cases' lay around. Sexuality In December the police acquired a belonging to a Daily Mail journalist, concerning rumours of between 'a police officer, a and a doctor'. The last directly implied Adams. This information had come, according to the reporter, directly from Hannam. The 'magistrate' was Sir, of Eastbourne (1929–31) and brother of, MP for Eastbourne (1910–24).
Gwynne was Adams's patient and known to visit every day at 9am. They went on frequent holidays together and had spent three weeks in that September. The 'police officer' was the Deputy Chief Constable of Eastbourne, Alexander Seekings.
Hannam ignored this line of inquiry (despite homosexual acts being a criminal offence in 1956), and the police instead gave the journalist a dressing-down. The memo is testament to Adams's close connections to those of power in Eastbourne at the time.
There were rumours of Adams having three ', but these were probably just 'covers' to avoid suspicion. Adams became engaged about 1933 to Norah O'Hara but called it off in 1935 after her father had bought them a house and furnished it. Various explanations have been suggested: Surtees suggests that it was because Adams's mother did not want him to marry 'trade' though he also quotes a rumour that Adams wanted O'Hara's father to change his will to favour his daughters. Cullen suggests that, apart from being homosexual, Adams also did not want his being married to interfere with his relationship with his elderly female patients.
Adams remained friends with O'Hara his whole life and remembered her in his will. Arrest Adams was arrested on 19 December 1956. When told of the charges he said: Murder.
Can you prove it was murder?. I didn't think you could prove it was murder. She was dying in any event. Then, while he was being taken away from Kent Lodge, he reportedly gripped his receptionist's hand and told her: 'I will see you in heaven.' Hannam considered he had collected enough evidence in at least four of the cases for prosecution to be warranted: regarding Clara Neil Miller, Julia Bradnum, Edith Alice Morrell and Gertrude Hullett.
Of these, Adams was charged on one count: the murder of Morrell, but with the death of Hullett (and also that of her husband) being used to prove 'system'. Adams and Eves On 22 February 1957 the police were notified of a and potentially prejudicial poem about the case titled. It had been read at the Cavendish Hotel on the 13th by the manager in front of 150 guests. An officer spent ten days investigating and discovered a chain of hands through which the poem had passed and been recopied to be redistributed.
The original author was not discovered; an unnamed journalist was suspected. The poem finished:. It's the chapel If they touch an After parting with a as a fee So to liquidate your odd By the needle of the Send them down to sunny Eastbourne by the sea. Patients Edith Alice Morrell Morrell was a wealthy widow who suffered a stroke on 24 June 1948 while visiting her son in.
She was partially and was admitted to a hospital near, where she received morphine injections for nine days from 27 June, prescribed by a Dr Turner. Cullen suggests that Adams, supposedly her usual doctor, arrived there on 26 June, the day before she was first prescribed morphine for the pain.
However, the Attorney-General's opening speech states that Mrs Morrell was transferred to Eastbourne on 5 July 1948, only then becoming one of Adams' patients, who first prescribed morphine on 9 July, adding heroin on 21 July. Mrs Morrell was not expected to live more than six months or so, but survived her stroke for over two years, suffering also from: between July 1948 and August 1950, she received routine evening injections of morphine and heroin and her condition was stable but from then, as her condition deteriorated, the dosages increased. An expert witness for the prosecution claimed that Mrs Morrell would have become addicted, but the only apparent symptoms of this were attributed by the defence's expert to a second stroke Mrs Morrell left an estate of £157,000 and made eight cash bequests of between £300 and £1,000. Cullen claims that in some of the several wills she made Adams was bequeathed large sums of money and her (valued at £1,500 ).
This appears incorrect and, in her will of 5 August 1950, he was only awarded outright a chest of silver cutlery worth £276, with a contingent right to the car and a Jacobean court cupboard if Mrs Morrell's son pre-deceased her A codicil of 13 September 1950 cut Adams out of her will completely. She died on 13 November 1950 aged 81. Adams certified the cause of death as 'stroke' and on inspecting the body, slit her wrist to ensure she was dead. Despite the last codicil, Mrs Morrell's son gave Adams the Rolls-Royce which was 19 years old, and the chest of silver cutlery. After Mrs Morrell's death, he also took away an infrared lamp she had bought herself, worth £60. Adams billed Morrell's estate for 1,100 visits, costing £1,674 in total.
The police estimated that Adams had visited Morrell a total of 321 times during her treatment. On her cremation form, Adams stated that 'as far as I am aware' he had no pecuniary interest in the death, thereby avoiding the necessity of a. Gertrude Hullett On 23 July 1956 Gertrude Hullett, another of Adams' patients, died aged 50. She had been depressed since the death of her husband four months earlier and had been prescribed large amounts of sodium and also sodium. She had told Adams on frequent occasions of her wish to commit suicide. On 17 July Hullett wrote out a cheque for Adams for £1,000 – to pay for an car her husband had promised to buy him. Adams paid the cheque into his account the next day, and on being told that it would clear by the 21st, asked for it to be specially cleared – to arrive in his account the next day.
On 19 July Hullett is thought to have taken an and was found the next morning in a. Adams was unavailable and a colleague, Dr Harris, attended her until Adams arrived later in the day. Not once during their discussion did Adams mention her or her barbiturate medication. They decided a was most likely.
On 21 July Dr Shera, a pathologist, was called in to take a sample and immediately asked if her stomach contents should be examined in case of poisoning. Adams and Harris both opposed this.
After Shera left, Adams visited a colleague at the Princess Alice Hospital in Eastbourne and asked about the treatment for barbiturate poisoning. He was told to give doses of 10 cc of every five minutes, and was given 100 cc to use. The recommended dose in the instructions was 100 cc to 200 cc. Dr Cook also told him to put Hullett on an. Adams did not. The next morning, at 8.30, Adams called the to make an appointment for a private. The coroner asked when the patient had died and Adams said she had not yet.
Harris visited again that day and Adams still made no mention of potential barbiturate poisoning. When Harris had left, Adams gave a single injection of 10 cc of the Megimide. Hullett developed broncho-pneumonia and on the 23rd at 6.00 a.m. Adams gave Hullett oxygen.
She died at 7.23 a.m. The results of a sample taken on 21 July were received after Hullett's death, on the 24th. It showed she had 115 of in her body – twice the fatal dose. An was held into Hullett's death on 21 August. The coroner questioned Adams' treatment and in his summing up said that it was 'extraordinary that the doctor, knowing the past history of the patient' did not 'at once suspect barbiturate poisoning'. He described Adams's 10 cc dose of Megimide as another 'mere gesture'.
The inquest concluded that Hullett committed suicide. After the inquest, the cheque for £1,000 disappeared.
Hullett left Adams her (worth at least £2,900 ) in a will written five days before her overdose. Adams sold it six days before he was arrested. Before the trial Case selection Charles Hewett, Hannam's assistant was quoted as saying that both officers were astounded at Manningham-Buller's decision to charge Adams with the murder of Morrell, since her body had been cremated and therefore there was no evidence to present before a jury. This assertion was published after the deaths of both Hannam and Manningham-Buller. Hewett believed that there were other cases against the doctor, where traces of drugs had been found in exhumed remains, which were more compelling as proof Cullen also describes Morrell as 'the weakest' case of the four the police deemed most suspicious.
Hewett’s view was not shared by Devlin. In 1957, it was the job of the police to investigate reported crimes, to determine if one had been committed and arrest a suspect. It was then the job of the Director of Public Prosecutions, or in very serious cases of the Attorney-General or Solicitor-General, to review the police case and decide whether to prosecute and, in more serious cases, what offences to prosecute. What to prosecute depends on legal issues and Devlin states that, to succeed in the murder case against Adams, the prosecution had to show, firstly, there had been an unnatural death, secondly, an act by Adams was capable of being murderous (such as an injection so large as to cause death) and finally Adams’ intent to kill. The Attorney-General thought he had evidence that Adams had prescribed large quantities of opiates to Mrs Morrell, Adams' own admissions that he had used them all on Mrs Morrell and injected all or almost all of them himself, and a medical expert's testimony that the only possible reason to inject so much over a short time was to kill her. Cullen mentions Mrs.
Morell, Mr and Mrs Hullett, Clara Neil Miller and Julia Bradnum as cases that Hannam regarded as warranting prosecution. However, in the cases of Mr Hullett, Clara Neil Miller and Julia Bradnum there was no certainty of an unnatural death as there was evidence in the committal hearing that Mr Hullett died of a heart attack and, at their exhumations, the pathologist concluded Miller had died from pneumonia and the condition of Bradnum’s body did not allow a cause of death to be stated, so none were good cases. Mrs Hullett had died an unnatural death, of a barbiturate overdose, but there was no evidence or admission that Adams had persuaded her to take that overdose and, had Mrs Hullett’s case been brought to trial after Adams’ first acquittal, Devlin believed that a second acquittal was virtually certain In these five cases, Adams may have contributed to the deaths in some way, but this would not have been sufficient for a capital murder conviction Committal hearing The committal hearing opened in on 14 January 1957. In accordance with the legal rule applying in 1957, Adams was charged on the single count of murdering Mrs Morrell, but the prosecution also alleged he had killed Mr and Mrs Hullett in a similar fashion, and introduced evidence relating to them as evidence of system, which the prosecution also wished to refer to in the Morrell trial. Despite the objections of the defence that this evidence was inadmissible, the magistrates allowed it but, in cross examination, the defence forced an admission from the Crown's expert witness that Mr Hullett died of a coronary thrombosis.
The hearing concluded on 24 January when, after a five-minute deliberation, Adams was committed for trial on the Morrell charge. Devlin considered the police case that there were similarities in deaths of Mrs Morrell and Mrs Hullett was not well founded, as the claimed similarities were not distinctive.
Had the police found two recent cases similar to Mrs Hullett's, where a patient had died of an overdose of pills prescribed by Adams, that might have shown system, but the police found no such cases. The Chairman of the was Sir Roland Gwynne, but he stepped down because of his close friendship with Adams. A vital piece of evidence, a cheque written out for £1000, went missing after the hearing, instigating a further police investigation. While the culprit was not found, Scotland Yard suspected the local Deputy Chief Constable of Eastbourne, Seekings, of having misplaced it to help Adams.
Seekings was known to have taken holidays with Adams and Gwynne, and looked after Gwynne's finances while he was in hospital in January 1957. Following the committal hearing, the Attorney-General advised Devlin that he would not be using the evidence regarding the Hullets in the Morrell trial, but seeking a second indictment relating to Mrs Hullett, which he did on 5 March 1957. Had this been proceeded with, a second committal hearing would have been required. The trial on the indictment relating to Mrs Morrell started on 18 March 1957 at the, with that relating to Mrs Hullett held back for a possible second separate trial. Three days later, a new came into effect; a single murder by poison became a non. Adams, having been indicted on both charges before this date, would still face the death penalty if convicted. The would be less likely to grant in the case of a second murder conviction in the Hullett case, as this would make it far more difficult politically to sentence Adams to, particularly as a double murder could still be capital under the 1957 Homicide Act, and Devlin considered that the Attorney-General's aim in bringing forward a second indictment was to make it more likely that Adams would hang.
Female Serial Killers
Trial Adams was first tried for the murder of Morrell, with the Hullett charge to be prosecuted afterwards. The trial lasted 17 days, the longest murder trial in Britain up to that point. It was presided over by Mr Justice. Devlin summed up the tricky nature of the case thus: 'It is a most curious situation, perhaps unique in these courts, that the act of murder has to be proved by.' The leading Defence counsel, had been briefed by the with the additional task of obtaining a ruling on whether medical treatments that might shorten the life of a terminally ill patient were legal. Lawrence, a 'specialist in real estate and divorce cases and a relative stranger in criminal court', who was defending his first murder trial, convinced the jury that there was no evidence that a murder had been committed, much less that a murder had been committed by Adams.
He emphasised that the indictment was based mainly on testimonies from the nurses who tended Morrell – and that none of the witnesses' evidence matched the others'. Then, on the second day of the trial, he produced notebooks written by the nurses, detailing Adams' treatment of Morrell. The prosecution claimed never to have seen these notebooks (even though they are recorded in pretrial lists of evidence). These differed from the nurses' recollection of events, and showed that smaller quantities of drugs were given to the patient than the prosecution had thought, based on Adams' prescriptions. Furthermore, the prosecution's two expert medical witnesses gave differing opinions: was prepared to say that murder had definitely been committed (though he changed his mind in the middle of his testimony regarding the exact date), but was more reticent.
The defence witness and physician was adamant that Adams's treatment, though unusual, was not reckless. Finally, the prosecution was wrong-footed by the defence not calling the loquacious Adams to give evidence, and thereby avoiding him 'chatting himself to the '. This was unexpected, shocking the prosecution and the press, and even surprising the judge. Mr Justice Devlin received a phone call from, the, at the time defence and prosecution were making their closing speeches. In the event of Adams being acquitted, Lord Goddard suggested that Devlin might consider an application to release Adams on bail before the Hullett trial, which was due to start afterwards. Devlin was surprised because no one accused of murder had ever been granted bail in British legal history, but he was willing to entertain the idea.
During the committal hearing prior to the trial, Goddard had been seen dining with at The White Hart Hotel in Lewes. Goddard, as Lord Chief Justice, appointed Devlin to try the Adams case shortly after the end of the committal hearing and, in a meeting of 19th February 1957, Goddard gave Devlin his views on the case. On 9 April 1957, the jury returned after 44 minutes to find Adams not guilty. Use of the nolle prosequi After the not guilty verdict on the count of murdering Morrell, the normal process would have been to bring the indictment regarding Mrs Hullett to trial, either a full trial or, in view of the acquittal in Mrs Morrell's case, so that Adams would plead not guilty, the Attorney-General would offer no evidence and the judge would direct the jury to bring in a not guilty verdit, the course Devlin expected.
However the Attorney-General, as a minister of the Crown had the power to suspend an indictment through a, something which Devlin said had never been used to prevent an accused from an acquittal, suggesting this was done because Manningham-Buller did not want a second acquittal and the loss of both the cases he had indicted. Nolle prosequi could legitimately be used in cases to protect a guilty person granted immunity to turn or to save the lives of the innocent, or sometimes on compassionate grounds. Retrieved 18 February 2010. 537. ^, 22 April 1957.
The Times, 11 June 1985, p. 10. ^ Devlin, 1985. ^, p. 7. He left £500 in his will to Marine Hall, his local Brethren congregation. 554).
^, pp. 24.
Bank of England inflation calculator,. Archived from on 5 February 2014. Retrieved 2014-02-05. 55.
'Kelly's Directory of Eastbourne (1929)'. Kelly's Directories Ltd.
24. ^, 1984., p. 15–17. ^, p. 40. Devlin, p. Robins, pp.77-83.
593., p.588. Devlin, pp.
'almost designed to frustrate the investigation'. (, p.587). ^, p. 224.
The, wrote to Manningham-Buller that: 'The disclosure of this document is likely to cause me considerable embarrassment. As you know, police reports have always been treated as highly confidential documents and it has been the invariable practice to refuse to disclose their contents to Parliament or to individual Members.
Indeed I should have no hesitation in claiming privilege if their production were required in a court of law.' He ended: 'I can only hope that no harm will result.' (Quoted in, p.
230). 'It cannot be imagined that the Attorney General, a lawyer just one place below the rank of the Lord Chancellor of the realm could 'loan' a police report of such importance for Macrae to take it to his President and expect that only one particular paragraph would be read by them or that they would make no copy of the report.' 228. In court, the defence would accuse Hannam of intentionally 'waylaying' Adams in order to informally question him. Hannam denied this.
594. ^ Hallworth, 1983. Probably Rodney Hallworth (, p. 610). ^, pp.
243–244., pp. 622–635., p. 611.
She was the sister-in-law of one of Adams's Brethren friends (Norman Gray), and her father owned six butchers in the town. 23. He left her: 'in gratitude and memories of our long standing friendship any one item of furniture or personal or household or domestic use ornament or consumption belonging to me at the time of my death'. 250. Devlin, pp.
560. Devlin, pp. 2-3.
Devlin, pp. 83, 127. Devlin, pp. 113-4, 149.
^, p. 94. Devlin, p.
93., p.564. Devlin, pp. 97, 105., p. 156–159. Over 80 days 1512 of the former and 6¼ grains of the latter were prescribed. 185.
The inquest has been described as a 'travesty'. In the opinion of Cullen, with an ongoing police investigation, the inquest should have been adjourned until the investigation had concluded.
577. Devlin, pp. 122-3. Devlin, p. 123. Cite error: The named reference, pp.
250, 636 was invoked but never defined (see the ). 143. Devlin, p. 25.
Devlin, pp. 13-14, 218. Devlin, pp. 25, 179. Devlin, pp. 198, 218.
^, p. 249. Devlin, pp. 29-30. Devlin, p.
Devlin, pp. Devlin, p.33. Devlin, pp.43, 47-8. Devlin, pp.48-9., p.281. Furneaux, pp.
When asked by Lawrence whether it was possible 'to rule out the hypothesis that when the end came in that way at that time on that date, it was the result of natural causes?' , Ashby replied 'It cannot be ruled out'.
Devlin, pp. Devlin, pp. Devlin, pp. 599. Cullen, Pamela V., A Stranger in Blood: The Case Files on Dr John Bodkin Adams, London, Elliott & Thompson, 2006.
His reticence is especially perplexing since he was known for his doggedness. As Lord Devlin later said of him: 'He could be downright rude but he did not shout or bluster. Yet his disagreeableness was so pervasive, his persistence so interminable, the obstructions he manned so far flung, his objectives apparently so insignificant, that sooner or later you would be tempted to ask yourself whether the game was worth the candle: if you asked yourself that, you were finished.'
598–599., p. Macmillan, Harold. The Macmillan Diaries, The Cabinet Years, 1950–1957, ed. Peter Catterall (London, Macmillan, 2003). 124–126., pp. 109–111.
Documentation was found recording the purchase, though Adams denied it had taken place. 273). ^, pp. 102–108., pp. 80–81. ^, pp. 132–144., pp.
143–144., p. 126–131., pp.
145–147., p. 548. 's 1963 book The Ability to Kill originally contained a chapter on Adams. 50 promotional copies were produced before the publishers got cold feet and removed the chapter for fear of being sued. The book was finally published with an alternative chapter included., p.
550–552. ^ 10 August 2004 at the. At shycyberchamber.com., p. 553–554. 'Whenever the name of Dr John Bodkin Adams comes up, I am asked, 'Did he do it?' 'Was he guilty?'
And I always answer, 'No'. 199. Quoted in, p. 165. Though Hoskins and Hallworth did visit Eastbourne in 1956 and talked to local residents and the police. Surtees interviewed many local residents and Adams himself, though decades after the events.
He 'may have had more victims than Shipman – and he had a far more successful career as a serial killer' from., p. 637. Devlin, pp. 10, 199. Mahar, C. Easing the Passing: R v Adams and Terminal Care in Post-war Britain. Social History of Medicine Vol.
159-60. Mahar, pp. 161-2. Mahar, pp. 163-4. Devlin, p.
124. Mahar, p. 166.
Mahar, p. 167. Devlin, pp. 124, 170.
Devlin, p. 199. Devlin, p.
218. Mahar, pp. 169-70. The summing up affirmed 'that a doctor will be immune from criminal liability if his or her primary intention in these circumstances can be characterised as an intention to relieve pain, rather than an intention to hasten death.' ( 13 November 2008 at the.). 28 August 2007 at the. Sources.
(1989). The Best We Can Do. London: Penguin. Cullen, Pamela V. A Stranger in Blood: The Case Files on Dr John Bodkin Adams.
London: Elliott & Thompson. Easing the passing: The trial of Doctor John Bodkin Adams.
London: The Bodley Head. Famous Criminal Cases (4). London: Allan Wingate. Two men were acquitted: The trial and acquittal of Doctor John Bodkin Adams. London: Secker & Warburg.
Hallworth, Rodney; Williams, Mark (1983). Where there's a will. The sensational life of Dr John Bodkin Adams.
Jersey: Capstan Press. Robins, Jane (2013). The Curious Habits of Dr Adams: A 1950s Murder Mystery. John Murray. Surtees, John (2000).
The Strange Case of Dr. Bodkin Adams: The Life and Murder Trial of Eastbourne's Infamous Doctor and the Views of Those Who Knew Him. Further reading. Ambler, Eric, The Ability to Kill, 1963 (promotional edition with chapter on Adams only – subsequent editions had it removed due to libel fears).
Bedford, Sybille, The Trial of Dr. Simon and Schuster, Inc. Bedford 'contributes the reporter's arts of observation, selection and emphasis.' —From the 1962 editor's preface. Cavendish, Marshall. Murder Casebook 40 Eastbourne's Doctor Death, 1990. Chapman, D.
'Jill's Letter' in The Postmodern Malady, Concept, 2010. Gaute, J.H.H. And Robin Odell, The New Murderer's Who's Who, Harrap Books, London, 1996. External links Wikiquote has quotations related to:., Time, New York, 28 January 1957 (Account of the initial trial, which because of libel and contempt laws could not have been published in Britain at the time).